An AAV-based gene therapy for inherited antithrombin deficiency

Abstract

The present invention discloses a gene therapy for hereditary antithrombin deficiency based on AAV, belonging to the field of gene therapy technology. The present invention explores for the first time the long-term effect and safety of hSERPINC1 gene delivery mediated by AAV vector in the treatment of hereditary AT deficiency, and proves its efficacy in restoring the AT antigen level and activity in the plasma of AT-deficient mice, rebalancing the coagulation and anticoagulation systems in the body, and reducing the thrombosis tendency of mice. AAV gene therapy is expected to completely cure AT deficiency. This discovery provides a preclinical basis for the clinical transformation of AAV therapy for the treatment of AT deficiency, and provides a new treatment direction for the long-term radical cure of patients with thrombophilias such as hereditary antithrombin deficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene therapy, and in particular relates to a gene therapy for hereditary antithrombin deficiency based on AAV. Background Art

[0002] Hereditary antithrombin (AT) deficiency is an autosomal dominant 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 the known hereditary thrombotic susceptibility diseases, AT deficiency has a high risk of VTE. Patients with AT deficiency have an increased risk of first-time VTE (hazard 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 thromboembolic events in children. The main clinical manifestation of AT heterozygous deletion is venous thrombosis, and some patients may also have arterial thrombotic events such as myocardial infarction. Homozygous deletion of AT usually leads to embryonic death, or causes giant thrombosis, purpura fulminans, and disseminated intravascular coagulation in newborns, making it difficult for them to survive.

[0003] Considering the high incidence, recurrence rate and severity of early thromboembolic events in patients with AT deficiency, it is clinically recommended that symptomatic AT mutation gene carriers and asymptomatic AT mutation gene carriers with other high-risk factors for thrombosis use drugs such as vitamin K antagonists (VKA) for lifelong anticoagulation treatment, but some patients with severe AT deficiency still experience recurrent thrombosis under adequate anticoagulation. The direct oral anticoagulants currently widely used in clinical practice have certain limitations, and their preventive effect on thromboembolic events in patients with severe thrombophilia is limited. At the same time, AT-deficient patients who receive long-term anticoagulation treatment require continuous clinical monitoring and laboratory-related indicator monitoring, because such patients show resistance to heparin when receiving anticoagulation treatment, and may also be at risk of death from massive bleeding.

[0004] In order to solve the treatment dilemma of AT deficiency patients in the clinic and achieve a safe and effective long-term cure, we consider using gene therapy. The applicant has previously successfully used CRISPR-CAS9 gene editing technology to correct the SERPINC1 mutation in induced pluripotent stem cells (iPSCs) derived from homozygous patients with severe AT deficiency. Injecting these iPCS-differentiated hepatocytes into the spleen of AT-deficient mice successfully normalized the plasma AT concentration of mice and reduced thrombosis. The study has been published in Science.<Science Transalational Medicine> This study shows that gene therapy is a promising research direction for curing AT deficiency. However, the disadvantage is that gene editing methods need to be customized according to the different locations and types of gene mutations and cannot be universal. The supplementary treatment of normal gene delivery can overcome this disadvantage and is applicable to various types of AT deficiency gene mutations.

[0005] AAV has attracted extensive attention and application due to its advantages in effectively, continuously and safely expressing foreign genes in low-immunogenic host cells. It has become the preferred gene therapy vector and the most promising gene therapy tool. rAAV vectors have been put into clinical trials in phase II-III for the treatment of hemophilia, but there are no relevant research reports on their use in the treatment of hereditary thrombophilia. Summary of the invention

[0006] One of the purposes of the present invention is to provide a recombinant adeno-associated virus (rAAV) virus particle, wherein the recombinant adeno-associated virus (rAAV) virus particle comprises an AAV8 serotype capsid, an AAV2 type gene backbone, and a gene encoding SRPINC1;

[0007] The vector genome comprises, from 5' to 3' direction:

[0008] (1) TBGp promoter;

[0009] (2) SERPINC1 gene shown in SEQ ID NO.1;

[0010] (3) SV40 PolyA;

[0011] There are arbitrary linkers between (1) and (2), and between (2) and (3).

[0012] Preferably, the vector genome comprises, in 5' to 3' direction:

[0013] (1) TBGp promoter;

[0014] (2) SERPINC1 gene shown in SEQ ID NO.1;

[0015] (3) Linker shown in SEQ ID NO.2;

[0016] (4) Luc fluorescent tag shown in SEQ ID NO.3;

[0017] (5) SV40 PolyA;

[0018] There are arbitrary linkers between (1) and (2), and between (4) and (5).

[0019] Preferably, the AAV8 serotype capsid comprises AAV8 VP1, AAV8 VP2 and AAV8 VP3.

[0020] A second object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned recombinant adeno-associated virus (rAAV) virus particles.

[0021] Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable excipient.

[0022] More preferably, the recombinant adeno-associated virus (rAAV) virus particles are the only active ingredient in the pharmaceutical composition.

[0023] The third object of the present invention is to provide the use of the above-mentioned recombinant adeno-associated virus (rAAV) virus particles or the above-mentioned pharmaceutical composition in the preparation of a product for treating hereditary antithrombin deficiency.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention explores for the first time the long-term efficacy and safety of hSERPINC1 gene delivery mediated by AAV vector in the treatment of hereditary AT deficiency, and proves its efficacy in restoring the level and activity of AT antigen in the plasma of AT-deficient mice, rebalancing the coagulation and anticoagulation systems in the body, and reducing the thrombosis tendency of mice. AAV gene therapy is expected to completely cure AT deficiency. This discovery provides a preclinical basis for the clinical transformation of AAV therapy for the treatment of AT deficiency, and provides a new treatment direction for the long-term cure of patients with thrombophilias such as hereditary antithrombin deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the AAV vector in Example 1, including NM_000488-Linker-Luc, GV632:TBGp-MCS-SV40PolyA, and the element sequence is: TBGp-SERPINC1(NM_000488-Linker-Luc))-SV40 PolyA.

[0027] Figure 2 This is a flow chart of the experiment in Example 1.

[0028] Figure 3 These are the in vivo imaging results and the detection results of plasma AT activity and concentration at different time points after AAV-hSERPINC1 injection in mice in Example 1, wherein A: in vivo imaging results of mice; B: fluorescence quantitative analysis results of in vivo imaging of mice; C: AT activity detection results; D: AT antigen level detection results.

[0029] Figure 4 This is the result of immunofluorescence staining of liver tissue sections of mice at 8 weeks after AAV-hSERPINC1 injection in Example 1. The red fluorescence represents AT, and the blue fluorescence represents DAPI.

[0030] Figure 5 These are the test results of the thrombotic tendency of mice 8 weeks after the injection of AAV-hSERPINC1 in Example 1, wherein A: the results of the in vitro thrombin generation test of mouse plasma; B: the test results of the weight of the thrombus formed after the mouse model of deep vena cava stenosis was established; C: the test results of the length of the thrombus formed after the mouse model of deep vena cava stenosis was established.

[0031] Figure 6 These are the test results of liver, kidney and heart indicators of mice at different time points after AAV-hSERPINC1 injection in Example 1. DETAILED DESCRIPTION

[0032] Example 1

[0033] 1. AAV vector construction:

[0034] (1) An AAV2 / 8 chimeric vector (type 2 gene backbone, type 8 serotype) with liver-specific promoter TBG, hSERPINC1 target gene and Luc fluorescent tag was designed and constructed. For details, see Figure 1 .

[0035] (2) NM_000488-Linker-Luc sequence:

[0036]

[0037]

[0038] The horizontal line part SEQ ID NO.2 is the linker sequence, and the wavy line part SEQ ID NO.3 is the Luc sequence.

[0039] 2. Construction of knockout mice: Construction of AT using CRISPR / Cas9 genome editing tools + / -KO mice (the mouse SERPINC1 gene GenBank accession number is NM_080844.4).

[0040] 3. Experimental plan: Inject liver-specific AAV-hSERPINC1 with Luc tag into 6-week-old AT once through the tail vein in 2-fold gradient, divided into 3 doses (0.5×10^11vg / mouse; 1×10^11vg / mouse; 2×10^11vg / mouse). + / - In KO mice, live imaging was performed on each mouse at different time points to visualize the expression of the vector in the mouse. At the same time, blood was collected from the inner canthus of the eye at this time point, and the AT antigen level and activity of the mouse plasma were measured by antigen and activity kits. The observation time was about 11 months to evaluate whether the thrombotic tendency of AT-deficient mice was improved after AAV gene therapy.

[0041] 4. Experimental results: (1) Compared with untreated knockout mice, the plasma AT concentration and activity of knockout mice injected with a moderate dose of AAV (1×10^11 vg / mouse) remained at therapeutic levels (activity: increased from 43.3±3.9% to 89.5±5.3%, p<0.0001; concentration: increased from 133.6±12.5μg / mL to 347.8±19.6μg / mL, p<0.0001) and lasted for up to 40 weeks ( Figure 3 ). Tissue immunofluorescence results showed that AAV can significantly improve AT + / - KO mouse liver AT levels. This indicates that AAV2 / 8-hSERPINC1 can successfully target the liver and restore the liver's ability to produce AT ( Figure 4 ).

[0042] (2) The mouse inferior vena cava stenosis model showed that the weight of thrombus formed in knockout mice after medium-dose AAV treatment was significantly lower than that in untreated knockout mice (10.32±1.79milligrams VS17.02±4.52milligrams, P<0.001), but there was no significant difference compared with wild-type mice (10.88±1.61milligrams) ( Figure 5 ).

[0043] (3) After AAV treatment, the liver and kidney indicators of mice were tested and it was found that the liver enzymes of mice increased in a short period of time, reaching a peak in the 8th week, and then gradually decreased ( Figure 6 During the AAV treatment observation period, no abnormal phenomena such as death or bleeding occurred in the mice in all experimental groups.

[0044] 5. Conclusion: The study demonstrated that adeno-associated virus AAV2 / 8-hSERPINC1-mediated gene therapy can continuously correct the hypercoagulable state and thrombotic tendency of mice with hereditary antithrombin deficiency. The therapy was proven to be safe and effective, providing a potential radical application for hereditary antithrombin deficiency and a preclinical basis for the clinical transformation of AAV therapy for the treatment of AT deficiency.

[0045] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant adeno-associated virus (rAAV) virus particle, characterized in that The recombinant adeno-associated virus (rAAV) virus particle comprises an AAV8 serotype capsid, an AAV2 type gene backbone, and a vector genome encoding SRPINC1; The vector genome is composed of a TBG promoter, a SERPINC1 gene shown in SEQ ID NO.1, and SV40 PolyA connected in sequence from 5' to 3' direction.

2. The recombinant adeno-associated virus (rAAV) virus particle according to claim 1, characterized in that The vector genome is composed of a TBG promoter, a SERPINC1 gene shown in SEQ ID NO.1, a Linker shown in SEQ ID NO.2, a Luc fluorescent tag shown in SEQ ID NO.3, and SV40 PolyA connected in sequence from 5' to 3' direction.

3. The recombinant adeno-associated virus (rAAV) virus particle according to claim 1 or claim 2, characterized in that The AAV8 serotype capsid comprises AAV8 VP1, AAV8 VP2 and AAV8 VP3.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the recombinant adeno-associated virus (rAAV) virus particles described in claim 1 or claim 2.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also contains a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The recombinant adeno-associated virus (rAAV) virus particles in the pharmaceutical composition are the only active ingredient.

7. Use of the recombinant adeno-associated virus (rAAV) virus particles of claim 1 or claim 2 or the pharmaceutical composition of claim 5 in the preparation of a product for treating hereditary antithrombin deficiency.

8. Use of the recombinant adeno-associated virus (rAAV) virus particles according to claim 3 in the preparation of a product for treating hereditary antithrombin deficiency.

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