Patent Application
20220177919
The present disclosure belongs to the technical field of genetic engineering, and relates to a lentiviral vector, a lentiviral vector-transduced stem cell, and a preparation method and application thereof, and in particular to a CYBB lentiviral vector, a lentiviral vector-transduced stem cell, and a preparation method and application thereof.
Chronic granulomatous disease (CGD) is a hereditary and primary immunodeficiency disease caused by loss of function of NADPH oxidase in neutrophilic granulocytes and monocytes. It is characterized by repeated infection, inflammation and autoimmunity in patients. NADPH oxidase consists of p47phox, p40phox, p67phox, and gp91-phox, and loss of function of any part will cause CGD. Most patients are X-chromosome sex-linked recessive inherited due to gp91-phox mutation, and a few are autosomal recessive inherited. Patients often have a family history, and the symptoms are often found in children. Patients with mutation in the gp91-phox subunit gene of cytochrome b are the most common, accounting for about 65% of the total number of patients. The mutated gp91 gene is named CYBB (MIM306400) which contains 13 exons and is located on X chromosome xp21.1, occupying approximately 30 kb.
The NADPH oxidase complex consists of a membrane-binding protein and a cytoplasmic protein. They have a synergistic effect during phagocyte activation and assist the production of reactive oxygen species (ROS) to kill bacteria and fungi. In general, normal granulocytes phagocytize bacteria and de-granulate to produce hydrogen peroxide and release new ecological oxygen which oxidizes iodine and chlorine compounds to free iodine and chlorine, thereby achieving a complete hydrogen peroxide-peroxidase-iodide ion bactericidal system. However, CGD patients cannot produce hydrogen peroxide and cannot exert bactericidal effects in vivo due to NADPH oxidase deficiency, such that purulent infections occurs repeatedly in various parts of the body, which leads to purulent lymphadenitis, rhinitis, and sinusitis, and purulent inflammation in pericardium, lung, liver and nerve system. Patients may exhibit cutaneous granulomas, eczema dermatitis, hepatomegaly and splenomegaly, and granulomas formed by histiocytes containing pigment lipids in affected organs. Most of the patients die from severe infections at a young age. [Reference 7]
Currently, the only way to completely cure CGD is hematopoietic stem cell transplantation. However, finding a suitable donor is just one of the problems. The chemotherapy dose of transplant pretreatment, the infection status of the patient at the time of transplantation, the control of GVHD after transplantation, and the ability of CGD patients to rebuild the immune system after transplantation are the key factors that affect the success of the transplantation [Reference 8].
Since CGD is a disease caused by a single gene mutation, gene therapy is another potential treatment. At present, many studies in China and abroad have reported to gene therapy applications using viral vectors. However, different viral vectors or even different preparation methods of the same viral vector often have significantly different gene delivery efficiency, which directly affects the therapeutic effect of the gene therapy. At present, most cell and gene therapy methods for genetic diseases have an efficiency issue, and these methods are only applicable to blood stem cells, and the clinical effect is not as expected [Reference 8].
Phase I and II clinical trials in South Korea used retroviruses as vectors. Although no significant side effects were shown, genetically modified cells could not persist in patients for a long time [Reference 9]. Ravin et al. used CRISPR-Cas9 to repair mutant CYBB gene in CGD patients. However, the CRISPR-Cas9 system has targeting problems and potential safety hazards. Moreover, this method requires stringent conditions and high establishment costs, and results are unstable [Reference 10].
Lentiviral vector-mediated autologous stem cell gene therapy has been successfully applied to the treatment of diseases such as X-linked severe combined immunodeficiency (X-SCID), β-thalassemia, and sickle cell disease (SCD). Although the use of adenoviral vectors in gene therapy of hemophilia has been attempted since the 1990s, no animal experiment has reported positive results for lifelong continuous expression of coagulation factors. The difficulty of this method may be attributed to the immune response caused by the vector, the inability to express exogenous genes efficiently and continuously, and the inability to express exogenous genes in appropriate regions. In the past 10 years, many clinical gene therapy trials use gamma-oncoretroviral vectors whose viral characteristics include integration into the promoter region near oncogenes, and transgene silencing mechanism and thus have poor safety and lack of long term persistence.
Therefore, there is an urgent need for a viral vector that has high gene delivery efficiency and is suitable for targeting stem cells to improve the therapeutic effect for CGD.
In view of the shortcomings in the prior art, the present disclosure provides a CYBB lentiviral vector, a lentiviral vector-transduced stem cell, and a preparation method and application thereof. The hEF1α promoter in the lentiviral vector initiates the expression of CYBB that is the gene associated with CGD. The lentiviral vector has good safety and high gene transfer efficiency, thereby laying a foundation for improving the therapeutic effect for CGD.
To achieve this, the present disclosure adopts the following technical solutions:
In a first aspect, the present disclosure provides a lentiviral vector comprising a hEF1α promoter and CYBB that are organized in tandem.
In the present disclosure, under the initiation of the hEF1α promoter, the lentiviral vector carrying the CYBB gene achieves efficient gene delivery while ensuring safety, which is beneficial to increase the expression amount of the CYBB gene in transgenic cells.
Preferably, the hEF1α promoter has a nucleic acid sequence as shown in SEQ ID NO.1.
The nucleic acid sequence of SEQ ID NO. 1 is:
Preferably, the CYBB has an amino acid sequence as shown in SEQ ID NO. 2.
The amino acid sequence of SEQ ID NO. 2 is:
Preferably, the CYBB has a nucleic acid sequence as shown in SEQ ID NO. 3.
The nucleic acid sequence of SEQ ID NO. 3 is:
In a second aspect, the present disclosure provides a lentivirus which is introduced with the lentiviral vector as described in the first aspect.
In a third aspect, the present disclosure provides a host cell which is transduced with the lentivirus as described in the second aspect.
Preferably, the host cell includes a stem cell.
The stem cell of the present disclosure is used as a delivery vehicle to transport the lentiviral vector carrying the CYBB gene, thereby improving the expression efficiency and expression amount of the CYBB gene in differentiated or undifferentiated stem cells.
Preferably, the stem cell includes a hematopoietic stem cell.
According to the present disclosure, the hematopoietic stem cell is derived from blood or bone marrow and has the ability to differentiate into a series of somatic hematopoietic cells and the ability to renew various histiocytes. The hematopoietic stem cell of the present disclosure is used as a potential transport tool to carry the lentivirus containing the CYBB gene to achieve gene therapy for CGD.
In a fourth aspect, the present disclosure provides a method for preparing the host cell as described in the third aspect, which includes the following steps:
(1) constructing a lentiviral vector as described in the first aspect;
(2) performing lentivirus packaging by co-transducing the lentiviral vector obtained in step
(1) and a packaging plasmid into a mammalian cell, to obtain a lentivirus; and
(3) transforming the lentivirus obtained in step (2) into the genome of a host cell.
Preferably, the construction in step (1) is performed by inserting a hEF1α promoter and CYBB into TYF lentiviral vector through restriction enzyme digestion.
Preferably, the packaging plasmid in step (2) includes pNHP and pHEF-VSVG.
Preferably, the mammalian cell in step (2) includes a 293T cell.
Preferably, the method further comprises a step of purifying the lentivirus after step (2).
Preferably, the purification is performed by filtering and concentrating the lentivirus to high-titer virus.
In the present disclosure, the purification, filtration and concentration significantly improve the titer and concentration of the lentivirus.
Preferably, the host cell in step (3) includes a stem cell.
Preferably, the stem cell include a hematopoietic stem cell.
As a preferred technical solution, the present disclosure provides a method for preparing a host cell as described in the third aspect, which comprises the following steps:
In a fifth aspect, the present disclosure provides a pharmaceutical composition which includes any one or a combination of at least two of a group consisting of the lentiviral vector as described in the first aspect, the lentivirus as described in the second aspect, and the host cell as described in the third aspect.
Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a group consisting of a pharmaceutically acceptable carrier, an excipient and a diluent.
The pharmaceutical composition of the present disclosure repairs the mutant CYBB gene of a CGD patient at genetic level, which is beneficial to the repair of the patient's autologous stem cells with potential for long-term stable treatment of CGD.
In a sixth aspect, the present disclosure provides the use of the lentiviral vector as described in the first aspect, the lentivirus as described in the second aspect, the host cell as described in the third aspect, or the pharmaceutical composition as described in the fifth aspect in the preparation of a medicament for treating a disease.
Preferably, the disease includes chronic granulomatous disease.
In a seventh aspect, the present disclosure provides a method for treating chronic granulomatous disease using the lentiviral vector as described in the first aspect, the lentivirus as described in the second aspect, the host cell as described in the third aspect, or the pharmaceutical composition as described in the fifth aspect.
According to the present disclosure, the method comprises the following steps:
Preferably, the pretreatment in step (3′) is performed with busulfan 40 mg per kg of body weight and fludarabine 60 mg per m2 of body surface area.
Preferably, the washing in step (5′) is performed by washing the cells twice with normal saline containing 1% of human serum protein.
Preferably, the suspension in step (5′) is performed with normal saline containing 2.5% of human serum albumin protein.
Compared with the prior art, the present disclosure has the following beneficial effects:
In order to further illustrate the technical measures adopted by the present application and the effects thereof, the technical solutions of the present application are further described below with reference to the accompanying drawings and specific embodiments, and however, the present application is not limited to the scope of the embodiments. In the examples, techniques or conditions, which are not specifically indicated, are performed according to techniques or conditions described in the literature of the art, or according to product instructions.
The reagents or instruments used herein, which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.
Normal CYBB gene sequence (amino acid sequence as shown in SEQ ID NO.2, and nucleic acid sequence as shown in SEQ ID NO.3) was synthesized via whole gene synthesis and ligated into TYF-EF1α lentiviral vector (NHP/TYF lentiviral vector system) through restriction enzyme digestion, behind a human EF1α (hEF1α) promoter sequence (nucleic acid sequence as shown in SEQ ID NO.1). The obtained product was identified by methods such as sequencing and double-digestion (cloned at BamHI site for 5′ and cloned at SpeI site for 3′, referring to NEB Manufacturer's recommendation for the reaction conditions) to obtain a properly linked lentiviral vector carrying CYBB gene under the hEF1α promoter.
A multi-plasmid packaging system was used in this example. The lentiviral vector carrying the CYBB gene was packaged into a complete lentivirus via 293T cells. The specific steps are:
The purification and concentration of lentivirus is performed as follows:
(1) Lentivirus Purification
The packaged lentivirus was centrifuged at 1000 g for 5 min to remove cell debris. The resulting supernatant was filtered using a 0.45 μm low protein binding filter, dispensed and stored at −80° C.
(2) Lentivirus Concentration
The lentivirus supernatant was added to a Centricon filter tube and centrifuged at 2500 g for 30 min. The filter tube was shaken and centrifuged at 400 g for 2 min. The concentrated virus was collected into a collection cup.
Hematopoietic stem cells (HSCs) were inoculated into a culture vessel. The concentrated lentivirus carrying the CYBB target gene was added, centrifuged at 100 g for 100 min, and incubated at 37° C. for 24 h. Medium containing stem cell growth factor was added and incubated for 2-3 days to obtain stem cells carrying normal CYBB genes.
The collected peripheral blood was stained with dihydrorhodamine 123 (DHR123), and CD14 and CD15 were additionally stained. Oxidase function in neutrophilic granulocytes and monocytes in peripheral blood was analyzed by flow cytometry. These two cells mainly use oxidase to perform immune functions. Dihydrorhodamine 123 is oxidized by hydrogen peroxide to rhodamine 123 which emits yellow-green fluorescence at 515 nm when excited by 488 nm laser. When dihydrorhodamine 123 is co-cultured with cells stimulated with phorbol ester (PMA), the fluorescence intensity represents the functional strength of oxidase [Reference 6].
X-linked chronic granulomatous disease is mainly caused by oxidase deficiency in neutrophilic granulocytes. The functional strength and expression level of oxidase in neutrophilic granulocytes were observed weekly after the infusion. The results are shown in
Because the patient received myeloablation pretreatment before the infusion, the inventors monitored continuously the number of neutrophilic granulocytes and monocytes in CD45-positive cells in the patient after the infusion. As shown in
In addition to molecular and cytological evidence, clinical symptoms of the patient were also improved significantly.
In summary, the lentiviral vector of the present disclosure achieves efficient delivery of CYBB gene under the initiation of the EF1α promoter. Lentivirus carrying the CYBB gene is used to transduce stem cells, which are together serve as a delivery vector for the treatment of CGD disease, such that CYBB gene expression is increased in differentiated or undifferentiated stem cells. Infection of CD34 stem cells with lentivirus carrying the CYBB gene has therapeutic potential for X-CGD.
The applicant states that detailed methods of the present application are demonstrated in the present application through the above embodiments, however, the present application is not limited to the above detailed methods, and does not mean that the present application must rely on the above detailed methods to implement. It should be apparent to those skilled in the art that, for any improvement of the present application, the equivalent replacement of the raw materials of the present application, the addition of auxiliary components, and the selection of specific modes, etc., will all fall within the protection scope and the disclosure scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910310154.3 | Apr 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/085243 | 4/17/2020 | WO | 00 |
