An Epstein-Barr virus lytic cycle inducing construct based on BRLF1 gene, its preparation method and application

Abstract

The application discloses an Epstein-Barr virus lytic cycle induction construct based on a BRLF1 gene, a preparation method and application thereof, relates to the technical field of biological medicines, and comprises a humanized codon-optimized BRLF1 coding sequence; compared with a wild-type BRLF1 coding sequence, the codon adaptation index of the optimized BRLF1 coding sequence is increased to above 0.90, and the GC content is adjusted to 50%-55%; the Epstein-Barr virus lytic cycle induction construct based on the BRLF1 gene, the preparation method and application thereof are characterized in that the BRLF1 gene is subjected to humanized codon optimization, an enhanced nuclear localization signal is fused, and a Tet-On inducible expression system is introduced, so that an Epstein-Barr virus lytic cycle induction construct with high expression efficiency, strong nuclear localization ability and precise spatiotemporal regulation of expression is constructed, and the problems of low expression efficiency, insufficient nuclear transport and lack of a safe regulation mechanism in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an EB virus lysis cycle inducible construct based on the BRLF1 gene, its preparation method, and its application. Background Technology

[0002] Epstein-Barr virus (EBV) is closely associated with various human malignancies, including nasopharyngeal carcinoma, EBV-associated gastric cancer, and Burkitt lymphoma. In tumor cells, EBV mainly exists in a latent infection form. The viral genome exists in the cell nucleus as a circular episome outside the host chromosome, expressing only a small number of latent proteins. These proteins have immune evasion capabilities, making it difficult for the immune system to effectively recognize and eliminate infected cells.

[0003] The Rta protein encoded by the BRLF1 gene has a transactivation function, which can induce EBV to enter the lysis cycle from its latent phase, ultimately leading to tumor cell lysis and death. This strategy, known as "lytic induction therapy," provides a new approach for the treatment of EBV-positive tumors. However, in existing BRLF1 gene therapy strategies, the expression efficiency of the wild-type BRLF1 gene in human cells is low, and there is a lack of precise regulatory mechanisms, resulting in limited therapeutic efficacy and potential safety risks.

[0004] Specifically, the wild-type BRLF1 gene originates from the Epstein-Barr virus (EBV) genome, and its codon usage bias differs significantly from that of human cells. It has a low codon fitness index (CAI) and contains a large number of rare human codons, resulting in low translation efficiency and insufficient protein expression in human cells, making it difficult to effectively activate downstream cleavage genes. Furthermore, the Rta protein requires entry into the cell nucleus to exert its transactivation function, but the nuclear localization efficiency of the wild-type Rta protein is limited, further weakening its biological activity. In addition, most existing gene therapy constructs use constitutive promoters to drive BRLF1 expression, failing to achieve tissue-specific regulation of expression and posing a risk of ectopic expression in non-target tissues.

[0005] Therefore, we propose an EB virus lysis cycle inducible construct based on the BRLF1 gene, its preparation method and application, which can be efficiently expressed in human cells, significantly enhance nuclear localization efficiency and controllable expression. Summary of the Invention

[0006] The purpose of this invention is to provide an EB virus lysis cycle inducible construct based on the BRLF1 gene, its preparation method and application, so as to solve the problem of the lack of EB virus lysis cycle inducible constructs with high expression efficiency and good safety in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: an EB virus lysis cycle inducible construct based on the BRLF1 gene, comprising a BRLF1 coding sequence optimized with humanized codons, the optimized BRLF1 coding sequence being shown in SEQ ID NO:2.

[0008] Furthermore, compared with the wild-type BRLF1 coding sequence, the optimized BRLF1 coding sequence has a codon fitness index of over 0.90 and a GC content of 50%-55%.

[0009] Furthermore, the 5' end of the BRLF1 coding sequence is also fused with a nucleotide sequence encoding a nuclear localization signal, the amino acid sequence of which is shown in SEQ ID NO:3.

[0010] Furthermore, it also includes a Tet-On inducible expression system, wherein the BRLF1 coding sequence is placed under the control of the TRE promoter, and the construct simultaneously expresses the rtTA protein.

[0011] A method for preparing an EBV lysis cycle-inducing construct based on the BRLF1 gene includes the following steps:

[0012] S1. Humanized codons are optimized into the wild-type BRLF1 coding sequence to obtain the optimized sequence shown in SEQ ID NO:2;

[0013] S2. Synthesize or amplify the optimized sequence;

[0014] S3. The optimized sequence is cloned into a eukaryotic expression vector;

[0015] S4. Fuse a nucleotide sequence encoding the nuclear localization signal as shown in SEQ ID NO:3 to the 5' end of the optimized sequence;

[0016] S5. Introduce the Tet-On inducible expression system, place the BRLF1 coding sequence under the control of the TRE promoter, and express the rtTA protein on the same vector.

[0017] A pharmaceutical composition comprising:

[0018] (a) Construct;

[0019] (b) A delivery carrier, wherein the delivery carrier is a thermosensitive hydrogel matrix composed of chitosan and sodium β-glycerophosphate.

[0020] Application of an EBV lysis cycle inducing construct based on the BRLF1 gene in the preparation of drugs for treating EBV-positive tumors.

[0021] The use of a pharmaceutical composition in the preparation of a drug for treating EBV-positive tumors.

[0022] Furthermore, the EBV-positive tumors include nasopharyngeal carcinoma, EBV-positive gastric cancer, and Burkitt lymphoma.

[0023] Furthermore, the pharmaceutical composition is administered via intratumoral injection, and a single dose can induce sustained expression of the BRLF1 gene.

[0024] Compared with existing technologies, the EB virus lysis cycle inducible construct based on the BRLF1 gene, its preparation method, and its application provided by this invention have the following beneficial effects:

[0025] 1. This invention constructs an EB virus lysis cycle inducible construct with high expression efficiency, strong nuclear localization ability and precise expression temporal and spatial control by optimizing humanized codons of the BRLF1 gene, fusing enhanced nuclear localization signals and introducing the Tet-On inducible expression system. This solves the problems of low expression efficiency, insufficient nuclear transport and lack of safe regulatory mechanism in the prior art.

[0026] 2. This invention combines the construct with a chitosan / β-glycerophosphate sodium thermosensitive hydrogel. This composition has thermosensitive gelation and pH-responsive release properties, and can achieve continuous release of the BRLF1 gene through a single intratumoral injection, which significantly simplifies the administration regimen and improves patient compliance and treatment convenience.

[0027] 3. The constructs and pharmaceutical compositions of this invention have shown significant EBV-dependent antitumor effects in both in vitro cell experiments and in vivo animal models. They can effectively activate EBV from the latent phase into the lysis cycle, induce tumor cell-specific apoptosis, have a high tumor inhibition rate against EBV-positive nasopharyngeal carcinoma, and have no obvious toxic effects on normal cells and major organs, demonstrating good therapeutic specificity and in vivo safety. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below.

[0029] Example 1:

[0030] The BRLF1 coding sequence optimized with humanized codons was obtained and verified as follows:

[0031] 1. Wild-type BRLF1 sequence analysis

[0032] Referring to the BRLF1 gene sequence of the EBV standard strain B95-8 (GenBank accession number: V01555.2, interval 103789-105006), the sequence is 1218 bp in length, encoding a 405-amino acid Rta protein. Analysis showed that the wild-type BRLF1 coding sequence has a codon adaptation index (CAI) of 0.72 and a GC content of 58%, containing several rare human codons, such as the CGG and AGG codons for arginine, suggesting low expression efficiency in human cells.

[0033] 2. Humanized Codon Optimization Design

[0034] Humanized codons were optimized for the wild-type BRLF1 coding sequence using GeneOptimizer software (Thermo Fisher Scientific). Optimization strategies included:

[0035] (1) Replace rare codons with human preferred codons, referring to the human high-frequency codon usage table (KazusaDNA Research Institute).

[0036] (2) Adjust the GC content to 52% to make it closer to the average level of human genes;

[0037] (3) Eliminate potential elements that may interfere with transcription, such as 5' and 3' UTR splicing sites, internal TATA boxes, Chi sites, and ribosome entry sites;

[0038] (4) Optimize the secondary structure of mRNA to avoid the formation of stable hairpin structures that may affect translation initiation;

[0039] (5) Increase the CAI value of the sequence without changing the amino acid sequence.

[0040] The optimized BRLF1 coding sequence was synthesized in its entirety by a biotechnology company, cloned into the pUC57 vector, and verified to be correct by sequencing. The optimized BRLF1 coding sequence is shown in SEQ ID NO:2, with a CAI value increased to 0.94 and a GC content of 52%.

[0041] 3. Construction of expression carrier

[0042] The wild-type BRLF1 coding sequence (PCR amplified from the genome of EBV-positive cells B95-8) and the optimized BRLF1 coding sequence (SEQ ID NO:2) were cloned into the Hind III and EcoRI sites of the pcDNA3.1(+) eukaryotic expression vector (Invitrogen), respectively, to construct pcDNA3.1-wtBRLF1 and pcDNA3.1-optBRLF1. After verification of the plasmids by enzyme digestion and sequencing, the plasmids were extracted using an endotoxin-free large-scale extraction kit (Qiagen) for later use.

[0043] 4. Cell transfection and expression detection

[0044] EBV-positive nasopharyngeal carcinoma cell line C666-1 was seeded in 6-well plates (2×10⁻⁶). 5 Cells were cultured overnight in wells. pcDNA3.1-wtBRLF1 and pcDNA3.1-optBRLF1 were transfected using Lipofectamine 3000 transfection reagent (Invitrogen), with 2 μg of plasmid per well. Forty-eight hours after transfection, cells were collected, total protein was extracted, and protein concentration was determined using the BCA method. 20 μg of total protein was subjected to SDS-PAGE electrophoresis. After transfer, the cells were incubated with anti-Rta mouse monoclonal antibody (Argene, clone number 5A11) and anti-GAPDH rabbit polyclonal antibody (Cell Signaling Technology), respectively, and detected by ECL chemiluminescence. ImageJ software was used for quantitative analysis of band gray values, and the relative expression level of Rta protein was calculated using GAPDH as an internal control.

[0045] 5. Results

[0046] The expression level of Rta protein in the pcDNA3.1-optBRLF1 transfection group was 4.5 ± 0.3 times that in the pcDNA3.1-wtBRLF1 group (n=3, p<0.001). In summary, using the humanized codon-optimized BRLF1 coding sequence shown in SEQ ID NO:2 of this invention, its CAI value was increased to 0.94 and GC content was adjusted to 52%, resulting in significantly improved expression efficiency in human cells compared to wild-type BRLF1, thus laying a solid foundation for effective EBV cleavage induction.

[0047] Example 2:

[0048] Construction and verification of the BRLF1 construct incorporating nuclear localization signals:

[0049] 1. Design of nuclear localization signal sequence

[0050] Based on the classic sequence of the SV40 large T antigen nuclear localization signal (PKKKRKV), an enhanced nuclear localization signal (PKKKRKVEAAA) was designed, with its amino acid sequence shown in SEQ ID NO:3. The corresponding nucleotide sequence is: 5'-CCGAAGAAAAAGCGCAAGGTGGAAGCGGCGGCT-3', with restriction enzyme sites introduced at both ends (5' Hind III and 3' flexible linker peptide coding sequences).

[0051] 2. Merging Builder Construction

[0052] The NLS sequence was fused to the 5' end of the optimized BRLF1 coding sequence using overlap extension PCR. In the first round of PCR, the NLS fragment (using synthetic primers as templates) and the optBRLF1 fragment (using pUC57-optBRLF1 from Example 1) were amplified separately. In the second round of PCR, equal amounts of the two fragments were used as templates, and fusion PCR was performed using outer primers. The fusion product was digested with Hind III and EcoRI and cloned into the pcDNA3.1(+) vector to construct pcDNA3.1-NLS-optBRLF1. Sequencing verified the correct fusion sequence.

[0053] 3. Immunofluorescence detection of nuclear localization

[0054] C666-1 cells were seeded in confocal culture dishes (1×10⁻⁶). 5 Cells were cultured overnight in dishes. pcDNA3.1-optBRLF1 (without NLS) and pcDNA3.1-NLS-optBRLF1 were transfected, with 1 μg of plasmid per dish. 48 h after transfection, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. Anti-Rta mouse monoclonal antibody (1:500 dilution) was added, and the cells were incubated overnight at 4°C. After washing, Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution) was added, and the cells were incubated at room temperature in the dark for 1 h. DAPI staining was performed for 5 min. Images were observed and acquired using a Leica TCS SP8 confocal microscope. The fluorescence intensity in the nucleus and cytoplasm was analyzed using ImageJ software, and the nucleus / cytoplasm ratio was calculated.

[0055] 4. Results

[0056] In the pcDNA3.1-optBRLF1 group (without NLS), Rta protein was distributed in both the cytoplasm and nucleus, with a nucleus / cytoplasm ratio of 1.2 ± 0.2; in the pcDNA3.1-NLS-optBRLF1 group, Rta protein was mainly concentrated in the nucleus, with a nucleus / cytoplasm ratio of 4.8 ± 0.4 (n = 20 cells, p < 0.001). In summary, fusing the nuclear localization signal shown in SEQ ID NO:3 to the 5' end of the BRLF1 coding sequence can increase the nuclear transport efficiency of Rta protein by approximately 4-fold, thereby more effectively exerting its transactivation function in the nucleus.

[0057] Example 3:

[0058] Construction and validation of the Tet-On inducible expression system construct:

[0059] 1. Construction of inducible expression vectors

[0060] Using the pTRE-Tight vector (Clontech) as a backbone, a dual-gene vector simultaneously expressing BRLF1 and rtTA was constructed. Specific steps:

[0061] (1) The NLS-optBRLF1 fusion sequence of Example 2 was cloned into the downstream of the TRE promoter of the pTRE-Tight vector through EcoRI and SalI sites to construct pTRE-NLS-optBRLF1;

[0062] (2) Using the pTet-On vector (Clontech) as a template, the rtTA coding sequence was amplified by PCR and cloned into another expression cassette of the same vector through the BamHI and XhoI sites (driven by the CMV promoter) to construct pTRE-NLS-optBRLF1-CMV-rtTA (abbreviated as "pBRLF1-Tet").

[0063] 2. Validation of induced expression

[0064] C666-1 cells were seeded in 24-well plates (5 × 10⁻⁶ cells per well). 4 Cells were cultured overnight in wells. The pBRLF1-Tet plasmid was transfected using Lipofectamine 3000, 0.5 μg per well. 24 h after transfection, different concentrations of doxycycline (0, 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 μg / mL) were added. Cells were cultured for another 24 h, then collected, and Rta protein expression was detected by Western blotting (method as in Example 1).

[0065] 3. Results

[0066] Without doxycycline, the Rta protein was almost undetectable; with increasing doxycycline concentration, Rta expression increased in a dose-dependent manner, reaching a plateau at 1.0 μg / mL. This embodiment demonstrates that the construct of the present invention, which includes a Tet-On inducible expression system, places the BRLF1 coding sequence under the control of the TRE promoter and simultaneously expresses the rtTA protein, enabling precise regulation of BRLF1 expression and significantly improving the safety of gene therapy.

[0067] Example 4:

[0068] Preparation of EBV lysis cycle inducible constructs based on the BRLF1 gene:

[0069] S1. Humanize the wild-type BRLF1 coding sequence to obtain the optimized sequence as shown in SEQ ID NO:2; the specific operation is the same as in Example 1.

[0070] S2. Synthesize or amplify the optimized sequence; commission a biotechnology company to synthesize the whole genome of the optimized sequence shown in SEQ ID NO:2, or perform PCR amplification using the synthesized sequence as a template;

[0071] S3. The optimized sequence was cloned into a eukaryotic expression vector. The synthesized optimized sequence was digested with Hind III and EcoRI, ligated into a pcDNA3.1(+) eukaryotic expression vector digested with the same enzymes, transformed into DH5α competent cells, screened for positive clones, and the plasmid was extracted for enzyme digestion and sequencing verification.

[0072] S4. A nucleotide sequence encoding the nuclear localization signal as shown in SEQ ID NO:3 is fused to the 5' end of the optimized sequence; according to the method of Example 2, the nucleotide sequence encoding the nuclear localization signal as shown in SEQ ID NO:3 is fused to the 5' end of the optimized sequence using the overlap extension PCR method to obtain the NLS-optBRLF1 fusion sequence.

[0073] S5. Introduce the Tet-On inducible expression system, place the BRLF1 coding sequence under the control of the TRE promoter, and express the rtTA protein on the same vector to construct the final inducible expression plasmid pBRLF1-Tet. Specifically, following the method in Example 3, firstly, the NLS-optBRLF1 fusion sequence is cloned downstream of the TRE promoter in the pTRE-Tight vector to form a TRE-regulated expression cassette; subsequently, the rtTA expression cassette containing the CMV promoter and the rtTA coding sequence is cloned into another suitable site on the vector. The resulting pBRLF1-Tet plasmid is a bicistronic vector, and its core structure from one end to the other is as follows: TRE promoter - NLS-optBRLF1 fusion sequence - polyA signal, and CMV promoter - rtTA coding sequence - polyA signal. This plasmid constitutes the EB virus lysis cycle inducible construct based on the BRLF1 gene described in this invention.

[0074] Example 5:

[0075] Preparation and characterization of pharmaceutical compositions containing the construct:

[0076] 1. Reagents and Materials

[0077] Chitosan (degree of deacetylation 95%, molecular weight 100 kDa): Zhejiang Jinke Pharmaceutical Co., Ltd.

[0078] Sodium β-glycerophosphate (β-GP): Sigma-Aldrich;

[0079] Hyaluronic acid (molecular weight 100 kDa): Shandong Freda Biomedical Co., Ltd.

[0080] pBRLF1-Tet plasmid: prepared according to the method provided in Example 3.

[0081] 2. Solution preparation

[0082] (1) Chitosan solution: Dissolve chitosan in 0.1M acetic acid solution to prepare a 2.5% (w / v) chitosan solution, stir overnight at 4℃ until completely dissolved, and adjust the pH to 6.2 with 1M NaOH;

[0083] (2) β-GP solution: Dissolve sodium β-glycerophosphate in deionized water to prepare a 20% (w / v) β-GP solution, and filter it through a 0.22 μm filter membrane for sterilization;

[0084] (3) Hyaluronic acid solution: Dissolve hyaluronic acid in deionized water to prepare a 1% (w / v) hyaluronic acid solution;

[0085] (4) Plasmid solution: Dissolve pBRLF1-Tet plasmid in sterile TE buffer at a concentration of 5 mg / mL.

[0086] 3. Preparation of pharmaceutical compositions

[0087] Mix the components in the following proportions under ice bath conditions:

[0088] Component (a): 0.5 mL of a therapeutically effective amount of an EB virus lysis cycle inducible construct based on the BRLF1 gene, wherein the construct is provided in plasmid form and contains 2.5 mg of pBRLF1-Tet plasmid;

[0089] Component (b): Pharmaceutically acceptable delivery carrier;

[0090] Chitosan solution: 4.0 mL, hyaluronic acid solution: 0.5 mL, sterile water: 3.0 mL, β-GP solution: 2.0 mL. Mix well and adjust the pH to 7.2 to obtain the thermosensitive hydrogel formulation loaded with pBRLF1-Tet plasmid. Aliquot into 2 mL vials, 1 mL per vial, and store at 4°C for later use.

[0091] 4. Gelation performance testing

[0092] Add 2 mL of the gel precursor solution to a test tube and place it in a constant temperature water bath. Gradually increase the temperature from 25℃, equilibrating for 5 minutes at 0.5℃ increments. The gelation state is determined using the inverted test tube method—no liquid flow within 30 seconds of inversion is considered gelled. Simultaneously, the gelation time is measured in a 37℃ constant temperature water bath. Results: The gelation temperature of this formulation is 36.5±0.3℃, and the gelation time at 37℃ is 8.5±1.2 min, meeting the requirements for in vivo injection. This confirms that the thermosensitive hydrogel matrix composed of chitosan and β-glycerophosphate has good thermosensitive properties.

[0093] 5. In vitro release study

[0094] Add 1 mL of gel precursor solution to a 2 mL vial and incubate at 37°C for 30 min until complete gelation. Add 2 mL of PBS release medium (pH 7.4 or pH 6.5) and shake at 37°C (50 rpm). Take 0.5 mL samples at predetermined time points (1 h, 4 h, 8 h, 1 d, 2 d, 3 d, 5 d, 7 d, 10 d, 14 d), and simultaneously add an equal volume of fresh medium. Determine the plasmid DNA content in the release solution using the PicoGreen fluorescent dye method (Invitrogen).

[0095] Results: At pH 7.4, the cumulative release rate after 14 days was 68.5±4.2%; at pH 6.5, the cumulative release rate after 14 days was 84.6±3.8%, which was significantly higher than that of the pH 7.4 group (p<0.05), indicating that the hydrogel has pH-responsive release characteristics, which is beneficial for accelerating drug release in the acidic tumor microenvironment; the released plasmid was detected by agarose gel electrophoresis and its conformation was intact, indicating that the release process did not destroy the plasmid structure.

[0096] Example 6:

[0097] Evaluation of in vitro antitumor effects:

[0098] 1. Cell lines and culture

[0099] EBV-positive nasopharyngeal carcinoma cell line C666-1 and EBV-negative nasopharyngeal carcinoma cell line CNE-1 were used as controls;

[0100] Culture conditions: RPMI-1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 incubator.

[0101] 2. Experimental Grouping

[0102] Group 1: Blank control group (PBS); Group 2: Empty vector hydrogel group (hydrogel without plasmid); Group 3: Wild-type BRLF1 plasmid group (pcDNA3.1-wtBRLF1, liposome transfection); Group 4: Optimized BRLF1 plasmid group (pcDNA3.1-optBRLF1, liposome transfection); Group 5: pBRLF1-Tet+DOX group (liposome transfection); Group 6: pBRLF1-Tet+DOX+GCV group (liposome transfection); Group 7: pBRLF1-Tet hydrogel + DOX + GCV group (the drug composition provided in Example 5).

[0103] 3. Handling methods

[0104] C666-1 cells were seeded in 24-well plates (5 × 10⁻⁶ cells per well). 4Cells / well), cultured overnight. For the hydrogel group (Group 7: pBRLF1-Tet hydrogel + DOX + GCV), the gel precursor solution prepared in Example 5 was added to Transwell chambers (0.4 μm pore size), 100 μL per chamber, and incubated at 37°C for 30 min to allow gelation. The chambers were then placed in culture plates for co-culture with cells. For the liposome transfection groups (Groups 3-6: pcDNA3.1-wtBRLF1, pcDNA3.1-optBRLF1, pBRLF1-Tet+DOX, pBRLF1-Tet+DOX+GCV), transfection was performed at 1 μg plasmid and 2 μL Lipofectamine 3000 per well.

[0105] After 24 hours of treatment, groups 5-7 were treated with doxycycline (final concentration 1 μg / mL) to induce BRLF1 expression. After another 24 hours of culture, groups 6-7 were treated with ganciclovir (GCV, final concentration 50 μM). After another 48 hours of culture, cells were collected for the following assays:

[0106] (1) Western Blot detection: The method is the same as in Example 1, and the expression of Rta protein and EBV lysis phase protein BMRF1 (anti-BMRF1 antibody, Millipore, clone number 5B11) is detected.

[0107] (2) Cell viability assay: Add 10 μL of CCK-8 reagent to each well, incubate at 37℃ for 2 h, and measure the absorbance at 450 nm. Cell viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%;

[0108] (3) Apoptosis detection: Annexin V-FITC / PI apoptosis detection kit (BD Biosciences), flow cytometry detection.

[0109] 4. Results

[0110] (1) Protein expression: The expression level of Rta protein in group 7 (pBRLF1-Tet hydrogel + DOX + GCV) was the highest, which was 2.1 times that of group 6 (pBRLF1-Tet + DOX + GCV) and 3.2 times that of group 5 (pBRLF1-Tet + DOX) (grayscale scanning quantification); the expression level of BMRF1 protein in EBV lysis phase in group 7 (pBRLF1-Tet hydrogel + DOX + GCV) was also the highest, indicating that BRLF1 successfully activated EBV to enter the lysis cycle.

[0111] (2) Cell viability:

[0112] Table 1

[0113] Group C666-1 cell survival rate (%) CNE-1 cell survival rate (%) Blank control 100.0±5.2 100.0±4.8 Empty hydrogel 96.8±4.7 97.2±5.1 wtBRLF1 72.1±6.1 94.5±4.3 optBRLF1 56.7±5.3 93.8±5.0 pBRLF1-Tet+DOX 51.2±4.8 92.1±4.6 pBRLF1-Tet+DOX+GCV 42.1±4.2 91.5±4.9 pBRLF1-Tet hydrogel + DOX + GCV 28.3±3.5 90.2±5.3

[0114] As shown in Table 1, the cell survival rate of group 7 (pBRLF1-Tet hydrogel + DOX + GCV) was significantly lower than that of the other groups; compared with group 6 (pBRLF1-Tet + DOX + GCV), p < 0.01; compared with group 5 (pBRLF1-Tet + DOX), p < 0.001. In EBV-negative CNE-1 cells, the cell survival rate of each group was >90%, indicating that the killing effect was EBV-dependent, confirming that the constructs and pharmaceutical compositions of this invention can be used to prepare drugs for treating EBV-positive tumors.

[0115] Example 7:

[0116] Evaluation of in vivo anti-tumor effects:

[0117] 1. Establishment of animal models

[0118] BALB / c-nu nude mice, female, 4-6 weeks old, weighing 18-22g, were subcutaneously inoculated with C666-1 cells (1×10⁻⁶) under the right axilla. 7 A nasopharyngeal carcinoma xenograft model was established by suspending cells / cell in 0.1 mL PBS. The xenograft was established when the tumor volume reached 100-150 mm. 3 The experiment will begin at that time.

[0119] 2. Experimental grouping and dosing regimen

[0120] Tumor-bearing mice were randomly divided into 5 groups of 10 mice each;

[0121] PBS control group: 100 μL of PBS was injected intratumorally twice a week for 3 weeks;

[0122] Empty hydrogel group: 100 μL of blank hydrogel without plasmids was injected into the tumor in a single injection.

[0123] Wild-type BRLF1 plasmid group: intratumoral injection of pcDNA3.1-wtBRLF1 plasmid (50 μg / time, dissolved in 100 μL PBS), twice a week for 3 weeks;

[0124] pBRLF1-Tet liposome group: pBRLF1-Tet plasmid (50 μg) was mixed with Lipofectamine 3000 and injected intratumorally twice a week for 3 weeks; at the same time, doxycycline (50 mg / kg once a day) and GCV (50 mg / kg once a day) were injected intraperitoneally.

[0125] pBRLF1-Tet hydrogel group (the present invention group): 100 μL of pBRLF1-Tet plasmid-loaded hydrogel (containing 50 μg plasmid) prepared in Example 5 was injected intratumorally; at the same time, doxycycline (50 mg / kg, once daily) and GCV (50 mg / kg, once daily) were injected intraperitoneally.

[0126] 3. Observation indicators

[0127] Tumor volume: The long axis (a) and short axis (b) of the tumor were measured with calipers every 3 days, and the tumor volume V = a × b was calculated. 2 / 2, plot the tumor growth curve;

[0128] Mouse body weight: Weighed every 3 days to assess systemic toxicity;

[0129] Tumor inhibition rate: Mice were sacrificed on day 21, tumors were removed and weighed, and the tumor inhibition rate was calculated as (1 - average tumor weight of experimental group / average tumor weight of control group) × 100%;

[0130] Histological examination: Tumor tissue was stained with hematoxylin and eosin (HE), and immunohistochemical staining was used to detect the expression of Rta, BMRF1, and Ki-67. TUNEL staining was used to detect cell apoptosis.

[0131] Safety evaluation: HE staining of major organs (heart, liver, spleen, lungs, and kidneys) was performed to observe pathological changes.

[0132] 4. Results

[0133] (1) Tumor growth curve

[0134] Table 2

[0135] Group <![CDATA[Average tumor volume on day 21 (mm 3 )]]> Tumor inhibition rate (%) PBS 1245.6±156.3 - Empty hydrogel 1186.3±142.8 4.8 wtBRLF1 612.5±72.1 48.5 Liposomes 412.7±58.3 62.3 pBRLF1-Tet hydrogel 235.4±42.6 78.5

[0136] As shown in Table 2, within 21 days after a single injection of the pBRLF1-Tet hydrogel, tumor growth was significantly inhibited, with a tumor inhibition rate of 78.5%, which was significantly better than the wild-type BRLF1 group (48.5%) and the liposome multiple injection group (62.3%) (p<0.05 compared with the liposome group; p<0.01 compared with the wtBRLF1 group). This example demonstrates that the use of the drug composition for intratumoral injection can achieve sustained expression of the BRLF1 gene and significant antitumor effects with a single administration.

[0137] (2) Immunohistochemical staining

[0138] The pBRLF1-Tet hydrogel group showed the highest rates of Rta and BMRF1 positive cells in tumor tissue, indicating that BRLF1 was successfully expressed and effectively activated EBV lysis and replication; the Ki-67 positive rate was the lowest, indicating that tumor cell proliferation was inhibited; and the TUNEL positive rate was the highest, indicating that apoptosis was the most significant.

[0139] (3) Safety evaluation

[0140] There was no significant difference in body weight among the groups of mice (p>0.05), and no obvious pathological damage was observed in the major organs by HE staining, indicating that the construct and drug composition of the present invention have good in vivo safety.

[0141] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above description is illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An EBV lysis cycle inducing construct based on the BRLF1 gene, characterized in that, The optimized BRLF1 coding sequence includes humanized codons, as shown in SEQ ID NO:

2.

2. The EB virus lysis cycle inducing construct based on the BRLF1 gene according to claim 1, characterized in that, Compared with the wild-type BRLF1 coding sequence, the optimized BRLF1 coding sequence has a codon fitness index of over 0.90 and a GC content of 50%-55%.

3. An EB virus lysis cycle inducing construct based on the BRLF1 gene according to claim 1 or 2, characterized in that, The 5' end of the BRLF1 coding sequence is also fused with a nucleotide sequence encoding a nuclear localization signal, the amino acid sequence of which is shown in SEQ ID NO:

3.

4. The EB virus lysis cycle inducing construct based on the BRLF1 gene according to claim 3, characterized in that, It also includes a Tet-On inducible expression system, wherein the BRLF1 coding sequence is placed under the control of the TRE promoter, and the construct simultaneously expresses the rtTA protein.

5. A method for preparing the EB virus lysis cycle inducible construct based on the BRLF1 gene as described in claim 3, characterized in that, Includes the following steps: S1. Humanized codons are optimized on the wild-type BRLF1 coding sequence to obtain the optimized sequence shown in SEQ ID NO:2; S2. Synthesize or amplify the optimized sequence; S3. The optimized sequence is cloned into a eukaryotic expression vector; S4. Fuse a nucleotide sequence encoding the nuclear localization signal as shown in SEQ ID NO:3 to the 5' end of the optimized sequence; S5. Introduce the Tet-On inducible expression system, place the BRLF1 coding sequence under the control of the TRE promoter, and express the rtTA protein on the same vector.

6. A pharmaceutical composition, characterized in that, include: (a) The construct according to any one of claims 1-4; (b) A delivery carrier, wherein the delivery carrier is a thermosensitive hydrogel matrix composed of chitosan and sodium β-glycerophosphate.

7. The use of an EBV lysis cycle inducing construct based on the BRLF1 gene according to any one of claims 1-4 in the preparation of a drug for treating EBV-positive tumors.

8. The use of the pharmaceutical composition according to claim 6 in the preparation of a drug for treating EBV-positive tumors.

9. The application according to claim 7 or 8, characterized in that, The EBV-positive tumors include nasopharyngeal carcinoma, EBV-positive gastric cancer, and Burkitt lymphoma.

10. The application according to claim 8, characterized in that, The drug composition is administered via intratumoral injection, and a single dose can induce sustained expression of the BRLF1 gene.