Tissue-specific promoters and their use

Abstract

Provided is a tissue-specific promoter and its use. The tissue-specific promoter has a nucleic acid sequence comprising more than 80% of the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. The tissue-specific promoter can promote the specific expression of the coding gene in endothelial cells (EC) or megakaryocyte-platelet cells, and can be applied to gene therapy where the gene is required to be specifically expressed in EC or megakaryocyte-platelet cells, ensuring the therapeutic effect, reducing the risk of immune rejection and saving the therapeutic cost.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to tissue-specific promoters and their use.

Background Art

[0002] Gene therapy refers to introducing a foreign gene into target cells in order to correct or supplement a defective gene or abnormal gene for the purpose of treating diseases caused by the defective gene or abnormal gene. However, non-specific expression often occurs, that is, the foreign gene is widely expressed in the human body, causing immune rejection in the human body, which is one of the difficult problems to solve clinically.

[0003] For example, hemophilia A (HA), also known as hereditary antihemophilia globulin deficiency or FVIII (F8) deficiency, is a blood coagulation disorder caused by a genetic defect in the coagulation factor VIII gene (FVIII gene or F8 gene). Current treatments for HA mainly include protein replacement therapy with plasma-derived coagulation factors or exogenously produced recombinant proteins, and gene therapy. Analysis of the human F8 gene revealed the domain structure of the protein represented by A1-A2-B-A3-C1-C2. The B domain is encoded by a large exon that has a highly conserved region consisting of asparagine (N)-linked oligosaccharides. Miao et al. showed that partially deleting the B domain, while retaining the 226 amino acids at the N-terminus containing six complete asparagine-binding glycosylation sites, could increase F8 secretion tenfold in vitro (see Miao, HZ, Sirachainan, N., Palmer, L., Kucab, P., Cunningham, MA et al., Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004, 103(9), pp. 3412-3419). However, gene therapy using the B domain-deleted F8 gene (F8-BDD) currently has problems such as low protein secretion and function, low efficiency of F8 viral vector introduction, and antibody formation associated with inhibitory reactions (immune rejection). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Miao, HZ, Sirachainan, N., Palmer, L., Kucab, P., Cunningham, MA et al., Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004, 103(9), pp. 3412-3419. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, in order to avoid immune rejection, achieving tissue-specific in vivo expression of therapeutic genes is an urgent issue that needs to be addressed in the field of gene therapy. [Means for solving the problem]

[0006] (Summary) Considering the shortcomings of existing technologies and practical requirements, this application provides a tissue-specific promoter and its use. The tissue-specific promoter can promote the specific expression of coding genes in endothelial cells (ECs), megakaryocytes, or platelets, and reduce ectopic expression in unrelated tissue cells. The tissue-specific promoter can be applied to gene therapies where the gene needs to be specifically expressed in ECs, effectively reducing antibody and inhibitor responses.

[0007] To achieve the above objectives, this application employs the following technical solutions.

[0008] In a first embodiment, the application provides a tissue-specific promoter having a nucleic acid sequence comprising more than 80% of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0009] [ka] TIFF0007884286000002.tif105165

[0010] This application creatively designs tissue-specific promoters, including promoters VEC (SEQ ID NO: 1 or having at least 80% homology to SEQ ID NO: 1) and KDR (SEQ ID NO: 2 or having at least 80% homology to SEQ ID NO: 2) that promote specific gene expression in EC, and promoters ITGA (SEQ ID NO: 3 or having at least 80% homology to SEQ ID NO: 3) and Gp (SEQ ID NO: 4 or having at least 80% homology to SEQ ID NO: 4) that promote specific gene expression in megakaryocyte-platelet cells. By using the tissue-specific promoters of this application, coding genes can be specifically expressed in EC or megakaryocyte-platelet cells. Therefore, the tissue-specific promoters of this application can be effectively applied to gene therapies that require the specific expression of target coding genes in EC or megakaryocyte-platelet cells, reducing the risk of immune rejection and saving treatment costs, such as in HA gene therapy.

[0011] Preferably, the tissue-specific promoter has the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0012] In a second embodiment, the present application provides a gene expression cassette comprising a tissue-specific promoter and coding gene as described in the first embodiment.

[0013] Preferably, the coding gene includes the coding gene for recombinant coagulation factor VIII.

[0014] Preferably, the coding gene for recombinant coagulation factor VIII contains the nucleic acid sequence shown in Sequence ID No. 5.

[0015] [ka] TIFF0007884286000004.tif96166

[0016] In a specific example of the present application, the tissue-specific promoter of the present application is used to initiate the expression of the coding gene of recombinant coagulation factor VIII. By expressing the FVIII gene in ECs (such as hepatic sinusoidal ECs) or megakaryocytes, the ectopic expression of FVIII protein in vivo is reduced, or the antibody response and inhibitor response are reduced, and the gene therapy of HA is effectively carried out.

[0017] In a third aspect, the present application provides a recombinant expression vector. The recombinant expression vector contains the tissue-specific promoter described in the first aspect.

[0018] Preferably, the recombinant expression vector includes a viral vector or a plasmid vector containing the tissue-specific promoter described in the first aspect.

[0019] Preferably, the viral vector includes the lentiviral vector pEGWI.

[0020] Preferably, the recombinant expression vector further contains a coding gene.

[0021] Preferably, the coding gene includes the coding gene of recombinant coagulation factor VIII.

[0022] Preferably, the 5' splice donor site of the lentiviral vector pEGWI is mutated.

[0023] Preferably, the enhancer in the U3 region of the lentiviral vector pEGWI is deleted.

[0024] Preferably, the U3 region of the lentiviral vector pEGWI contains an insulator.

[0025] In this application, the lentiviral vector pEGWI has been modified. The wild-type 5' splice donor site has been mutated, the enhancer in the U3 region has been deleted, and an insulator (cHS4 insulator) has been added to the U3 region. This effectively improves the introduction and expression efficiency of pEGWI, reducing the cost of using the vector and also improving its safety.

[0026] In a specific example of this application, a tissue-specific promoter and the coding gene for recombinant coagulation factor VIII are co-inserted into the lentiviral vector pEGWI. The lentiviral vector is then introduced into the body by direct intravenous injection, and the gene for coagulation factor VIII is efficiently delivered and specifically expressed, thereby effectively ensuring therapeutic efficacy, reducing the risk of immune rejection, saving on treatment costs, and realizing highly efficient HA gene therapy.

[0027] In a fourth embodiment, the application provides a recombinant lentivirus comprising the recombinant expression vector described in the third embodiment.

[0028] In a fifth embodiment, the application provides recombinant cells containing the tissue-specific promoter described in the first embodiment.

[0029] Preferably, the gene expression cassette described in the second embodiment is incorporated into the genome of recombinant cells.

[0030] Preferably, the recombinant cells contain the recombinant expression vector described in the third embodiment.

[0031] In a sixth embodiment, the present application provides a method for preparing recombinant cells as described in the fifth embodiment. The method comprises the following steps: The method includes introducing a tissue-specific promoter according to the first embodiment, a gene expression cassette according to the second embodiment, a recombinant expression vector according to the third embodiment, or a recombinant lentivirus according to the fourth embodiment into a host cell to obtain recombinant cells.

[0032] Preferably, the introduction is carried out by a method comprising one of the following: electrical introduction, viral vector system, non-viral vector system, or direct gene injection.

[0033] Preferably, the host cells include hematopoietic stem cells.

[0034] In a seventh aspect, the application provides a pharmaceutical composition comprising one or at least two of the following: a tissue-specific promoter as described in the first aspect, a gene expression cassette as described in the second aspect, a recombinant expression vector as described in the third aspect, a recombinant lentivirus as described in the fourth aspect, or a recombinant cell as described in the fifth aspect.

[0035] Preferably, the pharmaceutical composition further comprises one or at least two combinations of pharmaceutically acceptable carriers, excipients, or diluents.

[0036] In an eighth aspect, the application provides the use of a tissue-specific promoter according to the first aspect, a gene expression cassette according to the second aspect, a recombinant expression vector according to the third aspect, a recombinant lentivirus according to the fourth aspect, a recombinant cell according to the fifth aspect, or a pharmaceutical composition according to the seventh aspect in the preparation of a drug for tissue-specific gene therapy. [Effects of the Invention]

[0037] Compared to existing technologies, this application has the following beneficial effects: (1) The tissue-specific promoter of this application can promote the specific expression of coding genes in EC or megakaryocyte-platelet cells and can be applied to gene therapies in which the gene is required to be specifically expressed in EC or megakaryocyte-platelet cells, thereby ensuring therapeutic efficacy, reducing the risk of immune rejection, and saving on treatment costs.

[0038] (2) In this application, an expression vector is constructed using a tissue-specific promoter, the coding gene for coagulation factor VIII, and a lentiviral vector. The expression vector can be expressed normally in HA mice, can modify the hemorrhagic phenotype of HA mice to some extent, and has a low antibody response, which is very important in ensuring the effectiveness of gene therapy and lays the foundation for achieving faster relief of HA symptoms and more comprehensive and durable gene therapy. [Brief explanation of the drawing]

[0039] [Figure 1] Structural diagram of the lentiviral vector pEGWI. [Figure 2] These are structural diagrams of different tissue-specific promoters and the F8-BDD gene in lentiviral vectors. [Figure 3] This figure shows the vector copy number (VCN) of recombinant lentiviruses in ECs and megakaryocytes. [Figure 4] This figure shows the results of analyzing the amount of fluorescence expressed in cells transduced with recombinant lentivirus LV-wasabi. [Figure 5] This figure shows the results of the amount of protein expressed in cells transduced with recombinant lentivirus LV-F8-BDD. [Figure 6] This figure shows the detection results of the in vitro plasma substrate luminescence method. [Figure 7] This diagram shows the treatment methods for HA mice. [Figure 8] This figure shows the detection results of positive coagulation factor VIII in mouse blood cells. [Figure 9] This figure shows the activity of coagulation factor VIII in mice. [Figure 10] This figure shows the detection results of enzyme-linked immunosorbent assay (ELISA) in mouse plasma. [Modes for carrying out the invention]

[0040] To further explain the technical means employed and the effects achieved in this application, this application will be described below with examples and drawings. It should be understood that the following specific examples are for illustrative purposes only and are not intended to limit this application. [Examples]

[0041] Experiments in which specific techniques or conditions are not described in the examples shall be carried out in accordance with the techniques or conditions described in the relevant technical literature or product specifications. Reagents or equipment used herein that are not specified by manufacturer are conventional products commercially available through legitimate channels.

[0042] Although HA is used as an example in the embodiments of this application, the tissue-specific promoter of this application has been shown to be effectively applicable to gene therapy and to be able to effectively reduce antibody and inhibitory responses to coagulation factors.

[0043] [Example 1] A lentiviral vector possessing the specific promoter and F8 gene of this application was constructed. Specifically, this embodiment includes the following steps.

[0044] (1) The structural diagram of the lentivirus vector pEGWI is shown in Figure 1. The wild-type 5' splice donor site was mutated, the enhancer in U3 was deleted, and an insulator (cHS4 insulator) was added to U3. For details on the modification method, please refer to "Contributions of Viral Splice Sites and cis-Regulatory Elements to Lentivirus Vector Function," Cui et al., Journal of Virology, July 1999, pp. 6171-6176.

[0045] (2) Insertion of different tissue-specific promoters and the F8-BDD gene The Wasabi gene sequence (expressing a fluorescent protein), the B-domain deletion F8 gene (F8-BDD) sequence (SEQ ID NO: 5), and the nucleic acid sequences of tissue-specific promoters EF1α (SEQ ID NO: 6), VEC (SEQ ID NO: 1), KDR (SEQ ID NO: 2), ITGA (SEQ ID NO: 3), and Gp (SEQ ID NO: 4) were synthesized by whole-genome synthesis. Each of the above promoters and F8-BDD were co-ligated to the lentiviral vector pEGWI through restriction enzyme sites. The resulting products were identified by sequencing and dual enzyme digestion, following the optimal reaction conditions recommended by New England Biolabs (NEB). The BamHI cloning site (ggatccacc)-AUG was used at the 5' end, and the SpeI cloning site (actagt) was used at the 3' end. Properly ligated lentiviral vectors pEGWI-EF1α-F8-BDD, pEGWI-VEC-F8-BDD, pEGWI-KDR-F8-BDD, pEGWI-ITGA-F8-BDD, or pEGWI-Gp-F8-BDD were obtained by driving the F8-BDD gene under promoters containing EF1α, VEC, KDR, ITGA, or Gp, respectively. The specific ligation sites and compositions of the lentiviral vectors are shown in Figure 2. Each promoter and the Wasabi gene were co-inserted into pEGWI to obtain lentiviral vectors pEGWI-EF1α-Wasabi, pEGWI-VEC-Wasabi, pEGWI-KDR-Wasabi, pEGWI-ITGA-Wasabi, or pEGWI-Gp-Wasabi, which were used as controls in subsequent experiments.

[0046] [ka]

[0047] [Example 2] In this example, the lentiviral vector constructed in Example 1 was further packaged, purified, and concentrated to obtain recombinant lentivirus. For experimental methods, please refer to "[1] Chang LJ, Urlacher V, Iwakuma T et al., Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system [J]. Gene Therapy, 1999, 6(5):715-728" and "[2] Chang LJ, Zaiss AK. Chang, LJ and Zaiss, AK. Lentiviral vectors. Preparation and use. Methods Mol Med 69:303-318 [J]. Methods in Molecular Medicine, 2002, 69:303-318".

[0048] For specific steps, please refer to the above-mentioned literature. A brief explanation of the specific steps follows below.

[0049] (1) The lentiviral vector constructed in Example 1, along with the packaged helper plasmids pNHP and pHEF-VSV-G, were co-introduced into mammalian cells HEK293T and cultured for 48 hours, after which the supernatant was collected.

[0050] (2) The recovered lentiviruses were purified and concentrated to obtain recombinant lentiviruses named LV-EF1α-F8-BDD, LV-VEC-F8-BDD, LV-KDR-F8-BDD, LV-ITGA-F8-BDD, LV-Gp-F8-BDD, LV-EF1α-Wasabi, LV-VEC-Wasabi, LV-KDR-Wasabi, LV-ITGA-Wasabi, and LV-Gp-Wasabi, respectively.

[0051] (3) Lentivirus VCNs were detected, and the detection results are shown in Figure 3. Lentiviruses LV-EF1α-F8-BDD, LV-VEC-F8-BDD, LV-KDR-F8-BDD, LV-ITGA-F8-BDD, and LV-Gp-F8-BDD showed essentially similar VCNs at the same MOI (Moment of Infection).

[0052] [Example 3] In this example, recombinant lentiviruses containing different promoters and Wasabi genes prepared in Example 2 were tested in vitro. Promoter specificity in different cells was detected by detecting the amount of fluorescent protein expressed by the Wasabi gene.

[0053] The five lentiviruses (LV-EF1α-Wasabi, LV-VEC-Wasabi, LV-KDR-Wasabi, LV-ITGA-Wasabi, and LV-Gp-Wasabi) containing the normal Wasabi gene, prepared in Example 2, were separately transduced into two cell lines: endothelial cells (EC) and megakaryocytes. The method of lentivirus introduction is described below.

[0054] Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution was added to 6-well plates (Corning Incorporated, USA). 3 × 10 4 EC or 1 × 10 5 Each megakaryocyte was inoculated into a well and cultured at 37°C under 5% CO2 for 18 hours. Then, lentivirus was introduced at a MOI of 200, and polyblen (8 μg / mL, Sigma-Aldrich) was added up to a final volume of 600 μL, followed by transduction for 24 hours. Subsequently, the medium was replaced with fresh medium daily. When the cells reached 90% confluence, the cells were transferred to a T75cm well. 2 It was transferred to a culture flask (Corning Incorporated, USA).

[0055] The amount of expressed fluorescent protein was detected to determine the expression of the Wasabi gene in cells. The results are shown in Figure 4. Both ECs transduced with LV-EF1α-Wasabi and megakaryocytes transduced with LV-EF1α-Wasabi showed high fluorescence intensity, indicating that the EF1α promoter efficiently promotes Wasabi gene expression in these two cell types and is not tissue-specific. ECs transduced with LV-VEC-Wasabi and LV-KDR-Wasabi, respectively, showed higher fluorescence intensity than ECs transduced with LV-ITGA-Wasabi and LV-Gp-Wasabi, respectively. Megakaryocytes transduced with LV-VEC-Wasabi and LV-KDR-Wasabi, respectively, showed lower fluorescence intensity than megakaryocytes transduced with LV-ITGA-Wasabi and LV-Gp-Wasabi, respectively. In conclusion, the VEC and KDR promoters are EC-specific, while the ITGA and Gp promoters are megakaryocyte-specific.

[0056] [Example 4] In this example, the recombinant lentivirus containing the F8-BDD gene prepared in Example 2 was tested in vitro.

[0057] The five lentiviruses (LV-EF1α-F8-BDD, LV-VEC-F8-BDD, LV-KDR-F8-BDD, LV-ITGA-F8-BDD, and LV-Gp-F8-BDD) possessing the F8-BDD gene, prepared in Example 2, were separately transduced into two cell lines: endothelial cells (EA-hy926) and megakaryocytes (DAMI). The method of lentivirus introduction was the same as in Example 3.

[0058] The supernatants secreted from transduced EA-hy926 and DAMI cells were collected and concentrated. Intracellular extracts were also collected. Protein expression levels were detected by ELISA. Cells not transduced with lentivirus were used as negative controls (NC). The results are shown in Figure 5. The universal EF1α promoter efficiently promoted F8 expression in both cell types. In megakaryocytes, the ITGA promoter efficiently promoted F8 expression. In EC cells, the VEC promoter was more effective at promoting F8 expression than other tissue-specific promoters, but F8 was expressed at significantly lower levels (10 times lower) compared to the EF1α promoter.

[0059] The method for evaluating coagulation function is based on a substrate luminescence assay, which measures activity using a human F8 colorimetric assay kit (Hyphen BioMed, France). The substrate luminescence assay is performed as follows: The plasma to be tested and the blank control group are diluted 40-fold with Tris-BSA buffer (R4+), and 50 μL of each system is added to a microplate. 50 μL of factor X (R1), a mixture of activated factor IX (R2), and 50 μL of SXa-11 substrate (R3) are added to the system. The system is incubated at 37°C for 5 minutes, and the reaction is stopped by adding 50 μL of 20% acetic acid. The absorbance value at 405 nm is read.

[0060] The supernatants of EA-hy926 and DAMI transduced with the virus were taken out at -80°C, thawed on ice, and each supernatant was mixed with plasma from F8-deficient individuals. Only the plasma from F8-deficient individuals was used as the non-container (NC), while plasma from healthy volunteers was used as the positive control (PC). Substrate luminescence was used for detection.

[0061] Figure 6 shows the detection results of human F8 by substrate luminescence assay. In the supernatants of EA-hy926 cells in which F8 expression was promoted by EF1α and EA-hy926 cells in which F8 expression was promoted by VEC, human F8 activity within the therapeutic range was detected, approximately 6 times and 2.5 times the normal level, respectively. On the other hand, human F8 activity was not detected in the supernatants of cells containing other promoters. In the supernatants of DAMI cells in which F8 expression was promoted by EF1α and DAMI cells in which F8 expression was promoted by ITGA, human F8 activity 5 times higher than the normal level was detected.

[0062] In conclusion, in this application, the expression of the F8 gene is successfully promoted by the tissue-specific promoter of the lentiviral vector to express normal human F8 protein in cells, and the VEC promoter and ITGA promoter have relatively good specificity and promote the expression of human F8 protein which may have high activity and coagulation function.

[0063] [Example 5] The lentiviruses containing F8-BDD prepared in Example 2 were separately injected directly into HA mice via the tail vein, and therapeutic experiments were conducted.

[0064] A schematic diagram of the treatment process for HA mice is shown in Figure 7. The HA mice used were C57BL / 6 female mice (6 weeks old, purchased from Beijing Biosubstrate Technologies) with the F8 gene knocked out. All mice were placed in a pathogen-free environment and irradiated using an X-ray irradiation device (Faxitron, Tucson, Arizona, USA) (600 cGy / mouse). Lentiviruses LV-EF1α-F8-BDD, LV-VEC-F8-BDD, LV-ITGA-F8-BDD, and LV-Gp-F8-BDD were separately injected directly into HA mice by intravenous injection to treat the disease, with a viral injection dose of 1 x 10⁻⁶. 7 The study was conducted using a TU (Triple Uncontrolled) approach. Phosphate-buffered saline (PBS) (200 μL per mouse) was used as a control (mock).

[0065] Human F8 gene expression in peripheral blood was detected by flow cytometry on days 7, 15, 30, 45, 60, and 120 post-treatment days. The results are shown in Figure 8. Human F8 was stably expressed in the blood of mice treated with LV-VEC-F8-BDD (10% to 30% of normal plasma levels). Human F8 was also stably expressed in the blood of mice treated with LV-Gp-F8-BDD at approximately 15%. On the other hand, human F8 expression in the blood of mice treated with LV-EF1α-F8-BDD and LV-ITGA-F8-BDD gradually decreased (from 30% to 5%).

[0066] Blood was collected from mice on days 7, 15, 30, 45, 60, and 120 post-treatment days, and plasma was separated from the blood. Untreated hemophilic mice (Mock) and wild-type mice (WT) were used separately as controls, and F8 activity was measured by substrate luminescence spectroscopy. The results are shown in Figure 9. The results were consistent with those measured by flow cytometry. In the LV-VEC-F8-BDD and LV-Gp-F8-BDD treatment groups, human F8 activity in mouse plasma steadily increased, reaching a positive rate of 25% at day 60, and further increasing to 80% (LV-VEC-F8-BDD group) and 25% (LV-Gp-F8-BDD group) at day 120. In the LV-EF1α-F8-BDD and LV-ITGA-F8-BDD treatment groups, human F8 activity in mouse plasma gradually decreased after 30 days (less than 3%).

[0067] In conclusion, both flow cytometry and substrate luminescence detection results demonstrate that tail vein injection of LV-Gp-F8-BDD or LV-VEC-F8-BDD significantly improves F8 levels, maintaining stable levels in the plasma of HA mice. In other words, LV-Gp-F8-BDD or LV-VEC-F8-BDD can effectively treat HA in mice.

[0068] In addition, for antibody responses, orbital peripheral blood was collected from the treated mice described above and centrifuged at 3000 rpm for 15 minutes to obtain plasma. The plasma was diluted 1:200 with Tris-BSA buffer and placed in polyvinyl chloride (PVC) microplates. Peroxidase-labeled goat anti-mouse total IgG was added. Subsequently, the luminescent substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added for ELISA, and the antibody response to coagulation factor VIII (F8) was evaluated. HA mice injected with anticoagulation factor VIII monoclonal antibody were used as positive controls (Ctrl+). The results are shown in Figure 10. The IgG antibody response in mice from the LV-VEC-F8-BDD, LV-Gp-F8-BDD, and LV-ITGA-F8-BDD groups was relatively low, while the antibody response in mice from the LV-EF1α-F8-BDD group was the highest, demonstrating that gene therapy using the tissue-specific promoters VEC, Gp, and ITGA of this application can effectively reduce immune rejection.

[0069] In conclusion, the tissue-specific promoter is creatively designed in this application. The tissue-specific promoter can promote the specific expression of coding genes in EC or megakaryocyte-platelet cells and effectively reduce ectopic expression, and can be applied to gene therapies requiring specific gene expression in EC or megakaryocyte-platelet cells, such as gene therapy for HA. The tissue-specific promoter and lentivirus possessing F8-BDD are prepared in this application. HA mice, upon direct intravenous injection, show effectively improved transduction efficiency of the F8-BDD gene and improved expression levels of the F8-BDD gene in mice, and the hemorrhagic phenotype of HA mice is modified to some extent. The VEC promoter has the best therapeutic effect by promoting F8-BDD gene expression and showing less antibody response, which is crucial for ensuring the effectiveness of gene therapy and lays the foundation for achieving faster relief of HA symptoms and more comprehensive and durable gene therapy.

[0070] The applicant has described the detailed methods of this application through the embodiments described above, but states that this application is not limited to the detailed methods described above, and that the implementation of this application is not necessarily dependent on the detailed methods described above. It will be apparent to those skilled in the art that improvements made to this application, equivalent substitutions of raw materials in the products of this application, addition of adjuvant components to the products of this application, and selection of specific methods are all included within the scope of protection and disclosure of this application.

Claims

1. A recombinant expression vector comprising a tissue-specific promoter and an encoding gene, The tissue-specific promoter consists of the sequence shown in Sequence ID No. 1, and The coding gene includes the coding gene for recombinant coagulation factor VIII, and the coding gene has the sequence shown in Sequence ID No. 5, The recombinant expression vector wherein the coding gene is under the control of the tissue-specific promoter.

2. The recombinant expression vector according to claim 1, comprising a viral vector or a plasmid vector.

3. The recombinant expression vector according to claim 2, wherein the viral vector comprises the lentiviral vector pEGWI.

4. A recombinant lentivirus containing the recombinant expression vector described in claim 1.

5. Recombinant cells containing the recombinant expression vector described in claim 1.

6. A method for preparing recombinant cells according to claim 5 in vitro, comprising introducing the recombinant expression vector into a host cell to obtain the recombinant cells.

7. The method according to claim 6, wherein the introduction is carried out by a method comprising one of the following: electrical introduction, a viral vector system, a non-viral vector system, or direct gene injection.

8. The method according to claim 6, wherein the host cell includes hematopoietic stem cells.

9. A pharmaceutical composition comprising a recombinant expression vector according to any one of claims 1 to 3, a recombinant lentivirus according to claim 4, or a recombinant cell according to claim 5, The pharmaceutical composition further comprises one or at least two combinations of pharmaceutically acceptable carriers, excipients, or diluents.

10. A recombinant expression vector according to any one of claims 1 to 3, a recombinant lentivirus according to claim 4, or a recombinant cell according to claim 5, for use in the treatment of hemophilia A.

11. The pharmaceutical composition according to claim 9 for use in the treatment of hemophilia A.

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