METHODS FOR PREPARING AND USING HIGHLY ACTIVE BLOOD COAGULATION FACTOR XI MUTANT AND GENE THERAPY/EDITING VECTOR AND RECOMBINANT/FUSION PROTEIN THEREOF

Abstract

Methods of preparing and using a highly active blood coagulation factor XI mutant and a gene therapy/editing vector thereof and a recombinant/fusion protein thereof. The nucleotide sequence of the mutant is as shown in SEQ ID NOs: 1-6, and the amino acid sequence is as shown in SEQ ID NO: 7.

Claims

1. A highly active blood coagulation factor XI mutant, comprising: (1) the nucleotide sequence thereof is as shown in SEQ ID NO: 1, the nucleotide at position 1189 is A rather than G; or (2) the nucleotide sequence thereof is as shown in SEQ ID NO: 2, the nucleotide at position 1189 is A rather than G, and the nucleotide at position 1191 is C rather than T; or (3) the nucleotide sequence thereof is as shown in SEQ ID NO: 3, the nucleotide at position 1189 is T rather than G, and the nucleotide at position 1190 is C rather than G; or (4) the nucleotide sequence thereof is as shown in SEQ ID NO: 4, the nucleotide at position 1189 is T rather than G, the nucleotide at position 1190 is C rather than G, and the nucleotide at position 1191 is C rather than T; or (5) the nucleotide sequence thereof is as shown in SEQ ID NO: 5, the nucleotide at position 1189 is T rather than G, the nucleotide at position 1190 is C rather than G, and the nucleotide at position 1191 is A rather than T; or (6) the nucleotide sequence thereof is as shown in SEQ ID NO: 6, the nucleotide at position 1189 is T rather than G, the nucleotide at position 1190 is C rather than G, and the nucleotide at position 1191 is G rather than T; or there is a combination of any other nucleotide mutation at positions 1189, 1190, and 1191.

2. A highly active blood coagulation factor XI mutant protein according to claim 1, wherein the amino acid sequence thereof is as shown in SEQ ID NO: 7, the amino acid at position 397 in the mutant (FXI G397S) is Ser rather than Gly in human wild-type FXI (hFXI), or there is any other amino acid change at position 397.

3. A nucleic acid encoding the highly active blood coagulation factor XI mutant protein according to claim 2, or a nucleic acid having the same length as and being completely complementary to the encoding nucleic acid.

4. A vector expressing the highly active blood coagulation factor XI mutant protein according to claim 2.

5. A method for preparing a highly active blood coagulation factor XI mutant protein, comprising the steps of: (1) inserting a human coagulation factor IX gene of human wild-type or blood coagulation factor XI Gly397Ser mutant into a vector to obtain a recombinant vector; (2) transforming a host cell with the above recombinant vector to obtain a cell clone expressing the recombinant blood coagulation factor XI Gly397Ser mutant; (3) cultivating the above recombinant cell clone in a serum-free medium by continuous perfusion to induce expression of the recombinant blood coagulation factor XI Gly397Ser mutant protein; (4) performing isolation, purification, filtration, final filling and lyophilization to obtain the expressed highly active blood coagulation factor XI Gly397Ser mutant protein.

6. A method of using a highly active blood coagulation factor XI mutant protein and its encoding nucleic acid sequence, wherein the method of using the nucleotide sequence according to claim 2 for preparing a gene therapy drug, comprising ligating the nucleotide sequence with a promoter and/or a termination sequence to construct an expression plasmid, a viral or non-viral vector for gene therapy, expressing the highly active blood coagulation factor XI protein according to claim 2.

7. A method of using the highly active blood coagulation factor XI mutant protein according to claim 2 and its encoding nucleic acid sequence, characterized by the use wherein the method of using in a viral or non-viral vector or template for gene editing.

8. A method of using the highly active blood coagulation factor XI mutant protein according to claim 2, wherein the method of using is for preparing a recombinant protein therapy drug for the treatment of hemophilia or other hemorrhagic diseases.

9. A method of using the highly active blood coagulation factor XI mutant protein according to claim 2, wherein the method of using is for preparing a fusion protein of the blood coagulation factor XI Gly397Ser mutant, and applying to a recombinant protein therapy drug for the treatment of hemophilia or other hemorrhagic diseases.

10. The method of using the highly active blood coagulation factor XI mutant protein according to claim 9, wherein the fusion protein is human albumin, immunoglobulin Fc, transferrin or alpha 1 antitrypsin.

11. A method of using the highly active blood coagulation factor XI mutant protein according to claim 3 and its encoding nucleic acid sequence, wherein the method of using in a viral or non-viral vector or template for gene editing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 to FIG. 7 are schematic diagrams of the sequences of nucleic acids and an encoding protein of a highly active blood coagulation factor XI mutant of the present invention;

[0037] FIG. 8 is a schematic diagram of the vector structure of the present invention;

[0038] FIG. 9 is a schematic diagram of the activity of the recombinant blood coagulation factor XI mutant Gly397Ser;

[0039] FIG. 10 is a schematic diagram of the plasma thrombin generation via the blood coagulation factor XI mutant Gly397Ser remedying in vitro the blood coagulation factor VIII deficiency;

[0040] FIG. 11 is a schematic diagram of the human blood coagulation factor XI antigen level in hemophilia mice after gene transduction of the blood coagulation factor XI mutant Gly397Ser;

[0041] FIG. 12 is a schematic diagram of the activity of the blood coagulation factor XI in hemophilia mice after gene transduction of the blood coagulation factor XI mutant Gly397Ser;

[0042] FIG. 13 is a schematic diagram of the bleeding volume of hemophilia mice with tails amputated after gene transduction of the blood coagulation factor XI mutant Gly397Ser;

[0043] FIG. 14 is a schematic diagram of thromboelastogram for detecting the gene transduced blood coagulation factor XI mutant Gly397Ser remedying the plasma coagulation deficiency in hemophilia mice, in which a represents a group treated with blood coagulation factor XI mutant

[0044] Gly397Ser, b represents a group of normal mice without blood coagulation factor VIII deficiency, and c represents a group of hemophilia mice with blood coagulation factor VIII deficiency.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is further described below in conjunction with specific Examples. It should be understood that these Examples are only used to illustrate the present invention rather than to limit the scope of the present invention. In addition, it should be understood that, upon reading the disclosure of the present application, persons skilled in the art can make various changes or modifications to the present invention, and these equivalents also fall within the scope defined by the appended claims of the present application.

EXAMPLE 1

[0046] The amino acid sequence of a highly active blood coagulation factor XI mutant protein is as shown in SEQ ID NO: 7.

[0047] The method for preparing a highly active blood coagulation factor XI mutant protein includes the steps of:

[0048] (1) inserting a human blood coagulation factor IX encoding gene of human wild-type or blood coagulation factor XI mutant Gly397Ser into a vector to obtain a recombinant vector;

[0049] (2) transforming a host cell with the above recombinant vector to obtain a recombinant expression cell clone;

[0050] (3) cultivating the above cell clone in a serum-free medium to express the highly active blood coagulation factor XI mutant protein;

[0051] The serum-free medium was SAFC Biosciences EX-CELL 302 (commercial reagent). In order to ensure product safety and prevent blood-derived preparations from transmitting infectious diseases, a serum-free medium was used for mammalian cell cultivation and protein expression. When the cells reached a steady state after logarithmic growth, the cell density was kept within the target interval for maintaining high expression of blood coagulation factor XI;

[0052] (4) performing isolation, purification and lyophilization to obtain the expressed highly active coagulation factor XI mutant protein and relevant fusion protein;

[0053] After the medium was collected, it was clarified and filtered by a deep bed filter and further isolated and purified. The purification step was separated into two stages, namely, primary purification and refined purification. The primary purification involved subjecting the filtered and clarified culture solution to 10-fold ultrafiltration concentration, and then inactivating lipid-enveloped viruses such as HIV1/2, HCV and HBV by an organic solvent/detergent method. The refined purification involved further removing residual impurities mainly composed of other proteins secreted by the host cell from the product with chromatography methods such as ion exchange (anion and cation) and molecular sieve. The purified proteins were subjected to ultrafiltration with the medium changed and the formula adjusted. Then, they were filtered through a 20nm nanomembrane to remove the viruses, and lyophilized. The lyophilization process involved quick freezing, quenching, freezing, vacuuming, main drying, and post-drying. The lyophilization formula was mainly composed of inert sugars and inorganic salts, such as, glycine, mannitol, sodium chloride, calcium chloride and the like (the components are glycine, mannitol, sodium chloride, calcium chloride, etc.; the time for lyophilization was 30 hours).

[0054] (5) Method for detecting the blood coagulation factor XI mutant activity and antigen. The specific blood coagulation activity of the blood coagulation factor XI was deduced by comparing the blood coagulation activity and antigen, see FIG. 9. As illustrated in FIG. 9, the blood coagulation factor XI mutant Gly397Ser exhibited higher blood coagulation activity than the wild type.

[0055] Method for detecting the blood coagulation factor XI activity and antigen:

[0056] {circumflex over (1)} Detecting the blood coagulation factor XI activity with the coagulation method:

[0057] The normally mixed plasma was diluted with OV Buffer at the ratios of 1:10, 1:20, 1:40, 1:80, 1: 160, and 1: 320 respectively; the plasma sample to be tested was diluted at the ratios of 1:10 and 1:20 respectively; the cell supernatant solution was kept untreated. 50 l of the diluted normal mixed plasma, the plasma sample to be tested or the cell supernatant solution of the transfected blood coagulation factor XI-expressing vector was added with 50 l of blood coagulation factor XI substrate plasma, and added with an APTT reagent, incubated at 37 C. for 3 minutes, and then added with 500 of calcium chloride. The coagulation time was recorded on the ST4 semi-automatic coagulometer (Stago, France). On the basis that the activity of the blood coagulation factor XI of the normally mixed plasma diluted at the ratio of 1:10 was 100%, a standard curve was established according to the log value of the coagulation time at different dilution rates and the log value of corresponding activity. If the correlation coefficient R2 was greater than 0.95, the value of the sample to be tested would be brought into calculation to obtain the blood coagulation factor XI activity of the sample to be tested.

[0058] {circumflex over (2)} Detecting the blood coagulation factor XI antigen with the double-antibody sandwich method:

[0059] A coating antibody (F9 ELISA kit, Affinity Biologicals, EIA9-0035R1) was diluted with a coating solution (1.59 g/L sodium carbonate and 2.94 g/L sodium bicarbonate, pH 9.6) at the ratio of 1: 100. Then, 100 .sub.itil of the diluted antibody was added per well, incubated at room temperature for 2 hours, and washed for 3 times. The normally mixed plasma sample was doubly diluted with a sample dilute (23.8 g HEPES (free acid)/L, 5.84 g/L NaCl, 3.72g/L Na2EDTA, 10 g/L BSA, 0.1% Tween-20, Ph 7.2) at the ratio from 1: 100 until 1: 3200. The plasma sample to be tested was diluted at the ratios of 1: 200, 1: 400 and 1: 800, and the cell supernatant was kept as the original solution, and diluted at the ratios of 1:10 and 1: 100 respectively. 1000 of the diluted normally mixed plasma or the sample to be tested was added into each well, left at room temperature for 90 minutes, and washed for 3 times. A detection antibody was diluted with a sample diluent at the ratio of 1:100. Then, 1000 of the diluted detection antibody was added to each well, left at room temperature for 90 minutes, and washed for 3 times. 1001 of OPD substrate was added to each well. When a stable yellow color appeared (about 5-10 minutes), 100 l of a stop solution was added to each well. The absorbance was read with a microplate reader at a wavelength of 450 nm. A standard curve was established and the antigen value of the sample to be tested was calculated.

[0060] (6) Using the blood coagulation factor XI mutant to remedy the plasma thrombin generation deficiency in hemophilia A patients

[0061] Thrombin generation test (TGT): a comprehensive test for monitoring the capability of thrombin generation in plasma. The activator (including a tissue factor and phospholipid) was added into the plasma to initiate the coagulation reaction. Then, a thrombin-specific fluorescent substrate was added. The resulting thrombin-catalyzed substrate released fluorescent groups. The fluorescent signal generated was dynamically monitored by a FLUOROSKAN fluorescence reader, and converted into a thrombin generation curve with a matched thrombin generation lab software. The thrombin generation capability was mainly evaluated by several parameters of the curve: (1) lag time, i.e. the time spanning from the start of reaction to the start of thrombin generation; (2) peak, i.e. the maximum amount of thrombin generated; (3) time to peak (ttpeak), i.e. the time spanning from the start of reaction to the peak of thrombin generation; (4) endogenous thrombin potential (ETP), i.e. the area under the thrombin generation curve, which reflects the total amount of thrombin generated.

[0062] The blood coagulation factor XI or its mutant (at the concentration of 5 ug/mL normal physiological concentration) was added into the plasma of hemophilia A patients (deficient in blood coagulation factor VIII) to detect the thrombin generation, see FIG. 10. As illustrated in FIG. 10, the blood coagulation factor XI mutant Gly397Ser could partially remedy the thrombin generation disorder caused by blood coagulation factor VIII deficiency.

EXAMPLE 2

[0063] The method for preparing a plasmid vector of the highly active blood coagulation factor XI for gene therapy comprises the steps of:

[0064] (1) Constructing a plasmid vector by inserting FXI Gly397Ser-encoding cDNA into pcDNA3.1 plasmid containing a CMV promoter.

[0065] (2) Injecting a purified vector into hemophilia A mice. Hemophilia mice of 4-8 weeks old were selected, 6-7 of them were injected with 150 g of plasmid vector dissolved in 2 mL of normal saline through caudal vein, and 6 of them were injected with PBS as a negative control.

[0066] (3) Detecting the blood coagulation factor XI activity with aPTT. 48 hours after the injection, the orbital blood was collected to detect the blood coagulation factor XI activity and antigen, wherein the activity referred to the sum of the activity of the original blood coagulation factor XI in mice and the activity of the human wild-type or mutant blood coagulation factor XI introduced in mice, and the antigen only referred to the level of the human blood coagulation factor XI introduced in mice. The blood coagulation factor XI antigen in mice's plasma after vector gene transduction was shown in FIG. 11. As illustrated in FIG. 11, the expression of blood coagulation factor XI mutant Gly397Ser in the gene-transduced human plasma is similar with the wild-type blood coagulation factor XI. The blood coagulation factor XI activity in mice's plasma after gene transduction of human plasma blood coagulation factor XI mutant Gly397Ser was shown in FIG. 12. As illustrated in FIG. 12, the gene-transduced and expressed human plasma blood coagulation factor XI mutant Gly397Ser exhibited higher blood coagulation activity.

[0067] (4) After the mice were anesthetized, their tails were amputated at the place where the diameter was 2 mm and immersed in PBS. Immediately start time counting for 10 minutes. Then the hemoglobin was detected, and the degree of hemorrhage was estimated according to the amount of hemoglobin (see FIG. 13). As illustrated in FIG. 13, the gene-transfected and expressed human plasma blood coagulation factor XI mutant Gly397Ser could remedy in vivo the blood coagulation deficiency due to blood coagulation factor VIII deficiency, and reduce hemorrhage.

[0068] (5) Drawing blood from the heart of mice to detect thromboelasticity (see FIG. 14)

[0069] Thromboelastogram (TEG): a comprehensive test for monitoring the entire clotting process of the whole blood. Without processing blood specimens, it can accurately provide the patient's coagulation profile by using a small amount of whole blood to monitor interactions between coagulation factors, platelets, fibrinogen, fibrinolysis system, and other cellular components. During the test, the anticoagulated blood was first added into a reagent bottle for activation monitoring; then a certain volume of the blood was sucked out and added into a special cylindrical cup (pre-added with CaCl.sub.2). The cup was rotated at an angle of 445 and a constant speed of 1 round/9 s. The coagulation state of the blood was monitored by a needle suspended via a spiral wire and soaked in the blood, and the curve of coagulation speed and intensity was drawn by a computer. The coagulation process was mainly evaluated by the following curve parameters: (1) the reaction time R value, i.e. the time required from the start of detection to the rise of the curve amplitude to 2mm, which also referred to the time required from the start of specimen detection to the start of fibrin clot formation; (2) the agglutination time K value and the clot formation rate a angle, wherein the agglutination time K value referred to the time required from the end of clotting time to the time when the curve amplitude reached 20 mm, and the clot formation rate a angle referred to the angle between the tangent line from the point of blood clot formation to the maximum arc of the tracing diagram and the horizontal line. They reflected the result of joint action of fibrin and platelets when the blood clot started to form, which was mainly affected by the function of fibrinogen; (3) the MA value which indicated the maximum amplitude of the tracing diagram, i.e. the maximum shear force coefficient. It reflected the strongest kinetic characteristics of the forming fibrin and platelets bonding together and the stability of blood clot formation, wherein the platelets played a greater role than fibrinogen, accounting for about 80%; (4) the coagulation index (CI) value which was deduced in view of the reaction time, agglutination time, clot formation rate, and maximum amplitude in the thromboelastogram curve. It reflected the overall coagulation state of the sample under various conditions, wherein a CI value below 3 indicated low coagulation, a CI value above 3 indicated high coagulation, and a CI value between 3 and 3 indicated normal coagulation.

[0070] As illustrated in FIG. 14, the gene-transduced and expressed human plasma blood coagulation factor XI mutant Gly397Ser could remedy in vivo the blood coagulation deficiency due to blood coagulation factor VIII deficiency.