Adeno-associated virus targeting sfpq and use thereof in the manufacture of a medicament for treating neuroblastoma

Abstract

The application provides a guide RNA which targets SFPQ, primer sequences of the guide RNA are as follows: SFPQ sgRNA-FWD: 5'-CGTACTCAAACGTGCCATG-3'; SFPQ sgRNA-REV: 5'-CATGGCACGTTTGAGTACG-3'. The application also provides an adeno-associated virus which targets SFPQ and contains the guide RNA. The application also provides use of the adeno-associated virus which targets SFPQ in preparation of a drug for treating neuroblastoma. The adeno-associated virus which targets SFPQ provided by the application can not only inhibit tumor growth, but also promote NB cell differentiation, thereby providing a new strategy for NB treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to an adeno-associated virus targeting SFPQ, in particular, an adeno-associated virus targeting SFPQ and use thereof in the preparation of a medicament for treating neuroblastoma. BACKGROUND

[0002] Neuroblastoma (NB) is one of the most common malignant tumors in children, accounting for about 10% of all childhood malignancies, with an incidence of about one in ten thousand, and is therefore known as the "king of childhood malignant tumors". Due to the diversity of its clinical manifestations and tumor behavior, NB is considered a highly heterogeneous disease. The tumor of some patients can spontaneously regress without any intervention, while the survival rate of high-risk patients is still less than 60% even after multidisciplinary treatment. At present, with the continuous development of genomics, proteomics and single-cell analysis, we have a deeper understanding of the biological mechanisms of NB, but few of these research results can be directly applied to clinical diagnosis and treatment.

[0003] NB is caused by the differentiation arrest of neural crest-derived sympatho-adrenal lineage cells, which usually occurs in neural crest cells migrating to different sites and their differentiated daughter cells, of which the most common sites are the adrenal medulla and paraspinal ganglia. The developmental arrest of neural crest cells can effectively explain the various biological characteristics of the disease, including cell heterogeneity, mutation spectrum, spontaneous regression and response to drugs that induce tumor cell differentiation.

[0004] Differentiation therapy, which promotes tumor cells to transform into non-invasive mature phenotype by reactivating cell differentiation pathways, shows significant therapeutic potential. Currently, RA (retinoic acid) is widely considered as the most effective differentiation inducer for NB in clinic, and RA derivative 13-CRA (13-cis-retinoic acid) has become a commonly used clinical treatment drug. 13-CRA has good pharmacokinetic characteristics, but its affinity for RA receptor is relatively weak, and it is prone to drug resistance in high-risk patients. Other RA derivatives, such as 9-cRA (9-cis-retinoic acid), have stronger efficacy, but their higher toxicity, shorter half-life and lower oral bioavailability limit their clinical application potential. In addition to RA derivatives, other differentiation induction strategies have gradually attracted attention. MYCN proto-oncogene plays a key role in maintaining the undifferentiated tumor phenotype, and targeting MYCN and its regulatory pathways has become an important strategy to drive differentiation. BET inhibitor JQ1 can effectively inhibit MYCN expression, successfully induce NB differentiation in vitro, and reduce tumor burden in mouse models. BET inhibitors IBET-762 and OTX-015 have also been used in clinical trials for the treatment of hematological tumors and solid tumors, but due to significant side effects, the clinical use of these inhibitors has been temporarily suspended. MYCN can also be destroyed by small molecule inhibitors 10058-F4 or 10074-G5 to bind to its interacting protein MAX, thereby inducing in vitro differentiation of MYCN-amplified NB cells and significantly improving the survival rate of TH-MYCN mice. In addition to MYCN targeting strategies, histone deacetylase inhibitors (such as valproic acid, vorinostat and sodium phenylbutyrate) and small molecule compounds (such as niklochloramidol) have also been found to be able to induce NB differentiation, however, these strategies still need further clinical verification.

[0005] Current differentiation therapy, especially RA, is usually used in the maintenance phase of NB treatment, mainly by inducing differentiation of residual tumor cells in patients after receiving chemotherapy, surgical resection and bone marrow transplantation, thereby reducing disease recurrence. However, differentiation therapy in the early stage of tumor development may be more effective. Recent studies have shown that after all-trans retinoic acid (ATRA) induces differentiation of NB cell lines, significant changes in the proteome and phosphoproteome of the cells occur, identifying multiple signaling pathways related to cytoskeleton, cell division, chaperone function, protein folding and one-carbon metabolism. These results provide a new molecular mechanism perspective for differentiation therapy. Although differentiation therapy has less damage to normal cells and has certain advantages in the treatment of childhood tumors, high toxicity, drug resistance and patient stratification still limit its widespread application. Therefore, future research should focus on combining biomarker analysis, optimizing patient stratification strategies, exploring combination therapy regimens and improving drug delivery systems to further improve the efficacy of treatment and overcome existing limitations.

[0006] The splicing factor proline / glutamine-rich protein (SFPQ), also known as PSF, is involved in the formation of early spliceosomes as a member of the DBHS protein family. SFPQ has two isoforms, with the major isoform SFPQ-A consisting of 707 amino acids, and the short isoform SFPQ-F containing 669 amino acids and lacking a nuclear localization signal. Abnormal expression and localization of SFPQ can lead to RNA splicing disorders, causing the occurrence of tumors and neurodegenerative diseases. In metastatic or advanced prostate cancer, SFPQ is involved in the regulation of androgen receptor (AR) splicing, promoting the production of AR and variants such as AR-V7, thereby driving tumor malignancy and hormone resistance. In addition, SFPQ can interact with other splicing factors or regulate the expression of splicing factor genes, thereby affecting the splicing process of prostate cancer and activating various oncogenic pathways. In bladder cancer, SFPQ interacts with another splicing factor NONO, regulates SEMA variable splicing, leads to the upregulation of H3K27me3, and inhibits the transcription of metastasis-related oncogenes. In addition, SFPQ is involved in tumor immune regulation, regulating the alternative splicing of transcription factor IRF1 in the tumor microenvironment through a TGFβ-dependent mechanism, thereby regulating the anti-tumor response of T helper 1 cells (Th1). Although SFPQ plays a key regulatory role in various tumors and neurodegenerative diseases, its function in NB has not been reported.

[0007] Currently, the treatment of NB is mainly based on patient risk classification: low-risk group: (1) surgical treatment + observation (2) chemotherapy combined with or without surgery. Medium-risk group: (1) elective surgery before or during chemotherapy (about 4 courses). High-risk group: the treatment plan includes three stages, namely induction period (chemotherapy and surgery), consolidation period (sequential transplantation and radiotherapy for primary tumors and residual metastatic sites), and maintenance treatment after consolidation period (immunotherapy and 13-CRA). After 2 cycles of chemotherapy, autologous peripheral blood stem cell collection is performed, followed by 2 cycles of chemotherapy and elective surgery. Postoperative chemotherapy for 2 courses, with a total course of no more than 6 courses. After conventional chemotherapy, autologous stem cell transplantation and radiotherapy with a dose of 21.6 Gy for tumor bed radiotherapy are performed. Finally, GD2 monoclonal antibody immunotherapy combined with GM-CSF and 13-CRA treatment is performed.

[0008] Current treatment deficiencies:

[0009] (1) Although the five-year event-free survival (EFS) of low-risk and medium-risk NB patients is usually between 80% and 90%, and the five-year overall survival (OS) is more than 95%, the survival rate of high-risk patients is still less than 60% even after multidisciplinary treatment.

[0010] (2) Surgical operation is an important part of the treatment of neuroblastoma, especially in low-risk or part of the medium-risk cases, surgical resection of the tumor is the preferred strategy. However, this method has significant limitations in practice: current surgical treatment is only a local treatment method, which cannot control the distant metastasis or circulating tumor cells in the blood that has occurred, cannot cure advanced NB, and is prone to recurrence after surgery. Some tumor locations are complex and difficult to remove.

[0011] (3) Chemotherapy is the main treatment for medium-risk and high-risk neuroblastoma patients, especially in the preoperative (neoadjuvant) and postoperative (adjuvant) stages. Commonly used drugs include: cisplatin, etoposide, vincristine, cyclophosphamide, etc. However, this treatment method also has many serious shortcomings: severe side effects, affecting growth and development; rapid development of drug resistance, affecting efficacy; difficult to completely remove stem cell-like cells; chemotherapy lacks specificity, killing normal cells.

[0012] (4) Differentiation therapy reactivates cell differentiation pathways, promotes tumor cells to transform into non-invasive mature phenotypes, and shows significant therapeutic potential. Currently, RA (retinoic acid) is widely recognized as the most effective NB differentiation inducer in clinical practice, and RA derivative 13-CRA has become a commonly used clinical treatment drug. 13-CRA has good pharmacokinetic properties, but its affinity for RA receptors is relatively weak, and it is prone to drug resistance in high-risk patients (especially MYCN amplification patients).

[0013] In recent years, adeno-associated virus (AAV, Adeno-Associated Virus) has become one of the most widely used viral vectors in gene therapy due to its good safety, low immunogenicity, tissue targeting and long-term gene expression ability. Different serotypes of AAV can achieve targeted delivery to specific tissues or organs, and are widely used in the treatment of various genetic diseases, especially showing significant efficacy in ophthalmic diseases, nervous system diseases and muscle diseases. A variety of engineered AAV vectors have been developed in the prior art to improve transduction efficiency, expand packaging capacity or reduce host immune response, thereby laying an important foundation for the clinical translation of AAV-mediated gene therapy. However, there is no literature or public report that AAV gene therapy is applied to neuroblastoma. SUMMARY

[0014] In view of the above technical problems in the prior art, the present application provides a SFPQ-targeted adeno-associated virus and its use in the preparation of a drug for treating neuroblastoma. The SFPQ-targeted adeno-associated virus and its use in the preparation of a drug for treating neuroblastoma solve the technical problem of poor effect of existing drugs for treating neuroblastoma.

[0015] The application provides a guide RNA (sgRNA) which targets SFPQ, a primer sequence of the guide RNA is shown as follows:

[0016] SFPQ sgRNA-FWD: 5'-CGTACTCAAACGTGCCATG-3' (SEQ ID NO. 1);

[0017] SFPQ sgRNA-REV: 5'-CATGGCACGTTTGAGTACG-3' (SEQ ID NO. 2).

[0018] The application also provides an adeno-associated virus targeting SFPQ, containing the guide RNA.

[0019] The application also provides the use of the adeno-associated virus targeting SFPQ in the preparation of a drug for treating neuroblastoma.

[0020] The application also provides the use of SFPQ as a target in the preparation of a drug for screening a drug for treating neuroblastoma.

[0021] The application also provides a CRISPR-SauriCas9 system, which comprises a SauriCas9 protein and the guide RNA; the guide RNA can combine with the SauriCas9 protein to form a complex and guide the complex to contact with SFPQ genomic DNA (target DNA).

[0022] The application provides an AAV vector targeting SFPQ, which can not only inhibit tumor growth, but also promote NB differentiation, and provides a new strategy for NB treatment.

[0023] The application provides the use of SFPQ as a target in the preparation of an AAV virus for treating high-risk neuroblastoma, and the AAV virus can effectively inhibit the expression of SFPQ in vivo. Inhibiting SFPQ can effectively inhibit tumor cell growth and promote NB differentiation in vitro. The adeno-associated virus targeting SFPQ can significantly inhibit NB tumor growth in mice.

[0024] Compared with the prior art, the technical effect of the application is positive and obvious. Targeting and inhibiting SFPQ can effectively promote NB cell differentiation in vitro. The adeno-associated virus targeting SFPQ can significantly inhibit NB tumor growth in mice. The adeno-associated virus vector targeting SFPQ provided by the application is used for inhibiting tumor proliferation and promoting tumor cell differentiation, so as to improve the problems of poor effect, easy recurrence and strong drug resistance of existing treatment methods. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 a. Immunoblotting showed that the present invention successfully knocked down SFPQ in SK-N-BE(2) and CHP-134 neuroblastoma cells. Figure 1 b. Proliferation experiments showed that SFPQ knockdown significantly inhibited tumor cell proliferation.

[0026] Figure 2 The results show that SFPQ deficiency leads to differentiated cell morphology. Figure 2 In the 'a' pattern, cell volume increases, accompanied by elongation of nerve-like processes. Figure 2 b and c in the data show that SFPQ knockdown significantly promoted NB differentiation; Figure 2 The values ​​d and e in the figure show that SFPQ deficiency leads to a significant downregulation of the stemness marker SOX2, while the expression of the differentiation markers NDRG1 and TUJ1 is significantly upregulated.

[0027] Figure 3 The study showed that orthotopic injection of AVV virus targeting SFPQ into mouse tumors significantly inhibited tumor growth. Detailed Implementation

[0028] Example 1: In neuroblastoma, SFPQ deficiency inhibits tumor proliferation.

[0029] Using a liposome nucleic acid transfection kit (Yisheng Biotechnology, 40802ES03), the two independent shRNAs targeting SFPQ (SFPQ shRNA-1-FWD) were transfected according to the instructions.

[0030] 5'-CCGGAGCGCCAAAGAGAGGAAAGTTACTCGAGTAACTTTCCTCTCTTTGGCGCTTTTTTG-3';

[0031] SFPQ shRNA-1- REV:

[0032] 5'-AATTCAAAAAAGCGCCAAAGAGAGGAAAGTTACTCGAGTAACTTTCCTCTCTTTGGCGCT-3';

[0033] SFPQ shRNA-2-FWD:

[0034] 5'-CCGGAGACCAACAAATCTCAAGCCCTCTCGAGAGGGCTTGAGATTTGTTGGTCTTTTTTG-3';

[0035] SFPQ shRNA-2- REV:

[0036] 5'-AATTCAAAAAAGACCAACAAATCTCAAGCCCTCTCGAGAGGGCTTGAGATTTGTTGGTCT-3') with psPAX2 (packaging plasmid, Addgene, Plasmid 12260), pMT24G (envelope plasmid, Addgene, Plasmid 12259) respectively into SK-N-BE(2) (Manassas, VA, USA, CRL-2271) and CHP-134 (Braunschweig, Germany, ACC-653) cells. Subsequently, the proliferation ability of the constructed control and SFPQ knockdown SK-N-BE(2) or CHP-134 cells was evaluated, respectively.

[0037] 1.1, Targeting SFPQ shRNA plasmid construction

[0038] 1) Oligonucleotide synthesis and annealing:

[0039] A. Primer design: The target sequence of shRNA was derived from the official website of Merck (https: / / www.sigmaaldrich.cn). The appropriate knockdown sequence was selected according to the target gene to ensure its specificity and efficiency.

[0040] SFPQ shRNA-1-FWD:

[0041] 5'-CCGGAGCGCCAAAGAGAGGAAAGTTACTCGAGTAACTTTCCTCTCTTTGGCGCTTTTTTG-3' (SEQ ID NO. 1)

[0042] SFPQ shRNA-1-REV:

[0043] 5'-AATTCAAAAAAGCGCCAAAGAGAGGAAAGTTACTCGAGTAACTTTCCTCTCTTTGGCGCT-3' (SEQ ID NO. 2)

[0044] SFPQ shRNA-2-FWD:

[0045] 5'-CCGGAGACCAACAAATCTCAAGCCCTCTCGAGAGGGCTTGAGATTTGTTGGTCTTTTTTG-3' (SEQ ID NO. 3)

[0046] SFPQ shRNA-2-REV:

[0047] 5'-AATTCAAAAAAGACCAACAAATCTCAAGCCCTCTCGAGAGGGCTTGAGATTTGTTGGTCT-3' (SEQ ID NO. 4)

[0048] B. Primer dilution and annealing reaction preparation: Dilute the primer powder to 100 μΜ using sterilized deionized water and prepare a single-stranded oligonucleotide solution. Prepare the following reaction system for primer annealing preparation (10 μΐ, total system):

[0049]

[0050] C. Annealing program setting: After gently mixing and centrifuging the prepared system, place it in a gradient PCR instrument and perform annealing according to the following program: 37°C for 30 minutes, 95°C for 5 minutes, 95°C to 25°C (5°C / min).

[0051] D. Dilution of product: Dilute the annealed double-stranded oligonucleotide product 200 times using sterilized deionized water for subsequent vector ligation experiments.

[0052] 2) Linearization of plasmid vector: This experiment uses pLKO.1 vector (Addgene Plasmid, 10878) for linearization treatment by Age I (NEB, R3552) and EcoRI (NEB, R3101) restriction enzymes. Incubate in a 37°C water bath for 1 hour to ensure complete linearization of the plasmid. Then prepare a 1% agarose gel (Shanghai Shengong Bioengineering Co., Ltd.) and perform gel running verification. Next, use a clean blade to cut the DNA band corresponding to the linearized vector, and place the gel in a 1.5 mL centrifuge tube for later use. Then follow the instructions of the agarose gel recovery kit (OMEGA, D2500) to recover the gel.

[0053] 3) Ligation of linearized vector and double-stranded oligonucleotide:

[0054] A. According to the T4 ligation kit (NEB, M0202), prepare the following reaction system for ligation reaction:

[0055]

[0056] Calculation of the amount of vector and insert:

[0057] Cloning vector (corresponding to about 0.03 pmol of molar amount): Vector amount (ng) = 0.02 x number of base pairs (bp) of the vector

[0058] Insert (corresponding to the molar mass of about 0.06 pmol): insert amount (ng) = 0.04 x insert base pair number (bp)

[0059] After calculation according to the above formula, the volume of x and y in the reaction system is adjusted to ensure that the molar ratio of vector to insert is close to 1:2, so as to improve the ligation efficiency.

[0060] B. Place the prepared ligation reaction mixture at room temperature for 1-2 hours.

[0061] 4) Transformation and plating of ligation products:

[0062] A. Add the ligation reaction product to 100 μL competent cells (Yikang Bio), gently shake the tube wall several times to mix, and place in ice bath for 30 minutes.

[0063] B. Place the competent cells in a 42°C water bath for 90 seconds, then immediately place in an ice bath for 2 minutes.

[0064] C. Then add 1 mL of antibiotic-free LB medium (Yikang Bio) and incubate in a bacterial shaking incubator for 40 minutes.

[0065] D. Centrifuge the culture at 1500 rpm for 3 minutes, resuspend with 100 μL of LB medium, and evenly spread on plates containing 100 μg / mL ampicillin (Yikang Bio). Invert the plate and incubate at 37°C overnight.

[0066] 5) Clone sequencing:

[0067] A. The next day, pick single colonies into 1 mL of liquid LB containing ampicillin and incubate in a bacterial shaking incubator for 4-5 hours.

[0068] B. Take 500 μL of bacterial solution for sequencing, and store the remaining bacterial solution at 4°C.

[0069] 6) Endotoxin-free plasmid extraction:

[0070] According to the instructions of the Endotoxin-free Plasmid Miniprep Kit (Tiangen, DP118):

[0071] A. Inoculate the correct bacterial solution into 20 mL of liquid LB medium containing ampicillin and incubate at 37°C, 250 rpm for 12-16 hours.

[0072] B. The next day, centrifuge the cultured bacterial solution at 13400 x g for 1 minute, discard the supernatant, and retain the bacterial precipitate.

[0073] C. Resuspend the bacterial pellet with 500 μL of solution P1 containing RNase A, mix well using a vortex.

[0074] D. Add 500 μL of solution P2, mix well by inverting the tube 7-8 times, make sure this step is completed within 5 minutes.

[0075] E. Add 500 μL of solution P4, mix well by inverting the tube 7-8 times, let it stand at room temperature for 10 minutes.

[0076] F. Centrifuge at 12000 x g for 10 minutes at room temperature, collect the supernatant and load it into the filter column CS in 700 μL aliquots, centrifuge at 12000 rpm for 2 minutes, collect the filtrate in a clean 2 mL tube.

[0077] G. Add 0.3 volume of isopropanol to the filtrate, mix well and load it into the adsorption column CP4 in 700 μL aliquots. Centrifuge at 12000 rpm for 1 minute at room temperature, discard the waste in the collection tube and put the adsorption column back into the collection tube.

[0078] H. Add 600 μL of rinse PW, pre-mixed with absolute ethanol, to the adsorption column CP4, centrifuge at 12000 rpm for 1 minute, discard the waste in the collection tube and put the adsorption column CP4 into a new collection tube.

[0079] I. Add another 600 μL of rinse PW to the adsorption column CP4, centrifuge at 12000 rpm for 1 minute, discard the waste in the collection tube and put the adsorption column CP4 into the collection tube.

[0080] J. Put the adsorption column CP4 back into the collection tube, centrifuge at 12000 rpm for 2 minutes to remove residual rinse from the adsorption column.

[0081] K. Put the adsorption column CP4 into a clean tube, add 100-300 μL of elution buffer TB to the middle of the adsorption membrane, let it stand for 2 minutes at room temperature, then centrifuge at 12000 rpm for 1 minute to collect the plasmid solution.

[0082] L. Measure the concentration and purity of the plasmid using a UV spectrophotometer.

[0083] 1.2. Lentivirus packaging and construction of stable cell lines

[0084] 1) Lentivirus packaging

[0085] A. One day before packaging virus, digest and passage the well-grown HEK293 (Manassas, VA, USA, CRL-3216) cells, replate to 10 cm culture dish, so that the cell density reaches 70%~80%, and is in logarithmic growth phase.

[0086] B. The next day, prepare reaction system 1 as follows, and mix gently, and stand at room temperature for 5 minutes:

[0087]

[0088] Prepare reaction system 2 as follows:

[0089]

[0090] C. Slowly add reaction system 2 to reaction system 1, mix gently, and stand at room temperature for 15 minutes.

[0091] D. Take the HEK293 cells out of the incubator, gently remove the supernatant, add 7~8 mL of fresh DMEM complete medium (Thermo Fisher), gently add the mixture in C to the culture dish, mix gently using the cross method, and then put the cells back into the incubator for further culture.

[0092] E. 48 hours and 72 hours after transfection, collect the supernatant, and use a 0.45 μm filter to remove residual cells in the supernatant. Then, transfer the filtered liquid to a virus concentration column, centrifuge at 4000 rpm, 4°C for 30 minutes, aliquot the virus liquid, and store in a -80°C refrigerator.

[0093] 2) Construction of stable cell lines:

[0094] A. One day before transfection, digest the target cells and inoculate them on the culture plate, so that the cell density reaches 40%~60%, and is in logarithmic growth phase.

[0095] B. The next day, take the target cells out of the incubator, and add an appropriate amount of control lentivirus or experimental lentivirus (about 20 μL of virus liquid per well) to each well of the 6-well plate. Then put the target cells back into the incubator for further culture.

[0096] C. 48 hours after transfection, perform cell passage treatment.

[0097] D. The next day, remove the supernatant in the well plate, and add fresh complete medium containing an appropriate amount of antibiotic. At the same time, add the same concentration of antibiotic to the control cells that are not transfected with virus, as a negative control.

[0098] E. After 48 hours, observe the cell growth status. Generally, the cells in the negative control group without virus infection should be completely dead under the antibiotic selection, while most of the cells in the experimental group should survive.

[0099] F. To evaluate the transfection effect and verify the expression of the target gene, Western blotting is used to detect the level of the target protein in each sample. Figure 1 a) The following stable transfection cell lines are finally constructed: SK-N-BE(2) / shC, SK-N-BE(2) / shSFPQ-1, SK-N-BE(2) / shSFPQ-2, CHP-134 / shC, CHP-134 / shSFPQ-1, CHP-134 / shSFPQ-2.

[0100] 1.3, Cell proliferation detection

[0101] A. Digest the control cell line and SFPQ knockdown cell line of neuroblastoma in the logarithmic growth phase into a single cell suspension, and count the cells.

[0102] B. According to the cell count results, inoculate the cells into a 96-well plate at an appropriate density (usually 4000 cells / well, 100 μL complete culture medium per well). After inoculation, place the well plate in an incubator for further culture.

[0103] C. Count the cells of each group at the same time point every day on the 1st, 2nd, 3rd, 4th and 5th day after inoculation. At least 3 repeated wells should be set for each time point and each group of samples.

[0104] D. According to the counting results at each time point, draw a cell proliferation curve. Through statistical analysis, compare the differences in cell number at different time points to evaluate the cell proliferation.

[0105] Figure 1 (a, b) The results show that knocking down SFPQ in MYCN-amplified NB cell lines SK-N-BE(2) and CHP-134 can significantly inhibit tumor cell proliferation.

[0106] Example 2 Knocking down SFPQ in neuroblastoma causes cell differentiation phenotype

[0107] 1.1, Immunofluorescence staining:

[0108] 1) Cell crawling sheet:

[0109] A. Digest the control cell line or SFPQ knockdown cell line of neuroblastoma in the logarithmic growth phase into a single cell suspension, and count the cells.

[0110] B. Inoculate 5 x 10 4The cells were subcultured at a ratio of 1:3 when the cell confluence reached 50%-70%. The cells were evenly inoculated into a 24-well plate containing cell climbing sheets (Univivi) and placed in an incubator for overnight culture.

[0111] 2) Cell fixation:

[0112] A. The next day, the target cells were taken out of the incubator, the upper culture solution was removed, and the cells were washed twice with PBS buffer (HyClone), and the supernatant was removed.

[0113] B. An appropriate amount of 4% paraformaldehyde solution (Shenguo Bioengineering Co., Ltd.) was added, and the cells were fixed at room temperature for 10-20 minutes.

[0114] C. The cells were washed with PBS phosphate buffer for 3 times, each for 5 minutes.

[0115] 3) Cell permeation:

[0116] A. 0.25% Triton X-100 (Shenguo Bioengineering Co., Ltd.) solution was added, and incubated at room temperature for 15 minutes.

[0117] B. The cells were washed with PBS phosphate buffer for 3 times, each for 5 minutes.

[0118] 4) Blocking:

[0119] An appropriate amount of 10% goat serum (Aibisheng Biological Co., Ltd.) was added, and incubated at room temperature for 1 hour.

[0120] 5) Primary antibody incubation:

[0121] A. According to the antibody instruction, an appropriate amount of primary antibody solution (TUJ1, Biolegend, 801213) was prepared, and 10% goat serum was used for dilution.

[0122] B. An appropriate amount of primary antibody solution was added to the target cells, and incubated at 4°C overnight.

[0123] 6) Secondary antibody incubation:

[0124] A. The target cells were taken out, the primary antibody solution was recovered, and the cells were washed with PBST (0.1% Tween 20 in PBS) for 2 times, each for 5 minutes.

[0125] B. According to the antibody instruction, a secondary antibody solution (CST, 8890) was prepared, and PBST solution was used for dilution.

[0126] C. An appropriate amount of secondary antibody solution was added to the target cells, and incubated at room temperature for 1 hour in the dark.

[0127] D. Wash cells with PBST for 3 times, 5 minutes each time.

[0128] 7) Nuclei staining, mounting and microscopic observation:

[0129] A. Add DAPI (ThermoFisher, D1306) nuclei staining solution, incubate at room temperature for 5 minutes.

[0130] B. Wash cells with PBST for 3 times, 5 minutes each time.

[0131] C. Add anti-fade reagent (ThermoFisher) in the slide, and invert the cell slide on the slide, pay attention to avoid bubbles.

[0132] D. Use appropriate glue to fix the cover glass.

[0133] E. Use fluorescence microscope to observe the sample. Choose appropriate filter, observe according to the wavelength of fluorescence label, and ensure that the sample avoids overexposure or fading, and record the image in time.

[0134] 1.2, RNA extraction, reverse transcription, real-time quantitative PCR

[0135] 1) RNA extraction:

[0136] A. After the target cells are digested into single cell suspension using trypsin, centrifuge at 1500 rpm for 5 minutes, and wash with PBS phosphate buffer twice to remove the supernatant, add 1 mL Trizol solution (ThermoFisher), and mix well.

[0137] B. Then add 200 μL chloroform (Merck), mix well, and stand at room temperature for 2~3 minutes, then centrifuge at 12000 rpm, 4°C for 15 minutes.

[0138] C. Transfer the supernatant (about 500 μL) to a new 1.5 mL EP tube, add an equal volume of isopropanol solution (Merck), mix well by inverting the centrifuge tube. Stand at room temperature for 10 minutes or store at -20°C for 2 hours, then centrifuge at 12000 rpm, 4°C for 15 minutes.

[0139] D. Discard the supernatant, add 800 μL 75% ethanol (Merck) for cleaning, centrifuge at 7500 rpm, 4°C for 5 minutes.

[0140] E. Discard the supernatant, and remove the residual ethanol after short-time high-speed centrifugation.

[0141] F. Open the lid and let it stand for 5-10 minutes until the precipitate is transparent. Then add 20-100 μL of DEPC water or nuclease-free water to dissolve the precipitate and ensure that it is fully dissolved.

[0142] G. RNA concentration should be determined using a spectrophotometer, with an OD260 / 280 value between 1.8 and 2.2.

[0143] 2) RNA reverse transcription

[0144] Follow the instructions for the reverse transcription kit (Yisheng Bio, 11149ES10) to operate.

[0145] A. Removal of genomic DNA: The reaction system is prepared as follows:

[0146]

[0147] After gently mixing the prepared system and briefly centrifuging it, incubate it at 42°C for 2 minutes.

[0148] B. Prepare the reverse transcription reaction system as follows:

[0149]

[0150] After gently mixing the prepared reaction mixture and briefly centrifuging, place it in a gradient PCR instrument for reverse transcription. The reverse transcription program is as follows:

[0151]

[0152] After inversion, the cDNA is diluted 5-fold and can be used for subsequent RT-qPCR or conventional PCR detection.

[0153] 3) Real-time quantitative PCR

[0154] Follow the instructions for the Real-Time PCR Amplification Kit (Yisheng Bio, 11184ES03)

[0155] A. Prepare the reaction volume as follows:

[0156]

[0157] B. After gently mixing the prepared mixture and briefly centrifuging, place it in a gradient PCR instrument for amplification. The amplification program is as follows:

[0158]

[0159] C. Data Analysis: Using 2 (-ΔΔCt) Methods for analyzing qPCR data.

[0160] 1.3 Western blot assay

[0161] 1) Preparation of reagents:

[0162] 6 × SDS loading buffer (all reagents are purchased from Shenguo Bioengineering Co., Ltd.)

[0163]

[0164] 10 × Running buffer (pH 8.3) (all reagents are purchased from Shenguo Bioengineering Co., Ltd.)

[0165]

[0166] 10 × Transfer buffer (all reagents are purchased from Shenguo Bioengineering Co., Ltd.)

[0167]

[0168] 10 × TBST (pH 7.6) (all reagents are purchased from Shenguo Bioengineering Co., Ltd.)

[0169]

[0170] 2) Sample preparation: After digesting the target cells, wash the cells twice with PBS phosphate buffer and remove the supernatant. According to the number of cells, add an appropriate volume of 1 × Loading buffer (usually 1e 6 Add 40 μL of 1 × Loading buffer to the cells, mix well on a shaker, and incubate at 100℃ in a metal bath for 10 minutes to denature the proteins.

[0171] 3) Gel preparation:

[0172] A. Preparation of lower protein electrophoresis gel (total volume 20 mL) (all reagents are purchased from Shenguo Bioengineering Co., Ltd.):

[0173]

[0174] B. Slowly add anhydrous ethanol on top of the lower gel and let it stand at room temperature until the gel solidifies.

[0175] C. After discarding the anhydrous ethanol solution, prepare the upper protein electrophoresis gel as follows:

[0176]

[0177] D. Insert the comb and let it stand until the upper gel solidifies.

[0178] 4) Protein electrophoresis:

[0179] A. Clamped the two sides of the gel plate with a gel plate clamp and placed it into the electrophoresis tank, added 1 × electrophoresis solution to the gel plate layer, and removed the comb.

[0180] B. Added protein standards and samples to the gel wells in turn, and added an appropriate amount of 1 × electrophoresis solution to the outside of the gel layer.

[0181] C. Started the electrophoresis device, set the constant voltage to 120 V, started the gel running, and stopped the electrophoresis when the position of the protein standard at the bottom of the gel and the bromophenol blue dye was observed to reach the bottom of the gel plate.

[0182] 5) Membrane transfer:

[0183] A. Soaked the NC membrane (Cytiva, 10600001), sponge and filter paper in 1 × transfer solution in advance.

[0184] B. Removed the gel from the gel plate and soaked it in 1 × transfer solution.

[0185] C. Placed each layer in the following order: negative electrode-sponge-thick filter paper-thin filter paper-gel-membrane-thin filter paper-thick filter paper-sponge-positive electrode, and ensured that there were no air bubbles between the layers.

[0186] D. Placed the transfer clamp and ice bag into the transfer tank, added 1 × transfer solution, started the electrophoresis device, set the constant current to 250 mA, and performed ice bath transfer for 1.5 hours.

[0187] 6) Blocking:

[0188] A. Prepared a 5% milk solution (Shenguo Bioengineering Co., Ltd.).

[0189] B. Removed the NC membrane and soaked it in milk, and blocked it at room temperature for 1-2 hours at a speed of 30-40 rpm.

[0190] C. After blocking was completed, washed the NC membrane with 1 × TBST solution at a speed of 50-60 rpm for 2 times, each for 5 minutes.

[0191] 7) Primary antibody binding:

[0192] A. Prepared an appropriate volume of primary antibody solution according to the antibody instructions (TUJ1, Biolegend, 801213; NDRG1, Proteintech, 26902-4-1g; SFPQ, Sigma, SAB4200501; SOX2, Abeam, ab92494; GAPDH, Proteintech, 10494-1-AP).

[0193] B. Soak the NC membrane completely in the primary antibody solution and incubate overnight at 4°C with shaking at 30-40 rpm.

[0194] C. Recover the primary antibody solution and wash the NC membrane twice with 1 x TBST solution for 5 minutes each at 50-60 rpm.

[0195] 8) Secondary antibody binding:

[0196] A. Prepare the appropriate volume of secondary antibody solution (Anti-rabbit IgG-HRP-linked Antibody, CST, 7074; Anti-mouse IgG-HRP-linked Antibody, CST, 7076) according to the antibody instructions.

[0197] B. Soak the NC membrane completely in the secondary antibody solution and incubate for 1 hour at room temperature with shaking at 30-40 rpm.

[0198] C. Discard the secondary antibody solution and wash the NC membrane three times with 1 x TBST for 5 minutes each at 50-60 rpm.

[0199] 9) Development:

[0200] A. Prepare the necessary tools such as disposable gloves, absorbent paper, a marker, luminescent solution, a pipette, tweezers, etc.

[0201] B. Prepare the luminescent solution (A and B in a 1:1 ratio) (Nanjing Sunny Biotech) and use it immediately after preparation.

[0202] C. Use tweezers to remove the membrane, gently drain the TBST on the membrane, place it on the disposable gloves, evenly add the luminescent solution, and wait for 30 seconds to 1 minute before taking the chemiluminescence photo.

[0203] According to the method described in Example 1 (1.1 and 1.2), SK-N-BE(2) and CHP-134 control group (shC) and SFPQ knockdown group (shSFPQ-1 or shSFPQ-2) cell lines were constructed. It was observed under white light that SFPQ loss led to significant changes in cell morphology, specifically an increase in cell volume accompanied by elongation of neural-like processes, which are typical characteristics of NB differentiation. Figure 2 a).

[0204] According to Example 2 (1.1), immunofluorescence staining was performed using TUJ1 antibody, and quantitative analysis of neural-like process length was performed, finding that SFPQ knockdown significantly promoted NB differentiation (Fig. 2b and c).

[0205] Real-time quantitative PCR and Western blotting experiments were performed according to Example 2 (1.2 and 1.3), and it was found that SFPQ deletion led to significant down-regulation of the stemness marker SOX2 expression, while the expression of the differentiation markers NDRG1 and TUJ1 was significantly up-regulated (Fig. 2d and e), suggesting that SFPQ deletion promotes neuroblastoma differentiation.

[0206] Example 3 Targeting SFPQ adeno-associated virus plasmid construction and virus packaging

[0207] 1.1, Adeno-associated virus vector construction:

[0208] The sgRNA targeting SFPQ was designed by CRISPR RGEN Tools (http: / / www.rgenome.net / ) and synthesized by Jinsheng Biotechnology Co., Ltd. and cloned into the empty plasmid pAAV-CMV-SauriCas9.

[0209] The sgRNA primer sequences are as follows:

[0210] SFPQ sgRNA-FWD: 5'-CGTACTCAAACGTGCCATG-3' (SEQ ID NO. 5);

[0211] SFPQ sgRNA-REV: 5'-CATGGCACGTTTGAGTACG-3' (SEQ ID NO. 6).

[0212] 1.2, Adeno-associated virus packaging:

[0213] A. Preparation of reagents:

[0214] PEI MAX transfection reagent: Transfection reagent PEI MAX (24765, Polysciences) was prepared into a 1 mg / mL stock solution with sterile deionized water and aliquoted and stored in a -20°C refrigerator.

[0215] B. The day before packaging the virus, digest and subculture the well-grown HEK293 cells, replate to 500 mL culture bottles containing 10% FBS in DMEM solution, so that the cell density reaches 70%-80%, and is in the logarithmic growth phase.

[0216] C. The next day, transfection reagent PEI MAX was prepared into a 1 mg / mL stock solution with sterile deionized water and aliquoted and stored in a -20°C refrigerator.

[0217] D. Add total amount of 2 mg plasmid according to pAAV-CMV-SauriCas9 (Addgene, 135964): pRC2-mi342: pHelper (packaging plasmid pRC2-mi342 and pHelper, TAKARA, 6234) = 1:1:1 molar ratio into 176 mL Opti-MEM (ThermoFisher, 31985070) solution.

[0218] E. Add 6 mL PEI MAX transfection reagent into the plasmid mixture in step D, shake vigorously for 30 seconds, and let stand at room temperature for 15 minutes.

[0219] F. Remove the HEK293 cells from the incubator and remove the culture supernatant, then add a DMEM solution containing 2% FBS to the cells.

[0220] G. Gently add the plasmid / PEI mixture solution after standing to the culture bottle, mix gently, and then return the cells to the incubator for further culture for 72 hours.

[0221] 1.3, Adeno-associated virus extraction, concentration and purification:

[0222] Follow the TAKARA instructions (TAKARA, 6675):

[0223] 1) Adeno-associated virus extraction:

[0224] A. Add 0.5 M EDTA (pH 8.0) (Thermo Fisher) to the AAV infected cells at a volume of 1 / 80 of the culture solution, mix well, and let stand at room temperature for 10 minutes.

[0225] B. Peel off the cells and transfer them to a sterile centrifuge tube, centrifuge at 1,750 x g at 4°C for 10 minutes, and discard the supernatant.

[0226] C. Tap the tube wall or shake gently to loosen the cell pellet. Make sure there are no clumps.

[0227] D. Add 25 mL AAV Extraction Solution A, shake for 15 seconds to suspend the cells, let stand at room temperature for 5 minutes, and vortex again for 15 seconds. Then centrifuge at 14,000 x g at 4°C for 10 minutes.

[0228] E. Collect the supernatant into a new sterile tube and add 2.5 mL AAV Extraction Solution B. 2) Adeno-associated virus concentration

[0229] A. Add 1 / 100 volume of Cryonase Cold-active Nuclease to the AAV solution and incubate at 37°C for 1 hour.

[0230] B. Add 1 / 10 volume of Precipitator A, shake for 10 seconds, incubate at 37°C for 30 minutes, and shake for another 10 seconds.

[0231] C. Add 1 / 20 volume of Precipitator B, shake rapidly for 10 seconds, centrifuge at 5,000-9,000 x g at 4°C for 5 minutes.

[0232] D. Collect the supernatant and filter through a Millex-HV 0.45 μm filter.

[0233] E. Add the filtrate to an Amicon Ultra-4 (100 kDa), centrifuge at 2,000 x g at 15°C for 5 minutes, and confirm that the volume is <0.4 ml. Repeat the centrifugation if the volume is >0.4 ml.

[0234] F. Remove the filtrate, add 1 mL of Suspension Buffer, mix by pipetting, centrifuge at 2,000 x g at 15°C for 5 minutes, and repeat the centrifugation if the volume is >1.5 mL.

[0235] G. Remove the filtrate, mix by pipetting or shaking for 30 seconds, transfer the AAV solution to a sterile centrifuge tube, aliquot, and store at -80°C.

[0236] Example 4 Adeno-Associated Virus (AAV) Treatment

[0237] The adeno-associated virus (AAV)-mediated CRISPR / SauriCas9 system prepared by the method of Example 3 was used to knock down SFPQ in vivo by intratumoral injection.

[0238] Sixteen NSG female mice (Jiangsu Jizhuangkang Biotechnology Co., Ltd.) were used, and the mice were randomly divided into two groups. Each mouse was subcutaneously inoculated with 5 x 10 6 On the 7th day after tumor implantation, the mice were randomly divided into groups and were given intratumoral injection of AAV virus targeting SFPQ (AAV-sgSFPQ) or control AAV virus (AAV-sgC), respectively.

[0239] During the feeding of the mice, the tumor volume was measured every two days, and the state of the mice was observed. When the tumor volume of the control mice reached 1000 mm 3At the end of the experiment, mice were sacrificed and the subcutaneous tumor tissues of the control group (AAV-sgC) and the SFPQ knockdown group (AAV-sgSFPQ) were photographed and weighed. The experimental results showed that the inhibition of SFPQ could significantly inhibit tumor growth Figure 3 a and b). The above results suggest that targeted inhibition of SFPQ expression can provide a new idea for the treatment of high-risk NB patients.

Claims

1. A guide RNA, characterized in that: The guide RNA targets SFPQ, and the primer sequence of the guide RNA is shown below: SFPQ sgRNA-FWD: 5'-CGTACTCAAACGTGCCATG-3'; SFPQ sgRNA-REV: 5'-CATGGCACGTTTGAGTACG-3'.

2. An adeno-associated virus targeting SFPQ, characterized in that: It contains the guide RNA as described in claim 1.

3. Use of the adeno-associated virus targeting SFPQ as described in claim 2 in the preparation of a medicament for treating neuroblastoma.

4. A CRISPR-SauriCas9 system, characterized in that: The CRISPR-SauriCas9 system comprises the SauriCas9 protein and the guide RNA as described in claim 1; the guide RNA is capable of binding to the SauriCas9 protein to form a complex and guiding the complex to contact the SFPQ genomic DNA.

Images