CYBB LENTIVIRAL VECTOR, LENTIVIRAL VECTOR-TRANSDUCED STEM CELL, AND PREPARATION METHOD AND APPLICATION THEREOF

Abstract

Provided are a CYBB lentiviral vector, a lentiviral vector-transduced stem cell, a preparation method and application thereof. The lentiviral vector includes a hEF 1α promoter and CYBB that are organized in tandem. The lentiviral vector carries the CYBB gene which under the initiation of the hEF 1α promoter, and expresses the carried CYBB gene in differentiated or undifferentiated stem cells. Stem cells serve as a delivery vector.

Claims

1. A lentiviral vector, comprising a hEF1α promoter and CYBB that are organized in tandem.

2. The lentiviral vector according to claim 1, wherein the hEF1α promoter has a nucleic acid sequence as shown in SEQ ID NO.1.

3. The lentiviral vector according to claim 1, wherein the CYBB has an amino acid sequence as shown in SEQ ID NO. 2 and a nucleic acid sequence as shown in SEQ ID NO.3.

4. A lentivirus that is introduced with the lentiviral vector according to claim 1.

5. A host cell that is transduced with the lentivirus according to claim 4.

6. The host cell according to claim 5, wherein the host cell comprises a stem cell.

7. The host cell according to claim 6, wherein the stem cell comprises a hematopoietic stem cell.

8. A method for preparing a host cell according to claim 5, comprising the following steps: (1) constructing a lentiviral vector comprising a hEF1α promoter and CYBB that are organized in tandem; (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) transferring the lentivirus obtained in step (2) into the genome of a host cell.

9. The method according to claim 8, wherein the construction in step (1) is performed by inserting a hEF1α promoter and CYBB into a TYF lentiviral vector through restriction enzyme digestion.

10. The method according to claim 8, wherein the packaging plasmid in step (2) comprises pNHP and pHEF-VSVG.

11. The method according to claim 8, further comprising a step of purifying the lentivirus after step (2); preferably, the purification is performed by filtering, centrifuging and concentrating the lentivirus.

12. The method according to claim 8, comprising the following steps: (1) inserting a hEF1α promoter and CYBB into a TYF lentiviral vector through restriction enzyme digestion to construct a lentiviral vector; (2) performing lentivirus packaging by co-transducing the lentiviral vector obtained in step (1) and packaging plasmids pNHP and pHEF-VSVG into a 293T cell to obtain a lentivirus, and purifying the lentivirus to obtain a concentrated lentivirus; and (3) transforming the lentivirus obtained in step (2) into the genome of a hematopoietic stem cell.

13. A pharmaceutical composition, comprising the lentiviral vector according to claim 1.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition further comprises any one or a combination of at least two of a group consisting of a pharmaceutically acceptable carrier, excipient and diluent.

15. (canceled)

16. A method for treating a disease, comprising administering to a patient in need thereof an effective amount of the lentiviral vector according to claim 1, wherein the disease comprises chronic granulomatosis.

17. The method according to claim 10, wherein the mammalian cell in step (2) comprises a 293T cell.

18. The method according to claim 11, further comprising a step of purifying the lentivirus after step (2).

19. The method according to claim 11, wherein the purification is performed by filtering, centrifuging and concentrating the lentivirus.

20. The method according to claim 11, wherein the host cell in step (3) comprises a stem cell.

21. The method according to claim 11, wherein the stem cell comprises a hematopoietic stem cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 is a schematic diagram showing the composition of the lentiviral vector;

[0063] FIG. 2 is a diagram showing a treatment procedure;

[0064] FIG. 3 (a) shows the analysis of peripheral blood from the patient's father who has a normal genotype, FIG. 3 (b) shows the analysis of peripheral blood from the patient's mother who has a heterozygous genotype, FIG. 3 (c) shows the analysis of peripheral blood from the patient before infusion, and FIG. 3 (d) shows the analysis of peripheral blood from the patient on day 28 after treatment;

[0065] FIG. 4 (a) shows the average fluorescence intensity multiple stained with rhodamine 123, and FIG. 4 (b) shows the proportion of neutrophilic granulocytes that emit fluorescence;

[0066] FIG. 5 (a) shows the number of neutrophilic granulocytes in CD45-positive cells after infusion, and FIG. 5 (b) shows the number of monocytes in CD45-positive cells after infusion;

[0067] FIG. 6 shows the change of CYBB gene copy number in peripheral blood from the patient after infusion;

[0068] FIG. 7 shows CT scan images of the lung of the patient before and after infusion.

DETAILED DESCRIPTION

[0069] 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.

[0070] The reagents or instruments used herein, which are not indicated with manufacturers, are conventional products that are commercially available from formal sources.

Example 1 Construction of a Lentiviral Vector Carrying a CYBB Gene

[0071] 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. FIG. 1 shows the NHP/TYF lentiviral vector system, including virus packaging plasmids (NHP, EF-VSV-G) and a vector plasmid (pTYF-EF-CYBB). The packaging plasmids include pNHP and pHEF-VSV-G(env). pNHP expresses Gag-Pol protein, and pHEF-VSV-G expresses a coat protein. The gene delivery plasmid pTYF-EF carries a chimeric CMV-IE promoter at the 5′-end in combination with HIV-1 virus TAR-mutation U5 plus an attachment sequence at the right end (CMV-IE-TAR-dl.U5/attR), which is followed by a primer binding site (PBS) and a lentiviral vector packaging signal (psi) and a mutated gag sequence. EF1a-CYBB is followed by a mutated 3′LTR (self-inactivating SIN LTR), a polypurine track sequence (PPT), an attachment site at the left end (attL), and a bovine growth hormone polyA (bGHpA). See references [1]-[3] for details.

Example 2 Lentivirus Packaging

[0072] 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: [0073] (1) A 293T cell strain was cultured for 17-18 hours. Fresh DMEM containing 10% of FBS was added to the culture. [0074] (2) DMEM, pNHP, pHEF-VSV-G and the lentiviral vector constructed in Example 1 were added to a sterile centrifuge tube successively, and vortexed. [0075] (3) Superfect transduction reagent (QIAGEN) was added to the centrifuge tube and let stand at room temperature for 7-10 min. [0076] (4) The lentiviral vector-Superfect mixture in the centrifuge tube was added dropwise to the 293T cells, vortexed, and incubated at 37° C. and 5% CO.sub.2 for 4-5 hours. [0077] (5) The cell culture medium was removed, the cells were rinsed, and culture medium was added to continue the incubation. [0078] (6) The culture medium was returned to the 5% CO.sub.2 incubator and incubated overnight, and then the transduction efficiency was observed with a fluorescence microscope.

Example 3 Purification and Concentration of Lentivirus

[0079] The purification and concentration of lentivirus is performed as follows:

[0080] (1) Lentivirus Purification

[0081] 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.

[0082] (2) Lentivirus Concentration

[0083] 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.

Example 4 Transduction of Hematopoietic Stem Cells with Lentivirus

[0084] 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.

Example 5 Treatment of a Patient with X-Linked Chronic Granulomatous Disease (X-CGD) by Infecting CD34 Stem Cells with Lentivirus Carrying CYBB Gene (TYF-EF1a-CYBB)

[0085] FIG. 2 shows the treatment procedure. [0086] (1) CD34 Stem cells of the patient was mobilized by granulocyte colony-stimulating factor (G-CSF). The patient was collected for peripheral blood or bone marrow twice, with the first collection performed on day 37 before the infusion and the second collection on day 4 before the infusion. Because CGD patients generally have a poor response to mobilization, two bone marrow collections are beneficial to obtain sufficient CD34 stem cells [Reference 4]. [0087] (2) In the laboratory on day 4 before the infusion, CD34-positive cells were isolated from both bone marrow or peripheral blood stem cells collected from the patient using Miltenyi CD34 beads and incubated overnight in HSC culture medium (Sigma Stemline II HSC expansion medium). [0088] (3) On day 3 and 2 before the infusion, the patient was pre-treated with 40 mg/kg of body weight of busulfan and 60 mg/m.sup.2 of body surface area of fludarabine, separately. According to the literature, proper pretreatment can effectively extend the survival of transgenic CD34 stem cells in patients [Reference 5]. [0089] (4) In the laboratory on day 3 and day 2 before the infusion, gene transduction was performed twice by infecting CD34 cells with lentivirus carrying the CYBB gene. Then the infected cells were incubated for one day. [0090] (5) On the day of infusion, the cells were washed twice with normal saline containing 1% of human serum albumin protein, and suspended in normal saline containing of 2.5% human serum albumin protein, and infused back to the patient. [0091] (6) After infusion to the patient, follow up was taken every week. The patient was collected for peripheral blood and measured for oxidase function, immune cell ratio and gene copy number.

Result Analysis

[0092] 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].

[0093] FIG. 3 (a) shows the analysis of peripheral blood from the patient's father who has a normal genotype. Both the proportions of monocytes (CD14+) and neutrophilic granulocytes (CD15+) expressing oxidase are greater than 90%. FIG. 3 (b) shows the analysis of peripheral blood from the patient's mother who has a heterozygous genotype. 70% of monocytes express oxidase and 57% of neutrophilic granulocytes express oxidase, both of which are slightly lower than the proportion of healthy people. FIG. 3 (c) shows the analysis of peripheral blood from the patient before infusion. It can be seen that the patient did not express oxidase at all before treatment. FIG. 3 (d) shows the analysis of peripheral blood from the patient on day 28 after treatment. 35% of monocytes and 47% of neutrophilic granulocytes in peripheral blood express oxidase, which are greatly improved compared with that before infusion.

[0094] 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 FIG. 5. FIG. 4 (a) shows the average fluorescence intensity multiple, that is the functional strength of oxidases in cells after stimulation, which is calculated by dividing the average fluorescence intensity of cells stimulated with phorbol ester (PMA) by the average fluorescence intensity of cells not stimulated with phorbol ester when rhodamine 123 was used for staining. Before the treatment, the patient's ratio was 1.08, suggesting that no oxidase response occurred in cells after received stimulation. After the treatment, the function of oxidase fluctuated with time, but improved overall, wherein day 28 and day 73 after the infusion showed the highest values, reaching 1.44-fold and 1.62-fold respectively. FIG. 4 (b) shows the proportion of neutrophilic granulocytes that emit fluorescence, that is, the proportion of neutrophilic granulocytes that express oxidase. The patient did not express any oxidase before the infusion. On day 28 and day 73 after the infusion, 47% and 66% of the patient's neutrophilic granulocytes expressed oxidase, respectively. The proportion of cells expressing oxidase has maintained above 20% since day 49 after the infusion (the patient received blood transfusion on day 7 after the infusion, which is not discussed herein).

[0095] 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 FIG. 5 (a) on day 14 after infusion, the number of neutrophilic granulocytes reached the lowest point, accounting for only 0.2% of CD45-positive cells, which is obviously affected by the myeloablation pretreatment compared with the case in the normal genotype father whose neutrophilic granulocytes maintained at 60%. But after 14 days, the percentage of neutrophilic granulocytes gradually increased, and on day 49 after the infusion, to the normal proportion of healthy people and maintained till the latest follow-up, that is, day 103 after the infusion. As shown in FIG. 5 (b), the number of monocytes reached the lowest point on day 7 after the infusion, which accounts for only 2% of CD45-positive cells.

[0096] FIG. 6 shows the change of CYBB gene copy number in peripheral blood from the patient after infusion. On day 21 and day 35 after the infusion, the copy number reached 3.75% and 2.41%, respectively, which were the highest values monitored. It can be seen that CYBB gene was stably retained in peripheral blood cells. 0.23% of peripheral blood cells still carried CYBB gene even on day 103 after infusion.

[0097] In addition to molecular and cytological evidence, clinical symptoms of the patient were also improved significantly. FIG. 7 shows pulmonary CT scan images of the patient before and after infusion. On day 39 before the infusion, the patient had a severe infection in the lungs and received antifungal and antibacterial drugs as adjuvant therapy. On day 55 after the infusion, the pulmonary infection situation was improved significantly. On day 105 after the infusion, the pulmonary infection had relived and no drug support was required.

[0098] 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.

REFERENCES

[0099] [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. [0100] [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. [0101] [3] Iwakuma T., Y. Cui, and L.-J. Chang (1999). Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261, 120-132. [0102] [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. [0103] [5] Manuel Grez, Janine Reichenbach, Joachim Schwable, Reinhard Seger, Mary C Dinauer, and Adrian J Thrasher. (2011) Gene Therapy of Chronic Granulomatous Disease: The Engraftment Dilemma. Molecular Therapy 19, 28-35. [0104] [6] Yu Chen and Wolfgang G. Junger. (2012) Measurement of Oxidative Burst in Neutrophilic granulocytes. Methods in Molecular biology 844, 115-144. [0105] [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. [0106] [8] Danielle E. Arnold and Jennifer R. Heimall. (2017) A Review of Chronic Granulomatous Disease. Advances in Therapy 34, 2543-2557. [0107] [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. [0108] [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.

[0109] 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.