Recombinant tissue plasminogen activator (tPA) fragments and their use

Abstract

This technology relates to recombinant polypeptides containing tissue plasminogen activator (tPA) fragments, or nucleotides encoding them, and their use for treating cardiovascular diseases. In some embodiments, the tPA fragment comprises the kringle 2 domain of tPA.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 63 / 467,450, filed on 18 May 2023, the contents of which are incorporated by reference in whole for any and all purposes.

[0002] Technical field This technology provides recombinant polypeptides containing tissue plasminogen activator (tPA) fragments, or nucleotides encoding them, and their use for treating cardiovascular diseases. In some embodiments, the tPA fragment comprises the kringle 2 domain of tPA (tPA-K2).

[0003] Statement on federally sponsored research or development This invention was created with government support, based on HL163516, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0004] Sequence List An informal sequence listing is provided herein. [Background technology]

[0005] background Apolipoprotein B (apoB)-containing lipoproteins initiate and promote atherosclerotic cardiovascular disease (CVD) [1, 2]. Examples of apoB-containing lipoproteins include very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and lipoprotein(a) [Lp(a)] derived from hepatocytes, as well as chylomicrons and chylomicron remnants derived from intestinal cells. Hepatocytes and intestinal cells produce and secrete VLDL and chylomicrons into the bloodstream, respectively. Circulating VLDL is then progressively hydrolyzed in the blood to form IDL and LDL. Similarly, circulating chylomicrons are hydrolyzed to form chylomicron remnants [2]. Current recommended lipid interventions to help prevent CVD primarily use statins or proteolytic enzyme subtilisin / kexin type 9 (PCSK9) inhibitors, both of which lower LDL by increasing hepatic LDL receptor (LDLR)-mediated LDL clearance [3]. However, these treatments have little effect on other atherogenic apoB-containing lipoproteins such as VLDL, IDL, Lp(a), chylomicrons, and chylomicron remnants [4, 5], which contribute to the residual CVD risk in populations with well-controlled LDL cholesterol [6, 7]. Therefore, therapies that inhibit hepatic VLDL and Lp(a) production and intestinal chylomicron production may be useful in reducing CVD risk because they reduce all atherogenic apoB lipoproteins. Thus, there is a need in the art for new therapeutic approaches to reduce atherogenic apoB lipoproteins and treat cardiovascular disease. [Overview of the project]

[0006] overview In one embodiment, the disclosure provides an isolated polynucleotide molecule comprising: (a) the nucleotide sequence described in SEQ ID NO: 6, (b) the nucleotide sequence described in SEQ ID NO: 10, (c) the nucleotide sequence described in SEQ ID NO: 15, (d) the nucleotide sequence described in SEQ ID NO: 39, (e) the nucleotide sequence described in SEQ ID NO: 2, (f) a nucleotide sequence that is at least about 85% identical to any one of (a) to (f), and encoding a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptide, wherein the recombinant tPA-K2-containing polypeptide can bind to apolipoprotein B (apoB) and / or inhibit apoB secretion from hepatocytes and / or intestinal cells and / or inhibit apoB protein lipidization; (g) a nucleotide sequence that is a complement to any one of (a) to (f); and (h) an RNA sequence encoded by any one of (a) to (g), wherein the nucleotide sequence is operably linked to a heterologous nucleic acid. In some embodiments, the heterologous nucleic acid includes an endoplasmic reticulum (ER) localized sequence. In some embodiments, the ER localized sequence encodes an amino acid having the sequence KDEL (SEQ ID NO: 11). In some embodiments, the nucleotide sequence is described in SEQ ID NO: 8. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 34. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 16. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 4. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 36. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 38. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 32. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 40. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 14.In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 35. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 30. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 37. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO: 31. In some embodiments, the disclosure provides an expression vector comprising any one of the polynucleotide molecules operably linked to one or more regulatory sequences suitable for leading expression in eukaryotic cells. In some embodiments, the one or more regulatory sequences comprises a promoter. In some embodiments, the promoter is a thyroxine-binding globulin (TBG) promoter, or the promoter comprises the nucleic acid sequence described in SEQ ID NO: 17. In some embodiments, the disclosure provides a cell comprising either the polynucleotide molecule or the expression vector. In some embodiments, the cell is selected from hepatocytes or intestinal cells. In some embodiments, the disclosure provides an infection particle comprising any one of the polynucleotide molecules. In some embodiments, the infection particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the AAV is AAV8. In some embodiments, the disclosure provides lipid nanoparticles comprising any one of polynucleotide molecules. In some embodiments, the lipid nanoparticles are lyophilized, present in a suspension, or emulsified. In some embodiments, the disclosure provides a composition comprising any one of polynucleotide molecules, vectors, infected particles, or lipid nanoparticles and a pharmaceutically acceptable carrier.In some embodiments, the Disclosure provides a method for reducing plasma apoB lipoprotein in subjects requiring a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of a composition to the subject. In some embodiments, the plasma apoB lipoprotein is selected from very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof. In some embodiments, the Disclosure provides a method for treating diseases associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount of a composition to the subject. In some embodiments, the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes. In some embodiments, the Disclosure provides a method for reducing plasma triglyceride and / or cholesterol levels in subjects requiring a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of a composition to the subject. In some embodiments, the method further comprises administering therapeutically effective doses of plasminogen activator inhibitor-1 (PAI-1) inhibitors to a subject simultaneously, separately, or sequentially. In some embodiments, the PAI-1 inhibitor is selected from the group consisting of: MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, Aleplasinin, Loureirin B, Diaplasinin, Toddalolactone, SK-216, Geodin, Fendosal, AZ3976, TM5007, and any combination thereof. In some embodiments, the composition is administered to the subject intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly. In some embodiments, the subject is human.

[0007] In one embodiment, the disclosure provides recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptides, or pharmaceutically acceptable salts, tautomers, hydrates, and / or solvates thereof, wherein the (tPA-K2 polypeptide) comprises (a) an amino acid sequence selected from the group consisting of (i) the amino acid sequence shown in SEQ ID NO: 5, (ii) the amino acid sequence shown in SEQ ID NO: 13, (iii) the amino acid sequence shown in SEQ ID NO: 9, (iv) the amino acid sequence shown in SEQ ID NO: 30, (v) the amino acid sequence shown in SEQ ID NO: 1, (vi) the amino acid sequence shown in SEQ ID NO: 37, and (vii) an amino acid sequence that is at least about 85% identical to any one of (i) to (vi), and can bind to apolipoprotein B (apoB) and / or can inhibit apoB secretion from hepatocytes and / or intestinal cells and / or can inhibit apoB lipoproteinization, and (b) a heterologous amino acid sequence. In some embodiments, the heterologous amino acid sequence includes an endoplasmic reticulum localization motif. In some embodiments, the endoplasmic reticulum localization motif is KDEL. In some embodiments, the amino acid sequence is described in SEQ ID NO: 7. In some embodiments, the amino acid sequence is described in SEQ ID NO: 14. In some embodiments, the amino acid sequence is described in SEQ ID NO: 35. In some embodiments, the amino acid sequence is described in SEQ ID NO: 37. In some embodiments, the amino acid sequence is described in SEQ ID NO: 3. In some embodiments, the polypeptide does not have serine protease function. In some embodiments, the polypeptide is not fibrinolytic. In some embodiments, the polypeptide binds to apoB. In some embodiments, administration of a therapeutically effective amount of the polypeptide to a subject reduces plasma triglyceride and / or cholesterol levels in the subject. In some embodiments, administration of a therapeutically effective amount of the polypeptide to a subject reduces plasma levels of one or more apoB lipoproteins in the subject.In some embodiments, one or more apoB lipoproteins are selected from very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof. In some embodiments, the Disclosure provides a composition comprising one of the recombinant polypeptides and a pharmaceutically acceptable carrier. In some embodiments, the Disclosure provides a method for reducing plasma apoB lipoprotein in a subject requiring a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of the recombinant polypeptide or composition to the subject. In some embodiments, the Disclosure provides a method for treating a disease associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount of one of the recombinant polypeptides or composition to the subject. In some embodiments, the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes. In some embodiments, the method further comprises administering therapeutically effective doses of plasminogen activator inhibitor-1 (PAI-1) inhibitors to a subject simultaneously, separately, or sequentially. In some embodiments, the PAI-1 inhibitor is selected from the group consisting of: MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, aleprasinin, laurelin B, diaprasinin, todarolactone, SK-216, geodin, fendosal, AZ3976, TM5007, and any combination thereof. In some embodiments, the recombinant polypeptide or composition is administered to the subject intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly. In some embodiments, the subject is human.

[0008] In one embodiment, the Disclosure provides a polynucleotide molecule comprising the nucleotide sequence described in Sequence ID No. 8. In some embodiments, the polynucleotide is formulated in an infectious particle for delivery to a subject. In some embodiments, the infectious particle is an adeno-associated virus (AAV). In some embodiments, the polynucleotide is formulated in lipid nanoparticles (LNPs) for delivery to a subject. In some embodiments, the Disclosure provides a composition comprising one of the polynucleotides and a pharmaceutically acceptable carrier. In some embodiments, the Disclosure provides a method for reducing plasma apoB lipoprotein in a subject that requires a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, the Disclosure provides a method for reducing plasma triglyceride and / or cholesterol levels in a subject that requires a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of the composition to the subject.

[0009] In one embodiment, the Disclosure provides a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence described in SEQ ID NO: 34, (b) the nucleotide sequence described in SEQ ID NO: 10, and (c) the nucleotide sequence described in SEQ ID NO: 36. In some embodiments, the polynucleotide is formulated in an infectious particle for delivery to a subject. In some embodiments, the infectious particle is an adeno-associated virus (AAV). In some embodiments, the polynucleotide is formulated in lipid nanoparticles (LNPs) for delivery to a subject. In some embodiments, the Disclosure provides a composition comprising one of the polynucleotides and a pharmaceutically acceptable carrier. In some embodiments, the Disclosure provides a method for reducing plasma apoB lipoprotein in a subject that requires a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, the Disclosure provides a method for reducing plasma triglyceride and / or cholesterol levels in a subject that requires a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 34. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 10. In some embodiments, the nucleotide sequence is described in SEQ ID NO: 36.

[0010] In one embodiment, the Disclosure provides recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptides, or pharmaceutically acceptable salts, tautomers, hydrates, and / or solvates thereof (tPA-K2 polypeptides), comprising an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NO: 9 and SEQ ID NO: 35. In some embodiments, the Disclosure provides compositions comprising a recombinant polypeptide and a pharmaceutically acceptable carrier. In some embodiments, the Disclosure provides a method for reducing plasma apoB lipoprotein in subjects requiring a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of any one of the recombinant polypeptide compositions to the subject. In some embodiments, the Disclosure provides a method for treating diseases associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount of any one of the recombinant polypeptides or compositions to the subject. In some embodiments, the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.

[0011] In one embodiment, the disclosure provides a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) polypeptide or a fragment thereof. The recombinant tPA-K2 polypeptide comprises a sequence having at least 80% identity with SEQ ID NO: 5. In some embodiments, the polypeptide does not have serine protease function. In some embodiments, the polypeptide is not fibrinophilic. In some embodiments, the recombinant tPA-K2 polypeptide binds to apolipoprotein B (apoB). In some embodiments, the recombinant tPA-K2 polypeptide comprises a sequence selected from SEQ ID NOs: 13-14. In some embodiments, the recombinant tPA-K2 polypeptide comprises SEQ ID NO: 14. In some embodiments, the disclosure provides a method for producing the recombinant tPA-K2 polypeptide. In some embodiments, the disclosure provides a polynucleotide comprising a nucleotide sequence encoding the recombinant tPA-K2 polypeptide. In some embodiments, the nucleotide sequence encoding the recombinant tPA-K2 polypeptide comprises a nucleotide sequence selected from SEQ ID NOs: 15-16. In some embodiments, the polynucleotide further comprises at least one regulatory sequence operably linked to the nucleotide sequence encoding the recombinant tPA-K2 polypeptide. In some embodiments, at least one regulatory sequence comprises a promoter, an enhancer, or both a promoter and an enhancer. In some embodiments, at least one regulatory sequence comprises a promoter. In some embodiments, the promoter is a thyroxine-binding globulin (TBG) promoter, or the promoter comprises SEQ ID NO: 17. In some embodiments, the polynucleotide comprises two or more nucleotide sequences encoding a recombinant tPA-K2 polypeptide. In some embodiments, the disclosure provides nanoparticles comprising a recombinant tPA-K2 polypeptide or a polynucleotide. In some embodiments, the disclosure provides infectious particles comprising a polynucleotide. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the AAV is AAV8.In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant tPA-K2 polypeptide. In some embodiments, the present disclosure provides a pharmaceutical composition comprising nanoparticles. In some embodiments, the present disclosure provides a pharmaceutical composition comprising infectious particles. In some embodiments, the present disclosure provides a method comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject. In some embodiments, the subject has hypercholesterolemia or hyperlipidemia. In some embodiments, the subject has been diagnosed with a cardiovascular disease. In some embodiments, the cardiovascular disease includes atherosclerosis. In some embodiments, the subject has been diagnosed with type 2 diabetes.

[0012] In one aspect, the present disclosure provides a method of treating a cardiovascular disease in a subject who needs to treat a cardiovascular disease, the method comprising administering to the subject a tissue plasminogen activator (tPA) or a fragment thereof to treat the cardiovascular disease in the subject. In some embodiments, the tPA fragment comprises a tPA-K2 domain.

[0013] In one aspect, the present disclosure provides a method of reducing blood cholesterol levels in a subject who needs to reduce blood cholesterol levels, the method comprising administering to the subject a tissue plasminogen activator (tPA) or a fragment thereof to reduce blood cholesterol levels in the subject. In some embodiments, the tPA fragment comprises a tPA-K2 domain. In some embodiments, the administration comprises oral administration or intravenous administration. In some embodiments, the method reduces the levels of intermediate density lipoprotein (IDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), lipoprotein (a) [Lp(a)], chylomicron, or chylomicron remnant in the serum of the subject. In some embodiments, the tPA comprises a pharmaceutical composition.

[0014] In one aspect, the present disclosure provides a method for reducing plasma lipid and / or apolipoprotein B (apoB) levels in a subject that requires reducing plasma lipid and / or apolipoprotein B (apoB) levels, the method comprising administering a composition comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of a therapeutically effective amount of: (a) the nucleotide sequence set forth in SEQ ID NO: 2; (b) the nucleotide sequence encoding the polypeptide sequence set forth in SEQ ID NO: 1; (c) a nucleotide sequence that is at least about 85% identical to any one of the nucleotide sequences of (a)-(b); (d) a nucleotide sequence that is a complement of any one of (a)-(c); and (e) an RNA sequence encoded by any one of (a)-(d), wherein the nucleotide sequence is operably linked to a heterologous nucleic acid comprising an endoplasmic reticulum localization sequence, and the subject has or is at increased risk of having an atherosclerotic thrombotic event. In some embodiments, the endoplasmic reticulum localization sequence encodes a polypeptide having the amino acid sequence KDEL (SEQ ID NO: 11). In some embodiments, the polynucleotide molecule comprises the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encodes the polypeptide sequence set forth in SEQ ID NO: 3. In some embodiments, the composition is formulated in lipid nanoparticles or adeno-associated virus for delivery to the subject. In some embodiments, administration of the composition reduces plasma apoB and / or lipid levels in the subject. In some embodiments, the subject has received, is scheduled to receive, or has previously received thrombolytic therapy. In some embodiments, the method further comprises administering to the subject tissue plasminogen activator simultaneously, sequentially, or separately. In some embodiments, the method further comprises administering to the subject an anticoagulant simultaneously, sequentially, or separately.

[0015] In some embodiments, any of the treatment methods disclosed herein may involve subjects who have suffered from or are at increased risk of an atherothrombotic event. In some embodiments, subjects are receiving, are scheduled to receive, or have previously received treatment with thrombolytic agents, anticoagulants, catheter thrombolysis, and / or thrombectomy. [Brief explanation of the drawing]

[0016] [Figure 1A]Figures 1A–1E show that silencing hepatocyte tPA increases atherosclerotic apoB lipoprotein cholesterol and apoB, independently of LDLR or ApoE. Figure 1A is a series of charts showing the results for Ldlr- / - mice given a Western diet (WD) treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr). Ldlr- / - mice were treated with sh-tPA or scr and then given a WD for 8 weeks. Mouse liver was assayed for tPA protein, and plasma samples were assayed for total cholesterol and apoB-100 concentrations, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100 (n=9–10 mice per group). Figure 1B is a series of charts showing the results for Apoe- / - mice given a Western diet (WD) treated with AAV8-shPlat (sh-tPA) or AAV8-scrambled control (scr). Apoe- / - mice were treated with sh-tPA or scr and then given the WD for 8 weeks. Liver was assayed for tPA protein, and plasma samples were assayed for total cholesterol and apoB-100 concentrations, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100 (n=5 mice per group). Figure 1C is a series of charts showing the results for Platfl / fl mice given a Western diet (WD) treated with AAV8-TBG-cre (Cre) or AAV8-TBG-GFP (GFP). Platfl / fl mice were treated with Cre or GFP and then given the WD for 8 weeks. Liver was assayed for tPA protein, and plasma samples were assayed for total cholesterol and apoB-100 concentrations, as well as for FPLC profiles of cholesterol, triglycerides, and apoB-100. Cholesterol in the VLDL fraction is shown in a small enlarged graph (n=6 mice per group). Figure 1D is a series of charts and immunoblot images showing the results of human primary hepatocytes treated with siRNA (si-tPA) or scrambled RNA against tPA mRNA for 24 hours.apoB in the cell culture medium was quantified by immunoblotting. The VLDL fraction was isolated by ultracentrifugation, and the cholesterol and triglyceride concentrations in the VLDL fraction were assayed. Figure 1E shows a series of charts and immunoblot images of McA-RH7777 cells treated with siRNA (si-tPA) or scrambled RNA against tPA mRNA for 24 hours. VLDL was isolated from the medium by ultracentrifugation, and the cholesterol and triglyceride concentrations in the VLDL were assayed. apoB in the cell culture medium was assayed by immunoblotting. Data are shown as mean ± SEM, *P<0.05 (by two-sided Student's t-test). [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 1E] See the explanation in Figure 1A. [Figure 2A]Figures 2A-2K show that tPA restricts apoB lipidization in the endoplasmic reticulum (ER). Figure 2A: Wild-type (WT) mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a 14-week Western diet (WD). P407 was intraperitoneally injected into the mice to assess VLDL secretion. Plasma triglyceride concentrations were measured (n=4-5 mice per group). Figure 2B: WT mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a 14-week WD. P407 was intraperitoneally injected into the mice to assess VLDL secretion. Plasma apoB concentrations were measured by ELISA (n=4-5 mice per group). Figure 2C: Ldlr- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by 8 weeks of WD. VLDL was isolated by ultracentrifugation and visualized by transmission electron microscopy. The diameter of VLDL (n=100 for each group) was measured and analyzed using Image-Pro Plus 10.0. Scale bar: 100 nm. Figure 2D: Ldlr- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by 8 weeks of WD (n=9-10 mice per group). VLDL was isolated by ultracentrifugation, and the diameter of VLDL was measured by dynamic light scattering. Figure 2E: Ldlr- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), and then fed WD for 8 weeks (n=9-10 mice per group). VLDL was isolated by ultracentrifugation and assayed for the triglyceride to apoB-100 ratio (n=9-10 mice per group). Figure 2F: Whole-body tPA knockout mice (holo-tPA-KO) were treated with AAV8-TBG-Plat (tPA) or AAV8-TBG-lacZ (LacZ), and then fed a normal diet for 8 weeks. Plasma samples were assayed for VLDL cholesterol, LDL cholesterol, and apoB-100 concentrations (n=6 mice per group).Figure 2G: Human primary hepatocytes were transduced with tPA and a C-terminal HA tag (tPA-HA, SEQ ID NO: 27) or a plasmid encoding GFP (SEQ ID NO: 29). After 48 hours, apoB secretion was measured using [3H]leucine-containing medium, followed for 3 hours in [3H]leucine-free medium, and then the radioactivity associated with apoB in the cell medium was quantified by scintillation counting. Figure 2H: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled (scr)RNA against tPA mRNA for 24 hours. apoB secretion was measured using [3H] labeling, as in G. The radioactivity associated with apoB in the cell medium was quantified by scintillation counting. Figure 2I: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled (scr)RNA against tPA mRNA for 24 hours. VLDL was isolated by ultracentrifugation, and the diameter of the VLDL was measured by dynamic light scattering. Figure 2J: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled (scr)RNA against tPA mRNA for 24 hours. VLDL was isolated by ultracentrifugation and assayed for the triglyceride to apoB-100 ratio. Figure 2K: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled (scr)RNA against tPA mRNA for 24 hours. The endoplasmic reticulum (ER) fraction was isolated, and proteins were extracted from the ER. The apoB lipoprotein extracted from the ER was further separated by density gradient ultracentrifugation and divided into six fractions with increasing density from fraction 1 to 6. apoB from each fraction was measured by immunoblotting. Data are shown as mean ± SEM, *P<0.05 (by two-sided Student's t-test). [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 2E] See the explanation in Figure 2A. [Figure 2F] See the explanation in Figure 2A. [Figure 2G] See the explanation in Figure 2A. [Figure 2H] See the explanation in Figure 2A. [Figure 2I] See the explanation in Figure 2A. [Figure 2J] See the explanation in Figure 2A. [Figure 2K] See the explanation in Figure 2A. [Figure 3A]Figures 3A-3J show that tPA blocks apoB-VLDL assembly. Figure 3A: Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA for 24 hours. Cell lysates (input) and anti-MTP immunoprecipitation (IP:MTP) were assayed for apoB and MTP by immunoblotting. Figure 3B: Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA (group 1) or tPA mRNA (si-tPA) (groups 2 and 3) for 24 hours. Microsomal fractions were isolated and assayed for neutral lipid transfer activity using DMSO (groups 1 and 2) or without the MTP inhibitor CP-346086 (10nM) (group 3). Figure 3C: Human primary hepatocytes were transduced for 48 hours with a plasmid encoding tPA (tPA-HA, SEQ ID NO: 27) or GFP (SEQ ID NO: 29) having a C-terminal HA tag. Cell lysates (input) and anti-MTP precipitates (IP:MTP) were assayed for apoB and MTP by immunoblotting. Figure 3D: Human primary hepatocytes were transduced for 48 hours with a plasmid encoding tPA-HA (SEQ ID NO: 27) or GFP (SEQ ID NO: 29). Microsomal fractions were isolated and assayed for neutral lipid transfer activity. Figure 3E: The effect of recombinant human tPA on lipid transfer from donor vesicles to human LDL was assayed. Figure 3F: Human primary hepatocytes were transduced for 48 hours with a plasmid encoding tPA-HA (SEQ ID NO: 27) or GFP (SEQ ID NO: 29). Cell lysates (input) and anti-HA immunoprecipitates (IP:HA) were assayed for apoB and tPA. Figure 3G: ApoB-tPA interaction in human primary hepatocytes was measured using a proximity ligation assay. Scale bar, 10 μm. Figure 3H: Intracellular localization of tPA and apoB in human primary hepatocytes was measured using confocal microscopy immunofluorescence imaging. Scale bar, 10 μm.Figure 3I: Human primary hepatocytes were transduced for 48 hours with either wild-type tPA (tPA-WT (SEQ ID NO: 1)), an enzymatically inactive mutant of tPA (tPA-S513A (SEQ ID NO: 9)), tPA with an endoplasmic reticulum-retained signal sequence (tPA-KDEL (SEQ ID NO: 3)), or a plasmid encoding GFP. apoB secretion was measured by [3H] labeling, as shown in Figures 2G and 2H. Figure 3J: The effect of recombinant wild-type tPA (tPA-WT) or an enzymatically inactive mutant of tPA (tPA-S513A) on lipid transfer from donor vesicles to human LDL was assayed. Data are shown as mean ± SEM, *P<0.05 (by two-sided Student's t-test (Figure 3D), or one-way ANOVA followed by Dunnett's test (Figures 3B, 3E, 3I, 3J)). [Figure 3B] See the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 3D] See the explanation in Figure 3A. [Figure 3E] See the explanation in Figure 3A. [Figure 3F] See the explanation in Figure 3A. [Figure 3G] See the explanation in Figure 3A. [Figure 3H] See the explanation in Figure 3A. [Figure 3I] See the explanation in Figure 3A. [Figure 3J] See the explanation in Figure 3A. [Figure 4A]Figures 4A–4I show that the kringle 2 (K2) domain of tPA interacts with the N-terminus of apoB. Figure 4A: The interaction between LDL and recombinant human wild-type tPA (tPA-WT) or enzymatically inactive tPA-S513A was measured using a solid-phase assay. Binding of tPA-WT or tPA-S513A to wells without LDL was also measured under the same conditions as the control. Figure 4B: The interaction between recombinant human tPA and purified human MTP complexes was measured using a solid-phase assay. Figure 4C: The ability of tPA to inhibit the binding of MTP to LDL was measured using a solid-phase assay. Figure 4D: The interaction between human recombinant tPA and LDL was measured using surface plasmon resonance. Figure 4E: The anti-apoB N-terminal antibody (1D1) and control IgG (produced against the β3 domain of apoB) were tested using a solid-phase assay to determine whether they block the binding of human recombinant tPA to LDL. Figure 4F: Primary human hepatocytes were transduced with plasmids encoding wild-type tPA (tPA-WT (SEQ ID NO: 1)), tPA variants lacking the K2 domain (tPA-Δ-K2 (SEQ ID NOs: 19 and 20)), or tPA mutated at the K2 domain lysine binding site (tPA-D236, 238N (SEQ ID NOs: 21 and 22)). apoB secretion was measured by [3H] labeling, as shown in Figures 2G and 2H. Figure 4G: A solid-phase assay was used to test whether antibodies against the tPA-K2 domain interfered with the interaction between recombinant human tPA and purified LDL. Figure 4H: A solid-phase assay was used to measure whether tranexamic acid (TXA) interfered with the interaction between recombinant human tPA and purified LDL. Figure 4I: Schematic diagram showing the interaction between the N-terminus of apoB and the K2 domain of tPA. Figure generated using biorender.com. Data are presented as mean ± SEM, *P<0.05 (one-way ANOVA followed by Dunnett's test). ns, not significant (P≧0.05). [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 4E] See the explanation in Figure 4A. [Figure 4F] See the explanation in Figure 4A. [Figure 4G] See the explanation in Figure 4A. [Figure 4H] See the explanation in Figure 4A. [Figure 4I] See the explanation in Figure 4A. [Figure 5A]Figures 5A–5J show that PAI-1 sequesters tPA from apoB, resulting in increased VLDL assembly in hepatocytes. Figure 5A: Human primary hepatocyte lysates (input) and anti-PAI-1 immunoprecipitate (IP:PAI-1) were assayed for tPA and PAI-1 by immunoblotting. Figure 5B: tPA-PAI-1 interaction in human primary hepatocytes was measured using proximity ligation assay. Scale bar, 10 μm. Figure 5C: Human primary hepatocytes were treated for 6 hours with oleate complexed with 0.4 mM fatty acid-free BSA (oleate group) or fatty acid-free BSA alone (vehicle control). Cell lysates were assayed for tPA by immunoblotting using the Jess Simple Western System. Figure 5D: Human primary hepatocytes were treated with oleate complexed with 0.4 mM fatty acid-free BSA (oleate group) or fatty acid-free BSA alone (vehicle control). Cell lysates were assayed by ELISA for tPA concentrations without the tPA-PA-1 complex and without PAI-1. Figure 5E: Human primary hepatocytes were treated with siRNA (si-PAI1) or scrambled RNA against PAI-1 mRNA, and then incubated in a medium containing either 0.4 mM oleate (oleate group) complexed with fatty acid-free BSA or fatty acid-free BSA alone (vehicle control). Cell lysates were assayed by ELISA for tPA concentrations without PAI1. Figure 5F: Human primary hepatocytes were treated with siRNA (si-PAI1) or scrambled RNA against PAI-1 mRNA, and then incubated in a medium containing either 0.4 mM oleate (oleate group) complexed with fatty acid-free BSA or fatty acid-free BSA alone (vehicle control). apoB secretion was measured by [3H] labeling as shown in Figures 2G and 2H. Figure 5G: Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or siRNA against PAI-1 mRNA (si-PAI1) or scrambled RNA. apoB secretion was measured by [3H] labeling, as shown in Figures 2G and 2H.Figure 5H: The interaction between LDL and recombinant human tPA or tPA-PAI-1 complexes was measured using a solid-phase binding assay. Figure 5I: The effect of recombinant human tPA and tPA-PAI-1 complexes on lipid transfer from donor vesicles to human LDL was assayed. Figure 5J: Wild-type mice fed a normal solid diet were fasted for 5 hours, then orally administered olive oil and euthanized at 0, 1, 2, and 6 hours. Liver lysates were assayed by ELISA for tPA concentrations without PAI-1. Data are shown as mean ± sem, *P<0.05 (one-way ANOVA, followed by Dunnett's test (Figures 5D-5G, 5I, 5J)). ns, not significant (P≧0.05). [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 5E] See the explanation in Figure 5A. [Figure 5F] See the explanation in Figure 5A. [Figure 5G] See the explanation in Figure 5A. [Figure 5H] See the explanation in Figure 5A. [Figure 5I] See the explanation in Figure 5A. [Figure 5J] See the explanation in Figure 5A. [Figure 6A]Figures 6A-6J show that PAI-1 deficiency leads to decreased plasma apoB and apoB cholesterol concentrations in mice and humans. Figure 6A: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or the control AAV8-TBG-LacZ (control), and then fed a high-fat diet for 8 weeks. Plasma total cholesterol concentration was measured (n=6 mice per group). Figure 6B: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or the control AAV8-TBG-LacZ (control), and then fed a high-fat diet for 8 weeks. Plasma apoB was measured by immunoblotting (n=6 mice per group). Figure 6C: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or the control AAV8-TBG-LacZ (control), and then fed a high-fat diet for 8 weeks. Plasma samples were subjected to FPLC fractionation and cholesterol concentration was assayed. In the FPLC profile of cholesterol, cholesterol in the VLDL fraction is shown as a smaller, magnified graph (n=6 mice per group). Figure 6D: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or control AAV8-TBG-LacZ (control), and then fed a high-fat diet for 8 weeks. Liver lysates were assayed by ELISA for tPA concentration without PAI-1 (n=6 mice per group). Figure 6E: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or control AAV8-TBG-GFP (control), and then fed a normal solid diet for 4 weeks. P407 was intraperitoneally injected into the mice to evaluate VLDL secretion (n=10 mice per group). Figure 6F: Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or control AAV8-TBG-GFP (control), and then fed a normal solid diet for 6 weeks. The mice were fasted for 5 hours, then orally administered olive oil, and euthanized at 0, 2, and 4 hours. Plasma apoB-100 concentrations were measured by ELISA.Figure 6G: Plasma samples from SERPINE1-deficient humans and age / sex / BMI-matched non-affected individuals from the same community were assayed for VLDL cholesterol, LDL cholesterol, and apoB-100 concentrations (n=10 per group). Figure 6H: tPA concentrations were measured in VLDL isolated by ultracentrifugation from the plasma of subjects in panel Figure 6G (n=10 per group). Figure 6I: VLDL derived from the plasma of subjects in panel Figure 6G was analyzed by dynamic light scattering (n=10 per group). The correlation between VLDL-bound tPA and VLDL diameter was calculated (n=10 per group). Figure 6J: Schematic diagram illustrating how tPA-PAI-1 interaction in hepatocytes determines VLDL assembly. Without lipid stimulation, tPA interacts with apoB, inhibiting MTP-apoB interaction in the ER, thereby limiting MTP-mediated apoB lipidization and VLDL assembly. When hepatocytes are loaded with lipids, PAI-1 sequesters free tPA from apoB, increasing VLDL assembly. Figures were generated using biorender.com. Data are shown as mean ± sem, and p-values ​​were calculated by two-tailed Student's t-test (Figures 6A, 6D, 6E, 6F), paired Student's t-test (Figures 6G, 6H), or Pearson correlation analysis (Figure 6I). *P<0.05. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 6D] See the explanation in Figure 6A. [Figure 6E] See the explanation in Figure 6A. [Figure 6F] See the explanation in Figure 6A. [Figure 6G] See the explanation in Figure 6A. [Figure 6H] See the explanation in Figure 6A. [Figure 6I] See the explanation in Figure 6A. [Figure 6J] See the explanation in Figure 6A. [Figure 7A]Figures 7A-7D show that regulating hepatocyte tPA expression alters plasma tPA levels in mice. Figure 7A: Ldlr- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Plasma tPA levels were measured by ELISA (n=9-10 mice per group). Figure 7B: Apoe- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Plasma tPA levels were measured by ELISA (n=5 mice per group). Figure 7C: C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Figure 7D: Platfl / fl mice were treated with AAV8-TBG-cre (Cre) or AAV8-TBG-GFP (GFP), followed by a Western diet for 8 weeks. Plasma tPA was measured by ELISA (n=6 mice per group). Plasma tPA was measured by ELISA (n=9-10 mice per group). Data are shown as mean ± sem, *P<0.05 (two-sided Student's t-test). [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8]Silencing hepatocyte tPA in mice increases plasma apoB-lipoprotein cholesterol and apoB. Wild-type mice were treated with AAV8-H1-sh-tPA (sh-tPA) or AAV8-H1-scrambled (scr) and then fed a Western diet for 8 weeks. Liver was assayed for tPA protein, and plasma samples were assayed for tPA, total cholesterol, and apoB-100 concentrations, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100. Cholesterol in the VLDL fraction is shown in a smaller, magnified graph. (n=6 mice per group). Data are shown as mean ± sem, *P<0.05 (by two-tailed Student's t-test). [Figure 9A] Figures 9A-9C show that silencing hepatocyte tPA in mice does not alter liver apoB mRNA levels. Figure 9A: Ldlr- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Liver apoB mRNA was measured by real-time PCR. Figure 9B: Apoe- / - mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Liver ApoB mRNA was measured by real-time PCR. Figure 9C: C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Liver ApoB mRNA was measured by real-time PCR. Data are presented as mean ± sem, and statistical analysis was performed by a two-tailed Student's t-test. ns, not significant (P≧0.05). [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 10A]Figures 10A-10B demonstrate that silencing hepatocyte tPA does not alter plasma apoE and hepatic LDLR levels. Figure 10A: C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Plasma apoE concentrations were measured by ELISA. Figure 10B: C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr), followed by a Western diet for 8 weeks. Hepatic LDLR was measured by immunoblotting. Data are shown as mean ± sem, and statistical analysis was performed by two-sided Student's t-test. ns, not significant (P≧0.05). [Figure 10B] See the explanation in Figure 10A. [Figure 11] Figures 11A-11D show that silencing hepatocyte tPA does not alter apoB mRNA levels. Figure 11A: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled RNA against tPA mRNA for 24 hours. Cellular tPA mRNA was measured by real-time PCR. Figure 11B: Human primary hepatocytes were treated with siRNA (si-tPA) or scrambled RNA against tPA for 24 hours. Cellular apoB mRNA was measured by real-time PCR. Figure 11C: McA-RH7777 cells were treated with siRNA (si-tPA) or scrambled RNA against tPA for 24 hours. Cellular tPA mRNA was measured by real-time PCR. Figure 11D: McA-RH7777 cells were treated with siRNA (si-tPA) or scrambled RNA against tPA for 24 hours. Cellular apoB mRNA was measured by real-time PCR. Data are presented as mean ± sem, *P<0.05 (by two-tailed Student's t-test), ns, not significant (P≧0.05). [Figure 12]In McA-RH7777 cells, silencing tPA increases apoB secretion. McA-RH7777 cells were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA for 24 hours. apoB secretion was measured by [3H] labeling, as shown in Figures 2G and 2H. Data are shown as mean ± sem, *P<0.05 (two-sided Student's t-test or two-way ANOVA, followed by Dunnett's test). ns, not significant (P≧0.05). [Figure 13] This study demonstrates that incubation of primary human hepatocytes with recombinant tPA does not alter apoB levels in cell culture medium. Primary human hepatocytes were incubated in culture medium containing recombinant human tPA (10 ng / ml) for 1, 6, and 24 hours, respectively. Hepatocytes incubated in medium without recombinant tPA were used as a control. ApoB-100 in cell culture medium was measured by ELISA. Very low-volume (VLDL) cholesterol was isolated by ultracentrifugation, and cholesterol and triglycerides in VLDL were measured. Data are shown as mean ± sem;*Statistical analysis was performed by two-way analysis of variance (ANOVA), followed by Dunnett's test. ns, not significant (P≧0.05). [Figure 14] Silencing tPA increases MTP-dependent lipid transfer in the microsomal fraction of McA-RH7777 cells. McA-RH7777 cells were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA for 24 hours. Microsomal fractions were assayed for neutral lipid transfer activity with or without 10 nM CP-346086, an MTP inhibitor. Data are shown as mean ± sem, *P<0.05 (two-sided Student's t-test or one-way ANOVA, followed by Dunnett's test). [Figure 15]Figures 15A and 15B show the purification of human recombinant tPA and LDL by size exclusion chromatography. Figure 15A: Further purification of human LDL isolated by ultracentrifugation using size exclusion chromatography. Figure 15B: Further purification of recombinant human tPA purified by affinity chromatography using size exclusion chromatography. [Figure 16] This study demonstrates that tPA interacts with defatted apoB-100. Purification of human recombinant tPA and LDL was performed by size exclusion chromatography. Solid-binding was used to assay the interaction between defatted apoB-100 and recombinant tPA. Data are presented as mean ± sem. [Figure 17] This study demonstrates that tranexamic acid partially reduces the interaction between tPA and solid-phase bound LDL. The interaction between LDL and recombinant human tPA in the presence of tranexamic acid was measured using a solid-phase assay. Data are presented as mean ± sem, *P<0.05 (one-way ANOVA, followed by Dunnett's test). ns, not significant (P≧0.05). [Figure 18] Silencing tPA PAI-1 in McA-RH7777 cells reduces apoB secretion. McA-RH7777 cells were treated for 24 hours with siRNA against PAI-1 mRNA (si-PAI1) or scrambled RNA. Cells were treated with 0.4 mM oleate complexed with fatty acid-free BSA (oleate group) or fatty acid-free BSA alone (oleate-free group). ApoB secretion was measured by [3H] labeling as shown in Figure 2. Data are shown as mean ± sem, *P<0.05 (by two-sided Student's t-test), ns, not significant (P≧0.05). [Figure 19]This study demonstrates that oral force-feeding with olive oil does not alter total tPA and total PAI-1 levels in the liver of mice. C57BL / 6 mice fed a normal solid diet were fasted for 5 hours, then orally force-fed with olive oil and sacrificed at 0, 1, 2, and 6 hours. Liver lysates were assayed for tPA and PAI-1 concentrations by ELISA. Data are shown as mean ± sem, *P<0.05 (two-sided Student's t-test or two-way ANOVA, followed by Dunnett's test). ns, not significant (P≧0.05). [Figure 20A] Figures 20A-20B show that silencing tPA in hepatocytes increases apo(a) levels in cell culture. Cell culture apo(a) levels were measured by ELISA. Figure 20A is a chart showing apo(a) levels in scrambled siRNA-treated or si-tPA-treated human primary hepatocytes. [Figure 20B] Figures 20A-20B show that silencing tPA in hepatocytes increases apo(a) levels in cell culture. Cell culture apo(a) levels were measured by ELISA. Figure 20B is a chart showing apo(a) levels in HepG2 cells transduced with apo(a) expression plasmid and then treated with scrambled siRNA or si-tPA. [Figure 21A] Figures 21A–21C show that knocking out intestinal tPA in mice increases plasma cholesterol levels. The following measurements were taken by treating intestinal tPA knockout mice (e-tPA-KO) or littermates (controls): Figure 21A: tPA mRNA levels in intestinal cells and liver normalized to Rplp0 mRNA; Figure 21B: total plasma cholesterol; and Figure 21C: plasma cholesterol profiled by FPLC (controls, pooled plasma from 8 mice; e-tPA-KO, pooled plasma from 11 mice). [Figure 21B] See the explanation in Figure 21A. [Figure 21C] See the explanation in Figure 21A. [Figure 22A] Figures 22A-22B show that knockout of intestinal tPA in mice promoted chylomicron production. Intestinal tPA knockout mice (e-tPA-KO) or littermates (control) were examined after oral forced administration of olive oil. Figure 22A: Changes in plasma triglyceride levels over time after oral forced administration (n=9 for control (lower line in the chart), n=11 for e-tPA-KO (upper line in the chart)). [Figure 22B] Figures 22A-22B show that knockout of intestinal tPA in mice promoted chylomicron production. Intestinal tPA knockout mice (e-tPA-KO) or littermates (controls) were tested after oral forced administration of olive oil. Figure 22B: Immunoblot analysis of apoB-48 levels in isolated chylomicron fractions and albumin in plasma, collected 2 hours after forced administration. [Figure 23A] Figures 23A-23B show that tPA-K2 domain expression reduces ApoB secretion in cultured hepatocytes. Cultured McA-RH7777 cells were treated with a plasmid encoding tPA-K2 (SEQ ID NO: 31) (SEQ ID NO: 33) or a GFP control. Figure 23A: tPA-K2 domain expression was detected by immunoblotting. [Figure 23B] Figures 23A-23B show that tPA-K2 domain expression reduces ApoB secretion in cultured hepatocytes. Cultured McA-RH7777 cells were treated with a plasmid encoding tPA-K2 (SEQ ID NO: 31) (SEQ ID NO: 33) or a GFP control. Figure 23B: ApoB levels in cell culture medium were detected by immunoblotting. [Figure 24A]Figures 24A-24B show that tPA-K2 domain expression reduces plasma lipids in mice. Male C57 wild-type mice were fed a high-fat diet for 4 weeks. Mice were intravenously injected with LNPs carrying mRNA encoding a tPA-K2 domain with an endoplasmic reticulum localization motif, or with control luciferase (1 mg / kg body weight). Blood was collected at 6, 24, and 48 hours after injection, as well as immediately before injection. Plasma triglyceride (Figure 24A) and cholesterol (Figure 24B) levels were measured. [Figure 24B] See the explanation in Figure 24A. [Figure 25] This study demonstrates that tPA-K2 domain expression does not alter bleeding time in mice. Male C57 wild-type mice were fed a high-fat diet for 5 weeks. Mice were intravenously injected with either LNPs carrying mRNA encoding a tPA-K2 domain with an endoplasmic reticulum localization motif or control luciferase (1 mg / kg body weight). The tail bleeding test was performed 24 hours after LNP injection. [Modes for carrying out the invention]

[0017] Detailed explanation This disclosure is not limited in terms of the specific embodiments described in this application, which are intended as single examples of individual aspects of this disclosure. Not all of the various embodiments of this disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variations of this disclosure can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This disclosure should be limited only by the conditions of the appended claims, together with the entire scope of equivalents to which such claims are entitled.

[0018] This disclosure is not limited to any particular use, method, reagent, compound, composition, or biological system, and it should be understood that these are, of course, subject to change. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to be limiting.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0020] Outline Both statins and proteolytic enzyme subtilisin / kexin type 9 (PCSK9) inhibitors are currently used to prevent or treat cardiovascular disease by lowering low-density lipoprotein (LDL) levels by increasing hepatic LDL receptor (LDLR)-mediated LDL clearance. However, despite their ability to reduce LDL, these treatments only slightly alter the levels of other atherogenic apolipoprotein B (apoB) containing apolipoproteins, such as intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), lipoprotein(a)[Lp(a)], chylomicrons, and chylomicron remnants.

[0021] ApoB is a large amphipathic protein that serves as a structural scaffold for the formation of VLDL and chylomicrons [8]. VLDL and chylomicrons are assembled in hepatocytes and intestinal cells by incorporating triglycerides, cholesteryl esters, and phospholipids, respectively, into apoB to form spherical particles [9]. This process, known as apoB lipidization, depends on both lipid availability and the neutral lipid transporter, microsomal triglyceride transfer protein (MTP) [10, 11]. MTPs bind to apoB in the hepatocyte endoplasmic reticulum (ER) and transfer lipids to apoB

[10] . If MTPs and / or lipids are unavailable, VLDL and chylomicrons cannot be synthesized, and newly translated apoB becomes a target for degradation [12, 13]. While the essential role of MTPs in apoB lipidization is well established, little is known about the apoB-MTP interaction and the regulation of MTP-mediated lipid transfer to apoB.

[0022] Tissue plasminogen activator (tPA) is a serine protease that plays a crucial role in fibrinolysis, the process of dissolving blood clots

[14] . Previous studies have shown that low plasma tPA activity is associated with a higher risk of atherosclerotic CVD [15-17], although it remains unclear whether reduced fibrinolysis contributes to CVD in this setting. Another plausible mechanism linking low tPA to CVD is elevated plasma cholesterol, as seen in humans with reduced tPA activity [18-20]. However, little is known about how tPA affects circulating atherosclerotic lipoproteins. Given the central role of hepatocytes in apoB lipoprotein production and our recent research showing that hepatocytes are an important source of tPA [21, 22], we attempted to identify potential relationships between tPA and apoB lipoprotein assembly and secretion in hepatocytes and intestinal cells. Our investigations have revealed that endogenous hepatocyte tPA restricts VLDL and chylomicron production by directly interacting with apoB, interfering with the interaction between apoB and MTP, and thereby impairing MTP-dependent neutral lipid transfer and apoB lipidization.

[0023] Plasminogen activator inhibitor 1 (PAI-1), encoded by the SERPINE1 gene, is a major serine protease inhibitor of tPA and is also expressed in hepatocytes. In this disclosure, we demonstrate that PAI-1 binds to tPA in hepatocytes and neutralizes the effect of tPA on limiting VLDL assembly. Furthermore, the regulatory pathway of VLDL assembly by the hepatocyte PA1-1 / tPA system is physiologically relevant to postprandial lipid load-related VLDL production. These findings suggest a novel therapeutic strategy to reduce atherosclerotic apoB lipoprotein production.

[0024] The inventors of this technology discovered that tPA directly interacts with apoB, preventing the transfer of neutral lipids to apoB by MTP, leading to the degradation of apoB, and thereby reducing the level of atherogenic apolipoprotein.

[0025] Furthermore, the inventors of this technology discovered that the serine protease function of tPA (specific cleavage of the Arg-Val bond in plasminogen to form plasmin: EC: 3.4.21.68) is not required for tPA to reduce apoB secretion by hepatocytes. See Figures 3I, 3J, and 4A, for example, which demonstrate that expression of the protease-deficient tPA S513A mutant in hepatocytes still results in reduced apoB secretion in conditionally tPA knockout animals.

[0026] Recombinant tPA (also known as rtPA and ACTIVASE (alteplase)) is used for its "thrombolytic" activity, which helps dissolve blood clots, and is used to treat heart attacks, strokes, and pulmonary thrombosis. However, these functions depend on the serine protease activity of tPA.

[0027] The disclosure of this technology is based, at least in part, on the discovery that the tPA-K2 polypeptide of this technology is effective when administered to subjects in need of reducing apoB secretion and lowering plasma triglyceride and total cholesterol levels, despite lacking serine protease and proteolytic activity. Furthermore, because the tPA-K2 fragment of this technology (e.g., including the amino acid sequence described in SEQ ID NO: 7) does not possess serine protease and proteolytic activity, administration of the peptide of this technology does not increase the risk of bleeding in subjects. The examples provided herein demonstrate that expression of recombinant tPA-K2 peptides containing only the tPA kringle 2 domain and endoplasmic reticulum localized sequences can reduce apoB secretion from hepatocytes (Figures 23A-23B) and lower plasma lipid levels in an in vivo mouse model (Figures 24A-24B), but does not alter bleeding time in mice (Figure 25). These data support the usefulness of compositions comprising recombinant tPA-K2 polypeptide (or nucleic acids encoding it, e.g., cDNA, mRNA) in methods for treating diseases or conditions associated with elevated plasma apoB lipoprotein levels. In some embodiments, compositions of the present technology are useful in methods for reducing one or more of the following plasma levels: VLDL, IDL, LDL, Lp(a), chylomicrons, chylomicron remnants, triglycerides, or total cholesterol levels.

[0028] composition One aspect of this disclosure provides recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) polypeptides, or nucleic acids (e.g., mRNA, cDNA) encoding them. As used herein, “recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) polypeptide” or “recombinant tPA-K2 polypeptide” (also referred to as “recombinant polypeptide” in this disclosure) means a recombinant polypeptide comprising the kringle 2 domain of tissue plasminogen activator, fragments of the kringle 2 domain, fragments thereof, or nucleic acids encoding them, wherein the recombinant polypeptide has sequence identity of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of full-length wild-type human tPA (SEQ ID NO: 1). Accordingly, the present technology provides recombinant tPA-K2 polypeptides having an amino acid sequence that is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the amino acid sequence described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. Recombinant tPA-K2 polypeptides suitable for use in the methods described herein also include variants comprising polypeptides having amino acid changes, e.g., amino acid substitutions, deletions, or additions, compared to the amino acid sequence of any tPA-K2 polypeptide described herein. Such sequence-mutant proteins are suitable for the methods described herein, insofar as the modified amino acid sequence retains sufficient biological activity to function in the compositions and methods described herein. When amino acid substitutions are performed, the substitutions may be conservative amino acid substitutions.Among commonly occurring naturally occurring amino acids, for example, "conservative amino acid substitutions" are represented by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0029] This technology also provides nucleic acids (e.g., mRNA, cDNA) encoding recombinant tPA-K2 polypeptides, such as polynucleotides, which have nucleic acid sequences that are at least approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% identical to the nucleic acid sequence described in any of SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 32, 34, 36, 38, 39, or 40, or their complement, or the nucleic acid sequence encoding the polypeptide described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. In some embodiments, the technology provides RNA transcripts encoded by any of SEQ ID NOs: 2, 4, 6, 10, 15, 16, 34, 36, 38, or 39. In some embodiments, the nucleic acids can reduce apoB lipoprotein blood (e.g., plasma or serum) levels when administered to a subject.

[0030] Examples of recombinant tPA-K2 polypeptides (or nucleic acids encoding them) of the Technology include, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. In some embodiments, the recombinant tPA-K2 polypeptides (or nucleic acids encoding them) of the Technology include an endoplasmic reticulum (ER) localized sequence. One non-limiting example of an ER localized sequence is KKXX (wherein "K" represents lysine and "X" can be any amino acid). Another non-limiting example of an ER localized sequence is KDEL (SEQ ID NO: 11). Thus, for example, SEQ ID NO: 7 is a recombinant tPA-K2 polypeptide containing the sequences SEQ ID NO: 5 and KDEL (SEQ ID NO: 11). In some embodiments, the recombinant tPA-K2 polypeptides (or nucleic acids encoding them) of the Technology include, for example, SEQ ID NO: 9, which is a tPA S513A mutant lacking serine protease function. In some embodiments, the tPA S513A variant further comprises an ER localization sequence such as KDEL (SEQ ID NO: 11) and the amino acid sequence described in SEQ ID NO: 35. In some embodiments, the recombinant tPA-K2 polypeptide (or the nucleic acid encoding it) of the Art comprises, for example, SEQ ID NO: 5 and SEQ ID NO: 7, which includes KDEL (SEQ ID NO: 11). In some embodiments, the recombinant tPA-K2 polypeptide may comprise a sequence having 100% identity, at least about 99% identity, at least about 98% identity, at least about 97% identity, at least about 96% identity, or at least about 95% identity with the full-length wild-type tPA sequence (SEQ ID NO: 1), but further comprises one or more additional heterologous amino acid sequences.

[0031] The recombinant tPA-K2 polypeptide of this technology can be constructed by methods well known in the art. For example, the nucleotide sequence encoding the recombinant tPA-K2 polypeptide can be generated by PCR. In some embodiments, the tPA-K2 sequence includes an in-frame C-terminal endoplasmic reticulum localization sequence, such as the sequence encoding KDEL (e.g., SEQ ID NO: 12). In some embodiments, the recombinant tPA-K2 polypeptide of this technology may be referred to as a “fusion protein” comprising the tPA-K2 polypeptide or a fragment thereof, a linker, and an endoplasmic reticulum localization motif. In some embodiments, the recombinant tPA-K2 polypeptide of this technology has the following non-limiting formula: tPA-K2 polypeptide (or fragment thereof)-X-endoplasmic reticulum localization motif (Formula I), where -X- is a linker. In some embodiments, -X- is one or more amino acids. In some embodiments, the amino acid sequence of the linker includes GGGGS (SEQ ID NO: 41).

[0032] In some embodiments, the recombinant tPA-K2 polypeptide of the technology includes a protein tag domain comprising one or more amino acid sequences that facilitate immunoprecipitation, purification, and / or detection of the exogenously expressed fusion protein. In some embodiments, the protein tag domain comprises an epitope tag and / or a polyhistidine tag. Examples, but not limited to, include one or more of the following tags: HA (hemagglutinin) tag, histidine tag (e.g., 6-histidine tag), FLAG tag, CBP (calmodulin-binding peptide), CYD (covalently bonded but dissociable NorpD peptide), Strepll, or HPC (heavy chain of protein C). In some embodiments, the protein tag domain comprises about 10 to 20 amino acids in length. In some embodiments, the protein tag domain comprises 2 to 40 amino acids in length, e.g., 6 to 20 amino acids in length. In some embodiments, the epitope tag is an HA tag. In some embodiments, the HA tag comprises the amino acid sequence YPYDVPDYA (SEQ ID NO: 42). Those skilled in the art will understand that the addition of epitope tags, such as HA tags, may be used to facilitate immunoprecipitation, purification, and / or detection of expressed proteins, and that the inclusion of tags does not in any way imply that it is limited to recombinant tPA-K2 polypeptides or the nucleic acids encoding them as described herein, or their functions.

[0033] In some embodiments, the recombinant tPA-K2 polypeptide (or nucleic acid encoding it) of the present invention is a therapeutic polypeptide (or nucleic acid) that inhibits the interaction between MTP and apoB, blocks PAI-I binding to endogenous tPA, inhibits the formation of VLDL and chylomicrons, and promotes the degradation of apoB; and is useful in reducing the likelihood of CVD and related diseases and conditions associated with VLDL, IDL, LDL, Lp(a), chylomicrons, and chylomicron remnants, such as, but not limited to, hyperlipidemia, atherosclerosis, increased risk of thrombosis, angina pectoris, heart attack, heart failure, stroke, transient ischemic attack (TIA), peripheral artery disease, and hypertension. The recombinant tPA-K2 polypeptide of the present disclosure comprises a tissue plasminogen kringle 2 domain, e.g., SEQ ID NO: 5, or a fragment thereof. The tPA kringle 2 domain may contain a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 5. While we do not wish to be bound by theory, the compositions of this disclosure block the interaction between MTP and apoB. The tPA kringle 2 domain (tPA-K2) may contain the sequence NPDGDAKPWCHVLKNRRLTWEY (SEQ ID NO: 13). tPA-K2 may further contain a KDEL (sequence number 11) sequence that localizes tPA-K2 to the endoplasmic reticulum, such as NPDGDAKPWCHVLKNRRLTWEYKDEL (sequence number 14).

[0034] The recombinant polypeptides of this disclosure may also include tPA lacking serine protease activity, i.e., tPA that is not fibrinolytic. As discussed above, serine protease activity is necessary for "thrombolytic" or fibrinolytic tPA. Loss of serine protease activity may be achieved in the recombinant polypeptide by mutation of amino acid residues important for the serine protease function of tPA, for example, serine 513 may be mutated to alanine (S513A), where the position of 513 is relative to SEQ ID NO: 1. Alternatively, the entire peptidase domain of tPA, or an important portion of the peptidase domain of tPA, may be removed from the recombinant polypeptide. The peptidase domain of tPA contains amino acids 311-561 relative to SEQ ID NO: 1. Therefore, recombinant polypeptides may include sequences having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with sequences, and may include sequences having a suitable mutation, for example, having S513A to disrupt the serine protease function of tPA, or having a cleavage of sequences 1 to remove the peptidase domain (i.e., amino acids 311-561) from sequences 1.

[0035] Recombinant polypeptides may contain sequences that are at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to sequence number 9.

[0036] The recombinant polypeptide may contain sequences that are at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to sequence number 35.

[0037] Recombinant polypeptides may contain sequences that are at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to sequence number 7.

[0038] Recombinant polypeptides may contain sequences that are at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identical to sequence number 5.

[0039] tPA lacking a peptidase domain, i.e., tPA lacking amino acids 311-516 relative to SEQ ID NO: 1, may contain a sequence having at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with SEQ ID NO: 30.

[0040] In some embodiments, the delivery of the disclosed polypeptide, or the nucleic acid (mRNA, cDNA) encoding it, can be efficiently facilitated by nanoparticles. Accordingly, the disclosure further provides nanoparticles comprising recombinant polypeptides, or nucleic acids encoding them. The nanoparticles may comprise one or more polymers, such as poly(lactide) (PLA), poly(lactide-coglycolide) (PLGA) copolymer, poly(ε-caprolactone) (PCL), and poly(amino acids), arginate, chitosan, gelatin, and albumin.

[0041] In some embodiments, the nanoparticles may comprise, for example, one or more lipids, such as 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), which can be suitably formed into liposomes. Additional or alternative exemplary lipids for incorporation into the nanoparticles are known in the art, such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate](PDP-PE), 306O i10, Tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazandiyl)bis(propane-3,1-diyl))bis(azantriyl))tetrapropionate; 9A1P9, Decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, Ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azandiyl)bis(hex San-6,1-diyl)bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-ol; BAME-O16B, bis(2-(dodecyl (Disulfanyl)ethyl)3,3′-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azandiyl)dipropionate; BHEM-cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)carbonyl)amino)-N,N-bi Su(2-hydroxyethyl)-N-methylethane-1-aminium bromide; C12-200, 1,1′-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azandiyl)bis(dodecane-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol;DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate; DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane; DOTMA, 1,2-di-O-octadecenyl-3- Rimethylammonium propane; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanegyl))tris(propane-3,1-diyl))tris(azanegyl))hexanonaate; lipid H (SM-102), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azantriyl))tetrakis(ethane-2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate); PEG2000-DMG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, N; 1 , N 3 , N 5 -Includes, but is not limited to, tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide.

[0042] Polynucleotides As stated above, the Disclosure also provides polynucleotides encoding the recombinant polypeptides of the Disclosure. The polynucleotides of the Disclosure are nucleotide sequences described in one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 32, 34, 36, 38, 39, or 40, or SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 32, 34, 36, 38, 39, or 40, and at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% The polynucleotides may include sequences having at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity, or nucleotide sequences encoding a polypeptide described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. The polynucleotides of this disclosure also include RNA sequences encoded by any one of SEQ ID NOs: 2, 4, 6, 10, 15, 16, 34, 36, 38, or 39. In some embodiments, the polynucleotide includes the mRNA sequence described in SEQ ID NO: 8. In some embodiments, the polynucleotide includes the mRNA sequence described in SEQ ID NO: 40. In some embodiments, the polynucleotide includes the mRNA sequence described in SEQ ID NO: 32.

[0043] In some embodiments, the polynucleotide includes a nucleic acid sequence encoding the tPA kringle 2-KDEL described in SEQ ID NO: 34, or a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 34.

[0044] In some embodiments, the polynucleotide includes a nucleic acid sequence encoding the tPA kringle 2 domain described in SEQ ID NO: 6, or a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 6.

[0045] In some embodiments, the polynucleotide includes a nucleic acid sequence encoding tPAS513A as described in SEQ ID NO: 10, or a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 10.

[0046] In some embodiments, the polynucleotide includes a nucleic acid sequence encoding tPA-S513A-KDEL as described in SEQ ID NO: 36, or a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with SEQ ID NO: 36.

[0047] In some embodiments, the polynucleotides of the Art include nucleotide sequences encoding protein tags (e.g., epitope tags) that facilitate immunoprecipitation, purification, and / or detection of exogenously expressed proteins. Those skilled in the art will understand that the inclusion of nucleotide sequences encoding epitope tags, such as HA tags, may be used to facilitate immunoprecipitation, purification, and / or detection of expressed proteins, and that the inclusion of tags is by no means intended to limit to nucleic acids encoding recombinant tPA-K2 polypeptides or to their functions as described herein.

[0048] The polynucleotides of this disclosure may further comprise at least one regulatory region operably ligated to a nucleotide sequence encoding a recombinant polypeptide of this disclosure. The at least one regulatory region may comprise, for example, an enhancer, a promoter, or both an enhancer and a promoter. The regulatory region may comprise a tissue-specific promoter, such as a hepatocyte-specific promoter, such as a thyroxine-binding globulin (TBG) promoter (SEQ ID NO: 17). As used herein, a polynucleotide is said to be “operably ligated” or “operably linked” if it is functionally related to a second polynucleotide sequence.

[0049] The disclosed polynucleotides are intended to be used to express the disclosed polypeptides in cells, for example, human cells, for example, hepatocytes, or in human subjects. Therefore, the disclosed polynucleotides may be, for example, plasmids, minicircles, and may include regulatory regions that enable viral delivery of the polynucleotides.

[0050] For example, the disclosed polynucleotides may include viral regulatory regions, such as adeno-associated virus (AAV) inverted terminal repeat sequences (ITRs).

[0051] As used herein, the term “inverted end repeat” (ITR) sequence refers to a sequence of DNA adjacent to a portion of the AAV genome that enables insertion of the genome into a host cell. ITRs are the only cis-acting elements necessary for AAV formation. Therefore, additional elements of the AAV genome may be added trans to facilitate the formation of a complete viral particle. Thus, the only portions of the viral genome that need to be included in the DNA carried by the AAV are the 5' ITR and the 3' ITR. Preferred ITRs are known in the art and may include, for example, a 5' or 3' ITR having the sequence of SEQ ID NO: 18.

[0052] infectious particles Suitable infecting particles are known in the art. See, for example, Deverman et al. (Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain, Nature Biotechnology, 34(2):204-209, 2016) and Chan et al. (Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous system, Nature Neuroscience, 20(8):1172-1179, 2017), which are incorporated herein in their entirety by reference. Those skilled in the art will be familiar with the elements and configurations required for vector construction to encode the constructs described herein. Exemplary infecting particles include adeno-associated virus (AAV) particles, adenovirus particles, herpesvirus particles, lentivirus particles, baculovirus particles, virus-like particles (VLPs), or any other suitable viral particles that can be used to deliver the polypeptides and / or nucleic acids (e.g., mRNA, DNA) encoding them to cells or tissues.

[0053] The disclosed infectious particles may include adeno-associated virus AAV containing the aforementioned viral regulatory regions, which direct the expression of products from the disclosed polynucleotides, either contained within or associated with the virus, in a host cell, and the host cell expresses the polynucleotides to produce the disclosed recombinant polypeptide. In some cases, the virus is selected from, among others, AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the virus is AAV8 (AAV8) virus. See, for example, Figures 1A, 1B, 2A-2F, and 6A-6F.

[0054] Pharmaceutical composition The disclosed polynucleotides and recombinant polypeptides and / or nucleic acids encoding them are intended to be administered to a subject. Accordingly, in one aspect of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises one or more disclosed polynucleotides, polypeptides and / or nucleic acids encoding them, and a pharmaceutically acceptable carrier or excipient, or disclosed infectious particles and a pharmaceutically acceptable carrier or excipient. In some embodiments, the polynucleotides of the present technology are formulated to be delivered to a subject via infectious particles (e.g., adeno-associated virus (AAV)) or lipid nanoparticles (LNPs).

[0055] As used herein, “effective dose” or “therapeutic effective dose” means an amount or dosage of the disclosed composition that, upon single or multiple dose administration to a subject, results in a desired effect in the subject being diagnosed or treated, such as a reduction in serum apoB lipoprotein. A reduction in serum apoB lipoprotein includes one or more of the following: a reduction in serum LDL, a reduction in serum IDL, a reduction in serum VLDL, a reduction in serum Lp(a), a reduction in serum chylomicrons, a reduction in serum chylomicron remnants, or an improvement in one or more metrics related to cardiovascular disease, including a reduction in total cholesterol, a decrease in blood pressure, a decrease in fasting blood glucose, or a decrease in hemoglobin A1C. The effective dose can be readily determined by a person skilled in the art by the use of known techniques and by observing the results obtained under similar circumstances. When determining the effective amount or dose of a compound to be administered, several factors are considered by the attending physician, including, for example, the species, size, age, and general health status of the subject, the degree or severity of involvement of any associated disease or disorder, the individual patient's response, the specific compound to be administered, the mode of administration, the bioavailability characteristics of the preparation to be administered, the dose regimen selected, the use of concomitant medications, and other relevant circumstances. The composition may also be administered in combination with one or more additional therapeutic compounds / medicines (e.g., "concurrent administration," where additional or other therapeutic agents may be administered simultaneously, sequentially, or separately).

[0056] As will be apparent to those skilled in the art, the therapeutically effective amount of one or more recombinant tPA-K2 polypeptides of the present technology, and / or the nucleic acid encoding it (e.g., mRNA, cDNA), depends on factors such as the subject's disease state, age, gender, weight, and general condition, as well as the desired response (the subject's response to treatment) of the recombinant tPA-K2 polypeptide of the present technology, and / or the nucleic acid encoding it (e.g., mRNA, cDNA) in a particular subject. It can vary. When delivering a recombinant tPA-K2 polypeptide, or the nucleic acid encoding it (e.g., mRNA, cDNA), to a subject, the dosage will also vary depending on factors such as the general medical condition, previous medical history, disease type and progression.

[0057] Generally, typical dosages of the therapeutically effective amount of the recombinant tPA-K2 polypeptide of the present technology, or the nucleic acid encoding it (e.g., mRNA, cDNA), can contain from about 0.01 mg / kg to about 100 mg / kg (e.g., from about 0.05 mg / kg to about 50 mg / kg, and / or from about 0.1 mg / kg to about 25 mg / kg, and / or from about 1 mg / kg to about 10 mg / kg) of the disclosed recombinant polypeptide, or the nucleic acid encoding it.

[0058] Typical dosages of the disclosed pharmaceutical compositions containing virus particles can contain from about 5 μg of viral DNA to about 100 μg of viral DNA, from about 10 μg of viral DNA to about 50 μg of viral DNA. The dosage of the disclosed virus particles administered to a subject is from about 1×10 10 virus particles to about 1×10 20 virus particles, e.g., can contain from 1×10 14 to 1×10 16 virus particles, or can contain about 1×10 14 particles, about 2×10 14 particles, about 3×10 14 particles, about 4×10 14 particles, about 5×10 14 particles, about 6×10 14 particles, about 7×10 14 particles, about 8×10 14 particles, about 9×10 14 particles, about 1×1015 pieces, approximately 2×10 15 pieces, about 3×10 15 pieces, about 4×10 15 pieces, about 5×10 15 pieces, about 6×10 15 pieces, about 7×10 15 pieces, about 8×10 15 pieces, approximately 9×10 15 pieces, about 1×10 16 The individual units may be administered to the subject as a single dose. The compositions comprising the disclosed recombinant polypeptides can be formulated into unit dosage forms, each dose containing about 1 to about 500 mg of each polypeptide individually or in a single unit dosage form, for example, about 5 to about 300 mg, about 10 to about 100 mg, and / or about 25 mg. The term "unit dosage form" refers to a physically separated unit suitable as a unit dose for a patient, each unit containing a predetermined amount of the active substance calculated to produce a desired therapeutic effect, accompanied by a suitable pharmaceutical carrier, diluent, or excipient.

[0059] In some embodiments, the Disclosure provides compositions comprising one or more recombinant tPA-K2 polypeptides of the Art as pharmaceutically acceptable salts, tautomers, hydrates, and / or solvates thereof. In some embodiments, a mixture of two or more recombinant tPA-K2 polypeptides may be used as a therapeutic agent. Polypeptides may be synthesized by any method known in the Art. In some embodiments, polypeptides may be formulated as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” means a salt prepared from a base or acid that is acceptable for administration to a patient, such as a mammal (e.g., a salt that is mammalian safety acceptable for a given administration regime). However, it should be understood that the salt does not have to be a pharmaceutically acceptable salt, such as a salt of an intermediate compound not intended for administration to a patient. Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. Furthermore, if the polypeptide contains both a basic moiety such as an amine, pyridine, or imidazole and an acidic moiety such as a carboxylic acid or tetrazole, an amphoteric ion may be formed, which falls within the scope of the term “salt” as used herein. Certain compound(s) / polypeptide(s) of this disclosure may exist in solvated forms, including both unsolvated and hydrated forms. For example, solvated forms may exist because it is difficult or impossible to remove all solvent from the polypeptide after synthesis. Generally, solvated forms are equivalent to unsolvated forms and are included within the scope of this disclosure.

[0060] Certain compound(s) / polypeptides(s) of this disclosure may exist in crystalline form, multiple crystalline form, amorphous form, or any combination thereof. Certain compound(s) / polypeptides(s) of this disclosure may exist in various tautomer forms. Certain compound(s) / polypeptides(s) of this disclosure may exist in various salt forms or mixtures of salt forms. In general, all physical forms of compound(s) / polypeptides(s) disclosed herein are intended to be equivalent and within the scope of this disclosure for the applications envisioned herein.

[0061] Combination therapies are also disclosed herein. The disclosed compositions, e.g., recombinant polypeptides and / or nucleic acids encoding them, nanoparticles, infected particles, or pharmaceutical compositions may be administered in combination with statins, PCSK9 inhibitors, plasminogen activator-1 (PAI-1) inhibitors, and ACE inhibitors, insulin sensitizers, hormone replacement therapy in women, or any combination thereof, or in combination with standard therapies currently known or unknown for the treatment of dyslipidemia or high cholesterol. Exemplary, non-limiting PAI-1 inhibitors include MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, alepracinin, laurelin B, diapracinin, todarolactone, SK-216, geodin, fendosal, AZ3976, and TM5007. Other PAI-1 inhibitors are described in U.S. Patents 8,759,327, 9,096,501, 9,230,744, and 9,527,878, each incorporated in whole by reference. Intravenous administration is an exemplary route for administering the compounds used in the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, subarachnoid, intracerebral, oral, or rectal routes. The route of administration may be modified in any way, limited by the physical properties of the compound used and / or the convenience of the subject and / or caregiver.

[0062] As those skilled in the art will understand, preferred formulations include those suitable for two or more routes of administration. For example, a formulation may be suitable for both intravenous and intramuscular administration. Alternatively, preferred formulations may include those suitable for only one route of administration, and those suitable for one or more routes of administration but unsuitable for one or more other routes. For example, a formulation may be suitable for oral, transdermal, transcutaneous, intravenous, intramuscular, intranasal, buccal, and / or subarachnoid administration, but unsuitable for intracerebral administration.

[0063] The inactive components and formulation methods of the pharmaceutical compositions may be conventional. In this specification, conventional formulation methods used in pharmacy may be used. All of the conventional types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, lozenges, suppositories, transdermal patches, and suspensions. Generally, compositions contain about 0.5% to about 50% of the compound in total, depending on the desired dose and the type of composition used. However, the amount of compound is best defined as the “effective dose,” i.e., the amount of compound that provides the desired dose to a patient requiring such treatment. The activity of the compounds used in the compositions and methods disclosed herein is not considered to depend largely on the properties of the composition; therefore, compositions can be selected and formulated primarily or simply for convenience and economic reasons.

[0064] Pharmaceutical compositions and preparations comprising the recombinant tPA-K2 polypeptide of the present technology, or nucleic acids encoding it (e.g., mRNA, cDNA), may be manufactured by conventional mixing, dissolution, granulation, emulsification, encapsulation, mounting, or lyophilization processes. The pharmaceutical compositions can be formulated in conventional methods using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries to facilitate the formulation of preparations suitable for in vitro, in vivo, or ex vivo use. The compositions can be combined with one or more additional activators and formulated with pharmaceutically acceptable carriers, diluents, or excipients to produce pharmaceutical (including biological) or veterinary compositions of the present disclosure suitable for parenteral administration.

[0065] As will be understood by those skilled in the art, many types of formulations are possible. The specific type selected depends on the chosen route of administration, as is well recognized in the art. For example, systemic formulations are generally designed for administration by injection, e.g., intravenous administration. In some embodiments, systemic formulations are sterile.

[0066] Sterile injectable preparations are prepared by incorporating recombinant tPA-K2 polypeptide, or the nucleic acid encoding it (e.g., mRNA, cDNA), in the required amount of a suitable solvent containing various other components listed herein, followed, if necessary, by a suitable sterilization method. Generally, dispersion preparations are prepared by incorporating various sterilizing active compounds into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for preparing sterile injectable preparations, preferred preparation methods are vacuum drying and freeze-drying, thereby obtaining a powder with any additional desired components added from its pre-sterilized filtered solution.

[0067] In some embodiments, compositions comprising recombinant tPA-K2 polypeptide, or nucleic acids encoding it (e.g., mRNA, cDNA), may be formulated in aqueous solution or in a physiologically suitable solution or buffer, such as Hanks solution, Ringer's solution, mannitol solution, or physiological saline buffer. In certain embodiments, any of the recombinant tPA-K2 polypeptides, or the nucleic acids encoding them (e.g., mRNA, cDNA), may include a formulation agent, such as a suspension agent, stabilizer, penetrant or dispersant, buffer, lyoprotectant or preservative, such as polyethylene glycol, polysorbate 80, 1-dodecylhexahydro-2H-azepine-2-one (Laurocapran), oleic acid, sodium citrate, Tris-HCl, dextrose, propylene glycol, mannitol, polysorbate polyethylene sorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol, sucrose, trehalose, and other substances commonly known in the art that may be used in any composition of the present disclosure. (Pramanick et al., Pharma Times 45(3):65-76 (2013)).

[0068] Capsules are prepared by mixing a compound with a suitable diluent and filling capsules with an appropriate amount of the mixture. Common diluents include inert powders (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol, and sucrose), cereal flour, and similar edible powders.

[0069] Tablets are prepared by direct compression, wet granulation, or dry granulation. Their formulations typically incorporate diluents, binders, lubricants, and disintegrants (in addition to the compound). Typical diluents include, for example, various starches, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose). Natural and synthetic gums, including acacia, arginate, methylcellulose, and polyvinylpyrrolidine, can also be used. Polyethylene glycol, ethylcellulose, and waxes can also be used as binders.

[0070] Tablets can be coated with sugar, for example, as a flavor enhancer and sealant. The compound may also be formulated as a chewable tablet by using a large amount of a pleasant-tasting substance, such as mannitol, in the formulation. For example, a fast-dissolving tablet-like formulation may be used to ensure that the patient takes the dosage form and to avoid the difficulties some patients experience when swallowing solid objects.

[0071] To prevent tablets and punches from sticking together in the die, lubricants can be used in tablet formulations. Lubricants can be selected from slippery solids such as talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0072] Tablets may also contain disintegrants. Disintegrants are substances that swell when wet, causing the tablet to disintegrate and release compounds. These include starch, clay, cellulose, algin, and gum. Further examples of disintegrants that can be used include corn starch and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered sponge, cation exchange resin, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose.

[0073] The composition can be formulated, for example, as an enteric-coated formulation to protect the active ingredient from the highly acidic contents of the stomach. Such formulations can be produced by coating a solid dosage form with a polymer film that is insoluble in acidic environments and soluble in basic environments. Exemplary films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0074] Transdermal patches can also be used to deliver compounds. A transdermal patch may include a resin composition in which the compound dissolves or partially dissolves, and a film that protects the composition and holds it in contact with the skin. Other, more complex patch compositions can also be used, such as those having a membrane perforated with multiple pores, allowing the drug to be delivered by penetration.

[0075] As those skilled in the art will also understand, a formulation can be prepared from materials having properties (e.g., purity) that make the formulation suitable for administration to humans (e.g., active substance excipients, carriers (e.g., cyclodextrins), diluents, etc.). Alternatively, a formulation can be prepared from materials having purity and / or other properties that make it suitable for administration to non-human subjects but unsuitable for administration to humans.

[0076] method As shown herein, the disclosed polynucleotides, recombinant tPA-K2 fragments, or nucleic acids encoding them are effective in a method for reducing the assembly and secretion of atherosclerotic apoB lipoprotein from hepatocytes (Figures 23A-23B and 24A-24B). Accordingly, another aspect of the present disclosure provides a method comprising administering a therapeutically effective amount of one or more of the disclosed polynucleotides, recombinant polypeptides, or nucleic acids encoding them to a subject in need thereof. The subject may have hypercholesterolemia or hyperlipidemia, or may be diagnosed with a cardiovascular disease, such as atherosclerosis or arteriosclerosis, or may be diagnosed with type 2 diabetes.

[0077] The disclosed methods can reduce the levels of total cholesterol, or intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), lipoprotein(a) [Lp(a)], chylomicrons, or chylomicron remnants in the subject's blood (e.g., serum or plasma). Methods for measuring lipoproteins, such as VLDL, LDL, IDL, Lp(a), and HDL, are known in the art and are considered routine medical clinical tests. The disclosed methods can be used to treat hyperlipidemia in subjects requiring such treatment.

[0078] In some embodiments, the method includes reducing plasma lipid and / or apoB levels in a subject who has suffered from or is at increased risk of an atherothrombotic event. In some embodiments, the method includes administering to a subject a composition comprising a polynucleotide including a tPA polypeptide (e.g., full-length tPA and / or any of the recombinant tPA-K2 polypeptides disclosed herein or the nucleic acid encoding it) or an endoplasmic reticulum localized motif (e.g., KDEL). In some embodiments, the composition is formulated in LNPs or in infectious particles such as adeno-associated viruses for delivery to the subject. In some embodiments, the subject has suffered from or is at risk of suffering from a severe atherothrombotic event. In some embodiments, administration of the composition to the subject reduces plasma lipid levels in the subject. In some embodiments, the subject is receiving or has received standard treatment for atherothrombotic disease or thrombosis. In some embodiments, atherothrombosis therapy or standard treatment for thrombosis may include one or more of the following: anticoagulants, thrombolytic agents, catheter thrombolysis, or thrombectomy. In some embodiments, the subject is receiving or has received tissue plasminogen activator therapy. In some embodiments, the subject is receiving or has received anticoagulants. In some embodiments, the subject is receiving, is scheduled to receive, or has received treatment with thrombolytic agents, anticoagulants, catheter thrombolysis, and / or thrombectomy.

[0079] As used herein, “hyperlipidemia” refers to abnormally elevated levels of any or all of the lipids or lipoproteins in the blood, such as fat, cholesterol, or triglycerides. Hyperlipidemia encompasses hypercholesterolemia.

[0080] The inventors have demonstrated that administration of a viral genome encoding the disclosed recombinant polypeptide, i.e., AAV containing the disclosed polynucleotide, is effective in reducing VLDL and LDL in animals lacking tPA expression. See Figure 2F (mice treated with AAV8-TBG-Plat encoding full-length mouse tPA). Thus, in other aspects of the present disclosure, the method comprises administering the disclosed pharmaceutical composition containing infectious particles to a subject requiring it.

[0081] The disclosed polynucleotides may be administered using any suitable delivery vehicle. For example, in some cases, the nucleic acid encoding the recombinant polypeptide of the present technology may be incorporated into a delivery vehicle capable of driving nucleic acid expression. Examples of delivery vehicles include, but are not limited to, non-viral vectors (e.g., plasmids (e.g., expression plasmids), liposomes, and polymerosomes) and viral vectors (e.g., adeno-associated virus (AAV) vectors, HSV vectors, and lentiviral vectors). In some embodiments, the disclosed polynucleotides may be delivered using an AAV vector. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV, as well as any other AAV currently known or to be discovered later. The genome sequences of various AAVs and autonomous parvoviruses, as well as the sequences of ITR, Rep proteins, and capsid subunits, are known in the art. Such sequences may be found in the literature or in public databases, such as the GenBank database.

[0082] In some embodiments, but not limited to, subjects administered a therapeutically effective dose show a reduction in total cholesterol or a reduction in levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), Lp(a), chylomicrons, or chylomicron remnants in the subject's serum within approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, compared to an untreated control.

[0083] Treatment may be administered multiple times a day, multiple times a week, or monthly, or in some cases, treatment may only need to be performed once, for example, in the case of infectious particle-mediated delivery of the disclosed tPA-K2 polypeptide.

[0084] Method for producing recombinant tPA-K2 polypeptide Methods for producing recombinant tPA-K2 polypeptides are also disclosed herein. The disclosed polypeptides can be produced according to known methods for producing recombinant polypeptides. For example, the polynucleotides of this disclosure can be introduced into suitable cells, such as animal cells or human cell lines, according to standard procedures, such as transfection, thereby expressing the polynucleotides to produce recombinant tPA-K2 polypeptides. The recombinant tPA-K2 polypeptides can be isolated and further purified according to methods known in the art, such as column purification or liquid chromatography.

[0085] Furthermore, methods for generating rAAV virions are well known. See, for example, K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745 (both incorporated herein by reference in their entirety).

[0086] Kits, systems, and platforms In other aspects of this disclosure, kits, systems, and platforms are provided. These kits, systems, and platforms may, for example, comprise one or more of the disclosed recombinant polypeptides, one or more of the disclosed polynucleotides, and / or one or more of the disclosed infectious particles. In some embodiments, the kits also provide instructions for use.

[0087] This technology will be described using several definitions, which are set forth below and throughout this application.

[0088] definition The subject matter disclosed may be further described using the following definitions and terms. The definitions and terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0089] As used herein, the singular forms “a,” “an,” and “the” include the plural form unless explicitly indicated otherwise in the context. For example, the term “a substituent” should be interpreted as “one or more substituents” unless explicitly indicated otherwise in the context.

[0090] As used herein, “about,” “generally,” “substantially,” and “significantly” are to be understood by those skilled in the art and vary to some extent depending on the context in which they are used. Where there are terms used in a given context that are not obvious to those skilled in the art, “about” and “generally” mean up to plus or minus 10% of a particular term, and “substantially” and “significantly” mean more than plus or minus 10% of a particular term.

[0091] As used herein, “administering” or “dosing” an agent (i.e., a therapeutic agent) or compound / pharmaceutical (including a composition (i.e., a formulation or pharmaceutical)) to a subject includes any route through which the compound / pharmaceutical is introduced or delivered to the subject to perform its intended function. Dosage may be by any preferred route, such as oral administration. Dosage may be subcutaneous. Dosage may be intravenous. Dosage may be intraocular. Dosage may be systemic. Alternatively, dosage may be local, intranasal, intraperitoneal, intradermal, ophthalmically, subarachnoid, intravenous, iontophoresis, transmucosal, intravitreous, or intramuscular. Dosage may include self-administration, administration by another person, or administration using a device (e.g., an infusion pump).

[0092] As used herein, the term “excipient” refers to a natural or synthetic substance formulated alongside the active ingredient of a drug, and is included for purposes such as long-term stabilization, increase in solid formulation volume, or providing therapeutic enhancement to the active ingredient in the final dosage form, such as enhancing drug absorption, reducing viscosity, or improving solubility.

[0093] As used herein, “heterogeneous” means a sequence, such as an amino acid or nucleotide sequence, that is not naturally present as part of the genome in which it exists, or is found at one or more locations within the genome or vector that differ from its naturally occurring location.

[0094] "Homologie," "identity," "percentage of identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homologie can be determined by comparing the positions in each sequence that can be aligned for comparison. If the positions in the comparison sequences are occupied by the same bases or amino acids, the molecules are homologous at those positions. The degree of homology between sequences correlates with the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage of "sequence identity" (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of another sequence means that when the two sequences are aligned, that percentage of bases (or amino acids) are the same. This alignment and percentage of homology or sequence identity can be determined using software programs known in the art.

[0095] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as “non-restrictive” transitional terms that permit the inclusion of additional components beyond those enumerated in the claims. The terms “consist” and “consisting of” should be interpreted as “restrictive” transitional terms that do not permit the inclusion of additional components other than those enumerated in the claims. The term “essentially consisting of” should be interpreted as partially restrictive and permitting the inclusion of only additional components that do not fundamentally alter the nature of the claimed subject matter.

[0096] As used herein, “operably linked” with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to one another. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, thereby allowing the nucleic acid to be transcribed and translated to express a functional fusion protein, in which case the leader peptide influences the secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) can be operably linked to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in the expression of one of the two polypeptides. For example, an amber stop codon can be placed between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, and as a result, when introduced into partial amber suppressor cells, the resulting single mRNA transcript can be translated to produce a fusion protein containing both the first and second polypeptides, or it can be translated to produce only the first polypeptide. In another example, a promoter may be operably ligated to a nucleic acid encoding a polypeptide, thereby allowing the promoter to regulate or mediate the transcription of the nucleic acid.

[0097] As used herein, the term “pharmaceutically acceptable carrier” refers to any diluent, excipient, or carrier that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier includes, is essentially composed of, or further consists of, nanoparticles such as polymer nanoparticle carriers or lipid nanoparticles (LNPs). Further or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolinic tallow. Suitable pharmaceutical carriers are listed in Remington's Pharmaceutical Sciences, Mack Publishing Company, the standard reference literature in this field. They can be selected in accordance with the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and in accordance with conventional pharmaceutical practice.

[0098] As used herein, “prevention” or “prevention” of a disease, disorder, or condition means, in a statistical sample, a reduction in the incidence of the disease, disorder, or condition in a sample or subject administered with the therapeutic agent compared to a control sample or subject. Such prevention may be referred to as prophylactic treatment.

[0099] As used herein, “recombinant” with respect to polynucleotides or polypeptides means polynucleotides or polypeptides modified in vitro by techniques known in the art. For example, an expression vector or expression plasmid, or its expression product, is considered recombinant. In some embodiments, polynucleotides or polypeptides are modified by the introduction of heterologous nucleic acids or proteins, or by the modification of native nucleic acids or proteins, or the material is derived from modified cells. Thus, for example, recombinant cells express genes not found in the cell’s native (non-recombinant) form, or native genes that would otherwise be abnormally expressed, underexpressed, or not expressed at all.

[0100] As used herein, the term “separate” therapeutic use means administering at least two active ingredients simultaneously or substantially simultaneously via different routes.

[0101] As used herein, the term “sequential” therapeutic use means administering at least two active ingredients at different times, either via the same or different routes of administration. More specifically, sequential use refers to the total administration of one of the active ingredients before the administration of one or more other active ingredients is initiated. Thus, it is possible to administer one of the active ingredients over several minutes, hours, or days before administering one or more other active ingredients. In this case, concurrent therapy is not performed.

[0102] As used herein, the term “concurrent” therapeutic use means administering at least two active ingredients simultaneously or substantially simultaneously via the same route.

[0103] As used herein, “synergistic therapeutic effect” refers to a therapeutic effect that is greater than an additive therapeutic effect, resulting from a combination of at least two drugs and exceeding the results that would be obtained from individual administrations of the drugs.

[0104] As used herein, the terms “to treat” or “to cure” refer to a therapeutic action whose purpose is to reduce, alleviate, or delay (reduce) an existing disease or disorder, or its associated signs, symptoms, or condition. For example, if, after receiving an effective amount of the composition, the subject exhibits an observable and / or measurable reduction or absence of one or more signs, symptoms, or conditions associated with the disease, disorder, or condition, then the “treatment” for the disease is successful. Furthermore, the various modes of treatment for medical conditions as described are intended to mean “substantial,” and it should be understood that this includes not only the complete relief of the condition, signs, or symptoms of the disease or disorder, but also “partial” relief resulting in some biological or medically relevant outcome.

[0105] The phrase "etc." should be interpreted as "including, for example." Furthermore, any and all use of illustrative language, including but not limited to "etc.," is intended merely to better illustrate the technology and, unless otherwise claimed, does not limit the scope of the technology.

[0106] Furthermore, where conventions similar to “at least one of A, B, and C” are used, such interpretations are generally intended to be understood by those skilled in the art (for example, “a system having at least one of A, B, and C” includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or systems having both A, B, and C). It will be further understood by those skilled in the art that any de facto disjunct word and / or phrase for which two or more alternative terms exist, whether in a specification or drawing, should be understood as intended to include the possibility of including one of those terms, either one of those terms, or both of those terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0107] As will be understood by those skilled in the art, for any or all purposes, particularly in terms of providing written explanations, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any enumerated scope can be readily recognized as sufficiently descriptive, allowing the same scope to be equally divided into at least half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each scope considered herein can be readily divided into a lower third, a middle third, and an upper third, etc. Also, as will be understood by those skilled in the art, all language such as “maximum,” “at least,” “greater than,” “less than,” etc., includes the enumerated number and refers to a scope that can later be divided into subscopes as considered above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, a group having members 1 to 3 refers to a group having members 1, 2, or 3. Similarly, a group having members 1 to 5 refers to a group having members 1, 2, 3, 4, or 5, and so on. The modal verb "may" refers to a preferred use or choice of one or more of several described embodiments or features contained within. If no choice or choice is disclosed with respect to a particular embodiment or feature contained within, the modal verb "may" refers to a positive action relating to the method and manner of creating or using a described embodiment or feature contained within, or a definitive decision to use a particular skill relating to a described embodiment or feature contained within. In this latter context, the modal verb "may" has the same meaning and implications as the auxiliary verb "can". [Examples]

[0108] The following embodiments are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0109] Example 1 - Intracellular tPA-PAI-1 interaction determines apoB lipidization and VLDL assembly in hepatocytes. Methods and materials Plasma derived from a human with PAI-1 deficiency. The plasma sample contains a frameshift mutation in SERPINE1 (SERPINE1 - / - Data were collected from members of the Bern Amish community with PAI-1 deficiency (n=10) (55), and from age, sex, and BMI-matched controls (n=10) from the same community. Age and sex information for both subjects with PAI-1 deficiency and controls is listed in Table 2. The Institutional Review Boards of the Indiana Center for Hemophilia and Thrombosis and the Medical College of Wisconsin (MCW) approved the study protocol. Study participants provided written informed consent.

[0110] Mouse. Plat fl / fl Mice were generated using wild-type C57BL6 / J mice via homologous recombination in an embryonic stem cell-based approach (Biocytogen, Wakefield, MA). Briefly, the target construct is designed to introduce the loxP sequence (flox) by inserting the loxP site into introns 3 and 6 of the Plat gene. This design conditionally knocks out exons 4-6 of Plat by the Cre-loxP system. Hepatocyte tPA knockout mice were created by introducing AAV8, AAV8-TBG-cre (Cre), which expresses Cre recombinase driven by the thyroxine-binding globulin (TBG) promoter into Plat. fl / fl The AAV8-TBG-GFP (GFP) was produced by administering it to mice and then the resulting plate was treated with AAV8-TBG-GFP. fl / fl Mice were used as a control. To silence tPA expression in hepatocytes, mice were intravenously injected with AAV8 virus containing shPlat (AAV8-H1-shPlat) (19). Age-matched control mice were injected with AAV8-H1-scramble silencing control. Ldlr was used to silence hepatocyte tPA. - / - Apoe - / -The WT C57BL / 6J mice were purchased from Jackson Laboratory (JAX) (catalog numbers 002207, 002052, and 000664, respectively). Ldlr - / - and Apoe - / - Mice were fed WD (Teklad, catalog number TD88137). WT mice were fed standard solid diet (Lab diet, catalog number 5053), DIO diet (Research Diets, catalog number 12492), or WD (Teklad, catalog number TD88137). To express tPA in hepatocytes, WT C57BL / 6J mice and holo-tPA-KO mice (Jax, catalog number 002508) fed standard solid diet received intravenous injection of AAV8-TBG-Plat. For all experiments, mice were housed in a 12-hour light / 12-hour dark cycle with ad libitum access to standard solid diet, WD, or DIO diet and water. Mice of the same age and similar body weight were randomly assigned to experimental and control groups. Plasma lipids were assayed in blood collected after 5 hours of fasting. Power calculation was used to determine the number of mice in each experiment. Both male and female mice were included. Mice of the same age and weight were randomly assigned to groups. Exclusion criteria were death, injury requiring euthanasia, or weight loss exceeding 10%, which were assumed to be rare events not statistically different between groups. All endpoint assays and analyses were performed by researchers blinded to cohort identity. All mouse experiments were conducted with the approval of the Institutional Animal Care and Use Committee at the Biomedical Resource Center of MCW and the Institutional Animal Care and Use Committee at Columbia University Irving Medical Center.

[0111] Vector construction. AAV8-TBG-cre and AAV8-TBG-GFP were purchased from Addgene. As previously described (19, 20), AAV8-H1-short hairpin RNA (shRNA) constructs targeting mouse Plat were constructed by annealing complementary oligonucleotides and then ligating them into the pAAV-RSV-GFPH1 vector. AAV8-TBG-Plat was purchased from Vector Biolabs. The plasmid expressing wild-type human tPA, pCMV3-tPA-HA, was purchased from Sino Biologic (Beijing, China). Plasmid constructs expressing human tPA variants were constructed using Versiti BRI Core based on pCMV3-tPA-HA. Constructed tPA variants included tPA-S513A, tPA-KDEL, tPA-Δ-K2-HA, and tPA-D236, 238N. In particular, tPA-Δ-K2 refers to a tPA mutant in which K2 is replaced with K1, resulting in two copies of K1 but no K2. The purpose of this design is to create a mutant tPA that lacks K2 but mimics the structure of normal tPA.

[0112] Collection and analysis of mouse plasma. Blood obtained by cardiac puncture was placed in 10% volume of sodium citrate (3.8%, w / v) and centrifuged at 2300g for 15 minutes at room temperature. Plasma was carefully collected from the supernatant fraction. Plasma samples were divided into aliquots, flash-frozen, and stored at -80°C until analysis. Total plasma antigen levels of tPA and apoB-100 were measured by ELISA using a kit according to the manufacturer's instructions.

[0113] Human primary hepatocyte experiment. Human primary hepatocytes were obtained from the Liver Tissue Cell Distribution System of the University of Pittsburgh (Pittsburgh, Pennsylvania, USA). All cells were cultured in Williams medium E supplemented with hepatocyte maintenance supplement pack (Thermo Fisher Scientific, catalog number CM4000). The experiment was performed as described in the legend of the figures. Cells were harvested, the culture medium was collected, flash-frozen in liquid nitrogen, and stored at -80°C until processing. Age and sex information of the human donors are listed in Table 3.

[0114] McA-RH7777 cell experiment. Rat liver cancer McA-RH7777 cells were obtained from the American Type Culture Collection (Manassas, Virginia, USA). The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, catalog number 12430054) containing 10% fetal bovine serum (FBS). The experiment was performed as described in the legend in the figure. Cells were collected, the culture medium was recovered, flash-frozen in liquid nitrogen, and stored at -80°C until processing.

[0115] Transfection of cultured hepatocytes with a plasmid encoding tPA-K2-HA-KDEL. McA-RH7777 cells were seeded in culture plates. Transfection mixtures were prepared by pre-incubating plasmids encoding tPA-K2-HA-KDEL or a GFP control with the transfection reagent Lipofectamine 3000 in Opti-MEM for 10 minutes at a culture density of approximately 30-40%. The transfection mixtures were then added to the culture medium. After 72 hours of transfection, hepatocytes and culture medium were harvested and collected. The expression efficiency of tPA-K2-HA-KDEL in cell lysates and apoB in cell medium was detected by immunoblotting (Figures 23A-23B).

[0116] LNP injection in mice. Mice were intravenously injected with LNPs (1 mg / kg body weight) carrying mRNA encoding tPA-K2-HA-KDEL or a control luciferase (SEQ ID NO: 32). The same experiment was performed with LNPs carrying mRNA encoding tPA-K2-KDEL (SEQ ID NO: 8). Blood samples were collected 6, 24, and 48 hours after injection, and immediately before injection. Plasma triglyceride and cholesterol levels were measured (Figures 24A-24B).

[0117] Protein extraction and immunoblotting. Liver tissue samples and cultured hepatocytes were homogenized in RIPA buffer (Thermo Fisher Scientific, catalog no. 89900) supplemented with Halt® protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog no. 78444). Protein extracts were electrophoresed on an SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% (w / v) BSA. The membrane was then incubated overnight at 4°C with a primary antibody in TBST containing 5% BSA, followed by incubation with a suitable secondary antibody conjugated to horseradish peroxidase. Proteins were detected by ECL chemiluminescence.

[0118] Quantitative RT-PCR was performed. Total RNA was extracted using the RNeasy kit (QIAGNE, catalog number 74004). cDNA was synthesized using the iScript® cDNA synthesis kit (BIO-RAD, catalog number 1708891). Quantitative RT-PCR was performed using the Quant Studio 6 system (Applied Biosystems). To normalize relative expression, the expression levels of each gene were normalized to 36B4 (housekeeping). The primer sequences used for quantitative RT-PCR are listed in Table 4.

[0119] Mouse hepatic VLDL production rate. To measure the hepatic VLDL secretion rate, mice were administered poloxamer-407 (1,000 mg / kg body weight) intraperitoneally (96). Tail vein blood samples were collected 60, 90, and 120 minutes after poloxamer-407 administration, and plasma triglyceride levels were measured by assay according to the manufacturer's instructions (Wako, Fujifilm). The rate of plasma triglyceride elevation for 60–90 minutes, or 90–120 minutes, was calculated by dividing the increased triglyceride level by the corresponding time. This reflects the VLDL secretion rate within this time frame.

[0120] A pulse-chase assay of apoB secretion. As mentioned above. 3 Hepatocyte apoB-100 secretion was assayed using the [H] labeling method (31). Human primary hepatocytes or McA-RH7777 cells were washed in leucine-free medium. 3 The samples were pulsed with [H]leucine (80 uCi / ml; 160 Ci / mmol, Perkin Elmer, catalog number NET1166005MC) for 20 minutes. 3 [H] Leucine-containing medium was removed, and cells were incubated with fresh DMEM for a further 0.5, 1, or 3 hours. ApoB was immunoprecipitated from cell homogenates and medium using an anti-apoB antibody (Sigma-Aldrich, catalog no. AB742). Unlabeled apoB-100 standard was added to the precipitate, and the samples were separated by SDS-PAGE gel. The gel was stained with silver, the band corresponding to apoB-100 was excised, and the radioactivity associated with apoB-100 was quantified by a scintillation counter.

[0121] Isolation and protein extraction of the endoplasmic reticulum (ER). The ER fraction from hepatocytes was isolated as previously described (97). Cells were washed in ice-cold PBS and then collected in ER extraction buffer (20 mM HEPES, 250 mM sucrose, pH=7.4). The cell membrane was sheared by passing the cells through a 29 gauge needle. The cell debris was pelleted by centrifugation at 3000 g for 10 minutes for two rounds. The supernatant was overlaid on a discontinuous sucrose gradient of 580, 880, and 1100 mM sucrose in ER extraction buffer and centrifuged at 100,000 g at 4°C for 2 hours. This pellet contained purified ER membrane. Protein extraction from the ER was performed as described (21). The ER pellet was dissolved in 1 M sodium carbonate at pH 11.5 containing 250 mM sucrose and 0.0625% deoxycholic acid in 2 ml of 3 M KCl. The mixture was incubated at room temperature for 30 minutes. After centrifugation, the supernatant contains proteins extracted from the ER.

[0122] Ultracentrifugation of apoB-lipoprotein extracted from the ER in a sucrose gradient. The ER protein extract was prepared in 12.5% ​​sucrose. The sucrose gradient was formed by layering from the bottom of the tube: 1 ml of 47% sucrose, 1 ml of 25% sucrose, the sample in 2.5 ml of 12.5% ​​sucrose, and 1.5 ml of phosphate-buffered saline. This gradient solution was rotated in a Beckman SW40 rotor at 35,000 rpm at 12°C for 65 hours and separated into six fractions from top to bottom (unloaded) (21, 98).

[0123] MTP expression and purification. As previously (99), Cos-7 cells were transfected with a plasmid (pcDNA3-hMTP-FLAG) (Addgene, catalog number 138335) carrying complementary DNA (cDNA) of hMTP cDNA. 48 hours after transfection, cells were collected in 1 ml of buffer K containing 150 mM NaCl (10 mM Tris-Cl, 1 mM MgCl2, and 1 mM EGTA, pH 7.4) and lysed by sonication on ice. Cell lysates were rotated at 13,500 g at 4°C for 10 minutes to remove undestroyed cells and cell debris. FLAG-tagged MTP was purified using a column packed with anti-FLAG M2 affinity agarose beads. Removal of the FLAG peptide and concentration of the purified protein were performed by ultrafiltration using a cutoff centrifugal filter (Am Amicon Ultra, Merck Millipore, catalog number UFC9010).

[0124] Neutral lipid transfer activity assay. Neutral lipid transfer activity in the microsomal fraction of cultured hepatocytes was measured using a commercially available kit (Sigma-Aldrich, MAK110) as previously described (36). Specifically, hepatocytes were homogenized in hypotonic buffer (10 mM Tris-HCl, 1 mM EGTA, and 1 mM MgCl2, pH 7.4) using a Polytron homogenizer. Microsomes were isolated by ultracentrifugation (SW55 Ti rotor, 50,000 rpm, 1 hour). Neutral lipid transfer activity was assayed using the kit according to the manufacturer's manual. Isolated microsomes were incubated with fluorescent lipid-containing "donor vesicles" and "acceptor vesicles" - LDL. The fluorescence signal self-quenched when the labeled lipid was present in the "donor vesicles," but was detectable after the lipid was transferred to the "acceptor vesicles."

[0125] Immunofluorescence imaging and proximity ligation assay (PLA). Human primary hepatocytes were seeded on collagen-coated coverslips. After rinsing with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% TritonX-100 in PBS for 5 minutes, and then blocked at room temperature for 1 hour using PBS containing 5% BSA. Blocked slides were incubated overnight at 4°C with a suitable primary antibody, followed by incubation with a species-specific fluorophore conjugate secondary antibody at room temperature for 1 hour. For primary antibodies, rabbit anti-tPA antibody (ProteinTech, catalog no. 10147-1-AP), goat polyclonal anti-apoB (Sigma-Aldrich, catalog no. AB742), and rabbit antibodies against each intracellular organelle marker—Calnexin for the endoplasmic reticulum (ER) and TGN46 for the trans-Golgi network—were used. For secondary antibodies, we used Alexa Fluor 488-conjugate donkey anti-goat IgG (Thermo Fisher Scientific, catalog number A21206), Alexa Fluor 568-conjugate donkey anti-rabbit IgG (Thermo Fisher Scientific, catalog number A11057), and Alexa Fluor 647-conjugate donkey anti-mouse IgG (Thermo Fisher Scientific, catalog number A31571). Each coverslip was washed three times with PBS for 5 minutes, followed by incubation with DAPI nuclear stain (Invitrogen) for 2 minutes. The coverslips were then rinsed one last time with PBS and mounted in slow anti-fade mounts.

[0126] For PLA, the Duolink in situ kit (Sigma-Aldrich) was used according to the manufacturer's protocol. The primary anti-tPA and anti-apoB antibodies were the same as those used in the immunofluorescence experiments described above. All reactions were performed in a humidified chamber at 37°C. After fixation and permeabilization, hepatocytes were incubated with primary antibodies for 40 minutes and with a pair of PLA probes (anti-rabbit Minus and anti-goat Plus) for 60 minutes, followed by ligase addition for 30 minutes and signal amplification for 100 minutes using a red detection reagent. The coverslips were then mounted using Duolink in situ mounting medium with DAPI and sealed with clear nail polish.

[0127] Fluorescence images were acquired at 40x magnification using a Nikon A1R confocal laser scanning system and digitally zoomed using NIS-Elements analysis software. Blue, green, red, and far-red lasers and filters were used to detect Alexa fluorophores. Texas Red settings were used for PLA detection.

[0128] Solid-phase protein binding assay. Solid-phase binding was performed using an enzyme-linked immunosorbent assay on a polystyrene microtiter plate. Microtiter plate wells were coated overnight at 4°C with 5 μg / ml LDL in coating buffer (TBS). Unbound sites were blocked at 37°C for 1 hour with 3% skim milk in TBS. After washing with TBS containing 0.05% Tween20 (TBS-Tween), tPA was added to the wells at a concentration of 0-20 μg / ml in TBS-Tween. After incubation at 37°C for 1 hour, the wells were washed with TBS-Tween. Binding proteins were reacted with anti-tPA (1 μg / mL IgG in TBS-Tween in the presence of 3% skim milk), followed by goat anti-rabbit IgG conjugated with horseradish peroxidase. TMB substrate was added. After stopping the reaction, absorbance was measured at 450 nm.

[0129] Surface plasmon resonance (SPR). The binding of recombinant tPA to purified LDL was studied using a CM5 sensor tip (Cytiva, catalog no. 29149603) with a Biacore S200 SPR instrument (Biacore) (94). LDL was attached to the tip using amine coupling chemistry according to the manufacturer's instructions. In summary, the tip surface was prepared by exposing a carboxylated dextran matrix to an aqueous solution containing 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.1 M N-hydroxysuccinimide (10 μl / min, 7 minutes). Next, LDL (500 μg / ml in 10 mM sodium acetate buffer, pH 5.5) was flowed over the tip surface at the same rate for 7 minutes, followed by flowing 1 M ethanolamine-HCl (pH 8.5) at 19 μl / min for 7 minutes to inactivate excess reactive groups and remove any non-covalently bound LDL. This procedure immobilized approximately 6,000 response units (RUs) of LDL. To monitor LDL binding with tPA, tPA solutions in HBS-E buffer (Biacore) (0.01 M Hepes, 0.15 M NaCl, 3 mM EDTA, pH 7.4) at concentrations ranging from 10 to 500 μg / mL were flowed through the tip at 20 μl / min for 6 minutes at room temperature. Next, tPA dissociation was monitored by washing the surface with HES buffer alone for 6 minutes. tPA was then flowed through an activated but uncoated CM5 tip under the same conditions as the nonspecific binding control.

[0130] Plasma lipoprotein isolation by FPLC. FPLC was performed at 4°C using an AKTA purifier 10 with two Superose 6 increase10 / 300GL columns (Cytiva, catalog number 29091596) connected in tandem. 300 μl (1.5 times the volume of the sample loop) of pooled plasma was injected. Tris-buffered saline (25 mM Tris, 150 mM NaCl, 2 mM EDTA, pH=7.4) was used as the running buffer. Elution was collected from 12 ml to 47 mL fractions into 1 mL fractions. Cholesterol, triglyceride (Wako, Fujifilm), and apoB-100 (Abcam, catalog number ab230932; Mabtech, catalog number 3715-1HP) levels from the fractions were measured according to the manual.

[0131] Plasma lipoprotein isolation by continuous ultracentrifugation. Plasma lipoproteins were separated by KBr (potassium bromide) density ultracentrifugation, as previously (95, 100). Equal volumes of mouse plasma were used for continuous density ultracentrifugation to separate very low-density lipoproteins (d<1.006 g / mL), low-density lipoproteins (d=1.006~1.063 g / mL), and high-density lipoproteins (d=1.063~1.21 g / mL) in a TLA100 rotor.

[0132] VLDL particle size analysis. VLDL particles (d<1.006 g / mL) were isolated by KBr density ultracentrifugation (95), and then two methods were used to measure their diameter. First, the isolated VLDL particles were negatively stained with 20 g / L phosphotungstic acid (pH 7.0) for 2 minutes and then observed under a Philips CM10 electron microscope. The mean diameter of the VLDL particles was determined using Image-Pro Plus 5.0 image analysis software. Second, the hydrodynamic diameter of the isolated VLDL particles was measured at 633 nm using a Zetasizer μV dynamic laser light scattering instrument (Malvern Instruments). The VLDL sample was transferred to a quartz cuvette, and light scattering readings were performed at 20°C (25-27).

[0133] Mouse tail bleeding assay. Mice were anesthetized with isoflurane and placed horizontally on a platform so that their tails could be lowered approximately 2 cm from the top of the platform. A portion of the distal tail tip was cut with a No. 11 surgical scalpel to create a wound approximately 2 mm in diameter. Bleeding was monitored by gently applying the tip of a Whatman paper to the tail at 10-second intervals until bleeding stopped. The time to stable cessation of bleeding was defined as the time interval between tail incision and cessation of bleeding without signs of rebleeding for 60 seconds. Bleeding exceeding 15 minutes was stopped by applying pressure.

[0134] Statistical analysis. The data presented in this study were generated from biological replicates. The number of human participant studies and mouse experiments is indicated in the legend of the applicable figures. In vitro cell experiments were repeated at least three times. All results are presented as mean ± SEM. P-values ​​were calculated using a two-tailed Student's t-test for data that passed the normality test, and the Mann-Whitney rank-sum U test for data that were not normally distributed. When analyses were performed with three or more groups, one-way ANOVA with post-hoc Tukey test was used to assess differences between groups.

[0135] Study Approval. All mouse experiments were conducted with the approval of the Institutional Biosafety Committees of IACUC and MCW, and the IACUC at Columbia University Medical Center. The use of human cell and plasma samples in this study was approved by the IRBs of MCW and the Indiana Hemophilia and Thrombosis Center. All participants provided written informed consent.

[0136] (Table 1) Main Resources TIFF2026520004000002.tif232168TIFF2026520004000003.tif83167

[0137] (Table 2) Age and sex information for homozygous individuals and corresponding controls for PAI-1 deficiency. TIFF2026520004000004.tif66167

[0138] (Table 3) Age and sex information of human participants who provided primary hepatocytes TIFF2026520004000005.tif78167

[0139] (Table 4) Primers for quantitative PCR TIFF2026520004000006.tif81157

[0140] result Silencing hepatocyte tPA increases atherogenic apoB-lipoprotein-cholesterol and apoB independently of LDLR or ApoE. Adenovirus-associated virus-8 (AAV8) expressing hairpin RNA (AAV8-H1-shPlat, short as sh-tPA) against Plat mRNA (encoding tPA) driven by the H1 promoter was administered to Ldlr, an established hypercholesterolemia mouse model given a Western diet (WD). - / - It was administered to mice to specifically silence tPA expression in hepatocytes [21, 22]. Hepatocyte tPA-silencing mice showed 47% higher plasma total cholesterol levels (p<0.01) and 28% higher apoB-100 (p<0.05) than mice treated with AAV8-H1-scrambled RNA (scr) (Figure 1A). High-performance protein liquid chromatography (FPLC) plasma lipoprotein fraction profiling showed higher cholesterol and apoB in the VLDL and LDL fractions, as well as higher triglycerides in the VLDL, in hepatocyte tPA-silencing mice (Figure 1A). Similarly, WD-supplied Apoe knockout (Apoe - / -Silencing hepatocyte tPA in mice resulted in a 30% increase in plasma total cholesterol (p<0.05), a 25% increase in apoB-100 (p<0.05), and a 27% increase in triglycerides (p<0.05) in a consistent distribution in the lipoprotein fraction compared to scrambled silencing controls (Figure 1B). Similar results were observed in WD-supplied C57BL / 6J wild-type (WT) mice without alteration of hepatic ApoB mRNA (Figures 9A-9C), suggesting that the increased apoB levels were not due to increased apoB synthesis. Furthermore, silencing hepatocyte tPA did not alter hepatic LDLR and plasma apoE levels in WT mice (Figures 10A-10B). In summary, silencing hepatocyte tPA results in higher plasma apoB lipoprotein cholesterol through a mechanism independent of LDLR or apoE.

[0141] Consistently, tPA silencing in primary human hepatocytes using siRNA (si-tPA) against PLAT mRNA resulted in higher apoB-100 levels in serum-free medium (Figure 1D) without altering hepatocyte APOB mRNA levels (Figure 11B). Cholesterol and triglycerides in isolated VLDL were elevated in tPA-silencing human hepatocytes (Figure 1D). Similar findings were observed in cultured McA-RH7777 cells (Figure 1E; Figures 11C-11D), a rat hepatoma cell line that is an established model system for studying VLDL production because it synthesizes VLDL with a size similar to human VLDL particles [23, 24].

[0142] Silencing hepatocyte tPA increases VLDL production and apoB lipidization in the ER. Following injection with the nonionic surfactant detergent poloxamer 407 (P407) [25, 26], which inhibits lipoprotein lipase activity and VLDL lipolysis, hepatocyte tPA-silencing mice exhibited a faster triglyceride elevation rate (Figure 2A), suggesting that tPA silencing in hepatocytes increases apoB-VLDL production. Furthermore, plasma apoB-100 derived solely from hepatocytes increased to a lager degree during hepatocyte tPA silencing compared to plasma apoB-48 produced by both mouse hepatocytes and intestines

[27] (Figure 2B).

[0143] Lipidization is a crucial factor in determining the fate of intrahepatic apoB. Insufficiently lipid-laden apoB undergoes intracellular degradation, while fully lipid-laden apoB is efficiently secreted as particles of larger size and lower density

[28] . Electron microscopy scanning of VLDL particles isolated by density ultracentrifugation revealed hepatocyte tPA silencing Ldlr - / - A shift distribution to a larger diameter was observed in mice (Figure 2C). Dynamic light scattering (DLS) revealed a consistent larger hydrodynamic diameter of isolated VLDL due to their slower dispersed particle velocities (Brownian motion) [29-32] (Figure 2D), suggesting a higher lipid content, which was validated by a higher TG / apoB ratio (Figure 2E).

[0144] Consistently, silencing tPA in human primary hepatocytes results in higher apoB-related radioactivity in the cell culture medium, and higher 3[H]-leucine-labeled apoB

[33] secretion was observed (Figure 2H). Similar results were observed in McA-RH7777 cells (Figure 12). Silencing tPA increased the diameter and TG / apoB ratio of VLDL isolated from cell culture (Figures 2I, 2J). ER-related apoB levels were higher in tPA-silencing human primary hepatocytes than in controls (Figure 2K), but tPA-silencing hepatocytes accumulated more apoB in the less dense ER fractions (fractions 1 and 2, Figure 2K). Since density and lipidization are inversely correlated, this finding was consistent with the hypothesis that tPA limits apoB lipidization.

[0145] Hepatocyte tPA disrupts the MTP-apoB interaction and inhibits MTP-dependent triglyceride transfer. MTP is a key chaperone that promotes intrahepatic apoB lipidization by transferring and incorporating triglycerides, particularly triglycerides and cholesteryl esters, into apoB to assemble VLDL [10,11]. tPA silencing in primary human hepatocytes did not alter MTP protein levels (Figure 3A, input), but anti-MTP immunoprecipitated from tPA-silencing cells showed higher apoB levels compared to control hepatocytes (Figure 3A), suggesting that tPA silencing increased apoB-MTP interaction. Consistently, microsomal fractions isolated from tPA-silencing hepatocytes showed twice the triglyceride transfer activity compared to microsomes from control cells (Figure 3B, groups 1-2). The transfer activity was indeed mediated by MTP, and the MTP inhibitor CP-346086

[34]

[35] completely abolished the increased neutral lipid transfer activity in tPA-silencing hepatocytes (Figure 3B, Group 3). Similar findings were observed in McA-RH7777 cells (Figure 14). MTP-mediated lipid transfer to apoB is accompanied by direct binding to apoB

[10] , and inhibition of the apoB-MTP interaction reduces apoB lipidization and secretion

[11] .

[0146] In primary human hepatocytes transfected with a plasmid expressing tPA with a C-terminal HA tag, apoB was detected in the anti-HA precipitate eluate (Figure 3F). Proximity ligation assay (PLA), showing punctate fluorescence signals (Figure 3G), demonstrated proximity between tPA and apoB in hepatocytes, suggesting intracellular interactions between endogenous tPA and apoB in hepatocytes, further supported by co-localization of tPA and apoB in the ER by immunofluorescence staining (Figure 3H). Purified recombinant tPA dose-dependently interacted directly with purified apoB-containing LDL bound to the surface of a microtiter plate in a solid-phase protein binding assay (Figure 4A). Consistent interactions were observed between purified recombinant tPA and purified apoB-100 (Figure 16). In contrast, purified tPA did not interact with surface-bound MTP (Figure 3B). Pre-incubation of LDL with tPA inhibited binding between MTP and surface-bound LDL (Figure 3C) and reduced MTP-mediated transfer of neutral lipids to LDL (Figure 3J). Surface plasmon resonance (SPR) studies suggested non-covalent bonding between tPA and LDL particles (Figure 4D). Quantitative analysis of sensorgrams using a two-state coupling model yielded a Kd of approximately 260 nM for the LDL-tPA interaction. Taken together, tPA directly interacts with lipoprotein-containing apoB, reducing the availability of apoB to MTP for neutral lipid incorporation, thereby inhibiting VLDL assembly.

[0147] Transduction of tPA into hepatocytes reduces VLDL assembly and apoB secretion in the hepatocyte ER, independently of tPA's serine protease activity. In tPA knockout mice (holo-tPA-KO), increasing tPA expression only in hepatocytes carrying the AAV8 virus under the TBG promoter (AAV8-TBG-tPA) [21, 22, 36-38] reduced plasma apoB-100, VLDL-, and LDL cholesterol compared to AAV8-TBG-LacZ controls (Figure 2F). Consistently, tPA expression by plasmids (e.g., pCMV3-tPA-HA) in primary human hepatocytes reduced the neutral lipid transfer activity and apoB-MTP interaction of newly synthesized apoB secretion using pulse-chase assays (Figures 2G, 3C, 3D). In summary, tPA restricts apoB lipidization and subsequent secretion by reducing apoB's accessibility to MTP.

[0148] MTP-apoB interactions occur in the hepatocyte ER

[39] . Using confocal immunofluorescence microscopy, tPA and apoB colocalize in the ER (Figure 3H), and apoB interacts with MTP for its lipidization and VLDL assembly. Retaining tPA in the ER by adding a KDEL sequence [40-43] to the C-terminus of the human tPA protein (tPA-KDEL, SEQ ID NO: 3) reduced newly synthesized apoB secretion by pulse-chase assay in human primary hepatocytes transduced with the plasmid encoding tPA-KDEL (SEQ ID NO: 3) (pCMV3-tPA-KDEL, described in SEQ ID NO: 24) (Figure 3I). In summary, these results indicate that tPA interacts with apoB in the ER and limits the assembly and secretion of apoB-VLDL.

[0149] The protease activity of tPA is dependent on serine at position 513, and serine substitution to alanine (S513A) completely inactivates the serine protease activity of tPA

[44] . Similar to WTtPA, the serine protease mutant tPA (S513A, SEQ ID NO: 9) reduced apoB secretion by pulse-chase assay in human primary hepatocytes transduced with the plasmid encoding tPA S513A (pCMV3-tPA-S513A, SEQ ID NO: 23) (Figure 3I). Recombinant tPA-S513A protein bound to surface-bound LDL with similar affinity to WTtPA (Figure 4A) and impaired MTP-mediated lipid transfer activity (Figure 3J), suggesting that tPA binds to apoB and reduces apoB lipidation and secretion independently of tPA serine protease activity.

[0150] The lysine-binding site in the kringle 2 domain of tPA interacts with the lysine-rich region at the N-terminus of apoB. The kringle 2 domain of tPA has a lysine-binding site

[45] which is required for its interaction with fibrin. The load-charged residues aspartate-236 and -238 within the kringle 2 domain of human tPA are involved in the binding of tPA to positively charged lysine

[46] . The surface exposed at the N-terminus of apoB

[47] has a lysine-rich region which is involved in binding to MTP and MTP-mediated lipidization activity

[48] . Expression of either a tPA mutant lacking the kringle 2 domain (tPA-Δ-K2) or a tPA mutant in which aspartate at positions 236 and 238 is replaced with asparagine (tPA-D236, 238N) did not alter apoB secretion by pulse-chase assay (Figure 4F) compared to wild-type tPA. Similarly, in a solid-phase protein binding assay, an antibody against the tPA kringle 2 domain (Sigma, HPA003412) inhibited the binding of surface-bound tPA to LDL compared to a rabbit IgG control (Figure 4G). Tranexamic acid (TXA), a lysine analog, reduced the interaction between tPA and surface-bound LDL (Figure 4H). Therefore, tPA blocks the apoB-MTP interaction by competitively binding to the lysine-rich region of apoB via lysine-binding sites at aspartate-236 and -238 on the kringle 2 domain of tPA, thereby reducing the accessibility of MTP to apoB for lipidization.

[0151] PAI-1 sequesters tPA from apoB, resulting in increased VLDL assembly within hepatocytes. Studies by numerous groups have characterized lipid loading-induced postprandial cholesterolemia as enhancing hepatic production of VLDL, although the underlying mechanisms remain largely unknown. Obesity, often associated with dyslipidemia, increases tPA synthesis in hepatocytes

[21] , which is then overcompensated by a greater increase in its serpine inhibitor, PAI-1, resulting in a decrease in net free functional tPA in the liver and plasma

[21] . PAI-1 covalently binds to tPA, forming a stable complex that inactivates the serine protease function of tPA

[49] . Therefore, we hypothesize that postprandial lipid loading increases intracellular interactions between tPA and PAI-1, leading to a reduction in free tPA that suppresses the availability of apoB accessed by MTP for lipidization, ultimately causing increased VLDL assembly and secretion into the bloodstream.

[0152] Probing of tPA (approximately 70 kd) after immunoprecipitation of PAI-1 (approximately 50 kd) elution from human primary hepatocytes via SDS-PAGE gel showed a band located at approximately 120 kd, representing a covalently bound SDS-stable tPA-PAI-1 complex, which was further investigated by PLA for their intrinsic interactions in proximity in living human primary hepatocytes (Figure 5A-5B). When human primary hepatocytes were treated with oleate, a well-established stimulant for apoB lipidization and VLDL production

[50] , tPA-PAI-1 complexation increased and free tPA decreased as early as 1 hour later (Figure 5C-5D). This is similar to the time to the stimulating effect of oleate on VLDL production

[51] . This rapid complexation from PAI-1 to tPA, without the need for newly synthesized tPA or PAI-1 protein, and the subsequent sequestering of tPA by PAI-1, suggest timely and fine-tuned regulation of apoB lipidization and VLDL production when hepatocytes are lipid-loaded. Extending oleate treatment to 6 and 24 hours further reduced free tPA, resulting in increased PAI-1-tPA complex formation (Figure 5D). Consistent results were observed in McA-RH7777 cells (Figure 18).

[0153] Silencing PAI-1 in primary human hepatocytes resulted in higher free tPA and lower apoB secretion via pulse chase (Figures 5E-5F). The inhibitory effect of si-PAI-1 on apoB secretion was more pronounced after oleate treatment, resulting in an approximately 400% increase in free tPA and an approximately 60% decrease in apoB secretion, compared to only an approximately 66% increase in free tPA and a 25% decrease in apoB secretion without oleate. These observations are consistent with the hypothesis that under basal conditions, the majority of tPA is free and not bound by PAI-1. Silencing PAI-1 under these basal conditions results in only a moderate increase in free tPA and a subsequent mild decrease in apoB secretion. However, under oleate overload conditions, more tPA is bound to PAI-1, and silencing PAI-1 results in a robust increase in free tPA and a decrease in apoB secretion. Consistently, silencing both tPA and PAI-1 simultaneously did not further reduce apoB secretion compared to silencing tPA alone, supporting the idea that PAI-1 promotes apoB lipidization not through PAI-1 itself, but by sequestering tPA from apoB (Figure 5G). Consistent results were observed in McA-RH7777 hepatocytes (Figure 18).

[0154] Complexation of tPA and PAI-1 causes a structural change in the tPA protein structure

[52] , causing it to lose its fibrin-binding ability mediated by the lysine-binding site within the kringle 2 domain

[53] , suggesting that binding of PAI-1 to tPA blocks the lysine-binding site within the kringle 2 domain, which may be mediated by steric hindrance or structural changes in the kringle 2 domain. Unlike purified tPA alone, the purified PAI-1-tPA complex is unable to bind to LDL or inhibit MTP-mediated neutral lipid transfer activity (Figure 5H-5I), indicating that complexation of tPA and PAI-1 prevents interaction between tPA and apoB.

[0155] After force-feeding C57BL / 6J mice with olive oil to increase dietary fatty acid intake, followed by increased blood fatty acid and lipid load on hepatocytes, hepatic free tPA decreased compared to baseline at 2 or 6 hours later, without altering hepatic total tPA and PAI-1 levels (Figure 5J, Figure 19). As expected, obese hepatocyte-specific PAI-1 knockout mice (H-PAI-1KO) [21, 54] had higher free tPA in both liver and plasma, lower plasma apoB, lower total cholesterol, and lower cholesterol in the VLDL and LDL fractions compared to littermates and controls (Figure 6A-6F).

[0156] In summary, the PAI1-tPA intracellular interaction maintains the balance of VLDL production. When fatty acids are loaded into hepatocytes, PAI-1 rapidly complexes with tPA within the hepatocytes, reducing the amount of available free tPA that directly interacts with apoB, thus limiting its lipidization and ultimately leading to increased VLDL assembly and secretion into the bloodstream.

[0157] PAI-1 deficiency in humans leads to decreased plasma apoB and apoB cholesterol levels. - / -Individuals with homozygous PAI-1 deficiency due to a specific loss-of-function mutation (n=10) had 22% lower LDL cholesterol (p<0.05), a borderline significant 21% lower apoB (p=0.07), 18% lower cholesterol in the VLDL fraction (p=0.06), and 16% lower triglycerides (p=0.07) compared to unaffected individuals in the same community (age, sex, and BMI-matched controls, n=10) (Figure 6G). None of these individuals were taking lipid-lowering agents or had a known history of cardiovascular disease. Compared to unaffected controls, PAI-1 deficiencies had higher tPA levels on isolated VLDL particles (Figure 6H). Furthermore, VLDL-bound tPA levels were inversely correlated with VLDL diameter (r=-0.59, p<0.01) (Figure 6I). Along with data from hepatocyte-PAI-1-deficient mice and PAI-1-silencing human primary hepatocytes (Figures 5F and 6D), PAI-1 deficiency results in higher levels of free tPA interacting with apoB, limiting apoB lipidization in hepatocytes.

[0158] In summary, the above results demonstrate that tPA directly interacts with apoB via its K2 domain within the ER of hepatocytes, and that this interaction reduces MPT-mediated VLDL assembly. Lipid loading to hepatocytes induces the formation of the tPA-PAI-1 complex, sequestering tPA from apoB, thereby promoting apoB lipidization and VLDL assembly (Figure 6J).

[0159] Notably, in cultured hepatocytes, transduction of a plasmid encoding a tPA-K2 domain operably linked to an endoplasmic reticulum localized motif resulted in exogenous expression of only the tPA-K2 domain, which reduced apoB secretion (Figures 23A-23B). Furthermore, intravenous injection of an LNP containing tPA-K2 mRNA reduced plasma lipids in mice (Figures 24A-24B). These findings demonstrate the usefulness of the recombinant tPA-K2 polypeptide of the present invention in compositions and methods for treating hyperlipidemia.

[0160] Silencing tPA resulted in a reduction of Lp(a) in cell culture medium from both cultured human primary hepatocytes (Figure 20A) and HepG2 cells (Figure 20B), indicating that tPA also limits the production of Lp(a), another atherosclerotic apoB-containing lipoprotein, in hepatocytes. Furthermore, knocking out tPA in intestinal cells increased plasma cholesterol levels (Figures 21A-21C) and chylomicron production (Figures 22A-22B) in mice, demonstrating that the regulatory effect of tPA on apoB lipoprotein production also exists in intestinal cells, which absorb dietary lipids and transport them into the body. These findings demonstrate that the recombinant tPA-K2 polypeptide of this technology is useful in compositions and methods for reducing plasma Lp(a) and chylomicron levels and further reducing cardiovascular risk.

[0161] Consideration Our findings reveal a novel mechanism by which tPA in hepatocytes fine-tunes the rate of apoB-lipoprotein assembly. For example, although we do not wish to be constrained by theory, the assembly of VLDL in the ER of hepatocytes is achieved in two steps [9,55]. In the first step, co-translational lipidization of apoB allows MTP to transfer lipids to the growing apoB polypeptide, including its surface-exposed N-terminus, on the ER membrane to form primordial VLDL particles of roughly the same size as plasma HDL

[11] . The primordial VLDL then detach from the ER membrane to become luminal particles [9], which undergo further lipidization into mature VLDL particles in the second step of VLDL assembly

[55] . MTP also promotes the fusion of primordial VLDL with lipid droplets in the ER lumen

[56] . MTP inhibitors disrupt apoB lipidization and reduce VLDL production

[57] . In hepatocytes, tPA competes with MTP for interaction with apoB, similar to MTP inhibitors (Figure 6J). The kringle domain is an autonomous protein domain that folds into a large loop stabilized by three disulfide bonds responsible for protein-protein interactions

[58] . The kringle 2 domain of tPA has a lysine binding site

[46] and interacts with a lysine-rich region [45,59]. The N-terminus of apoB contains a lysine-rich region necessary for interaction with MTP for lipidation

[10] . Our competitive binding assays suggested that antibodies against the tPA kringle 2 domain, or the lysine analog TXA, inhibit the interaction between tPA and LDL, and that this interaction may be mediated by the lysine binding site in the kringle 2 domain of tPA. The newly synthesized apoB takes approximately 40 minutes to be secreted from hepatocytes

[51] , and the apoB-related radioactivity in the early follow-up phase (5-10 minutes) is used to represent the amount of newly synthesized apoB after pulse labeling

[51] . The inventors found no difference in apoB-related radioactivity in the cell lysates after 10 minutes of follow-up (Figure 18).This suggests that silencing tPA does not increase the rate of apoB protein synthesis, which is supported by the observation that silencing tPA did not alter apoB mRNA levels in WT mouse liver, cultured human primary hepatocytes, and McA-RH7777 cells (Figures 9A-9C and 11A-11D).

[0162] Oleic acid influx into hepatocytes stimulates apoB lipidization and VLDL assembly

[51] , but the mechanism is not fully understood. Our finding that oleate treatment increases intracellular tPA-PAI-1 complex formation and leads to a decrease in free tPA in hepatocytes provides new insights into the molecular mechanism of the stimulating effect of oleates on apoB production. The tPA-PAI-1 complex increased only 1 hour after oleate treatment, with no effect on either total tPA or PAI-1 levels. In contrast, extended oleate treatment for more than 6 hours also increased total PAI-1 protein levels in cultured human primary hepatocytes. Consistently, mice orally administered olive oil, in which approximately 70% of the fatty acids are oleic acid, showed decreased hepatic tPA-PAI-1 after 2 hours, but no changes in total tPA or PAI-1 (Figure 19). These results suggest that oleate influx into hepatocytes rapidly induces complexation between tPA and PAI-1 within 1 hour in vitro or 2 hours in vivo. In summary, lipid loading to hepatocytes induces PAI-1 complex formation with tPA, allowing tPA to cleave from apoB and apoB to bind to MTP, thus enhancing apoB lipidization and VLDL production, and revealing the physiological significance of the tPA-PAI-1 interaction in the fine-tuning of apoB lipidization after lipid loading to hepatocytes.

[0163] Elevated serum PAI-1 levels and decreased tPA activity lead to impaired fibrinolysis and are independent risk factors for atherothrombotic disease

[60] . Clinical observational data show that plasma PAI-1 levels are positively associated with apoB cholesterol, while tPA activity is negatively associated with apoB cholesterol [57, 60, 61].

[0164] overview In summary, these results demonstrate that the tPA-K2 polypeptide of this technology is effective in reducing apoB secretion and lowering plasma triglyceride and total cholesterol levels when administered to subjects, despite lacking serine protease and proteolytic activity. Furthermore, because the tPA-K2 fragment of this technology (e.g., including the amino acid sequence described in SEQ ID NO: 7) does not possess serine protease and proteolytic activity, administration of the peptide of this technology does not increase the risk of bleeding in subjects. In this example, we demonstrate that expression of recombinant tPA-K2 peptides containing the tPA kringle 2 domain alone and the endoplasmic reticulum localized sequence reduces apoB secretion from hepatocytes (Figures 23A-23B) and lowers plasma lipid levels in an in vivo mouse model (Figures 24A-24B), but does not alter bleeding time in mice (Figure 25). Therefore, these data support that compositions comprising recombinant tPA-K2 polypeptide (or the nucleic acid encoding it (e.g., cDNA, mRNA)) are useful in methods for treating diseases or conditions associated with elevated plasma apoB-lipoprotein levels, and in methods for reducing one or more of the following plasma levels: VLDL, IDL, LDL, Lp(a), chylomicrons, chylomicron remnants, triglycerides, or total cholesterol. These data also demonstrate that compositions of this technology are useful in methods for reducing plasma lipids and / or apoB levels in subjects requiring and / or undergoing thrombolytic therapy.

[0165] Example 2 - Therapy of a target with the recombinant polypeptide of the present disclosure For example, a subject suffering from cardiovascular disease, such as hyperlipidemia, atherosclerosis, increased risk of thrombosis or atherothrombotic events, angina pectoris, heart attack, heart failure, stroke, transient ischemic attack (TIA), peripheral artery disease, or hypertension, is administered a therapeutically effective dose of a pharmaceutical composition comprising the disclosed recombinant tPA-K2 polypeptide or the nucleic acid encoding it. The subject may be receiving standard thrombolytic therapy (e.g., tissue plasminogen activator therapy). The disclosed recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., polypeptides comprising the amino acid sequences described in SEQ ID NOs: 3, 5, and 7, and / or polynucleotide molecules comprising the nucleic acid sequences described in SEQ ID NOs: 4, 6, 8, or 34) may be administered via any route indicated by the subject's specific therapeutic needs, such as oral, transdermal, intravenous, intramuscular, intranasal, buccal, subarachnoid, intracerebral, or rectal routes. Signs and symptoms of cardiovascular disease may be reduced by administration of a pharmaceutical composition comprising the disclosed recombinant tPA-K2 polypeptide or the nucleic acid encoding it. Treatment may be administered daily, every other day, every three days, or on a schedule determined by the patient's progression, at the discretion of the physician. Subjects are expected to experience reductions in triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, chylomicron remnants, blood pressure, or other metrics associated with the reduction of signs or symptoms of cardiovascular disease, compared to untreated subjects. Methods for measuring the reduction of signs and symptoms of cardiovascular disease are known in the art.

[0166] These results are expected to demonstrate that compositions containing the recombinant tPA-K2 polypeptide or the nucleic acid encoding it are useful in methods for treating subjects with cardiovascular disease.

[0167] Example 3 - Treatment of hypercholesterolemia in subjects using recombinant tissue plasminogen activator In one example, a subject suffering from hyperlipidemia is administered a therapeutically effective dose of a pharmaceutical composition containing one or more recombinant tPA-K2 polypeptides of the Technology or nucleic acids encoding them. The subject may be at risk of or suffering from an atherothrombotic event and may be receiving standard thrombolytic therapy (e.g., tissue plasminogen activator therapy). Recombinant tPA-K2 or nucleic acids encoding it (e.g., polypeptides containing the amino acid sequences described in SEQ ID NOs: 3, 5, and 7, and / or polynucleotide molecules containing the nucleic acid sequences described in SEQ ID NOs: 4, 6, 8, or 34) may be administered via any route indicated by the subject's specific therapeutic needs, e.g., orally, percutaneously, intravenously, intramuscularly, intranasally, buccally, subarachnoidally, intracerebrally, or rectally. Levels of triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, or chylomicron remnants may be reduced by administration of recombinant tPA-K2 or the nucleic acid encoding it. Treatment may be administered daily, every other day, every three days, or on a schedule determined by the patient's progression, at the discretion of the physician. Subjects are expected to experience a reduction in triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, chylomicron remnants, or other metrics associated with a reduction in signs or symptoms of hyperlipidemia, compared to untreated subjects. Methods for measuring the reduction in signs and symptoms of hyperlipidemia are known in the art.

[0168] These results are expected to demonstrate that compositions containing the recombinant tPA-K2 polypeptide or the nucleic acid encoding it are useful in methods for treating patients with hyperlipidemia.

[0169] Example 4 - Treatment with recombinant tPA-K2 reduces Lp(a) assembly and production in hepatocytes. This experiment demonstrates the effectiveness of the recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., polypeptides containing the amino acid sequences described in SEQ ID NOs. 5 and 7 and / or polynucleotide molecules containing the nucleic acid sequences described in SEQ ID NOs. 6, 8, or 34) in reducing Lp(a) assembly and production in hepatocytes. Briefly, human primary hepatocytes are treated with plasmids, cDNA, and / or mRNA encoding tPA-K2 (e.g., tPA-K2-HA-KDEL). Cells and cell culture media are harvested and recovered. Lp(a) levels in cell lysates and cell culture media are measured by immunoblotting.

[0170] These results are expected to demonstrate that compositions containing the recombinant tPA-K2 polypeptide of this technology, or nucleic acids encoding it, are useful in methods for reducing plasma Lp(a) levels in subjects requiring such methods.

[0171] Example 5 - Treatment with recombinant tPA-K2 reduces chylomicron assembly and production in intestinal cells. This experiment demonstrates the effectiveness of the recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., polypeptides containing the amino acid sequences described in SEQ ID NOs. 5 and 7 and / or polynucleotide molecules containing the nucleic acid sequences described in SEQ ID NOs. 6, 8, or 34) in reducing chylomicron assembly and production in intestinal cells. Briefly, Caco-2 cells, an intestinal epithelial cell line, are treated with plasmids, cDNA, and / or mRNA encoding tPA-K2 (e.g., tPA-K2-HA-KDEL). Cells and cell culture media are harvested and collected. ApoB48 levels in cell lysates and cell culture media are measured by immunoblotting.

[0172] These results are expected to demonstrate that compositions containing the recombinant tPA-K2 polypeptide or the nucleic acid encoding it are useful in methods for reducing chylomicron production.

[0173] Example 6 - Treatment with recombinant tPA-K2 does not significantly increase the risk of fatty liver or liver injury in the subjects. This experiment demonstrates that treatment of subjects with the recombinant tPA-K2 polypeptide of this technology or the nucleic acid encoding it (e.g., polypeptides containing the amino acid sequences described in SEQ ID NOs. 5 and 7 and / or polynucleotide molecules containing the nucleic acid sequences described in SEQ ID NOs. 6, 8, or 34) does not increase the risk of fatty liver or liver damage. In short, mice are intravenously injected with an LNP carrying mRNA encoding tPA-K2-HA-KDEL (SEQ ID NO. 32) or a control luciferase (1 mg / kg body weight). Blood is collected 24 hours after injection and immediately before injection. Mice are euthanized 24 hours after injection, and their livers are collected. Blood liver damage markers are measured. Liver histological analysis is performed.

[0174] These results are expected to demonstrate that treatment of subjects with recombinant tPA-K2 or the nucleic acid encoding it using this technology does not increase the risk of fatty liver or liver damage in the subjects.

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[0176] As will be readily apparent to those skilled in the art, various substitutions and modifications can be made to the technologies disclosed herein without departing from the scope and spirit of the art. The inventions described exemplary in this art can be suitably practiced even without any elements(s) or limitations(s) not specifically disclosed herein. The terms and expressions used are for illustrative purposes only, not limitations, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or any part thereof, but it should be recognized that various modifications are possible within the scope of the art. Therefore, although the art is described by specific embodiments and optional features, those skilled in the art should understand that modifications and / or variations of the concepts disclosed herein can be made, and such modifications and variations are considered to be within the scope of the art.

[0177] Numerous patent and non-patent references may be cited herein. Cited references are incorporated herein by reference in their entirety. If there is any conflict between the definition of a term in a cited reference and the definition of a term herein, that term should be interpreted according to the definition herein.

[0178] Equal portions This technology is not limited to the specific embodiments described in this application, which are intended as single examples of individual aspects of this technology. Many modifications and variations of the technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of this technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This technology should be limited only by the conditions of the appended claims, along with the entire scope of equivalents to which such claims are entitled. This technology is not limited to any particular method, reagent, compound composition, or biological system, which may, of course, vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0179] In addition, if any feature or aspect of the present disclosure is described from the perspective of the Markush group, a person skilled in the art will recognize that the present disclosure is also described from the perspective of any individual member or subgroup of a member of the Markush group.

[0180] As will be understood by those skilled in the art, for any or all purposes, and particularly in terms of providing written explanations, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. As will also be understood by those skilled in the art, all language such as “maximum,” “at least,” etc., includes the enumerated numbers.

[0181] List of sequences TIFF2026520004000007.tif186160TIFF2026520004000008.tif240160TIFF2026520004000009.tif240160TIFF2026520004000010.tif245160TIFF2026520004000011.tif246160TIFF2026520004000012.tif246160TIFF2026520004000013.tif242160TIFF2026520004000014.tif243160TIFF2026520004000015.tif243160TIFF2026520004000016.tif243160TIFF2026520004000017.tif243160TIFF2026520004000018.tif243160TIFF2026520004000019.tif246160TIFF2026520004000020.tif244160TIFF2026520004000021.tif244160TIFF2026520004000022.tif244160TIFF2026520004000023.tif244160TIFF2026520004000024.tif244160TIFF2026520004000025.tif244160TIFF2026520004000026.tif244160TIFF2026520004000027.tif244160TIFF2026520004000028.tif244160TIFF2026520004000029.tif244160TIFF2026520004000030.tif244160TIFF2026520004000031.tif240160TIFF2026520004000032.tif244160TIFF2026520004000033.tif244160TIFF2026520004000034.tif244160TIFF2026520004000035.tif244160TIFF2026520004000036.tif244160TIFF2026520004000037.tif245160TIFF2026520004000038.tif244160TIFF2026520004000039.tif244160TIFF2026520004000040.tif243160TIFF2026520004000041.tif245160TIFF2026520004000042.tif240160TIFF2026520004000043.tif192160.

Claims

1. Isolated polynucleotide molecules, (a) Nucleotide sequence described in Sequence ID No. 6, (b) Nucleotide sequence described in Sequence ID No. 10, (c) Nucleotide sequence described in Sequence ID No. 15, (d) Nucleotide sequence described in Sequence ID No. 39, (e) Nucleotide sequence described in Sequence ID No. 2, (f) A nucleotide sequence that is at least about 85% identical to any one of the nucleotide sequences of (a) to (f), and which encodes a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) containing polypeptide, wherein the recombinant tPA-K2 containing polypeptide can bind to apolipoprotein B (apoB) and / or inhibit apoB secretion from hepatocytes and / or intestinal cells and / or inhibit apoB lipoproteinization, (g) A nucleotide sequence that is a complement of any one of (a) to (f), and RNA sequence encoded by any one of (h)(a) to (g) It includes a nucleotide sequence selected from the group consisting of, The nucleotide sequence is operably linked to a different nucleic acid. The isolated polynucleotide molecule.

2. The polynucleotide molecule according to claim 1, wherein the heterogeneous nucleic acid includes an endoplasmic reticulum localized sequence.

3. The polynucleotide molecule according to claim 2, wherein the endoplasmic reticulum localization sequence encodes an amino acid having sequence KDEL (SEQ ID NO: 11).

4. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is as described in Sequence ID No.

8.

5. The polynucleotide molecule described in 3, wherein the nucleotide sequence is as described in Sequence ID No.

34.

6. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is as described in Sequence ID No.

16.

7. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is as described in Sequence ID No.

4.

8. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is as described in Sequence ID No.

36.

9. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is described in Sequence ID No.

38.

10. The polynucleotide molecule according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in Sequence ID No.

7.

11. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO:

14.

12. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO:

35.

13. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO:

30.

14. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO:

3.

15. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in Sequence ID No.

9.

16. The polynucleotide according to claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence described in SEQ ID NO:

37.

17. An expression vector comprising a polynucleotide molecule according to any one of claims 1 to 16, operably linked to one or more regulatory sequences suitable for inducing expression in eukaryotic cells.

18. The expression vector according to claim 17, wherein the one or more regulatory sequences include a promoter.

19. The expression vector according to claim 18, wherein the promoter is a thyroxine-binding globulin (TBG) promoter, or the promoter comprises the nucleic acid sequence described in SEQ ID NO:

17.

20. A cell comprising a polynucleotide molecule according to any one of claims 1 to 16 or an expression vector according to any one of claims 17 to 19.

21. The cells according to claim 20, selected from hepatocytes or intestinal cells.

22. An infectious particle comprising a polynucleotide molecule according to any one of claims 1 to 16.

23. The infectious particle according to claim 22, which is a virus.

24. The infectious particle according to claim 23, wherein the virus is adeno-associated virus (AAV).

25. The infectious particle according to claim 24, wherein the AAV is AAV8.

26. Lipid nanoparticles comprising a polynucleotide molecule according to any one of claims 1 to 16.

27. The lipid nanoparticles according to claim 26, wherein the lipid nanoparticles are freeze-dried, present in a suspension, or emulsified.

28. A composition comprising a polynucleotide molecule according to any one of claims B1 to B16, a vector according to any one of claims 17 to 19, an infectious particle according to any one of claims B22 to B25, or lipid nanoparticles according to claim 26 or 27, and a pharmaceutically acceptable carrier.

29. A method for reducing plasma apoB lipoprotein in a subject requiring a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of the composition according to claim 28 to the subject.

30. The method according to claim 29, wherein the plasma apoB lipoprotein is selected from very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof.

31. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount of the composition according to claim 28 to the subject.

32. The method according to claim 31, wherein the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.

33. A method for lowering plasma triglyceride and / or cholesterol levels in a subject requiring a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of the composition according to claim 28 to the subject.

34. The method according to any one of claims 29 to 33, further comprising administering a therapeutically effective amount of a plasminogen activator inhibitor-1 (PAI-1) inhibitor to the subject simultaneously, separately, or sequentially.

35. The method according to claim 34, wherein the PAI-1 inhibitor is selected from the group consisting of MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, Aleplasinin, Loureirin B, Diaplasinin, Todarolactone, SK-216, Geodin, Fendosal, AZ3976, TM5007, and any combination thereof.

36. The method according to any one of claims 29 to 35, wherein the composition is administered to the subject intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly.

37. The method according to claim 29 or 30, wherein the subject is a human.

38. Recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptide, or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof, wherein (tPA-K2 polypeptide) is (a) Below: (i) The amino acid sequence described in Sequence ID No. 5, (ii) The amino acid sequence described in Sequence ID No. 13, (iii) Amino acid sequence described in Sequence ID No. 9, (iv) Amino acid sequence described in SEQ ID NO: 30, (v) The amino acid sequence described in Sequence ID No. 1, (vi) Amino acid sequence described in SEQ ID NO: 37, (vii) An amino acid sequence that is at least approximately 85% identical to any one of the amino acid sequences of (i) to (vi), and that can bind to apolipoprotein B (apoB), and / or can inhibit apoB secretion from hepatocytes and / or intestinal cells, and / or can inhibit apoB lipoproteinization. An amino acid sequence selected from the group consisting of, (b) Heterogeneous amino acid sequences and A recombinant tPA-K2-containing polypeptide, or a pharmaceutically acceptable salt thereof, tautomer, hydrate, and / or solvate thereof.

39. The recombinant tPA-K2 polypeptide according to claim 38, wherein the heterologous amino acid sequence includes an endoplasmic reticulum localized motif.

40. The recombinant tPA-K2 polypeptide according to claim 39, wherein the endoplasmic reticulum localized motif is KDEL.

41. The recombinant tPA-K2 polypeptide according to claim 40, wherein the amino acid sequence is as described in Sequence ID No.

7.

42. The recombinant tPA-K2 polypeptide according to claim 40, wherein the amino acid sequence is as described in Sequence ID No.

14.

43. The recombinant tPA-K2 polypeptide according to claim 40, wherein the amino acid sequence is described in Sequence ID No.

35.

44. The recombinant tPA-K2 polypeptide according to claim 40, wherein the amino acid sequence is described in SEQ ID NO:

37.

45. The recombinant tPA-K2 polypeptide according to claim 40, wherein the amino acid sequence is as described in Sequence ID No.

3.

46. The recombinant tPA-K2 polypeptide according to any one of claims 38 to 45, wherein the polypeptide does not have serine protease function.

47. The recombinant tPA-K2 polypeptide according to any one of claims 38 to 46, wherein the polypeptide is not fibrin-soluble.

48. The recombinant polypeptide according to any one of claims 38 to 47, wherein the polypeptide is bound to apoB.

49. The recombinant polypeptide according to any one of claims 38 to 48, wherein administration of a therapeutically effective amount of the polypeptide to a subject reduces plasma triglyceride and / or cholesterol levels in the subject.

50. The recombinant polypeptide according to any one of claims 38 to 49, wherein administration of a therapeutically effective amount of the polypeptide to a subject reduces the plasma level of one or more apoB lipoproteins in the subject.

51. The recombinant polypeptide according to claim 50, wherein the one or more apoB lipoproteins are selected from very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof.

52. A composition comprising a recombinant polypeptide according to any one of claims 38 to 51 and a pharmaceutically acceptable carrier.

53. A method for reducing plasma apoB lipoprotein in a subject for whom reduction of plasma apoB lipoprotein is required, comprising administering to the subject a therapeutically effective amount of a recombinant polypeptide according to any one of claims 38 to 51 or a composition according to claim 52.

54. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount to a recombinant polypeptide according to any one of claims 38 to 51 or the composition according to claim 52.

55. The method according to claim 54, wherein the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.

56. The method according to any one of claims 53 to 55, further comprising administering a therapeutically effective amount of a plasminogen activator inhibitor-1 (PAI-1) inhibitor to the subject simultaneously, separately, or sequentially.

57. The method according to claim 56, wherein the PAI-1 inhibitor is selected from the group consisting of MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, alepracinin, laurelin B, diapracinin, todarolactone, SK-216, geodin, fendosal, AZ3976, TM5007, and any combination thereof.

58. The method according to any one of claims 53 to 57, wherein the recombinant polypeptide or composition is administered to the subject intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly.

59. The method according to any one of claims 53 to 58, wherein the subject is a human.

60. A polynucleotide molecule containing the nucleotide sequence described in Sequence ID No.

8.

61. The polynucleotide according to claim 60, formulated within an infectious particle for delivery to a target.

62. The polynucleotide according to claim 61, wherein the infectious particle is adeno-associated virus (AAV).

63. The polynucleotide according to claim 60, formulated in lipid nanoparticles (LNPs) for delivery to a target.

64. A composition comprising a polynucleotide according to any one of claims 60 to 63 and a pharmaceutically acceptable carrier.

65. A method for reducing plasma apoB lipoprotein in a subject for whom reduction of plasma apoB lipoprotein is required, comprising administering a therapeutically effective amount of the composition according to claim 64 to the subject.

66. A method for lowering plasma triglyceride and / or cholesterol levels in a subject requiring a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of the composition according to claim 64 to the subject.

67. (a) Nucleotide sequence described in Sequence ID No. 34, (b) The nucleotide sequence described in Sequence ID No. 10, and (c) Nucleotide sequence described in Sequence ID No. 36 A polynucleotide molecule containing a nucleotide sequence selected from the group consisting of the following.

68. The polynucleotide according to claim 67, formulated within an infectious particle for delivery to a target.

69. The polynucleotide according to claim 68, wherein the infectious particle is adeno-associated virus (AAV).

70. The polynucleotide according to claim 67, formulated in lipid nanoparticles (LNPs) for delivery to a target.

71. A composition comprising a polynucleotide according to any one of claims 67 to 70 and a pharmaceutically acceptable carrier.

72. A method for reducing plasma apoB lipoprotein in a subject requiring a reduction in plasma apoB lipoprotein, comprising administering a therapeutically effective amount of the composition according to claim 71 to the subject.

73. A method for lowering plasma triglyceride and / or cholesterol levels in a subject requiring a reduction in plasma triglyceride and / or cholesterol levels, comprising administering a therapeutically effective amount of the composition according to claim 71 to the subject.

74. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is described in Sequence ID No.

34.

75. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is described in Sequence ID No.

10.

76. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is described in Sequence ID No.

36.

77. Recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptide, or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof, wherein the (tPA-K2 polypeptide) comprises an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NO: 9 and SEQ ID NO:

35.

78. A composition comprising the recombinant polypeptide according to claim 77 and a pharmaceutically acceptable carrier.

79. A method for reducing plasma apoB lipoprotein in a subject for whom reduction of plasma apoB lipoprotein is required, comprising administering to the subject a therapeutically effective amount of the recombinant polypeptide according to claim 77 or the composition according to claim 78.

80. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, comprising administering a therapeutically effective amount to the subject of the recombinant polypeptide according to claim 77 or the composition according to claim 78.

81. The method according to claim 80, wherein the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.

82. Recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) polypeptide, or a fragment thereof.

83. The recombinant tPA-K2 polypeptide according to claim 82, comprising a sequence having at least 80% identity with sequence number 5.

84. The recombinant tPA-K2 polypeptide according to claim 82 or 83, wherein the polypeptide does not have serine protease function.

85. The recombinant tPA-K2 polypeptide according to claim 82 or 83, wherein the polypeptide is not fibrin-soluble.

86. A recombinant tPA-K2 polypeptide according to any one of claims 82 to 85, which binds to apolipoprotein B (apoB).

87. A recombinant tPA-K2 polypeptide according to any one of claims 82 to 86, comprising a sequence selected from sequence numbers 13 to 14.

88. The recombinant tPA-K2 polypeptide according to claim 87, comprising sequence number 14.

89. A method for producing a recombinant tPA-K2 polypeptide according to any one of claims 82 to 88.

90. A polynucleotide comprising a nucleotide sequence encoding a recombinant tPA-K2 polypeptide according to any one of claims 82 to 88.

91. The polynucleotide according to claim 90, wherein the nucleotide sequence encoding the recombinant tPA-K2 polypeptide includes a nucleotide sequence selected from SEQ ID NOs: 15 to 16.

92. The polynucleotide according to claim 91, further comprising at least one regulatory sequence operably linked to the nucleotide sequence encoding the recombinant tPA-K2 polypeptide.

93. The polynucleotide according to claim 92, wherein the at least one regulatory sequence comprises a promoter, an enhancer, or both a promoter and an enhancer.

94. The polynucleotide according to claim 93, wherein the at least one regulatory sequence includes a promoter.

95. The polynucleotide according to claim 94, wherein the promoter is a thyroxine-binding globulin (TBG) promoter, or the promoter comprises Sequence ID No.

17.

96. The polynucleotide according to any one of claims 90 to 95, comprising two or more nucleotide sequences encoding the recombinant tPA-K2 polypeptide.

97. Nanoparticles comprising a recombinant tPA-K2 polypeptide according to any one of claims 1 to 6 or a polynucleotide according to any one of claims 90 to 96.

98. Infected particle comprising a polynucleotide according to any one of claims 90 to 96.

99. An infectious particle according to claim 98, which is a virus.

100. The infectious particle according to claim 99, wherein the virus is adeno-associated virus (AAV).

101. The infectious particle according to claim 100, wherein the AAV is AAV8.

102. A pharmaceutical composition comprising the recombinant tPA-K2 polypeptide described in any one of claims 82 to 88.

103. A pharmaceutical composition comprising the nanoparticles described in claim 97.

104. A pharmaceutical composition comprising an infectious particle according to any one of claims 98 to 100.

105. A method comprising administering a therapeutically effective amount of the pharmaceutical composition described in any one of claims 102 to 104 to a target.

106. The method according to claim 105, wherein the subject has hypercholesterolemia or hyperlipidemia.

107. The method according to claim 105, wherein the subject has been diagnosed with a cardiovascular disease.

108. The method according to claim 107, wherein the cardiovascular disease includes atherosclerosis.

109. The method according to any one of claims 105 to 108, wherein the subject has been diagnosed with type 2 diabetes.

110. A method for treating a cardiovascular disease in a subject requiring treatment for the cardiovascular disease, comprising administering tissue plasminogen activator (tPA) or a fragment thereof to the subject in order to treat the cardiovascular disease.

111. The method according to claim 110, wherein the tPA fragment comprises a tPA-K2 domain.

112. A method for reducing blood cholesterol levels in a subject that requires a reduction in blood cholesterol levels, the method comprising administering tissue plasminogen activator (tPA) or a fragment thereof to the subject in order to reduce blood cholesterol levels in the subject.

113. The method according to claim 112, wherein the tPA fragment comprises a tPA-K2 domain.

114. The method according to any one of claims 110 to 113, wherein the administration includes oral administration or intravenous administration.

115. The method according to any one of claims 105 to 114, for reducing the levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), lipoprotein (a) [Lp(a)], chylomicrons, or chylomicron remnants in the serum of the subject.

116. The method according to any one of claims 105 to 115, wherein the tPA comprises the pharmaceutical composition according to any one of claims 102 to 104.

117. A method for reducing plasma lipid and / or apolipoprotein B (apoB) levels in subjects for whom it is necessary to reduce plasma lipid and / or apolipoprotein B (apoB) levels, (a) Nucleotide sequence described in Sequence ID No. 2, (b) A nucleotide sequence encoding the polypeptide sequence described in Sequence ID No. 1, (c) A nucleotide sequence that is at least approximately 85% identical to any one of the nucleotide sequences in (a) to (b), (d) A nucleotide sequence that is a complement of any one of (a) to (c), and (e) RNA sequence encoded by any one of (a) to (d) The method involves administering a therapeutically effective amount of a composition comprising a polynucleotide molecule containing a nucleotide sequence selected from the group consisting of the following: The nucleotide sequence is operably linked to a heterologous nucleic acid including an endoplasmic reticulum localized sequence. The aforementioned subjects are suffering from or at increased risk of an atherothrombotic event. The aforementioned method.

118. The method according to claim 117, wherein the endoplasmic reticulum localization sequence encodes a polypeptide having the amino acid sequence KDEL (SEQ ID NO: 11).

119. The method according to claim 118, wherein the polynucleotide molecule comprises the nucleotide sequence described in SEQ ID NO:

4.

120. The method according to claim 119, wherein the nucleotide sequence encodes the polypeptide sequence described in Sequence ID No.

3.

121. The method according to any one of claims 117 to 120, wherein the composition is formulated in lipid nanoparticles or in adeno-associated virus for delivery to the target.

122. The method according to any one of claims 117 to 121, wherein administration of the composition reduces the plasma apoB and / or lipid levels in the subject.

123. The method according to any one of claims 117 to 122, wherein the subject is currently receiving, scheduled to receive, or has previously received thrombolytic therapy.

124. The method according to claim 123, further comprising administering tissue plasminogen activators to the subject simultaneously, sequentially, or separately.

125. The method according to any one of claims 117 to 124, further comprising administering an anticoagulant to the subject simultaneously, sequentially, or separately.

126. The method according to any one of claims 29-37, 53-59, 65-66, 72-73, 79-81, or 105-116, wherein the subject is suffering from or at increased risk of an atherothrombotic event.

127. The method according to claim 126, wherein the subject is receiving, is scheduled to receive, or has received treatment with a thrombolytic agent, an anticoagulant, catheter thrombolysis, and / or thrombectomy.

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