Serpinc1 gene mutant and its application
By identifying and correcting the c.506C>T mutation in the serpinc1 gene, using iPSC-mediated gene therapy to generate and transplant liver cells that can secrete normal antithrombin, the radical problem of hereditary antithrombin deficiency was solved and safe and efficient treatment effects were achieved.
Patent Information
- Application Number
- CN202211270382.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-18
- Publication Date
- 2025-05-13
- Estimated Expiration
- 2042-10-18
AI Technical Summary
Hereditary antithrombin deficiency leads to recurrence of venous thromboembolic, long-term anticoagulation treatment has side effects and drug resistance problems, and there is a lack of effective radical cure solutions.
Through gene mutant technology, c.506C>T mutations in the serpinc1 gene were identified and corrected, and gene therapy mediated by inducing pluripotent stem cells (iPSCs) were used to generate mature liver cells that can stably secrete normal antithrombin, and autotransplant was performed to restore AT levels.
Radical treatment of antithrombin deficiency is achieved, the side effects of anticoagulant therapy are avoided, the risk of thrombosis recurrence is significantly reduced, and new diagnostic and therapeutic pathways are provided.
Smart Images
Figure CN115725595B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of gene technology, and in particular relates to a serpinc1 gene mutant and an application thereof. Background Art
[0002] Hereditary antithrombin (AT) deficiency is an autosomal dominant genetic disease with a prevalence of approximately 0.02%-0.2% in the general population and approximately 1%-5% of patients with venous thromboembolism (VTE). Among patients with known hereditary thrombophilias, AT deficiency has a very high risk of VTE. Patients with AT deficiency have an increased risk of first venous thromboembolic events (odds ratio 14.0; 95% CI, 5.5-29.0), and the annual risk of VTE recurrence is 8.8% (95% CI, 4.6-14.1). In addition, AT deficiency is also one of the most common causes of thrombosis in children. The main 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 usually embryonic lethal, or causes the embryo to have neonatal giant thrombi or purpura fulminans and is difficult to survive. Families with multiple severe thromboembolic events have also been reported in the West with combined deficiencies in SERPINC1 and factor V Leiden. 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.
[0003] Combined with the characteristics of AT deficiency disease, symptomatic patients with AT deficiency caused by SERPINC1 mutations are encouraged to use vitamin K antagonists (VKA) for lifelong anticoagulation therapy, and asymptomatic carriers are encouraged to take antithrombotic prophylaxis in dangerous situations. A considerable number of patients with severe AT deficiency will still have recurrence of venous thromboembolism even after appropriate anticoagulation therapy. At the same time, AT deficiency patients who receive long-term anticoagulation therapy need continuous clinical monitoring. They may develop heparin resistance and are also at risk of major bleeding. Hereditary AT deficiency is caused by pathogenic mutations in the antithrombin gene (SERPINC1). More than 400 different variants, mainly single nucleotide variants or small fragment insertions / deletions, have been identified and reported, but there are still some cases where the cause of the disease is unknown. In this framework, gene therapy offers the possibility of a radical cure for AT deficiency, which will avoid all the side effects of classical anticoagulation therapy. Therefore, it is of great clinical significance to explore the feasibility and safety of gene therapy. Summary of the invention
[0004] In view of the above problems, the present invention provides serpinc1 gene mutants and applications, mainly discovering new pathogenic genes for antithrombin deficiency diseases and providing new directions for subsequent diagnosis and treatment.
[0005] In order to solve the above problems, the present invention adopts the following technical solutions:
[0006] A nucleic acid comprising the following target fragment, wherein the target fragment has a c.506C>T mutation compared with a wild-type serpinc1 gene, wherein the wild-type serpinc1 sequence is shown in SEQ ID NO: 1.
[0007] A polypeptide, wherein 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).
[0008] A gene mutation, compared with a wild-type serpinc1 gene, the gene mutation has a c.506C>T mutation, wherein the wild-type serpinc1 sequence is shown in SEQ ID NO: 1.
[0009] 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, when other sites change relative to SEQ ID NO: 1 or SEQ ID NO: 2, it should also be within the scope of the present invention.
[0010] 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
[0011] a. the aforementioned nucleic acid having a mutation,
[0012] b. a polypeptide having a mutated nucleic acid as described above,
[0013] c. Gene mutation of the aforementioned gene mutation;
[0014] Preferably, the antithrombin deficiency disease includes AT deficiency, AT deficiency-induced venous thrombosis and post-thrombotic syndrome; AT deficiency is hereditary antithrombin deficiency.
[0015] When used in the preparation of screening reagents, refer to the subsequent specific uses; when used in the preparation of preventive and therapeutic reagents, some methods are used 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 effects of inhibitors in the pharmaceutical process.
[0016] Use of a detection reagent in the preparation of a screening reagent for antithrombin deficiency diseases, wherein the detection reagent is at least one of the following
[0017] a. the aforementioned detection reagent for nucleic acid having mutation;
[0018] b. a detection reagent for the aforementioned polypeptide having a mutation;
[0019] c. The aforementioned gene mutation detection reagent.
[0020] 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
[0021] a. the aforementioned nucleic acid having a mutation,
[0022] b. the aforementioned polypeptide having a mutation,
[0023] c. the aforementioned gene mutations;
[0024] In some embodiments, the reagent is a nucleic acid probe or primer or other equivalent detection kit, and the primer is designed as Figure 4 As shown in, or according to the mutation site, a targeted design is made based on the existing technology. Alternatively, some other product technologies that can directly detect the mutation site, when used to detect the mutation site and to determine whether there is an antithrombin deficiency disease, should be regarded as using the solution of the present invention.
[0025] The construct comprises the aforementioned nucleic acid or the aforementioned gene mutation.
[0026] 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 may also be only one base. Among them, c.506 mainly refers to the form of the 506th base in the sequence relative to the SEQ ID NO: 1 sequence. Antithrombin deficiency diseases include antithrombin deficiency, and its induced deep vein thrombosis (DVT) and pulmonary embolism (PE) of the lower extremities, and the occurrence of post-thrombotic syndrome: such as chronic pulmonary hypertension and chronic ulcers of the lower extremities.
[0027] Drugs for the prevention and treatment of antithrombin deficiency diseases, containing at least one of the following
[0028] a. Inhibitors of the aforementioned gene mutations,
[0029] b. Inhibitors of the serpinc1 polypeptide p.S169F mutation,
[0030] c. A gene editing fragment, wherein the gene editing fragment is a fragment that can replace T at position c.506 in the serpinc1 gene mutant with 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 T at position 506 of the serpinc1 gene in cells derived from patients to C.
[0031] The gene fragment can be used by loading it on a vector, and some forms of gene vectors are human-derived cells (such as mature liver cells that can secrete antithrombin), plasmids, and adenovirus vectors. By replacing the mutant gene, it can be restored to a normal gene sequence to achieve treatment, which can achieve the purpose of prevention and treatment, or the gene vector can directly secrete normal AT in the body for a long time. At present, gene vectors are preferably known and mature, and of course new ones that can be recognized in the future should also be within the scope of the present invention.
[0032] The use of human-derived nuclear cells in the preparation of drugs for the prevention and treatment of 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 induce differentiation into mature hepatocytes. The human-derived nuclear cells are mainly derived from patients with antithrombin deficiency diseases themselves, such as peripheral blood of patients. And the single nucleotide C at the 506th position 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 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.
[0033] The beneficial effects of the present invention are:
[0034] Serpinc1 (c.506C>T, p.S169F) is related to AT. The present invention broadens the understanding of the etiology 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 AT deficiency mediated by induced pluripotent stem cells (iPSC). BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 This is a chart showing the status of thrombin in patients in the research project.
[0036] Figure 2-3 This is the Sanger sequencing map.
[0037] Figure 4 To test the primer table,
[0038] Figure 5 For pathogenicity analysis,
[0039] Figure 6 for a gene therapy program diagram;
[0040] Figure 7 This is a species-conservative 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 admitted to Wuhan Union Hospital. The patient had his first thromboembolic event at the age of 19 without obvious cause: deep vein thrombosis (DVT) and pulmonary embolism (PE) in the lower extremities, and developed post-thrombotic syndrome: such as chronic pulmonary hypertension and chronic ulcers in the lower extremities. Although the patient received appropriate long-term anticoagulant therapy such as oral warfarin or rivaroxaban, he still needed to be hospitalized every year to treat 1-2 recurrent acute venous thrombotic events.
[0044] 2. Genome 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).
[0046] Groups Lag time (min) TTP (min)_ Peak (nM) ETP (nM×min) Start to Tail_(min) Proband_AT 3 5.33 531.56 2917.62 25.67 Control 2.33 5 205 985.49 19.33
[0047] First-generation Sanger sequencing: primer design, specific design as follows Figure 4 PCR amplification. Sequencing results showed that the proband's parents had no base deletion in the gene, and it was speculated that the proband's mutation was a new mutation.
[0048] Gene sequencing showed a homozygous mutation in the SERPINC1c.506C>T (p.S169F) gene encoding antithrombin AT ( Figure 2 ; Figure 3 The patient's AT activity was 25% (reference value, 80-120%) and the concentration was 64.5% (reference value, 60-135%). Both the patient's father and mother carried heterozygous mutations at this site ( Figure 3(B). The patient's AT activity was 25% (reference value, 80-120%), and the concentration was 64.5% (reference value, 60-135%). The AT activity of the patient's father and mother was 54% and 60%, respectively, and the AT concentration was 71% and 64%, respectively.
[0049] 3. Mutation Analysis
[0050] The mutation is located in exon 2 of serpinc1, at the 169th protein position of 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 it 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 is found that the mutation is harmful and will cause disease after mutation. For species conservation results, see Figure 7 , which can also lead to abnormal protein function.
[0051] Prediction software Prediction results Polyphen2 D PROVEAN D
[0052] AT deficiency can be divided into two types of mutations: type I (quantitative), where both antigen and AT activity are decreased; and type II (qualitative), where the antigen is normal but AT activity is absent or impaired. The patient had an AT activity of 25 and an antigen of 64.5, which was type II.
[0053] IV. Treatment Plan
[0054] In this study, a severe AT-deficient patient with a newly discovered SERPINC1 mutation was found. The patient had early-onset and frequently recurrent deep vein thrombosis, and was poorly responsive to drug anticoagulation therapy, making him an ideal candidate for gene therapy. Peripheral blood mononuclear cells from the patient were collected and reprogrammed to generate iPSCs. CRISPR-Cas9 technology was used for in vitro gene correction, and the fluorescent tag originally used for screening was deleted using the Cre / loxP gene editing tool to achieve precise and specific editing of the site. The edited iPSCs were then induced into mature hepatocytes that can stably secrete normal human AT protein. These hepatocytes were transplanted into AT-deficient mice (AT KO+ / -) and successfully rescued the thrombophilic phenotype of the mice. In contrast, transplantation of hepatocytes differentiated from unedited iPSCs did not improve the expression of AT in the plasma and the tendency of thrombosis in AT+ / - KO mice. The results showed that in AT-deficient mice transplanted with hepatocytes differentiated from edited iPSCs, plasma AT antigen and AT activity remained above the normal level of AT within 3 weeks after transplantation, and decreased to the initial level in the fourth week after transplantation. It is speculated that the decrease in AT levels in mice in the late stage is due to human-mouse heterologous immune rejection, as shown by the results of mouse liver immunofluorescence staining experiments. Some specific technical details can be found in the prior art based on the technical terms in this paragraph.
[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 expression level of AT 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 normal iPSCs induced differentiated hepatocytes after autologous transplantation editing will not have obvious rejection reactions. In theory, the corrected cells can stably express AT in vivo for a long time, restore AT levels to normal, and achieve the purpose of permanent cure. Therefore, this treatment method has great application value.
[0056] Human-derived cells are edited in vitro to correct mutations and restore normal function. They are then induced to mature hepatocytes that secrete antithrombin (AT) in the human body. Hepatocytes are transplanted into the body and can effectively secrete normal levels of AT for a long time, avoiding the side effects of classical anticoagulation (drug resistance, heavy bleeding, long-term monitoring) or the ineffectiveness of common drugs. The cells are derived from the patient himself, so the possibility of immune rejection is reduced. This technical idea can be tried in the clinic, which is a new treatment idea for patients with severe hereditary thrombophilia. The development of gene editing has made this idea possible, and the novelty is to provide long-term treatment from patient-derived iPS. Currently, among the diseases related to bleeding and hemostasis, only the bleeding disease hemophilia has relevant research and is immature, and there is no such research in all thrombophilias. In addition, previous gene therapy was more through viruses such as AAV, and iPSCs were less. This gene therapy has shown good efficacy and safety. This principle can also be applied to hereditary thrombophilias such as protein C and protein S deficiency. It provides a potential treatment strategy that may cure patients with severe hereditary thrombophilia who require lifelong anticoagulation. Of course, when this type of gene therapy is used on humans, it must also comply with relevant legal regulations.
[0057] It will be clear 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. All of these modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.
Claims
1. Use of gene editing fragments in the preparation of drugs for preventing and treating hereditary antithrombin deficiency, characterized in that: The gene editing 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 and express it; the gene editing fragment is used to edit and repair the T at position 506 of the serpinc1 gene in the patient-derived cells to C, wherein the serpinc1 gene mutant has only the c.506C>T mutation compared with the wild-type serpinc1 gene with the sequence of SEQ ID NO:1.