Methods and compositions for modulating plasminogen activator inhibitor-1 (PAI-1)

Abstract

Disclosed are siRNA molecules that inhibit the expression of plasminogen activator inhibitor 1 (PAI-1), and the siRNA molecules may have a sense strand and an antisense strand. The sense strand may have a sequence identity of at least 80% to any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. In some embodiments, the antisense strand may have a sequence identity of at least 80% to any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10. The sense strand and / or antisense strand may comprise one or more modified nucleotides. The sense strand and / or antisense strand may comprise a ligand. Also disclosed is a lipid nanoparticle comprising the siRNA molecule. Also disclosed are methods of treating a subject having at least one PAI-1 associated condition, disease, or disorder using the siRNA molecule or lipid nanoparticles comprising the siRNA molecule.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to nucleic acids for targeting PAI-1 and pharmaceutical formulations thereof. BACKGROUND

[0002] Plasminogen activator inhibitor-1 (PAI-1) is a blood protein that slows the breakdown of blood clots, thus promoting clot stabilization and thrombosis. High PAI-1 levels are associated with increased risk of thrombosis, and PAI-1 levels also increase with the progression of several conditions, diseases, and disorders, including atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive disorders, sepsis, pulmonary fibrosis, Alzheimer’s disease, and inflammatory diseases. PAI-1 plays a crucial role in the progression of metabolic syndrome and adipose deposition. In mouse models of thrombosis and Alzheimer’s disease, reducing PAI-1 significantly reduces thrombosis and brain plaques, respectively. People lacking PAI-1 have lower cardiovascular morbidity and longer life spans. These individuals can suffer from prolonged bleeding after severe trauma, but they do not experience spontaneous bleeding and are not at risk for internal bleeding. Reducing circulating PAI-1 levels has experimental and therapeutic value, but there are currently no suitable methods for clinical use that are long-lasting. RNA gene therapy represents an alternative option for modulating target protein levels in those who cannot be modulated by traditional small molecule- or protein-based therapies. SUMMARY

[0003] The present invention relates to small interfering RNA (siRNA) molecules for use in reducing or inhibiting PAI-1 gene expression in a cell with a treatment comprising a composition of siRNA molecules.

[0004] In a first aspect, the present invention provides a siRNA molecule for use in inhibiting PAI-1 expression in a cell, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region. In some embodiments, the antisense strand comprises a region that is complementary to a mRNA encoding PAI-1. According to one embodiment of the present disclosure, the PAI-1 is human PAI-1.

[0005] 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.

[0006] 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 NO: 10.

[0007] 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 NO: 10.

[0008] 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 NO: 10.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] In some embodiments, the sense strand and / or the antisense strand of the siRNA molecule comprises one or more modified nucleotides. For example, about 10% to about 50% of the nucleotides in the sense and / or antisense strand comprise modified nucleotides.

[0014] In such embodiments, the one or more modified nucleotides can be 2'-0-alkyl modified nucleotides, 2'-halogen modified nucleotides, phosphorothioate-containing nucleotides, deoxy nucleotides, 3'-terminal deoxythymidine nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-0-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, methylphosphonate-containing nucleotides, 5'-phosphate group-containing nucleotides, 5'-phosphate mimic-containing nucleotides, glycol modified nucleotides, 2-0-(N-methylacetamide) modified nucleotides, or any combination thereof. For example, the one or more modified nucleotides are 2'-0-methyl modified nucleotides and / or 2'-fluoro modified nucleotides.

[0015] In 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 can 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.

[0016] In 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 can 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.

[0017] The siRNA molecules of the application comprise a double-stranded region of at least 15 nucleotides in length. For example, the double-stranded region can be 15 to 35 nucleotides in length, 18 to 35 nucleotides in length, or 20 to 30 nucleotides in length.

[0018] The siRNA molecules of the application can comprise a ligand conjugated to the 3' end, 5' end, and / or one or more nucleotides of the sense strand and / or the antisense strand.

[0019] In some embodiments, the ligand conjugated to the siRNA molecule can 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.

[0020] In other embodiments, the sugar group comprises a sugar monomer, an oligosaccharide, and / or a derivative thereof. For example, the sugar group can be N-acetylgalactosamine.

[0021] In another aspect, the sense strand and / or the antisense strand of the siRNA molecule comprises a 3' and / or 5' overhang of 1 to 6 nucleotides (or any number of nucleotides therebetween).

[0022] In some embodiments, the sense strand and the antisense strand of the siRNA molecule form a double-stranded region without overhangs and comprise a blunt end at both the 5' end and the 3' end.

[0023] In another aspect, the present application provides a lipid nanoparticle (LNP) comprising an siRNA molecule for reducing or inhibiting PAI-1 expression in a cell. The LNP of the present application comprises: an siRNA molecule directed to a human PAI-1 mRNA; an ionizable cationic lipid having a pKa of 5.5 to 7.0, and which is present at 10 mol% to 85 mol% or any mol% therebetween. In some embodiments, the ionizable cationic lipid is present at 30 mol% to 55 mol% or any mol% therebetween. The LNP further comprises a neutral vesicle-forming lipid, including a phospholipid and / or a triglyceride; a sterol; and a hydrophilic polymer-lipid conjugate, the neutral vesicle-forming lipid being present at 0.5 mol% to 5 mol% or any mol% therebetween.

[0024] In some embodiments, the neutral vesicle-forming lipid is present at 15 mol% to 60 mol% or any mol% therebetween. In other embodiments, the sterol is cholesterol or a derivative thereof. In other embodiments, the sterol is present at 15 mol% to 65 mol% or any mol% therebetween.

[0025] In another aspect, the present application provides a pharmaceutical composition comprising an siRNA molecule for reducing or inhibiting PAI-1 expression, thereby treating and / or preventing one or more conditions, diseases, or disorders requiring modulation of PAI-1 levels, such as thrombosis, atherosclerosis, coronary artery disease, obesity, sepsis, pulmonary fibrosis, diabetes, cancer, liver necrosis, neurocognitive disorders, inflammatory diseases, or any combination thereof. In some examples, the pharmaceutical composition comprises a lipid nanoparticle having a lipid component as described herein and an encapsulated siRNA targeting PAI-1 mRNA to achieve a controllable and / or sustained reduction in PAI-1 levels in the blood or other body part.

[0026] In some embodiments, a pharmaceutical composition comprising an siRNA molecule as described herein is provided formulated in an unbuffered solution. For example, the unbuffered solution can be saline and / or water.

[0027] In some embodiments, a pharmaceutical composition comprising an siRNA molecule as described herein is provided formulated in a buffered solution. For example, the buffered solution can include acetate, zein, carbonate, and / or phosphate. In one embodiment, the buffered solution can be phosphate buffered saline.

[0028] According to another aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA molecule or a lipid nanoparticle as described in any aspect or embodiment herein, and wherein the pharmaceutical composition comprises a pharmaceutically acceptable salt and / or an excipient.

[0029] In another aspect, there is provided a method of inhibiting PAI-1 expression in a cell by contacting the cell with a lipid nanoparticle or pharmaceutical composition comprising a PAI-1 siRNA molecule described herein. In one embodiment, the cell is in a subject. In another embodiment, the subject is a human.

[0030] In another aspect, there is provided a method of inhibiting PAI-1 expression in a human having a PAI-1 related condition, disease, or disorder. In one embodiment, the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, sepsis, pulmonary fibrosis, liver necrosis, neurocognitive disorders, Alzheimer’s disease, inflammatory diseases, or any combination thereof. In one embodiment, the PAI-1 related condition, disease, or disorder is aging and increased lifespan. In another embodiment, the thrombotic disease is venous thrombosis, arterial thrombosis, senile thrombosis, microorganism infection related thrombosis, viral infection related thrombosis, cancer related thrombosis, post-trauma thrombosis, post-surgery thrombosis, geriatric related thrombosis, inflammation exacerbation related thrombosis, aging related thrombosis, tissue plasminogen activator deficiency related thrombosis, antithrombin deficiency related thrombosis, protein C deficiency related thrombosis, protein S deficiency related thrombosis, Factor V Leiden related thrombosis, or any combination thereof.

[0031] Further embodiments include the use of a lipid nanoparticle or pharmaceutical composition described above for inhibiting the expression of PAI-1 in a human having a PAI-1 related condition, disease, or disorder as described herein.

[0032] In another aspect, methods of treating a patient having a PAI-1 related condition, disease, or disorder are provided. In one embodiment, the PAI-1 related condition, disease, or disorder can be thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive disorders, Alzheimer’s disease, sepsis, pulmonary fibrosis, inflammatory diseases, or any combination thereof. In one embodiment, the PAI-1 related condition, disease, or disorder is aging and increased lifespan. In some embodiments, the thrombotic disorder can be venous thrombosis, arterial thrombosis, senile thrombosis, microorganism infection related thrombosis, viral infection related thrombosis, cancer related thrombosis, post-trauma thrombosis, post-surgery thrombosis, inflammation exacerbation related thrombosis, aging related thrombosis, tissue plasminogen activator deficiency related thrombosis, antithrombin deficiency related thrombosis, protein C deficiency related thrombosis, protein S deficiency related thrombosis, Factor V Leiden related thrombosis, or any combination thereof.

[0033] Further embodiments include the use of the above-described pharmaceutical compositions or lipid nanoparticles for treating a PAI-1 related condition, disease, or disorder as described herein by modulating the levels of PAI-1 in the blood or other body sites of a patient in need of such treatment.

[0034] Another embodiment includes the use of the above-described pharmaceutical compositions or lipid nanoparticles in the manufacture of a medicament for treating a PAI-1 related condition, disease, or disorder as described herein by modulating the levels of PAI-1 in the blood or other body sites. In one embodiment, the PAI-1 related condition, disease, or disorder is aging and increased lifespan. In some embodiments, the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive disorders, sepsis, pulmonary fibrosis, inflammatory diseases, or any combination thereof. In one embodiment, the thrombotic disease can be venous thrombosis, arterial thrombosis, microorganism infection related thrombosis, viral infection related thrombosis, cancer related thrombosis, post-trauma thrombosis, post-surgery thrombosis, inflammation exacerbation related thrombosis, aging related thrombosis, tissue plasminogen activator deficiency related thrombosis, antithrombin deficiency related thrombosis, protein C deficiency related thrombosis, protein S deficiency related thrombosis, Factor V Leiden related thrombosis, or any combination thereof.

[0035] In addition, methods are provided for treating patients with PAI-1-related conditions, diseases, or disorders by regulating the level of PAI-1 in the blood or other body parts, the methods comprising applying the pharmaceutical compositions or lipid nanoparticles described herein to patients in need of such treatment.

[0036] According to any further example of any aspect or embodiment of this document, the pharmaceutical compositions or lipid nanoparticles described herein are used to inhibit the expression of PAI-1, thereby treating and / or preventing one or more coagulation disorders.

[0037] Further examples of any aspect or embodiment of the present invention provide the use of the pharmaceutical composition in the preparation of a medicament for treating coagulation disorders in patients requiring such treatment.

[0038] According to another example of any aspect or embodiment of this document, a method for treating a patient with a coagulation disorder is provided, the method comprising administering to a patient requiring such treatment a pharmaceutical composition as described in any aspect of the embodiments herein. Attached Figure Description

[0039] Figure 1 A shows the plasma levels of PAI-1 protein in mice treated one week after injection with a control luciferase-targeting siRNA (siLuc) and a PAI-1-targeting siRNA (siPAI-1) encapsulated in an LNP containing either MC3 or ALC-0315 (ALC) as an ionizable lipid. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0040] Figure 1 B shows the liver levels (%) of PAI-1-targeting siRNA relative to the control PAI-1 mRNA one week after injection for the luciferase siRNA control (siLuc) and the double-stranded siRNA corresponding to the msPAI-1 siRNA sequences (SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0041] Figure 1 C shows the plasma levels of PAI-1 protein in mice treated with a control siRNA targeting luciferase (siLuc) and a siRNA targeting PAI-1 (siPAI-1) at 3, 10, 14, and 20 days post-injection. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0042] Figure 1 D shows representative rotational thromboelastography (ROTEM) curves from mouse blood treated with either control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0043] Figure 1 E shows the percentage of clot dissolution in mouse whole blood at 30, 45, and 60 minutes after treatment with either the control siRNA luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0044] Figure 1 F shows the α-horn of mice treated one week after injection with either the control siRNA luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0045] Figure 1 G shows the clotting time of mice treated with either the control siRNA luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0046] Figure 1 H shows the maximum clot hardness (MCF) of mice treated one week after injection with either the control siRNA luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0047] Figure 2A shows the clot weights of 9- to 12-week-old mice treated with either a control luciferase-targeting siRNA (siLuc) or a PAI-1-targeting siRNA (siPAI-1) four days after inferior vena cava (IVC) ligation. siPAI-1, corresponding to the double-stranded siRNA sequences of msPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) (Table 2), was administered to mice three days prior to IVC ligation.

[0048] Figure 2 B shows the plasma PAI-1 levels relative to body weight in mice treated with a control luciferase-targeting siRNA (siLuc) and a PAI-1-targeting siRNA (siPAI-1) four days after IVC ligation. siPAI-1 (Table 2), corresponding to the msPAI-1 siRNA sequences (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12), was administered to mice three days prior to IVC ligation.

[0049] Figure 2 C shows representative histological sections of mice treated with either control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) 4 days after IVC ligation. siPAI-1 (Table 2) corresponding to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) was administered to mice 3 days before IVC ligation.

[0050] Figure 2 D shows the survival probability plot of 80-week-old mice treated with either a control siRNA targeting luciferase (siLuc) or a siRNA targeting PAI-1 (siPAI-1) up to 4 days after IVC ligation. siPAI-1 corresponding to the msPAI-1 siRNA sequences (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2) was administered to mice 3 days before IVC ligation.

[0051] Figure 2 E shows the clot weights of 80-week-old mice treated with either a control luciferase-targeting siRNA (siLuc) or a PAI-1-targeting siRNA (siPAI-1) four days after IVC ligation. siPAI-1, corresponding to the msPAI-1 siRNA sequences (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2), was administered to mice three days prior to IVC ligation.

[0052] Figure 2F shows the cytokine levels in 80-week-old mice treated with either the control siRNA luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1) 4 days after ligation, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin-1-α (IL-1α), interleukin-6 (IL-6), and interleukin-12 (IL-12). siPAI-1 corresponding to the msPAI-1 siRNA sequences (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2) was administered to mice 3 days prior to IVC ligation.

[0053] Figure 3 A shows the plasma levels of PAI-1 protein in mice fed a high-fat diet (HFD) or a low-fat diet (LFD).

[0054] Figure 3 B shows the percentage of liver PAI-1 mRNA expression in high-fat diet mice (HFD) compared to low-fat diet mice (LFD).

[0055] Figure 3 C shows the relationship between liver PAI-1 mRNA levels and mouse body weight.

[0056] Figure 3 D shows the plasma levels of PAI-1 protein in high-fat diet mice treated one week after injection with a control siRNA targeting luciferase (siLuc) and a siRNA targeting PAI-1 (siPAI-1). siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0057] Figure 3 E shows the liver PAI-1 mRNA levels (%) of high-fat diet mice treated with luciferase siRNA control (siLuc) and double-stranded siRNAs corresponding to the msPAI-1 siRNA sequences (SEQ ID No: 11 and SEQ ID No: 12) (Table 2) one week after injection, relative to the control.

[0058] Figure 4Table A shows the serum levels of alkaline phosphatase (ALP) in mice treated 5 hours post-injection with phosphate-buffered saline (PBS) or a PAI-1-targeting siRNA (siPAI-1) encapsulated in an LNP containing either MC3 or ALC-0315 (ALC) as an ionizable lipid. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the lower and upper thresholds of normal ALP levels.

[0059] Figure 4 B shows the serum levels of aspartate aminotransferase (AST) in mice treated 5 hours post-injection with phosphate-buffered saline (PBS) or a siRNA targeting PAI-1 (siPAI-1) encapsulated in an LNP containing either MC3 or ALC-0315 (ALC) as an ionizable lipid. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the upper and lower thresholds of normal AST levels.

[0060] Figure 4 C shows the serum alanine aminotransferase (ALT) levels in mice treated 5 hours post-injection with phosphate-buffered saline (PBS) or a PAI-1-targeting siRNA (siPAI-1) encapsulated in an LNP containing either MC3 or ALC-0315 (ALC) as an ionizable lipid. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the upper and lower limits of normal ALT levels.

[0061] Figure 4 D shows the blood urea nitrogen (BUN) levels in mice treated 5 hours post-injection with phosphate-buffered saline (PBS) or a PAI-1-targeting siRNA (siPAI-1) encapsulated in an LNP containing either MC3 or ALC-0315 (ALC) as an ionizable lipid. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the lower and upper limits of normal BUN levels.

[0062] Figure 4E shows the white blood cell (WBC) counts in mice treated with either the control siRNA luciferase (siLuc) or the PAI-1-targeting siRNA (siPAI-1) at doses of 1 mg / kg or 3 mg / kg 7 days after injection. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the lower and upper thresholds for normal WBC counts.

[0063] Figure 4 F shows the red blood cell (RBC) counts in mice treated with either the control siRNA luciferase (siLuc) or the PAI-1-targeting siRNA (siPAI-1) at doses of 1 mg / kg or 3 mg / kg 7 days after injection. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the lower and upper thresholds for normal RBC counts.

[0064] Figure 4 G shows platelet counts in mice treated with control siRNA luciferase (siLuc) or siRNA targeting PAI-1 (siPAI-1) at doses of 1 mg / kg or 3 mg / kg 7 days post-injection. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2). The two dashed lines represent the lower and upper limits of normal platelet count.

[0065] Figure 4 A shows weekly plasma PAI-1 levels in aged mice (72 to 76 weeks old) starting on day 3 after injection of the first dose of either the control siRNA targeting luciferase (siLuc) or the siRNA targeting PAI-1 (siPAI-1), followed by weekly injections. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0066] Figure 4 A shows the apolipoprotein E gene knockout (ApoE) on days 3 and 7 after injection of either a control siRNA targeting luciferase (siLuc) or a siRNA targeting PAI-1 (siPAI-1). - / -Plasma levels of PAI-1 in mice. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0067] Figure 4 B shows the apolipoprotein E gene knockout (ApoE) on days 3 and 7 after injection of either a control siRNA targeting luciferase (siLuc) or a siRNA targeting PAI-1 (siPAI-1). - / - Plasma cholesterol levels in mice. siPAI-1 corresponds to the msPAI-1 siRNA sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) (Table 2).

[0068] Figure 4 The table shows the percentage of LNPs containing control siRNA luciferase (siLuc) or LNPs containing double-stranded human PAI-1 siRNA sequences relative to control human PAI-1 mRNA after in vitro addition to HEPG2 cells at concentrations of 0.3 μg / mL, 1 μg / mL, or 3 μg / mL, wherein the double-stranded human PAI-1 siRNA sequences are sequences A (SEQ ID No: 13 and SEQ ID No: 14), B (SEQ ID No: 15 and SEQ ID No: 16), C (SEQ ID No: 17 and SEQ ID No: 18), and D (SEQ ID No: 19 and SEQ ID No: 20) (Table 2). Detailed Implementation

[0069] In this specification, unless the context otherwise requires, the words “including”, “contains”, etc., shall be interpreted as having an inclusive meaning, the opposite of an exclusive meaning, that is, the meaning of “including but not limited to”.

[0070] Small interfering RNA

[0071] As used herein, “siRNA molecules targeting plasminogen activator inhibitor-1 (PAI-1)” include single-stranded RNAs (e.g., mature miRNAs) or double-stranded RNAs (i.e., double-stranded RNAs, such as siRNAs, aiRNAs, or pre-miRNAs) capable of reducing or inhibiting PAI-1 expression, for example, mediating the degradation of mRNAs complementary to the siRNA sequence or inhibiting the translation of said mRNAs to reduce or inhibit PAI-1 expression, as measured in vitro or in vivo. The siRNA may be substantially or completely identical to the gene or sequence encoding PAI-1, or may contain mismatched regions (i.e., mismatched motifs). The sequence of the siRNA may correspond to the full-length target sequence or a subsequence of it.

[0072] In one embodiment, the siRNA molecule reduces or inhibits the expression of a coagulation factor to alter coagulation. In one embodiment, the coagulation factor is PAI-1. The siRNA may be a double-stranded siRNA. In such an 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 strand and antisense strand having at least partial complementarity. In another embodiment, the siRNA is single-stranded. Further non-limiting examples of this disclosure are described in more detail below.

[0073] In some implementations, siRNA comprises a double-stranded region of 15 to 35 nucleotides in length. For example, the length of the double-stranded region can be 18 to 35 nucleotides, or 20 to 30 nucleotides. Because siRNA is double-stranded, the nucleotide length corresponds to the length of the shorter strand of the antisense or sense strand.

[0074] The siRNA described herein may contain "mismatch motifs" or "mismatch regions," which refer to portions of the siRNA sequence that are not 100% complementary to their target sequence. siRNA may have at least one, two, three, four, five, six, or more mismatch regions. Mismatch regions may be continuous or separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. Mismatch motifs or regions may contain a single nucleotide or may contain two, three, four, five, or more nucleotides.

[0075] In some implementations, as measured in vitro or in vivo, siRNA reduces or inhibits PAI-1 expression. Inhibition or reduction of PAI-1 expression is achieved when the value obtained with siRNA relative to a relevant control (e.g., buffer or empty lipid nanoparticles) is approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, inhibition or reduction of PAI-1 expression in cells transfected with PAI-1-specific siRNA is achieved by reducing it by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0076] Suitable assays for measuring the expression of a target gene or target sequence include, for example, the detection of protein or RNA levels using techniques known to those skilled in the art, such as quantitative PCR (qPCR), Western blotting, dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function assays, and phenotypic assays known to those skilled in the art. Decreases in in vitro expression and activity can be measured using assays as described in the Examples section.

[0077] The expression "inhibition or reduction of PAI-1 expression" is achieved when the value obtained using interfering RNA is approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the relevant control. In other words, "inhibition or reduction of PAI-1 expression" is achieved when the levels of PAI-1 protein and / or mRNA decrease by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, mRNA or protein levels can be measured.

[0078] The nucleotides of siRNA can be modified. Examples of modifications include, but are not limited to, nucleotides modified with 2'-O-alkyl groups, nucleotides modified with 2'-halogens, nucleotides containing thiophosphate groups, deoxynucleotides, 3'-terminal deoxythymidine nucleotides, nucleotides modified with 2'-deoxy groups, nucleotides modified with 2'-amino groups, nucleotides modified with 2'-O-allyl groups, nucleotides modified with 2'-C-alkyl groups, nucleotides modified with 2'-hydroxy groups, nucleotides modified with 2'-methoxyethyl groups, nucleotides containing methylphosphonic acid groups, nucleotides containing 5'-phosphate groups, nucleotides containing 5'-phosphate mimics, nucleotides modified with ethylene glycol, nucleotides modified with 2-O-(N-methylacetamide), and combinations thereof.

[0079] The sense and antisense strands of the siRNA may have sequence identity with any of the nucleotide sequences shown in Tables 1 and 2 below. More generally, the siRNA has sequence identity with the human nucleotide sequences shown in Table 1 or Table 2. When referring to two nucleic acids herein, the expression "sequence identity" means that the two sequences or subsequences are identical or have a specified percentage of identical nucleotides when compared and aligned in a comparison window or using a known comparison algorithm or by manual alignment and visual inspection to obtain the maximum correspondence. Sequence identity is expressed as a percentage. When referring to sequence identity of at least a certain number, it should be understood to include any value between the number and 100%. As a non-limiting example, when the siRNA molecule contains a nucleotide sequence having at least 90% sequence identity with a reference nucleotide sequence, this includes siRNA molecules containing nucleotide sequences having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a reference nucleotide sequence. To determine sequence identity, a reference sequence is typically used, and the test sequence is compared to that reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Default program parameters can be used, or optional parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters. Sequence identity is typically measured using BLAST (Camacho C, et al TL. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15;10:421), which is well known to those skilled in the art.

[0080] In some embodiments, the siRNA molecule used to inhibit PAI-1 expression in cells comprises a sense strand and an antisense strand forming a double-stranded region. In some embodiments, the antisense strand comprises a region complementary to the mRNA encoding PAI-1. According to one embodiment of this disclosure, PAI-1 is human PAI.

[0081] In other embodiments, the sense strand of the siRNA molecule has at least 80% sequence identity with 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 with 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 with 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 with any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

[0082] In one embodiment, the sense strand of the siRNA molecule has at least 70% sequence identity with SEQ ID NO: 3, and the antisense strand has a nucleotide sequence that contains at least 70% sequence identity with 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 with SEQ ID NO: 3, and the antisense strand may have at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 4.

[0083] In one embodiment, the sense strand of the siRNA comprises a nucleotide sequence differing from any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 by no more than 4 nucleotides. In some embodiments, the antisense strand of the siRNA comprises a nucleotide sequence differing from any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 by no more than about 4 nucleotides.

[0084] In other embodiments, the sense strand of the siRNA molecule may contain one or more modified nucleotides and have at least 80% identity with 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 contain one or more modified nucleotides and have at least 80% sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20. For example, the sense strand may contain one or more modified nucleotides and have at least 85%, at least 90%, or at least 95% identity with any 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 contain one or more modified nucleotides and have at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20.

[0085] In one embodiment, the sense strand of the siRNA molecule comprises a nucleotide sequence differing from any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19 by no more than about 4 nucleotides. In some embodiments, the antisense strand of the siRNA molecule comprises a nucleotide sequence differing from any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20 by no more than about 4 nucleotides. In some embodiments, the sense strand and / or antisense strand comprise, respectively, all 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, but this is not mandatory. The sense strand and / or antisense strand may contain one or more 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, or may not contain the above nucleotide modifications.

[0086] In one embodiment, the sense or antisense strand of the siRNA molecule has at least 30% to 100% sequence identity with any one of SEQ ID NOs: 1-20 in Tables 1 and 2 below. For example, the sense or 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%, or at least 98% sequence identity with any one of SEQ ID NOs: 1-20. In one embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence that differs from any one of SEQ ID NOs: 1-20 by no more than about 4 nucleotides.

[0087] In another embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence differing from the sequence shown in SEQ ID NO: 1-20 by no more than about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In one embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence differing by no more than about 10 nucleotides or no more than about 5 nucleotides.

[0088] In another embodiment, this disclosure provides one or more exemplary siRNA sequences selected from SEQ ID NO: 3-10 or SEQ ID NO: 13-20 (human sequences) or their double strands to inhibit or reduce PAI-1 expression.

[0089] In one embodiment, the sense or antisense strand of the siRNA molecule has at least 30% to 100% sequence identity with any one of SEQ ID NO: 3-10 in Table 1 or SEQ ID NO: 13-20 in Table 2. For example, the sense or 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%, or at least 98% sequence identity with any one of SEQ ID NO: 3-10 in Table 1 or SEQ ID NO: 13-20 in Table 2. In one embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 3-10 in Table 1 or SEQ ID NO: 13-20 in Table 2 by no more than about 4 nucleotides.

[0090] It should be understood that sequence identity in this article does not require an exact match between two nucleotides. For example, a given nucleotide can be methylated and will be considered to have identity with an unmethylated nucleotide.

[0091] In a further embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence differing from the sequences shown in SEQ ID NO: 3-10 of Table 1 and SEQ ID NO: 13-20 of Table 2 by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In one embodiment, the sense or antisense strand of the siRNA molecule comprises a nucleotide sequence differing from the sequences shown in SEQ ID NO: 3-10 of Table 1 and SEQ ID NO: 13-20 of Table 2 by no more than about 10 nucleotides or no more than about 8, 7, 6, or 5 nucleotides.

[0092] In another embodiment, this disclosure provides one or more siRNA sequences selected from SEQ ID NO: 3-10 (Table 1) and SEQ ID NO: 13-20 (Table 2) or their double strands to inhibit or reduce PAI-1 expression.

[0093] In another embodiment, this disclosure provides an siRNA molecule that inhibits or reduces PAI-1 expression. The siRNA molecule may be a double-stranded siRNA molecule comprising a sense strand and an antisense strand. The sense strand or antisense strand may comprise the nucleotide sequence shown in any one of SEQ ID NO: 3-10 (Table 1). In some embodiments, the sense strand or antisense strand comprises a nucleotide sequence differing from the sequence shown in SEQ ID No: 3-10 by no more than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, one or more nucleotides of the sense strand and / or antisense strand comprise nucleotide modifications. In some embodiments, about 10% to about 100% of the sense strand and / or antisense strand, including any range therebetween, such as 20% to 90%, 30% to 80%, 40% to 70%, 50% to 60%, comprises nucleotide modifications. Non-limiting examples of nucleotide modifications include nucleotides modified with 2'-O-alkyl groups, nucleotides modified with 2'-halogens, nucleotides containing thiophosphate groups, deoxynucleotides, 3'-terminal deoxythymidine nucleotides, nucleotides modified with 2'-deoxy groups, nucleotides modified with 2'-amino groups, nucleotides modified with 2'-O-allyl groups, nucleotides modified with 2'-C-alkyl groups, nucleotides modified with 2'-hydroxy groups, nucleotides modified with 2'-methoxyethyl groups, nucleotides containing methylphosphonic acid groups, nucleotides containing 5'-phosphate groups, nucleotides containing 5'-phosphate mimics, nucleotides modified with ethylene glycol, nucleotides modified with 2-O-(N-methylacetamide), and any combination thereof. In some example embodiments, the nucleotide modification includes 2'-O-alkyl modification. In some example embodiments, the 2'-O-alkyl modification and 2'-halogen modification include 2'-O-Me modification and / or 2'-halogen modification.

[0094] Without restrictions, siRNA sequences can exhibit modification patterns similar to those shown in Table 2 below.

[0095] Table 1. Base composition of the double-stranded siRNA sequence targeting plasminogen activator inhibitor-1 (PAI-1) mRNA

[0096] Table 2. Base modifications of the double-stranded siRNA sequence targeting PAI-1 mRNA. “r” indicates an unmodified base, “m” indicates a 2'-O-methylated base, and bold indicates a DNA base.

[0097] It should be understood that siRNAs having sequences similar to those shown in the sequence listing may optionally be conjugated to another part, such as, but not limited to, ligands, as described below.

[0098] Within siRNA, the antisense and sense strands can be programmed to anneal without overhangs when they form a double helix due to base pair complementarity, resulting in blunt ends at both the 3' and 5' ends of the double helix, or overhangs at one or more of the following locations: the 3' end of the sense strand, the 3' end of 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 no 3' antisense overhangs, but there are 3' sense overhangs. In other aspects, there are no 5' overhangs, but there are both 3' antisense and 3' sense overhangs.

[0099] When overhangs are present, they can be, for example, 1 to 6 nucleotides long. In some aspects, the overhangs are dinucleotides. As a non-limiting example, in one aspect, there is a 3' sense overhang, which is dTdT, and there is no overhang on the antisense strand and no 5' sense overhang. As another non-limiting example, in another aspect, there is a 3' sense overhang dTdT and a 3' antisense overhang, also dTdT, but there is no 5' overhang on either the antisense or sense strand. As another non-limiting example, in one aspect, there is a 3' sense overhang dTdT and a 3' dinucleotide antisense overhang, which are complementary to two nucleotides on the target molecule, said two nucleotides being adjacent to a region on the target molecule complementary to the antisense region within the double strand. In this aspect, there is no 5' overhang on either the antisense or sense strand. When overhangs are present, the nucleotides within the overhangs are included in the range of 18 to 30 nucleotides for each of the aforementioned strands.

[0100] In some respects, siRNA molecules covalently bind to one or more ligands to form conjugates. In other respects, ligands are selected because they facilitate the delivery of siRNA to an organism or cell. siRNA can bind to the conjugate at, for example, the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand, the 3' end of the sense strand, or at a location not at the 3' or 5' end of the sense strand and / or antisense strand.

[0101] Examples of ligands include, but are not limited to, one or more of antibodies, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell-penetrating peptide polymers, and glycosides and their derivatives. In one embodiment, the glycoside comprises a glycomonomer, an oligosaccharide, and / or its derivatives. In one aspect, the ligand comprises N-acetylgalactosamine (GalNAc).

[0102] Figure 4

[0103] In one embodiment, this disclosure provides a nucleic acid targeting PAI-1 mRNA encapsulated within lipid nanoparticles. In one embodiment, the nucleic acid is used to inhibit or reduce the expression of PAI-1.

[0104] It should be understood that this invention is not limited to the location or nature of nucleic acid incorporation within lipid nanoparticles. That is, the term "encapsulation" does not imply limitation to any specific interaction between nucleic acids and lipid nanoparticles. Nucleic acids can be incorporated into the aqueous portion, any lipid layer, or both.

[0105] The lipid nanoparticles (LNPs) described herein may comprise ionizable cationic lipids that can associate with or complex with nucleic acids. The term "ionizable cationic lipid" refers to any of many lipid species that carry a net positive charge at a selected pH below their 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 protonable tertiary amine (e.g., pH-titrile) head group, a C16 to C18 alkyl chain, an ether bond between the head group and the alkyl chain, 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.

[0106] In some embodiments, ionizable cationic lipids may be present in the lipid nanoparticles at 20 mol% to 70 mol%, 30 mol% to 55 mol%, or 35 mol% to 55 mol% of the total lipids present in the lipid nanoparticles. 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% between these values.

[0107] In addition to ionizable cationic lipids, the lipid nanoparticles (LNPs) described herein may also comprise “vesicle-forming lipids” or “auxiliary lipids.” In the context of this disclosure, the term “vesicle-forming lipid” includes any vesicle-forming lipid (e.g., bilayer-forming lipids) selected from phosphatidylcholine lipids, sphingomyelins, or mixtures thereof. In some embodiments, the auxiliary lipid is selected from sphingomyelins, distearate phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and dipalmitoylphosphatidylcholine (DPPC). In some embodiments, the vesicle-forming lipid is DOPC, DSPC, or sphingomyelin. In one embodiment, the vesicle-forming lipid is DSPC. In other embodiments, the vesicle-forming lipid content may include a mixture of two or more different types of different auxiliary lipids.

[0108] In some embodiments, vesicle-forming lipids 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 the total lipids present in the lipid nanoparticles. The vesicle-forming lipid content is determined based on the total amount of lipids (including sterols) in the lipid nanoparticles.

[0109] In some embodiments, 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 the total lipids present in the lipid nanoparticles. The lipid content of phosphatidylcholine is determined based on the total amount of lipids (including sterols) in the lipid nanoparticles.

[0110] In one embodiment, the LNP comprises sterols, hydrophilic polymer-lipid conjugates, or both. Examples of sterols include cholesterol or cholesterol derivatives, such as cholesterolanol, cholesterol ketones, cholesterol ketones, fecal alcohol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, β-sitosterol, fucosterol, etc. In one embodiment, based on the total lipids present in the lipid nanoparticles, the sterols are present in a range of 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% therebetween. In another embodiment, based on the total lipids and sterols present in the lipid nanoparticles, the sterols comprising cholesterol or derivatives thereof, and said cholesterol or derivatives being present in a range of 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% therebetween.

[0111] In one embodiment, the hydrophilic polymer lipid conjugate comprises (i) a vesicle-forming lipid having a polar head group, and (ii) a hydrophilic polymer chain covalently linked to the head group. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyethylene methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyasparagine. In one embodiment, the hydrophilic polymer lipid conjugate is a PEG-lipid conjugate.

[0112] The hydrophilic polymer-lipid conjugate may be present in the nanoparticles in any mol% range of total lipids, from 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% therebetween. In another embodiment, the PEG-lipid conjugate is present in the nanoparticles in any mol% range of total lipids, from 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% therebetween. In some embodiments, the PEG-lipid conjugate may be present in the nanoparticles in any mol% range of total lipids, from 0 mol% to 5 mol%, from 0 mol% to 3 mol%, from 0 mol% to 2.5 mol%, from 0 mol% to 2.0 mol%, from 0 mol% to 1.8 mol%, or in any mol% range therebetween.

[0113] Figure 4

[0114] In another aspect, this disclosure provides a method for treating subjects suffering from any disorder or condition that would benefit from reduced PAI-1 expression.

[0115] In one implementation, the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, Alzheimer's disease, neurocognitive impairment, sepsis, pulmonary fibrosis, inflammatory disease, or any combination thereof.

[0116] In another implementation, PAI-1-related conditions, diseases, or disorders are associated with aging and increased lifespan.

[0117] This includes "thrombotic diseases," which, as used herein, encompasses any condition of any severity that causes abnormal clot formation in the subject, such as, but not limited to, coagulation disorders. Thrombotic diseases include, but are not limited to, venous thrombosis, arterial thrombosis, age-related thrombosis, microbial infection-related thrombosis, viral infection-related thrombosis, cancer-related thrombosis, post-traumatic thrombosis, post-operative thrombosis, inflammation-related thrombosis, tissue plasminogen activator deficiency-related thrombosis, antithrombin deficiency-related thrombosis, protein C deficiency-related thrombosis, protein S deficiency-related thrombosis, factor V Leiden-related thrombosis, or any combination thereof. The method comprises administering a therapeutically effective amount of siRNA to the subject, said RNA optionally encapsulated in lipid nanoparticles or conjugated with a ligand, thereby treating the subject or providing a preventative (protective) effect.

[0118] As used herein, the term "subject" includes any human or non-human mammal that would benefit from reduced PAI-1 expression relative to the absence of said treatment. In some embodiments, this includes a preventative (protective) benefit. In some embodiments, the subject is a human.

[0119] In one embodiment, this disclosure provides a method for preventing at least one symptom in a subject suffering from a PAI-1-related condition, disease, or disorder, the subject benefiting from reduced PAI-1 expression. The method includes administering a therapeutically effective amount of siRNA to the subject to prevent at least one symptom in the subject suffering from a condition, disease, or disorder, the subject benefiting from reduced PAI-1 expression. Non-limiting examples of symptoms include clot formation, arterial thickening, arteriosclerosis, intra-arterial plaque formation, exacerbated weight gain, increased blood glucose levels, cancer cell metastasis, cancer cell growth, tissue necrosis, any cognitive decline, increased levels of systemic inflammatory markers, or any combination thereof. The method includes administering a therapeutically effective amount of siRNA to the subject to prevent at least one symptom in the subject suffering from a condition, disease, or disorder, the subject benefiting from reduced PAI-1 expression.

[0120] In one embodiment, this disclosure provides a method for preventing at least one symptom (e.g., thrombosis) in a subject suffering from a thrombotic disease, the subject benefiting from reduced PAI-1 expression. The method includes administering a therapeutically effective amount of siRNA to the subject to prevent at least one symptom in the subject suffering from a condition, disease, or disorder, the patient benefiting from reduced PAI-1 expression.

[0121] In one implementation, administration of siRNA to a subject induces a reduction in symptoms of PAI-1-related disorders, diseases, or impairments.

[0122] In one implementation, administration of siRNA to the subject resulted in a reduction in thrombus formation and / or a reduction in PAI-1 expression and / or accumulation.

[0123] In another embodiment, this disclosure provides a method for treating a patient by modulating coagulation, the method comprising: administering siRNA to a subject in need to inhibit the expression of PAI-1. PAI-1 expression or activity may be evaluated as described in the Examples section herein.

[0124] Further methods for assessing knockdown, inhibition, and / or reduction of PAI-1 expression include quantifying hepatic PAI-1 mRNA levels, plasma PAI-1 protein concentrations, and / or PAI-1 activity. Inhibition of target gene or target sequence expression is achieved when the values ​​obtained using siRNA are approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to relevant controls. In other words, inhibition or reduction of PAI-1 expression is achieved when hepatic PAI-1 mRNA levels, plasma PAI-1 protein concentrations, and / or PAI-1 activity are reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0125] In another implementation, siRNA is used to treat cells in vitro or in vivo. The cells may be within an object, such as a mammalian object, or a human object suffering from a PAI-1 related condition, disease, or disorder.

[0126] Figure 4

[0127] In some embodiments, siRNA or lipid nanoparticles comprising nucleic acids that reduce PAI-1 expression are part of a pharmaceutical composition, and said siRNA or lipid nanoparticles are administered to treat and / or prevent disease conditions. The treatment may provide preventative (proprioceptive), ameliorative, or therapeutic benefits for treating hemorrhagic diseases. The pharmaceutical composition will be administered at any suitable dosage.

[0128] In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intra-arterial, intravenous, subcutaneous, or intramuscularly. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinally, intrathecally, or via other local routes.

[0129] The pharmaceutical composition contains a pharmaceutically acceptable carrier. For example, a "pharmaceutically acceptable carrier" means a component of the pharmaceutical preparation that is non-toxic to the target substance, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0130] Pharmaceutical compositions contain pharmaceutically acceptable salts and / or excipients. As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt derived from a variety of organic and inorganic counterions well known in the art, and by way of example only, said counterions include sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functional group, said pharmaceutically acceptable salt is derived from a salt of an organic or inorganic acid, such as hydrochloride, hydrobromide, tartrate, methanesulfonate, 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.

[0131] As used herein, the term "excipient" means a substance used to formulate an active pharmaceutical ingredient (API) into a pharmaceutical preparation. Non-limiting examples include mannitol, Captisol®, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, etc. Acceptable excipients are non-toxic and can be any solid, liquid, or semi-solid excipient commonly available to those skilled in the art.

[0132] The examples are intended to illustrate the preparation and properties of the invention and not to limit the scope of the invention.

[0133] Example

[0134] Materials and methods

[0135] Figure 4

[0136] Rodent research is approved by each institution's respective animal care committee. Rodents aged 8 to 10 weeks... Figure 4 ( Jackson Labs (Bar Harbor, ME, stock #000664) mice and aged mice aged 72 to 80 weeks were used for adult and aged PAI-1 knockdown studies, respectively. Diet-induced obesity in 17-week-old mice... Figure 4Mice (Jackson Labs, Bar Harbor, ME, stock#380050) were used for obesity PAI-1 knockdown studies. For other obesity PAI-1 studies, C57BL / 6J mice were fed a high-fat diet (HFD 60% fat; catalog number D12492, Research Diets) or a random low-fat diet (LFD, 10% to 13% fat; D12450, Research Diets).

[0137] siRNA-LNP formulations and injections

[0138] siRNAs targeting PAI-1 or luciferase (negative, out-of-order siRNA control) (Integrated DNA Technologies, Coralville, USA) were encapsulated in LNPs. Briefly, the siRNA was dissolved in sodium acetate (pH 4) and mixed with a lipid solution at an amine / phosphate (N / P) ratio of 3. The lipid formulation contained DSPC, cholesterol, and PEG-DMG in a molar ratio of 10:38.5:1.5% and was enriched with 50% DLin-MC3-DMA or ALC-0315 (Avanti PolarLipids, Birmingham, AL). The LNPs were dialyzed overnight with a 500-fold excess volume of Dulbecco phosphate-buffered saline (PBS) at pH 7.4. To determine siRNA concentration and encapsulation efficiency, RiboGreen assays (Quant-IT RiboGreen RNA Assay Kit, ThermoFisher) were performed with or without Triton X-100 detergent. Cholesterol E assay kit (Wako Chemicals, Mountain View, CA USA) was used to measure cholesterol levels. LNP was diluted to a final concentration of 0.1 mg siRNA / ml or 0.3 mg siRNA / ml in PBS (pH 7.4) or cryobuffer (10% sucrose, 10 mM L-histidine, pH 7.4). Mice were administered a single dose of 1 mg siRNA / kg body weight or 3 mg siRNA / kg body weight via tail vein, or repeated every other week.

[0139] Extracts of mouse liver, serum and plasma

[0140] Endpoint blood samples were collected via cardiac puncture, and liver tissue was surgically removed under isoflurane anesthesia. Blood was collected into a syringe containing sodium citrate (0.32% final concentration), and platelet-rich plasma (PRP) was separated from whole blood by rotation at 1500×g for 10 minutes. Serum for toxicological analysis was separated in the same manner as plasma, but blood was collected without sodium citrate and allowed to coagulate for 40 minutes before centrifugation. For non-endpoint blood samples, blood was collected retro-orbital.

[0141] mRNA extraction and quantification

[0142] Liver tissue was homogenized in Trizol (ThermoFisher, Waltham, USA). DNA and RNA were separated by precipitation with chloroform and isopropanol. DNA was digested by incubating samples with TURBO Dnase and 10-fold TURBO Dnase Buffer (ThermoFisher, Waltham, USA). DNase was removed by repeated 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 SYBR Green Master Mix (ThermoFisher, Waltham, USA) and DNA primers targeting PAI-1 (IntegratedDNA Technologies). PAI-1 expression was quantified relative to the expression of the housekeeping gene peptidylprolyl isomerase A using the DDCt method. Data were collected and analyzed using QuantStudio 6.

[0143] Analysis of PAI-1 levels in plasma

[0144] Total plasma PAI-1 concentrations were analyzed using the Mouse Total PAI-1 ELISA Kit (IMSPAI1KTT, Innovative Research) following the manufacturer's instructions. For optimal results, modifications to the manufacturer's protocol included loading 100 μL of plasma into each sample and extending the primary antibody incubation time to 45 minutes.

[0145] Coagulation function test

[0146] Rotational thromboelastography (ROTEM) (Rotem Delta, Werfen SA, Spain) was performed according to the manufacturer's instructions. Whole mouse blood was mixed with 20 μL of 0.2M CaCl2, 20 μL of EXTEM reagent containing tissue factor (Werfen SA, Spain), and recombinant human tPA (IHUTPA85SC100UG, Innovative Research) to a final concentration of 500 ng / ml. All reagents were incubated until they reached 37°C. Each test was performed for 1.5 hours.

[0147] IVC ligation

[0148] Mice were intravenously injected with 3 mg / kg siPAI-1 or siLuc. Complete IVC ligation was performed 3 days post-injection. Briefly, anesthesia was induced in 100% oxygen with 4% isoflurane until the righting reflex disappeared. Mice were transferred to an operating table and anesthesia was maintained in 100% oxygen with 1% to 2% isoflurane. Adequate depth of anesthesia was confirmed by the absence of pain response during paw pinching. After aseptic preparation of the abdomen, laparotomy was performed. The intestines were externalized and protected with saline-soaked gauze to limit non-obvious fluid loss. The IVC was carefully exposed and ligated directly below the renal branch using 7-0 prismin sutures. Any other venous branches present between the renal vein and inferior iliac vein were similarly ligated. After ligation, the intestines were returned to the abdominal cavity, and the laparotomy site was sutured. Postoperative analgesia was administered subcutaneously with buprenorphine. Mice were then allowed to recover from anesthesia. Four days post-ligation, mice were euthanized to collect blood, liver tissue, and IVC thrombi for subsequent analysis.

[0149] Toxicological analysis

[0150] Mice were intravenously injected with 3 mg / kg of PBS or siPAI-1. Five hours after injection, the mice were euthanized, and serum samples were collected as described above. Serum samples were subjected to comprehensive toxicological analysis using Idexx BioAnalytics (North Grafton, MA, USA). This included measurements of liver and kidney function biochemical parameters, including aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), gamma-glutamyl transferase (GGT), and biomarkers of metabolic health, such as blood lipids, blood urea nitrogen (BUN), and creatine (CREA).

[0151] Blood cell count analysis

[0152] Whole blood was collected and blood cell counts were performed using Heska Element HT5.

[0153] Blood cholesterol analysis

[0154] Cholesterol levels in plasma samples were assessed using the Total Cholesterol E Kit from Fujifilm.

[0155] Screening for siRNAs targeting PAI-1 in human hepatocyte (HEPG2) cell cultures.

[0156] HepG2 cells were transfected with different siRNA sequences targeting PAI-1 encapsulated in LNPs at single doses (0.3 ng / ml, 1 ng / ml, or 3 ng / ml). PAI-1 mRNA levels were quantified at 24 hours as described above.

[0157] Example 1: In vivo PAI-1 siRNA knockdown and its effect on coagulation under in vitro conditions

[0158] This example demonstrates that siRNA knockdown of PAI-1 mRNA in mice leads to depletion of circulating PAI-1 protein and reduces PAI-1 activity in vivo.

[0159] The control siRNA (siLuc) targeting luciferase or the siRNA (siPAI-1) targeting the mouse PAI-1 sequence (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) was encapsulated in lipid nanoparticles containing MC3 or ALC-0315 (ALC) as ionizable lipids and administered intravenously to mice as described in Materials and Methods.

[0160] The ELISA method described in Materials and Methods was used to quantify the PAI-1 protein level in mouse plasma after administration of siLuc or mouse siPAI-1, and blood samples were collected one week later.

[0161] The results are as follows Figure 4 As shown in A. Compared with siLuc-treated mice, one week after administration of mouse siPAI-1 (double-stranded siRNA of SEQ ID No: 11 and SEQ ID No: 12) encapsulated in lipid nanoparticles containing MC3 (siPAI-1 MC3) and lipid nanoparticles containing ALC-0315 (siPAI-1 ALC), the plasma PAI-1 protein level in mice was significantly reduced.

[0162] One week after administration of mouse siPAI-1 (double-stranded siRNAs of SEQ ID No: 11 and SEQ ID No: 12) to mice, liver tissue was collected, and liver PAI-1 mRNA levels were quantified by PCR (qPCR) as described in Materials and Methods and compared with control siRNAs targeting luciferase (siLuc).

[0163] The results are as follows Figure 4 As shown in B. Compared with siLuc-treated mice, the level of hepatic PAI-1 mRNA in the liver of mice was significantly reduced one week after administration of mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12).

[0164] Time-course studies were conducted by using ELISA as described in Materials and Methods to quantify plasma PAI-1 protein levels at 3, 10, 14, and 20 days after a single injection of 3 mg / kg of control siLuc or mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) as shown in Table 2.

[0165] The results are as follows Figure 4 As shown in C. Compared with siLuc-treated mice, the plasma PAI-1 levels in mice injected with siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) in Table 2 were significantly reduced on days 3 and 10.

[0166] The rotational thromboelastography (ROTEM) assay, as described in Materials and Methods, was used to measure the ex vivo coagulation properties of mouse blood treated with siLuc and mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) as shown in Table 2. The percentage of clot dissolution, clot formation rate, clotting time, and clot stiffness were quantified based on the ROTEM.

[0167] Representative ROTEM curves are shown Figure 4 D.

[0168] Figure 4 E compared the percentage of clot dissolution in mouse blood treated with siLuc and siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) in Table 2 at 30, 45 and 60 minutes before the assay, showing that siPAI-1 treated mouse blood was protected from clot dissolution compared with siLuc treated mice.

[0169] Figure 4 F and Figure 4G compared the rate (α angle) and time of clot formation in the blood of mice treated with siLuc and siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) as shown in Table 2, and showed that mice treated with siLuc and siPAI-1 formed clots at similar rates.

[0170] Figure 4 H compared the maximum clot stiffness (MCF) in mouse blood treated with siLuc and mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) as shown in Table 2, and showed that clots formed from mouse blood treated with siPAI-1 had the same stiffness as clots formed from mouse blood treated with siLuc.

[0171] Example 2: Using siRNA to deplete plasma PAI-1 to reduce thrombus formation

[0172] This example demonstrates that siRNA knockdown of PAI-1 can reduce thrombus formation in vivo.

[0173] As described in Materials and Methods, siLuc or mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) listed in Table 2 was administered to adult (9 to 12 weeks) and aged (80 weeks) mice, inducing inferior vena cava (IVC) stasis three days later. As described in Materials and Methods, the endpoint was four days after IVC ligation, at which time clots and blood were collected and analyzed.

[0174] like Figure 4 As shown in A, adult mice treated with siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) in Table 2 formed significantly smaller and lighter clots compared with mice treated with siLuc.

[0175] Figure 4 B shows that, compared with siLuc-treated mice, adult mice treated with the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) listed in Table 2 had significantly reduced plasma PAI-1 protein levels.

[0176] Figure 4 C shows representative trichrome hematoxylin and eosin (H & E) staining of clots formed in the IVC of adult mice treated with siLuc and the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) of Table 2. The clots in mice treated with siLuc were significantly larger compared with those in mice treated with the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) of Table 2.

[0177] Figure 4 D shows the survival probability of aged mice treated with siLuc or the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) from Table 2 after IVC ligation, demonstrating that depletion of circulating PAI-1 increases survival probability after induction of IVC thrombosis. The black line represents the survival probability of mice treated with siLuc, and the gray line represents the survival probability of mice treated with siPAI-1.

[0178] Figure 4 E shows that, compared with mice treated with siLuc, aged mice treated with the siPAI-1 mice (SEQ ID No: 11 and SEQ ID No: 12) listed in Table 2 formed significantly smaller and lighter clots.

[0179] Serum levels of several cytokines were quantified as described in Materials and Methods, including granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interleukin-1-α (IL-1α), interleukin-6 (IL-6), and interleukin-12 (IL-12). Results are as follows: Figure 4 As shown in F.

[0180] like Figure 4 As shown in Figure F, mice treated with siPAI-1 had lower levels of various cytokines compared to mice treated with siLuc.

[0181] Example 3: Depletion of circulating PAI-1 alleviated obesity-induced upregulation of circulating PAI-1 levels.

[0182] As described in Materials and Methods, mice were fed a high-fat diet (HFD) to induce obesity, or a low-fat diet (LFD) as a control, and blood and liver samples were collected for PAI-1 protein and mRNA quantification.

[0183] Figure 4 A showed that mice with HFD had significantly higher levels of circulating PAI-1 protein compared to mice with LFD.

[0184] Figure 4 B shows that mice with HFD had significantly higher levels of PAI-1 mRNA in their livers.

[0185] Figure 4 C correlated PAI-1 mRNA levels with corresponding mouse body weight, showing that heavier mice had higher PAI-1 mRNA levels.

[0186] As described in Materials and Methods, separate groups of mice were fed a high-fat diet (HFD) to induce obesity and treated with siLuc or the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) listed in Table 2. Blood and liver samples were collected for quantification of PAI-1 protein and mRNA, respectively.

[0187] Figure 4 D and Figure 4 E showed that siPAI-1 significantly reduced circulating PAI-1 protein and hepatic PAI-1 mRNA levels in obese mice.

[0188] Example 4: The use of LNP-delivered siRNA targeting PAI-1 did not induce hepatotoxicity or abnormal blood cell counts.

[0189] As described in Materials and Methods, mice were administered phosphate-buffered saline (PBS) or LNPs containing MC3 or ALC-0315 (ALC) as shown in Table 2, and serum was collected 5 hours later for toxicological analysis.

[0190] Figure 4 A shows that serum alkaline phosphatase (ALP) levels were comparable between mice treated with PBS and siPAI-1.

[0191] Figure 4 B shows that serum aspartate aminotransferase (AST) levels were comparable between mice treated with PBS and siPAI-1.

[0192] Figure 4 C shows that serum alanine aminotransferase (ALT) levels were comparable between mice treated with PBS and siPAI-1.

[0193] Figure 4 D shows that serum blood urea nitrogen (BUN) levels were comparable between mice treated with PBS and siPAI-1.

[0194] In general, Figure 4 A to Figure 4 D shows that mice treated with mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) encapsulated in LNP containing MC3 or ALC-0315 (ALC) as ionizable lipids did not induce hepatotoxicity.

[0195] Mice were administered siLuc or the mouse siPAI-1 listed in Table 2 (SEQ ID No: 11 and SEQ ID No: 12) at 1 mg / kg or 3 mg / kg. Blood was collected 7 days later for complete blood cell count analysis as described in Materials and Methods.

[0196] Figure 4 E showed that the white blood cell (WBC) counts were comparable between siLuc and siPAI-1 treated mice.

[0197] Figure 4 F shows that the red blood cell (RBC) counts were comparable between siLuc and siPAI-1 treated mice.

[0198] Figure 4 G showed that platelet (WBC) counts were comparable between siLuc and siPAI-1 treated mice.

[0199] In general, Figure 4 E to Figure 4 G shows that mice treated with the mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) in Table 2 at doses of 1 mg / kg or 3 mg / kg did not induce abnormal blood cell counts.

[0200] Example 5: Knockdown of PAI-1 siRNA in aged mice reduced PAI-1 levels to those found in healthy young adult mice.

[0201] As described in Materials and Methods, aged mice (72 to 76 weeks old) were administered siLuc or mouse siPAI-1 (SEQ ID No: 11 and SEQ ID No: 12) listed in Table 2 weekly until the study endpoint. Blood samples were collected from the mice starting 3 days after the first injection and then weekly until the study endpoint.

[0202] Figure 4 A showed that, compared with mice treated with siPAI-1, aged mice treated with siPAI-1 had significantly lower plasma PAI-1 levels 10 days after the first dose of siPAI-1, and this reduction persisted until the study endpoint of weekly siPAI-1 injections.

[0203] Example 6: Apolipoprotein E knockout (ApoE) - / - PAI-1 siRNA knockdown in mice reduces blood cholesterol.

[0204] As described in Materials and Methods, to ApoE - / -Mice (72 to 76 weeks old) were administered siLuc or the mouse siPAI-1 listed in Table 2 (SEQ ID No: 11 and SEQ ID No: 12). Blood samples were collected from mice starting on days 3 and 7 post-treatment to quantify plasma levels of PAI-1 and cholesterol.

[0205] Figure 4 A shows the results on days 3 and 7 post-injection, compared to ApoE treated with siLuc. - / - Compared to mice, ApoE treated with siPAI-1 - / - Plasma levels of PAI-1 were significantly reduced in mice.

[0206] Figure 4 B shows the results on day 3 post-injection, compared to ApoE treated with siLuc. - / - Compared to mice, ApoE treated with siPAI-1 - / - Plasma cholesterol levels were significantly reduced in mice. On day 7 post-injection, ApoE mice treated with siLuc and siPAI-1 showed significantly reduced cholesterol levels. - / - The mice had relatively high levels of cholesterol in their plasma.

[0207] Example 7: In vitro knockdown of human PAI-1 using siRNA

[0208] This example demonstrates that siRNA can knock down PAI-1 in human hepatocytes in vitro.

[0209] After administering LNPs containing siLuc and human siPAI-1 corresponding to sequences A (SEQ ID No: 13 and SEQ ID No: 14), B (SEQ ID No: 15 and SEQ ID No: 16), C (SEQ ID No: 17 and SEQ ID No: 18), and D (SEQ ID No: 19 and SEQ ID No: 20) to cultured human hepatocytes (HEPG2), the levels of human PAI-1 mRNA were measured using quantitative PCR as described in Materials and Methods.

[0210] like Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 Figure As shown, compared with siLuc-treated cells, treatment with LNP containing double-stranded siRNA corresponding to sequence A (SEQ ID No: 13 and SEQ ID No: 14) resulted in significant depletion of PAI-1 mRNA in HEPG2 cells.

[0211] Although the present invention has been described and illustrated with reference to the foregoing specific embodiments and examples, it is obvious that various modifications and variations can be made without departing from the present invention.

Claims

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

10.

2. The siRNA molecule of claim 1, wherein the sense strand has a nucleotide sequence comprising at least 85% sequence identity with 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 with any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:

10.

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

10.

4. The siRNA molecule of claim 1, wherein the sense strand has a nucleotide sequence comprising at least 95% sequence identity with 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 with any one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:

10.

5. A siRNA molecule for inhibiting the expression of plasminogen activator inhibitor-1 (PAI-1) in cells, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to the mRNA encoding PAI-1, wherein the sense strand has a nucleotide sequence comprising at least 70% sequence identity with SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 70% sequence identity with 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 with SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 80% sequence identity with 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 with SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 90% sequence identity with 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 with SEQ ID NO: 3, and the antisense strand has a nucleotide sequence comprising at least 95% sequence identity with SEQ ID NO:

4.

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

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

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

13. The siRNA molecule according to any one of claims 9 to 12, wherein the modified nucleotide is selected from: 2'-O-alkyl modified nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-halogen modified nucleotides, nucleotides containing a thiophosphate group, deoxynucleotides, 3'-terminal deoxythymidine nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate group, nucleotides containing a 5'-phosphate ester mimic, ethylene glycol modified nucleotides, 2-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

14. A siRNA molecule for inhibiting the expression of plasminogen activator inhibitor-1 (PAI-1) in cells, wherein the siRNA molecule comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to the mRNA encoding PAI-1, wherein the sense strand has a nucleotide sequence comprising at least 80% sequence identity with 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 with 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 with 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 with 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 with any one of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, and the antisense strand comprises a nucleotide sequence comprising at least 90% sequence identity with 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 with 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 with any one of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO:

20.

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

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

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

21. The siRNA molecule according to 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 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 5' end of the antisense strand.

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

25. The siRNA molecule according to any one of claims 21 to 24, wherein the ligand comprises one or more of antibodies, peptides, amino acids, aptamers, phosphate groups, cholesterol moieties, lipids, cell-penetrating peptide polymers, glycosides, and their derivatives.

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

27. The siRNA molecule of claim 26, wherein the glycosylation group comprises a glycomonomer, an oligosaccharide, and / or a derivative 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 comprises a 5' overhang of at least one 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 comprises a 3' overhang of at least one nucleotide.

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

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

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

34. Lipid nanoparticles, comprising: The siRNA molecule targeting PAI-1 mRNA as described in any one of claims 1 to 33; Approximately 20 mol% to approximately 70 mol% of ionizable cationic lipids with pKa values ​​of 5.5 to 7.0; Neutral vesicle formation assisting lipids, which include phospholipids and / or triglycerides; Sterols; and Hydrophilic polymer-lipid conjugates, approximately 0.5 mol% to approximately 5 mol%.

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

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

37. The lipid nanoparticles of any one of claims 34 to 36, wherein the neutral vesicle-forming assisting lipids comprise phospholipids.

38. The lipid nanoparticles of any one of claims 34 to 37, wherein the lipid nanoparticles comprise about 20 mol% to about 60 mol% of the neutral vesicle-forming assisting lipids.

39. The lipid nanoparticles of any one of claims 34 to 38, wherein the sterols include cholesterol or derivatives thereof.

40. The lipid nanoparticles of any one of claims 34 to 39, wherein the lipid nanoparticles comprise about 15 mol% to 65 mol% of the sterols.

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

42. A pharmaceutical composition for inhibiting PAI-1 expression, comprising lipid nanoparticles as described in 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 buffer solution.

46. ​​The pharmaceutical composition of claim 45, wherein the buffer solution comprises one or more of acetic acid, citrate, alcohol-soluble gluten, carbonate, and phosphate.

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

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

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

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

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

52. The method of claim 51, wherein the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive impairment, Alzheimer's disease, sepsis, pulmonary fibrosis, inflammatory disease, or any combination thereof.

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

54. The method of claim 51, wherein the PAI-1 related condition, disease, or disorder is a coagulation disorder.

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

56. The method of claim 53, wherein the thrombotic disease is venous thrombosis, arterial thrombosis, senile thrombosis, microbial infection-related thrombosis, viral infection-related thrombosis, cancer-related thrombosis, post-traumatic thrombosis, post-operative thrombosis, inflammation-related thrombosis, tissue plasminogen activator deficiency-related thrombosis, antithrombin deficiency-related thrombosis, protein C deficiency-related thrombosis, protein S deficiency-related thrombosis, factor V Leiden-related thrombosis, or any combination thereof.

57. A method of treating a subject suffering from a PAI-1 related condition, disease, or disorder, the method comprising administering to the subject a therapeutically effective amount of any one of claims 1 to 33, any one of claims 34 to 40, or any one of claims 41 to 47, thereby treating the subject suffering from a PAI-1 related condition, disease, or disorder.

58. A method for preventing at least one symptom in a subject suffering from a PAI-1-related condition, disease, or disorder, the subject benefiting from reduced PAI-1 mRNA expression, the method comprising administering to the subject a preventatively effective amount of any siRNA molecule of any one of claims 1 to 33, any lipid nanoparticle of any one of claims 34 to 40, or any pharmaceutical composition of any one of claims 41 to 47, thereby preventing at least one symptom in a subject suffering from a PAI-1-related condition, disease, or disorder, the subject benefiting from reduced PAI-1 mRNA expression.

59. The method of claim 57 or 58, wherein the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive impairment, Alzheimer's disease, sepsis, pulmonary fibrosis, inflammatory disease, or any combination thereof.

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

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

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

63. The method of claim 60, wherein the thrombotic disease is venous thrombosis, arterial thrombosis, senile thrombosis, microbial infection-related thrombosis, viral infection-related thrombosis, cancer-related thrombosis, post-traumatic thrombosis, post-operative thrombosis, inflammation-related thrombosis, tissue plasminogen activator deficiency-related thrombosis, antithrombin deficiency-related thrombosis, protein C deficiency-related thrombosis, protein S deficiency-related thrombosis, factor V Leiden-related thrombosis, or any combination thereof.

64. Use of the siRNA molecule of any one of claims 1 to 33, the lipid nanoparticle of any one of claims 34 to 40, or the pharmaceutical composition of any one of claims 41 to 47 in the treatment of PAI-1 related conditions, diseases, or disorders.

65. Use of the siRNA molecule of any one of claims 1 to 33, the lipid nanoparticle of any one of claims 34 to 40, or the pharmaceutical composition of any one of claims 41 to 47 in the prevention of at least one symptom in a subject suffering from a PAI-1 related condition, disease, or disorder, said subject benefiting from a reduction in PAI-1 mRNA expression.

66. The use as described in claim 64 or 65, wherein the PAI-1 related condition, disease, or disorder is thrombosis, atherosclerosis, coronary artery disease, obesity, diabetes, cancer, liver necrosis, neurocognitive impairment, Alzheimer's disease, sepsis, pulmonary fibrosis, inflammatory disease, or any combination thereof.

67. The use as described in claim 64 or 65, wherein the PAI-1 related condition, disease, or disorder is a thrombotic disease.

68. The use as described in claim 64 or 65, wherein the PAI-1 related condition, disease, or disorder is a coagulation disorder.

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

70. The use as described in claim 67, wherein the thrombotic disease is venous thrombosis, arterial thrombosis, senile thrombosis, microbial infection-related thrombosis, viral infection-related thrombosis, cancer-related thrombosis, post-traumatic thrombosis, post-operative thrombosis, inflammation-related thrombosis, tissue plasminogen activator deficiency-related thrombosis, antithrombin deficiency-related thrombosis, protein C deficiency-related thrombosis, protein S deficiency-related thrombosis, factor V Leiden-related thrombosis, or any combination thereof.

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