A cybb lentivirus vector, a lentivirus vector transfected stem cell and a preparation method and application thereof

Abstract

The application provides a CYBB lentivirus vector, a stem cell transfected by the lentivirus vector and a preparation method and application thereof. The lentivirus vector comprises an hEF1 alpha promoter and CYBB tandem co-expression. The lentivirus vector of the application carries a CYBB gene, realizes safe and efficient expression of the carried CYBB gene in differentiated or undifferentiated stem cells under the start of an hEF1 alpha promoter, and meanwhile, the stem cell serves as a potential delivery carrier, and the expression amount of the CYBB gene in a transgenic cell is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to a lentivirus vector, a stem cell transfected by the lentivirus vector, and a preparation method and application thereof, in particular to a CYBB lentivirus vector, a stem cell transfected by the lentivirus vector, and a preparation method and application thereof. BACKGROUND

[0002] Chronic granulomatous disease (CGD) is a genetic primary immunodeficiency disease caused by the functional deficiency of NADPH oxidase in neutrophilic granulocytes and monocytes, and is characterized by repeated infections, inflammation and autoimmunity. NADPH oxidase is composed of p47phox, p40phox, p67phox and qp91-phox, and the functional deficiency of any part thereof will trigger CGD. The disease is mostly sex-linked recessive, and a small number of cases are autosomal recessive, and there is usually a family history, and the disease is mostly seen in children. The most common is the mutation of the qp91-phox subunit gene of cytochrome b, and the number of patients accounts for about 65% of the total patients. The mutated gene is named CYBB (MIM306400), contains 13 exons, and is located on the X chromosome xp21.1, and accounts for about 30 kb.

[0003] The NADPH oxidase complex is composed of membrane-bound proteins and cytoplasmic proteins, which have a synergistic effect when phagocytes are activated, and assist in producing reactive oxygen species (ROS) to kill bacteria and fungi. Under normal circumstances, normal granulocytes produce hydrogen peroxide after phagocytosis of bacteria, release nascent oxygen, oxidize iodine and chlorine compounds into free iodine and chlorine, and form a complete hydrogen peroxide-peroxidase-iodine ion bactericidal system. CGD patients lack NADPH oxidase and cannot produce hydrogen peroxide, thus cannot exert the bactericidal effect in vivo, which causes repeated pyogenic infections in various parts of the body, leading to pyogenic inflammation of the heart, lung, liver, nervous system, etc. Patients can show skin granuloma, eczematous dermatitis, hepatosplenomegaly, and granulomas formed by histiocytes containing lipid pigments in various affected organs, and most patients die of severe infection at a young age.[Reference 7]

[0004] At present, the only means to completely cure CGD is to receive hematopoietic stem cell transplantation. However, finding a suitable donor for matching is only one of the problems. The dose of transplantation conditioning, the infection condition of the patient at the time of transplantation, the control of GVHD after transplantation, and the ability of immune reconstruction of CGD patients after transplantation are all key factors affecting the success of transplantation[Reference 8].

[0005] CGD is a disease caused by single gene mutation, so gene therapy is another possibility for cure. At present, there are many research reports on gene therapy using viral vectors at home and abroad, however, different viral vectors, even the same viral vectors due to different preparation methods often have obviously different gene delivery efficiency, which directly affects the treatment effect of the disease. Most of the current cell therapy methods for genetic diseases have the problem of low efficiency, and these methods are only suitable for blood stem cells, and the clinical effect does not meet the expected [reference 8].

[0006] A phase I / II clinical trial conducted in South Korea uses retrovirus as a carrier, although there are no obvious side effects, however, the gene modified cells cannot exist in the patient's body for a long time [reference 9]. Ravin et al. repaired the mutant CYBB gene of CGD patients using CRISPR-Cas9, however, the CRISPR-Cas9 system has targeting problems, has safety hazards, and the method has strict conditions, high establishment cost, and unstable results [reference 10].

[0007] Lentiviral vector-mediated autologous stem cell gene therapy has been successfully applied to the treatment of X-linked severe combined immunodeficiency (X-SCID), β-thalassemia, sickle cell disease (SCD) and other diseases. Although since the 1990s, adenovirus vectors have been tried for gene therapy of hemophilia, however, so far no animal experiment has reported positive results of lifelong expression of coagulation factors, and the difficulty can be attributed to immune response caused by the vector, inability of the exogenous gene to be expressed efficiently and continuously, and inability of the exogenous gene to be expressed in the appropriate area. The clinical gene therapy method in the past 10 years mostly uses retroviral vectors (gamma-oncoretroviral vectors), which have the characteristics of inserting the promoter region of the oncogene, have a start-up silencing mechanism, and have safety concerns, and cannot be expressed for a long time.

[0008] Therefore, there is an urgent need for a viral vector with high gene delivery efficiency and suitable for various stem cells to improve the treatment effect of CGD. SUMMARY

[0009] In view of the shortcomings of the prior art, the present application provides a CYBB lentiviral vector, a lentiviral vector transfected stem cell and a preparation method and application thereof, wherein the hEF1 alpha promoter in the lentiviral vector starts the expression of the CGD related gene CYBB, has good safety, high gene delivery efficiency, and lays a foundation for improving the treatment effect of CGD.

[0010] To achieve the above object, the present application adopts the following technical solutions:

[0011] In a first aspect, the present application provides a lentiviral vector comprising a hEF1 alpha promoter and a CYBB tandem co-expression.

[0012] In the present application, under the action of the hEF1 alpha promoter, the lentiviral vector carrying the CYBB gene realizes efficient gene delivery while ensuring safety, which is conducive to improving the expression amount of the CYBB gene in the transgenic cells.

[0013] Preferably, the nucleic acid sequence of the hEF1 alpha promoter is shown in SEQ ID NO. 1.

[0014] The nucleic acid sequence of SEQ ID NO. 1 is as follows:

[0015]

[0016] Preferably, the amino acid sequence of CYBB is as set forth in SEQ ID NO. 2;

[0017] The amino acid sequence of SEQ ID NO. 2 is:

[0018] MGNWAVNEGLSIFVILVWLGLNVFLFVWYYRVYDIPPKFFYTRKLLGSALALARAPAACLNFNCMLILLPVCRNLLSFLRGSSACCSTRVRRQLDRNLTFHKMVAWMIALHSAIHTIAHLFNVEWCVNARVNNSDPYSVALSELGDRQNESYLNFARKRIKNPEGGLYLAVTLLAGITGVVITLCLILIITSSTKTIRRSYFEVFWYTHHLFVIFFIGLAIHGAERIVRGQTAESLAVHNITVCEQKISEWGKIKECPIPQFAGNPPMTWKWIVGPMFLYLCERLVRFWRSQQKVVITKVVTHPFKTIELQMKKKGFKMEVGQYIFVKCPKVSKLEWHPFTLTSAPEEDFFSIHIRIVGDWTEGLFNACGCDKQEFQDAWKLPKIAVDGPFGTASEDVFSYEVVMLVGAGIGVTPFASILKSVWYKYCNNATNLKLKKIYFYWLCRDTHAFEWFADLLQLLESQMQERNNAGFLSYNIYLTGWDESQANHFAVHHDEEKDVITGLKQKTLYGRPNWDNEFKTIASQHPNTRIGVFLCGPEALAETLSKQSISNSESGPRGVHFIFNKENF.

[0019] Preferably, the nucleic acid sequence of CYBB is as set forth in SEQ ID NO. 3;

[0020] The nucleic acid sequence of SEQ ID NO. 3 is:

[0021]

[0022] In a second aspect, the present application provides a lentivirus into which the lentiviral vector of the first aspect is introduced.

[0023] In a third aspect, the present application provides a host cell transfected with the lentivirus of the second aspect.

[0024] Preferably, the host cell comprises a stem cell.

[0025] The stem cell of the present application serves as a delivery vehicle to transport the lentiviral vector carrying the CYBB gene, and improves the expression efficiency and expression amount of the CYBB gene in the differentiated or undifferentiated stem cell.

[0026] Preferably, the stem cell comprises a hematopoietic stem cell.

[0027] According to the present application, the hematopoietic stem cell is derived from blood or bone marrow, and has the ability to differentiate into a series of somatic cells and to renew various tissue cells. The hematopoietic stem cell of the present application serves as a potential delivery vehicle to carry the lentivirus containing the CYBB gene to achieve gene therapy for CGD.

[0028] In a fourth aspect, the present application provides a method for preparing the host cell of the third aspect, the method comprising the following steps:

[0029] (1) constructing the lentiviral vector of the first aspect;

[0030] (2) co-transfecting the lentiviral vector of step (1) and a packaging plasmid into a mammalian cell to perform lentivirus packaging;

[0031] (3) transforming the lentivirus packaged in step (2) into the genome of a host cell to obtain the host cell.

[0032] Preferably, the method for constructing in step (1) is to link the hEF1a promoter and CYBB into the TYF lentiviral vector through a restriction enzyme cutting site.

[0033] Preferably, the packaging plasmid of step (2) comprises pNHP and pHEF-VSVG.

[0034] Preferably, the mammalian cell of step (2) comprises 293T cells.

[0035] Preferably, step (2) is followed by a step of purifying the lentivirus.

[0036] Preferably, the purification is to concentrate the lentivirus into high-titer virus through filtration.

[0037] In the present application, the purification, filtration and concentration steps significantly improve the titer and concentration of the lentivirus.

[0038] Preferably, the host cell of step (3) comprises stem cells.

[0039] Preferably, the stem cells comprise hematopoietic stem cells.

[0040] As a preferred technical solution, the present application provides a preparation method of the host cell according to the third aspect, comprising the following steps:

[0041] (1) connecting the hEF1a promoter and CYBB into the TYF lentivirus vector through restriction enzyme digestion for vector construction;

[0042] (2) co-transfecting the lentivirus vector of step (1) with pNHP and pHEF-VSVG packaging plasmids into 293T cells for lentivirus packaging, removing cell debris by centrifugation at 1000-1100g for 3-5min, filtering the obtained supernatant with a 0.45-0.5μm low protein binding filter, centrifuging at 2000-2500g for 30-40min, shaking the filter tube, and centrifuging at 300-400g for 2-5min;

[0043] (3) transforming the lentivirus of step (2) into the genome of hematopoietic stem cells to obtain the hematopoietic stem cells.

[0044] In the fifth aspect, the present application provides a pharmaceutical composition comprising any one or a combination of at least two of the lentivirus vector according to the first aspect, the lentivirus according to the second aspect or the host cell according to the third aspect.

[0045] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0046] The pharmaceutical composition of the present application repairs the mutant CYBB gene of CGD patients at the gene level, is beneficial to realize the repair of autologous stem cells of patients, and has the potential for long-term stable treatment of CGD.

[0047] In the sixth aspect, the present application provides a use of the lentivirus vector according to the first aspect, the lentivirus according to the second aspect, the host cell according to the third aspect or the pharmaceutical composition according to the fifth aspect in the preparation of a disease treatment drug.

[0048] Preferably, the disease comprises chronic granulomatous disease.

[0049] In a seventh aspect, the present application provides a method for treating chronic granulomatous disease by using the lentiviral vector of the first aspect, the lentivirus of the second aspect, the host cell of the third aspect or the pharmaceutical composition of the fifth aspect.

[0050] According to the present application, the method comprises the following steps:

[0051] (1') mobilize CD34 stem cells from the patient by granulocyte colony-stimulating factor and perform multiple bone marrow collection;

[0052] (2') isolate CD34 positive cells from the bone marrow collected from the patient by the laboratory side before reinfusion and culture;

[0053] (3') perform pretreatment on the patient by the clinical side before reinfusion;

[0054] (4') infect CD34 cells with lentivirus carrying CYBB gene by the laboratory side before reinfusion, perform twice gene transduction, and perform cell culture;

[0055] (5') on the day of reinfusion, wash and suspend the cells, and then reinfuse the patient;

[0056] (6') after reinfusing the patient, perform follow-up tracking every week, collect peripheral blood of the patient, and detect oxidase function, immune cell proportion and gene copy number.

[0057] Preferably, the pretreatment of step (3') is performed by using busulfan 40 mg per kilogram of body weight and fludarabine 60 mg per square meter of body surface area.

[0058] Preferably, the washing of step (5') is performed by using physiological saline containing 1% human serum protein to wash the cells twice.

[0059] Preferably, the suspension of step (5') is performed by using physiological saline containing 2.5% human serum protein.

[0060] Compared with the prior art, the present application has the following beneficial effects:

[0061] (1) the lentiviral vector of the present application realizes safe and efficient expression of the carried CYBB gene in differentiated or undifferentiated stem cells under the initiation of the hEF1 alpha promoter;

[0062] (2) in the present application, the lentivirus carrying CYBB gene is used to transfect stem cells, which realizes efficient repair of autologous stem cells of the patient, and at the same time, the stem cells as potential delivery carriers improve the expression amount of CYBB gene in the transgenic cells;

[0063] (3) The stem cells transfected with the CYBB lentivirus are re-input into the patient, the expression amount of the oxidase in the mononuclear cells and the neutrophils of the patient is significantly improved, the number of the neutrophils and the number of the mononuclear cells expressing the oxidase can be maintained at a high level, the CYBB gene has good stability in the peripheral blood cells, the long-term stable treatment effect of the chronic granulomatous disease is achieved, the safety is high, and the method has application potential in the treatment of the chronic granulomatous disease. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 A schematic diagram of a lentivirus vector is shown, wherein Lentiviral Vector-lentivirus vector, Packaging Plasmids-packaging plasmid, Transducing Lentivector-gene delivery lentivirus vector;

[0065] Figure 2 A treatment flowchart is shown;

[0066] Fig. 3(a) is a peripheral blood analysis of a normal genotype father of the patient, Fig. 3(b) is a peripheral blood analysis of a heterozygote mother of the patient, Fig. 3(c) is a peripheral blood analysis before re-input of the patient, and Fig. 3(d) is a peripheral blood analysis on the 28th day after treatment of the patient;

[0067] Fig. 4(a) is the average fluorescence intensity fold of rhodamine 123 staining, and Fig. 4(b) is the proportion of fluorescent neutrophils;

[0068] Fig. 5(a) is the number of neutrophils in CD45 positive cells after re-input, and Fig. 5(b) is the number of mononuclear cells in CD45 positive cells after re-input;

[0069] Figure 6 The change of the CYBB gene copy number in the peripheral blood of the patient after re-input is shown;

[0070] Figure 7 The lung CT scan images of the patient before and after re-input are shown. DETAILED DESCRIPTION

[0071] The present application mainly improves the structure and production method of the lentivirus, and the technical means and effects adopted by the present application are further described below in combination with the embodiments and the drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

[0072] The specific techniques or conditions not mentioned in the embodiments are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be commercially available through regular channels.

[0073] Example 1: Construction of a lentiviral vector carrying the CYBB gene

[0074] The normal CYBB gene sequence (amino acid sequence as shown in SEQ ID NO.2, nucleic acid sequence as shown in SEQ ID NO.3) was synthesized from the whole genome and ligated into the TYF-EF1α lentiviral vector (NHP / TYF lentivirus vector system) via restriction enzyme sites. After ligating into the human EF1α (hEF1α) promoter sequence (nucleic acid sequence as shown in SEQ ID NO.1), the obtained product was identified by sequencing and double enzyme digestion (cloning at the 5' BamHI site and the 3' SpeI site; optimal reaction conditions were based on NEB manufacturer recommendations). This yielded a correctly ligated lentiviral vector carrying the CYBB gene under the hEF1α promoter. Figure 1 The image shows the NHP / TYF lentiviral vector system, which includes a viral packaging plasmid (NHP, EF-VSV-G) and a vector plasmid (pTYF-EF-CYBB). The packaging plasmid includes pNHP and pHEF-VSV-G (env). pNHP expresses the Gag-Pol protein, and pHEF-VSV-G expresses the mantle protein. The gene delivery plasmid pTYF-EF has a chimeric CMV-IE promoter at its 5' end, which binds to the HIV-1 virus TAR-mutant U5 plus a right-hand appendage sequence (CMV-IE-TAR-dl.U5 / attR), followed by a primer binding site (PBS), a lentiviral vector packaging signal (psi), and a mutant gag sequence. EF1a-CYBB is followed by a mutated 3'LTR (self-inactivating SIN LTR), a polypurine track sequence (PPT), a left attachment site (attL), and bovine growth hormone polyA (bGHpA) signaling. For details, see references [1]-[3].

[0075] Example 2 Lentiviral Packaging

[0076] This embodiment employs a multi-plasmid packaging system to package a lentiviral vector carrying the CYBB gene into a complete lentivirus using 293T cells. The specific steps are as follows:

[0077] (1) Culture the 293T cell line for 17-18 hours, then add fresh DMEM containing 10% FBS;

[0078] (2) Add DMEM, pNHP, pHEF-VSV-G and the lentivirus vector constructed in Example 1 into a sterile centrifuge tube in turn, and vortex;

[0079] (3) Add Superfect transfection reagent (QIAGEN) into the centrifuge tube, and stand at room temperature for 7-10 min;

[0080] (4) Add the lentivirus vector-Superfect mixture in the centrifuge tube dropwise into 293T cells, vortex, and culture at 37℃ and 5% CO2 for 4-5 h;

[0081] (5) Remove the cell culture solution, rinse the cells, and add the culture solution for continued culture;

[0082] (6) Place the culture medium back into the 5% CO2 incubator for overnight culture, and then observe the transfection efficiency by fluorescence microscopy.

[0083] Example 3 Purification and concentration of lentivirus

[0084] The process of purification and concentration of lentivirus is as follows:

[0085] (1) Lentivirus purification

[0086] Centrifuge the packaged lentivirus at 1000 g for 5 min to remove cell debris, filter the obtained supernatant with a 0.45 μm low protein binding filter, and store the aliquots at -80℃;

[0087] (2) Lentivirus concentration

[0088] Add the lentivirus supernatant into a Centricon filter tube, centrifuge at 2500 g for 30 min; shake the filter tube, centrifuge at 400 g for 2 min, and collect the concentrated virus into a collection cup.

[0089] Example 4 Purification and concentration of lentivirus

[0090] The process of purification and concentration of lentivirus is as follows:

[0091] (1) Lentivirus purification

[0092] Centrifuge the packaged lentivirus at 1100 g for 3 min to remove cell debris, filter the obtained supernatant with a 0.5 μm low protein binding filter, and store the aliquots at -80℃;

[0093] (2) Lentivirus concentration

[0094] Add the lentivirus supernatant into a Centricon filter tube, centrifuge at 2000 g for 40 min; shake the filter tube, centrifuge at 300 g for 5 min, and collect the concentrated virus into a collection cup.

[0095] Example 5 Lentivirus transfection of hematopoietic stem cells

[0096] Hematopoietic stem cells (HSCs) were seeded into culture dishes, concentrated lentivirus carrying CYBB target genes were added, centrifuged at 100g for 100min, incubated at 37°C for 24h, and then medium containing stem cell growth factors was added. After 2-3 days of culture, stem cells carrying normal CYBB genes were obtained.

[0097] Example 6 Lentivirus carrying CYBB gene infection of CD34 stem cells for treatment of X-linked chronic granulomatous disease (X-CGD) patients

[0098] As shown in Figure 2 , it is a treatment flow chart.

[0099] (1) The patient mobilized CD34 stem cells with granulocyte colony-stimulating factor (G-CSF) and underwent two bone marrow collections, the first on day 37 before reinfusion and the second on day 4 before reinfusion. Since CGD patients generally have a poor response to mobilization, two bone marrow collections are beneficial for obtaining sufficient CD34 stem cells [Reference 4];

[0100] (2) On day 4 before reinfusion, CD34-positive cells (Miltenyi CD34 beads) were isolated from the patient's two collections of bone marrow in the laboratory and cultured overnight in HSC culture medium (Sigma Stemline II HSC expansion medium);

[0101] (3) On day 3 and day 2 before reinfusion, the patient was pre-treated with busulfan 40mg / kg and fludarabine 60mg / m 2 , respectively, in the clinic. According to the literature, appropriate conditioning can effectively prolong the survival of transgenic CD34 stem cells in patients [Reference 5];

[0102] (4) On day 3 and day 2 before reinfusion, the CD34 cells were infected with lentivirus carrying CYBB gene in the laboratory, and two times of gene transduction were performed, and then the infected cells were cultured for one day;

[0103] (5) On the day of reinfusion, the cells were washed twice with physiological saline containing 1% human serum protein, suspended in physiological saline containing 2.5% human serum protein, and reinfused into the patient;

[0104] (6) After transfusion, the patient was followed up every week, and the peripheral blood was collected to detect the oxidase function, the immune cell ratio and the gene copy number.

[0105] Result analysis

[0106] The collected peripheral blood was subjected to dihydro rhodamine 123 (DHR123) staining experiment and CD14 and CD15 staining, and the oxidase function in the peripheral blood neutrophils and monocytes was analyzed by flow cytometry. The dihydro rhodamine 123 is oxidized to rhodamine 123 by hydrogen peroxide, and emits yellow-green fluorescence at 515 nm after being excited by 488 nm laser. After the dihydro rhodamine 123 is incubated with phorbol ester (PMA)-stimulated cells, the fluorescence intensity represents the oxidase function intensity [Reference 6].

[0107] Fig. 3(a) is the peripheral blood analysis of the patient's normal genotype father, and the proportion of monocytes (CD14+) and neutrophils (CD15+) expressing oxidase is greater than 90%; Fig. 3(b) is the peripheral blood analysis of the patient's heterozygous mother, and 70% of the monocytes express oxidase, and 57% of the neutrophils express oxidase, both of which are slightly lower than the proportion of healthy people; Fig. 3(c) is the peripheral blood analysis of the patient before transfusion, and it can be seen that the patient does not express oxidase at all before treatment; Fig. 3(d) is the peripheral blood analysis of the patient 28 days after treatment, and 35% of the monocytes express oxidase, and 47% of the neutrophils express oxidase, which is greatly improved compared with before transfusion.

[0108] X-linked chronic granulomatous disease is mainly caused by the deficiency of oxidase in neutrophils. The function intensity and expression degree of oxidase in neutrophils were observed every week after transfusion, and the results are shown in Fig. 5. Fig. 4(a) shows the average fluorescence intensity multiple of rhodamine 123 staining. The multiple is the average fluorescence intensity of cells stimulated by phorbol ester (PMA) divided by the average fluorescence intensity of cells without phorbol ester stimulation, that is, the function intensity of oxidase after cell stimulation. Before treatment, the patient's ratio was 1.08, and the cells had almost no oxidase response after stimulation. After treatment, the function of oxidase fluctuated over time, but overall improved, with the highest on the 28th day and the 73rd day after transfusion, reaching 1.44 times and 1.62 times, respectively; Fig. 4(b) is the proportion of fluorescent neutrophils, that is, the proportion of neutrophils expressing oxidase. Before transfusion, the patient did not express any oxidase. On the 28th day and the 73rd day after transfusion, the patient had 47% and 66% of neutrophils expressing oxidase, respectively. Since the 49th day after transfusion, the proportion of cells expressing oxidase has been maintained above 20% (the patient received blood transfusion on the 7th day after transfusion, which is not discussed).

[0109] Since the patient was subjected to myeloablative conditioning before the reinfusion, the inventors monitored the number of neutrophils and monocytes in the CD45-positive cells after the reinfusion. As shown in Fig. 5(a), on day 14 after the reinfusion, the number of neutrophils reached a minimum of 0.2% of the CD45-positive cells, which was significantly affected by the myeloablative conditioning compared to the normal genotype father whose neutrophils were maintained at 60%, but the percentage of neutrophils gradually increased after 14 days and returned to the normal proportion of healthy people on day 49 after the reinfusion and was maintained until the latest follow-up on day 103 after the reinfusion; as shown in Fig. 5(b), on day 7 after the reinfusion, the number of monocytes reached a minimum of 2% of the CD45-positive cells.

[0110] Figure 6 Fig. 4 shows the change in the copy number of the CYBB gene in the peripheral blood of the patient after the reinfusion. On day 21 and day 35 after the reinfusion, the copy number reached 3.75% and 2.41%, respectively, which were the highest values monitored. It can be seen that the CYBB gene stably exists in the peripheral blood cells, and even on day 103 after the reinfusion, there are still 0.23% of the peripheral blood cells carrying the CYBB gene.

[0111] In addition to the molecular and cytological evidence, the patient's clinical symptoms of infection have also been greatly improved. Figure 7 Fig. 5 shows the lung CT scan images of the patient before and after the reinfusion. The patient had a severe lung infection on day 39 before the reinfusion and was treated with antifungal and antibacterial drugs; on day 55 after the reinfusion, the lung infection was significantly improved; on day 105 after the reinfusion, the lung infection was relieved and did not require heavy drug support.

[0112] In summary, the lentiviral vector of the present application realizes efficient delivery of the CYBB gene under the initiation of the EF1a promoter; the stem cells are transfected with the lentivirus carrying the CYBB gene and are used as a delivery carrier for the treatment of CGD disease, so that the expression amount of the CYBB gene in the differentiated or undifferentiated stem cells is increased; the CD34 stem cells are infected with the lentivirus carrying the CYBB gene, which has a therapeutic potential for X-CGD.

[0113] References:

[0114] [1] Chang, L.-J., V. Urlacher, T. Iwakuma, Y. Cui, and J. Zucali (1999). Efficacy and safety analyses of a recombinant human immunodeficiency virus derived vector system. Gene Therapy 6, 715-728.

[0115] [2] Cui, Y., T. Iwakuma and L.-J. Chang (1999). Contributions of viral splice sites and cis-regulatory elements to lentivirus vector functions. J Virol 73, 6171-6176.

[0116] [3] Iwakuma T., Y. Cui, and L.-J. Chang (1999). Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261, 120-132.

[0117] [4] Sandhya R. Panch, Yu Ying Yau, Elizabeth M. Kang, Suk See De Ravin, Harry L. Malech and Susan F. Leitman (2014). Mobilization characteristics and strategies to improve hematopoietic progenitor cell mobilization and collection in patients with chronic granulomatous disease and severe combined immunodeficiency. Transfusion 55, 265-274.

[0118] [5] Manuel Grez, Janine Reichenbach, Joachim Reinhard Seger, Mary C Dinauer, and Adrian J Thrasher. (2011) Gene Therapy of Chronic Granulomatous Disease: The Engraftment Dilemma. Molecular Therapy 19, 28-35.

[0119] [6] Yu Chen and Wolfgang G. Junger. (2012) Measurement of Oxidative Burst in Neutrophils. Methods in Molecular biology 844, 115-144.

[0120] [7] Douglas B. Kuhns et al. (2010) Residual NADPH Oxidase and Survival in Chronic Granulomatous Disease. The New England Journal of Medicine 363, 2600-2610.

[0121] [8] Danielle E. Arnold and Jennifer R. Heimall. (2017) A Review of Chronic Granulomatous Disease. Advances in Therapy 34, 2543-2557.

[0122] [9] Hyoung Jin Kang et al. (2011) Retroviral Gene Therapy for X-linked Chronic Granulomatous Disease: Results From Phase I / II Trial. Molecular Therapy 19, 2092-2101.

[0123]

[10] De Ravin et al. (2017) CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Science Translational Medicine 9, eaah3480.

[0124] Applicants declare that the present application is illustrated by the above detailed method through the above examples, but the present application is not limited to the above detailed method, i.e. it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. all fall within the protection scope and disclosure scope of the present application. SEQUENCE LISTING <110> Beijing Meikangji Biotechnology Co., Ltd. <120> CYBB lentivirus vector, lentivirus vector transfected stem cells and preparation method and application thereof <130> 20190404 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1536 <212> DNA <213> Artificial Sequence <400> 1 gctagcatgc ctaggtcgac caattctcat gtttgacagc ttatcatcga taagctttgg 60 agctaagcca gcaatggtag agggaagatt ctgcacgtcc cttccaggcg gcctccccgt 120 caccaccccc cccaacccgc cccgaccgga gctgagagta attcatacaa aaggactcgc 180 ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag aacccagaga 240 tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg gagaagagca 300 tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 360 gttgggggga ggggtcggca attgaaccgg tgcctagaga aagtggcgcg gggtaaactg 420 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 480 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 540 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 600 ttgaattact tccacgcccc tggctgcagt acgtgattct tgatcccgag cttcgggttg 660 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag 720 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct 780 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc 840 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg 900 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc 960 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg 1020 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc 1080 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct 1140 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa 1200 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca 1260 ggcacctcga ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt 1320 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc 1380 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca 1440 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga aaactctaga 1500 gcggccgcgg aggccgaatti ccgtcgagga tccacc 1536 <210> 2 <211> 570 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Asn Trp Ala Val Asn Glu Gly Leu Ser Ile Phe Val Ile Leu 1 5 10 15 Val Trp Leu Gly Leu Asn Val Phe Leu Phe Val Trp Tyr Tyr Arg Val 20 25 30 Tyr Asp Ile Pro Pro Lys Phe Phe Tyr Thr Arg Lys Leu Leu Gly Ser 35 40 45 Ala Leu Ala Leu Ala Arg Ala Pro Ala Ala Cys Leu Asn Phe Asn Cys 50 55 60 Met Leu Ile Leu Leu Pro Val Cys Arg Asn Leu Leu Ser Phe Leu Arg 65 70 75 80 Gly Ser Ser Ala Cys Cys Ser Thr Arg Val Arg Arg Gln Leu Asp Arg 85 90 95 Asn Leu Thr Phe His Lys Met Val Ala Trp Met Ile Ala Leu His Ser 100 105 110 Ala Ile His Thr Ile Ala His Leu Phe Asn Val Glu Trp Cys Val Asn 115 120 125 Ala Arg Val Asn Asn Ser Asp Pro Tyr Ser Val Ala Leu Ser Glu Leu 130 135 140 Gly Asp Arg Gln Asn Glu Ser Tyr Leu Asn Phe Ala Arg Lys Arg Ile 145 150 155 160 Lys Asn Pro Glu Gly Gly Leu Tyr Leu Ala Val Thr Leu Leu Ala Gly 165 170 175 Ile Thr Gly Val Val Ile Thr Leu Cys Leu Ile Leu Ile Ile Thr Ser 180 185 190 Ser Thr Lys Thr lie Arg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr 195 200 205 His His Leu Phe Val lie Phe Phe lie Gly Leu Ala lie His Gly Ala 210 215 220 Glu Arg lie Val Arg Gly Gin Thr Ala Glu Ser Leu Ala Val His Asn 225 230 235 240 lie Thr Val Cys Glu Gin Lys lie Ser Glu Trp Gly Lys lie Lys Glu 245 250 255 Cys Pro lie Pro Gin Phe Ala Gly Asn Pro Pro Met Thr Trp Lys Trp 260 265 270 lie Val Gly Pro Met Phe Leu Tyr Leu Cys Glu Arg Leu Val Arg Phe 275 280 285 Trp Arg Ser Gin Gin Lys Val Val lie Thr Lys Val Val Thr His Pro 290 295 300 Phe Lys Thr lie Glu Leu Gin Met Lys Lys Lys Gly Phe Lys Met Glu 305 310 315 320 Val Gly Gin Tyr lie Phe Val Lys Cys Pro Lys Val Ser Lys Leu Glu 325 330 335 Trp His Pro Phe Thr Leu Thr Ser Ala Pro Glu Glu Asp Phe Phe Ser 340 345 350 Ile His Ile Arg Ile Val Gly Asp Trp Thr Glu Gly Leu Phe Asn Ala 355 360 365 Cys Gly Cys Asp Lys Gln Glu Phe Gln Asp Ala Trp Lys Leu Pro Lys 370 375 380 Ile Ala Val Asp Gly Pro Phe Gly Thr Ala Ser Glu Asp Val Phe Ser 385 390 395 400 Tyr Glu Val Val Met Leu Val Gly Ala Gly Ile Gly Val Thr Pro Phe 405 410 415 Ala Ser Ile Leu Lys Ser Val Trp Tyr Lys Tyr Cys Asn Asn Ala Thr 420 425 430 Asn Leu Lys Leu Lys Lys Ile Tyr Phe Tyr Trp Leu Cys Arg Asp Thr 435 440 445 His Ala Phe Glu Trp Phe Ala Asp Leu Leu Gln Leu Leu Glu Ser Gln 450 455 460 Met Gln Glu Arg Asn Asn Ala Gly Phe Leu Ser Tyr Asn Ile Tyr Leu 465 470 475 480 Thr Gly Trp Asp Glu Ser Gln Ala Asn His Phe Ala Val His His Asp 485 490 495 Glu Glu Lys Asp Val Ile Thr Gly Leu Lys Gln Lys Thr Leu Tyr Gly 500 505 510 Arg Pro Asn Trp Asp Asn Glu Phe Lys Thr Ile Ala Ser Gin His Pro 515 520 525 Asn Thr Arg Ile Gly Val Phe Leu Cys Gly Pro Glu Ala Leu Ala Glu 530 535 540 Thr Leu Ser Lys Gin Ser Ile Ser Asn Ser Glu Ser Gly Pro Arg Gly 545 550 555 560 Val His Phe Ile Phe Asn Lys Glu Asn Phe 565 570 <210> 3 <211> 1713 <212> DNA <213> Artificially synthesized <400> 3 atggggaact gggctgtgaa tgaggggctc tccatttttg tcattctggt ttggctgggg 60 ttgaacgtct tcctctttgt ctggtattac cgggtttatg atattccacc taagttcttt 120 tacacaagaa aacttcttgg gtcagcactg gcactggcca gggcccctgc agcctgcctg 180 aatttcaact gcatgctgat tctcttgcca gtctgtcgaa atctgctgtc cttcctcagg 240 ggttccagtg cgtgctgctc aacaagagtt cgaagacaac tggacaggaa tctcaccttt 300 cataaaatgg tggcatggat gattgcactt cactctgcga ttcacaccat tgcacatcta 360 tttaatgtgg aatggtgtgt gaatgcccga gtcaataatt ctgatcctla ttcagtagca 420 ctctctgaac ttggagacag gcaaaatgaa agttatctca attttgctcg aaagagaata 480 aagaaccctg aaggaggcct gtacctggct gtgaccctgt tggcaggcat cactggagtt 540 gtcatcacgc tgtgcctcat attaattatc acttcctcca ccaaaaccat ccggaggtct 600 tactttgaag tcttttggta cacacatcat ctctttgtga tcttcttcat tggccttgcc 660 atccatggag ctgaacgaat tgtacgtggg cagaccgcag agagtttggc tgtgcataat 720 ataacagttt gtgaacaaaa aatctcagaa tggggaaaaa taaaggaatg cccaatccct 780 cagtttgctg gaaaccctcc tatgacttgg aaatggatag tgggtcccat gtttctgtat 840 ctctgtgaga ggttggtgcg gttttggcga tctcaacaga aggtggtcat caccaaggtg 900 gtcactcacc ctttcaaaac catcgagcta cagatgaaga agaaggggtt caaaatggaa 960 gtgggacaat acatttttgt caagtgccca aaggtgtcca agctggagtg gcaccctttt 1020 acactgacat ccgcccctga ggaagacttc tttagtatcc atatccgcat cgttggggac 1080 tggacagagg ggctgttcaa tgcttgtggc tgtgataagc aggagtttca agatgcgtgg 1140 aaactaccta agatagcggt tgatgggccc tttggcactg ccagtgaaga tgtgttcagc 1200 tatgaggtgg tgatgttagt gggagcaggg attggggtca cacccttcgc atccattctc 1260 aagtcagtct ggtacaaata ttgcaataac gccaccaatc tgaagctcaa aaagatctac 1320 ttctactggc tgtgccggga cacacatgcc tttgagtggt ttgcagatct gctgcaactg 1380 ctggagagcc agatgcagga aaggaacaat gccggcttcc tcagctacaa catctacctc 1440 actggctggg atgagtctca ggccaatcac tttgctgtgc accatgatga ggagaaagat 1500 gtgatcacag gcctgaaaca aaagactttg tatggacggc ccaactggga taatgaattc 1560 aagacaattg caagtcaaca ccctaatacc agaataggag ttttcctctg tggacctgaa 1620 gccttggctg aaaccctgag taaacaaagc atctccaact ctgagtctgg ccctcgggga 1680 gtgcatttca ttttcaacaa ggaaaacttc taa 1713

Claims

1. A lentiviral vector, characterized in that, The lentiviral vector is a TYF lentiviral vector that includes the hEF1α promoter and CYBB tandem co-expression. The nucleic acid sequence of the hEF1α promoter is shown in SEQ ID NO.1; The amino acid sequence of the CYBB is shown in SEQ ID NO.2; The nucleic acid sequence of the CYBB is shown in SEQ ID NO.

3.

2. A lentivirus, characterized in that, The lentivirus is introduced using the lentivirus vector as described in claim 1.

3. A host cell, characterized in that, The host cell is transfected with the lentivirus as described in claim 2, and the host cell is a hematopoietic stem cell.

4. A method for preparing host cells as described in claim 3, characterized in that, The method includes the following steps: (1) The hEF1α promoter and CYBB were ligated into the TYF lentiviral vector via restriction enzyme sites to construct the lentiviral vector as described in claim 1; (2) The lentiviral vector described in step (1) was co-transfected with pNHP and pHEF-VSVG packaging plasmids into 293T cells to perform lentiviral packaging. The packaged lentiviral vector was then purified to obtain concentrated lentiviral. (3) The lentivirus obtained in step (2) is converted into the hematopoietic stem cell genome to obtain the hematopoietic stem cells.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one or a combination of at least two of the lentiviral vector as described in claim 1, the lentivirus as described in claim 2, or the host cell as described in claim 3.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

7. The use of a lentiviral vector as described in claim 1, a lentivirus as described in claim 2, a host cell as described in claim 3, or a pharmaceutical composition as described in claim 5 in the preparation of a medicament for treating chronic granulomatous disease.

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