Recombinant tissue plasminogen activator (TPA) fragments and uses thereof
Patent Information
- Application Number
- HK62026126861
- Authority / Receiving Office
- HK · HK
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2023-05-18
- Filing Date
- 2026-07-30
- Publication Date
- 2026-10-09
- Estimated Expiration
- 2044-05-16
Smart Images
Figure 00000000_0000_ABST 
Figure 00000000_0003_ABST 
Figure 00000000_0002_ABST 
Figure 00000000_0001_ABST
Abstract
Description
This technology relates to recombinant polypeptides or nucleotides encoding such polypeptides, which contain a tissue-type plasminogen activator (tPA) fragment, and for the treatment of cardiovascular diseases. In one embodiment, the tPA fragment contains the kringle 2 domain of tPA. Abstract
Claims
CLAIMSWhat is claimed is:
1. An isolated polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence set forth in SEQ ID NO: 6;(b) a nucleotide sequence set forth in SEQ ID NO: 10;(c) a nucleotide sequence set forth in SEQ ID NO: 15;(d) a nucleotide sequence set forth in SEQ ID NO: 39;(e) a nucleotide sequence set forth in SEQ ID NO: 2;(f) a nucleotide sequence that is at least about 85% identical to the nucleotide sequences of any one of (a) - (f), and which encodes a recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptide that is capable of binding apolipoprotein B (apoB), and / or inhibiting apoB secretion from a hepatocyte and / or an enterocyte, and / or inhibiting apoB lipoprotein lipidation;(g) a nucleotide sequence that is the complement of any one of (a) - (f); and(h) an RNA sequence encoded by any one of (a) - (g); wherein the nucleotide sequence is operably linked to a heterologous nucleic acid.
2. The polynucleotide molecule of claim 1, wherein the heterologous nucleic acid comprises an endoplasmic reticulum localization sequence.
3. The polynucleotide molecule of claim 2, wherein the endoplasmic reticulum localization sequence encodes for an amino acid having the sequence KDEL (SEQ ID NO: H).
4. The polynucleotide molecule of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 8.
5. The polynucleotide molecule of 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 34.
6. The polynucleotide molecule of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 16.
7. The polynucleotide molecule of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 4.
8. The polynucleotide molecule of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 36.
9. The polynucleotide molecule of claim 3, wherein the nucleotide sequence is set forth in SEQ ID NO: 38.
10. The polynucleotide molecule of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7.
11. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14.
12. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 35.
13. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 30.
14. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.
15. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 9.
16. The polynucleotide of claim 1, wherein the nucleotide sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 37.
17. An expression vector comprising the polynucleotide molecule of any one of claims 1-16, operably linked to one or more regulatory sequences suitable for directing expression in a eukaryotic cell.
18. The expression vector of claim 17, wherein the one or more regulatory sequences comprises a promoter.
19. The expression vector of claim 18, wherein the promoter is a thyroxine binding globulin (TBG) promoter or wherein the promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 17.
20. A cell comprising the polynucleotide molecule of any one of claims 1-16 or the expression vector of any one of claims 17-19.
21. The cell of claim 20, wherein the cell is selected from a hepatocyte or an enterocyte.
22. An infectious particle comprising the polynucleotide molecule of any one of claims 1-16.
23. The infectious particle of claim 22, wherein the infectious particle is a virus.
24. The infectious particle of claim 23, wherein the virus in an adeno-associated virus (AAV).
25. The infectious particle of claim 24, wherein the AAV is AAV8.
26. A lipid nanoparticle comprising the polynucleotide molecule of any one of claims 1- 16.
27. The lipid nanoparticle of claim 26, wherein the lipid nanoparticle is lyophilized, in a suspension, or emulsified.
28. A composition comprising the polynucleotide molecule of any one of claims Bill 16, the vector of any one of claims 17-19, the infectious particle of any one of claims B22- B25, or the lipid nanoparticle of claim 26 or claim 27, and a pharmaceutically acceptable carrier.
29. A method for lowering plasma apoB lipoproteins in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 28 to the subject.
30. The method of claim 29, wherein the plasma apoB lipoproteins are selected from very low density lipoproteins (VLDLs), intermediate density lipoproteins (IDLs), low density lipoproteins (LDLs), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof.
31. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering a therapeutically effective amount of the composition of claim 28 to the subject.
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 lowering plasma triglyceride and / or cholesterol levels in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 28 to the subject.
34. The method of any one of claims 29-33, further comprising simultaneously, separately, or sequentially administering a therapeutically effective amount of a 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, Aleplasinin, Loureirin B, Diaplasinin, Toddalolactone, SK-216, Geodin, Fendosal, AZ3976, TM5007; and any combination thereof.
36. The method of any one of claims 29-35, wherein the composition is administered intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly to the subject.
37. The method of any one of claims 29-30, wherein the subject is human.
38. A recombinant tissue plasminogen activator kringle 2 domain (tPA-K2)-containing polypeptide, or a pharmaceutically acceptable salt, tautomer, hydrate, and / or solvate thereof, (tPA-K2 polypeptide) comprising:(a) an amino acid sequence selected from the group consisting of:(i) the amino acid sequence set forth in SEQ ID NO: 5;(ii) the amino acid sequence set forth in SEQ ID NO: 13;(iii) the amino acid sequence set forth in SEQ ID NO: 9;(iv) the amino acid sequence set forth in SEQ ID NO: 30;(v) the amino acid sequence set forth in SEQ ID NO: 1;(vi) the amino acid sequence set forth in SEQ ID NO: 37;(vii) an amino acid sequence that is at least about 85% identical to the amino acid sequence of any one of (i)-(vi), and which is capable of binding apolipoprotein B (apoB), and / or inhibiting apoB secretion from a hepatocyte and / or an enterocyte, and / or inhibiting apoB lipoprotein lipidation; and(b) a 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 of claim 39, wherein the endoplasmic reticulum localization motif is KDEL.
41. The recombinant tPA-K2 polypeptide of claim 40, wherein the amino acid sequence is set forth in SEQ ID NO: 7.
42. The recombinant tPA-K2 polypeptide of claim 40, wherein the amino acid sequence is set forth in SEQ ID NO: 14.
43. The recombinant tPA-K2 polypeptide of claim 40, wherein the amino acid sequence is set forth in SEQ ID NO: 35.
44. The recombinant tPA-K2 polypeptide of claim 40, wherein the amino acid sequence is set forth in SEQ ID NO: 37.
45. The recombinant tPA-K2 polypeptide of claim 40, wherein the amino acid sequence is set forth in SEQ ID NO: 3.
46. The recombinant tPA-K2 polypeptide of any one of claims 38-45, wherein the polypeptide does not comprise serine protease function.
47. The recombinant tPA-K2 polypeptide of any one of claims 38-46, wherein the polypeptide is not fibrinolytic.
48. The recombinant polypeptide of any one of claims 38-47, wherein the polypeptide binds to apoB.
49. The recombinant polypeptide of any one of claims 38-48, wherein administration of a therapeutically effective amount of the polypeptide to a subject reduces plasma triglyceride and / or cholesterol levels in the subject.
50. The recombinant polypeptide of any one of claims 38-49, 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 the one or more apoB lipoproteins is selected from very low density lipoproteins (VLDLs), intermediate density lipoproteins (IDLs), low density lipoproteins (LDLs), lipoprotein a (Lp(a)), chylomicrons, chylomicron remnants, or any combination thereof.
52. A composition comprising the recombinant polypeptide of any one of claims 38-51 and a pharmaceutically acceptable carrier.
53. A method for lowering plasma apoB lipoproteins in a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant polypeptide of any one of claims 38-51 or the composition of claim 52 to the subject.
54. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering a therapeutically effective amount of the recombinant polypeptide of any one of claims 38-51 or the composition of claim 52 to the subject.
55. The method of claim 54, wherein the disease is selected from atherosclerotic cardiovascular disease, hypercholesterolemia, hyperlipidemia, or type 2 diabetes.
56. The method of any one of claims 53-55, further comprising simultaneously, separately, or sequentially administering a therapeutically effective amount of a 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, Aleplasinin, Loureirin B, Diaplasinin, Toddalolactone, SK-216, Geodin, Fendosal, AZ3976, TM5007; and any combination thereof.
58. The method of any one of claims 53-57, wherein the recombinant polypeptide or composition is administered intravenously, intraperitoneally, subcutaneously, intrabuccally, intradermally, intrahepatically, or intramuscularly to the subject.
59. The method of any one of claims 53-58, wherein the subject is human.
60. A polynucleotide molecule comprising a nucleotide sequence set forth 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 an adeno- associated virus (AAV).
63. The polynucleotide of claim 60, wherein the polynucleotide is formulated for delivery to a subject in a lipid nanoparticle (LNP).
64. A composition comprising the polynucleotide of any one of claims 60-63, and a pharmaceutically acceptable carrier.
65. A method for lowering plasma apoB lipoproteins in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 64 to the subject.
66. A method for lowering plasma triglyceride and / or cholesterol levels in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 64 to the subject.
67. A polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence set forth in SEQ ID NO: 34;(b) a nucleotide sequence set forth in SEQ ID NO: 10; and(c) a nucleotide sequence set forth 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 an adeno- associated virus (AAV).
70. The polynucleotide of claim 67, wherein the polynucleotide is formulated for delivery to a subject in a lipid nanoparticle (LNP).
71. A composition comprising the polynucleotide of any one of claims 67-70, and a pharmaceutically acceptable carrier.
72. A method for lowering plasma apoB lipoproteins in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 71 to the subject.
73. A method for lowering plasma triglyceride and / or cholesterol levels in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of claim 71 to the subject.
74. The polynucleotide molecule of any one of claims 67-73, wherein the nucleotide sequence is set forth in SEQ ID NO: 34.
75. The polynucleotide molecule of any one of claims 67-73, wherein the nucleotide sequence is set forth in SEQ ID NO: 10.
76. The polynucleotide molecule of any one of claims 67-73, wherein the nucleotide sequence is set forth in SEQ ID NO: 36.
77. A recombinant tissue plasminogen activator kringle 2 domain (tPA-K2) )-containing polypeptide, 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 sequence set forth in SEQ ID NO: 9 and SEQ ID NO: 35.
78. A composition comprising the recombinant polypeptide of claim 77 and a pharmaceutically acceptable carrier.
79. A method for lowering plasma apoB lipoproteins in a subject in need thereof, the method comprising administering a therapeutically effective amount of the recombinant polypeptide of claim 77 or the composition of claim 78 to the subject.
80. A method for treating a disease associated with elevated plasma apoB lipoprotein levels, the method comprising administering a therapeutically effective amount of the recombinant polypeptide of claim 77 or the composition of claim 78 to the subject.
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 of claim 82, wherein the recombinant tPA-K2 polypeptide comprises a sequence with at least 80% identity to SEQ ID NO: 5.
84. The recombinant tPA-K2 polypeptide of claim 82 or 83, wherein the polypeptide does not comprise serine protease function.
85. The recombinant tPA-K2 polypeptide of claim 82 or 83, wherein the polypeptide is not fibrinolytic.
86. The recombinant tPA-K2 polypeptide of any one of claims 82-85, wherein the recombinant tPA-K2 polypeptide binds to apolipoprotein B (apoB).
87. The recombinant tPA-K2 polypeptide of any one of claims 82-86, wherein the recombinant tPA-K2 polypeptide comprises a sequence selected from SEQ ID NOs: 13-14.
88. The recombinant tPA-K2 polypeptide of claim 87, wherein the recombinant tPA-K2 polypeptide comprises SEQ ID NO: 14.
89. A method of making the recombinant tPA-K2 polypeptide of any one of claims 82- 88.
90. A polynucleotide comprising a nucleotide sequence encoding the recombinant tPA- K2 polypeptide of any one of claims 82-88.
91. The polynucleotide of claim 90, wherein the nucleotide sequence encoding the recombinant tPA-K2 polypeptide comprises a nucleotide sequence selected from SEQ ID NOs: 15-16.
92. The polynucleotide of claim 91, further comprising at least one regulatory sequence operably 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 thyroxine binding globulin (TBG) promoter or wherein the promoter comprises SEQ ID NO: 17.
96. The polynucleotide of any one of claim 90-95, wherein the polynucleotide comprises more than one nucleotide sequence encoding the recombinant tPA-K2 polypeptide.
97. A nanoparticle comprising the recombinant tPA-K2 polypeptide of any one of claims 1-6 or the polynucleotide of any one of claims 90-96.
98. An infectious particle comprising the polynucleotide of any one of claims 90-96.
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 an adeno-associated virus (AAV).
101. The infectious particle of claim 100, wherein the AAV is an AAV8.
102. A pharmaceutical composition comprising the recombinant tPA-K2 polypeptide of any one of claims 82-88.
103. A pharmaceutical composition comprising the nanoparticle of claim 97.
104. A pharmaceutical composition comprising the infectious particle of any one of claims 98-100.
105. A method comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 102-104 to a subject.
106. The method of claim 105, wherein the subject has hypercholesterolemia or hyperlipidemia.
107. The method of claim 105, wherein the subject has been diagnosed with a cardiovascular disease.
108. The method of claim 107, wherein the cardiovascular disease comprises atherosclerosis.
109. The method of any one of claims 105-108, wherein the subject has been diagnosed with type 2 diabetes.
110. A method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering tissue plasminogen activator (tPA), or a fragment thereof, to the subject to treat the cardiovascular disease in the subject.
111. The method of claim 110, wherein the tPA fragment comprises a tPA-K2 domain.
112. A method of reducing blood cholesterol levels in a subject in need thereof, the method comprising administering tissue plasminogen activator (tPA), or a fragment thereof, to the subject to reduce blood cholesterol levels in the subject.
113. The method of claim 112, wherein the tPA fragment comprises a tPA-K2 domain.
114. The method of any one of claims 110-113, wherein administration comprises oral administration or intravenous administration.
115. The method of any one of claims 105-114, wherein the method reduces a level of intermediate density lipoproteins (IDLs), low density lipoproteins (LDLs), very low density lipoproteins (VLDLs), lipoprotein (a) [Lp(a)], chylomicron, or chylomicron remnants in the serum of the subject.
116. The method of any one of claims 105-115, wherein the tPA comprises the pharmaceutical composition of any one of claims 102-104.
117. A method for lowering plasma lipids and / or apolipoprotein B (apoB) levels in a subject in need thereof, 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 set forth in SEQ ID NO: 2;(b) a nucleotide sequence that encodes for the polypeptide sequence set forth in SEQ ID NO: 1;(c) a nucleotide sequence that is at least about 85% identical to the nucleotide sequences of any one of (a) - (b);(d) a nucleotide sequence that is the complement of any one of (a) - (c); and(e) an RNA sequence encoded by any one of (a) - (d); wherein the nucleotide sequence is operably linked to a heterologous nucleic acid comprising an endoplasmic reticulum localization sequence; and wherein the subject is suffering from or at an increased risk for atherothrombotic events.
118. The method of claim 117, wherein the endoplasmic reticulum localization sequence encodes for a polypeptide having the amino acid sequence KDEL (SEQ ID NO: 11).
119. The method of claim 118, wherein the polynucleotide molecule comprises a nucleotide sequence set forth in SEQ ID NO: 4.
120. The method of claim 119, wherein the nucleotide sequence encodes for a polypeptide sequence set forth in SEQ ID NO: 3.
121. The method of any one of claims 117-120, wherein the composition is formulated for delivery to the subject in a lipid nanoparticle or adeno-associated virus.
122. The method of any one of claims 117-121, wherein the administration of the composition lowers plasma apoB and / or lipid levels in the subject.
123. The method of any one of claims 117-122, wherein the subject is undergoing, will undergo, or has undergone thrombolytic therapy.
124. The method of claim 123, further comprising simultaneously, sequentially, or separately administering a tissue plasminogen activator to the subject.
125. The method of any one of claims 117-124, further comprising simultaneously, sequentially, or separately administering an anticoagulant to the subject.
126. The method of any one of claims 29-37, 53-59, 65-66, 72-73, 79-81, or 105-116, wherein the subject is suffering from or at an increased risk for atherothrombotic events.
127. The method of claim 126, wherein the subject is undergoing, will undergo, or has undergone treatment with a thrombolytic agent, an anticoagulant, catheter-directed thrombolysis, and / or thrombectomy.