CN115725595A - serping 1 gene mutant and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genes, and particularly relates to a serpinc1 gene mutant and application thereof. A nucleic acid comprising a fragment of interest having a c.506c > T mutation as compared to a wild-type serping 1 gene, wherein said wild-type serping 1 sequence is as defined by SEDIQ:1 is shown. A polypeptide having a p.s1699 f mutation compared to a wild-type serpinc1, wherein said wild-type serpinc1 sequence is as defined by SEDIQ:2, respectively. The medicine for preventing and treating antithrombin deficiency disease at least contains an editing gene segment, wherein the editing gene segment is a segment capable of replacing mononucleotide T at position c.506 in a serpinc1 gene mutant with mononucleotide C. The present invention provides a broadening of the recognition of antithrombin deficient diseases; provides a new diagnosis mode for antithrombin deficiency, provides a new way for preventing and treating antithrombin deficiency, and discusses the possibility of treating AT deficiency based on Induced Pluripotent Stem Cell (iPSC) mediated gene.

Description

serping 1 gene mutant and application thereof

Technical Field

The invention belongs to the technical field of genes, and particularly relates to a serpinc1 gene mutant and application thereof.

Background

Genetic Antithrombin (AT) deficiency is an autosomal dominant genetic disease with a prevalence of about 0.02% to 0.2% in the general population and about 1% to 5% in patients with Venous Thromboembolism (VTE). The risk of VTE for AT deficiency is very high in known patients with genetic predisposition to thrombosis. Patients with AT deficiency have an increased risk of developing a primary venous thromboembolic event (odds ratio 14.0, 95% CI, 5.5-29.0), and a VTE risk of annual relapse of 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 major clinical manifestation of AT deficiency in heterozygotes is venous thromboembolism, but some patients may develop arterial thrombotic events such as myocardial infarction. Homozygous AT defects are often embryonic lethal or result in embryos that have great neonatal thrombosis or purpura fulminans and are difficult to survive. The combined deficiency of SERPINC1 and VLeiden factors in families with multiple thromboembolic events has also been reported in western countries. AT defects can be divided into two types of mutations: type I (quantitative), i.e. both antigenic and AT activity decreased; type II (qualitative), i.e., normal antigen, mutation with absent or impaired AT activity.

In combination with the disease profile of AT deficiency, symptomatic patients with AT deficiency due to SERPINC1 mutations are clinically encouraged to use Vitamin K Antagonists (VKA) for life-long anticoagulation and asymptomatic carriers to prevent antithrombotic treatment in AT-risk situations. A significant number of patients with severe AT deficiency have a recurrence of venous thromboembolism, even after appropriate anticoagulation. Meanwhile, AT-deficient patients who undergo long-term anticoagulation therapy need continuous clinical monitoring, and they may develop heparin resistance and also risk heavy bleeding. Genetic AT deficiency is caused by a pathogenic mutation of the antithrombin gene (SERPINC 1). There are more than 400 different variations, mainly single nucleotide variations or insertions/deletions of small fragments, which 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 would avoid all the side effects of classical route anticoagulant therapy. Therefore, it is of great clinical significance to investigate the feasibility and safety of gene therapy.

Disclosure of Invention

Aiming at the problems, the invention provides a serpinc1 gene mutant and application thereof, mainly finds a new pathogenic gene aiming at antithrombin deficiency diseases, and provides a new direction for subsequent diagnosis and treatment.

In order to solve the problems, the invention adopts the following technical scheme:

a nucleic acid comprising a fragment of interest having a c.506c > T mutation as compared to a wild-type serping 1 gene, wherein said wild-type serping 1 sequence is as defined by SEDIQ:1 is shown.

A polypeptide having a p.s1699 f mutation compared to a wild-type serpinc1, wherein said wild-type serpinc1 sequence is as defined by SEDIQ:2 (GenBank: CAA 48690.1).

A gene mutation having a c.506c > T mutation as compared to a wild-type serpinc1 gene, wherein the wild-type serpinc1 sequence is as defined in SEDIQ:1 is shown.

When mononucleotide or amino acid sequence sites of other sites of the serpinc1 gene are mutated, the protection of the detection site related to the invention is not influenced, and on the premise that the mutation of the target site is the same, the other sites are compared with the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2 should also be within the scope of the present invention.

Use of a biological model carrying at least one of the following for the preparation of an antithrombin deficient disease screening formulation

a. The nucleic acid having a mutated nucleic acid as described above,

b. the aforementioned polypeptide having a mutated nucleic acid,

c. a gene mutation of the aforementioned gene mutation;

preferably, the antithrombin deficiency disorder includes AT deficiency, AT deficiency-induced venous thrombosis, and post-thrombotic syndrome; AT deficiency is hereditary antithrombin deficiency.

When used in the preparation of screening reagents, reference is made to the subsequent specific use; when the biological inhibitor is used in preparation of a prevention and treatment agent, some modes are used as action targets of the medicine, or other biological medicines are used in steps of testing, extracting and the like in the preparation process, and the biological inhibitor is mainly used as auxiliary agents in the pharmaceutical process, for example, in the test of the action effect of the inhibitor in the pharmaceutical process.

Use of a detection reagent in the preparation of an antithrombin deficiency disease screening reagent, wherein the detection reagent is at least one of

a. A detection reagent for the nucleic acid having a mutation as described above;

b. a detection reagent for the aforementioned polypeptide having a mutation;

c. the aforementioned gene mutation detection reagent.

An antithrombin deficiency disease screening reagent comprising a reagent capable of detecting a serpinc1 gene mutation, wherein the serpinc1 gene mutation comprises at least one of

a. The aforementioned nucleic acid having a mutation is,

b. the aforementioned polypeptide having a mutation is a polypeptide having,

c. mutations of the aforementioned genes;

in some embodiments, the reagent is a nucleic acid probe or primer or other equivalent detection kit, and the primer is designed as shown in fig. 4, or is designed specifically according to the mutation site in the prior art. Alternatively, other product technologies capable of directly detecting the mutation site, when used for detecting the mutation site and determining whether the antithrombin deficiency disease is present, should be regarded as using the scheme of the present invention.

A construct comprising a nucleic acid as described above or a mutation in a gene as described above.

The application of the gene editing segment in preparing the medicines for preventing and treating the antithrombin deficiency diseases is characterized in that the gene editing segment can replace the mononucleotide T at the c.506 position in the serpinc1 gene mutant into the mononucleotide C. The antithrombin deficiency disease is mainly hereditary antithrombin deficiency, and the editing gene segment can be only one base. Wherein c.506 is predominantly directed to the gene relative to SEQ ID NO:1, the 506 th base form in the sequence is mainly considered. Antithrombin deficiency disorders include antithrombin deficiency, and its induced Deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) of the lower extremities, and the development of post-thrombotic syndrome: such as chronic pulmonary hypertension and chronic ulcers of the lower extremities.

A medicine for preventing and treating antithrombin deficiency diseases contains at least one of the following components

a. An inhibitor of the aforementioned mutation of a gene,

an inhibitor of the serpinc1 polypeptide p.s19f mutation,

c. a gene editing segment, wherein the gene editing segment can replace C with C at c.506 in a serpinc1 gene mutant; specifically, the fragment can be expressed by replacing the mononucleotide T at the 506 th site of the serpinc1 gene with the mononucleotide C, and preferably, the antithrombin deficiency disease is antithrombin deficiency. For example, the gene editing fragment is used for repairing T editing at position 506 of serping 1 gene in cells from a patient to C.

The gene fragment can be used by being carried on a vector, and some expression forms of the gene vector are human-derived cells (such as mature hepatocytes capable of secreting antithrombin), plasmids, and adenovirus vectors. The mutant gene is replaced to restore the normal gene sequence to realize treatment, thereby achieving the purposes of prevention and treatment, or the gene vector directly effectively secretes normal AT in vivo for a long time, and the like. At present, the gene vector is preferably considered to be known and mature, although future emergence will gain approval and should be within the scope of the present invention.

The application of the human nuclear cells in preparing the medicines for preventing and treating the antithrombin deficiency diseases is characterized in that the 506 th site of a serpinc1 editing gene in the human nuclear cells is mononucleotide C, and the human nuclear cells can be induced and differentiated into mature liver cells. Wherein the human-derived nuclear cells are mainly derived from the patient suffering from the antithrombin deficiency disease, such as peripheral blood of the patient. And the mononucleotide C at 506 th site of serpinc1 editing gene in the human-derived nuclear cell is obtained by editing and correcting, and the mononucleotide C at 506 th site of the serpinc1 editing gene in the original pathogenic human-derived nuclear cell is not mononucleotide C. Of course, the human nuclear cell nucleus is mainly used for treating antithrombin deficiency diseases induced by serpinc1 gene c.506C > T, c.506C > G or c.506C > A type mutation.

The invention has the beneficial effects that:

serpinc1 (c.506C > T, p.S1699F) is related to AT, and the invention widens the understanding of the causes of antithrombin deficiency diseases; provides a new diagnosis mode for antithrombin deficiency, provides a new way for preventing and treating antithrombin deficiency, and discusses the possibility of treating AT deficiency based on Induced Pluripotent Stem Cell (iPSC) mediated gene.

Drawings

FIG. 1 is a chart of thrombin status in patients in a study,

FIGS. 2-3 are Sanger sequencing charts,

FIG. 4 is a table of detection primers,

FIG. 5 is a diagram showing the pathogenicity analysis,

FIG. 6 is a gene therapy program diagram;

FIG. 7 shows the results of species conservation.

Detailed Description

The invention is further illustrated below with reference to specific research projects:

1. sample collection

Wuhan harmonizes with a male patient who is accepted by hospital. Patients were first free of obvious causes of thromboembolic events at age 19: deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) of the lower limbs, and post-thrombotic syndrome: such as chronic pulmonary hypertension and chronic ulcers of the lower extremities. Despite the fact that patients receive appropriate long-term anticoagulant therapy such as oral warfarin or rivaroxaban, hospitalization is required to treat 1-2 recurrent acute venous thrombotic episodes each year.

2. Genomic detection

Study analysis was performed after obtaining informed consent from all family members. It was found by examination that thrombin generation was significantly increased in the patients compared to normal healthy persons (fig. 1, table 1).

Generation Sanger sequencing: designing a primer, wherein the specific design is shown in figure 4; and (4) PCR amplification. Sequencing results show that parents of the proband have no base deletion on genes, and the mutation of the proband is presumed to be a new mutation.

Gene sequencing revealed that the coding gene SERPINc1c.506C > T (p.S1699F) homozygote mutation of antithrombin AT (FIG. 2; A in FIG. 3) resulted in the mutation of the original serine to phenylalanine, and no mutation was found in the genes of protein C, protein S, VLeiden and prothrombin G20210A. This mutation was not reported in the GnomAD or HGMD databases. The patient had an AT activity of 25% (reference, 80-120%) and a concentration of 64.5% (reference, 60-135%). Both the patient father and mother carry a heterozygous mutation at this site (B in fig. 3). The patient had an AT activity of 25% (reference, 80-120%) and a concentration of 64.5% (reference, 60-135%). The patient's father and mother had 54% and 60% AT activity, respectively, and 71% and 64% AT concentration, respectively.

3. Mutation analysis

The mutation is located in exon 2 of serpinc1 and in position 169 of the serpinc1 protein. The mutation NM _000488.3: c.506C > T | NP _000479.1: s1699F, in which the base C of the cDNA was mutated to the base T at 506, in accordance with the mutation sequence in comparison with the normal sequence. This region is the heparin binding region, resulting in ThrombHaemost. The mutation was found to be harmful and pathogenic after mutation, according to the analysis of the harmfulness prediction software including Polyphen2, PROVEAN, etc., see Table 2 below. Species conservation results see figure 7, which also results in protein dysfunction.

AT defects can be divided into two types of mutations: type I (quantitative), i.e. both antigenic and AT activity decreased; type II (qualitative), i.e. mutations where the antigen is normal, AT activity is absent or impaired. The patient had AT activity 25, antigen 64.5, which was type II.

4. Treatment regimens

In this study, a severe AT-deficient patient was found with a newly discovered SERPINC1 mutation. The patient has early and recurrent deep venous thrombosis, and the medicine has poor anticoagulation treatment effect, so the medicine is an ideal candidate suitable for a gene therapy method. Peripheral blood mononuclear cells from the patient were collected and reprogrammed to generate iPSCs. And (3) carrying out in-vitro gene correction by using a CRISPR-Cas9 technology, and deleting the fluorescent label originally used for screening by using a Cre/loxP gene editing tool to realize accurate and specific editing of the site. And then inducing the edited iPSCs into mature hepatocytes capable of stably secreting human normal AT protein. These hepatocytes were transplanted into AT-deficient mice (ATKO +/-), successfully rescuing the thrombosis-prone phenotype of the mice. In contrast, liver cell transplantation with unedited iPSCs induced differentiation did not improve AT expression and thrombophilia in the plasma of AT +/-KO mice. The results show that plasma AT antigen and AT activity of the liver cell transplanted AT-deficient mice induced and differentiated by iPSCs after the editing are maintained above the AT normal level within 3 weeks after the transplantation and are reduced to the initial level in the fourth week after the transplantation. The later AT level decrease of the mice is presumed to be caused by the xenorejection of the human and the mice, as shown by the immunofluorescence staining experimental results of the liver of the mice. Some of the specific technical details may be found in the prior art based on the technical terms in this paragraph.

Using patient-derived cells, it was demonstrated that human AT mutant cells can treat diseases by in vitro gene correction and induction of differentiation. After the human cells are transplanted into a mouse body, immune rejection is inevitable, and the AT expression level is gradually reduced. It is well known that xenotransplantation is more susceptible to irreversible immunological rejection than xenotransplantation. Activation of mouse macrophages after xenotransplantation of human hepatocytes into mice is a major obstacle to long-term survival of human hepatocytes. However, it is believed that normal iPSCs induced differentiated hepatocytes after autograft editing, and there was no significant rejection. Theoretically, the corrected cells can stably express AT in vivo for a long time, so that the AT level is recovered to be normal, and the aim of permanent cure is fulfilled. Therefore, the treatment method has good application value.

Cells from human bodies are subjected to gene editing and mutation correction in vitro to restore the normal functions of the cells, then are directionally induced into mature hepatocytes mainly secreting Antithrombin (AT) in human bodies, and are transplanted into the human bodies, so that the hepatocytes are planted in the human bodies, normal-level AT can be effectively secreted for a long time, and the side effects (drug resistance, heavy bleeding and long-term monitoring) of classical path anticoagulation or the condition that common drugs are ineffective are avoided. The cell is derived from the patient, so the possibility of immunological rejection is reduced, the technical idea can be tried in clinic, and the cell is a new treatment idea for the patient with serious heredity and easy thrombosis. The development of gene editing enabled this idea to be newly aimed at long-term therapy from patient-derived ips. Currently, among bleeding and hemostasis-related diseases, only bleeding disease hemophilia has related research and is immature, and not among all thrombosis-prone diseases. Moreover, more of the previous gene therapy was by viruses such as AAV, iPSCs. The gene therapy shows good efficacy and safety. The principle can also be applied to genetic thrombosis-prone diseases such as deficiency of protein C and protein S. Provides a potential treatment strategy, and has the possibility of curing serious hereditary thrombosis patients needing lifetime anticoagulation. Of course, when the gene therapy mode is applied to human bodies, relevant legal requirements also need to be met.

It will be apparent to those skilled in the art that various modifications to the above embodiments can be made without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims (10)

1. A nucleic acid comprising a fragment of interest having a c.506c > T mutation as compared to a wild-type serpinc1 gene, wherein the wild-type serpinc1 sequence is such as SED IQ:1 is shown.

2. A polypeptide having a p.s1699 f mutation compared to a wild-type serping 1 sequence, such as SED IQ:2, respectively.

3. A genetic mutation characterized in that it has a c.506c > T mutation as compared to a wild-type serping 1 gene, wherein the wild-type serping 1 sequence is as defined in SED IQ:1 is shown.

4. Use of a biological model for the preparation of an antithrombin deficient disease screening formulation, characterized in that the biological model carries at least one of the following

a. The nucleic acid according to claim 1, wherein said nucleic acid is a nucleic acid,

b. the polypeptide of claim 2, wherein said polypeptide is,

c. the gene mutation of claim 3.

5. Use of a detection reagent in the preparation of an antithrombin deficiency disease screening reagent, wherein the detection reagent is at least one of

a. A detection reagent for the nucleic acid of claim 1;

b. a detection reagent for the polypeptide of claim 2;

c. a reagent for detecting a mutation in the gene according to claim 3.

6. An antithrombin deficiency disease screening reagent, comprising a reagent capable of detecting serpinc1 gene mutation, wherein the serpinc1 gene mutation includes at least one of

a. The nucleic acid according to claim 1, wherein said nucleic acid is,

b. the polypeptide according to claim 2, wherein said polypeptide,

c. the gene of claim 3.

7. The reagent for screening of antithrombin deficient disease according to claim 6 wherein said reagent is a nucleic acid probe or primer.

8. A construct comprising the nucleic acid of claim 1 or the genetic mutation of claim 3.

9. The application of the gene editing segment in preparing the medicine for preventing and treating the antithrombin deficiency disease is characterized in that the gene editing segment is a segment which can replace the mononucleotide T at the c.506 position in the serpinc1 gene mutant into the mononucleotide C.

10. A pharmaceutical composition for preventing and treating antithrombin deficiency diseases, characterized by comprising at least one of the following

an inhibitor of c.506C > T mutation of serpinc1 gene,

an inhibitor of the serpinc1 polypeptide p.s19f mutation,

c. and editing a gene segment, wherein the gene segment is a segment capable of replacing the mononucleotide T at the c.506 position in the serpinc1 gene mutant with the mononucleotide C.

The application of the human nuclear cells in preparing the medicines for preventing and treating the antithrombin deficiency diseases is characterized in that c.506 of serpinc1 editing genes in the human nuclear cells is mononucleotide C, and the human nuclear cells can be induced and differentiated into mature liver cells.

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