Double-stranded siRNA, shRNA, pharmaceutical composition and application for regulating protease activated receptor 4 gene expression

Abstract

The present invention discloses a double-stranded siRNA, shRNA, pharmaceutical composition and application for regulating the expression of protease activated receptor 4 gene, belonging to the field of gene biomedicine, wherein the double-stranded siRNA is selected from one of SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12 and SEQ ID NO.13-14. The present invention designs siRNA and shRNA with excellent protease activated receptor 4 expression inhibitory activity, and has good in vitro preparation of PAR4-deficient platelets by combining with iPSC and other technologies. The PAR4-deficient platelets prepared by the present invention have good hemostatic ability and low risk of thrombosis, making platelet transfusion therapy safer.

Description

Technical Field

[0001] The present invention belongs to the field of gene biomedicine, and specifically relates to a double-stranded siRNA and shRNA pharmaceutical composition for regulating the expression of protease activated receptor 4 (PAR4) gene and its application. Background Art

[0002] Platelets are anucleated blood cells in mammals, produced by megakaryocytes (MKs), and are mainly involved in coagulation, thrombosis, maintaining vascular integrity, immunity, and wound healing. Abnormalities in platelet function and / or quantity in patients may be life-threatening. Platelet transfusion is the most effective method for clinical treatment of thrombocytopenia, and is an indispensable means in emergency hemostasis and emergency response situations. Currently, blood donation is the only way to collect platelet preparations for transfusion, and platelets obtained through traditional blood donation routes are in short supply.

[0003] The technology of using human induced pluripotent stem cells (hiPSC) to differentiate immortalized megakaryocyte cell lines (imMKCL) and then using these megakaryocytes to generate platelets in vitro has made great progress and is expected to solve the problem of insufficient platelet supply. Clinical trials of HLA-homologous platelets derived from human iPSC have been launched, and the first patient has been treated without any adverse reactions, proving the feasibility of producing functional platelets in vitro. On this basis, researchers have developed a universal platelet for the HLA gene. This platelet has no human leukocyte antigen on its surface and will not produce immune rejection when injected into the human body, thus expanding the population suitable for artificially produced platelets as much as possible. This shows that it is feasible to develop "functional" platelets using iPSC and other means such as in vitro gene editing.

[0004] Platelet transfusions may induce thrombosis in some patients with diseases associated with increased platelet activation and consumption, and may aggravate their condition. A retrospective analysis of patients who received clinical platelet transfusions showed that patients with thrombotic thrombocytopenic purpura (TTP), heparin-induced thrombocytopenia (HIT), and immune thrombocytopenic purpura (ITP) are prone to thrombotic complications, but platelet transfusions are still required when uncontrollable active bleeding occurs and threatens their lives. Statistical analysis suggests that in these patients, platelet transfusions are closely related to arterial thrombosis and death. Currently, there is no targeted research on the use of platelets in these special patients.

[0005] Protease-activated receptor 4 (PAR4) is a thrombin receptor on the surface of platelets, and its function has been investigated by using PAR4 knockout (PAR4 - / - ) mice. - / -The mouse model exhibited an anti-thrombotic phenotype, with a three-fold increase in vessel occlusion time in a ferric chloride-induced mouse mesenteric artery injury model. Other studies have also found that PAR4 - / - Mice are similarly protected against laser-induced ablation of mesenteric arteriolar endothelial cells, electrolyte-induced, and trauma-induced carotid artery injury. PAR4 deficiency also protects against pulmonary embolism and cerebral infarction following cerebral artery occlusion. PAR4 deficiency does not affect the integrity of the hemostatic response in mice, and there is no evidence that PAR4 - / - Mice will experience spontaneous bleeding. Therefore, PAR4 is considered a safe and effective antithrombotic target, and two small molecule inhibitors are in Phase II clinical trials. There is still a lack of RNA-type molecules or drugs targeting protease-activated receptor 4 for antithrombotic fields and in vitro platelet production, which has great application prospects and significance. Summary of the invention

[0006] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a double-stranded siRNA, shRNA, and a pharmaceutical composition for regulating the expression of protease-activated receptor 4, which can effectively inhibit the expression of PAR4 protein and be used to generate PAR4-deficient platelets in vitro, and develop new platelet products. Such platelets retain basic hemostatic ability while having lower thrombosis ability, and can be used for platelet transfusion. When special populations such as elderly patients or patients with platelet-consuming diseases need platelet transfusion, the risk of thrombosis is reduced, thereby exerting a safer therapeutic effect.

[0007] The present invention also provides the application of the double-stranded siRNA, shRNA and pharmaceutical composition. The present invention proposes a novel platelet product which retains the hemostatic ability but does not have the thrombogenic ability, making platelet transfusion safer.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a double-stranded siRNA for regulating the expression of protease-activated receptor 4 gene, wherein the double-stranded siRNA comprises a positive strand and an antisense strand, and the double-stranded siRNA is selected from any one of SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, and SEQID NO.13-14.

[0009] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 001) is 5'-UGACAGCACGCCCUCAAUC -3' (SEQ ID NO: 1), and the antisense strand is 5'-GAUUGAGGGCGUGCUGUCA-3' (SEQ ID NO: 2).

[0010] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 002) is 5'-GUGCCAAUGACAGUGACAC-3' (SEQ ID NO: 3), and the antisense strand is 5'-GUGUCACUGUCAUUGGCAC-3' (SEQ ID NO: 4).

[0011] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 003) is 5'-CUCCACCAUGCUGCUGAUG-3' (SEQ ID NO: 5), and the antisense strand is 5'-CAUCAGCAGCAUGGUGGAG-3' (SEQ ID NO: 6).

[0012] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 004) is 5'-GUCACAUGUAUGGCUCAGU-3' (SEQ ID NO: 7), and the antisense strand is 5'-ACUGAGCCAUACAUGUGAC-3' (SEQ ID NO: 8).

[0013] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 005) is 5'-UUGGACUCUGCAUGGCUGC-3' (SEQ ID NO: 9), and the antisense strand is 5'-GCAGCCAUGCAGAGUCCAA-3' (SEQ ID NO: 10).

[0014] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 006) is 5'-CAACCUCUAUGGUGCCUAC-3' (SEQ ID NO: 11), and the antisense strand is 5'-GUAGGCACCAUAGAGGUUG-3' (SEQ ID NO: 12).

[0015] The sense strand of one of the double-stranded siRNAs (double-stranded number: SD 007) is 5'-GCGUGGAUCCCUUCAUCUA-3' (SEQ ID NO: 13), and the antisense strand is 5'-UAGAUGAAGGGAUCCACGC-3' (SEQ ID NO: 14).

[0016] A more preferred double-stranded siRNA (double-stranded number: SD 007) has a sense strand of 5'-GCGUGGAUCCCUUCAUCUA-3' (SEQ ID NO: 13) and an antisense strand of 5'-UAGAUGAAGGGAUCCACGC-3' (SEQ ID NO: 14).

[0017] The shRNA for regulating the expression of the protease activated receptor 4 gene of the present invention comprises the double-stranded siRNA, a stem-loop structure connecting the sense strand and the antisense strand of the siRNA, a sequence complementary to the sticky end of the vector restriction site, and a terminator.

[0018] The stem-loop structure sequence connecting the sense strand and the antisense strand of the siRNA includes any one of 5'-TTCAAGAGA-3', 5'-TCAAGAG-3', 5'-CTCGAG-3', and 5'-TAGTGAAGCCACAGATGTA-3'.

[0019] Among them, the sequence complementary to the sticky end added to the 5' end of the sense chain of the shRNA includes one of the following: 5'-GATCC-3', 5'-CCGG-3'; the sequence complementary to the sticky end added to the 5' end of the antisense chain of the shRNA includes one of the following: 5'-AATTC-3', 5'-AATT-3'; the terminator sequence added to the 3' end of the sense chain of the shRNA includes one of the following: 5'-TTTTTT-3', 5'-TTTTT-3', 5'-TTTTTG-3'.

[0020] The sense strand sequence of the shRNA (double-strand number: SD 008) is: 5'-CCGGGCGTGGATCCCTTCATCTATCAAGAGTAGATGAAGGGATCCACGCTTTTTTG-3' (SEQ ID NO: 15); the antisense strand sequence of the shRNA is: 5'-AATTCAAAAAAGCGTGGATCCCTTCATCTACTCTTGATAGATGAAGGGATCCACGCG-3' (SEQ ID NO: 16).

[0021] In the double-stranded siRNA or the shRNA of the present invention, each base may be modified by one or more modifications including but not limited to 2'-O-methyl (2'-O-methyl, 2'-OMe), 2'-fluoro (2'-fluoro, 2'-F), thiophosphate modification, cholesterol modification, etc.

[0022] During synthesis, in order to improve the stability and efficiency of siRNA, an additional TT sequence can be added to the 3' end of the sequence.

[0023] The pharmaceutical composition of the present invention contains the double-stranded siRNA or the shRNA and a pharmaceutically acceptable carrier or excipient.

[0024] Among them, the pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, including but not limited to liposomes, lipid nanoparticles, polymer nanoparticles, magnetic nanoparticles, calcium phosphate nanoparticles, mesoporous silica, etc., and one or more of their derivatives. In addition, the vector can also be an expression vector of shRNA, including but not limited to one of a plasmid vector, a lentiviral vector, an adenoviral vector, an AAV viral vector, a piggyBac vector, etc. The excipient can be various preparations or compounds conventionally used in the art, such as one or more of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0025] Furthermore, the double-stranded siRNA, or the above-mentioned shRNA or pharmaceutical composition is used to regulate the expression of protease activated receptor 4.

[0026] Furthermore, the double-stranded siRNA, or the above-mentioned shRNA or pharmaceutical composition is used in the preparation of an inhibitor of protease activated receptor 4 (PAR4) expression.

[0027] The double-stranded siRNA or the shRNA or the pharmaceutical composition of the present invention is used in preparing engineered platelets lacking PAR4.

[0028] Furthermore, the double-stranded siRNA or the shRNA or the pharmaceutical composition of the present invention is used in the production of engineered platelets lacking PAR4 in vitro.

[0029] The PAR4-deficient engineered platelets are engineered platelets with hemostatic ability and lower thrombogenicity ability.

[0030] Among them, PAR4-deficient engineered platelets can be obtained by transfecting platelet source cells with an expression system containing shRNA; wherein the expression system can be a pharmaceutically acceptable vector such as a lentiviral vector, and the platelet source cells can be engineered into a cell library that can stably express shRNA through pressure screening, and megakaryocytes can be induced to differentiate into platelets in a platelet bioreactor.

[0031] The source cells of platelets include, but are not limited to, primary hematopoietic stem cells and their progenitor cells, megakaryocytes and their progenitor cells, iPSCs, hematopoietic stem cells or megakaryocytes and their progenitor cells or immortalized megakaryocytes derived from iPSCs, and various megakaryocytes derived from reprogramming or transdifferentiation technologies, etc. In addition, platelet-like particles PLP produced by MEG-01 cells can also be used.

[0032] The PAR4-deficient engineered platelets produced in vitro by using specific double-stranded siRNA or the shRNA or the pharmaceutical composition of the present invention have hemostatic ability and low thrombosis ability. They can be used for platelet transfusion, especially for patients with clinical diseases related to enhanced platelet activation and increased consumption, and have a low risk of thrombosis while treating bleeding, greatly increasing the safety of platelet transfusion.

[0033] The present invention designs siRNA for regulating protease activated receptor 4, and obtains sequences with different interference effects. At the same time, the above siRNA sequence is designed into shRNA, and is used to produce PAR4-deficient platelets, which have comparable hemostatic ability and lower thrombosis ability compared to existing platelets, and are safer for clinical platelet transfusion. The present invention designs and obtains siRNA with the best interference efficiency for PAR4, and at the same time proposes for the first time to prepare PAR4-deficient platelet products for platelet transfusion, and the prepared PAR4-deficient platelets are safer.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] The present invention has designed siRNA and shRNA sequences with excellent protease activated receptor 4 (PAR4) expression inhibition activity, and has good in vitro ability to prepare PAR4-deficient platelets by combining with iPSC and other technologies. The PAR4-deficient platelets prepared by the present invention have good hemostatic ability and low risk of thrombosis. Compared with the existing donor-derived platelets or iPSC-derived platelets, the hemostatic ability is equivalent but the safety is greatly improved. The platelets prepared by the present invention reduce the risk of thrombosis when special populations such as elderly patients or patients with platelet-consuming diseases need platelet transfusion, thereby playing a safer therapeutic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the relative expression level of PAR4 gene in MEG-01 cells after 24h and 48h of transfection with siRNA of Example 1. Compared with NC, P<0.05, P<0.01, P<0.001, ns means no significance, P>0.05.

[0037] Figure 2 The cellular calcium mobilization after PAR4 activation 72 hours after the siRNA of Example 1 was transfected into MEG-01 cells was used as an indicator for evaluating platelet activation.

[0038] Figure 3 is the relative expression level of PAR4 gene after the shRNA of Example 2 acted on MEG-01 cells. Compared with the NC group, P<0.001.

[0039] Figure 4 The aggregation rate of washed platelets from donors in Example 4, unedited iPSC-platelets, and PAR4-deficient iPSC-platelets after activation with thrombin and PAR4 agonist peptide AYP in vitro, respectively. P<0.001, ns means no significance, P>0.05.

[0040] Figure 5 is the tail bleeding time of mice after platelet transfusion statistically measured in Example 5. Wherein ns means no significance, P>0.05.

[0041] Figure 6 This is the time of mesenteric thrombosis induced by ferric chloride after platelet transfusion statistically described in Example 5. P<0.001, ns means no significance, P>0.05. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with embodiments and drawings.

[0043] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0044] The sequences designed in the present invention are all directly synthesized by biological companies. The siRNA sequences are synthesized by a common chemical synthesis method.

[0045] The cationic lipids (ALC-0315), auxiliary lipids (DSPC), cholesterol (CHO-HP) and polyethylene glycol lipids (DMG-PEG2000) used to prepare LNPs required for in vitro transfection were all purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., with product numbers ACL-0315: O02008, DSPC: S01005, CHO-HP: O01001, and DMG-PEG2000: O02005.

[0046] Lentiviral plasmid vector (pLKO-1 vector) Merck, SHC016V.

[0047] Example 1

[0048] Synthesis of siRNA, preparation of vectors and analysis of silencing ability of different siRNAs

[0049] Using the FASTA sequence of PAR4 mRNA as a template, siRNA was designed, the base ratio and quality were evaluated, and adjustments were made to obtain double-stranded siRNA, including a sense strand and an antisense strand, consisting of 19 to 22 base pairs.

[0050] The sense strand of the double-stranded siRNA (double-stranded number: SD 001) is 5'-UGACAGCACGCCCUCAAUC -3' (SEQ ID NO: 1), and the antisense strand is 5'-GAUUGAGGGCGUGCUGUCA-3' (SEQ ID NO: 2).

[0051] The sense strand of the double-stranded siRNA (double-stranded number: SD 002) is 5'-GUGCCAAUGACAGUGACAC-3' (SEQ ID NO: 3), and the antisense strand is 5'-GUGUCACUGUCAUUGGCAC-3' (SEQ ID NO: 4).

[0052] The sense strand of the double-stranded siRNA (double-stranded number: SD 003) is 5'-CUCCACCAUGCUGCUGAUG-3' (SEQ ID NO: 5), and the antisense strand is 5'-CAUCAGCAGCAUGGUGGAG-3' (SEQ ID NO: 6).

[0053] The sense strand of the double-stranded siRNA (double-stranded number: SD 004) is 5'-GUCACAUGUAUGGCUCAGU-3' (SEQ ID NO: 7), and the antisense strand is 5'-ACUGAGCCAUACAUGUGAC-3' (SEQ ID NO: 8).

[0054] The sense strand of the double-stranded siRNA (double-stranded number: SD 005) is 5'-UUGGACUCUGCAUGGCUGC-3' (SEQ ID NO: 9), and the antisense strand is 5'-GCAGCCAUGCAGAGUCCAA-3' (SEQ ID NO: 10).

[0055] The sense strand of the double-stranded siRNA (double-stranded number: SD 006) is 5'-CAACCUCUAUGGUGCCUAC-3' (SEQ ID NO: 11), and the antisense strand is 5'-GUAGGCACCAUAGAGGUUG-3' (SEQ ID NO: 12).

[0056] The sense strand of the double-stranded siRNA (double-stranded number: SD 007) is 5'-GCGUGGAUCCCUUCAUCUA-3' (SEQ ID NO: 13), and the antisense strand is 5'-UAGAUGAAGGGAUCCACGC-3' (SEQ ID NO: 14).

[0057] The sense strand sequence of shRNA (double-strand number: SD 008) is: 5'-CCGGGCGTGGATCCCTTCATCTATCAAGAGTAGATGAAGGGATCCACGCTTTTTTG-3' (SEQ ID NO: 15); the antisense strand sequence of shRNA is: 5'-AATTCAAAAAAGCGTGGATCCCTTCATCTACTCTTGATAGATGAAGGGATCCACGCG-3' (SEQ ID NO: 16).

[0058] The siRNA delivery vector for cells can use commercial transfection reagents, or prepare LNPs according to the disclosed LNP formula. In this example, LNPs were prepared for siRNA delivery as follows:

[0059] Four materials, namely cationic lipid (ALC-0315), auxiliary lipid (DSPC), cholesterol (CHO-HP) and polyethylene glycol lipid (DMG-PEG2000), were mixed at a molar ratio of 50:10:38.5:1.5. Weigh 6.44mg of CHO-HP, 3.42mg of DSPC, and 1.63mg of DME-PEG2000 and dissolve them in 1mL of anhydrous ethanol respectively. Take 18μL of ALC-0315 and dilute it in 1mL of anhydrous ethanol, and shake it thoroughly to mix evenly. Take the same volume of lipid solution, and its molar ratio satisfies the ratio of 50:10:38.5:1.5.

[0060] siRNA was diluted to the desired concentration using 10 mM sodium citrate buffer.

[0061] When preparing LNP, the four lipids and siRNA are mixed at a volume ratio of 1:3. Take the preparation of 100μL of LNP as an example: After the four lipids are prepared in the above manner, 6.25μL of each is mixed evenly (to ensure that the molar ratio of the four lipids is 50:10:38.5:1.5), placed on a vortex oscillator, 75μL of siRNA solution is added dropwise, and taken out after shaking for about 10s to prepare LNPs encapsulated with different groups of siRNA. The Tyndall effect can be used to preliminarily judge whether the nanoparticles are successfully formed.

[0062] MEG-01 cells were used to analyze the silencing ability of siRNA. The specific operation was as follows:

[0063] MEG-01 cells were cultured at 5×10 5 Cells were plated in 12-well plates. LNPs encapsulated with different groups of siRNA were prepared according to the above LNP preparation method 30 min before transfection and added to MEG-01 cells, with 30 pmol of each siRNA. After continued culture for 24 h or 48 h, qRT-PCR was used to detect the expression of the PAR4 gene, with β-actin as the internal reference.

[0064] The primer sequences used in this example are:

[0065] PAR4: upstream primer 5'-CTCTATGGTGCCTACGTGCC-3', downstream primer: 5'-CACCTTGTCCCTGAACTCGG-3'.

[0066] β-actin: upstream primer 5'-ACTCTTCAGCCTTCCTTCC -3', downstream primer: 5'-CAATGCCAGGGTACATGGTG -3'.

[0067] Results: Figure 1 Compared with NC, the silencing ability of SD 007 sequence reached 85%~90% after 24h and 48h, and it had the best effect compared with the other 6 sequences, and the action time was longer and more stable, with significant differences.

[0068] Evaluation of the ability of siRNA to inhibit platelet activation:

[0069] The dynamic changes of calcium signal induced by PAR4 activation, namely calcium mobilization, are the basis of platelet activation. In platelet function, prolonged calcium signal can drive procoagulant reactions. Therefore, the effect of siRNA on PAR4 agonist peptide AYP (AYPGKF-NH2)-induced cellular calcium mobilization was studied to evaluate its potential role in inhibiting platelet function.

[0070] MEG-01 cells were cultured at 3×10 5 cells were plated in a 12-well plate. 30 min before transfection, LNPs coated with different groups of siRNA were prepared according to the above-mentioned LNP preparation method, and added to MEG-01 cells, using 30 pmol of each siRNA. 36 h after transfection, LNPs coated with siRNA were prepared again by the above-mentioned LNP preparation method, and added to MEG-01 cells again, using 30 pmol of each siRNA. Calcium mobilization was performed 72 h after the first siRNA transfection. Specifically, the transfected MEG-01 cells were inoculated in a black-bottomed 96-well plate pre-coated with PDL (polylysine, to promote cell adhesion) (i.e., 70 μL of PDL was added to each well and discarded after 45 min), and 1.5×10 5 The cells were inoculated at a density of 100 μL. After the cells adhered to the wall, 5 μM Fluo-4 AM was used to load the cells as a fluorescent indicator of calcium ions. After incubation for 45 minutes, the fuel was discarded and the cells were washed 3 times with Locke's buffer. Finally, 175 μL of Locke's buffer was retained in each well. The FLIPR high-throughput real-time fluorescence imaging plate reader was used to detect the changes in intracellular fluorescence before and after the addition of AYP. The detection was started 1 minute before the addition of AYP, and the detection was continued for at least 5 minutes after the addition of AYP. The instrument operation process is as follows:

[0071] (1) Turn on the FLIPR instrument in advance according to the regulations and complete the self-test; then set the FLIPR temperature to 37°C;

[0072] (2) Preparation of AYP agonist (drug plate): Prepare different concentrations of AYP according to experimental requirements. The AYP concentration is 8 times the final concentration. Use Locke's buffer to dilute the solution to ensure that the proportion of solvents at different concentrations is consistent. After mixing, add the solution to a V-bottom transparent 96-well plate. The volume per well should be at least 70 μL to ensure that the instrument can absorb the solution. The final volume of the solution is 25 μL. Since the sample is added automatically by the instrument, the concentration of the agonist added to each well of the drug plate should correspond to the concentration of the agonist to be added to the planned cell plate (well to well, for example, if the A-1 well of the cell plate is planned to use 50 μM AYP agonist, then the corresponding A-1 well of the drug plate needs to add 70 μL of 400 μM AYP solution). The blank control group does not use AYP agonist, that is, physiological saline can be added.

[0073] (3) After the incubation, remove the cell plate, add 140 μL Locke's buffer / well, then discard 150 μL / well, and then add 150 μL new Locke's buffer / well. Repeat the washing process for about 6 times. After discarding 150 μL of the solution for the last time, add 125 μL new Locke's buffer to make the volume of each well 175 μL.

[0074] (4) After washing, place the cell plate in the READ position of the FLIPR and stabilize it for 5 minutes;

[0075] (5) Before starting the experiment, vortex the drug plate to ensure that the drug solution is uniform; place the cell plate, drug plate and required pipette tip boxes, make sure that the plate covers are open, and close the hatch;

[0076] (6) Open the software, click Instrument-Manua1 Operation-Loadtips, install the tip, and re-confirm Stage temperature, Camera-status, Chillerstatus and Protocol1. Click ProtocolSigna1 Test to detect the background fluorescence of the cell plate, adjust Exposure-Time, Exc.Intensity and Camera·Gain to make the background fluorescence value between 500-600, and save the modified parameters.

[0077] (7) Click Run to start the test and monitor continuously for about 10 minutes (during this process, the instrument automatically completes the addition of the agonist AYP or saline. As mentioned above, place the cell plate and drug plate with the sample pipette tip in the specified position of the instrument. There is no need to add the sample yourself). After the test, Unload Tips, remove the cell plate, drug plate and pipette tip box, save the data, and then shut down the computer and FLIPR, then Chiller, and finally UPS.

[0078] Locke's buffer: weigh 9.0g NaCl, 0.2g NaHCO3, 0.42g KCl in a beaker, add 980mL ultrapure water to dissolve, dissolve 0.24g CaCl2 in 20mL ultrapure water, then add dropwise to the above solution, stir evenly, adjust the pH to 7.4, filter through a 0.22μm pore size filter membrane, and store in aliquots.

[0079] The results are as follows Figure 2, cells stimulated without agonist peptides did not produce a transient increase in calcium ion fluorescence signals, indicating that no cellular calcium mobilization reaction occurred; cells stimulated with 100μM AYP, the calcium ion fluorescence signal of the control NC group increased transiently and slowly decreased, showing the calcium signal dynamics after PAR4 activation. Comparing the maximum calcium ion signal peak, compared with the NC group, the calcium signals of siRNA sequences SD 002 to SD 005 and SD 007 were significantly reduced, which basically corresponded to the gene expression level, indicating that cellular calcium mobilization was inhibited. Among them, the SD 007 sequence group did not show a calcium signal peak after activation with 100μM AYP, and no calcium mobilization reaction mediated by PAR4 activation occurred, indicating that SD 007 completely inhibited the expression of PAR4 protein, thereby inhibiting the corresponding functions mediated by PAR4. Compared with other sequences, SD 007 showed the best gene silencing efficiency and inhibition function.

[0080] Example 2

[0081] shRNA design, synthesis and analysis of its silencing ability

[0082] The SD 007 sequence with the best gene silencing effect was selected as a template to design shRNA. The stem loop (loop) connecting the sense strand and antisense strand of the siRNA selected the best 5'-GATCC-3' sequence, the sticky end complementary sequence of the restriction site was selected according to the expression vector, and 5'-TTTTTT-3' was added as the terminator sequence. A shRNA (double-stranded number: SD008) was obtained, wherein the sense strand sequence was: 5'-CCGGGCGTGGATCCCTTCATCTATCAAGAGTAGATGAAGGGATCCACGCTTTTTTG-3' (SEQ ID NO: 15); the antisense strand sequence was: 5'-AATTCAAAAAAGCGTGGATCCCTTCATCTACTCTTGATAGATGAAGGGATCCACGCG-3' (SEQ ID NO: 16). It was synthesized by chemical synthesis.

[0083] Select a lentiviral plasmid vector (pLKO-1 vector) containing a U6 promoter and a puromycin resistance selection tag to construct a recombinant lentiviral vector. Anneal the synthesized shRNA oligo single-stranded DNA oligo to obtain a double-stranded shRNA. Dilute the oligonucleotide to 100 μM with water. Prepare the annealing reaction system according to the following system:

[0084] Sense oligonucleotide (100 μM) 2 μL;

[0085] Antisense oligonucleotide (100 μM) 2 μL;

[0086] 10×T4 DNA ligase buffer 1 μL;

[0087] Add 15 μl of water to make up to 20 μL;

[0088] Incubate in a PCR machine at 95°C for 4 minutes, 70°C for 10 minutes, and then slowly cool to room temperature over several hours.

[0089] After obtaining ds oligo, the plasmid vector was digested with AgeⅠ and EcoRⅠ. The reaction system was:

[0090] 6 μg pLKO.1;

[0091] 5 μL 10×cut smart buffer;

[0092] 1 μL AgeI-HF;

[0093] 1 μL EcoRI-HF;

[0094] Add ddH2O to 50 μL;

[0095] Digest the cells at 37°C for more than two hours with interval shaking to obtain a linear vector.

[0096] The annealed fragments are connected to the enzyme digestion products, and the annealed oligonucleotides are diluted 100 times with water for later use. The connection reaction system is prepared according to the following system:

[0097] T4 DNA ligase 5U;

[0098] 2 μL of linearized vector;

[0099] 2 μL of diluted oligonucleotide;

[0100] 10× Ligase Buffer 1 μL;

[0101] Add water to make up to 10 μL;

[0102] The ligation was carried out at 4°C overnight to obtain a ligation product.

[0103] The subsequent products can be transformed (DH5α), screened for positive clones, extracted from plasmids, and then used to obtain shRNA lentiviral plasmids for virus packaging, infection, stable strain screening, and interference experiments.

[0104] MEG-01 cells were used to analyze the silencing ability of shRNA (SD 008). The specific operation was as follows (no stable transfection screening was required, only the interference efficiency of shRNA was verified, and the screening experiment of stable transfection cells was conducted in the later stage):

[0105] (1) Plasmid transformation

[0106] Take out a DH5α competent cell from -80℃ and thaw it on ice; add 2μL of the above-obtained shRNA lentiviral plasmid to the competent cell in a clean bench, flick to mix, and incubate on ice for 30min;

[0107] The competent cells were placed in a 42°C constant temperature water bath, and after heat shock for 45 seconds, they were immediately placed on ice for 2 minutes;

[0108] Add 900 μL of LB liquid medium (without resistance) to the competent cells, mix well, and recover at 37°C 220 rpm / min for 1 h;

[0109] Centrifuge at 5000 rpm / min at room temperature for 3 min, discard 900 μL of supernatant, resuspend the bacteria with the remaining liquid, add to the Amp-resistant plate, spread evenly with a sterile coating rod, let it dry for 10 min, and invert in a 37°C constant temperature incubator for 12-16 h.

[0110] (2) Screening of positive clones

[0111] Select a single colony on the transformed plate and transfer it to a sterile test tube containing 4 mL of LB liquid culture medium (containing Amp resistance, 1:1000, v / v). Incubate at 37°C in a shaker at 220 rpm / min for 12-16 h until obvious turbidity occurs.

[0112] (3) Cultivation and preservation of bacterial strains

[0113] Culture: After the monoclonal colony proliferates, take 200 μL of bacterial solution and add it to a culture bottle containing 20 mL of LB liquid medium (containing Amp resistance 1:1000, v / v), and culture at 220 rpm / min and 37°C for 10-12 hours. It can be used for preservation or plasmid extraction.

[0114] Storage: Store in a sterile 1.5 mL EP tube at a volume ratio of 1:3: fresh bacterial solution: 40% sterile glycerol. Blow well, label all information, and store at -20°C or in a -80°C refrigerator for long-term storage.

[0115] (4) Plasmid extraction

[0116] The glycerol bacteria containing the lentiviral plasmid containing the target shRNA (SD 008) sequence (obtained by the above transformation and other operations) and the glycerol bacteria containing the two auxiliary plasmids psPAX2 and pMD2.G (preserved in the laboratory) were inoculated into 20 mL of Amp-resistant liquid LB medium at a ratio of 1:100, and the bacteria were shaken at 37°C and 220 rpm / min for 12-16 hours. The bacterial solution was then transferred to a 15 mL centrifuge tube and centrifuged at 8000 rpm / min for 10 min, and the supernatant was discarded to obtain the bacterial precipitate; the plasmid extraction operation was performed using an endotoxin-free small extraction kit (Beijing Tiangen, DP118-02) according to the instructions to obtain three plasmids.

[0117] (5) Lentiviral packaging

[0118] HEK293T cells were passaged twice and pre-plated 36 h before transfection. The cells were digested and collected and counted. The cell density was adjusted and 1.2×10 7 cells, and ensure that the cell density is controlled at about 80%-90% for the best transfection effect;

[0119] Take out the target plasmid, auxiliary plasmid and transfection reagent LipoFiter from the -20℃ refrigerator in advance and melt them;

[0120] Prepare serum-free DMEM medium in two 15 mL centrifuge tubes, 2 mL in each tube;

[0121] The amount of plasmid used to transfect a 15 cm dish of HEK293T cells is as follows: according to the number of cell dishes to be transfected, calculate and prepare the mixed solution, add psPAX2, pMD2.G, and the target plasmid to one tube respectively, and the transfection mass ratio is 1:1:1. Add LipoFiter staining reagent (Hanheng Bio, HB-LF3-1000) to another tube. Mix the two tubes and let them stand for 5 min. The usage is as follows: 24 μg of target plasmid, 24 μg of psPAX2, 24 μg of pMD2.G, and 108 μL of transfection reagent.

[0122] Gently mix the plasmid and transfection reagent in the same centrifuge tube and let it stand at room temperature for 20 min;

[0123] Reduce the culture system of HEK293T cells to 12 mL, and add 4 mL of the plasmid-transfection reagent mixture to each dish of cells. Note that it needs to be added slowly, and the dish should be gently shaken to mix thoroughly;

[0124] After culturing in a 37°C incubator for 6 h, replace with 20 mL of new DMEM medium (containing 10% FBS) and continue culturing; collect the culture supernatant at 48 h, 72 h, and 96 h, which is the original virus solution, and replace with 20 mL of new DMEM complete medium containing 10% FBS. Mix the collected virus solutions and store them briefly at 4°C.

[0125] (6) Lentivirus Concentration

[0126] The original virus solution was collected in a 50 mL centrifuge tube and centrifuged at 8000 g for 10 min at 4°C. The cell debris was centrifuged at the bottom of the 50 mL centrifuge tube;

[0127] Use a 0.2 μM PVDF filter to filter the virus stock solution to remove protein molecules and impurities, and collect the supernatant in a new 50 mL centrifuge tube;

[0128] Add the virus concentrate to the lentivirus solution at a ratio of 1:9, and place it on a rotary mixer to mix thoroughly overnight;

[0129] Centrifuge at 8000×g for 10 min, and the white lentivirus precipitate will be centrifuged on the tube wall. Perform subsequent operations in a biosafety cabinet: add 1640 (containing 10% FBS) culture medium to completely dissolve the lentivirus, retain a small amount of virus liquid for titer determination, and aliquot and freeze the rest at -80°C to obtain lentivirus containing shRNA (SD 008).

[0130] (7) Lentivirus titer detection

[0131] HEK293T cells were revived from -80°C freezer and adjusted to logarithmic growth phase;

[0132] Adjust the cell density to 5 × 10 5 / mL, spread 200 μL of cells per well into a 24-well plate, and add DMEM complete medium to 400 μL, and pipette to mix thoroughly;

[0133] After the HEK293T cells were cultured for about 6 h and the cells were completely attached, the reserved lentiviral concentrate was diluted 20 times and 1 μL and 2 μL were added to the HEK293T cells and cultured in a 37°C, 5% CO2 incubator for 48 h;

[0134] The 24-well plate was removed from the incubator, and the cells were digested and collected in a 1.5 mL EP tube and centrifuged at 300 g at 4°C for 5 min;

[0135] Discard the supernatant and add 1 mL of pre-cooled PBS wash solution to each cell sample to wash the cells twice;

[0136] Resuspend the cell pellet with 100 μL PBS, add 1 μL Biotin-Fc protein to each well, gently pipette to mix, place on ice, and incubate for 30 min;

[0137] Add 1 mL of PBS to each sample, centrifuge at 300 × g, 4°C for 5 min, discard the supernatant, and repeat the wash;

[0138] Discard the supernatant, add secondary antibody Streptavidin-PE, 1 μL per well, gently pipette to mix, place on ice, and incubate for 30 min; Add 1 mL PBS wash solution to each sample, centrifuge at 300×g, 4℃ for 5 min, discard the supernatant, and repeat the wash;

[0139] Resuspend the cells with 800 μL PBS and transfer to a new 1.5 mL EP tube. Detect the positive rate of lentivirus expression on a flow cytometer. Use HEK293T cells that are not infected with the virus as a blank control to set the gate, and calculate the virus titer according to the following formula: Lentivirus titer (TU / mL) = (number of positive rate HEK293T cells × dilution factor) / (volume of added lentivirus (μL)) × 1000; the lentivirus titer used in this experiment is 1×10 8 TU / mL, used for subsequent lentiviral infection experiments.

[0140] (8) Lentivirus infection of MEG-01 cells

[0141] MEG-01 cells were cultured at 3×10 5 MEG-01 cells were plated in a 12-well plate. MEG-01 cells were infected with lentivirus at an MOI of 50 and a virus titer of 1×10 8 TU / mL, transfection 3×10 5 The required virus volume is 150 μL, that is, add 150 μL of lentivirus to the wells of the cell plate.

[0142] (9) Detection of PAR4 gene expression

[0143] 72h after lentiviral infection, the expression of PAR4 gene was detected by qRT-PCR to evaluate the interference efficiency of shRNA.

[0144] The primer sequences used in this example are:

[0145] PAR4: upstream primer 5'-CTCTATGGTGCCTACGTGCC-3', downstream primer: 5'-CACCTTGTCCCTGAACTCGG-3'.

[0146] β-actin: upstream primer 5'-ACTCTTCAGCCTTCCTTCC -3', downstream primer: 5'-CAATGCCAGGGTACATGGTG -3'.

[0147] The gene detection results of the above qRT-PCR are as follows Figure 3 , indicating that the silencing efficiency of shRNA on the PAR4 gene can reach more than 80%.

[0148] Example 3

[0149] Generation of PAR4-deficient platelets

[0150] Editing was performed before platelet production based on the established iPSC-megakaryocyte progenitor line (imMKCL). The imMKCL were revived and cultured and passaged to 1×10 cells in basal medium containing 15% fetal bovine serum (FBS) and cocktail A. 5 -2×10 6 cell / mL. The basal medium was supplemented with L-glutamine (200mM), insulin-transferrin-selenium, ascorbic acid (100×, A4544) and 450μM 1-thioglycerol. Cocktail A consisted of 50 ng / mL recombinant human stem cell factor (SCF) without animal origin, 200 ng / mL TA-316 and 5 μg / mL doxycycline. Megakaryocytes were infected with the lentivirus containing shRNA (SD 008) constructed in Example 2. The infection process was the same as that of MEG-01 cells infected with the lentivirus in Example 2. 2μg / mL puromycin was added and cultured for 2 weeks to select stable strains (stable strains can continue to grow after two weeks), and the stable strains were expanded.

[0151] The differentiation stage of the selected stable strains was continued in a basal medium containing cocktail B and serum or plasma. The following were used as serum or plasma: 15% FBS or different concentrations of human plasma or human serum. In the case of human plasma, 10 U of sodium heparin was added. Cocktail B consisted of 50 ng / mL SCF, 200 ng / mL TA-316, 15 μM KP-457, 0.75 μM SR-1 or 0.5 μM GNF-351 (CABIOCHEM), and 10 μM Y27632 (WAKO) or 0.5 μM Y39983 (MedChemExpress). A platelet bioreactor (Ito Y, Nakamura S, Sugimoto N, et al. Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production. Cell. 2018;174(3):636-648.e18. doi:10.1016 / j.cell.2018.06.011) was used for culture at the differentiation stage to promote platelet production. In this example, a 2L system was used with 2×10 cells. 5 After 6 days of culture at 37°C, the culture suspension containing platelets was filtered through a hollow fiber (Micro-Invasive Medical Devices, device number 20143101838) to reduce the volume, and then washed and removed the remaining imMKCL using an ACP centrifuge. The platelets were resuspended in platelet storage solution to obtain a PAR4-deficient iPSC-platelet suspension.

[0152] Ringer's solution: 8.6 g sodium chloride, 0.3 g potassium chloride, 0.28 g calcium chloride, dissolve in distilled water and make up to 1000 mL;

[0153] Platelet preservation solution: 2.4 g citric acid, 6.6 g trisodium citrate, 7.35 g glucose, 2.5 g BSA, dissolved in Ringer's solution and adjusted to 50 mL.

[0154] Example 4

[0155] Testing of the Aggregation Ability of PAR4-Deficient Platelets in Vitro

[0156] Platelet aggregation rate was analyzed using a platelet aggregometer (PRECILBY-NJ4). Washed platelets from donors, unedited iPSC-platelets, and PAR4-deficient iPSC-platelets prepared in Example 3 were suspended in 70% human plasma containing 3 mM CaCl2 (platelets 3×10 8270 μL of sample was mixed with 30 μL of agonist containing 1 U / mL thrombin and 1 mM PAR4 agonist peptide AYP, reacted at 37°C for 5 min, and the aggregation rate was detected.

[0157] Donor-derived platelets: Venous blood was collected from healthy volunteers and placed in sodium citrate anticoagulant tubes, approximately 3 mL per tube. The human blood was centrifuged at 980 rpm / min for 5 minutes, and the upper plasma, i.e., platelet-rich plasma (PRP), was placed in a 15 mL silanized tube rinsed with saline and placed in a 37°C water bath for later use. PRP was resuspended and washed with Tyrode's buffer, centrifuged at 2700 rpm for 5 min, the supernatant was discarded and washed again, and finally resuspended with Tyrode's buffer to adjust the platelet count to 3.0×10 8 / mL and placed in a 37℃ water bath for experiments.

[0158] Unedited iPSC-platelets: Thaw and culture imMKCLs and passage to 1×10 in basal medium containing 15% fetal bovine serum (FBS) and cocktail A. 5 -2×10 6 cell / mL. Basal medium supplemented with L-glutamine, insulin-transferrin-selenium, ascorbic acid (A4544) and 450 μM 1-thioglycerol. Cocktail A consisted of 50 ng / mL animal-free recombinant human stem cell factor (SCF), 200 ng / mL TA-316 and 5 μg / mL doxycycline. The differentiation phase continued in basal medium supplemented with cocktail B and serum or plasma. The following were used as serum or plasma: 15% FBS or different concentrations of human plasma or human serum. In the case of human plasma, 10 U of sodium heparin was added. Cocktail B consisted of 50 ng / mL SCF, 200 ng / mL TA-316, 15 μM KP-457, 0.75 μM SR-1 or 0.5 μM GNF-351 (CABIOCHEM) and 10 μM Y27632 (WAKO) or 0.5 μM Y39983 (MedChemExpress). Use a platelet bioreactor to culture in the differentiation stage to promote platelet production (in this example, a 2L system with 2×10 cells was used). 5 After 6 days, the culture suspension containing platelets was filtered through a hollow fiber (Micro-Invasive, 20143101838) to reduce the volume, and then washed and removed the remaining imMKCL using an ACP centrifuge. The platelets were resuspended in platelet storage solution.

[0159] The results are as follows Figure 4: The aggregation ability of PAR4-deficient iPSC-platelets under AYP or thrombin stimulation was significantly reduced, which was significantly different from the platelets from the transfused donor and iPSC-platelets, indicating that their thrombosis ability was significantly reduced.

[0160] Example 5

[0161] Testing of the hemostatic and thrombotic abilities of PAR4-deficient platelets in vivo

[0162] NOG mice were irradiated with 2.4 Gy radiation 9 days before transfusion to induce thrombocytopenia. On the 9th day after irradiation, 2×10 8 Platelets (100 μL, 2 × 10 9 / mL), respectively, iPSC-platelets without editing (prepared in Example 4) and iPSC-platelets lacking PAR4 (prepared in Example 3). The tail artery was punctured with a 23G needle 2 cm from the tip, and the hemostatic effect was evaluated by bleeding from the tail.

[0163] The results are as follows Figure 5 The tail bleeding time of mice infused with PAR4-deficient platelets was not significantly prolonged, and there was no significant difference compared with the infusion of donor-derived platelets and iPSC-platelets, demonstrating its excellent hemostatic ability.

[0164] NOG mice were irradiated with 2.4 Gy radiation 9 days before transfusion, which caused thrombocytopenia. 2×10 8 Platelets (100 μL, 2 × 10 9 / mL), edited iPSC-platelets and PAR4-deficient iPSC-platelets, respectively. They were then used for in vivo imaging studies. In vivo analysis of thrombosis was based on the methods of Nakamura et al. and Nishimura et al. (References: Expandable megakaryocyte cell lines enableclinically applicable generation of platelets from human induced pluripotentstem cells. Cell Stem Cell 14, 535–548. and In vivo imaging visualizes discoidplatelet aggregations without endothelium disruption and implicatescontribution of inflammatory cytokine and integrin signaling. Blood 119, e45–e56.). In brief, a laser- or ferric chloride-induced mesenteric injury thrombosis model was performed in vivo, and images were collected using a resonant scanning confocal microscope to record the thrombus formation time. The collected images were analyzed to evaluate the effects of infusion of PAR4-deficient iPSC-platelets on the mesenteric thrombus formation time and thrombus stability compared with infusion of unedited iPSC-platelets.

[0165] The results are as follows Figure 6 : Infusion of PAR4-deficient iPSC-platelets prolonged the thrombosis time by approximately 2-fold, which was significantly different from infusion of donor-derived platelets and iPSC-platelets, indicating that PAR4-deficient platelets have significantly reduced thrombosis ability.

[0166] In summary, the PAR4-deficient platelets prepared by the present invention have hemostatic ability comparable to that of platelets and lower thrombosis ability, and are safer to be used for platelet transfusion in vivo.

Claims

1. A double-stranded siRNA for regulating the expression of protease activated receptor 4 gene, characterized in that: The double-stranded siRNA is selected from SEQ ID NO.13-14.

2. A shRNA for regulating the expression of protease activated receptor 4 gene, characterized in that: The shRNA comprises the double-stranded siRNA according to claim 1, a stem-loop structure connecting the sense strand and the antisense strand of the siRNA, a sequence complementary to the sticky end of the vector restriction site and a terminator, and the shRNA is selected from SEQ ID NO.15-16.

3. A pharmaceutical composition, characterized in that It contains the shRNA according to claim 2 and a pharmaceutically acceptable carrier or excipient.

4. Use of the double-stranded siRNA according to claim 1, the shRNA according to claim 2, or the pharmaceutical composition according to claim 3 in the preparation of an inhibitor of protease activated receptor 4 (PAR4) expression.

5. Use of the double-stranded siRNA according to claim 1, the shRNA according to claim 2, or the pharmaceutical composition according to claim 3 in preparing engineered platelets lacking PAR4.

6. The use according to claim 5, characterized in that: The PAR4-deficient engineered platelets are engineered platelets with hemostatic ability and low thrombogenicity ability.

7. The use according to claim 5, characterized in that: The platelet-derived cells are transfected with an expression system containing shRNA, wherein the expression system is a lentiviral vector or other pharmaceutically acceptable vector, and the platelet-derived cells are engineered into a cell bank that can stably express shRNA through pressure screening, and are induced to differentiate into platelets in a platelet bioreactor for platelet transfusion; the platelet-derived cells include primary hematopoietic stem cells and their progenitor cells, megakaryocytes and their progenitor cells, iPSCs, and iPSC-derived hematopoietic stem cells or megakaryocytes and their progenitor cells or immortalized megakaryocytes, various megakaryocytes derived from reprogramming or transdifferentiation techniques, or platelet-like particles PLP produced by MEG-01 cells.

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