Recombinant humanized 164.88° collagen type III for vascular restoration

Claims

1. The use of collagen, a fusion protein containing this collagen, a nucleic acid encoding this collagen or fusion protein, a vector containing this nucleic acid, a host cell containing this vector, or a composition containing these collagen, fusion protein, nucleic acid, vector and / or host cell for the manufacture of a medical device, or a drug, or a drug-device combination product, or a kit for the prevention and / or treatment of cardiovascular diseases, for the elimination of vascular damage or for the prevention and / or treatment of vascular damage in a subject, wherein the collagen comprises n repeating units and wherein the repeating unit contains: (1) the sequence indicated in SEQ ID NO: 1; (2) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO: 1; or (3) a sequence in which a substitution, addition, deletion and / or insertion of 1 to 5 amino acid residues is made in the sequence indicated in SEQ ID NO: 1; where n is an integer greater than or equal to 1, preferably an integer in the range from 1 to 32, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, wherein when n is an integer greater than or equal to 2, the repeating sequences are directly linked to each other; preferably the patient has skin damage caused by photoaging due to ultraviolet radiation or an infected wound due to diabetes; collagen preferably has an effect that prevents damage to endothelial cells; preferably, vascular damage is damage to the vascular endothelium; preferably the medical device or drug or drug-device combination product or kit is further capable of preventing and / or treating skin damage caused by ultraviolet radiation photoaging or a diabetic infected wound; preferably the collagen is recombinant humanized collagen type III, preferably 164.88° recombinant humanized collagen type III; preferably the recombinant humanized collagen type III is in trimeric form; preferably, the fusion protein comprises collagen and a protein that promotes the secretion, release and / or purification of collagen; preferably the protein is selected from an enzyme cleavage site sequence, a signal peptide and a purification tag sequence; preferably, the enzyme cleavage site sequence is a TEV protease cleavage site sequence; preferably, the protein tag sequence is a His tag, a GST tag, an MBP tag, a SUMO tag, a Cytiva Protein Select tag, or a NusA tag; preferably the collagen is linked to the protein directly or via a linker, wherein the linker is a flexible linker such as (G) a or (GGGGS) b , where a and b independently of each other represent an integer from 1 to 10, or from 1 to 5, or from 1 to 3; Preferably, the medical device is a gel, a dressing, an invasive device, or an implantable device.

2. The use according to claim 1, wherein the collagen comprises the sequence indicated in SEQ ID No. 2, or does not comprise the sequence indicated in SEQ ID NO:

2.

3. The use according to claim 1 or 2, wherein the collagen comprises: (a) the amino acid sequence SEQ ID NO: 3; (b) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3 while maintaining the effect of the amino acid sequence of SEQ ID NO: 3 in preventing endothelial cell injury; or (c) an amino acid sequence in which 1 to 80, such as 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5 amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3 while maintaining the effect of the amino acid sequence of SEQ ID NO: 3 in preventing endothelial cell damage.

4. The use according to any one of claims 1 to 3, wherein the cardiovascular disease is a cardiovascular disease associated with endothelial cell damage, preferably the endothelial cell damage is angiotensin II or ROS-induced endothelial cell damage, and / or the vascular damage is angiotensin II or ROS-induced vascular damage.

5. The use according to any one of claims 1 to 4, wherein the cardiovascular disease is selected from arteriosclerosis, atherosclerosis, hypertension, ischemic heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease; preferably wherein the hypertension is spontaneous hypertension; preferably the drug is a drug for lowering blood pressure; preferably the vascular endothelial damage is vascular endothelial damage in diabetes.

6. The use according to any one of claims 1 to 5, wherein the composition comprises a lipid regulating agent and / or an antihypertensive agent, such as statins, an angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a beta-receptor blocker.

7. A pharmaceutical composition comprising collagen, a fusion protein containing collagen, a nucleic acid encoding collagen or the fusion protein, a vector containing the nucleic acid, a host cell containing the vector according to any one of claims 1 to 3, and an agent for the prevention and / or treatment of cardiovascular disease or for the elimination of vascular damage, preferably an agent regulating lipid levels and / or an antihypertensive agent such as statins, an angiotensin-converting enzyme inhibitor, a calcium channel blocker and / or a beta-receptor blocker.

8. A medical device comprising collagen, a fusion protein comprising this collagen, a nucleic acid encoding this collagen or fusion protein, a vector comprising this nucleic acid, a host cell comprising this vector according to any one of claims 1 to 3, and a material or device for repairing a damaged vessel, wherein the medical device is preferably a gel, a dressing, an invasive device or an implantable device.

9. A method for blocking or eliminating damage to vascular endothelial cells caused by angiotensin II or ROS in vitro, comprising the step of providing contact of the vascular endothelial cell with collagen according to any one of claims 1 to 3, wherein preferably the vascular endothelial cell is a vascular endothelial cell of the umbilical vein, preferably a vascular endothelial cell of the human umbilical vein.

10. An in vitro method for (1) reducing angiotensin II-induced ROS production in a cell or (2) eliminating microtubule damage, preferably angiotensin II-induced microtubule damage in a cell, comprising the step of contacting the cell with collagen according to any one of claims 1 to 3, wherein the cell is preferably a vascular endothelial cell, preferably a human umbilical vein vascular endothelial cell, preferably a human umbilical vein vascular endothelial cell.

11. A method for screening an active ingredient for the prevention and / or treatment of cardiovascular diseases or for the restoration of vascular damage in a subject, comprising: (i) obtaining collagen containing n repeating units, wherein the repeating unit contains: (1) the sequence indicated in SEQ ID NO: 1; (2) a sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO: 1; or (3) a sequence in which a substitution, addition, deletion and / or insertion of 1 to 5 amino acid residues is made in the sequence indicated in SEQ ID NO: 1; where n is an integer equal to 1 or greater, preferably an integer in the range from 1 to 32, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, where if n is an integer greater than or equal to 2, the repeating sequences are directly linked to each other; (ii) addition of collagen to angiotensin II-treated HUVEC; (iii) selection of collagen having the property of: (a) reducing ROS formation / or (b) reducing microtubule damage, as an active ingredient; and preferably the subject has skin damage caused by photoaging due to ultraviolet radiation or an infected wound due to diabetes; collagen preferably has an effect that prevents damage to endothelial cells; preferably, the active ingredient is also capable of preventing and / or treating skin damage caused by photoaging due to ultraviolet radiation or diabetic infected wound; preferably the cardiovascular disease is a cardiovascular disease associated with endothelial cell injury, preferably the endothelial cell injury is angiotensin II or ROS-induced endothelial cell injury, and / or the vascular injury is angiotensin II or ROS-induced vascular injury; preferably wherein the cardiovascular disease is selected from atherosclerosis, hypertension, coronary heart disease, peripheral vascular disease, stroke, heart failure, arrhythmia, cerebrovascular disease, atrial fibrillation and thrombotic disease; Preferably, the selected collagen contains: (a) the amino acid sequence SEQ ID NO: 3; (b) an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 3; or (c) an amino acid sequence in which 1 to 80, such as 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 10, 1 to 8, or 1 to 5 amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence of SEQ ID NO: 3; preferably, the active ingredient can be used as a drug, or as a medical device, or as a combination product of a drug and a device.