Recombinant tissue plasminogen activator (TPA) fragments and uses thereof

Abstract

The present technology relates to recombinant polypeptides comprising tissue-type plasminogen activator (tPA) fragments or nucleotides encoding said recombinant polypeptides and their use for the treatment of cardiovascular diseases. In some embodiments, the tPA fragment comprises a kringle 2 domain of the tPA.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 467,450, filed May 18, 2023, the contents of which are incorporated by reference in their entirety for any and all purposes. TECHNICAL FIELD

[0003] The present technology provides recombinant polypeptides comprising a tissue-type plasminogen activator (tPA) fragment, or nucleotides encoding the same, and their use for treating cardiovascular disease. In some embodiments, the tPA fragment comprises the kringle 2 domain of tPA (tPA-K2).

[0004] Statement as to Federally Sponsored Research or Development

[0005] This application was made with government support under HL163516 awarded by the National Institutes of Health. The government has certain rights in the application.

[0006] SEQUENCE LISTING

[0007] An informal sequence listing is provided herein. BACKGROUND

[0008] Apolipoprotein B (apoB)-containing lipoproteins initiate and promote atherosclerotic cardiovascular disease (CVD) [1, 2]. ApoB-containing lipoproteins include hepatocyte-derived very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and lipoprotein (a) [Lp(a)], as well as enterocyte-derived chylomicrons and chylomicron remnants. Hepatocytes and enterocytes produce VLDL and chylomicrons, respectively, and secrete them into the blood. 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 intervention therapies to help prevent CVD primarily use statins or inhibitors of the protein convertase subtilisin / kexin type 9 (PCSK9), both of which lower LDL by enhancing hepatic LDL receptor (LDLR)-mediated clearance of LDL [3]. However, these treatments have only modest effects on other apoB-containing lipoproteins that are atherogenic, such as VLDL, IDL, Lp(a), chylomicrons, and chylomicron remnants [4, 5], which can lead to residual CVD risk in populations with good LDL cholesterol control [6, 7]. Therefore, therapies that inhibit hepatic VLDL and Lp(a) production, as well as enterocyte chylomicron production, can help reduce CVD risk because they would lower all atherogenic apoB lipoproteins. Thus, there is a need in the art for new therapeutic approaches to lower atherogenic apo-B lipoproteins and treat cardiovascular disease. SUMMARY

[0009] In one aspect, the present disclosure provides an isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 6; (b) the nucleotide sequence set forth in SEQ ID NO: 10; (c) the nucleotide sequence set forth in SEQ ID NO: 15; (d) the nucleotide sequence set forth in SEQ ID NO: 39; (e) the nucleotide sequence set forth in SEQ ID NO: 2; (f) a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any one of (a) to (f) and encodes a polypeptide comprising a recombinant tissue-type plasminogen activator kringle 2 domain (tPA-K2) that is capable of binding apolipoprotein B (apoB), and / or inhibiting apoB secretion from hepatocytes and / or enterocytes, and / or inhibiting apoB lipoprotein lipogenesis; (g) a nucleotide sequence that is a complement of 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 comprises an endoplasmic reticulum localization sequence. In some embodiments, the endoplasmic reticulum localization sequence encodes an amino acid having the sequence KDEL (SEQ ID NO: 11). In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 8. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 34. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 16. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 4. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 36. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 38. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 32. In some embodiments, the nucleotide sequence is set forth in SEQ ID NO: 40. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 37.In some embodiments, the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the disclosure provides an expression vector comprising any of the polynucleotide molecules operably linked to one or more regulatory sequences suitable for directing expression in a eukaryotic cell. In some embodiments, the one or more regulatory sequences comprise a promoter. In some embodiments, the promoter is a thyroxin binding globulin (TBG) promoter, or wherein the promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 17. In some embodiments, the disclosure provides a cell comprising any of the polynucleotide molecules or expression vectors. In some embodiments, the cell is selected from a hepatocyte or an intestinal epithelial cell. In some embodiments, the disclosure provides an infectious particle comprising any of the polynucleotide molecules. 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 disclosure provides a lipid nanoparticle comprising any of the polynucleotide molecules. In some embodiments, the lipid nanoparticle is lyophilized, in suspension, or emulsified. In some embodiments, the disclosure provides a composition comprising any of the polynucleotide molecules, vectors, infectious particles, or lipid nanoparticles and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a method for reducing plasma apoB lipoproteins in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition. In some embodiments, the plasma 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 method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering to the subject a therapeutically effective amount of the composition. 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 a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a plasminogen activator inhibitor 1 (PAI-1) inhibitor 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, Dragoninin B, Diaplasinin, Toddalolactone, SK-216, Geodin, Fendosal, AZ3976, TM5007; and any combination thereof. In some embodiments, the composition is administered intravenously, intraperitoneally, subcutaneously, buccally, intradermally, intrahepatically, or intramuscularly to the subject. In some embodiments, the subject is a human.

[0010] In one aspect, this disclosure provides a polypeptide containing a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof, the (tPA-K2 polypeptide) comprising: (a) an amino acid sequence selected from the group consisting of: (i) the amino acid sequence listed in SEQ ID NO: 5; (ii) the amino acid sequence listed in SEQ ID NO: 13; (iii) the amino acid sequence listed in SEQ ID NO: 9; (iv) the amino acid sequence listed in SEQ ID NO: 30; (v) the amino acid sequence listed in SEQ ID NO: 1; (vi) the amino acid sequence listed in SEQ ID NO: 37; and (vii) an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of (i) to (vi) and is capable of binding apolipoprotein B. (apoB), and / or inhibition of apoB secretion from hepatocytes and / or intestinal epithelial cells, and / or inhibition of apoB lipoprotein lipoprotein lipidation; and (b) a heterologous amino acid sequence. In some embodiments, the heterologous amino acid sequence comprises an endoplasmic reticulum (ER) localization motif. In some embodiments, the ER localization motif is KDEL. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 7. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 14. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 35. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 37. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 3. In some embodiments, the amino acid sequence is listed in SEQ ID NO: 3. In some embodiments, the polypeptide does not contain 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 the subject's plasma triglyceride and / or cholesterol levels. In some embodiments, administration of a therapeutically effective amount of the polypeptide to a subject reduces the subject's plasma levels of one or more apoB lipoproteins. In some embodiments, 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 residues, or any combination thereof. In some embodiments, this disclosure provides a composition comprising any one of a recombinant polypeptide and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a method for lowering plasma apoB lipoprotein in a subject of need, the method comprising administering a therapeutically effective amount of the recombinant polypeptide or composition to the subject.In some embodiments, this disclosure provides a method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering to a subject a therapeutically effective amount of either a recombinant polypeptide or a composition. 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 to a subject simultaneously, alone, or sequentially a therapeutically effective amount of a plasminogen activator inhibitor 1 (PAI-1) inhibitor. 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, draconin B, diaplasinin, toddalolactone, 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, buccally, intradermally, intrahepatically, or intramuscularly. In some embodiments, the subject is a human being.

[0011] In one aspect, the present invention provides a polynucleotide molecule comprising the nucleotide sequence listed in SEQ ID NO: 8. In some embodiments, the polynucleotide is formulated for delivery to a subject in an infectious particle. In some embodiments, the infectious particle is an adeno-associated virus (AAV). In some embodiments, the polynucleotide is formulated for delivery to a subject in lipid nanoparticles (LNPs). In some embodiments, this disclosure provides a composition comprising any one of a polynucleotide and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a method for lowering plasma apoB lipoprotein in a subject of need, the method comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, this disclosure provides a method for lowering plasma triglyceride and / or cholesterol levels in a subject of need, the method comprising administering a therapeutically effective amount of the composition to the subject.

[0012] In one aspect, this disclosure provides a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence listed in SEQ ID NO: 34; (b) the nucleotide sequence listed in SEQ ID NO: 10; and (c) the nucleotide sequence listed in SEQ ID NO: 36. In some embodiments, the polynucleotide is formulated for delivery to a subject in an infectious particle. In some embodiments, the infectious particle is an adeno-associated virus (AAV). In some embodiments, the polynucleotide is formulated for delivery to a subject in lipid nanoparticles (LNPs). In some embodiments, this disclosure provides a composition comprising any one of a polynucleotide and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a method for reducing plasma apoB lipoprotein in a subject of need, the method comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, this disclosure provides a method for reducing plasma triglyceride and / or cholesterol levels in a subject of need, the method comprising administering a therapeutically effective amount of the composition to the subject. In some embodiments, the nucleotide sequence is listed in SEQ ID NO: 34. In some embodiments, the nucleotide sequence is listed in SEQ ID NO: 10. In some embodiments, the nucleotide sequence is listed in SEQ ID NO: 36.

[0013] In one aspect, this disclosure provides a polypeptide containing a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) 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 listed in SEQ ID NO: 9 and SEQ ID NO: 35. In some embodiments, this disclosure provides a composition comprising the recombinant polypeptide and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a method for lowering plasma apoB lipoprotein in a subject of need, the method comprising administering to the subject a therapeutically effective amount of either the recombinant polypeptide or the composition. In some embodiments, this disclosure provides a method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering to the subject a therapeutically effective amount of either the recombinant polypeptide or the composition. In some embodiments, the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.

[0014] In one aspect, this disclosure provides a recombinant tissue-type plasminogen activator kringle 2 domain (tPA-K2) polypeptide or a fragment thereof. In some embodiments, the recombinant tPA-K2 polypeptide comprises a sequence at least 80% identical to SEQ ID NO: 5. In some embodiments, the polypeptide does not contain serine protease function. In some embodiments, the polypeptide is not fibrinolytic. 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 NO: 13 to 14. In some embodiments, the recombinant tPA-K2 polypeptide comprises SEQ ID NO: 14. In some embodiments, this disclosure provides a method for preparing the recombinant tPA-K2 polypeptide. In some embodiments, this 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 NO: 15 to 16. In some embodiments, the polynucleotide further comprises at least one regulatory sequence operatively 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 wherein the promoter comprises SEQ ID NO:17. In some embodiments, the polynucleotide comprises more than one nucleotide sequence encoding a recombinant tPA-K2 polypeptide. In some embodiments, this disclosure provides a nanoparticle comprising a recombinant tPA-K2 polypeptide or a polynucleotide. In some embodiments, this disclosure provides an infectious particle comprising a polynucleotide. In some embodiments, the infectious particle is a virus. In some embodiments, the virus is adeno-associated virus (AAV). In some embodiments, the AAV is AAV8. In some embodiments, this disclosure provides a pharmaceutical composition comprising a recombinant tPA-K2 polypeptide. In some embodiments, this disclosure provides a pharmaceutical composition comprising nanoparticles. In some embodiments, this disclosure provides a pharmaceutical composition comprising infectious particles. In some embodiments, this disclosure provides a method comprising administering a therapeutically effective amount of the pharmaceutical composition to a subject. In some embodiments, the subject has hypercholesterolemia or hyperlipidemia. In some embodiments, the subject has been diagnosed with cardiovascular disease. In some embodiments, cardiovascular disease includes atherosclerosis. In some embodiments, the subject has been diagnosed with type 2 diabetes.

[0015] In one aspect, this disclosure provides a method for treating cardiovascular disease in a subject of need, the method comprising administering to the subject a tissue-type plasminogen activator (tPA) or a fragment thereof to treat the subject's cardiovascular disease. In some embodiments, the tPA fragment comprises a tPA-K2 domain.

[0016] In one aspect, the present invention provides a method for lowering blood cholesterol levels in a subject in need, the method comprising administering to the subject a tissue plasminogen activator (tPA) or a fragment thereof to lower the subject's blood cholesterol levels. In some embodiments, the tPA fragment comprises a tPA-K2 domain. In some embodiments, administration comprises oral or intravenous administration. In some embodiments, the method lowers the levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), lipoprotein(a) [Lp(a)], chylomicrons, or chylomicron residues in the subject's serum. In some embodiments, the tPA comprises a pharmaceutical composition.

[0017] In one aspect, this disclosure provides a method for reducing plasma lipid and / or apolipoprotein B (apoB) levels in a subject of need, the method comprising administering a therapeutically effective amount of a composition comprising a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence listed in SEQ ID NO: 2; (b) a nucleotide sequence encoding a polypeptide sequence listed in SEQ ID NO: 1; (c) a nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any of (a) to (b); (d) a nucleotide sequence of complement of any of (a) to (c); and (e) an RNA sequence encoded by any of (a) to (d); wherein the nucleotide sequence is operatively linked to a heterologous nucleic acid comprising an endoplasmic reticulum localization sequence; and wherein the subject has had an atherosclerotic thrombotic event or is at increased risk of 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 listed in SEQ ID NO: 4. In some embodiments, the nucleotide sequence encodes the polypeptide sequence listed in SEQ ID NO: 3. In some embodiments, the composition is formulated for delivery to a subject in lipid nanoparticles or adeno-associated virus. In some embodiments, administration of the composition reduces the subject's plasma apoB and / or lipid levels. In some embodiments, the subject is undergoing, will undergo, or has already undergone thrombolytic therapy. In some embodiments, the method further comprises administering a tissue-type plasminogen activator to the subject concurrently, sequentially, or alone. In some embodiments, the method further comprises administering an anticoagulant to the subject concurrently, sequentially, or alone.

[0018] In some embodiments, for any of the treatments disclosed herein, the subject has experienced an atherosclerotic thrombotic event or is at increased risk of an atherosclerotic thrombotic event. In some embodiments, the subject is undergoing, will undergo, or has already undergone treatment with thrombolytic agents, anticoagulants, catheter-directed thrombolysis, and / or thrombectomy. Attached Figure Description

[0019] FIG. 1A to FIG. 1E The study showed that silencing hepatocytes tPA, independent of LDLR or ApoE, increases atherogenic apoB lipoprotein cholesterol and apoB. FIG. 1A This provides Ldlr (Low-Dystrophic) animals fed a Western diet (WD) and treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1-scrambled control (scr). - / - A set of images showing the results for the mice. Ldlr - / -Mice were treated with sh-tPA or scr and then fed with WD for 8 weeks. tPA protein in mouse liver was measured, and total cholesterol and apoB-100 concentrations, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100 in plasma samples were determined (n = 9 to 10 mice per group). FIG. 1B This provides Apoe (a Western diet) fed with AAV8-shPlat (sh-tPA) or AAV8 disordered control (scr) treatment. - / - A set of images showing the results in mice. Apoe - / - Mice were treated with sh-tPA or scr and then fed WD for 8 weeks. Liver tPA protein was measured, and total cholesterol and apoB-100 concentrations, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100 in plasma samples were measured (n = 5 mice per group). FIG. 1C It provides Plats fed a Western diet (WD) with AAV8-TBG-cre (Cre) or AAV8-TBG-GFP (GFP) treatment. fl / fl A set of images showing the results for the mice. Plat fl / fl Mice were treated with Cre or GFP and then fed WD for 8 weeks. Liver tPA protein was measured, and total cholesterol and apoB-100 concentrations in plasma samples, as well as FPLC profiles of cholesterol, triglycerides, and apoB-100, were determined. Cholesterol in the VLDL fraction is shown in magnified plots (n = 6 mice per group). FIG. 1D This is a set of figures and immunoblot images showing the results of treating human primary hepatocytes with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA for 24 hours. ApoB in the cell culture medium was quantified by immunoblotting. VLDL fractions were separated by ultracentrifugation, and the concentrations of cholesterol and triglycerides in the VLDL fractions were determined. FIG. 1E This is a set of figures and immunoblot images showing the results of treating McA-RH7777 cells with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA for 24 hours. VLDL was isolated from the culture medium by ultracentrifugation, and cholesterol and triglyceride concentrations in VLDL were determined. apoB in the cell culture medium was measured by immunoblotting. Data are shown as mean ± SEM; *P < 0.05 according to a two-tailed Student's t-test.

[0020] FIG. 2A to FIG. 2K This demonstrates tPA-restricted apoB lipidation in the endoplasmic reticulum (ER). FIG. 2AWild-type (WT) mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 randomized control (scr) and then fed a Western diet (WD) for 14 weeks. VLDL secretion was assessed by intraperitoneal (ip) injection of P407 in mice. Plasma triglyceride concentrations were measured (n = 4 to 5 mice per group). FIG. 2B WT mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 randomized control (scr) and then fed WD for 14 weeks. VLDL secretion was assessed by intraperitoneal injection of P407 in mice. Plasma apoB concentration was measured by ELISA (n = 4 to 5 mice per group). FIG. 2C Ldlr was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed a WD diet for 8 weeks. VLDLs were isolated by ultracentrifugation and observed by transmission electron microscopy. The diameter of VLDLs (n = 100 per group) was measured and analyzed using Image-Pro Plus 10.0. Scale bar, 100 nm. FIG. 2D Ldlr was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed WD for 8 weeks (n = 9 to 10 mice per group). VLDLs were isolated by ultracentrifugation and VLDL diameters were measured by dynamic light scattering. FIG. 2E Ldlr was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed WD for 8 weeks (n = 9 to 10 mice per group). VLDL was isolated by ultracentrifugation and the ratio of triglycerides to apoB-100 was determined (n = 9 to 10 mice per group). FIG. 2F Holo-tPA-KO mice were treated with AAV8-TBG-Plat (tPA) or AAV8-TBG-lacZ (LacZ) and then fed a normal diet for 8 weeks. The concentrations of VLDL cholesterol, LDL cholesterol, and apoB-100 in plasma samples were measured (n = 6 mice per group). FIG. 2G Human primary hepatocytes were transduced using plasmids encoding tPA and a C-terminal HA tag (tPA-HA; SEQ ID NO: 27) or GFP (SEQ ID NO: 29). 48 hours later, cells containing […] were transduced. 3apoB secretion was measured in a medium containing H]-leucine, and in the absence of [ 3 The cells were tracked in a culture medium containing H]-leucine for 3 hours, and then the radioactivity associated with apoB in the cell culture medium was quantified by scintillation counting. FIG. 2H Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or scrambled (scr) RNA for 24 hours. [Using [ 3 H] labeling measures apoB secretion, such as FIG. 2G As shown. The radioactivity associated with apoB in cell culture medium was quantified by scintillation counting. FIG. 2I Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or scrambled (scr) RNA for 24 hours. VLDLs were separated by ultracentrifugation, and VLDL diameters were measured by dynamic light scattering. FIG. 2J Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or scrambled RNA for 24 hours. VLDL was separated by ultracentrifugation, and the ratio of triglycerides to apoB-100 was determined. FIG. 2K Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or scrambled RNA for 24 hours. Endoplasmic reticulum (ER) fractions were isolated, and proteins were extracted from the ER. ApoB lipoproteins extracted from the ER were further separated by density gradient ultracentrifugation and divided into six fractions with progressively increasing density, from fraction 1 to fraction 6. ApoB from each fraction was measured by Western blotting. Data are shown as mean ± SEM; *P < 0.05 according to a two-tailed Student's t-test.

[0021] FIG. 3A to FIG. 3J The tPA-blocked apoB-VLDL assembly is shown. FIG. 3A Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA for 24 hours. ApoB and MTP in cell lysates (input) and anti-MTP immunoprecipitates (IP: MTP) were determined by Western blotting. FIG. 3B Human primary hepatocytes were treated for 24 hours with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA (Group 1) or tPA mRNA (si-tPA) (Groups 2 and 3). Microsomal fractions were isolated, and neutral lipid transfer activity was measured with DMSO (Groups 1 and 2) or without DMSO but with CP-346086 (10 nM) (MTP inhibitor) (Group 3). FIG. 3CHuman primary hepatocytes were transduced for 48 hours with plasmids encoding tPA (tPA-HA; SEQ ID NO: 27) or GFP with a C-terminal HA tag (SEQ ID NO: 29). ApoB and MTP in cell lysates (injected) and anti-MTP precipitates (IP: MTP) were determined by Western blotting. FIG. 3D Human primary hepatocytes were transduced for 48 hours using plasmids encoding tPA-HA (SEQ ID NO: 27) or GFP (SEQ ID NO: 29). Microsomal fractions were isolated, and neutral lipid transfer activity was measured. FIG. 3E The effect of recombinant human tPA on lipid transfer from donor vesicles to human LDL was determined. FIG. 3F Human primary hepatocytes were transduced for 48 hours using plasmids encoding tPA-HA (SEQ ID NO: 27) or GFP (SEQ ID NO: 29). The apoB and tPA of cell lysates (infused) and anti-HA immunoprecipitate (IP: HA) were measured. FIG. 3G : The apoB-tPA interaction in human primary hepatocytes was measured using a neighbor-to-neighbor connectivity assay. Scale bar, 10 μm. FIG. 3H Subcellular localization of tPA and apoB in human primary hepatocytes was measured using confocal microscopy and immunofluorescence imaging. Scale bar, 10 μm. FIG. 3I Human primary hepatocytes were transduced for 48 hours using plasmids encoding 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 retention signal sequence (tPA-KDEL (SEQ ID NO: 3)), or GFP. [The text abruptly ends here, likely due to an incomplete translation or missing information.] 3 H] labeling is used to measure apoB secretion, such as FIG. 2G and 2H As shown. FIG. 3J The effects of recombinant wild-type tPA (tPA-WT) or the enzymatically inactive mutant of tPA (tPA-S513A) on lipid transfer from donor vesicles to human LDL were determined. Data are shown as mean ± SEM; *Successfully analyzed by a two-tailed Student's t-test. FIG. 3D ) or one-way ANOVA, followed by Dunnett's test ( FIG. 3B , 3E (3I, 3J), P < 0.05.

[0022] FIG. 4A to FIG. 4I The interaction between the Kringle 2 (K2) domain of tPA and the N-terminus of apoB is shown. FIG. 4AThe interaction between LDL and recombinant human wild-type tPA (tPA-WT) or enzymatically inactive tPA-S513A was measured using a solid-binding assay. Binding of tPA-WT or tPA-S513A to wells without LDL was also measured under the same conditions as controls. FIG. 4B The interaction between recombinant human tPA and purified human MTP complex was measured using a solid-binding assay. FIG. 4C The ability of tPA to inhibit the binding of MTP to LDL was measured using a solid-binding assay. FIG. 4D : Surface plasmon resonance was used to measure the interaction between recombinant human tPA and LDL. FIG. 4E The solid-binding assay was used to test whether the anti-apoB N-terminal antibody (1D1) and control IgG (produced against the β3 domain of apoB) blocked the binding between human recombinant tPA and LDL. FIG. 4F Human primary hepatocytes were transduced with plasmids encoding wild-type tPA (tPA-WT (SEQ ID NO: 1)), tPA mutants lacking the K2 domain (tPA-Δ-K2 (SEQ ID NO: 19 and 20)), or tPA with mutations at the lysine binding site of the K2 domain (tPA-D236, 238N (SEQ ID NO: 21 and 22)). [...] 3 H] labeling is used to measure apoB secretion, such as FIG. 2G and 2H As shown. FIG. 4G The solid-binding assay was used to test whether antibodies targeting the tPA-K2 domain interfered with the interaction between recombinant human tPA and purified LDL. FIG. 4H The solid-state combination assay was used to measure whether tranexamic acid (TXA) interfered with the interaction between recombinant human tPA and purified LDL. FIG. 4I Schematic diagram depicting the interaction between the N-terminus of apoB and the K2 domain of tPA. This figure was generated using biorender.com. Data are shown as mean ± SEM; * by one-way ANOVA followed by Dunningt's test, P < 0.05, not significant (P ≥ 0.05).

[0023] FIG. 5A to FIG. 5J The study showed that PAI-1 isolates tPA from apoB, leading to increased VLDL assembly in hepatocytes. FIG. 5A : To determine tPA and PAI-1 in human primary hepatocyte lysate (infused) and anti-PAI-1 immunoprecipitate (IP: PAI-1) by immunoblotting. FIG. 5B : The tPA-PAI-1 interaction in human primary hepatocytes was measured using a neighbor-to-neighbor connectivity assay. Scale bar, 10 μm.FIG. 5C Human primary hepatocytes were treated for 6 hours with either 0.4 mM oleate (oleate group) combined with fatty acid-free BSA or fatty acid-free BSA alone (mediator control). tPA of cell lysates was determined by immunoblotting using the Jess Simple Western blotting system. FIG. 5D Human primary hepatocytes were treated with 0.4 mM oleate complexed with fatty acid-free BSA (oleate group) or fatty acid-free BSA alone (mediator control). The concentration of tPA-PA-1 complex in cell lysates and tPA without PAI-1 was determined by ELISA. FIG. 5E Human primary hepatocytes were treated with siRNA targeting PAI-1 mRNA (si-PAI1) or out-of-order RNA, and then incubated in medium containing 0.4 mM oleate (oleate group) in combination with fatty acid-free BSA or fatty acid-free BSA alone (mediator control). The concentration of PAI-1-free tPA in cell lysates was determined by ELISA. FIG. 5F Human primary hepatocytes were treated with siRNA targeting PAI-1 mRNA (si-PAI1) or out-of-order RNA, and then incubated in medium containing 0.4 mM oleate (oleate group) with BSA complexed with fatty acids-free BSA or BSA without fatty acids (mediator control). 3 H] labeling is used to measure apoB secretion, such as FIG. 2G and 2H As shown. FIG. 5G Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA), PAI-1 mRNA (si-PAI1), or disordered RNA. [The text abruptly ends here.] 3 H] labeling is used to measure apoB secretion, such as FIG. 2G and 2H As shown. FIG. 5H The interaction between LDL and recombinant human tPA or tPA-PAI-1 complex was measured using solid-binding assays. FIG. 5I The effects of recombinant human tPA and tPA-PAI-1 complex on lipid transfer from donor vesicles to human LDL were determined. FIG. 5J Wild-type mice fed a normal diet were withheld from food for 5 hours and then euthanized at 0, 1, 2, and 6 hours after oral gavage with olive oil. The concentration of tPA without PAI-1 in liver lysates was determined by ELISA. Data are shown as mean ± sem; * analyzed by one-way ANOVA followed by Dunningt's test, P < 0.05 ( ). FIG. 5D to 5G 5I, 5J). ns, not significant (P ≥ 0.05).

[0024] FIG. 6A to FIG. 6J The results showed that PAI-1 deficiency leads to decreased plasma apoB and apoB cholesterol concentrations in mice and humans. FIG. 6A Serpine 1fl / 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. Plasma total cholesterol concentration was measured (n=6 mice per group). FIG. 6B 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. Plasma apoB was measured by immunoblotting (n=6 mice per group). FIG. 6C Serpine 1fl / 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. Plasma samples were fractionated by plasma plasma chromatography (FPLC) and cholesterol concentrations were determined. For the FPLC profile of cholesterol, cholesterol in the VLDL fraction was magnified in a smaller plot. (n=6 mice per group). FIG. 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. The concentration of PAI-1-free tPA in liver lysates was determined by ELISA (n=6 mice per group). FIG. 6E Serpine1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or control AAV8-TBG-GFP (control) and then fed a normal diet for 4 weeks. P407 was injected intraperitoneally into the mice to assess VLDL secretion (n=10 mice per group). FIG. 6F Serpine 1fl / fl mice were treated with AAV8-TBG-Cre (Cre) or control AAV8-TBG-GFP (control) and then fed a normal diet for 6 weeks. Mice were withheld from food for 5 hours and then euthanized at 0, 2, and 4 hours after oral gavage with olive oil. Plasma apoB-100 concentrations were measured by ELISA. FIG. 6G : Determine the concentrations of VLDL cholesterol, LDL cholesterol, and apoB-100 in plasma samples from SERPINE1-deficient individuals and unaffected age / sex / BMI-matched individuals from the same population (n=10 per group). FIG. 6H : Using ultracentrifugation to extract from the assembly FIG. 6GThe concentration of tPA was measured in VLDL isolated from the plasma of the subjects (n=10 per group). FIG. 6I Analysis of the assembly through dynamic light scattering FIG. 6G VLDL levels in the plasma of the subjects were measured (n=10 per group). The correlation between VLDL-associated tPA and VLDL diameter was calculated (n=10 per group). FIG. 6J : A schematic diagram depicting how tPA-PAI-1 interaction in hepatocytes determines VLDL assembly. In the absence of lipid stimulation, tPA interacts with apoB and inhibits MTP-apoB interaction in the ER, thereby limiting MTP-mediated apoB lipidation and VLDL assembly. When hepatocytes are loaded with lipids, PAI-1 sequesters free tPA from apoB and increases VLDL assembly. This figure was generated using biorender.com. Data are shown as mean ± sem; analyzed by a two-tailed Student's t-test (…). FIG. 6A , 6D 6E, 6F), paired Student t-test ( FIG. 6G , 6H ) or Pearson correlation analysis ( FIG. 6I Use this to calculate the P-value. * P < 0.05.

[0025] FIG. 7A to FIG. 7D The study demonstrated how regulating hepatocyte tPA expression alters plasma tPA levels in mice. FIG. 7A Ldlr was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed a Western diet for 8 weeks. Plasma tPA was measured by ELISA (n=9 to 10 mice per group). FIG. 7B Apoe was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed a Western diet for 8 weeks. Plasma tPA was measured by ELISA (n=5 mice per group). FIG. 7C C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 randomized control (scr) and then fed a Western diet for 8 weeks. FIG. 7D Treat Plat with AAV8-TBG-cre (Cre) or AAV8-TBG-GFP (GFP) fl / flMice were then fed 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 to 10 mice per group). Data are shown as mean ± sem; *P < 0.05 by two-tailed Student's t-test.

[0026] FIG. 8 This study demonstrates the increase in plasma apoB lipoprotein cholesterol and apoB in mice by silencing hepatocyte tPA. Wild-type mice were treated with either AAV8-H1-sh-tPA (sh-tPA) or an AAV8-H1 randomized control (scr) and then fed a Western diet for 8 weeks. Liver tPA protein was measured, and plasma samples were analyzed 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 magnified in a smaller plot. (n=6 mice per group). Data are shown as mean ± sem; *P < 0.05 by two-tailed Student's t-test.

[0027] FIG. 9A to FIG. 9C The study showed that silencing hepatocyte tPA does not alter the level of hepatic apoB mRNA in mice. FIG. 9A Ldlr was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed a Western diet for 8 weeks. Liver apoB mRNA was measured by real-time PCR. FIG. 9B Apoe was processed using AAV8-H1-shPlat (sh-tPA) or AAV8-H1 out-of-order control (scr). - / - Mice were then fed a Western diet for 8 weeks. Liver Apob mRNA was measured by real-time PCR. FIG. 9C C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 scrambled control (scr) and then fed a Western diet for 8 weeks. Hepatic Apob mRNA was measured by real-time PCR. Data are shown as mean ± sem; statistical analysis was performed by a two-tailed Student's t-test. No significant difference was observed (P ≥ 0.05).

[0028] FIG. 10A to FIG. 10B The study showed that silencing hepatocyte tPA did not alter plasma apoE and hepatic LDLR levels. FIG. 10AC57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 randomized control (scr) and then fed a Western diet for 8 weeks. Plasma apoE concentrations were measured by ELISA. FIG. 10B C57BL / 6J mice were treated with AAV8-H1-shPlat (sh-tPA) or AAV8-H1 randomized control (scr) and then fed a Western diet for 8 weeks. Hepatic LDLR was measured by Western blotting. Data are shown as mean ± sem; statistical analysis was performed by two-tailed Student's t-test. No significant difference was found in ns (P ≥ 0.05).

[0029] FIG. 11A to FIG. 11D The study showed that silencing hepatocyte tPA did not alter apoB mRNA levels. FIG. 11A Human primary hepatocytes were treated with siRNA targeting tPA mRNA (si-tPA) or disordered RNA for 24 hours. Cellular tPA mRNA was measured by real-time PCR. FIG. 11B Human primary hepatocytes were treated with siRNA targeting tPA (si-tPA) or out-of-order RNA for 24 hours. Cellular apoB mRNA was measured by real-time PCR. FIG. 11C McA-RH7777 cells were treated with siRNA targeting tPA (si-tPA) or out-of-order RNA for 24 hours. Cellular tPA mRNA was measured by real-time PCR. FIG. 11D McA-RH7777 cells were treated with siRNA targeting tPA (si-tPA) or out-of-order RNA for 24 hours. Cellular apoB mRNA was measured by real-time PCR. Data are shown as mean ± sem; *P < 0.05 was not significant (P ≥ 0.05) according to a two-tailed Student's t-test.

[0030] FIG. 12 This study demonstrated that silencing tPA increases apoB secretion in McA-RH7777 cells. McA-RH7777 cells were treated with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA for 24 hours. [The text abruptly ends here, likely due to an incomplete translation or a formatting error.] 3 H] labeling is used to measure apoB secretion, such as FIG. 2G and 2H Data are shown as mean ± sem; *P < 0.05 was not significant (P ≥ 0.05) after two-tailed Student's t-test or two-way ANOVA followed by Dunningt's test.

[0031] FIG. 13This study demonstrated that incubation of primary human hepatocytes with recombinant tPA did not alter apoB levels in the cell culture medium. Primary human hepatocytes were incubated in medium containing recombinant human tPA (10 ng / ml) for 1, 6, and 24 hours, respectively. Hepatocytes incubated in medium without added recombinant tPA served as a control. ApoB-100 in the cell culture medium was measured by ELISA. VLDL was separated by ultracentrifugation, and cholesterol and triglycerides in VLDL were measured. Data are shown as mean ± sem; *Statistical analysis was performed by two-way ANOVA followed by Dunningt's test. No significant difference was observed (P ≥ 0.05).

[0032] FIG. 14 This study demonstrates that silencing tPA increases MTP-dependent lipid transfer in the microsomal fraction of McA-RH7777 cells. McA-RH7777 cells were treated with siRNA targeting tPA mRNA (si-tPA) or out-of-order RNA for 24 hours. Neutral lipid transfer activity of the microsomal fraction was measured with or without 10 nMCP-346086 (an MTP inhibitor). Data are shown as mean ± sem; *P < 0.05 was determined by two-tailed Student's t-test or one-way ANOVA followed by Dunningt's test.

[0033] FIG. 15A to FIG. 15B The purification of recombinant human tPA and LDL by size exclusion chromatography is shown. FIG. 15A Further purification of human LDL separated by ultracentrifugation using size exclusion chromatography. FIG. 15B Recombinant human tPA purified by affinity chromatography was further purified by size exclusion chromatography.

[0034] FIG. 16 The interaction between tPA and defatted apoB-100 is shown. Human recombinant tPA and LDL were purified by size exclusion chromatography. The interaction between defatted apoB-100 and recombinant tPA was determined using solid-state binding. Data are shown as mean ± sem.

[0035] FIG. 17 This study demonstrates the partial reduction of the interaction between tranexamic acid (tPA) and solid-bound LDL. The interaction between LDL and recombinant human tPA in the presence of tranexamic acid was measured using a solid-bound assay. Data are shown as mean ± sem; * by one-way ANOVA followed by Dunningt's test, P < 0.05, not significant (P ≥ 0.05).

[0036] FIG. 18Silencing tPA PAI-1 reduced apoB secretion in McA-RH7777 cells. McA-RH7777 cells were treated with siRNA targeting PAI-1 mRNA (si-PAI1) or out-of-order RNA for 24 hours. Cells were treated with 0.4 mM oleate compounded with fatty acid-free BSA (oleate group) or fatty acid-free BSA alone (oleate-free group). […] 3 [H] markers were used to measure apoB secretion, as shown in Figure 2. Data are presented as mean ± sem; *P < 0.05 was not significant (P ≥ 0.05) according to a two-tailed Student's t-test.

[0037] FIG. 19 Oral administration of olive oil via gavage did not alter the levels of total tPA and total PAI-1 in the liver of mice. C57BL / 6 mice fed a normal diet were fasted for 5 hours and sacrificed at 0, 1, 2, and 6 hours after oral administration of olive oil. The concentrations of tPA and PAI-1 in liver lysates were determined by ELISA. Data are shown as mean ± sem; *P < 0.05 was not significant (P ≥ 0.05) according to two-tailed Student's t-test or two-way ANOVA followed by Dunningt's test.

[0038] FIG. 20A to FIG. 20B The study showed that silencing tPA in hepatocytes increased apo(a) levels in the cell culture medium. Apo(a) levels in the cell culture medium were measured by ELISA. FIG. 20A This is a graph showing the apo(a) levels in human primary hepatocytes treated with disordered siRNA or si-tPA. FIG. 20B This is a graph showing the apo(a) levels in HepG2 cells transduced with an apo(a) expression plasmid and then treated with out-of-order siRNA or si-tPA.

[0039] FIG. 21A to FIG. 21C The study showed that tPA knockout in intestinal epithelial cells increased plasma cholesterol levels in mice. Treatment of intestinal epithelial cell tPA knockout mice (e-tPA-KO) or littermate controls (controls) was performed to measure: FIG. 21A The tPA mRNA levels in intestinal epithelial cells and liver were normalized to Rplp0 mRNA. FIG. 21B Total cholesterol in blood plasma; and FIG. 21C Plasma cholesterol as analyzed by FPLC (control, pooled plasma from 8 mice; e-tPA-KO, pooled plasma from 11 mice).

[0040] FIG. 22A to FIG. 22BThis study demonstrates that tPA in intestinal epithelial cells promotes chylomicron production in knockout mice. Intestinal epithelial cell tPA knockout mice (e-tPA-KO) or littermate controls (controls) received oral administration of olive oil via gavage. FIG. 22A Time course of plasma triglyceride levels after gavage (n=9 for control (bottom line in the figure) and n=11 for e-tPA-KO (top line in the figure)). FIG. 22B Immunoblot analysis was performed on the apoB-48 level in the isolated chylomicron fraction and the albumin in the plasma collected 2 hours after gavage.

[0041] FIG. 23A to FIG. 23B Expression of the tPA-K2 domain was shown to reduce apoB secretion in cultured hepatocytes. McA-RH7777 cells were treated with a plasmid encoding tPA-K2 (SEQ ID NO: 31) (SEQ ID NO: 33) or with a GFP control. FIG. 23A : Detection of tPA-K2 domain expression by immunoblotting. FIG. 23B The apoB level in cell culture medium was detected by immunoblotting.

[0042] FIG. 24A to FIG. 24B Expression of the tPA-K2 domain was shown to reduce plasma lipids in mice. Male C57 wild-type mice were fed a high-fat diet for 4 weeks. Mice were intravenously injected (1 mg / kg body weight) with an LNP carrying mRNA or a control luciferase, which encodes the tPA-K2 domain with an endoplasmic reticulum localization motif. Blood was collected at 6, 24, and 48 hours post-injection and before injection. Plasma triglycerides (P2P) were measured. FIG. 24A ) and cholesterol ( FIG. 24B )level.

[0043] FIG. 25 The expression of the tPA-K2 domain was shown not to alter the bleeding time in mice. Male C57 wild-type mice were fed a high-fat diet for 5 weeks. Mice were intravenously injected (1 mg / kg body weight) with an LNP carrying mRNA or a control luciferase, which encodes the tPA-K2 domain with an endoplasmic reticulum localization motif. Tail bleeding was tested 24 hours after LNP injection. Detailed Implementation

[0044] This disclosure is not limited to the specific embodiments described herein, which are intended as separate illustrations of a single aspect of this disclosure. Not all various embodiments of this disclosure will be described herein. Many modifications and variations can be made to this disclosure without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. In addition to those listed herein, functionally equivalent methods and apparatuses within the scope of this disclosure will be apparent to those skilled in the art based on the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. This disclosure is limited only by the terms of the appended claims and the full scope of equivalents obtained by granting those claims.

[0045] It should be understood that this disclosure is not limited to any particular use, method, reagent, compound, composition, or biological system, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0047] SUMMARY

[0048] Statins or PCSK9 inhibitors (both of which lower low-density lipoprotein (LDL) by enhancing LDL receptor (LDLR)-mediated LDL clearance) are currently used to prevent or treat cardiovascular disease. However, despite their ability to lower LDL, these treatments only slightly alter the levels of other atherosclerotic apolipoprotein B (apoB) containing apolipoproteins, such as intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), lipoprotein(a) [Lp(a)], chylomicrons, and chylomicron residues.

[0049] apoB (a large amphiphilic protein) is the structural scaffold for the formation of VLDL and chylomicrons [8]. VLDL and chylomicrons are assembled in hepatocytes and intestinal epithelial cells, respectively, by incorporating triglycerides, cholesterol esters, and phospholipids onto apoB to form globular particles [9]. This process (called apoB lipogenesis) depends on lipid availability and the neutral lipid transporter microsomal triglyceride transporter (MTP) [10, 11]. MTP binds to apoB in the endoplasmic reticulum (ER) of hepatocytes and transfers lipids to apoB

[10] . When MTP and / or lipids are unavailable, VLDL and chylomicrons cannot be synthesized, and newly translated apoB is targeted for degradation [12, 13]. Although the important role of MTP in apoB lipogenesis has been established, little is known about apoB-MTP interactions and the regulation of MTP-mediated lipid transfer to apoB.

[0050] Tissue plasminogen activator (tPA) is a serine protease that plays a key role in fibrinolysis (the process of thrombus lysis)

[14] . Previous studies have shown that low plasma tPA activity is associated with a higher risk of atherosclerotic CVD [15-17], but whether reduced fibrinolysis in this case leads to CVD remains unclear. Another plausible mechanism linking low tPA to CVD is elevated plasma cholesterol, as this is observed in individuals 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 the inventors’ recent research that hepatocytes are an important source of tPA [21,22], the inventors sought to identify a possible link between tPA and the assembly and secretion of apoB lipoproteins in hepatocytes and intestinal epithelial cells. The inventors’ research shows that endogenous hepatocyte tPA limits VLDL and chylomicron production by directly interacting with apoB, interrupting the interaction between apoB and MTP, and thus impairing MTP-dependent neutral lipid transfer and apoB lipidation.

[0051] Plasminogen activator inhibitor 1 (PAI-1), encoded by the SERPINE1 gene, is a key serine protease inhibitor of tPA and is also expressed in hepatocytes. In this disclosure, the inventors demonstrate that PAI-1 binds to tPA within hepatocytes and eliminates the role of tPA in limiting VLDL assembly. Furthermore, the hepatocyte PA1-1 / tPA axis regulatory pathway for VLDL assembly is physiologically relevant to postprandial lipid load-related VLDL production. These findings suggest novel therapeutic strategies for reducing the production of atherogenic apoB lipoproteins.

[0052] The inventors of this technology discovered that tPA directly interacts with apoB and prevents neutral lipids from being transferred to apoB via MTP, thereby leading to the degradation of apoB and thus reducing the level of atherogenic apolipoproteins.

[0053] Furthermore, the inventors of this technology have 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 essential for tPA to reduce apoB secreted by hepatocytes. See, for example... FIG. 3I , 3J Figure 4A shows that the expression of the protease-deficient tPA S513A mutant in hepatocytes still leads to reduced apoB secretion in conditional tPA knockout animals.

[0054] Recombinant tPA (also known as rtPA and ACTIVASE) is used for its "thrombus-breaking" activity (which helps dissolve blood clots) and is used to treat heart attacks, strokes, and blood clots in the lungs. However, these functions depend on the serine protease activity of tPA.

[0055] The disclosure of this technology is based, in part, on the discovery of the tPA-K2 peptide of this technology—which, despite lacking serine protease and proteolytic activity, is effective in methods for reducing apoB secretion and lowering plasma triglyceride and total cholesterol levels when administered to subjects in need. Furthermore, because the tPA-K2 fragment of this technology (e.g., containing the amino acid sequence listed in SEQ ID NO. 7) lacks serine protease and proteolytic activity, administration of the peptide of this technology does not increase the risk of bleeding in subjects. Examples provided herein demonstrate that expression of a recombinant tPA-K2 peptide containing only the tPA Kringle 2 domain and endoplasmic reticulum localization sequence can reduce apoB secretion from hepatocytes (…). FIG. 23A to FIG. 23B ), and reduced plasma lipid levels in mouse models in vivo ( FIG. 24A to FIG. 24B However, it did not change the bleeding time in mice. FIG. 25 These data support the usefulness of compositions comprising the recombinant tPA-K2 polypeptide (or a nucleic acid encoding it, such as cDNA or mRNA) in methods for treating diseases or symptoms associated with elevated plasma apoB lipoprotein levels. In some embodiments, compositions of this technology are useful in methods for reducing any one or more of plasma VLDL, IDL, LDL, Lp(a), chylomicrons, chylomicron residues, triglycerides, or total cholesterol levels.

[0056] COMPOSITIONS

[0057] In this disclosure, a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) polypeptide or a nucleic acid (e.g., mRNA, cDNA) encoding therein is provided. 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 a tissue plasminogen activator kringle 2 domain, a fragment of the kringle 2 domain, a fragment thereof, or a nucleic acid encoding therethe, wherein the recombinant polypeptide has a sequence identity of less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, or less than about 10% with full-length wild-type human tPA (SEQ ID NO: 1). Therefore, this technology provides a recombinant tPA-K2 polypeptide having 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 amino acid sequences to those described in any of SEQ ID NO: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. Recombinant tPA-K2 polypeptides suitable for the methods described herein also include variants, including polypeptides having amino acid alterations (e.g., amino acid substitutions, deletions, or additions) compared to the amino acid sequence of any tPA-K2 polypeptide described herein. Such sequence variant proteins are suitable for the methods described herein, provided that the altered amino acid sequence retains sufficient biological activity to function in the compositions and methods described herein. Where amino acid substitutions are made, the substitutions can be conserved amino acid substitutions. Among common, naturally occurring amino acids, for example, “conserved amino acid substitution” is illustrated by substitution between amino acids in 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.

[0058] This technology also provides nucleic acids (e.g., mRNA, cDNA) encoding recombinant tPA-K2 polypeptides, such as polynucleotides having a nucleic 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 100% identical to the nucleic acid sequence encoding the polypeptide described in any of SEQ ID Nos. 2, 4, 6, 8, 10, 15, 16, 32, 34, 36, 38, 39, or 40, or its complement, or identical to 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, this technology provides an RNA transcript encoded by any one of SEQ ID NO: 2, 4, 6, 10, 15, 16, 34, 36, 38, or 39. In some embodiments, when administered to a subject, the nucleic acid is able to reduce apoB lipoprotein blood (e.g., plasma or serum) levels.

[0059] Exemplary recombinant tPA-K2 peptides (or nucleic acids encoding them) of this technology include, for example, SEQ ID NO: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. In some embodiments, the recombinant tPA-K2 peptides (or nucleic acids encoding them) of this technology comprise endoplasmic reticulum (ER) localization sequences. A non-limiting example of an ER localization sequence is KKXX (where “K” represents lysine and “X” can be any amino acid). Another non-limiting example of an ER localization sequence is KDEL (SEQ ID NO: 11). Thus, for example, SEQ ID NO: 7 is a recombinant tPA-K2 peptide comprising the sequences SEQ ID NO: 5 and KDEL (SEQ ID NO: 11). In some embodiments, the recombinant tPA-K2 peptides (or nucleic acids encoding them) of this technology include, for example, SEQ ID NO: 9 (which is a tPA S513A mutant lacking serine protease function). In some embodiments, the tPA S513A mutant further comprises an ER-localizing sequence, such as KDEL (SEQ ID NO: 11), and contains an amino acid sequence as listed in SEQ ID NO: 35. In some embodiments, the recombinant tPA-K2 polypeptide (or the nucleic acid encoding it) of this technology comprises, for example, SEQ ID NO: 7 (which comprises SEQ ID NO: 5 and KDEL (SEQ ID NO: 11)). In some embodiments, the recombinant tPA-K2 polypeptide may comprise a sequence that is 100% identical, at least about 99% identical, at least about 98% identical, at least about 97% identical, at least about 96% identical, or at least about 95% identical to the full-length wild-type tPA sequence (SEQ ID NO: 1), but further comprises one or more additional heterologous amino acid sequences.

[0060] The recombinant tPA-K2 peptide of this technology can be constructed using methods well known in the art. As an example, but not limited to, PCR can be used to generate the nucleotide sequence encoding the recombinant tPA-K2 peptide. In some embodiments, the tPA-K2 sequence includes a C-terminal endoplasmic reticulum (ER) localization sequence that conforms to the reading frame, such as a sequence encoding KDEL (e.g., SEQ ID NO: 12). In some embodiments, the recombinant tPA-K2 peptide of this technology may be referred to as a "fusion protein" comprising the tPA-K2 peptide or a fragment thereof, a linker, and an ER localization motif. In some embodiments, the recombinant tPA-K2 peptide of this technology has the following non-limiting formula: tPA-K2 peptide (or a fragment thereof) – X – ER localization motif (Formula I), where – X – is the linker. In some embodiments, – X – is one or more amino acids. In some embodiments, the amino acid sequence of the linker comprises GGGGS (SEQ ID NO: 41).

[0061] In some embodiments, the recombinant tPA-K2 peptide of this technology comprises a protein tag domain containing 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 multiple histidine tags. Exemplary tags include, but are not limited to, HA (hemagglutinin) tags, histidine tags (e.g., 6-histidine tags), FLAG tags, CBP (calmodulin-binding peptide), CYD (covalently but dissociatively soluble NorpD peptide), StrepII, or HPC (heavy chain of protein C) or more. In some embodiments, the length of the protein tag domain comprises about 10 to 20 amino acids. In some embodiments, the length of the protein tag domain comprises 2 to 40 amino acids, for example, 6 to 20 amino acids. 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) can be used to facilitate the immunoprecipitation, purification, and / or detection of the expressed protein, and that the inclusion of tags in no way implies a limitation on the recombinant tPA-K2 polypeptide or the nucleic acid encoding it or their function as described herein.

[0062] In some embodiments, the recombinant tPA-K2 polypeptide (or the nucleic acid encoding it) of this technology is a therapeutic polypeptide (or nucleic acid) and can be used to inhibit the interaction between MTP and apoB, block the binding of PAI-I to endogenous tPA, inhibit the formation of VLDL and chylomicrons, enhance the degradation of apoB, and reduce the likelihood of CVD and related diseases and conditions (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, or hypertension) associated with VLDL, IDL, LDL, Lp(a), chylomicrons, and chylomicron residues. The recombinant tPA-K2 polypeptide of this disclosure contains a tissue-type plasminogen kringle 2 domain, such as SEQ ID NO: 5 or a fragment thereof. The tPA kringle 2 domain may contain a sequence that is 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% identical to that in SEQ ID NO: 5. Without being bound by theory, the disclosed compositions 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 include a KDEL (SEQ ID NO:11) sequence that localizes tPA-K2 to the endoplasmic reticulum, such as NPDGDAKPWCHVLKNRRLTWEYKDEL (SEQ ID NO:14).

[0063] The recombinant polypeptide disclosed herein may also contain tPA with no serine protease activity, i.e., tPA is not fibrinolytic. As discussed above, serine protease activity is essential for "thrombus rupture" or fibrinolytic activity of tPA. Loss of serine protease activity in the recombinant polypeptide can be accomplished by mutation of amino acid residues essential to the serine protease function of tPA, for example, serine 513 can be mutated to alanine (S513A), where the 513 position is relative to SEQ ID NO: 1. Alternatively, the entire peptidase domain of tPA or key portions of the peptidase domain of tPA can be removed from the recombinant polypeptide. The peptidase domain of tPA contains amino acids 311 to 561, see SEQ ID NO: 1. Therefore, the recombinant polypeptide may contain the sequence of SEQ ID NO: 1 or the sequence that is 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% identical to SEQ ID NO: 1, having a suitable mutation (e.g., S513A) to disrupt the serine protease function of tPA, or a sequence that truncates SEQ ID NO: 1 to remove the peptidase domain (i.e., amino acids 311 to 561, see SEQ ID NO: 1).

[0064] The recombinant polypeptide may contain the same sequence as SEQ ID NO: 9 (tPA-S513A) or 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%.

[0065] The recombinant polypeptide may contain the same sequence as SEQ ID NO: 35 (tPA-S513A-KDEL) or 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%.

[0066] The recombinant polypeptide may contain SEQ ID NO: 7 (tPA-K2 KDEL) or a sequence that is 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% identical to SEQ ID NO: 7.

[0067] The recombinant polypeptide may contain SEQ ID NO: 5 (tPA kringle 2 domain) or a sequence that is 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% identical to SEQ ID NO: 5.

[0068] A tPA lacking a peptidase domain (i.e., lacking amino acids 311 to 516, see SEQ ID NO: 1) may contain the same sequence as SEQ ID NO: 30 or 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%.

[0069] In some embodiments, delivery of the disclosed polypeptide or the nucleic acid (mRNA, cDNA) encoding it can be efficiently facilitated by nanoparticles. Therefore, this disclosure further provides nanoparticles comprising a recombinant polypeptide or a nucleic acid encoding the recombinant polypeptide. The nanoparticles may comprise one or more polymers, such as poly(lactide) (PLA), poly(lactide-co-glycolic acid) (PLGA) copolymer, poly(ε-caprolactone) (PCL), and poly(amino acids), alginate, chitosan, gelatin, and albumin.

[0070] In some embodiments, the nanoparticles may comprise, for example, one or more lipids, such as 1,2-bis-(9Z-octadecenoyl)-sn-glycerol-3-phosphocholine (DOPC), which may suitably form liposomes. Exemplary additional or alternative lipids for incorporation into the nanoparticles are known in the art and include, but are not limited to, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP-PE), 306O i10Tetra(8-methylnonyl)3,3',3'',3'''-(((methylazadiyl)bis(propane-3,1diyl))bis(azatriyl))tetrapropionate; 9A1P9, (2-(dioctylammonium)ethyl)decyl phosphate; A2-Iso5-2DC18, 5,5-di((Z)-heptadecyl-8-en-1-yl)-1-(3-(pyrrolidone-1-yl)propyl)-2,5-dihydro-1H-imidazolium-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecyl ester); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide; β- Sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptane-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopenta[a]phenanthrene-3-ol; BAME-O16B, 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexadecyl)azadiyl)dipropionate bis(2-(dodecyldithio)ethyl) ester; BHEM-cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptane-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,17-tetradecylhydro-1H-cyclopenta[a]phenanthrene-3-ol); BAME-O16B, 3,3'-((((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexadecyl)azadiyl)dipropionate bis(2-(dodecyldithio)ethyl) ester; BHEM-cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-17-((((3S,8S,9S,10R,13R,14S,17R)-17-((((3S,8S,9S 7R)-10,13-Dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentan[a]phenanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-amine bromide; C12-200,1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)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)-heptadodecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate; DOPE, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine; DOSPA, 2,3-dioleoyloxy-N-[2-(sperminecarbamoylamino)ethyl]-N,N-dimethyl-1-propylamine trifluoroacetate; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA, 1,2-di-O-octadecenyl-3-trimethylammonium-propane;DSPC, 1,2-distearyl-sn-glycerol-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexa(oct-3-yl)9,9',9'',9''',9''',9''''-((((benzene-1,3,5-tricarbonyl)tri(azadiyl))tri(propane-3,1-diyl))tri(azatriyl))hexononate; 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(azatriyl))tetra(ethane-2,1-diyl)(9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12''Z)-tetra(octadecane-9,12-dienoate); PEG2000-DMG, 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol-2000; TT3, N; 1 N 3 N 5 -Tris(3-(eicosylamino)propyl)benzene-1,3,5-tricarboxamide.

[0071] POLYNUCLEOTIDES

[0072] As described above, this disclosure also provides a polynucleotide encoding the recombinant polypeptide of this disclosure. The polynucleotide of this disclosure may comprise a nucleotide sequence listed in any one or more of SEQ ID NO: 2, 4, 6, 8, 10, 15, 16, 32, 34, 36, 38, 39, or 40, or a sequence identical to 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%, or encoding SEQ ID No: The nucleotide sequence of the polypeptide described in any one of SEQ ID NO: 1, 3, 5, 7, 9, 13, 14, 30, 31, 35, or 37. The polynucleotides of this disclosure also cover RNA sequences encoded by any one of SEQ ID NO: 2, 4, 6, 10, 15, 16, 34, 36, 38, or 39. In some embodiments, the polynucleotide comprises the mRNA sequence listed in SEQ ID NO: 8. In some embodiments, the polynucleotide comprises the mRNA sequence listed in SEQ ID NO: 40. In some embodiments, the polynucleotide comprises the mRNA sequence listed in SEQ ID NO: 32.

[0073] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding tPA kringle2-KDEL as listed in SEQ ID NO: 34, or a sequence that is 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% identical to SEQ ID NO: 34.

[0074] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the tPA kringle 2 domain as listed in SEQ ID NO: 6, or a sequence that is 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% identical to that in SEQ ID NO: 6.

[0075] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding tPA S513A as listed in SEQ ID NO: 10, or a sequence that is 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% identical to that in SEQ ID NO: 10.

[0076] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding tPA-S513A-KDEL as listed in SEQ ID NO: 36, or a sequence that is 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% identical to SEQ ID NO: 36.

[0077] In some embodiments, the polynucleotides of this technology comprise a nucleotide sequence encoding a protein tag (e.g., an epitope tag) that facilitates immunoprecipitation, purification, and / or detection of the exogenously expressed protein. Those skilled in the art will understand that the inclusion of a nucleotide sequence encoding an epitope tag (such as an HA tag) can be used to facilitate immunoprecipitation, purification, and / or detection of the expressed protein, and that the inclusion of the tag in no way implies a limitation on the nucleic acid encoding the recombinant tPA-K2 polypeptide or its function as described herein.

[0078] The polynucleotide of this disclosure may further include at least one regulatory sequence operatively linked to a nucleotide sequence encoding the recombinant polypeptide of this disclosure. At least one regulatory region may include, for example, an enhancer, a promoter, or both an enhancer and a promoter. The regulatory region may include a tissue-specific promoter, such as a hepatocyte-specific promoter, such as the thyroxine-binding globulin (TBG) promoter (SEQ ID NO: 17). As used herein, when a polynucleotide is placed in a functional relationship with a second polynucleotide sequence, the polynucleotide is “operably linked” or “operably connected.”

[0079] It is envisioned that the disclosed polynucleotide can be used to express the disclosed polypeptide in cells (e.g., human cells (e.g., hepatocytes)) or in human subjects. Therefore, the disclosed polynucleotide may be, for example, a plasmid, a microcircle, and may contain a regulatory region that allows viral delivery of the polynucleotide.

[0080] For example, the disclosed polynucleotides may contain viral regulatory regions, such as adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences.

[0081] As used herein, the term "inverted terminal repeat" (ITR) sequence refers to a DNA sequence that side-joins a portion of the AAV genome that allows for insertion of the genome into a host cell. The ITR is the only cis-acting element required for AAV formation. Therefore, additional elements of the AAV genome can be trans-added to promote the formation of complete viral particles. Thus, the only portions of the viral genome that need to be included in the DNA carried by AAV are the 5' ITR and the 3' ITR. Suitable ITRs are known in the art and may include, for example, a 5' ITR or a 3' ITR having the sequence SEQ ID NO: 18.

[0082] INFECTIOUS PARTICLE

[0083] Suitable infectious particles are known in the art. For example, see Deverman et al. (Cre-dependent selection yields AAV variants for widespread gene transfer to the adultbrain, 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 by reference in their entirety. Those skilled in the art will be familiar with the elements and configurations required to construct the vector to encode the constructs described herein. Exemplary infectious 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 peptides and / or nucleic acids (e.g., mRNA, DNA) encoding the present technology to cells or tissues.

[0084] The disclosed infectious particles may comprise adeno-associated virus (AAV) containing the aforementioned viral regulatory region, which directs the expression of a product derived from the disclosed polynucleotide in a host cell, the polynucleotide being contained within or associated with the virus, wherein the host cell expresses the polynucleotide to generate the disclosed recombinant polypeptide. In some cases, the virus is selected from AAV types 1, 2, 3 (including 3A and 3B), 4, 5, 6, 7, 8, 9, 10, and 11. In some embodiments, the virus is an AAV8 virus. See, for example, FIG. 1A , 1B ; 2A to 2F; 6A to 6F.

[0085] PHARMACEUTICAL COMPOSITIONS

[0086] It is envisioned that the disclosed polynucleotides and recombinant peptides and / or nucleic acids encoding the recombinant peptides can be administered to a subject. Therefore, pharmaceutical compositions are provided in this disclosure. In some embodiments, the pharmaceutical composition comprises the disclosed polynucleotide, peptide, and / or nucleic acid encoding the peptide, and a pharmaceutically acceptable carrier or excipient, or one or more of the disclosed infectious particles and a pharmaceutically acceptable carrier or excipient. In some embodiments, the polynucleotides of this technology are formulated for delivery to a subject via infectious particles (e.g., via adeno-associated virus (AAV)) or lipid nanoparticles (LNPs).

[0087] As used herein, "effective amount" or "therapeutic effective amount" means the amount or dose of the disclosed composition, administered to a subject in a single or multiple doses, that provides the desired effect in the subject under diagnosis or treatment, such as a reduction in serum apoB lipoprotein, including 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 residues, or an improvement in one or more indicators related to cardiovascular disease, including, for example, a reduction in total cholesterol, a reduction in blood pressure, a reduction in fasting blood glucose, and a reduction in hemoglobin A1C, or more of these. As a person skilled in the art, an attending physician can readily determine the effective amount by using known techniques and by observing results obtained in similar circumstances. In determining the effective amount or dosage of the compound administered, the attending physician may consider a variety of factors, such as: the species of the subject; their size, age, and general health; the extent or severity of the disease or condition involved; the individual subject's response; the specific compound administered; the manner of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; the use of concomitant medications; and other relevant circumstances. The composition may also be administered in combination with one or more additional therapeutic compounds / agents (“co-administration”, where, for example, additional or other therapeutic agents may be administered simultaneously, sequentially, or by means of individual administration).

[0088] As will be apparent to those skilled in the art, the therapeutically effective dose of one or more of the recombinant tPA-K2 peptide and / or the nucleic acid encoding it (e.g., mRNA, cDNA) of this technology can vary depending on factors such as the subject's disease state, age, sex, weight, and general condition, as well as the ability of the recombinant tPA-K2 peptide and / or the nucleic acid encoding it (e.g., mRNA, cDNA) of this technology to elicit the desired response (subject's response to the therapy) in a particular subject. The dosage will also vary depending on factors such as general medical condition, medical history, disease type, and progression when the recombinant tPA-K2 peptide or its encoding nucleic acid (e.g., mRNA, cDNA) is delivered to the subject.

[0089] Generally, a typical dose of the recombinant tPA-K2 peptide or the nucleic acid (e.g., mRNA, cDNA) encoding it in this technology may contain about 0.01 mg / kg to about 100 mg / kg (such as about 0.05 mg / kg to about 50 mg / kg, and / or about 0.1 mg / kg to about 25 mg / kg, and / or about 1 mg / kg to about 10 mg / kg) of the disclosed recombinant peptide or the nucleic acid encoding it.

[0090] Typical doses of the disclosed pharmaceutical compositions containing viral particles may include amounts from about 5 μg of viral DNA to about 100 μg of viral DNA, or from about 10 μg of viral DNA to about 50 μg of viral DNA. The dose of the disclosed viral particles administered to a subject may include about 1 x 102 times the dose administered as a single dose to the subject. 10 Virus particles approximately 1 x 10 20 Viral particles, for example, 1 x 10 14 Up to 1 x 10 16 Viral particles, or approximately 1 x 10 14 Approximately 2 x 10 14 Approximately 3 x 10 14 Approximately 4 x 10 14 Approximately 5 x 10 14 Approximately 6 x 10 14 Approximately 7 x 10 14 Approximately 8 x 10 14 Approximately 9 x 10 14 Approximately 1 x 10 15 Approximately 2 x 10 15 Approximately 3 x 10 15 Approximately 4 x 10 15 Approximately 5 x 10 15 Approximately 6 x 10 15 Approximately 7 x 10 15 Approximately 8 x 10 15 Approximately 9 x 10 15 Approximately 1 x 10 16 Compositions comprising the disclosed recombinant peptides can be formulated in unit dosage forms, each dose containing, individually or in a single unit dosage form, about 1 to about 500 mg of each peptide (such as 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 discrete unit suitable as a single dose to a patient, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, associated with a suitable drug carrier, diluent, or excipient.

[0091] In some embodiments, this disclosure provides a composition comprising one or more of the recombinant tPA-K2 polypeptide of the present technology as a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof. In some embodiments, a mixture of two or more of the recombinant tPA-K2 polypeptide can be used as a therapeutic agent. The polypeptide can be synthesized by any of the methods well known in the art. In some embodiments, the polypeptide can be formulated as a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” means a salt prepared from a base or acid that is acceptable for administration to patients (such as mammals) (e.g., a salt that has acceptable mammalian safety for a given dosage regimen). However, it is understood that the salt does not need to be a pharmaceutically acceptable salt, such as a salt of an intermediate compound not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. Additionally, when the polypeptide contains both a basic moiety (such as an amine, pyridine, or imidazole) and an acidic moiety (such as a carboxylic acid or tetrazolium), an zwitterion can be formed and included in the term “salt” as used herein. Some of the compounds / peptides disclosed in this disclosure may exist in both unsolvated and solvated forms (including hydrated forms). Solvated forms may exist, for example, because it is difficult or impossible to remove all the solvent from the synthesized peptide. Generally, solvated forms are equivalent to unsolvated forms and are covered within the scope of this disclosure.

[0092] Certain compounds / peptides disclosed herein may exist in crystalline form, various crystalline forms, amorphous form, or any combination thereof. Certain compounds / peptides disclosed herein may exist in various tautomer forms. Certain compounds / peptides disclosed herein may exist in various salt forms or mixtures of salt forms. Generally, the physical forms of all compounds / peptides disclosed herein are equivalent to those contemplated for the uses of this disclosure and are intended to be within the scope of this disclosure.

[0093] This document also discloses combination therapies. The disclosed compositions (e.g., recombinant peptides and / or nucleic acids encoding them, nanoparticles, infectious particles, or pharmaceutical compositions) may be administered in combination with statins, PCSK9 inhibitors, plasminogen activator inhibitor 1 (PAI-1) inhibitors and ACE inhibitors, insulin sensitizers, female hormone replacement therapy, or any combination thereof, or in combination with currently known or unknown standard care for the treatment of dyslipidemia or hypercholesterolemia. Exemplary non-limiting PAI-1 inhibitors may include MDI-2268, PAI-039, TM5441, TM5275 sodium, TM5441 sodium, CDE-096, alecithin, draconin B, dapoxetine, stigmaziin, SK-216, astragalus, fendusal, AZ3976, and TM5007. Other PAI-1 inhibitors have been described in U.S. Patent Nos. 8,759,327; 9,096,501, 9,230,744, and 9,527,878, each of which is incorporated herein by reference in its entirety. Intravenous administration is an illustrative route of administration of the compounds used in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, oral, intrathecal, intracerebral, oral, or rectal routes. The route of administration may vary in any way and is limited by the physical properties of the compounds used and / or the convenience of the subject and / or caregiver.

[0094] As those skilled in the art will understand, suitable formulations include those applicable to more than one route of administration. For example, the formulation may be suitable for both intravenous and intramuscular administration. Alternatively, suitable formulations include those applicable only to one route of administration, and those applicable to one or more routes of administration but not to one or more other routes of administration. For example, a formulation may be suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, oral, and / or intrathecal administration but not for intracerebral administration.

[0095] The inert components and formulation methods of the pharmaceutical composition can be conventional. Formulation methods commonly used in pharmaceutical science can be used. All common types of compositions can be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, nasal sprays or powders, lozenges, suppositories, transdermal patches, and suspensions. Generally, depending on the required dosage and the type of composition to be used, the composition contains approximately 0.5% to approximately 50% of the compound. However, the amount of the compound is preferably defined as the “effective amount,” i.e., the amount of compound that provides the required dose to a patient with such therapeutic need. It is believed that the activity of the compounds used in the compositions and methods disclosed herein does not depend extensively on the properties of the composition, and therefore, the compositions can be selected and formulated primarily or solely for convenience and economy.

[0096] Pharmaceutical compositions and formulations comprising the recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., mRNA, cDNA) of this technology can be manufactured using conventional methods of mixing, dissolving, granulating, emulsifying, capping, embedding, or lyophilizing. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants (to facilitate formulation suitable for in vitro, in vivo, or ex vivo use). The composition can be combined with one or more additional active agents and can be formulated with pharmaceutically acceptable carriers, diluents, or excipients to produce compositions suitable for parenteral administration of the pharmaceuticals of this disclosure, including biological or veterinary compositions.

[0097] As will be understood by those skilled in the art, many types of formulations are possible. The specific type chosen depends on the chosen route of administration, as is recognized in the art. For example, systemic formulations are typically designed for administration by injection (e.g., intravenous). In some embodiments, systemic formulations are sterile.

[0098] A sterile injectable solution is prepared by incorporating a recombinant tPA-K2 peptide or the nucleic acid encoding it (e.g., mRNA, cDNA) into a desired amount of an appropriate solvent, along with various other components listed herein (if desired), and then sterilizing it using a suitable sterilization method. Typically, a dispersion is prepared by incorporating various sterile active ingredients into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, a preferred preparation method is vacuum drying and freeze-drying techniques, which produce powder from a previously sterile filtered solution, with any additional desired components added.

[0099] In some embodiments, the composition comprising the recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., mRNA, cDNA) may be prepared in an aqueous solution or in a physiologically compatible solution or buffer (such as Hanks's solution, Ringer's solution, mannitol solution or physiological saline buffer). In some embodiments, either the recombinant tPA-K2 polypeptide or the nucleic acid encoding it (e.g., mRNA, cDNA) may contain formulations such as suspending agents, stabilizers, penetrants or dispersants, buffers, lyophilization protectants or preservatives (such as polyethylene glycol), polysorbate 80, 1-dodecyl hexahydro-2H-azaphen-2-one (laurate), oleic acid, sodium citrate, Tris HCl, dextran, propylene glycol, mannitol, polysorbate polyethylene glycol sorbitol monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropanol, ethanol, sucrose, trehalose, and other such formulations commonly known in the art that may be used in any of the compositions disclosed herein. (Pramanick et al., Pharma Times 45(3):65-76 (2013)).

[0100] Capsules are prepared by mixing a compound with a suitable diluent and filling an appropriate amount of the mixture into a capsule. Commonly used diluents include inert powdered substances (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol, and sucrose), cereal powders, and similar edible powders.

[0101] Tablets are prepared by direct compression, wet granulation, or dry granulation. Their formulations typically include diluents, binders, lubricants, and disintegrants (in addition to compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives may also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, etc.). Natural and synthetic gums may also be used, including gum arabic, alginate, methylcellulose, polyvinylpyrrolidone, etc. Polyethylene glycol, ethylcellulose, and waxes can also be used as binders.

[0102] Tablets can be coated with sugar, for example, as a flavor enhancer and sealant. Compounds can also be formulated into chewable tablets by using large amounts of substances with pleasant flavors, such as mannitol. Instantly dissolving tablet-like formulations can also be used, for example, to ensure patients consume the dosage form and avoid some of the difficulties patients experience when swallowing solid objects.

[0103] Lubricants can be used in tablet formulations to prevent tablets and punchers from sticking to the mold. Lubricants can be selected from smooth solids (such as talc, magnesium stearate, and calcium stearate), stearic acid, and hydrogenated vegetable oils.

[0104] Tablets may also contain disintegrants. Disintegrants are substances that swell when wetted to break down the tablet and release compounds. They include starch, clay, cellulose, alginate, and gum. As further examples, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginate, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethyl cellulose can be used.

[0105] The composition can be formulated as an enteric-coated formulation, for example, to protect the active ingredient from the highly acidic contents of the stomach. Such formulations can be produced by coating solid dosage forms with a film of a polymer that is insoluble in acidic environments but soluble in alkaline environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0106] Transdermal patches can also be used to deliver compounds. A transdermal patch may include a resin composition in which the compound will dissolve or partially dissolve; and a thin film that protects the composition and keeps the resin composition in contact with the skin. Other, more complex patch compositions may also be used, such as those with a membrane perforated with multiple pores, through which the drug is pumped by osmosis.

[0107] As those skilled in the art will also understand, formulations can be prepared using materials (e.g., active excipients, carriers (such as cyclodextrins), diluents, etc.) that make the formulation suitable for administration to humans (e.g., purity). Alternatively, formulations can be prepared using materials that have purity and / or other properties that make the formulation suitable for administration to non-human subjects but not for administration to humans.

[0108] METHODS

[0109] As demonstrated herein, the disclosed polynucleotide, recombinant tPA-K2 fragment, or nucleic acid encoding the recombinant tPA-K2 fragment is effective in methods for reducing the assembly and secretion of atherogenic apoB lipoprotein from hepatocytes. FIG. 23A to FIG. 23B and FIG. 24A to FIG. 24BTherefore, in another aspect of this disclosure, a method is provided. The method comprises administering to a subject in need a therapeutically effective amount of any or more of the disclosed polynucleotide, recombinant polypeptide, or nucleic acid encoding the recombinant polypeptide. The subject may have hypercholesterolemia or hyperlipidemia, may be diagnosed with cardiovascular disease (e.g., atherosclerosis or arteriosclerosis), or may be diagnosed with type 2 diabetes.

[0110] The disclosed method can lower the levels of total cholesterol in a subject, or the levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), lipoprotein(a) [Lp(a)], chylomicrons, or chylomicron residues in the subject's blood (e.g., serum or plasma). Methods for measuring lipoproteins (e.g., VLDL, LDL, IDL, Lp(a), and HDL) are known in the art and are considered routine medical laboratory tests. The disclosed method can be used to treat hyperlipidemia in subjects in need.

[0111] In some embodiments, the method includes reducing plasma lipid and / or apoB levels in a subject who has experienced an atherosclerotic thrombotic event or is at increased risk of such an event. In some embodiments, the method includes administering a composition to the subject comprising a tPA polypeptide (e.g., any of full-length tPA and / or the recombinant tPA-K2 polypeptide disclosed herein or a nucleic acid encoding such polypeptide) or a polynucleotide comprising an endoplasmic reticulum localization motif (e.g., KDEL). In some embodiments, the composition is formulated for delivery to the subject as an LNP or an infectious particle (such as adeno-associated virus). In some embodiments, the subject has experienced a severe atherosclerotic thrombotic event or is at risk of such an event. In some embodiments, administering the composition to the subject reduces the subject's plasma lipid levels. In some embodiments, the subject is undergoing or has undergone therapy for atherosclerotic thrombosis or standard care treatment for thrombosis. In some embodiments, therapy for atherosclerotic thrombosis or standard care treatment for thrombosis may include one or more of anticoagulants, thrombolytics, catheter-directed 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 an anticoagulant. In some embodiments, the subject is undergoing, will undergo, or has undergone treatment with thrombolytic agents, anticoagulants, catheter-directed thrombolysis, and / or thrombectomy.

[0112] As used in this article, "hyperlipidemia" refers to an abnormally high level of any or all lipids or lipoproteins (e.g., fats, cholesterol, or triglycerides) in the blood. Hyperlipidemia encompasses hypercholesterolemia.

[0113] The inventors have demonstrated that administration of AAV containing a viral genome encoding the disclosed recombinant polypeptide (i.e., comprising the disclosed polynucleotide) effectively reduces VLDL and LDL in animals lacking tPA expression. See also FIG. 2F (Mice treated with AAV8-TBG-Plat encoding full-length rat tPA). Therefore, in other aspects of this disclosure, the method includes administering the disclosed pharmaceutical composition containing infectious particles to a subject in need.

[0114] The disclosed polynucleotides can be administered using any suitable delivery medium. For example, in some cases, the nucleic acid encoding the recombinant polypeptide of this technology can be incorporated into a delivery medium that drives nucleic acid expression. Examples of delivery media include, but are not limited to, non-viral vectors (e.g., plasmids (e.g., expression plasmids), liposomes, and polymeric vesicles) and viral vectors (e.g., adeno-associated virus (AAV) vectors, HSV vectors, and lentiviral vectors). In some embodiments, the disclosed polynucleotides can be delivered using an AAV vector. As used herein, the term “adeno-associated virus” (AAV) includes, but is not limited to, AAV 1, AAV 2, AAV 3 (including 3A and 3B), AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV, as well as any other AAV now known or later discovered. 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 are available in literature or public databases such as GenBank.

[0115] As examples, but not limited to, in some embodiments, subjects who received a therapeutically effective dose will exhibit a reduction in total cholesterol, or a reduction in serum levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), Lp(a), chylomicrons, or chylomicron residues, at 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 untreated control subjects.

[0116] Treatment can be administered multiple times a day, multiple times a week, 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 peptide.

[0117] METHODS OF MAKING RECOMBINANT tPA-K2 POLYPEPTIDES

[0118] This document also discloses a method for preparing recombinant tPA-K2 peptides. The disclosed peptides can be prepared according to known methods for generating recombinant peptides. For example, the polynucleotides of this disclosure can be introduced into suitable cells (e.g., animal cells, human cell lines) according to standard procedures (e.g., transfection), thereby expressing the polynucleotides to produce recombinant tPA-K2 peptides. The recombinant tPA-K2 peptides can be isolated and further purified according to methods known in the art, such as column purification, liquid chromatography, etc.

[0119] Furthermore, methods for generating rAAV viral particles are well known. See, for example, K. Fisher et al., J.Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745, which is incorporated herein by reference in its entirety.

[0120] KITS, SYSTEMS, AND PLATFORM

[0121] In another aspect of this disclosure, kits, systems, and platforms are provided. These kits, systems, and platforms may comprise, for example, one or more of the disclosed recombinant peptides, one or more of the disclosed polynucleotides, and / or one or more of the disclosed infectious particles. In some embodiments, the kit also includes instructions for use.

[0122] This document uses the following and several definitions listed throughout the application to describe the technology.

[0123] DEFINITIONS

[0124] The disclosed subject matter may be further described using the following definitions and terms. The definitions and terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0125] Unless the context clearly specifies otherwise, as used herein, the singular forms “a,” “an,” and “the” include the plural forms. For example, the term “substituent” should be interpreted as “one or more substituents” unless the context clearly specifies otherwise.

[0126] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by those skilled in the art and will vary to some extent depending on the context in which they are used. If the use of a term is unclear to those skilled in the art, then, taking into account the context in which it is used, “about” and “approximately” will mean at most plus or minus 10% of a particular term, and “substantially” and “significantly” will mean more than plus or minus 10% of a particular term.

[0127] As used herein, “administering” or “administration” of an agent (i.e., a therapeutic agent) or compound / drug product (including compositions (i.e., formulations or drugs)) to a subject includes any route of introduction or delivery of the compound / drug product to the subject to exert its intended function. Administration may be performed via any suitable route, such as oral administration. Administration may be performed subcutaneously. Administration may be performed intravenously. Administration may be performed intraocularly. Administration may be performed systemically. Alternatively, administration may be performed topically, intranasally, intraperitoneally, intradermally, intraocularly, intrathecally, intraventricularly, via iontophoresis, transmucosally, intravitreal, or intramuscularly. Administration includes self-administration, administration by another person, or administration using a device (e.g., an infusion pump).

[0128] As used herein, the term "excipient" refers to a natural or synthetic substance formulated together with the active ingredient of a drug, including for the purpose of long-term stability, increasing the size of solid dosage forms, or imparting therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, or increasing solubility.

[0129] As used in this article, “heterologous” refers to 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 that is found in one or more locations in a genome or vector that are different from where it is naturally present.

[0130] "Homology," "identity," "same percentage," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparative purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of shared matching or homologous positions. "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence at a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, when aligned, a percentage of the bases (or amino acids) are the same in the comparison of the two sequences. This alignment and percentage homology or sequence identity can be determined using software programs known in the art.

[0131] 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 “open-ended” transitional terms that allow for the inclusion of additional components beyond those recited in the claims. The terms “compose” and “composed of…” should be interpreted as “closed-ended” transitional terms that do not allow for the inclusion of additional components beyond those recited in the claims. The term “substantially composed of…” should be interpreted as partially closed and allows for the inclusion of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0132] As used herein, “operably linked” in the context of a nucleic acid sequence, region, element, or domain means that the nucleic acid regions are functionally related to each other. 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, whereby 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 this nucleic acid is transcribed into a single mRNA transcript, but the translation of the mRNA transcript can result in the expression of one of the two polypeptides. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that when a partial amber-repressive cell is introduced, 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 instance, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of the nucleic acid.

[0133] As used herein, the term "pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, is substantially composed of, or is further composed of nanoparticles, such as polymer nanoparticle carriers or lipid nanoparticles (LNPs). Additionally or alternatively, pharmaceutically acceptable carriers include: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of metaglycerides 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, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are described in Remington Pharmaceutical Sciences, Mack Publishing, the standard reference text in the field. They can be selected based on the intended form of administration, such as oral tablets, capsules, elixirs, syrups, etc., in accordance with conventional pharmaceutical practices.

[0134] As used herein, “prevention” or “preventing” of a disease, condition, or symptom refers to a reduction in the incidence of the disease, condition, or symptom in a sample or subject receiving the treatment, relative to a control sample or subject. Such prevention is sometimes referred to as preventive treatment.

[0135] As used herein, “recombinant” in the context of polynucleotides or polypeptides refers to polynucleotides or polypeptides that have been modified in vitro using techniques known in the art. For example, expression vectors or expression plasmids, or their expression products, are considered recombinant. In some embodiments, polynucleotides or polypeptides are modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or the material is derived from cells that have been so modified. Thus, for example, recombinant cells express genes not found in native (non-recombinant) cell forms, or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

[0136] As used herein, the term “single” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes.

[0137] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, via the same or different routes of administration. More specifically, sequential use means the complete administration of one active ingredient before the administration of another or other active ingredient. Thus, an active ingredient may be administered minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no simultaneous treatment.

[0138] As used herein, the term “simultaneous” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via the same route.

[0139] As used herein, the term "synergistic therapeutic effect" refers to a therapeutic effect that is greater than the sum of its components, resulting from a combination of at least two agents and exceeding the effect of applying the agent alone.

[0140] As used herein, the term "treating" or "treatment" refers to therapeutic treatment in which the aim is to reduce, alleviate, or slow down (relieve) a pre-existing disease or condition or its associated signs, symptoms, or symptom. By example, but not limited to, a subject's disease is successfully "treated" 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 symptom associated with the disease, condition, or symptom. It should also be understood that the various modes of treatment for medical conditions described are intended to represent "fundamental," which includes complete improvement of the signs, symptoms, or symptom of the disease or condition, and "partial," in which some biological or medically relevant outcome is achieved.

[0141] The phrase “such as” should be interpreted as “for example, including”. Furthermore, the use of any and all exemplary language (including, but not limited to, “such as”) is intended only to better illustrate the technology and does not constitute a limitation on the scope of the technology unless otherwise required.

[0142] Furthermore, in cases where idioms such as "at least one of A, B, and C" are used, generally, the intention of such constructions is that those skilled in the art will understand the idioms (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, a system having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C). Those skilled in the art will further understand that any antonymous conjunctions and / or phrases that actually present two or more alternative terms, whether in the specification or figures, should be understood to cover the possibility of including one, any, or both of the terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0143] As those skilled in the art will understand, for any and all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficiently descriptive and such that the same scope can be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all language such as “up to,” “at least,” “greater than,” “less than,” etc., including the listed numbers, refers to a scope that can subsequently be decomposed into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1 to 3 members means a group having 1, 2, or 3 members. Similarly, a group having 1 to 5 members means a group having 1, 2, 3, 4, or 5 members, and so on. The modal verb "may" refers to the preferred use or selection of one or more options or choices among the described embodiments or features included in such embodiments. When no options or choices regarding a particular embodiment or feature included in such embodiment are disclosed, the modal verb "may" refers to an affirmative action regarding how to make or use the described embodiment or feature included in such embodiment, or a clear decision regarding a particular skill in using the described embodiment or feature included in such embodiment. In the latter case, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can".

[0144] Example

[0145] The following examples are illustrative and should not be construed as limiting the scope of the subject matter for which protection is sought.

[0146] Example 1 - Intracellular tPA-PAI-1 Interactions Determine apoB Lipidation and VLDL Assembly in Hepatocytes.

[0147] Methods and Materials

[0148] Plasma from individuals with PAI-1 deficiency. From individuals carrying SERPINE1 (SERPINE1 - / - Plasma samples were collected from members of the Berne Amish community (n=10) with frameshift mutations (55) and age-, sex-, and BMI-matched controls from the same community (n=10). Age and sex information for both subjects with PAI-1 deficiency and controls is listed in Table 2. The study protocol was approved by the Experimental Review Committee of the Indiana Center for Hemophilia and Thrombosis and the Medical College of Wisconsin (MCW). Written informed consent was provided by all study participants.

[0149] 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, Massachusetts). In short, a targeting construct was designed to insert the loxP site into introns 3 and 6 to flox-modify exons 4 through 6 of the Plat gene. This design conditionally knocked out exons 4 through 6 of Plat using the Cre-loxP system. fl / fl Hepatocyte tPA knockout mice were generated by administering AAV8 (AAV8-TBG-cre (Cre)) to mice expressing Cre recombinase driven by the thyroxine-binding globulin (TBG) promoter. Plasma cells receiving AAV8-TBG-GFP (GFP) were then used. fl / fl Mice were used as controls. 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 disordered silencing controls. Ldlr was used to silence tPA in hepatocytes. - / - Apoe - / - WTC57BL / 6J mice were purchased from Jackson Laboratory (JAX) (catalog numbers 002207, 002052, and 000664, respectively). Ldlr mice were fed WD (Teklad, catalog number TD 88137). - / - and Apoe - / -Mice. WT mice were fed a standard diet (laboratory diet, catalog 5053), a DIO diet (research diet, catalog 12492), or a WD diet (Teklad, catalog TD 88137). To express tPA in hepatocytes, WT C57BL / 6J mice and holo-tPA–KO mice (Jax, catalog 002508) were fed a standard diet and received intravenous injections of AAV8-TBG-Plat. For all experiments, mice were maintained on a 12-hour light / 12-hour dark cycle with free access to normal food, WD or DIO diet, and water. Mice of similar age and weight were randomly assigned to experimental and control groups. Plasma lipids in collected blood samples were measured 5 hours after fasting. We used power calculations to determine the number of mice for each experiment. We included both male and female mice. Mice of similar age and weight were randomly assigned to groups; exclusion criteria were death, injury requiring euthanasia, or weight loss >10%, assumed to be rare events with no statistically significant difference between groups. All endpoint determinations and analyses were performed by researchers who were blinded to the identity of the cohort. All mouse experiments were approved by the Laboratory Animal Management and Use Committee of the MCW Biomedical Resource Center and the Laboratory Animal Management and Use Committee of Columbia University Irving Medical Center.

[0150] Vector constructs. AAV8-TBG-cre and AAV8-TBG-GFP were purchased from Addgene. As previously described (19, 20), the AAV8-H1-short hairpin RNA (shRNA) construct targeting mouse plasma was prepared 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). The plasmid constructs expressing human tPA mutants were generated by Versiti BRI Core based on pCMV3-tPA-HA. The constructed tPA mutants included tPA-S513A, tPA-KDEL, tPA-Δ-K2-HA, and tPA-D236, 238N. Specifically, tPA-Δ-K2 refers to a tPA mutant in which K1 replaces K2, resulting in two copies of K1 but no K2. The aim of this design is to generate a mutant tPA that lacks K2 but mimics the structure of a normal tPA.

[0151] Mouse plasma collection and analysis. Blood obtained via cardiac puncture was added to 10% (3.8%, w / v) sodium citrate, centrifuged at 2300 g for 15 min at room temperature, and plasma was carefully collected from the supernatant fraction. Plasma samples were aliquoted, rapidly frozen, and stored at -80°C until analysis. Plasma total antigen levels of tPA and apoB-100 were measured by ELISA using the kit, according to the manufacturer's instructions.

[0152] Human primary hepatocyte experiments. Human primary hepatocytes were obtained from the liver tissue cell distribution system at the University of Pittsburgh (Pittsburgh, Pennsylvania, USA). All cells were cultured in Williams E medium supplemented with a hepatocyte maintenance supplement (Thermo Fisher Scientific, catalog number CM4000). The experiments were performed as depicted in the illustrations. Cells and culture medium were harvested, rapidly frozen in liquid nitrogen, and stored at –80°C until processing. The age and sex information of the human donors are listed in Table 3.

[0153] McA-RH7777 Cell Experiments. Rat liver cancer McA-RH7777 cells were obtained from the American Center for Type Culture Collection (Manassas, Virginia, USA). Cells were grown in Duchenne Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, catalog number 12430054) containing 10% fetal bovine serum (FBS). Experiments were performed as depicted in the illustrations. Cells and culture medium were harvested, rapidly frozen in liquid nitrogen, and stored at –80°C until processing.

[0154] Cultured hepatocytes were transfected with a plasmid encoding tPA-K2-HA-KDEL. McA-RH7777 cells were seeded in culture plates. At approximately 30% to 40% confluence, a transfection mixture was prepared by pre-incubating the plasmid encoding tPA-K2-HA-KDEL or a GFP control with Lipofectamine 3000 in Opti-MEM for 10 minutes. The transfection mixture was then added to the culture medium. Seventy-two hours post-transfection, hepatocytes and culture medium were harvested. The expression efficiency of tPA-K2-HA-KDEL in cell lysates and apoB in cell culture medium was detected by Western blotting. FIG. 23A to FIG. 23B ).

[0155] LNP injection in mice. Mice were intravenously injected (1 mg / kg body weight) with LNP carrying mRNA encoding tPA-K2-HA-KDEL (SEQ ID NO: 32) or a control luciferase. A similar experiment was performed with LNP carrying mRNA encoding tPA-K2-KDEL (SEQ ID NO: 8). Blood was collected at 6, 24, and 48 hours post-injection and before injection. Plasma triglyceride and cholesterol levels were measured. FIG. 24A to FIG. 24B ).

[0156] Protein extraction and immunoblotting. Liver tissue samples and cultured hepatocytes were subjected to Western blotting supplemented with Haltib. TM The protein extract was homogenized in RIPA buffer (ThermoFisher Scientific, catalog 89900) containing a mixture of protease and phosphatase inhibitors (ThermoFisher Scientific, catalog 78444). The protein extract was electrophoresed on an SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with tris-buffered saline containing 5% (w / v) BSA in 0.1% Tween 20 (TBST). The membrane was then incubated overnight at 4°C with 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.

[0157] Quantitative RT-PCR. Total RNA was extracted using the RNeasy kit (QIAGNE, catalog number 74004). iScript was used. TM cDNA was synthesized using a 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 level of each gene was normalized to 36B4 (housekeeper). Primer sequences used for quantitative RT-PCR are listed in Table 4.

[0158] Mouse hepatic VLDL production. To measure hepatic VLDL secretion rate, mice were administered poloxamer 407 (1,000 mg / kg body weight) via intraperitoneal injection (96). Blood samples were collected from the tail vein at 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 increase in plasma triglycerides between 60 and 90 minutes or between 90 and 120 minutes was calculated as the elevated triglyceride level divided by the corresponding time, reflecting the VLDL secretion rate within that time range.

[0159] Pulse-tracking assay for apoB secretion. As previously described, using 3 [H] labeling method was used to determine hepatocyte apoB-100 secretion (31). Human primary hepatocytes or McA-RH7777 cells were washed with leucine-free medium and then... 3 [H]leucine (80 uCi / ml; 160 Ci / mmol, Perkin Elmer, catalog number NET1166005MC) pulsed for 20 minutes. Removes [H]leucine. 3 Cells were cultured with [H]leucine and incubated with fresh DMEM for an additional 0.5, 1, or 3 hours. ApoB was immunoprecipitated from the cell homogenate and culture medium using an anti-apoB antibody (Sigma-Aldrich, catalog AB742). Unlabeled apoB-100 standards were added to the precipitate, and the sample was separated by SDS-PAGE gel electrophoresis. The gel was silver-stained, and the band corresponding to apoB-100 was excised. Radioactivity associated with apoB-100 was quantified using a scintillation counter.

[0160] Endoplasmic reticulum (ER) isolation and protein extraction. ER fractions were isolated from hepatocytes 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). Cell membranes were sheared by inserting a 29-gauge needle through the cells. Cell debris was precipitated by two rounds of centrifugation at 3000 g for 10 min each. The supernatant was separated on top of a discontinuous sucrose gradient of 580, 880, and 1100 mM sucrose in ER extraction buffer and centrifuged at 100,000 g for 2 h at 4 °C. The precipitate contained purified ER membranes. Proteins were extracted from the ER as described (21). ER particles were dissolved in 1 M sodium carbonate (pH 11.5) containing 250 mM sucrose in 3 M KCl and 2 ml of 0.0625% deoxycholate. The mixture was incubated at room temperature for 30 min. After centrifugation, the supernatant contained the proteins extracted from the ER.

[0161] Sucrose gradient ultracentrifugation of apoB lipoprotein extracted from ER. ER protein extract was adjusted to 12.5% ​​sucrose. A sucrose gradient was formed by stratification from the bottom of the tube: 1 ml of 47% sucrose, 1 ml of 25% sucrose, 2.5 ml of sample in 12.5% ​​sucrose, and 1.5 ml of phosphate-buffered saline. The gradient was rotated in a Beckman SW40 rotor at 12°C and 35,000 rpm for 65 hours and unloaded from top to bottom into 6 fractions (21, 98).

[0162] MTP Expression and Purification. As described previously (99), a plasmid (Addgene, catalog number 138335) carrying the complementary DNA (cDNA) of hMTP cDNA (pcDNA3-hMTP-FLAG) was transfected into Cos-7 cells. Forty-eight hours post-transfection, cells were collected in 1 ml buffer K (10 mM Tris-Cl, 1 mM MgCl2, and 1 mM EGTA, pH 7.4) containing 150 mM NaCl and lysed by sonication on ice. Cell lysates were rotated at 13,500 g for 10 minutes at 4°C to remove unlysed cells and cell debris. FLAG-labeled MTPs were purified using a column packed with anti-FLAG M2 affinity agarose beads. FLAG peptides were removed and the purified protein was concentrated by ultrafiltration using a cutoff centrifugation filter (Am Amicon Ultra, Merck Millipore, catalog number UFC9010).

[0163] Neutral lipid transfer activity assay. As described above (36), neutral lipid transfer activity in cultured hepatocyte microsomal fractions was measured using a commercially available kit (Sigma-Aldrich, MAK110). 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 separated by ultracentrifugation (SW55 Ti rotor, 50,000 rpm, 1 h). Neutral lipid transfer activity was determined using the kit according to the manufacturer's manual. The separated microsomes were incubated with “donor vesicles” and “recipient vesicles” containing fluorescent lipids-LDL. The fluorescence signal self-quenched when the labeled lipids were present in the “donor vesicles” but was detected after the lipids were transferred to the “recipient vesicles”.

[0164] Immunofluorescence imaging and adjacent junction assay (PLA). Human primary hepatocytes were seeded onto collagen-coated coverslips. After rinsing with PBS, cells were fixed with 4% paraformaldehyde for 15 min, infiltrated with 0.1% Triton X-100 in PBS for 5 min, and then blocked with PBS containing 5% BSA at room temperature for 1 h. The blocked slides were incubated overnight at 4°C with appropriate primary antibodies, and then incubated with species-specific fluorophore-conjugated secondary antibodies at room temperature for 1 h. For primary antibodies, we used rabbit anti-tPA antibody (ProteinTech, catalog 10147-1-AP), goat polyclonal anti-apoB (Sigma-Aldrich, catalog AB742), and rabbit antibodies against each intracellular organelle marker—calcin for the endoplasmic reticulum (ER) and TGN46 for the trans-Golgi network. For secondary antibodies, we used Alexa Fluor 488-conjugated donkey anti-goat IgG (Thermo Fisher Scientific, catalog number A21206), Alexa Fluor 568-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, catalog number A11057), and Alexa Fluor 647-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, catalog number A31571). The coverslips were washed three times with PBS for 5 minutes each time, and then incubated with DAPI nuclear staining (Invitrogen) for 2 minutes. The coverslips were then given a final rinse with PBS and mounted with a slow-fading anti-fluorescence mount.

[0165] For PLA, the Duolink in situ kit (Sigma-Aldrich) was used according to the manufacturer's protocol. The primary antibody tPA and anti-apoB antibody were the same as those used in the immunofluorescence assay described above. All reactions were performed in a humidified chamber at 37°C. After fixation and infiltration, hepatocytes were incubated with the primary antibody for 40 minutes and then with a pair of PLA probes (anti-rabbit Minus and anti-goat Plus) for 60 minutes; ligase was added for 30 minutes, followed by signal amplification using a red detection reagent for 100 minutes. Coverslips were then mounted using Duolink in situ mounting medium with DAPI and sealed with clear nail polish.

[0166] Fluorescence images were captured at ×40 magnification using a Nikon A1R confocal laser scanner, and digital zoom was performed using NIS-Elements analysis software. Blue, green, red, and far-red lasers and filters were used for Alexa fluorophore detection. Texas Red settings were used for PLA detection.

[0167] Solid-phase protein binding assay. Solid-phase binding was performed in polystyrene microtiter plates using enzyme-linked immunosorbent assay (ELISA). The wells of the microtiter plate 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% Tween 20 (TBS-Tween), tPA was added to the wells in the TBS-Tween at concentrations ranging from 0 to 20 μg / mL. After incubation at 37°C for 1 hour, the wells were washed with TBS-Tween. The bound protein reacted with anti-tPA (1 μg / mL IgG in TBS-Tween, in the presence of 3% skim milk) and then with goat anti-rabbit IgG conjugated to horseradish peroxidase. TMB substrate was added. After stopping the reaction, the absorbance was measured at 450 nm.

[0168] Surface plasmon resonance (SPR). The binding of recombinant tPA with purified LDL was studied using a Biacore S200 SPR instrument (Biacore) employing a CM5 sensor chip (Cytiva, catalog number 29149603) (94). LDL was attached to the chip using amine coupling chemistry, according to the manufacturer's instructions. Briefly, the chip 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, for 7 min). LDL (500 μg / ml, in 10 mM sodium acetate buffer, pH 5.5) was then flowed over the chip surface at the same rate for 7 min, followed by flow over 1 M ethanolamine-HCl (pH 8.5) at 19 μl / min for 7 min to inactivate excess reactive groups and remove any non-covalently bound LDL. This process resulted in the immobilization of LDL in approximately 6,000 response units (RUs). To monitor the association between LDL and 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 chip at 20 μl / min for 6 minutes at room temperature. Dissociation of tPA was then monitored by washing the surface with HES buffer alone for 6 minutes. Under the same conditions as the nonspecific binding control, tPA was flowed through an activated but uncoated CM5 chip.

[0169] Plasma lipoproteins were separated by FPLC. FPLC was performed at 4°C using an AKTA Purifier 10 and two tandem Superose 6 10 / 300 GL columns (Cytiva, catalog 29091596). 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 run buffer. Elution was collected in 1-mL fractions from 12-mL to 47-mL fractions. Cholesterol, triglyceride (Wako, Fujifilm), and apoB-100 (Abcam, catalog 230932; Mabtech, catalog 3715-1HP) levels from the fractions were measured according to the manual.

[0170] Plasma lipoproteins were separated by sequential ultracentrifugation. As previously described, plasma lipoproteins (95, 100) were separated by KBr (potassium bromide) density ultracentrifugation. Equal volumes of mouse plasma were used for sequential density ultracentrifugation to separate very low-density lipoprotein (d < 1.006 g / mL), low-density lipoprotein (d = 1.006 to 1.063 g / mL), and high-density lipoprotein (d = 1.063 to 1.21 g / mL) in a TLA 100 rotor.

[0171] VLDL particle diameter analysis. VLDL particles (d < 1.006 g / mL) were separated by KBr density ultracentrifugation (95), and their diameters were then measured using two methods. First, the separated VLDL particles were negatively stained with 20 g / L phosphotungstic acid (pH 7.0) for 2 min and then observed under a Philips CM10 electron microscope. The average diameter of the VLDL particles was determined using Image-Pro Plus 5.0 image analysis software. Second, the hydrodynamic diameter of the separated VLDL particles was measured at 633 nm using a Zetasizer µV dynamic laser scattering instrument (Malvern Instruments). The VLDL samples were transferred to quartz cuvettes and light scattering readings were taken at 20 °C (25 to 27).

[0172] Mouse tail hemorrhage assay. Mice were anesthetized with isoflurane and placed horizontally on a platform allowing the tail to descend approximately 2 cm from the top. A transverse incision of the tail tip was made at the distal end of the tail using a No. 11 scalpel, creating an incision approximately 2 mm in diameter. Bleeding was monitored by tapping the tail tip against Whatman paper at 10-second intervals until bleeding ceased. The time to stable cessation of bleeding was defined as the interval between the tail incision and cessation of bleeding, with no evidence of rebleeding within 60 seconds. Bleeding lasting longer than 15 minutes was stopped by applying pressure.

[0173] Statistical analysis. Data described in the study were generated from biological replication. The number of human participants and mouse experiments is depicted in applicable diagrams. In vitro cell experiments were repeated at least three times. All results are presented as mean ± SEM. For data that passed the normality test, p-values ​​were calculated using a two-tailed Student's t-test; or for non-normally distributed data, p-values ​​were calculated using a Mann-Whitney rank-sum U-test. When analyzing three or more groups, one-way ANOVA with a post-hoc Tukey test was used to assess differences between groups.

[0174] Research approval was granted. All mouse experiments were approved by the Laboratory Biosafety Committees of IACUC and MCW, as well as Columbia University Medical Center IACUC. Human cell and plasma samples used in this study were approved by MCW and the IRB of the Indiana Center for Hemophilia and Thrombosis. Written informed consent was provided by all participants.

[0175] Table 1. Key Resources.

[0176] Table 2. Age and sex information of homozygous individuals with PAI-1 deficiency and matched control individuals.

[0177]

[0178] Table 3. Age and gender information of participants who donated their primary hepatocytes.

[0179]

[0180] Table 4. Primers used for quantitative PCR.

[0181]

[0182] RESULTS

[0183] Silent hepatocytes of tPA, independent of LDLR or ApoE, increase atherogenic apoB lipoprotein cholesterol and apoB. Adenovirus-associated virus 8 (AAV8) expressing a hairpin RNA (AAV8-H1-shPlat, or sh-tPA) targeting a Plat mRNA (encoding tPA) driven by the H1 promoter was administered to Ldlr hepatocytes fed a Western diet (WD). - / -Mice (an established mouse model of hypercholesterolemia) were silencing tPA expression in hepatocytes [21, 22]. Compared with mice receiving AAV8-H1 disordered RNA (scr), mice with tPA-silenced hepatocytes showed 47% higher plasma total cholesterol levels (p<0.01) and 28% higher apoB-100 levels (p<0.05). FIG. 1A Plasma lipoprotein fractions analyzed by rapid protein liquid chromatography (FPLC) showed that hepatocyte tPA-silenced mice had higher levels of cholesterol and apoB in both VLDL and LDL fractions, and higher levels of triglycerides in VLDL. FIG. 1A Similarly, compared to the disordered silencing control, Apoe knockout (Apoe) fed with WD showed significantly higher levels of silencing. - / - Silencing hepatocyte tPA in mice led to 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), with consistent distribution among lipoprotein components. FIG. 1B Similar results were observed in wild-type (WT) C57BL / 6J mice fed with WD, while liver Apob mRNA remained unchanged. FIG. 9A to FIG. 9C This indicates that the increased apoB levels are not due to increased apoB synthesis. Furthermore, silencing hepatocyte tPA did not alter hepatic LDLR and plasma apoE levels in WT mice. FIG. 10A to FIG. 10B In summary, silencing hepatocyte tPA leads to higher plasma apoB lipoprotein cholesterol through a mechanism independent of LDLR or apoE.

[0184] Consistently, silencing tPA in primary human hepatocytes using siRNA (si-tPA) targeting PLAT mRNA resulted in higher apoB-100 levels in serum-free culture medium. FIG. 1D ), without altering hepatocyte APOB mRNA levels ( FIG. 11B In tPA-silenced human hepatocytes, isolated VLDL showed higher levels of cholesterol and triglycerides. FIG. 1D Similar findings were observed in cultured McA-RH7777 cells (a rat hepatocellular carcinoma cell line). FIG. 1E ; FIG. 11C to FIG. 11D This cell line is an established model system for studying VLDL production because it synthesizes VLDL particles of similar size to human VLDL particles [23, 24].

[0185] Silencing hepatocyte tPA increases VLDL production and apoB lipolysis in the ER. Following injection of the nonionic surfactant detergent poloxamer 407 (P407) (which inhibits lipoprotein lipase activity and VLDL lipolysis [25, 26]), mice with silenced hepatocyte tPA showed a faster rate of triglyceride elevation (P407). FIG. 2A This indicates that silencing tPA in hepatocytes increases apoB-VLDL production. Furthermore, plasma apoB-100 derived solely from hepatocytes increased to a greater extent after hepatocyte tPA silencing compared to plasma apoB-48 produced from both hepatocytes and intestines in mice.

[27] FIG. 2B ).

[0186] Lipidification is a key factor determining the fate of intrahepatic apoB. Hypolipidemic apoB is degraded intracellularly, while fully lipid-mediated apoB is efficiently secreted as larger and lower-density particles

[28] . Electron microscopy of VLDL particles separated by density ultracentrifugation showed that VLDL particles in hepatocytes silencing tPA were highly lipid-mediated. - / - In mice, the distribution of VLDL shifts towards larger diameters. FIG. 2C The hydrodynamic diameters of the separated VLDLs were consistently large, as observed by dynamic light scattering (DLS) methods, due to the slow movement of their dispersed particles (Brownian motion) [29 to 32]. FIG. 2D This indicates a high lipid content, which is confirmed by the high TG / apoB ratio. FIG. 2E ).

[0187] Consistently, silencing tPA in human primary hepatocytes led to higher apoB-related radioactivity in the cell culture medium, indicating that... 3 [H]-leucine-labeled apoB secretion is higher

[33] FIG. 2H Similar results were observed in McA-RH7777 cells. FIG. 12 Silencing tPA increased the diameter of VLDL isolated from cell culture medium and the TG / apoB ratio. FIG. 2I , 2J Compared with control human primary hepatocytes, tPA-silenced ER-associated apoB levels were higher ( FIG. 2K ), while tPA-silenced hepatocytes were classified in lower density ER grades (grades 1 and 2, FIG. 2K More apoB accumulated in tPA. Since density and lipidation are negatively correlated, this finding is consistent with the hypothesis that tPA limits apoB lipidation.

[0188] Hepatocyte tPA disrupts MTP-apoB interactions and inhibits MTP-dependent neutral lipid translocation. MTP is a key chaperone that promotes intrahepatic apoB lipidation by translocating and incorporating neutral lipids (particularly triglycerides and cholesterol esters) into apoB for VLDL assembly [10, 11]. Although tPA silencing in human primary hepatocytes does not alter MTP protein levels ( FIG. 3A (input), but compared with control hepatocytes, immunoprecipitated anti-MTP from tPA-silenced cells showed higher apoB ( FIG. 3A This indicates that silencing tPA increases apoB-MTP interaction. Consistently, microsomal fractions isolated from tPA-silenced hepatocytes showed twice the neutral lipid transfer activity compared to microsomes from control cells. FIG. 3B Group 1 to Group 2). Transfer activity was indeed due to MTP, as the MTP inhibitor CP-346086

[34] ,

[35] completely eliminated the elevated neutral lipid transfer activity in tPA-silenced hepatocytes ( FIG. 3B (Group 3). Similar findings were observed in McA-RH7777 cells ( FIG. 14 MTP-mediated lipid transfer to apoB involves its direct binding to apoB

[10] , as inhibition of apoB-MTP interaction reduces apoB lipidation and secretion

[11] .

[0189] In primary human hepatocytes transfected with a plasmid expressing tPA with a C-terminal HA tag, apoB was detected in the elution buffer containing anti-HA precipitation. FIG. 3F Ortho-linkage assay (PLA) showing punctate fluorescence signals. FIG. 3G The presence of tPA and apoB in hepatocytes indicates a close proximity, suggesting an intracellular interaction between endogenous tPA and apoB in hepatocytes. This is further supported by the co-localization of tPA and apoB in the ER observed by immunofluorescence staining. FIG. 3H In solid-phase protein binding assays, purified recombinant tPA interacted directly with purified apoB-containing LDL bound to the surface of a microtiter plate in a dose-dependent manner. FIG. 4A Consistent interactions were observed between purified recombinant tPA and purified apoB-100. FIG. 16 In contrast, purified tPA does not interact with surface-bound MTP. FIG. 3B Pre-incubation of LDL with tPA inhibited the binding between MTP and surface-bound LDL. FIG. 3C ), and reduced MTP-mediated transfer of neutral lipids to LDL ( FIG. 3JSurface plasmon resonance (SPR) studies indicate the existence of non-covalent bonding between tPA and LDL particles. FIG. 4D Quantitative analysis of the sensor map using a two-state binding model revealed that the Kd of the LDL-tPA interaction is approximately 260 nM. In summary, tPA directly interacts with apoB-containing lipoproteins to reduce the availability of apoB for MTP incorporation into neutral lipids, thereby inhibiting VLDL assembly.

[0190] Transduction of hepatocytes with tPA reduced VLDL assembly and apoB secretion in the hepatocyte ER, independent of tPA serine protease activity. In tPA knockout mice (holo-tPA-KO), tPA expression was increased only in hepatocytes with AAV8 virus under the TBG promoter (AAV8-TBG-tPA) [21, 22, 36 to 38], and plasma apoB-100, VLDL cholesterol, and LDL cholesterol were reduced compared to the AAV8-TBG-LacZ control. FIG. 2F Consistently, using pulse tracking assays, neutral lipid transfer activity, and apoB-MTP interaction, expression of tPA via plasmids (e.g., pCMV3-tPA-HA) in human primary hepatocytes reduced the secretion of newly synthesized apoB. FIG. 2G , 3C (3D). In summary, tPA restricts apoB lipidation and its subsequent secretion by reducing the accessibility of apoB to MTP.

[0191] MTP-apoB interaction occurs in the hepatocyte ER

[39] . Using confocal immunofluorescence microscopy, tPA and apoB were co-localized in the ER ( FIG. 3H ApoB interacts with MTP for its lipidation and VLDL assembly. By preserving 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: SEQ ID NO: 3), pulse tracking assays showed reduced secretion of newly synthesized apoB in human primary hepatocytes transduced with a plasmid encoding tPA-KDEL (SEQ ID NO: 3) (pCMV3-tPA-KDEL listed in SEQ ID NO: 24). FIG. 3I In summary, these results indicate that tPA interacts with apoB in the ER and restricts apoB-VLDL assembly and secretion.

[0192] The protease activity of tPA depends on serine at position 513, and the replacement of serine with alanine (S513A) completely eliminates the serine protease activity of tPA

[44] . Similar to WT tPA, the serine protease mutant tPA (S513A; SEQ ID NO: 9) showed reduced apoB secretion in human primary hepatocytes transduced with the plasmid encoding tPA S513A (pCMV3-tPA-S513A; SEQ ID NO: 23) as determined by pulse tracking. FIG. 3I The recombinant tPA-S513A protein binds to surface-bound LDL (which has a similar affinity to WT tPA). FIG. 4A ), and impairs MTP-mediated lipid transfer activity ( FIG. 3J This indicates that tPA binds to apoB and reduces apoB lipidation and secretion independent of tPA serine protease activity.

[0193] The lysine-binding site at the Kringle 2 domain of tPA interacts with the lysine-rich region at the N-terminus of apoB. The Kringle 2 domain of tPA contains a lysine-binding site

[45] , which is essential for its interaction with fibrin. The negatively charged residues aspartic acid 236 and 238 in the Kringle 2 domain of human tPA are responsible for the binding of tPA to the positively charged lysine

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

[47] has a lysine-rich region responsible for its binding to MTP and MTP-mediated lipidation activity

[48] . Pulse tracking assays showed that, compared with wild-type tPA, expression of the tPA mutant lacking the Kringle 2 domain (tPA-Δ-K2) or the tPA mutant with aspartic acid substitutions at positions 236 and 238 (tPA-D236, 238N) did not alter apoB secretion ( FIG. 4F Similarly, in solid-phase protein binding assays, compared to rabbit IgG controls, the antibody targeting the tPA Kringle 2 domain (Sigma, HPA003412) inhibited the binding between surface-bound tPA and LDL. FIG. 4G Tranexamic acid (TXA) is a lysine analog that reduces the interaction between tPA and surface-bound LDL. FIG. 4H Therefore, tPA competitively binds to the lysine-rich region of apoB via the lysine-binding sites at aspartic acid 236 and 238 on the Kringle 2 domain of tPA, thereby blocking apoB-MTP interaction and reducing the accessibility of MTP to apoB esterification.

[0194] PAI-1 sequesters tPA from apoB, leading to increased VLDL assembly in hepatocytes. Studies by numerous research groups have shown that lipid-load-induced postprandial lipid prolongation enhances hepatic VLDL production, but the underlying mechanisms remain unclear. Obesity, which is typically associated with dyslipidemia, increases tPA synthesis in hepatocytes

[21] , which is then overcompensated by a significant increase in its serine protease inhibitor PAI-1, resulting in a reduction in net free functional tPA in the liver and plasma

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

[49] . Therefore, the inventors hypothesize that postprandial lipid load increases the intracellular interaction between tPA and PAI-1, leading to a reduction in free tPA, which limits the availability of MTP for lipidation in apoB and ultimately results in increased VLDL assembly and secretion into the bloodstream.

[0195] After running an SDS-PAGE gel to elute immunoprecipitated PAI-1 (approximately 50 Kd) from human primary hepatocytes, tPA (approximately 70 Kd) was probed, revealing a band located at approximately 120 Kd. This band represents a covalently bound, SDS-stabilized tPA-PAI-1 complex, which was further validated by PLA for their adjacent endogenous interactions in live human primary hepatocytes. FIG. 5A to FIG. 5B As early as one hour after treatment of human primary hepatocytes with oleate (a recognized stimulant for apoB esterification and VLDL production

[50] ), tPA-PAI-1 complex increased while free tPA decreased ( FIG. 5C to FIG. 5D This is similar to the time-dependent stimulation of VLDL production by oleate

[51] . This rapid complexation of PAI-1 with tPA does not require newly synthesized tPA or PAI-1 protein, and the isolation of tPA by PAI-1 suggests that apoB lipidation and VLDL production are timely and finely regulated when hepatocytes are loaded with lipids. With prolonged oleate treatment for 6 and 24 hours, free tPA was further reduced due to the formation of more PAI-1-tPA complexes ( FIG. 5D Consistent results were observed in McA-RH7777 cells. FIG. 18 ).

[0196] Silencing PAI-1 in human primary hepatocytes resulted in higher free tPA and lower apoB secretion via pulse tracking. FIG. 5E to FIG. 5FOleate treatment significantly enhanced the inhibitory effect of si-PAI-1 on apoB secretion, increasing free tPA by approximately 400% and decreasing apoB secretion by approximately 60%, compared to an increase of only approximately 66% and a decrease of 25% in the absence of oleate. These observations are consistent with the hypothesis that, under basal conditions, most tPA is free and does not bind to PAI-1; silencing PAI-1 under these basal conditions only leads to a moderate increase in free tPA and a subsequent slight decrease in apoB secretion. However, under oleate overload conditions, more tPA binds to PAI-1, and silencing PAI-1 results in a significant increase in free tPA and a decrease in apoB secretion. Consistently, silencing both tPA and PAI-1 did not further reduce apoB secretion compared to silencing tPA alone, supporting the view that PAI-1 promotes apoB lipidation by isolating tPA from apoB, rather than acting through PAI-1 itself. FIG. 5G Consistent results were observed in McA-RH7777 hepatocytes. FIG. 18 ).

[0197] The complexation of tPA and PAI-1 causes a conformational change in the tPA protein structure

[52] and loses its fibrin-binding ability mediated by the lysine binding site in the Kringle 2 domain

[53] . This suggests that the binding of PAI-1 to tPA blocks the lysine binding site in the Kringle 2 domain, which may be mediated by steric hindrance or conformational changes in the Kringle 2 domain. Unlike purified tPA alone, the purified PAI-1-tPA complex cannot bind to LDL or inhibit MTP-mediated neutral lipid transfer activity ( FIG. 5H to FIG. 5I This indicates that the complexation of PAI-1 with tPA prevents the interaction between tPA and apoB.

[0198] Two or six hours after C57BL / 6J mice were administered olive oil via gavage (to increase dietary fatty acid intake, followed by increased blood fatty acids and lipid load on hepatocytes), free liver tPA was reduced compared to baseline. FIG. 5J , FIG. 19 However, it did not alter total tPA and PAI-1 levels in the liver. As expected, obese hepatocyte-specific PAI-1 knockout mice (H-PAI-1 KO) [21, 54] had higher levels of free tPA in both the liver and plasma, and lower levels of plasma apoB, total cholesterol, and cholesterol in VLDL and LDL fractions compared to their littermate controls. FIG. 6A to FIG. 6F ).

[0199] In summary, the intracellular interaction between PAI1 and tPA maintains the balance of VLDL production. When fatty acids are loaded into hepatocytes, PAI-1 rapidly complexes with intracellular tPA, reducing the amount of free tPA available to directly interact with apoB and thus limiting its lipidation, ultimately leading to increased VLDL assembly and secretion into the bloodstream.

[0200] PAI-1 deficiency in humans leads to decreased plasma apoB and apoB cholesterol levels. This is due to a unique loss-of-function mutation in SERPINE1 (SERPINE1...). - / - Compared with unaffected individuals from the same community (age, sex, and BMI matched controls, n=10), individuals with homozygous PAI-1 deficiency showed a 22% reduction in LDL cholesterol (p<0.05) and a significantly reduced apoB borderline (21% reduction, p=0.07), an 18% reduction in cholesterol (p=0.06), and a 16% reduction in triglycerides (p=0.07) on the VLDL fraction. FIG. 6G None of these individuals were taking lipid-lowering agents or had a known history of cardiovascular disease. Compared to unaffected controls, PAI-1 deficient individuals had higher tPA (tPA) on isolated VLDL particles. FIG. 6H Furthermore, VLDL-related tPA levels were negatively correlated with VLDL diameter (r = -0.59, p < 0.01). FIG. 6I ). Combining data from PAI-1-deficient mice and PAI-1-silenced human primary hepatocytes ( FIG. 5F and FIG. 6D ), PAI-1 deficiency leads to higher free tPA interaction with apoB and limits apoB lipidation in hepatocytes.

[0201] In summary, the results described above indicate that tPA interacts directly with apoB within the hepatocyte ER via its K2 domain, and this interaction reduces MPT-mediated VLDL assembly. Lipid loading in hepatocytes induces the formation of the tPA-PAI-1 complex, isolating tPA from apoB and thereby promoting apoB lipidation and VLDL assembly. FIG. 6J ).

[0202] Notably, in cultured hepatocytes, exogenous expression of only the tPA-K2 domain was reduced by transducing cells with a plasmid encoding a tPA-K2 domain that is operatively linked to an endoplasmic reticulum localization motif. FIG. 23A to FIG. 23B Furthermore, intravenous injection of LNPs containing tPA-K2 mRNA reduced plasma lipids in mice. FIG. 24A to FIG. 24BThese findings suggest that the recombinant tPA-K2 peptide of this technology is useful in compositions and methods for treating hyperlipidemia.

[0203] Silencing tPA will lead to the formation of cultured human primary hepatocytes ( FIG. 20A ) and HepG2 cells ( FIG. 20B Both tPA and tPA resulted in decreased Lp(a) levels in their cell culture media, indicating that tPA also limits the production of another atherosclerotic apoB-containing lipoprotein, Lp(a), in hepatocytes. Furthermore, knockout of tPA in intestinal epithelial cells increased plasma cholesterol levels in mice. FIG. 21A to FIG. 21C ) and chylomicrons produced ( FIG. 22A to FIG. 22B This indicates that the regulatory role of tPA in apoB lipoprotein production also exists in intestinal epithelial cells, which absorb and transport dietary lipids into the body. These findings suggest that the recombinant tPA-K2 peptide of this technology is useful in compositions and methods for reducing plasma Lp(a) and chylomicron levels, and further reducing cardiovascular risk.

[0204] DISCUSSION

[0205] The inventors’ findings reveal a novel mechanism for fine-tuning the rate at which apoB lipoproteins are assembled by tPA in hepatocytes. For example, not wanting to be bound by theory, VLDL assembly in the ER of hepatocytes is accomplished in two steps [9, 55]. In the first step, apoB co-translational lipidation allows MTP to transfer lipids to apoB polypeptides growing on the ER membrane (including their exposed N-terminus) to form primitive VLDL particles, which are approximately the same size as plasma HDL

[11] . The primitive VLDL then detaches from the ER membrane and becomes luminal particles [9], which are further lipidated in the second step of VLDL assembly to become mature VLDL particles

[55] . MTP also promotes the fusion of primitive VLDL with ER luminal lipid droplets

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

[57] . In hepatocytes, tPA, similar to MTP inhibitors, competes with MTP for interaction with apoB ( FIG. 6JKringle domains are autonomous protein domains that fold into a large ring stabilized by three disulfide bonds and are responsible for protein-protein interactions

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

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

[10] . The inventors’ competitive binding assays showed that antibodies against the Kringle 2 domain of tPA or the lysine analog TXA inhibited the interaction between tPA and LDL, suggesting that this interaction may be mediated by the lysine-binding site in the Kringle 2 domain of tPA. It takes about 40 minutes for newly synthesized apoB to be secreted from hepatocytes

[51] , and apoB-associated radioactivity during the early tracking phase (5 to 10 minutes) is used to represent the amount of newly synthesized apoB after pulse labeling

[51] . The inventors found no difference in apoB-associated radioactivity in cell lysates after 10 minutes of tracking ( FIG. 18 This indicates that silencing tPA does not increase the apoB protein synthesis rate, which is also supported by the observation that silencing tPA does not alter the apoB mRNA levels in WT mouse livers, cultured human primary hepatocytes, and McA-RH7777 cells. FIG. 9A to FIG. 9C and FIG. 11A to FIG. 11D ).

[0206] Oleic acid influx into hepatocytes stimulates apoB lipidation and VLDL assembly

[51] , the mechanism of which is not fully understood. The inventors found that oleate treatment increased the formation of the intracellular tPA-PAI-1 complex, leading to a decrease in free tPA in hepatocytes, providing new insights into the molecular mechanism of the stimulatory effect of oleic acid on apoB production. The tPA-PAI-1 complex increased only 1 hour after oleate treatment, with total tPA and PAI-1 levels unaffected. Conversely, prolonged oleate treatment for more than 6 hours also increased the total PAI-1 protein level in cultured human primary hepatocytes. Consistently, two hours after oral gavage administration of olive oil (of which approximately 70% of the fatty acids are oleic acid) to mice, hepatic tPA-PAI-1 decreased, while total tPA and PAI-1 remained unchanged. FIG. 19 These results indicate that oleate infusion into hepatocytes rapidly induced the complexation between tPA and PAI-1 within one hour in vitro or two hours in vivo. In summary, lipid loading into hepatocytes induces the complexation of PAI-1 and tPA, isolating tPA from apoB, making apoB available for MTP binding, and thereby enhancing apoB lipidation and VLDL production. This reveals the physiological significance of the tPA-PAI-1 interaction in the fine-tuning of apoB lipidation after lipid loading into hepatocytes.

[0207] Elevated blood PAI-1 levels and decreased tPA activity lead to impaired fibrinolysis, which is an independent risk factor for atherosclerotic thrombotic diseases

[60] . Clinical observation data show that plasma PAI-1 concentration is positively correlated with apoB cholesterol, while tPA activity is negatively correlated with apoB cholesterol [57, 60, 61].

[0208] SUMMARY

[0209] In summary, these results demonstrate that the tPA-K2 peptide of this technology—despite lacking serine protease and proteolytic activity—is effective in reducing apoB secretion and lowering plasma triglyceride and total cholesterol levels when administered to subjects. Furthermore, because the tPA-K2 fragment of this technology (e.g., containing the amino acid sequence listed in SEQ ID NO. 7) lacks serine protease and proteolytic activity, administration of the peptide of this technology does not increase the bleeding risk in subjects. This example demonstrates that expression of the recombinant tPA-K2 peptide containing only the tPA Kringle 2 domain and endoplasmic reticulum localization sequence reduces apoB secretion in hepatocytes (…). FIG. 23A-23B ) and reduced plasma lipid levels in mouse models in vivo ( FIG. 24A-24B However, it did not change the bleeding time in mice. FIG. 25 Therefore, these data support the usefulness of compositions comprising recombinant tPA-K2 peptides (or nucleic acids encoding them, such as cDNA or mRNA) in methods for treating diseases or symptoms associated with elevated plasma apoB lipoprotein levels, and in methods for reducing any or more of plasma VLDL, IDL, LDL, Lp(a), chylomicrons, chylomicron residues, triglycerides, or total cholesterol levels. These data also demonstrate the usefulness of compositions of this technology in methods for reducing plasma lipid and / or apoB levels in subjects who require and / or are undergoing thrombolytic therapy.

[0210] Example 2 - Treatment of a Subject with a Recombinant Polypeptide of the Disclosure.

[0211] In one instance, a therapeutically effective amount of a pharmaceutical composition comprising the disclosed recombinant tPA-K2 peptide or a nucleic acid encoding the recombinant tPA-K2 peptide is administered to a subject suffering from cardiovascular disease (e.g., hyperlipidemia, atherosclerosis, increased risk of thrombosis or atherosclerotic thrombotic events, angina, heart attack, heart failure, stroke, transient ischemic attack (TIA), peripheral artery disease, or hypertension). The subject may be receiving standard care of thrombolytic therapy (e.g., receiving tissue plasminogen activator therapy). The disclosed recombinant tPA-K2 peptide or the nucleic acid encoding it (e.g., peptides comprising amino acid sequences as listed in SEQ ID No. 3, 5, and 7 and / or polynucleotide molecules comprising nucleic acid sequences as listed in SEQ ID No. 4, 6, 8, or 34) may be administered via any route indicated by the subject's specific therapeutic needs (e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, oral, intrathecal, intracerebral, or rectal routes). Signs and symptoms of cardiovascular disease can be reduced by administration of a pharmaceutical composition comprising the disclosed recombinant tPA-K2 polypeptide or a nucleic acid encoding the recombinant tPA-K2 polypeptide. Treatment may be administered daily, every other day, every three days, or according to a schedule determined by the patient's progression, as determined by the physician. It is expected that, compared to untreated subjects, subjects will experience reductions in triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, chylomicron residues, blood pressure, or other indicators associated with a reduction in signs or symptoms of cardiovascular disease. Methods for measuring reductions in signs and symptoms of cardiovascular disease are known in the art.

[0212] These results are expected to demonstrate the usefulness of recombinant tPA-K2 peptides containing this technology or compositions encoding the nucleic acids thereof in methods for treating subjects with cardiovascular disease.

[0213] Example 3 - Treatment of subjects with high cholesterol with recombinant tissue plasminogen activator.

[0214] In one instance, a therapeutically effective amount of a pharmaceutical composition comprising one or more of the recombinant tPA-K2 polypeptide of the present technology or a nucleic acid encoding the recombinant tPA-K2 polypeptide is administered to a subject with hyperlipidemia. The subject may be at risk of or have experienced an atherosclerotic thrombotic event and is receiving standard care of thrombolytic therapy (e.g., receiving tissue plasminogen activator therapy). The recombinant tPA-K2 or the nucleic acid encoding it (e.g., a polypeptide comprising the amino acid sequences listed in SEQ ID No. 3, 5, and 7 and / or a polynucleotide molecule comprising the nucleic acid sequences listed in SEQ ID No. 4, 6, 8, or 34) may be administered via any route indicated by the subject's specific therapeutic needs (e.g., oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, oral, intrathecal, intracerebral, or rectal routes). Levels of triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, or chylomicron residues can be reduced by administration of recombinant tPA-K2 or the nucleic acid encoding that recombinant tPA-K2. Treatment may be administered daily, every other day, every three days, or according to a schedule determined by the patient's progression, depending on the physician's decision. It is expected that subjects will experience reductions in triglycerides, total cholesterol, VLDL, LDL, IDL, Lp(a), chylomicrons, chylomicron residues, or other indicators associated with a reduction in the signs or symptoms of hyperlipidemia compared to untreated subjects. Methods for measuring the reduction in the signs and symptoms of hyperlipidemia are known in the art.

[0215] These results are expected to demonstrate the usefulness of recombinant tPA-K2 peptides containing this technology or compositions encoding the nucleic acids thereof in methods for treating subjects with hyperlipidemia.

[0216] Example 4 - Treatment with recombinant tPA-K2 reduces Lp(a) assembly and production in hepatocytes.

[0217] This experiment will demonstrate the efficacy of the recombinant tPA-K2 peptide or its encoding nucleic acid (e.g., peptides containing amino acid sequences as listed in SEQ ID No. 5 and 7 and / or polynucleotide molecules containing nucleic acid sequences as listed in SEQ ID No. 6, 8, or 34) in reducing Lp(a) assembly and production in hepatocytes. Briefly, human primary hepatocytes will be treated with plasmids, cDNA, and / or mRNA encoding tPA-K2 (e.g., tPA-K2-HA-KDEL). Cells and cell culture medium will be harvested and collected. Lp(a) levels in cell lysates and cell culture medium will be measured by Western blotting.

[0218] These results are expected to demonstrate the usefulness of recombinant tPA-K2 peptides containing this technology or compositions encoding the nucleic acids thereof in methods for reducing plasma Lp(a) levels in subjects in need.

[0219] Example 5 - Treatment with recombinant tPA-K2 reduces chylomicron assembly and production in intestinal epithelial cells.

[0220] This experiment will demonstrate the efficacy of the recombinant tPA-K2 peptide or its encoding nucleic acid (e.g., peptides containing amino acid sequences as listed in SEQ ID No. 5 and 7 and / or polynucleotide molecules containing nucleic acid sequences as listed in SEQ ID No. 6, 8, or 34) in reducing chylomicron assembly and production in intestinal epithelial cells. In short, Caco-2 cells (an intestinal epithelial cell line) will be treated with plasmids, cDNA, and / or mRNA encoding tPA-K2 (e.g., tPA-K2-HA-KDEL). Cells and cell culture medium will be harvested and collected. ApoB48 levels in cell lysates and cell culture medium will be measured by Western blotting.

[0221] These results are expected to demonstrate the usefulness of recombinant tPA-K2 peptides containing this technology or compositions encoding the nucleic acids thereof in methods for reducing chylomicron production.

[0222] Example 6 - Treatment with recombinant tPA-K2 does not significantly increase the risk of liver steatosis or liver injury in subjects.

[0223] This experiment will demonstrate that treating subjects with the recombinant tPA-K2 peptide or its encoding nucleic acid (e.g., peptides containing amino acid sequences listed in SEQ ID No. 5 and 7 and / or polynucleotide molecules containing nucleic acid sequences listed in SEQ ID No. 6, 8, or 34) using this technology will not increase their risk of hepatic steatosis or injury. In short, mice will be administered an intravenous injection (1 mg / kg body weight) of an LNP carrying mRNA encoding tPA-K2-HA-KDEL (SEQ ID NO: 32) or a control luciferase. Blood will be collected 24 hours post-injection and before injection. Mice will be euthanized 24 hours post-injection, and livers will be collected. Blood biomarkers of liver injury will be measured. Liver histological analysis will be performed.

[0224] These results are expected to demonstrate that treatment of subjects with recombinant tPA-K2 or its encoding nucleic acid using this technology does not increase the risk of hepatic steatosis or liver damage.

[0225] References

[0226] 1. Brunner, F.J., et al., Application of non-HDL cholesterol for population-based cardiovascular risk stratification: results from the Multinational Cardiovascular Risk Consortium. Lancet, 2019. 394(10215): pp. 2173-2183.

[0227] 2. Marston, N.A., et al., Association of Apolipoprotein B-Containing Lipoproteins and Risk of Myocardial Infarction in Individuals With and Without Atherosclerosis: Distinguishing Between Particle Concentration, Type, and Content. JAMA Cardiol, 2022. 7(3): pp. 250-256.

[0228] 3. Stone, N.J., et al., 2013 ACC / AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology / American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol, 2014. 63(25 Pt B): pp. 2889-934.

[0229] 4. Mabuchi, H., et al., Effect of an inhibitor of 3-hydroxy-3-methyglutaryl coenzyme A reductase on serum lipoproteins and ubiquinone-10-levels in patients with familial hypercholesterolemia. N Engl J Med, 1981. 305(9): pp. 478-82.

[0230] 5. Horton, J.D., J.C. Cohen and H.H. Hobbs, PCSK9: a convertase that coordinates LDL catabolism. J Lipid Res, 2009. 50 Suppl: pp. S172-7.

[0231] 6. Reith, C. and J. Armitage, Management of residual risk after statin therapy. Atherosclerosis, 2016. 245: pp. 161-70.

[0232] 7. Wong, N.D., Residual Risk After Treatment of Patients With Atherosclerotic Cardiovascular Disease With Proprotein Convertase Subtilisin-Kexin Type 9 Monoclonal Antibody Therapy (from the Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk Trial). Am J Cardiol, 2017. 120(7): pp. 1220-1222.

[0233] 8. Sniderman, A.D., et al., Apolipoprotein B Particles and Cardiovascular Disease: A Narrative Review. JAMA Cardiol, 2019. 4(12): pp. 1287 - 1295.

[0234] 9. Sirwi, A. and M.M. Hussain, Lipid transfer proteins in the assembly of apoB - containing lipoproteins. J Lipid Res, 2018. 59(7): pp. 1094 - 1102.

[0235] 10. Davidson, N.O. and G.S. Shelness, APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation. Annu Rev Nutr, 2000. 20: pp. 169 - 93.

[0236] 11. Hussain, M.M., J. Shi and P. Dreizen, Microsomal triglyceride transfer protein and its role in apoB - lipoprotein assembly. J Lipid Res, 2003. 44(1): pp. 22 - 32.

[0237] 12. Rutledge, A.C., Q. Su and K. Adeli, Apolipoprotein B100 biogenesis: a complex array of intracellular mechanisms regulating folding, stability, and lipoprotein assembly. Biochem Cell Biol, 2010. 88(2): pp. 251 - 67.

[0238] 13. Fisher, E.A., The degradation of apolipoprotein B100: multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways. Biochim Biophys Acta, 2012. 1821(5): pp. 778 - 81.

[0239] 14. Cesarman-Maus, G. and K.A. Hajjar, Molecular mechanisms of fibrinolysis. Br J Haematol, 2005. 129(3): pp. 307 - 21.

[0240] 15. Munkvad, S., J. Gram and J. Jespersen, A depression of active tissue plasminogen activator in plasma characterizes patients with unstable angina pectoris who develop myocardial infarction. Eur Heart J, 1990. 11(6): pp. 525 - 8.

[0241] 16. Gram, J. and J. Jespersen, A selective depression of tissue plasminogen activator (t-PA) activity in euglobulins characterises a risk group among survivors of acute myocardial infarction. Thromb Haemost, 1987. 57(2): pp. 137 - 9.

[0242] 17. Gram, J., et al., On the usefulness of fibrinolysis variables in the characterization of a risk group for myocardial reinfarction. Acta Med Scand, 1987. 221(2): pp. 149 - 53.

[0243] 18. Jansson, J.H., et al., Hypo - fibrinolysis in patients with hypertension and elevated cholesterol. J Intern Med, 1991. 229(4): pp. 309 - 16.

[0244] 19. Yamada, R., et al., Association between tissue plasminogen activator and serum lipids in healthy volunteers. Ann Med, 1990. 22(5): pp. 313 - 8.

[0245] 20. Glueck, C.J., et al., Endogenous testosterone, fibrinolysis, and coronary heart disease risk in hyperlipidemic men. J Lab Clin Med, 1993. 122(4): pp. 412 - 20.

[0246] 21. Zheng, Z., et al., Interacting hepatic PAI - 1 / tPA gene regulatory pathways influence impaired fibrinolysis severity in obesity. J Clin Invest, 2020. 130(8): pp. 4348 - 4359.

[0247] 22. Zheng, Z., et al., An ATF6-tPA pathway in hepatocytes contributes to systemic fibrinolysis and is repressed by DACH1. Blood, 2019. 133(7): pp. 743-753.

[0248] 23. Tran, K., et al., Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells. J Biol Chem, 2002. 277(34): pp. 31187-200.

[0249] 24. Yamaguchi, J., et al., The conversion of apoB100 low density lipoprotein / high density lipoprotein particles to apoB100 very low density lipoproteins in response to oleic acid occurs in the endoplasmic reticulum and not in the Golgi in McA RH7777 cells. J Biol Chem, 2003. 278(43): pp. 42643-51.

[0250] 25. Millar, J.S., et al., Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339. J Lipid Res, 2005. 46(9): pp. 2023-8.

[0251] 26. Sehayek, E. and S. Eisenberg, The role of native apolipoprotein B-containing lipoproteins in atherosclerosis: cellular mechanisms. Curr Opin Lipidol, 1994. 5(5): pp. 350-353.

[0252] 27. Hinsdale, M.E., et al., ApoB-48 and apoB-100 differentially influence the expression of type-III hyperlipoproteinemia in APOE*2 mice. J Lipid Res, 2002. 43(9): pp. 1520-1528.

[0253] 28. Tiwari, S. and S.A. Siddiqi, Intracellular trafficking and secretion of VLDL. Arterioscler Thromb Vasc Biol, 2012. 32(5): pp. 1079-1086.

[0254] 29. Cheng, D., et al., Very Low Density Lipoprotein Assembly Is Required for cAMP-responsive Element-binding Protein H Processing and Hepatic Apolipoprotein A-IV Expression. J Biol Chem, 2016. 291(45): pp. 23793-23803.

[0255] 30. Weinberg, R.B., et al., ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins. J Lipid Res, 2012. 53(4): pp. 736-743.

[0256] 31. Blade, AM, et al., Biogenesis of apolipoprotein AV and its impacton VLDL triglyceride secretion. J Lipid Res, 2011, 52(2): 237-44.

[0257] 32. Sakurai, T., et al., Measurement of lipoprotein particle sizes using dynamic light scattering. Ann Clin Biochem, 2010. 47(Pt 5): 476-81.

[0258] 33.Sahoo, D., et al., ABCA1-dependent lipid efflux to apolipoprotein AI mediates HDL particle formation and decreases VLDL secretion from murinehepatocytes.J Lipid Res, 2004.45(6): Page 1122-31.

[0259] 34. Athar, H., et al., A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein. J Lipid Res, 2004, 45(4): 764-72.

[0260] 35. Chandler, CE, et al., CP-346086: an MTP inhibitor that lowersplasma cholesterol and triglycerides in experimental animals and in humans. JLipid Res, 2003.44(10): pp. 1887-901.

[0261] 36. Ozcan, L., et al., Hepatocyte DACH1 Is Increased in Obesity via Nuclear Exclusion of HDAC4 and Promotes Hepatic Insulin Resistance. Cell Rep, 2016. 15(10): pp. 2214 - 2225.

[0262] 37. Ghorpade, D.S., et al., Hepatocyte - secreted DPP4 in obesity promotes adipose inflammation and insulin resistance. Nature, 2018. 555(7698): pp. 673 - 677.

[0263] 38. Wang, X., et al., Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non - alcoholic Steatohepatitis. Cell Metab, 2020. 31(5): pp. 969 - 986 e7.

[0264] 39. Shelness, G.S., et al., Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly. J Nutr, 1999. 129(2S suppl): pp. 456S - 462S.

[0265] 40. Andres, D.A., I.M. Dickerson and J.E. Dixon, Variants of the carboxyl - terminal KDEL sequence direct intracellular retention. J Biol Chem, 1990. 265(11): pp. 5952 - 5.

[0266] 41. Gerondopoulos, A., et al., A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval. Elife, 2021. 10.

[0267] 42. Newstead, S. and F. Barr, Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins - SWEET talking the COPs. J Cell Sci, 2020. 133(19).

[0268] 43. Raykhel, I., et al., A molecular specificity code for the three mammalian KDEL receptors. J Cell Biol, 2007. 179(6): pp. 1193 - 204.

[0269] 44. Pu, H., et al., Protease-independent action of tissue plasminogen activator in brain plasticity and neurological recovery after ischemic stroke. Proc Natl Acad Sci U S A, 2019. 116(18): pp. 9115 - 9124.

[0270] 45. de Munk, G.A., et al., Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments. Biochemistry, 1989. 28(18): pp. 7318 - 25.

[0271] 46. Weening-Verhoeff, E.J., et al., Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis. Protein Eng, 1990. 4(2): pp. 191-8.

[0272] 47. Segrest, J.P., et al., Structure of apolipoprotein B-100 in low density lipoproteins. J Lipid Res, 2001. 42(9): pp. 1346-67.

[0273] 48. Hussain, M.M., A. Bakillah and H. Jamil, Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis. Biochemistry, 1997. 36(42): pp. 13060-7.

[0274] 49. Lin, H., et al., Therapeutics targeting the fibrinolytic system. Exp Mol Med, 2020. 52(3): pp. 367-379.

[0275] 50. White, A.L., et al., Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation. J Biol Chem, 1992. 267(22): pp. 15657 - 64.

[0276] 51. Dixon, J.L., S. Furukawa and H.N. Ginsberg, Oleate stimulates secretion of apolipoprotein B-containing lipoproteins from Hep G2 cells by inhibiting early intracellular degradation of apolipoprotein B. J Biol Chem, 1991. 266(8): pp. 5080 - 6.

[0277] 52. Gong, L., et al., Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1. J Biol Chem, 2015. 290(43): pp. 25795 - 804.

[0278] 53. Kaneko, M., et al., Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1. J Biochem, 1992. 111(2): pp. 244 - 8.

[0279] 54. Jiang, C., et al., Serpine 1 induces alveolar type II cell senescence through activating p53-p21-Rb pathway in fibrotic lung disease. Aging Cell, 2017. 16(5): pp. 1114-1124.

[0280] 55. Rustaeus, S., et al., Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation. J Nutr, 1999. 129(2S Suppl): pp. 463S-466S.

[0281] 56. Read, J., et al., A mechanism of membrane neutral lipid acquisition by the microsomal triglyceride transfer protein. J Biol Chem, 2000. 275(39): pp. 30372-7.

[0282] 57. Jovin, I.S. and G. Muller-Berghaus, Interrelationships between the fibrinolytic system and lipoproteins in the pathogenesis of coronary atherosclerosis. Atherosclerosis, 2004. 174(2): pp. 225-33.

[0283] 58. Wu, W., J.D. Bancroft, and J.W. Suttie, "Structural features of the kringle domain determine the intracellular degradation of under-gamma-carboxylated prothrombin: studies of chimeric rat / human prothrombin." Proc Natl Acad Sci U S A, 1997. 94(25): pp. 13654 - 60.

[0284] 59. Novokhatny, V., et al., "Tissue-type plasminogen activator (tPA) interacts with urokinase-type plasminogen activator (uPA) via tPA's lysine binding site. An explanation of the poor fibrin affinity of recombinant tPA / uPA chimeric molecules." J Biol Chem, 1995. 270(15): pp. 8680 - 5.

[0285] 60. Puccetti, L., et al., "Different mechanisms of fibrinolysis impairment among dyslipidemic subjects." Int J Clin Pharmacol Res, 2001. 21(3 - 4): pp. 147 - 55.

[0286] 61. Aso, Y., et al., Metabolic syndrome accompanied by hypercholesterolemia is strongly associated with proinflammatory state and impairment of fibrinolysis in patients with type 2 diabetes: synergistic effects of plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor. Diabetes Care, 2005. 28(9): pp. 2211-6.

[0287] 62. Khan, S.S., et al., A null mutation in SERPINE1 protects against biological aging in humans. Sci Adv, 2017. 3(11): pp. eaao1617.

[0288] 63. Ai, D., et al., Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice. J Clin Invest, 2012. 122(5): pp. 1677-87.

[0289] 64. Widenmaier, S.B., et al., NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis. Cell, 2017. 171(5): pp. 1094-1109 e15.

[0290] 65.Boren, J., S. Rustaeus and SO Olofsson, Studies on the assembly ofapolipoprotein B-100- and B-48-containing very low density lipoproteins inMcA-RH7777 cells. J Biol Chem, 1994.269(41): pp. 25879-88.

[0291] 66.Anaganti, N., S. Rajan and MM Hussain, An improved assay to measure the phospholipid transfer activity of microsomal triglyceride transportprotein. J Lipid Res, 2021.62: Page 100136.

[0292] 67. Basu, D., et al., Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor. Circ Res, 2018. 122(4): Pages 560-567.

[0293] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the techniques disclosed herein without departing from the scope and spirit of the art. The techniques illustratively described herein can be suitably practiced without the presence of any one or more elements or limitations not specifically disclosed herein. Terms and expressions used are used as illustrative rather than restrictive terms and are not intended to exclude any equivalents of the features shown and described or any parts thereof; however, it should be recognized that various modifications can be made within the scope of the art. Therefore, it should be understood that while the art has been described through specific embodiments and optional features, modifications and / or variations of the concepts disclosed herein can be made by those skilled in the art, and such modifications and variations are considered to fall within the scope of the art.

[0294] This document may cite numerous patent and non-patent references. All cited references are incorporated herein by reference in their entirety. If there is any inconsistency between the definition of a term in this specification and the definition of a term in a cited reference, the definition in this specification shall prevail.

[0295] Equivalent solution

[0296] This technology is not limited to the specific embodiments described in this application, which are intended as separate illustrations of a single aspect of the technology. Many modifications and variations can be made to the technology without departing from its spirit and scope, as will be apparent to those skilled in the art. Based on the above description, functionally equivalent methods and apparatuses within the scope of this technology, in addition to those methods and apparatuses listed herein, will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the appended claims. This technology is limited only by the terms of the appended claims and the full scope of equivalents enjoyed by such claims. It should be understood that the present invention is not limited to a particular method, reagent, compound, composition, or biological system, although such particular method, reagent, compound, composition, or biological system may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0297] Furthermore, when features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is also described in accordance with any individual member or subgroup of the Markush Group.

[0298] As those skilled in the art will understand, for any and all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also cover any and all possible subscopes and combinations thereof. As will also be understood by those skilled in the art, all language such as “up to,” “at least,” etc., includes the numbers listed.

[0299] sequence list

Claims

1. An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) The nucleotide sequence listed in SEQ ID NO: 6; (b) The nucleotide sequence listed in SEQ ID NO: 10; (c) The nucleotide sequence listed in SEQ ID NO: 15; (d) The nucleotide sequence listed in SEQ ID NO: 39; (e) The nucleotide sequence listed in SEQ ID NO: 2; (f) A nucleotide sequence that is at least about 85% identical to the nucleotide sequences of any of (a) to (f) and encodes a polypeptide containing a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2), the polypeptide being capable of binding apolipoprotein B (apoB) and / or inhibiting apoB secretion from hepatocytes and / or intestinal epithelial cells, and / or inhibiting apoB lipoprotein lipidation; (g) is the nucleotide sequence of the complement of any of (a) to (f); and (h) An RNA sequence encoded by any of (a) to (g); The nucleotide sequence therein is operatively linked to a heterologous nucleic acid.

2. The polynucleotide molecule according to claim 1, wherein the heterologous nucleic acid comprises an endoplasmic reticulum localization sequence.

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

4. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

8.

5. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

34.

6. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

16.

7. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

4.

8. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

36.

9. The polynucleotide molecule according to claim 3, wherein the nucleotide sequence is listed in SEQ ID NO:

38.

10. The polynucleotide molecule of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence listed in SEQ ID NO:

7.

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

14.

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

35.

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

30.

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

3.

15. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence listed in SEQ ID NO:

9.

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

37.

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

18. The expression vector of claim 17, wherein the one or more regulatory sequences comprise a promoter.

19. The expression vector according to claim 18, wherein the promoter is a thyroxine-binding globulin (TBG) promoter, or wherein the promoter comprises a nucleic acid sequence listed 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 cell of claim 20, wherein the cell is selected from hepatocytes or intestinal epithelial cells.

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

23. The infectious particle of claim 22, wherein the infectious particle is a virus.

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

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

26. A lipid nanoparticle comprising a polynucleotide molecule according to any one of claims 1 to 16.

27. The lipid nanoparticles of claim 26, wherein the lipid nanoparticles are lyophilized, in suspension, or emulsified.

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

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

30. The method of 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 residues, or any combination thereof.

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

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

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

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

35. The method of 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, alexinine, draconin B, dapoxetine, stigmaziin, SK-216, astragalus, fendus, 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 any one of claims 29 to 30, wherein the subject is a human.

38. A polypeptide containing a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof (tPA-K2 polypeptide), comprising: (a) Select the amino acid sequence from the group consisting of the following: (i) The amino acid sequence listed in SEQ ID NO: 5; (ii) The amino acid sequence listed in SEQ ID NO: 13; (iii) The amino acid sequence listed in SEQ ID NO: 9; (iv) The amino acid sequence listed in SEQ ID NO: 30; (v) The amino acid sequence listed in SEQ ID NO: 1; (vi) The amino acid sequence listed in SEQ ID NO: 37; (vii) an amino acid sequence that is at least about 85% identical to the amino acid sequences of any of (i) to (vi) and is capable of binding apolipoprotein B (apoB) and / or inhibiting apoB secretion from hepatocytes and / or intestinal epithelial cells, and / or inhibiting apoB lipoprotein lipoprotein esterification; and (b) Heterologous amino acid sequence.

39. The recombinant tPA-K2 polypeptide of claim 38, wherein the heterologous amino acid sequence comprises an endoplasmic reticulum localization motif.

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

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

7.

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

14.

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

35.

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

37.

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

3.

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

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

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 the subject's plasma triglyceride and / or cholesterol levels.

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 of claim 50, wherein 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 residues, 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 in need, the method comprising administering to the subject a therapeutically effective amount of the recombinant polypeptide according to any one of claims 38 to 51 or the composition according to claim 52.

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

55. The method of 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, simultaneously, alone or sequentially, a therapeutically effective amount of plasminogen activator inhibitor 1 (PAI-1) inhibitor to the subject.

57. The method of 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, alexinine, draconin B, dapoxetine, stigmaziin, SK-216, astragalus, fendusal, 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 comprising the nucleotide sequence listed in SEQ ID NO:

8.

61. The polynucleotide of claim 60, wherein the polynucleotide is formulated for delivery to a subject in an infectious particle.

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

63. The polynucleotide of claim 60, wherein the polynucleotide is formulated for delivery to a subject in lipid nanoparticles (LNPs).

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 in need, the method comprising administering to the subject a therapeutically effective amount of the composition according to claim 64.

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

67. A polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) The nucleotide sequence listed in SEQ ID NO: 34; (b) The nucleotide sequence listed in SEQ ID NO: 10; and (c) The nucleotide sequence listed in SEQ ID NO:

36.

68. The polynucleotide of claim 67, wherein the polynucleotide is formulated for delivery to a subject in an infectious particle.

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

70. The polynucleotide of claim 67, wherein the polynucleotide is formulated for delivery to a subject in lipid nanoparticles (LNPs).

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 in need, the method comprising administering to the subject a therapeutically effective amount of the composition according to claim 71.

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

74. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is listed in SEQ ID NO:

34.

75. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is listed in SEQ ID NO:

10.

76. The polynucleotide molecule according to any one of claims 67 to 73, wherein the nucleotide sequence is listed in SEQ ID NO:

36.

77. A polypeptide containing a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof (tPA-K2 polypeptide), comprising an amino acid sequence selected from the group consisting of the amino acid sequences listed 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 in need, the method comprising administering to the subject a therapeutically effective amount of the recombinant polypeptide of claim 77 or the composition of claim 78.

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

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

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

83. The recombinant tPA-K2 polypeptide according to claim 82, wherein the recombinant tPA-K2 polypeptide comprises a sequence that is at least 80% identical to that of SEQ ID NO:

5.

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

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

86. The recombinant tPA-K2 polypeptide according to any one of claims 82 to 85, wherein the recombinant tPA-K2 polypeptide is bound to apolipoprotein B (apoB).

87. The recombinant tPA-K2 polypeptide according to any one of claims 82 to 86, wherein the recombinant tPA-K2 polypeptide comprises a sequence selected from SEQ ID NO: 13 to 14.

88. The recombinant tPA-K2 polypeptide according to claim 87, wherein the recombinant tPA-K2 polypeptide comprises SEQ ID NO:

14.

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

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

91. The polynucleotide of claim 90, wherein the nucleotide sequence encoding the recombinant tPA-K2 polypeptide comprises a nucleotide sequence selected from SEQ ID NO: 15 to 16.

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

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

94. The polynucleotide of claim 93, wherein the at least one regulatory sequence comprises a promoter.

95. The polynucleotide of claim 94, wherein the promoter is a thyroxine-binding globulin (TBG) promoter, or wherein the promoter comprises SEQ ID NO:

17.

96. The polynucleotide according to any one of claims 90 to 95, wherein the polynucleotide comprises more than one nucleotide sequence encoding the recombinant tPA-K2 polypeptide.

97. A nanoparticle 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. An infectious particle comprising a polynucleotide according to any one of claims 90 to 96.

99. The infectious particle of claim 98, wherein the infectious particle is a virus.

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

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

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

103. A pharmaceutical composition comprising the nanoparticles according to claim 97.

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

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

106. The method of claim 105, wherein the subject suffers from hypercholesterolemia or hyperlipidemia.

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

108. The method of claim 107, wherein the cardiovascular disease comprises 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 of treating cardiovascular disease in a subject in need, the method comprising administering to the subject a tissue plasminogen activator (tPA) or a fragment thereof to treat the subject's cardiovascular disease.

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

112. A method for reducing blood cholesterol levels in a subject in need, the method comprising administering to the subject a tissue plasminogen activator (tPA) or a fragment thereof to reduce the subject's blood cholesterol levels.

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

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

115. The method according to any one of claims 105 to 114, wherein the method reduces the levels of intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), lipoprotein(a) [Lp(a)], chylomicrons, or chylomicron residues 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 a subject of need, the method comprising administering a therapeutically effective amount of a composition comprising a polynucleotide molecule, the polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) The nucleotide sequence listed in SEQ ID NO: 2; (b) The nucleotide sequence encoding the polypeptide sequence listed in SEQ ID NO: 1; (c) A nucleotide sequence that is at least about 85% identical to the nucleotide sequence of any of (a) to (b); (d) is the nucleotide sequence of the complement of any one of (a) to (c); and (e) An RNA sequence encoded by any of (a) through (d); The nucleotide sequence is operatively linked to a heterologous nucleic acid containing an endoplasmic reticulum localization sequence; and The subjects mentioned above had atherosclerotic thrombotic events or were at increased risk of atherosclerotic thrombotic events.

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

119. The method of claim 118, wherein the polynucleotide molecule comprises the nucleotide sequence listed in SEQ ID NO:

4.

120. The method of claim 119, wherein the nucleotide sequence encodes the polypeptide sequence listed in SEQ ID NO:

3.

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

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 of the subject.

123. The method according to any one of claims 117 to 122, wherein the subject is undergoing, will undergo, or has already undergone thrombolytic therapy.

124. The method of claim 123, further comprising administering a tissue plasminogen activator to the subject simultaneously, sequentially, or individually.

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

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

127. The method of claim 126, wherein the subject is undergoing, will undergo, or has undergone treatment with thrombolytic agents, anticoagulants, catheter-directed thrombolysis, and / or thrombectomy.

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