Application of SERPINC1 mutant in antithrombin deficiency screening
Through gene editing and iPSC induction of SERPINC1 mutants, the screening and treatment difficulties of antithrombin deficiency are solved, accurate diagnosis and long-lasting cure are achieved, and side effects and immune rejection of traditional treatment are avoided.
Patent Information
- Application Number
- CN202510588969.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-18
- Publication Date
- 2025-08-05
AI Technical Summary
The prior art is difficult to effectively screen and treat antithrombin deficiency, especially due to high-risk venous thromboembolism and arterial thrombosis events caused by mutations in SERPINC1 gene, and long-term anticoagulation treatment has side effects and risk of immune rejection.
Using SERPINC1 mutant nucleic acid, peptide and gene editing technology, the patient cells were gene-corrected in vitro through CRISPR-Cas9 and Cre/loxP tools to generate iPSCs and induce them into mature hepatocytes for gene therapy, avoiding immune rejection and restoring antithrombin function.
Accurate screening and potential cure for antithrombin deficiency is achieved, avoiding the side effects of long-term anticoagulant treatment, providing a safe and long-lasting treatment plan, and reducing the risk of immune rejection.
Smart Images
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Abstract
Description
[0001] This application is a divisional application of the Chinese invention patent whose parent case is entitled "serpinc1 gene mutants and applications", with an application date of October 18, 2022 and application number 202211270382.0. This application cites the full text of the parent case. Technical Field
[0002] The present invention belongs to the field of gene technology, and in particular relates to the application of SERPINC1 mutants in antithrombin deficiency screening. Background Art
[0003] Hereditary antithrombin (AT) deficiency is an autosomal dominant genetic disorder with a prevalence of approximately 0.02%-0.2% in the general population and accounts for approximately 1%-5% of venous thromboembolism (VTE) cases. Among patients with known inherited thrombophilias, antithrombin deficiency carries a particularly high risk of VTE. Patients with AT deficiency have an increased risk of a first VTE event (odds ratio, 14.0; 95% CI, 5.5-29.0) and an annual risk of VTE recurrence of 8.8% (95% CI, 4.6-14.1). AT deficiency is also one of the most common causes of thrombosis in children. The primary clinical manifestation of heterozygous AT deficiency is venous thromboembolism, but some patients may also experience arterial thrombotic events such as myocardial infarction. Homozygous AT deficiency is often embryonic lethal or results in neonatal giant thrombi or purpura fulminans, making survival difficult. Multiple severe thromboembolic events have also been reported in families with combined SERPINC1 and factor V Leiden deficiencies in the West. AT deficiency can be divided into two types of mutations: type I (quantitative), in which both antigen and AT activity are decreased; and type II (qualitative), in which the antigen is normal but AT activity is absent or impaired.
[0004] Given the characteristics of AT deficiency, symptomatic patients with AT deficiency caused by SERPINC1 mutations are encouraged to receive lifelong vitamin K antagonist (VKA) anticoagulation therapy, while asymptomatic carriers are encouraged to receive antithrombotic prophylaxis in critical situations. A significant number of patients with severe AT deficiency experience recurrent venous thromboembolism even with adequate anticoagulation. Furthermore, patients with AT deficiency who receive long-term anticoagulation therapy require continuous clinical monitoring, as they may develop heparin resistance and are at risk for major bleeding. Hereditary AT deficiency is caused by pathogenic mutations in the AT gene (SERPINC1). Over 400 different variants, primarily single nucleotide variants or small insertions / deletions, have been identified and reported. Within this framework, gene therapy offers the potential for a radical cure for AT deficiency, avoiding all the side effects of classical anticoagulation therapy. Therefore, exploring the feasibility and safety of gene therapy is of great clinical significance. Summary of the Invention
[0005] In response to the above problems, the present invention provides the application of SERPINC1 mutants in the screening of antithrombin deficiency, mainly discovering new pathogenic genes for antithrombin deficiency diseases and providing new directions for subsequent diagnosis and treatment.
[0006] In order to solve the above problems, the present invention adopts the following technical solutions:
[0007] A nucleic acid comprising the following target fragment, wherein the target fragment has a c.506C>T mutation compared with the wild-type SERPINC1 gene, wherein the wild-type SERPINC1 sequence is shown as SEQ ID NO.1.
[0008] A polypeptide, compared with a wild-type SERPINC1, the polypeptide has a p.S169F mutation, wherein the wild-type SERPINC1 sequence is shown as SEQ ID NO. 2 (GenBank: CAA48690.1).
[0009] The gene mutation comprises a c.506C>T mutation compared to the wild-type SERPINC1 gene, wherein the wild-type SERPINC1 sequence is shown in SEQ ID NO.1.
[0010] For the above-mentioned wild-type SERPINC1, when a single nucleotide or amino acid sequence site at other sites of the SERPINC1 gene mutates, it does not affect the protection of the detection site involved in the present invention. Under the premise that the target site mutation is the same, changes in other sites relative to SEQ ID NO.1 or SEQ ID NO.2 should also be within the scope of the present invention.
[0011] Use of a biological model in the preparation of a screening preparation for an antithrombin deficiency disease, wherein the biological model carries at least one of the following
[0012] a. the aforementioned nucleic acid having a mutation,
[0013] b. a polypeptide having a mutated nucleic acid,
[0014] c. Gene mutations of the aforementioned gene mutations;
[0015] Preferably, the antithrombin deficiency disease includes antithrombin deficiency, antithrombin deficiency-induced venous thrombosis and post-thrombotic syndrome; antithrombin deficiency is hereditary antithrombin deficiency.
[0016] When used in the preparation of screening reagents, refer to the subsequent specific use; when used in the preparation of preventive and therapeutic reagents, some methods are as drug targets, or used in testing, extraction and other steps in the preparation of other biological drugs, mainly as some auxiliary reagents in the pharmaceutical process, such as in testing the effect of inhibitors in the pharmaceutical process.
[0017] Use of a detection reagent in the preparation of a screening reagent for antithrombin deficiency disease, wherein the detection reagent is at least one of the following
[0018] a. a detection reagent for the aforementioned nucleic acid having a mutation;
[0019] b. a detection reagent for the aforementioned polypeptide having a mutation;
[0020] c. The aforementioned gene mutation detection reagent.
[0021] Antithrombin deficiency disease screening reagent, containing a reagent capable of detecting SERPINC1 gene mutation, wherein the SERPINC1 gene mutation includes at least one of the following
[0022] a. the aforementioned nucleic acid having a mutation,
[0023] b. the aforementioned polypeptide having a mutation,
[0024] c. the aforementioned gene mutations;
[0025] In some embodiments, the reagent is a nucleic acid probe or primer or other equivalent detection kit, and the primer design is as follows: Figure 2 As shown in , or targeted design based on the mutation site based on existing technology. Alternatively, some other product technologies that can directly detect the mutation site should be considered to adopt the solution of the present invention when used to detect the mutation site and determine whether antithrombin deficiency disease occurs.
[0026] The construct comprises the aforementioned nucleic acid or the aforementioned gene mutation.
[0027] The use of an edited gene fragment in the preparation of a drug for preventing and treating antithrombin deficiency disease, wherein the edited gene fragment is a fragment that can replace the single nucleotide T at position c.506 in the SERPINC1 gene mutant with a single nucleotide C. Antithrombin deficiency disease is mainly hereditary antithrombin deficiency, and the edited gene fragment can also be only a single base. Wherein c.506 mainly refers to the gene relative to the sequence of SEQ ID NO.1, and the main consideration is the form of the 506th base in the sequence.
[0028] Drugs for preventing and treating antithrombin deficiency diseases, containing at least one of the following
[0029] a. Inhibitors of the aforementioned gene mutations,
[0030] b. Inhibitors of the SERPINC1 polypeptide p.S169F mutation,
[0031] c. A gene editing fragment, wherein the gene editing fragment is a fragment that can replace the T at position c.506 in the SERPINC1 gene mutant with a C; specifically, a fragment that can replace the single nucleotide T at position 506 of the SERPINC1 gene with a single nucleotide C and express it. Preferably, the antithrombin deficiency disease is antithrombin deficiency. For example, the gene editing fragment is used to edit and repair the T at position 506 of the SERPINC1 gene to a C in patient-derived cells.
[0032] Gene fragments can be loaded onto vectors for use. Some forms of gene vectors include human-derived cells (e.g., mature hepatocytes capable of secreting antithrombin), plasmids, and adenoviral vectors. Treatment can be achieved by replacing the mutated gene and restoring it to its normal gene sequence, thereby achieving preventive or therapeutic purposes. Alternatively, the gene vector can directly secrete normal AT in vivo over the long term. Currently, known and mature gene vectors are preferred. However, new, approved vectors that emerge in the future should also fall within the scope of the present invention.
[0033] The use of human-derived nuclear cells in the preparation of drugs for preventing and treating antithrombin deficiency diseases, wherein the 506th position of the SERPINC1 edited gene in the human-derived nuclear cells is a single nucleotide C, and the human-derived nuclear cells can be induced to differentiate into mature hepatocytes. The human-derived nuclear cells are mainly derived from patients with antithrombin deficiency diseases themselves, such as the patient's peripheral blood. And the single nucleotide C at position 506 of the SERPINC1 edited gene in the human-derived nuclear cells is obtained through editing and correction, and the 506th position of the original pathogenic human-derived nuclear cell SERPINC1 edited gene is not a single nucleotide C. Of course, this type of human-derived nuclear cell is mainly used to treat antithrombin deficiency diseases induced by c.506C>T, c.506C>G or c.506C>A mutations in the SERPINC1 gene.
[0034] In the present disclosure, SERPINC1 (c.506C>T, p.S169F) is associated with AT. The present invention broadens the understanding of the cause of antithrombin deficiency diseases; provides a new diagnostic method for antithrombin deficiency, and also provides a new approach for its prevention and treatment, and explores the possibility of gene therapy for antithrombin deficiency mediated by induced pluripotent stem cells (iPSC). BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 A chart showing the thrombin status of patients in the research project;
[0036] Figure 2 Table of primers for detection;
[0037] Figures 3-4 This is the Sanger sequencing map;
[0038] Figure 5 The results of pathogenicity analysis;
[0039] Figure 6 A diagram of the gene therapy procedure;
[0040] Figure 7 This is a species conservation result. DETAILED DESCRIPTION
[0041] The present invention will be further described below in conjunction with specific research projects:
[0042] 1. Sample Collection
[0043] A male patient was admitted to Wuhan Union Hospital. At age 19, he experienced his first thromboembolic event without apparent cause: deep vein thrombosis (DVT) in the lower extremities and pulmonary embolism (PE). He also developed post-thrombotic syndrome, including chronic pulmonary hypertension and chronic leg ulcers. Despite receiving appropriate long-term anticoagulation therapy with oral warfarin or rivaroxaban, he still required hospitalization for one to two recurrent acute venous thrombotic events annually.
[0044] 2. Genomic Testing
[0045] The research and analysis were conducted after obtaining informed consent from all family members. The test found that compared with normal healthy people, the patient's thrombin generation was significantly increased ( Figure 1 , Table 1 below).
[0046]
[0047] First-generation Sanger sequencing: primer design, specific design such as Figure 2 PCR amplification was performed. Sequencing results showed that the proband's parents had no base deletion in the gene, suggesting that the proband's mutation was a de novo mutation.
[0048] Gene sequencing showed that the antithrombin AT encoding gene SERPINC1 c.506C>T (p.S169F) homozygous mutation ( Figure 3 , Figure 4 A), resulting in the original serine mutation to phenylalanine, protein C, protein S, V Leiden factor and prothrombin G20210A genes are not mutated. This mutation has not been reported in the GnomAD or HGMD database. The patient's AT activity is 25% (reference value, 80-120%) and the concentration is 64.5% (reference value, 60-135%). The patient's father and mother both carry heterozygous mutations at this site ( Figure 4The patient's AT activity was 25% (reference value, 80-120%) and concentration was 64.5% (reference value, 60-135%). The patient's father and mother had AT activities of 54% and 60%, respectively, and AT concentrations of 71% and 64%, respectively.
[0049] 3. Mutation Analysis
[0050] The mutation is located in exon 2 of SERPINC1, at the 169th protein position of the SERPINC1 protein. The mutation NM_000488.3: c.506C>T|NP_000479.1: p.S169F, where the mutation sequence is compared with the normal sequence, and is due to the mutation of the 506th base C of the cDNA to the base T. This region is the heparin binding region, which causes Thromb Haemost. According to the harmfulness prediction software, including Polyphen2, PROVEAN and other analyses, see Table 2 below, it was found that the mutation is harmful, and the results show that it will cause disease after the mutation. The species conservation results are shown in Figure 7 .
[0051]
[0052] AT deficiency can be divided into two types of mutations: Type I (quantitative), in which both antigen and AT activity are decreased; and Type II (qualitative), in which the antigen is normal but AT activity is absent or impaired. The patient's AT activity was 25 and the antigen was 64.5, which is a Type II mutation.
[0053] IV. Treatment Plan
[0054] In this study, a patient with severe AT deficiency was identified, harboring a newly discovered SERPINC1 mutation. This patient suffered from early-onset and frequently recurrent deep vein thrombosis (DVT) that was poorly responsive to pharmacological anticoagulation, making him an ideal candidate for gene therapy. Peripheral blood mononuclear cells (PBMCs) were collected from the patient and reprogrammed to generate iPSCs. CRISPR-Cas9 technology was used for in vitro gene correction, followed by Cre / loxP gene editing to remove the fluorescent tag originally used for selection, achieving precise and specific editing of the locus. The edited iPSCs were then induced to mature hepatocytes that stably secrete normal human AT protein. These hepatocytes were transplanted into AT-deficient mice (AT KO+ / -) and successfully rescued the mice's thrombophilic phenotype. In contrast, transplantation of hepatocytes derived from unedited iPSCs did not improve plasma AT expression or thrombotic propensity in AT+ / -KO mice. Results showed that in AT-deficient mice transplanted with hepatocytes derived from edited iPSCs, plasma AT antigen and AT activity remained above normal AT levels for three weeks after transplantation.
[0055] Using patient-derived cells, it was confirmed that human AT mutant cells can be used to treat the disease through in vitro gene correction and induced differentiation. After human cells are transplanted into mice, immune rejection is inevitable and the level of AT expression gradually decreases. It is well known that xenotransplantation is more prone to irreversible immune rejection than allogeneic transplantation. After human hepatocytes are xenotransplanted into mice, the activation of mouse macrophages is the main obstacle to the long-term survival of human hepatocytes. However, it is believed that autologous transplantation of normal iPSCs induced to differentiate into hepatocytes will not have obvious rejection reactions. In theory, the corrected cells can stably express AT in vivo for a long time, restoring AT levels to normal and achieving the goal of permanent cure. Therefore, this treatment method has great application value.
[0056] Human-derived cells undergo ex vivo gene editing to correct mutations and restore normal cell function. These cells are then targeted and induced to mature hepatocytes, the primary secretor of antithrombin (AT). Hepatocyte transplantation allows these cells to establish in vivo and effectively secrete normal levels of AT over the long term, avoiding the side effects of classical anticoagulation (drug resistance, severe bleeding, and long-term monitoring) or the ineffectiveness of conventional drugs. Because the cells are derived from the patient's own body, the possibility of immune rejection is reduced. This technological approach can be tested clinically and represents a new therapeutic approach for patients with severe inherited thrombophilias. Advances in gene editing have made this concept feasible, with the novelty of delivering long-term treatments from patient-derived iPS cells. Currently, research on bleeding and hemostasis-related diseases is limited to hemophilia, a bleeding disorder, and not to all thrombophilias. Furthermore, previous gene therapy approaches have primarily been conducted using viruses, such as AAV, with limited use of iPSCs. This gene therapy has demonstrated promising efficacy and safety. This principle can also be applied to inherited thrombophilias, such as protein C and protein S deficiencies. This provides a potential therapeutic strategy that could potentially cure patients with severe inherited thrombophilias who require lifelong anticoagulation. Of course, when this type of gene therapy is used on humans, it must comply with relevant legal regulations.
[0057] It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.
Claims
1. Use of the detection reagent in the preparation of a product for screening antithrombin deficiency; wherein, The detection reagent can detect the SERPINC1 c.506C>T mutation, in which the 506th base is mutated from C to T compared with the wild-type SERPINC1 with the sequence of SEQ ID NO.1; or, the detection reagent can detect the SERPINC1 gene mutant, in which the SERPINC1 gene mutant has a p.S169F mutation compared with the polypeptide encoded by the wild-type SERPINC1 with the sequence of SEQ ID NO.
2.
2. The use according to claim 1; wherein The antithrombin deficiency is hereditary antithrombin deficiency.
3. The use according to claim 1; wherein The detection reagent is a nucleic acid probe or a primer.
4. Use of human-derived nuclear cells in the preparation of drugs for preventing and treating antithrombin deficiency diseases; characterized in that, Compared with the wild-type SERPINC1 with the sequence of SEQ ID NO.1, the base at position 506 of the SERPINC1 of the human-derived nuclear cell is mutated from C to T, and after the SERPINC1 gene is edited, the base at position 506 can be changed from T to C, and the human-derived nuclear cell can be induced to differentiate into mature hepatocytes that can stably secrete normal human AT protein.
5. The use according to claim 4; wherein The drug for preventing and treating antithrombin deficiency disease is a drug for preventing and treating antithrombin deficiency in AT-deficient mice.
6. The use according to claim 5; wherein The AT-deficient mice are AT KO+ / - mice.
7. The use according to claim 5; wherein The effective period of the drug for preventing and treating antithrombin deficiency disease is 3 weeks.
8. The use according to claim 4; wherein The human-derived nuclear cells are peripheral blood mononuclear cells from the patient's peripheral blood.
9. Use of iPSCs in the preparation of drugs for preventing and treating antithrombin deficiency diseases; characterized in that, The iPSCs are derived from: peripheral blood mononuclear cells of AT-deficient patients with the SERPINC1 c.506C>T mutation are subjected to in vitro gene correction using CRISPR-Cas9 technology, and the edited iPSCs can be induced into mature hepatocytes that can stably secrete normal human AT protein.
10. Use of iPSCs in the preparation of a drug for preventing and treating antithrombin deficiency disease in mice; characterized in that: The iPSCs are derived from: peripheral blood mononuclear cells of AT-deficient patients with the SERPINC1 c.506C>T mutation are subjected to in vitro gene correction using CRISPR-Cas9 technology, and the edited iPSCs can be induced into mature hepatocytes that can stably secrete normal human AT protein; the mice are AT KO+ / - mice.