EP4720278A1 - Methods and compositions for modulating plasminogen activator inhibitor-1 (pai-1) - Google Patents

Abstract

A siRNA molecule for inhibiting expression of plasminogen activator inhibitor-1 (PAI-1) is disclosed and may have a sense strand and an antisense strand. The sense strand may have at least 80% sequence identity to any one of SEQ ID NOs: 3, 5, 7, and 9. In some embodiments, the antisense strand may have at least 80% sequence identity to any one of SEQ ID NOs: 4, 6, 8, and 10. The sense strand and/or the antisense strand may comprise one or more modified nucleotides. The sense strand and/or the antisense strand may comprise of a ligand. A lipid nanoparticle comprising the siRNA molecule is also disclosed. Methods of using the siRNA molecule or the lipid nanoparticle comprising the siRNA molecule to treat a subject who suffers from at least one PAI-1-associated condition, disease or disorder are also disclosed.

Description

METHODS AND COMPOSITIONS FOR MODULATING PLASMINOGEN ACTIVATOR INHIBITOR-1 (PALI)

TECHNICAL FIELD

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

BACKGROUND

Plasminogen activator inhibitor 1 (PAI-1) is a blood protein that slows the breakdown of blood clots, thereby promoting clot stabilization and thrombosis. High PALI levels are associated with increased thrombosis risk, and PALI levels also increase with the progression several conditions, diseases, and disorders, including atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive disorders, sepsis, lung fibrosis, Alzheimer’s disease and inflammatory disorders. PALI plays a crucial role in the metabolic syndrome and in the progression of fat deposition. In mouse models of thrombosis and Alzheimer’s disease, decreasing PALI significantly reduces thromboses and brain plaques, respectively. Humans with PAI-1 deficiency have a lower incidence of cardiovascular morbidity and a longer lifespan. These individuals may suffer prolonged bleeding following severe trauma, but they do not experience spontaneous bleedings nor are at risk of internal bleedings. Decreasing circulating PAI-1 levels has both experimental and therapeutic value, although no clinical suitable, long-duration approach is currently available. RNA gene therapy represents an alternate option to regulate the levels of target proteins that are not amenable to traditional small-molecule or protein-based therapies.

SUMMARY

The present invention relates to small interfering RNA (siRNA) molecules for reducing or inhibiting the expression of the PALI gene in cells treated with compositions comprising the siRNA molecules.

In a first aspect, the present invention provides an siRNA molecule for inhibiting expression of PALI in a cell, wherein said siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region. In some embodiments, the antisense strand comprises a region of complementarity to an mRNA encoding PAI-1. According to one embodiment of the disclosure, the PAI-1 is human.

In other embodiments, the sense strand of the siRNA molecule has at least 80% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has at least 80% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

In other embodiments, the sense strand of the siRNA molecule has at least 85% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has at least 85% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

In other embodiments, the sense strand of the siRNA molecule has at least 90% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has at least 90% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

In other embodiments, the sense strand of the siRNA molecule has at least 95% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has at least 95% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

In other embodiments, the sense strand of the siRNA molecule has at least 70% sequence identity to SEQ ID NO: 3, and the antisense strand has at least 70% sequence identity to SEQ ID NO: 4.

In other embodiments, the sense strand of the siRNA molecule has at least 80% sequence identity to SEQ ID NO: 3, and the antisense strand has at least 80% sequence identity to SEQ ID NO: 4.

In other embodiments, the sense strand of the siRNA molecule has at least 90% sequence identity to SEQ ID NO: 3, and the antisense strand has at least 90% sequence identity to SEQ ID NO: 4. In other embodiments, the sense strand of the siRNA molecule has at least 95% sequence identity to SEQ ID NO: 3, and the antisense strand has at least 95% sequence identity to SEQ ID NO: 4.

In some embodiments, the sense and/or antisense strand of the siRNA molecule comprise of one or more nucleotide. For example, about 10% to about 50% of the nucleotides in the sense and/or antisense strand comprises modified nucleotides.

In such embodiments, the one or more modified nucleotides may be a 2'-O-alkyl modified nucleotide, a 2’ -halogen modified nucleotide, a nucleotide comprising a phosphorothioate group, a deoxy-nucleotide, a 3 ’-terminal deoxy -thymine nucleotide, a 2'-deoxy-modified nucleotide, a 2’- amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’- hydroxyl-modified nucleotide, a 2’ -methoxy ethyl modified nucleotide, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5 ’-phosphate, a nucleotide comprising a 5’- phosphate mimic, a glycol modified nucleotide, a 2-O-(N-methylacetamide) modified nucleotide, or any combinations thereof. For example, the one or more modified nucleotides is a 2'-O-methyl modified nucleotide and/or a 2’ -fluoro modified nucleotide

In still other embodiments, the sense strand of the siRNA molecule comprises one or more modified nucleotides and has at least 80% identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. For example, the sense strand may comprise one or more modified nucleotides and have (a) at least 85% identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, (b) at least 90% identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, or (c) at least 95% identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19

In still other embodiments, the antisense strand comprises one or more modified nucleotides and has at least 80% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20. For example, the antisense strand of the siRNA molecule the antisense strand may comprise one or more modified nucleotides and have (a) at least 85% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, (b) at least 90% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, or (c) at least 95% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20. The siRNA molecule of the present invention comprises double-stranded region that is at least 15 nucleotides long. For example, the length of the double-stranded region may be 15 to 35 nucleotides in length, 18 to 35 nucleotides in length, or 20 to 30 nucleotides in length.

The siRNA molecule of the present invention may comprise a ligand that is conjugated to the 3’ end, the 5’ end, and/or to one or more nucleotides of the sense and/or antisense strand.

In some embodiments, the ligand conjugated to the siRNA molecule may be an antibody, a peptide, an amino acid, an aptamer, a phosphate group, a cholesterol moiety, a lipid, a cell-penetrating peptide polymer, a sugar group, and derivatives thereof.

In other embodiments, the sugar group comprises a sugar monomer, an oligosaccharide, and/or derivatives thereof. For example, the sugar group may be N-acetylgalactosamine.

In another aspect, the sense and/or antisense strand of the siRNA molecule comprise of a 3’ and/or 5’ overhang of 1 to 6 nucleotides, or any number of nucleotides therebetween.

In some embodiments, the sense strand and the antisense strand of the siRNA molecule forms a double-stranded region with no overhangs and comprises blunt ends at both 5’ and 3’ ends.

In another aspect, the present invention provides a lipid nanoparticle (LNP) comprising the siRNA molecule for reducing or inhibiting the expression of PAI-1 in a cell. The LNP of the present invention comprises: an siRNA molecule against human PAI-1 mRNA; an ionizable cationic lipid having a pKa of between 5.5 and 7.0 and that is present at between 10 mol% and 85 mol%, or any mol% therebetween. In some embodiments, the ionizable cationic lipid is present at between 30 mol% and 55 mol%, or any mol% therebetween. The LNP also comprises a neutral, vesicleforming lipid comprising of a phospholipid and/or a triglyceride; a sterol; and a hydrophilic polymer-lipid conjugate present at between 0.5 mol% and 5 mol%, or any mol% therebetween.

In some embodiments, the neutral, vesicle-forming lipid is present at between 15 mol% and 60 mol%, or any mol% therebetween. In still other embodiments, the sterol is cholesterol or a derivative thereof. In other embodiments, the sterol is present at between 15 ml% and 65 mol%, or any mol% therebetween. In another aspect, the present invention provides a pharmaceutical composition comprising an siRNA molecule for reducing or inhibiting PAI-1 expression and thereby treating and/or preventing one or more conditions, diseases or disorders for which it is desirable to modulate PAI- 1 levels, such as in thrombosis, atherosclerosis, coronary artery disease, obesity, sepsis, lung fibrosis , diabetes, cancer, liver necrosis, a neurocognitive disorder, an inflammatory disorder, or any combinations thereof. In some examples, the pharmaceutical composition comprises lipid nanoparticles having lipid components as described herein and encapsulating siRNA targeting PAI-1 mRNA to achieve controlled and/or sustained reduction of PAI-1 levels in the blood or other bodily sites.

In some embodiments, there is provided a pharmaceutical composition comprising an siRNA molecule as described herein, formulated in an unbuffered solution. For example, the unbuffered solution may be saline and/or water.

In some embodiments, there is provided a pharmaceutical composition comprising an siRNA molecule as described herein, formulated in a buffered solution. For example, the buffered solution may comprise acetic, prolamine, carbonate, and/or phosphate. In one embodiment, the buffered solution may be phosphate buffered saline.

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.

In another aspect, there is provided a method for inhibiting PAI-1 expression in a cell by contacting the cell with a lipid nanoparticle or pharmaceutical composition comprising the PAI-1 siRNA molecule described herein. In one embodiment, the cell is in a subject. In another embodiment, the subject is a human.

In another aspect, there is provided a method of inhibiting PAI-1 expression in a human suffering from a PAI-1 associated condition, disease or disorder. In one embodiment, the PAI-1 associated condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, sepsis, lung fibrosis, liver necrosis, a neurocognitive disorder, Alzheimer’s disease, an inflammatory disorder, or any combinations thereof. In one embodiment, the PAI-1 associated condition, disease or disorder is aging and increased lifespan. In another embodiment, the thrombotic disorder is venous thrombosis, arterial thrombosis, geriatric thrombosis, microbial infection-associated thrombosis, viral infection-associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with the elderly, thrombosis associated with increased inflammation, thrombosis associated with aging, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.

A further embodiment includes the use of lipid nanoparticle or pharmaceutical composition described above to inhibit the expression of PALI in a human suffering from a PAI-1 associated condition, disease or disorder as described herein.

In another aspect, there is provided a method of treating a patient having a PA I -associated condition, disease or disorder. In one embodiment, the PALI -associated condition, disease or disorder may be thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, a neurocognitive disorder, Alzheimer’s disease, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof. In one embodiment, the PALI associated condition, disease or disorder is aging and increased lifespan. In some embodiment, the thrombotic disorder may be venous thrombosis, arterial thrombosis, geriatric thrombosis, microbial infection- associated thrombosis, viral infection-associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with aging, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.

A further embodiment includes use of the pharmaceutical composition or lipid nanoparticle described above to treat a PALI -associated condition, disease or disorder as described herein, by modulating levels of PAI- 1 in the blood or other bodily sites in a patient in need of such treatment thereof.

Another embodiment includes use of the pharmaceutical composition or lipid nanoparticle described above in the manufacture of a medicament to treat a PAI- 1 -associated condition, disease or disorder as described herein, by modulating levels of PAI- 1 in the blood or other bodily sites. In one embodiment, the PAI-1 associated condition, disease or disorder is aging and increased lifespan. In some embodiments, the PAI- 1 -associated condition, disease or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, a neurocognitive disorder, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof. In one embodiment, the thrombotic disorder may be venous thrombosis, arterial thrombosis, microbial infection-associated thrombosis, viral infection-associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with aging, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.

Yet further, there is provided a method of treating a patient having a PAI- 1 -associated condition, disease, or disorder resulting by modulating levels of PALI in the blood or other bodily sites comprising administering the pharmaceutical composition or lipid nanoparticle described herein to a patient in need of such treatment thereof.

According to a further example of any aspect or embodiment herein, the pharmaceutical composition or lipid nanoparticle described herein is for inhibiting the expression of PALI, thereby treating and/or preventing one or more blood clotting 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 clotting 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 1A shows plasma levels of PAI- 1 protein in mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting PAI-1 (siPAI-1) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid one week post-injection. siPAI-1 corresponds to msPAI- 1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure IB shows hepatic levels of PAI- 1 mRNA relative to control (%) for a luciferase siRNA control (siLuc), and siRNA targeting PAI-1 corresponding to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) one week post injection.

Figure 1C shows plasma levels of PAI- 1 protein in mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting PALI (siPALl) three, ten, fourteen, and twenty days postinjection. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure ID shows a representative rotational thromboelastometry (ROTEM) curve of blood from mice treated with control siRNA luciferase (siLuc) or siRNA targeting PALI (siPALl). siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure IE shows the percent of clot lysis at 30, 45, and 60 min of whole blood of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PALI (siPALl). siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure IF shows the alpha angle of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PALI (siPALl) one week post-injection. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure 1G shows the clot time of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPALl). siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). Figure 1H shows the maximum clot firmness (MCF) of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) one-week post-injection. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure 2A shows the clot weight of 9-12 weeks old mice treated with control siRNA targeting luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) four days post-ligation of IVC. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

Figure 2B shows the plasma levels of PAI-1 relative to body weight in mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting PALI (siPALl) four days post IVC ligation. siPALl corresponding to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). was administered to mice 3 days prior to IVC ligation.

Figure 2C shows representative histology slides of clots in mice treated with control siRNA luciferrsase (siLuc) or siRNA targeting PAI-1 (siPALl) four days post IVC ligation. siPALl corresponding to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

Figure 2D shows a probability of survival plot of 80-week-old mice treated with control siRNA targeting luciferase (siLuc) or siRNA targeting PALI (siPALl) up to four days post IVC ligation. siPALl corresponding to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

Figure 2E shows the clot weight of 80 weeks old mice treated with control siRNA targeting luciferase (siLuc) or siRNA targeting PALI (siPALl) four days post IVC ligation. siPALl corresponding to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

Figure 2F shows the levels of cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-y), interleukin 1 -alpha (IL- 1 alpha), interleukin 6 (IL-6) and interleukin 12 (IL-12), 80 week elderly mice treated with control siRNA luciferase (siLuc) or siRNA targeting PALI (siPALl) four days post-ligation. siPALl corresponding to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

Figure 3A shows plasma levels of PAI-1 protein in mice fed a high fat diet (HFD) or low fat diet (LFD).

Figure 3B shows hepatic PAI-1 mRNA expression of high fat diet mice (HFD) relative to low fat diet mice (LFD) (%).

Figure 3C shows hepatic PALI mRNA levels in relation to the mouse body weight.

Figure 3D shows plasma levels of PALI protein in high fat diet mice treated with control siRNA targeting luciferase (siLuc) and siRNA targeting PALI (siPALl) one week post-injection. siPAL 1 corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure 3E shows hepatic PALI mRNA levels relative to control (%) for high fat diet mice treated with a luciferase siRNA control (siLuc), and siRNA targeting PALI corresponding to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2) one week post injection.

Figure 4A shows serum levels of alkaline phosphatase (ALP) 5 hours post-injection of mice treated with phosphate buffered saline (PBS) or siRNA targeting PALI (siPALl) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal levels of ALP.

Figure 4B shows serum levels of aspartate transaminase (AST) 5 hours post-injection of mice treated with phosphate buffered saline (PBS) or siRNA targeting PALI (siPALl) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal levels of AST.

Figure 4C shows serum levels of alanine transaminase (ALT) 5 hours post-injection of mice treated with phosphate buffered saline (PBS) or siRNA targeting PALI (siPALl) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal levels of ALT.

Figure 4D shows serum levels of blood urea nitrogen (BUN) 5 hours post-injection of mice treated with phosphate buffered saline (PBS) or siRNA targeting PAI-1 (siPAI-1) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal levels of BUN.

Figure 4E shows white blood cell (WBC) count 7 days post-injection of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) at 1 or 3 mg/kg dose. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal WBC count.

Figure 4F shows red blood cell (RBC) count 7 days post-injection of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) at 1 or 3 mg/kg dose. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal RBC count.

Figure 4G shows platelet count 7 days post-injection of mice treated with control siRNA luciferase (siLuc) or siRNA targeting PALI (siPALl) at 1 or 3 mg/kg dose. siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). The two dashed lines represent the lower and upper threshold of normal platelet count.

Figure 5A shows weekly plasma PAI-1 levels of elderly mice (72-76 weeks old) starting at 3 days post-injection with the first dose of control siRNA targeting luciferase (siLuc) or siRNA targeting PALI (siPALl), and weekly injections thereafter. siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure 6A shows plasma levels of PALI in apolipoprotein E knockout (ApoE '^A) mice at 3 and 7 days post-injection with control siRNA targeting luciferase (siLuc) or siRNA targeting PALI (siPALl). siPALl corresponds to msPALl siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2). Figure 6B shows plasma cholesterol levels in apolipoprotein E knockout (ApoE ’^/’) mice at 3 and 7 days post-injection of control siRNA targeting luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to msPAI-1 siRNA sequence (duplex siRNA of SEQ ID Nos 11 and 12) (Table 2).

Figure 7 shows human PAI-1 mRNA relative to control (%) for an LNP containing control siRNA luciferase (siLuc), or LNP containing duplex human PAI-1 siRNA sequences, sequence A (duplex siRNA of SEQ ID Nos 13 and 14), sequence B (duplex siRNA of SEQ ID Nos 15 and 16), sequence C (duplex siRNA of SEQ ID Nos 17 and 18), and sequence D (duplex siRNA of SEQ ID Nos 19 and 20) (Table 2) after addition to HEPG2 cells in vitro at 0.3, 1, or 3 pg/mL.

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”. siRNA

The expression “siRNA molecule against plasminogen activator inhibitor-1 (PALI)” as used herein comprise 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 PAI- 1 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 PALI 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 one embodiment, the siRNA molecule reduces or inhibits the expression of coagulation factors to alter clotting. In one embodiment, the coagulation factor is PALI. 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 singlestranded. Further non-limiting examples of the disclosure are described in more detail hereinafter. In some embodiments, the siRNA comprises a double-stranded region that is 15 to 35 nucleotides in length. For example, the double-stranded region may be 18 to 35 nucleotides in length, or 20 to 30 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 PAI-1 as measured in vitro or in vivo. Inhibition or reduction of expression of PAI- 1 is achieved when the value 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%. Expressed another way, inhibition or reduction of expression of PAI-1 is achieved in a cell transfected with a PAI-1 specific siRNA is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%.

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 PAI-1”, includes inhibition or reduction of PALI 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. Expressed another way, “inhibiting or reducing expression of PAI-1” is achieved when the levels of PALI protein and/or mRNA is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%. 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, a 2'-O-alkyl modified nucleotide, a 2’-halogen modified nucleotide, a nucleotide comprising a phosphorothioate group, a deoxy -nucleotide, a 3 ’-terminal deoxy -thymine nucleotide, a 2'-deoxy-modified nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl- modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’-hydroxyl-modified nucleotide, a 2’- methoxyethyl modified nucleotide, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5’-phosphate, a nucleotide comprising a 5’-phosphate mimic, a glycol modified nucleotide, a 2-O-(N-methylacetamide) modified nucleotide, and combinations thereof.

The sense and antisense strands of the siRNA may have sequence identity to any one of the nucleotide sequences set forth in Table 1, and Table 2 below. More typically, the siRNA has sequence identity to the human nucleotide sequences set forth in Table 1 or Table 2. 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. Sequence identity is expressed as a percentage. When referring to a sequence identity of at least a certain number, it is understood to include any value between the stated number and 100%. As one non-limiting example, where stated that the siRNA molecule comprises a nucleotide sequence having a sequence identity of at least 90% of a referenced nucleotide sequence, this includes siRNA molecules with nucleotide sequences which have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the referenced nucleotide sequence.

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 (Camacho C, et al TL. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15; 10:421 ), which is well-known to those of skill in the art.

In some embodiments, the siRNA molecule for inhibiting expression of PAI- 1 in a cell comprises a sense strand and an antisense strand forming a double-stranded region. In some embodiments, the antisense strand comprises a region of complementarity to an mRNA encoding PALI. According to one embodiment of the disclosure, the PALI is human.

In other embodiments, the sense strand of the siRNA molecule has at least 80% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has at least 80% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. For example, the sense strand of the siRNA molecule may have at least 85% , at least 90%, or at least 95% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand may have at least 85% , at least 90%, or at least 95%, sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

In one embodiments, the sense strand of the siRNA molecule has at least 70% sequence identity to SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 4. For example, the sense strand of the siRNA molecule may have at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 3, and the antisense strand may have at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 4.

In one embodiment, the sense strand of the siRNA comprise a nucleotide sequence which differs by no more than 4 nucleotides of any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. In some embodiments, the antisense strand of the siRNA comprise a nucleotide sequence which differs by no more than about 4 nucleotides of any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In still other embodiments, the sense strand of the siRNA molecule may comprise one or more modified nucleotides and have at least 80% identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand may comprises one or more modified nucleotides and have at least 80% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20. For example, the sense strand may comprise one or more modified nucleotides and have at least 85%, at least 90%, or at least 95%, identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand of the siRNA molecule may comprise one or more modified nucleotides and have at least 85%, at least 90%, or at least 95%, sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

In one embodiment, the sense strand of the siRNA molecule comprise a nucleotide sequence which differs by no more than about 4 nucleotides of any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. In some embodiments, the antisense strand of the siRNA molecule comprise a nucleotide sequence which differs by no more than about 4 nucleotides of any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:20. In some embodiments, the sense strand and/or the antisense strand comprise all of the nucleotide modifications of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 and SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:20, respectively, but this is not mandatory. The sense strand and/or the antisense strand may comprise one or more nucleotide modifications, or none of the nucleotide modifications, of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 and SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO:20, respectively.

In one embodiment, the sense strand or the antisense strand of the siRNA molecule has at least 30% to 100% sequence identity to any one of SEQ ID NOs: 1-20 in Table 1 and Table 2 below. For example, the sense strand or the antisense strand of the siRNA molecule 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: 1-20. In one embodiment, the sense strand or the antisense strand of the siRNA molecule comprise a nucleotide sequence which differs by no more than about 4 nucleotides of any one of SEQ ID NOs: 1-20. In a further embodiment, the sense strand or the antisense strand of the siRNA molecule comprises a nucleotide sequence that differs by no more than about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides from a sequence set forth SEQ ID NOs: 1-20. In one embodiment, the sense strand or the antisense strand of the siRNA molecule comprises a nucleotide sequence that differs by no more than about 10 nucleotides or no more than about 5 nucleotides.

In another embodiment the present disclosure provides one or more exemplary siRNA sequences or duplexes thereof selected from SEQ ID NOs: 3-10 or SEQ ID NOs: 13-20 (human sequences) to inhibit or reduce the expression of PAI- 1.

In one embodiment, the sense strand or antisense strand of the siRNA molecule has at least 30% to 100% sequence identity to any one of SEQ ID NOs: 3-10 in Table 1 or SEQ ID NOs 13-20 in Table 2 below. For example, the sense strand or the antisense strand of the siRNA molecule 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: 3-10 in Table 1 or SEQ ID NOs 13-20 in Table 2 below. In one embodiment, the sense strand or the antisense strand of the siRNA molecule comprises a nucleotide sequence that differs by no more than about 4 nucleotides from a sequence set forth in SEQ ID NOs: 3-10 in Table 1 or SEQ ID NOs 13-20 in Table 2.

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 sense strand or the antisense strand of the siRNA molecule comprises a nucleotide sequence that differs by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides from a sequence set forth in SEQ ID NOs: 3-10 in Table 1 and SEQ ID NOs: 13-20 in Table 2 below. In one embodiment, the sense strand or the antisense strand of the siRNA molecule comprises a nucleotide sequence that differs by no more than about 10 nucleotides or no more than about 8, 7, 6 or 5 nucleotides from a sequence set forth in SEQ ID NOs: 3-10 in Table 1 and SEQ ID Nos: 13-20 in Table 2 below. In another embodiment the present disclosure provides one or more siRNA sequences or duplexes thereof selected from SEQ ID NOs: 3-10 (Table 1) and SEQ ID NOs: 13-20 (Table 2) to inhibit or reduce the expression of PAI- 1.

In another embodiment, the present disclosure provides a siRNA molecule to inhibit or reduce the expression of PAI-1. The siRNA molecule may be a duplex siRNA molecule comprising a sense and an antisense strand. The sense strand or the antisense strand may comprise a nucleotide sequence set forth in one of SEQ ID NOs: 3-10 (Table 1). In some embodiments, the sense strand or the antisense strand comprises a nucleotide sequence that differs by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides from a sequence set forth in SEQ ID Nos: 3-10. In some embodiments, one or more nucleotides of the sense strand and/or the antisense strand comprise a nucleotide modification. In some embodiments, about 10% to about 100% of the nucleotide of the sense strand and/or the antisense strand, including any ranges therebetween, including 20% to 90%, 30% to 80%, 40% to 70%, 50% to 60%, etc., comprise nucleotide modifications. Non-limiting examples of nucleotide modifications include a 2'-O-alkyl modified nucleotide, a 2’ -halogen modified nucleotide, a nucleotide comprising a phosphorothioate group, a deoxy-nucleotide, a 3 ’-terminal deoxy -thymine nucleotide, a 2'-deoxy-modified nucleotide, a 2’- amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’- hydroxyl-modified nucleotide, a 2’ -methoxy ethyl modified nucleotide, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5 ’-phosphate, a nucleotide comprising a 5’- phosphate mimic, a glycol modified nucleotide, a 2-O-(N-methylacetamide) modified nucleotide, and any combinations thereof. In some example embodiments, the nucleotide modifications comprise 2'-O-alkyl modifications. In some example embodiments, the 2'-O-alkyl modifications and 2’-halogen modifications comprise 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 below. Table 1. Base composition of duplex siRNA sequence targeting plasminogen activator inhibitor 1 (PAI-1) mRNA.

Table 2. Base modification of duplex siRNA sequences targeting PAI-1 mRNA. “r” designates unmodified base, “m” designates 2’O-methylated base, BOLD designates a DNA 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 the 3’ end and the 5’ end 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 molecule is covalently bound to one or more ligand to form a conjugate. In some aspects, the ligand 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, one of 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 one or more nucleotides at a position that is not at the 3' end or 5' end of the sense and/or antisense strand. Examples of ligands 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, and derivatives thereof. In one embodiment, the sugar group comprises a sugar monomer, an oligosaccharide and/or derivatives thereof. In one aspect, the ligand comprises of N- Acetylgalactosamine (GalNAc).

Lipid nanoparticles

In one embodiment, the disclosure provides a nucleic acid against PAI-1 mRNA that is encapsulated within a lipid nanoparticle. In one embodiment, the nucleic acid is for inhibiting or reducing expression of PAI- 1.

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 cationic lipid that may associate or complex with the nucleic acid. The term “ionizable cationic 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 ionizable cationic lipid has a head group comprising an amino group. In some embodiments, the ionizable cationic lipid comprises a protonatable tertiary amine (e.g., pH titratable) head group, C16 to C18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. In some embodiments, the ionizable cationic lipid may be MC3, CL4H6, SM-102, ALC-0315, ALC-0317, CL1H6, CL15H6, CL1D6, ALC-0159, or any combination thereof.

In certain embodiments, the ionizable cationic lipid may be present at 20 mol% to 70 mol%, 30 mol% to 55 mol%, or 35 mol% to 55 mol%, of total lipid present in the lipid nanoparticle. For example, the cationic lipid content may be present in the LNP at 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, or 70 mol%, or any mol% therebetween. 1 The lipid nanoparticle (LNP) described herein may comprise a “vesicle-forming lipid” or “helper lipid” in addition to the ionizable cationic lipid. In the context of the present disclosure, the term “vesicle-forming 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), l-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC). In certain embodiments, the vesicleforming lipid is DOPC, DSPC or sphingomyelin. In one embodiment, the vesicle-forming lipid is DSPC. In other embodiments, the vesicle-forming The vesicle-forming lipid content may include mixtures of two or more different types of different helper lipids.

In certain embodiments, the vesicle-forming lipid may be present at 20 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, or 40 mol% to 60 mol%, or any mol% therebetween, of total lipid present in the lipid nanoparticle. The vesicle-forming lipid content is determined based on the total amount of lipid in the lipid nanoparticle, including the sterol.

In certain embodiments, the phosphatidylcholine may be present at 20 mol% to 60 mol%, 25 mol% to 60 mol%, 30 mol% to 60 mol%, 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'-hydroxyethyl 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%, or any mol% ranges therebetween, based on the total lipid present in the lipid nanoparticle. In another embodiment, the sterol comprises a cholesterol or a derivative thereof 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%, or any mol% ranges therebetween 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%, 0.5 mol% to 3 mol%, 0.5 mol% to 2.5 mol%, 0.5 mol% to 2.0 mol%, 0.5 mol% to 1.8 mol%, or any mol% ranges therebetween, of total lipid present in the lipid nanoparticle. In another embodiment, the PEG-lipid conjugate is present in the nanoparticle at 0 mol% to 5 mol%, 0.5 mol% to 3 mol%, 0.5 mol% to 2.5 mol%, 0.5 mol% to 2.0 mol%, 0.5 mol% to 1.8 mol%, or any mol% there between, of total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticle at 0 mol% to 5 mol%, 0 mol% to 3 mol%, 0 mol% to 2.5 mol%, 0 mol% to 2.0 mol%, 0 mol% to 1.8 mol%, or any mol% ranges therebetween, of total lipid in the lipid nanoparticle.

Methods to treat or prevent a PAI-l-associated condition, disease, or disorder

In another aspect, the present disclosure provides methods of treating a subject having any disorder or condition that would benefit from a reduction in PAI-1 expression.

In one embodiment, the PAI- 1 -associated condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, Alzheimer’s disease, a neurocognitive disorder, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof.

In another embodiment, the PAI-1 associated condition, disease or disorder is associated with aging and increased lifespan. This includes a “thrombotic disorder”, which as used herein includes any condition, of any severity, that results in abnormal formation of clots in a subject, such as but not limited to a blood clotting disorder. The thrombotic disorder includes but is not limited to venous thrombosis, arterial thrombosis, thrombosis associated with the elderly, microbial infection-associated thrombosis, viral infection-associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof. The methods include administering to the subject a therapeutically effective amount of the siRNA, optionally encapsulated in a lipid nanoparticle or conjugated to a ligand, thereby treating the subject or providing a prophylactic (preventive) effect.

As used herein, the term “subject” includes any human or non-human mammalian subject that would benefit from a reduction in PAI-1 expression relative to lack of treatment thereof. This includes a prophylactic (preventive) 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, in a subject having a PAI- 1 -associated condition, disease or disorder that would benefit from reduction in PAI-1 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 condition, disease, or disorder that would benefit from reduction in PAI-1 expression. Non-limiting examples of symptoms include clot formation, thickening of arteries, hardening of arteries, plaque buildup in arteries, increased weight gain, increased blood sugar levels, metastasis of cancerous cells, growth of cancerous cells, necrosis of tissues, decrease in any cognitive function, increased levels of systemic inflammatory markers, or any combinations thereof. 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 condition, disease, or disorder that would benefit from reduction in PAI-1 expression. In one embodiment, the disclosure provides methods of preventing at least one symptom, e.g., clot formation, in a subject having a thrombotic disorder that would benefit from reduction in PAI-1 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 condition, disease, or disorder that would benefit from reduction in PAI-1 expression.

In one embodiment, the administration of the siRNA to the subject causes a decrease in a symptom of a PAI- 1 -associated condition, disease or disorder.

In one embodiment, the administration of the siRNA to the subject causes a decrease in thrombosis, and/or a decrease in PAI-1 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 PAI- 1. PAI-1 expression or activity can be assessed as set forth in the Example section herein.

Further methods for assessing knockdown, inhibition and/or reduction in PAI-1 expression include quantifying hepatic PAI-1 mRNA level, plasma PAI-1 protein concentration, and/or PAI-1 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%. Expressed another way, inhibition or reduction in PAI-1 expression is achieved when hepatic PAI-1 mRNA level, plasma PAI-1 protein concentration, and/or PAI-1 activity is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%.

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 PAI-1- associated condition, disease, or disorder. Pharmaceutical formulations

In some embodiments, the siRNA or lipid nanoparticle comprising a nucleic acid reducing expression PAI-1 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 carrier. For example, a “pharmaceutically acceptable carrier” means an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

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

Mice

Murine studies were approved by the respective Animal Care Committee for each institution. C57BL/6J (Jackson Labs, Bar Harbor, ME, stock # 000664) mice of 8-10 weeks old and elderly mice of 72-80 weeks old were used in adult and elderly PAI-1 knockdown studies respectively. Diet-induced obese C57BL/6J mice (Jackson Labs, Bar Harbor, ME, stock#380050) age 17 weeks were used for the obese PAI-1 knockdown study. For other obese PAI-1 studies C57BL/6J mice were either fed a high fat diet (HFD 60% fat; catalog DI 2492, Research Diets) or ad libitum low- fat diet (LFD, 10-13% fat; D12450, Research Diets). siRNA-LNP formulation and injections siRNA (Integrated DNA Technologies, Coralville, USA) targeting PAI-1 or Luciferase (a negative, scramble siRNA control), were encapsulated in LNP. Briefly, siRNA 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 comprised ofDSPC, cholesterol and PEG-DMG at a 10:38.5: 1.5 % molar ratio, with either 50% DLin-MC3-DMA or ALC-0315 (Avanti Polar Lipids, Birmingham, AL). The LNP were dialyzed overnight against Dulbecco’s phosphate buffered saline (PBS) at pH 7.4 in 500-fold volume excess. To determine siRNA concentration and encapsulation efficiency, RiboGreen assay (Quant-IT Ribogreen RNA Assay Kit, ThermoFisher) was performed with or without Triton X-100 detergent. Cholesterol content was measured using the Cholesterol E Assay Kit (Wako Chemicals, Mountain View, CA USA). The LNP were diluted to a final concentration of 0.1 or 0.3 mg siRNA per mL in PBS (pH 7.4) or in freezing buffer (10 % sucrose, lOmM L- Histidine, pH 7.4). Mice were injected intravenously by tail vein injections with a single dose of 1 or 3 mg siRNA per kg body weight (mg/kg), or repeat dose at weekly intervals. Liver, serum, and plasma extraction from mice

Endpoint blood samples were collected via cardiac puncture and liver tissues were surgically excised under isoflurane anesthesia. Blood was collected into a syringe containing sodium citrate (0.32 % final) and platelet rich plasma (PRP) was separated from whole blood by spinning at 1500 x g for 10 minutes. Serum for toxicology analysis was isolated in the same way as the plasma, however the blood was collected without sodium citrate and was allowed to clot for 40 minutes before centrifuging. For non-endpoint blood samples, blood was collected retro-orbitally. mRNA extraction and quantification

Livers were homogenized in Trizol (ThermoFisher, Waltham, USA). DNA and RNA were isolated by chloroform and isopropanol precipitation. DNA was digested by incubating samples with TURBO Dnase and 10-times TURBO Dnase Buffer (ThermoFisher, Waltham, USA). DNAse was removed by repeating the precipitation of RNA in Trizol, chloroform and isopropanol. Reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA). Quantitative PCR (qPCR), was performed using the SYBR Green Master Mix (ThermoFisher, Waltham USA), and DNA primers against PAI-1 (Integrated DNA Technologies). PAI-1 expression was quantified using the DDCt method, relative to the expression of the housekeeping gene Peptidylprolyl isomerase A. Data was collected and analyzed using the QuantStudio 6.

Analysis of PALI levels in plasma

The total plasma PAI-1 concentration was analyzed with a mouse total PALI ELISA kit (IMSPAI1KTT, Innovative Research) following the manufacturer’s guidelines. For optimum results, alterations to the manufacturer’s protocol included loading 100 pL of plasma for each sample and primary antibody incubation time extended to 45 minutes.

Coagulation profile

Rotational thromboelastometry (ROTEM) (Rotem Delta, Werfen S.A., Spain) was performed according to the manufacturer’s instructions. Murine whole blood was mixed with 20 pL of 0.2 M CaCh, 20 pL EXTEM reagent containing tissue factor (Werfen S.A., Spain), and recombinant human tPA (H4UTPA85SC100UG, Innovative Research) at a final concentration of 500 ng/mL. All reagents were allowed to incubate until they reached 37 °C. Each test was allowed to proceed for 1.5 hours. IVC ligation

Mice were intravenously injected with 3 mg/kg siPAI-1 or siLuc. Three days post-injection, complete IVC ligation was performed. Briefly, anesthesia was induced using 4% isoflurane in 100% oxygen until loss of the righting reflex. Mice were transferred to the surgical board and anesthesia was maintained with 1-2% isoflurane in 100% oxygen. Adequate depth of anesthesia was confirmed with absent response to pain elicited by toe pinch. The abdomen was sterilely prepped and a laparotomy was created. The bowel was eviscerated and protected with saline soaked gauze to limit insensible fluid losses. The IVC was carefully exposed and ligated using 7- 0 prolene suture just inferior to the renal branches. Any additional venous branches between the renal and inferior iliac veins were similarly ligated. Following ligation, the bowel was repositioned into the abdominal cavity, and the laparotomy site was sutured closed. For post-operative analgesia, buprenorphine was administered subcutaneously. Mice were then allowed to recover from anesthesia. Four days post-ligation, the mice were euthanized for collection of blood, liver tissues, and the IVC thrombus for subsequent analysis.

Toxicological analysis

Mice were injected intravenously with either PBS or siPAI-1 at a dose of 3 mg/kg. Five hours postinjection, the mice were euthanized, and serum samples were collected as described above. Serum samples were to Idexx BioAnalytics (North Grafton, MA USA) for comprehensive toxicology profiling. The panel included measurements of biochemical indicators of liver and kidney fuction:Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), gamma-glutamyl transferase (GGT), alongside markers of metabolic health such as lipemia, blood urea nitrogen (BUN), creatine (CREA).

Blood cells count analysis

Whole blood was collected and blood count was performed using Heska Element HT5.

Blood cholesterol analysis

Cholesterol levels were assessed in plasma samples using the Total Cholesterol E Kit from Fujifilm. Screen siRNA targeting PALI in human hepatocyte (HEPG2) cell culture

HepG2 cells were transfected with a single dose (0.3, 1 or 3 ng/mL) of various siRNA sequences targeting PAI-1 encapsulated in LNPs. PAI-1 mRNA levels were quantified at 24 hours as described above.

Example 1: siRNA knock down of PAI-1 in vivo and effects on clotting ex vivo.

This example demonstrates that siRNA knocks down PAI-1 mRNA in mice, resulting in depletion of circulating PAI-1 protein and decreased PAI-1 activity in vivo.

Control siRNA targeting luciferase (siLuc), or siRNA targeting mouse PAI-1 sequences (siPAI-1) (duplex siRNA of SEQ ID Nos 11 and 12), was encapsulated in lipid nanoparticles containing MC3 or ALC-0315 (ALC) as the ionizable lipid, and administered to mice intravenously as described in the Material and Methods.

ELISA as described in the Materials and Methods was used to quantify PALI protein levels in mouse blood plasma after administration of siLuc or mouse siPAI-1 one week prior to blood sampling.

The results are shown in Figure 1 A. Compared to siLuc-treated mice, plasma PALI protein levels are significantly reduced in mice one week after administration with mouse siPALl (duplex siRNA of SEQ ID Nos 11 and 12 encapsulated in both lipid nanoparticle containing MC3 (siPAi- 1 MC3), and lipid nanoparticle containing ALC-0315 (siPALl ALC).

PCR (qPCR) as described in the Materials and Methods was used to quantify hepatic PALI mRNA levels after administration of mouse siPALl (duplex siRNA of SEQ ID Nos 11 and 12) of Table 2, to mice 1 week weeks prior to tissue collection, and compared to control siRNA targeting Luciferase (siLuc). The results are shown in Figure IB. Hepatic PAI-1 mRNA levels significantly reduced in liver from mice one week after administration with mouse siPAI-1 (SEQ ID Nos 11 and 12), compared to siLuc-treated mice.

A time course study was performed and ELISA as described in the Materials and Methods was used to quantify PAI-1 protein levels in blood plasma at 3, 10, 14 and 20 days post-injection with control siLuc or mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2 at a single dose of 3 mg/kg.

The results are shown in Figure 1C. Plasma PAI-1 levels are significantly depleted at day 3 and day 10 post-injection with mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, compared to siLuc- treated mice.

Rotational thromboelastometry (ROTEM) as described in the Materials and Methods was used to measure clot properties ex vivo in blood from mice treated with siLuc and mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2. Percent of clot lysis, rate of clot formation, clot time, and clot stiffness were quantified from ROTEM.

A representative ROTEM curve is shown in Figures ID.

Figure IE compares percent of clot lysis at 30, 45, and 60 minutes into the assay, in blood from mice treated with siLuc and mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, showing that blood from siPAI-1 treated mice is protected from clot lysis compared to siLuc-treated mice.

Figure IF and 1G compares the rate of (alpha angle) and time to clot formation, respectively, in blood from mice treated with siLuc and mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, showing that both siLuc and siPAI-1 treated mice formed a clot at similar rates.

Figure 1H compares maximum clot stiffness (MCF) in blood from mice treated with siLuc and mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, showing that blood from siPAI-1 treated mice forms a clot with the same stiffness as clot formed from blood from siLuc-treated mice. Example 2: Depletion of plasma PAI-1 with siRNA decreases thrombosis.

This example shows siRNA knockdown of PAI-1 can decrease thrombosis in vivo.

Adult (9-12 weeks) and elderly (80 weeks) mice were administered siLuc or mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2 three days prior to inducing inferior vena cava (IVC) stasis as described in the Materials and Methods. Endpoint was 4 days post-IVC ligation, and the formed clot and blood was collected and analyzed as described in the Materials and Methods.

As shown in Figure 2A, adult mice treated with mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2 forms a significantly smaller, less heavy clot compared to mice treated with siLuc.

Figure 2B shows that plasma PAI-1 protein levels are significantly depleted in adult mice treated with mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, compared to siLuc-treated mice.

Figure 2C shows a representative Trichrome Hematoxylin and eosin (H&E) stain of clots formed in the IVC of adult mice treated with siLuc, and mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2. Clots in mice treated with siLuc are significant larger compared to mice treated with mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2.

Figure 2D shows probability of survival post-IVC ligation, of elderly mice treated siLuc or mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2, demonstrating that depletion of circulating PAI-1 increases probability of survival when after IVC thrombosis is induced. The black line represents the probability of survival of mice treated with siLuc and grey line represents the probability of survival of siPAI-1 treated mice.

Figure 2E shows that elderly mice treated with mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2 forms a significantly smaller, less heavy clot compared to mice treated with siLuc.

Serum levels of several cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-y), interleukin 1 -alpha (IL- 1 alpha), interleukin 6 (IL-6) and interleukin 12 (IL-12), were quantified as described in the Materials and Methods, and results are shown in Figure 2F. As shown in Figure 2F, siPALl treated mice has lower levels of each cytokine, compared to siLuc- treated mice.

Example 3: Depletion of circulating PAI-1 attenuates obesity-induced upregulation of circulating PAI-1 levels.

As described in the Materials and Methods, mice were fed a or high fat diet (HFD) to induce obesity, or a low fat diet (LFD) as control, and blood and liver were collected for PAI-1 protein and mRNA quantification, respectively.

Figure 3 A shows that mice on a HFD have significantly higher levels of circulating PAI-1 protein levels compared to mice on a LFD.

Figure 3B shows that mice on a HFD have significantly higher levels of PALI mRNA in the liver.

Figure 3C correlates PALI mRNA levels to the corresponding mouse body weight, showing that heavier mice have higher PALI mRNA levels.

As described in the Materials and Methods, a separate cohort of mice were fed a high fat diet (HFD) to induce obesity, and treated with siLuc or mouse siPALl (SEQ ID Nos 11 and 12) of Table 2. Blood and liver were collected for PALI protein and mRNA quantification, respectively.

Figure 3D and 3E shows that siPALl significantly depletes circulating PALI protein and liver PALI mRNA levels, respectively, in obese mice.

Example 4: siRNA targeting PALI, delivered using LNP, does not induce liver toxicity or abnormal blood cell counts.

Mice were administered phosphate buffered saline (PBS), or mouse siPALl (SEQ ID Nos 11 and 12) of Table 2 encapsulated in LNP containing MC3 or ALC-0315 (ALC), and serum was collected 5 hours later for toxicological analyses as described in the Materials and Methods.

Figure 4A shows that serum levels of alkaline phosphatase (ALP) are comparable between PBS and siPALl treated mice. Figure 4B shows that serum levels of aspartate transaminase (AST) are comparable between PBS and siPALl treated mice.

Figure 4C shows that serum levels of alanine transaminase (ALT) are comparable between PBS and siPALl treated mice.

Figure 4D shows that serum levels of blood urea nitrogen (BUN) are comparable between PBS and siPALl treated mice.

Collectively, Figure 4A-D shows that mice treated with mouse siPAI-l (SEQ ID Nos 11 and 12) of Table 2, encapsulated in either LNP containing MC3 or ALC-0315 (ALC) as the ionizable lipid, does not induce liver toxicity.

Mice were administered siLuc, or mouse siPALl (SEQ ID Nos 11 and 12) of Table 2 at 1 or 3 mg/kg, and blood was collected 7 days later for complete blood count analyses as described in the Materials and Methods.

Figure 4E shows that white blood cell (WBC) counts are comparable between siLuc and siPAI-l treated mice.

Figure 4F shows that red blood cell (RBC) counts are comparable between siLuc and siPALl treated mice.

Figure 4G shows that platelet (WBC) counts are comparable between siLuc and siPALl treated mice.

Collectively, Figure 4E-G shows that mice treated with mouse siPALl (SEQ ID Nos 11 and 12) of Table 2 at either 1 or 3 mg/kg dose, does not induce abnormal blood cell counts.

Example 5: siRNA knockdown of PALI in elderly mice lowers PALI levels to healthy young adult mice concentrations

As described in the Materials and Methods, elderly mice (72-76 weeks old) were administered siLuc or mouse siPALl (SEQ ID Nos 11 and 12) of Table 2 at weekly intervals until study endpoint. Blood was collected from mice starting at 3 days after the first injection, and weekly thereafter until study endpoint.

Figure 5A shows that elderly mice treated with siPAI-1 has a significant decrease in plasma levels of PAI-1 by 10 days post injection with the first dose of siPAI-1, compared to mice treated with siLuc, and this lowered levels were sustained until study endpoint with regular, weekly injections of siPAI-1.

Example 6: siRNA knockdown of PAI-1 in apolipoprotein E knockout (ApoE ^_/ ) mice lowers blood cholesterol

As described in the Materials and Methods, ApoE" ' mice (72-76 weeks old) were administered siLuc or mouse siPAI-1 (SEQ ID Nos 11 and 12) of Table 2. Blood was collected from mice starting at 3 and 7 days after the post-treatment, and plasma levels of PAI- 1 and cholesterol was quantified.

Figure 6A shows that plasma levels of PAI-1 is significantly depleted in ApoE" ' mice treated with siPAI-1 at both 3 and 7 days post-injection, compared to ApoE'^/_ mice treated with siLuc.

Figure 6B shows that plasma levels of cholesterol is significantly decreased in ApoE" ' mice treated with siPAI-1 at 3 days post-injection, compared to ApoE'^/_mice treated with siLuc. At 7 days postinjection, ApoE'^/_ mice treated siLuc and siPAI-1 have comparable plasma levels of cholesterol.

Example 7: Human siRNA PAI-1 knock down in vitro

This example demonstrates that siRNA knocks down PALI in human hepatocytes in vitro.

Quantitative PCR as described in the Materials and Methods was used to measure human PALI mRNA levels after administration of LNP containing siLuc or human siPAIl corresponding to sequence A (duplex siRNA of SEQ ID Nos 13 and 14), sequence B (duplex siRNA of SEQ ID Nos 15 and 16), sequence C (duplex siRNA of SEQ ID Nos 17 and 18), and sequence D (duplex siRNA of SEQ ID Nos 19 and 20) of Table 1, to human hepatocyte cells (HEPG2) in culture. As shown in Figure 5, significant depletion of PAI- 1 mRNA was observed in HEPG2 cells after treatment with LNP containing siPAI-1 corresponding to sequence A (duplex siRNA of SEQ ID Nos 13 and 14), compared to siLuc-treated cells.

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. An siRNA molecule for inhibiting expression of plasminogen activator inhibitor-1 (PAI-1) in a cell, wherein said siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding PAI-1, wherein the sense strand has a nucleotide sequence comprising at least 80% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has a nucleotide sequence comprising at least 80% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

2. The siRNA molecule of claim 1, wherein the sense strand has a nucleotide sequence comprising at least 85% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has a nucleotide sequence comprising at least 85% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

3. The siRNA molecule of claim 1, wherein the sense strand has a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

4. The siRNA molecule of claim 1, wherein the sense strand has a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, and the antisense strand has a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID 10.

5. An siRNA molecule for inhibiting expression of plasminogen activator inhibitor 1 (PAI-1) in a cell, wherein said siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding PAI-1, wherein the sense strand has a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 70% sequence identity to SEQ ID NO: 4.

6. The siRNA molecule of claim 5, wherein the sense strand has a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO: 4.

7. The siRNA molecule of claim 5, wherein the sense strand has a nucleotide sequence comprising at least 90% sequence identity to SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 90% sequence identity to SEQ ID NO: 4.

8. The siRNA molecule of claim 5, wherein the sense strand has a nucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 4.

9. The siRNA molecule of any one of claims 1 through 8, wherein the sense strand comprises one or more modified nucleotides.

10. The siRNA molecule of any one of claims 1 through 9, wherein the antisense strand comprises one or more modified nucleotides.

11. The siRNA molecule of any one of claims 1 to 10, wherein about 10% to about 50% of the nucleotides in the sense strand comprises modified nucleotides.

12. The siRNA molecule of any one of claims 1 to 11, wherein about 10% to about 50% of the nucleotides in the antisense strand comprises modified nucleotides.

13. The siRNA molecule of any one of claims 9 to 12, wherein the modified nucleotides are selected from a 2'-O-alkyl modified nucleotide, a 2’O-methyl modified nucleotide, a 2’-fluoro modified nucleotide, a 2’ -halogen modified nucleotide, a nucleotide comprising a phosphorothioate group, a deoxy -nucleotide, a 3 ’-terminal deoxy -thymine nucleotide, a 2'- deoxy-modified nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’-hydroxyl-modified nucleotide, a 2’ -methoxy ethyl modified nucleotide, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5 ’-phosphate, a nucleotide comprising a 5 ’-phosphate mimic, a glycol modified nucleotide, a 2-O-(N-methylacetamide) modified nucleotide, and combinations thereof.

14. An siRNA molecule for inhibiting expression of plasminogen activator inhibitor 1 (PAI-1) in a cell, wherein said siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding PAI-1, wherein the sense strand has a nucleotide sequence comprising at least 80% sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand has a nucleotide sequence comprising at least 80% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

15. The siRNA molecule of claim 14, wherein the sense strand has a nucleotide sequence comprising at least 85% sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand has a nucleotide sequence comprising at least 85% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

16. The siRNA molecule of claim 14, wherein the sense strand has a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand has a nucleotide sequence comprising at least 90% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

17. The siRNA molecule of claim 14, wherein the sense strand has a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19, and the antisense strand has a nucleotide sequence comprising at least 95% sequence identity to any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

18. The siRNA molecule of any one of claims 1 to 17, wherein the sense strand and/or antisense strand is independently 15 to 35 nucleotides in length.

19. The siRNA molecule of any one of claims 1 to 17, wherein the sense strand and/or the antisense strand is independently 18 to 35 nucleotides in length.

20. The siRNA molecule of any one of claims 1 to 17, wherein the sense strand and/or the antisense strand is independently 20 to 30 nucleotides in length.

21. The siRNA molecule of any one of claims 1 to 20, further comprising a ligand.

22. The siRNA molecule of claim 21, wherein the ligand is conjugated to the 3’ end and/or the 5’ end of the sense strand.

23. The siRNA molecule of claim 21 or 22, wherein the ligand is conjugated to the 3’ end and/or the 5’ end of the antisense strand.

24. The siRNA molecule of any one of claims 21 to 23, wherein the ligand is conjugated to one or more nucleotides of the sense strand and/or the sense strand.

25. The siRNA molecule of any one of claims 21 to 24, wherein the ligand comprises 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, a sugar group, and derivatives thereof.

26. The siRNA molecule of any one of claims 21 to 25, wherein the ligand comprises a sugar group.

27. The siRNA molecule of claim 26 wherein the sugar group comprises a sugar monomer, an oligosaccharide, and/or derivatives thereof.

28. The siRNA molecule of claim 26 or 27, wherein the sugar group comprises N- Acetylgalactosamine (GalNac) or a derivative thereof.

29. The siRNA molecule of any one of claims 1 to 28, wherein at least one of the sense strand and the antisense strand comprise a 5’ overhang of at least 1 nucleotide.

30. The siRNA molecule of any one of claims 1 to 29, wherein at least one of the sense strand and the antisense strand comprise a 3’ overhang of at least 1 nucleotide.

31. The siRNA molecule of any one of claims 1 to 30, wherein at least one of the sense strand and the antisense strand comprise a 5’ overhang of between 1 and 6 nucleotides.

32. The siRNA molecule of any one of claims 1 to 31, wherein at least one of the sense strand and the antisense strand comprise a 3’ overhang of between 1 and 6 nucleotides.

33. The siRNA molecule of any one of claims 1 to 28, wherein the double-stranded region comprises blunt ends at both 5’ and 3’ ends.

34. A lipid nanoparticle comprising: an siRNA molecule against PAI-1 mRNA of any one of claims 1 to 33; about 20 mol% to about 70 mol% of ionizable cationic lipid having a pKa of between 5.5 and 7.0; a neutral, vesicle-forming helper lipid comprising a phospholipid and/or a triglyceride; a sterol; and about 0.5 mol% to about 5 mol% of a hydrophilic polymer-lipid conjugate.

35. The lipid nanoparticle of claim 34, wherein the PAI-1 mRNA is human PAI-1 mRNA.

36. The lipid nanoparticle of claim 34 or 35, wherein the lipid nanoparticle comprises about 30 mol% to about 55 mol% of the ionizable cationic lipid.

37. The lipid nanoparticle of any one of claims 34 to 36, wherein the neutral, vesicle-forming helper lipid comprises a phospholipid.

38. The lipid nanoparticle any one of claims 34 to 37, wherein the lipid nanoparticle comprises about 20 mol% to about 60 mol% of the neutral, vesicle-forming helper lipid.

39. The lipid nanoparticle of any one of claims 34 to 38, wherein the sterol comprises a cholesterol or a derivative thereof.

40. The lipid nanoparticle of any one of claims 34 to 39, wherein the lipid nanoparticle comprises about 15 mol% to 65 mol% of the sterol.

41. A pharmaceutical composition for inhibiting expression of PAI-1 comprising the siRNA molecule of any one of claims 1 to 33.

42. A pharmaceutical composition for inhibiting expression of PAI-1 comprising the lipid nanoparticle of any one of claims 34 to 40.

43. The pharmaceutical composition of claim 41 or 42, wherein the siRNA molecule or the lipid nanoparticle is formulated in an unbuffered solution.

44. The pharmaceutical composition of claim 43, wherein the unbuffered solution comprises saline and/or water.

45. The pharmaceutical composition of claim 41 or 42, wherein the siRNA molecule or the lipid nanoparticle is formulated in a buffered solution.

46. The pharmaceutical composition of claim 45, wherein the buffered solution comprises one or more of acetic, citrate, prolamine, carbonate, and phosphate.

47. The pharmaceutical composition of claim 45, wherein the buffered solution comprises phosphate buffered saline (PBS).

48. A method of inhibiting PAI-1 expression in a cell, the method comprising contacting the cell with the siRNA molecule of any one of claims 1 to 33, a lipid nanoparticle of any one of claims 34 to 40, or a pharmaceutical composition of any one of claims 41 to 47, thereby inhibiting expression of PAI- 1 in the cell.

49. The method of claim 48, wherein the cell is in a subject.

50. The method of claim 49, wherein the subject is a human.

51. The method of claim 50, wherein the human subject suffers from a PAI- 1 -associated condition, disease or disorder.

52. The method of claim 51, wherein the PAI- 1 -associated condition, disease or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, a neurocognitive disorder, Alzheimer’s disease, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof.

53. The method of claim 51, wherein the PAI- 1 -associated condition, disease or disorder is a thrombotic disorder.

54. The method of claim 51, wherein the PAI- 1 -associated condition, disease or disorder is a blood clotting disorder.

55. The method of claim 51, wherein the PAI- 1 -associated condition, disease or disorder is associated with aging and lifespan.

56. The method of claim 53, wherein the thrombotic disorder is venous thrombosis, arterial thrombosis, geriatric thrombosis, microbial infection-associated thrombosis, viral infection- associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.

57. A method of treating a subject having a PALI -associated condition, disease or disorder comprising administrating to the subject a therapeutically effective amount of the siRNA molecule of any one of claims 1 to 33, a lipid nanoparticle of any one of claims 34 to 40, or a pharmaceutical composition of any one of claims 41 to 47, thereby treating the subject suffering from a PAI 1 -associated condition, disease or disorder.

58. A method of preventing at least one symptom in a subject having a PA I -associated condition, disease or disorder which would benefit from reduction in expression of PALI mRNA, comprising administrating to the subject a prophylactically effective amount of the siRNA molecule of any one of claims 1 to 33, a lipid nanoparticle of any one of claims 34 to 40, or a pharmaceutical composition of any one of claims 41 to 47, thereby preventing at least one symptom in a subject having a PALLassociated condition, disease or disorder that would benefit from reduction in expression of PALI mRNA.

59. The method of claim 57 or 58, wherein the PAI- 1 -associated condition, disease or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, a neurocognitive disorder, Alzheimer’s disease, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof.

60. The method of claim 57 or 58, wherein the PAI- 1 -associated condition, disease or disorder is a thrombotic disorder.

61. The method of claim 57 or 58, wherein the PAI- 1 -associated condition, disease or disorder is a blood clotting disorder.

62. The method of claim 57 or 58, wherein the PAI- 1 -associated condition, disease or disorder is associated with aging and increased lifespan.

63. The method of claim 60, wherein the thrombotic disorder is venous thrombosis, arterial thrombosis, geriatric thrombosis, microbial infection-associated thrombosis, viral infection- associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.

64. Use of the siRNA molecule of any one of claims 1 to 33, a lipid nanoparticle of any one of claims 34 to 40, or a pharmaceutical composition of any one of claims 41 to 47 in the treatment of a PAI- 1 -associated condition, disease or disorder.

65. Use of the siRNA molecule of any one of claims 1 to 33, a lipid nanoparticle of any one of claims 34 to 40, or a pharmaceutical composition of any one of claims 41 to 47 in the prevention of at least one symptom in a subject having a PALI -associated condition, disease or disorder which would benefit from reduction in expression of PAI- 1 mRNA.

66. The use of claim 64 or 65, wherein the PALI -associated condition, disease or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, a neurocognitive disorder, Alzheimer’s disease, sepsis, lung fibrosis, an inflammatory disorder, or any combinations thereof.

67. The use of claim 64 or 65, wherein the PALLassociated condition, disease or disorder is a thrombotic disorder.

68. The use of claim 64 or 65, wherein the PAI- 1 -associated condition, disease or disorder is a blood clotting disorder.

69. The method of claim 64 or 65, wherein the PAI- 1 -associated condition, disease or disorder is associated with aging and increased lifespan.

70. The use of claim 67, wherein the thrombotic disorder is venous thrombosis, arterial thrombosis, geriatric thrombosis, microbial infection-associated thrombosis, viral infection-associated thrombosis, cancer-associated thrombosis, thrombosis following trauma, thrombosis following surgery, thrombosis associated with increased inflammation, thrombosis associated with deficiency in tissue plasminogen activator, thrombosis associated with antithrombin deficiency, thrombosis associated with protein C deficiency, thrombosis associated with protein S deficiency, thrombosis with factor V Leiden, or any combinations thereof.