Recombinant adeno-associated virus particle containing targeted transcriptional melittin gene as well as preparation method and application of recombinant adeno-associated virus particle

Abstract

The invention belongs to the technical field of biological medicines, and provides a recombinant adeno-associated virus particle containing a targeted transcriptional melittin gene as well as a preparation method and application of the recombinant adeno-associated virus particle. Wherein the expression cassette containing the targeted transcriptional melittin gene contains a human telomerase reverse transcriptase core promoter, a tetracycline response element, an optimized melittin gene, a ribosome insertion sequence and a human enterokinase light chain-melittin fusion gene. The invention aims to solve the problem of how to realize precise targeted therapy of tumors, effectively kill tumor cells, reduce damage to normal cells to the greatest extent, overcome the limitation of a traditional treatment method and solve the problem of packaging cell death caused by advanced transcription of melittin genes in a recombinant AAV packaging process. And it is ensured that melittin expressed in tumor cells has a natural sequence and activity, and a safer and more effective treatment choice is provided for tumor patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a recombinant adeno-associated virus particle containing a gene that targets the transcription of melitoxin, its preparation method, and its application. Background Technology

[0002] Cancer, as a disease that seriously threatens human health, has always been a key focus and challenge in medical research. Currently, common cancer treatments mainly include surgical resection, chemotherapy, radiotherapy, and immunotherapy, which has developed in recent years. However, each of these traditional treatments has significant limitations.

[0003] While surgical resection can directly remove tumor tissue, it often fails to achieve a radical cure for tumors in unusual locations that are difficult to completely remove, or for tumors that have metastasized. Furthermore, surgery is highly invasive, and the postoperative recovery process significantly impacts the patient's physical function, potentially leading to a series of complications.

[0004] Chemotherapy kills tumor cells using chemical drugs, but these drugs lack the ability to specifically recognize tumor cells. While killing tumor cells, they also severely damage normal cells, leading to a series of serious side effects such as hair loss, nausea, vomiting, and weakened immunity. Long-term chemotherapy can also cause tumor cells to develop drug resistance, reducing the effectiveness of treatment.

[0005] Radiation therapy uses high-energy rays to irradiate the tumor site, destroying the DNA of tumor cells and thus inhibiting their growth. However, in the process of killing tumor cells, radiation therapy also causes radiation damage to surrounding normal tissues, leading to adverse reactions such as radiation-induced inflammation. Furthermore, radiation therapy is not ideal for some types of tumors that are not sensitive to radiation.

[0006] While immunotherapy has brought new hope to cancer treatment, its efficacy varies from person to person, and not all patients can benefit from it. Furthermore, immunotherapy may trigger overactivation of the immune system, leading to serious adverse reactions such as autoimmune diseases.

[0007] Furthermore, most existing treatments struggle to achieve precise targeting of tumor cells, failing to effectively kill tumor cells while minimizing damage to normal tissues. Therefore, developing a safe, effective, and precise cancer treatment method is urgently needed. Finding a treatment that specifically targets tumor cells while causing minimal damage to normal cells has always been a hot topic and a challenge in the field of cancer treatment. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant adeno-associated virus (AAV) particle containing a gene for targeted transcription of melittin, its preparation method, and its application. This aims to address how to achieve precise targeted therapy for tumors, effectively killing tumor cells while minimizing damage to normal cells, overcoming the limitations of traditional treatment methods. It also solves the problem of pre-transcription of the melittin gene during the packaging process of recombinant AAV (recombinant adeno-associated virus), leading to cell death in the packaging cells, and ensures that the melittin expressed in tumor cells has its natural sequence and activity, providing a safer and more effective treatment option for cancer patients.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an expression cassette containing a gene for targeted transcription of melives venom peptides. The cassette includes a human telomerase reverse transcriptase core promoter (hTERTcp), a tetracycline response element (TRE), an optimized melives venom peptide gene (with a sequence optimized and methionine codon and the corresponding nucleic acid sequence for enterokinase recognition added sequentially to its N-terminus), a ribosomal insertion sequence (IRES), and a human enterokinase light chain-melives venom peptide fusion gene (hEK). L -hmel).

[0010] Preferably, the gene sequence of the expression cassette is hTERTcp-TRE-hmel-IRES-hEK. L -hmel, the nucleotide sequence of the expression cassette is shown in SEQ ID NO:1.

[0011] The present invention also provides the application of the expression cassette in the preparation of drugs for treating tumors.

[0012] The present invention also provides a recombinant adeno-associated virus particle containing a gene that targets the transcription of melitoxin, prepared from the expression cassette and the recombinant adeno-associated virus plasmid vector.

[0013] The present invention also provides a method for preparing the recombinant adeno-associated virus particles, comprising the following steps: (1) A DNA fragment containing the gene encoding the cytomegalovirus promoter (CMV promoter) and tetracycline repressor protein (TetR protein) was synthesized and cloned into a lentiviral vector to obtain the recombinant plasmid FUGW-CMV-TetR; (2) The recombinant plasmid FUGW-CMV-TetR, packaging plasmid and envelope plasmid were co-transfected into HEK293T cells. After 48-72 h, the supernatant was collected and concentrated to obtain concentrated lentivirus. (3) HEK293T cells were infected with concentrated lentivirus and screened with puromycin for 1-2 weeks to obtain a stable cell line HEK293T / TetR that stably expresses tetracycline repressor protein for later use. (4) Insert the expression cassette into the adeno-associated virus vector plasmid to obtain the recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel, for later use; (5) The recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel, packaging plasmid and helper plasmid were co-transfected into HEK293T / TetR cells. After culturing for 48-72 h, the cells were centrifuged, the precipitated cells were collected, and the cells were repeatedly frozen and thawed and lysed to collect the supernatant. (6) Degrade the cellular DNA and plasmid DNA in the supernatant to obtain recombinant adeno-associated virus particles.

[0014] Preferably, the cloning method in step (1) is a seamless cloning technique, and the lentivirus vector is FUGW.

[0015] Preferably, the packaging plasmid in step (2) is psPAX2, the coating plasmid is pMD2.G, and the concentration factor is 30 to 50 times.

[0016] Preferably, the concentration of puromycin in step (3) is 1~3 μg / mL.

[0017] Preferably, the packaging plasmid in step (5) is pAAV-RC8 and the auxiliary plasmid is pHelper.

[0018] The present invention also provides the application of the recombinant adeno-associated virus particles in the preparation of drugs for treating tumors.

[0019] To address the numerous problems of existing tumor treatment methods, such as lack of specificity, significant damage to normal cells, and easy development of drug resistance, this invention provides a comprehensive and precise regulation strategy for tumor treatment using recombinant AAV containing a gene that targets the transcription of melitoxin.

[0020] Traditional treatments such as chemotherapy and radiotherapy, lacking precise identification of tumor cells, inevitably damage a large number of normal cells during treatment, leading to severe toxic side effects for patients. This invention, however, utilizes recombinant AAV as a vector, combined with tumor-targeting transcriptional promoter regulation, to achieve specific expression of meliothionein within tumor cells. AAV exhibits good biocompatibility and tissue targeting; by utilizing the different tissue tropisms of different AAV serotypes, the impact on non-target tissues is reduced.

[0021] Melittin (mel) is a polypeptide with potent biological activity. Previous research on melittin has largely focused on its direct application; however, due to its lack of targeting, direct use may cause significant harm to the body. This invention innovatively utilizes the telomerase reverse transcriptase promoter to regulate the targeted transcription of the melittin gene. Telomerase is highly expressed in most tumor cells, while its activity is low or nonexistent in normal cells. Therefore, the telomerase reverse transcriptase promoter can specifically initiate the transcription of the melittin gene in tumor cells, allowing melittin to exert its effects within tumor cells and avoiding non-specific killing of normal cells.

[0022] From a mechanistic perspective, melittin expressed within tumor cells can disrupt the integrity of the tumor cell membrane, leading to leakage of cellular contents and subsequently inducing tumor cell apoptosis. Simultaneously, melittin may also activate apoptosis signaling pathways within tumor cells, inducing tumor cell death at multiple levels. Unlike traditional chemotherapy drugs, which readily induce drug resistance in tumor cells, the unique mechanism of action of melittin makes it difficult for tumor cells to develop drug resistance mechanisms, offering the possibility of long-term effective tumor treatment.

[0023] The beneficial effects of this invention are as follows: This invention modifies HEK293T cells using lentivirus to create a stable cell line expressing the tetracycline (Tet) repressor protein (TetR). Simultaneously, the Tet response element (TRE, composed of seven repeating TetO sequences) from the Tet resistance operon (TetO) is inserted between the telomerase reverse transcriptase promoter and the melittin gene. TetR binds to the TRE, inhibiting the transcription of the melittin gene, thus ensuring the normal growth and proliferation of the stable HEK293T cell line during recombinant AAV packaging. When the recombinant AAV enters tumor cells, because the tumor cells do not express TetR, the telomerase reverse transcriptase promoter can normally transcribe its downstream melittin gene, achieving targeted killing of tumor cells. This perfectly solves the problem that in the recombinant AAV packaging process, the HEK293T cells used are telomerase-positive cells, which can lead to premature transcription of the melittin gene in the packaging cells, causing HEK293T cell death and resulting in recombinant AAV packaging failure.

[0024] To ensure proper initiation of expression of the melipovetine gene after transcription and to obtain a sequence completely identical to that of natural melipovetine, this invention, when designing the gene expression cassette, sequentially added DNA sequences corresponding to the Met and enterokinase recognition sequences to the N-terminus of the melipovetine gene sequence. Simultaneously, human enterokinase light chain protein (hEK) was co-expressed using the ribosomal insertion sequence. LIn this way, when the melittin gene is transcribed and expressed in tumor cells, the human enterokinase light chain protein recognizes and cleaves the additional Met and enterokinase recognition sequences added at the N-terminus, resulting in a polypeptide sequence completely identical to the natural melittin sequence. This ensures that the melittin exerts its natural biological activity and further enhances its killing effect on tumor cells. This solves the problem that melittin requires two cleavage processes at the N-terminus and one at the C-terminus in bee venom glands to become a mature melittin, and that the first amino acid residue at the N-terminus of a mature melittin is not Met. Attached Figure Description

[0025] Figure 1 To detect the transcriptional activity of the hTERTcp promoter in HepG2 and B16 cells; Figure 2 Plasmid maps of pAAV-hTERTcp-TRE-2mel and FUGW-CMV-TetR; Figure 3 Nucleic acid electrophoresis detection of the TetR gene fragment; Figure 4 To determine the expression of EGFP in the stable cell line HEK293T / TetR; Figure 5 Changes in mouse body weight after modeling; Figure 6 The survival rate curve for mice; Figure 7 Changes in mouse body weight after drug administration; Figure 8 Images of tumor formation in the liver of mice and images of the liver of mice after treatment; Figure 9 Liver and spleen weights of mice in each group; Figure 10 HE staining of liver tissue from mice in each group. Detailed Implementation

[0026] This invention provides an expression cassette containing a gene for targeted transcription of melitoxin, the expression cassette comprising a human telomerase reverse transcriptase core promoter, a tetracycline response element, an optimized melitoxin gene, a ribosome insertion sequence, and a human enterokinase light chain-melitoxin fusion gene.

[0027] In this invention, the preferred gene sequence of the expression cassette is hTERTcp-TRE-hmel-IRES-hEK. L -hmel, the preferred nucleotide sequence of the expression cassette is shown in SEQ ID NO:1.

[0028] The present invention also provides the application of the expression cassette in the preparation of drugs for treating tumors.

[0029] The present invention also provides a recombinant adeno-associated virus particle containing a gene that targets the transcription of bee venom peptides, preferably prepared from the expression cassette and the recombinant adeno-associated virus plasmid vector.

[0030] The present invention also provides a method for preparing the recombinant adeno-associated virus particles, comprising the following steps: (1) A DNA fragment containing the gene encoding the cytomegalovirus promoter and tetracycline repressor protein was synthesized and cloned into a lentiviral vector to obtain the recombinant plasmid FUGW-CMV-TetR; (2) The recombinant plasmid FUGW-CMV-TetR, packaging plasmid and envelope plasmid were co-transfected into HEK293T cells. After 48-72 h, the supernatant was collected and concentrated to obtain concentrated lentivirus. (3) HEK293T cells were infected with concentrated lentivirus and screened with puromycin for 1-2 weeks to obtain a stable cell line HEK293T / TetR that stably expresses tetracycline repressor protein for later use. (4) Insert the expression cassette into the adeno-associated virus vector plasmid to obtain the recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel, for later use; (5) The recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel, packaging plasmid and helper plasmid were co-transfected into HEK293T / TetR cells. After culturing for 48-72 h, the cells were centrifuged, the precipitated cells were collected, and the cells were repeatedly frozen and thawed and lysed to collect the supernatant. (6) Degrade the cellular DNA and plasmid DNA in the supernatant to obtain recombinant adeno-associated virus particles.

[0031] In this invention, the cloning method in step (1) is preferably a seamless cloning technique, and the lentivirus vector is preferably FUGW.

[0032] In this invention, the packaging plasmid in step (2) is preferably psPAX2, the coating plasmid is preferably pMD2.G, and the concentration factor is preferably 30 to 50 times, and more preferably 40 times.

[0033] In this invention, the concentration of puromycin in step (3) is preferably 1~3 μg / mL, and more preferably 2 μg / mL.

[0034] In this invention, the packaging plasmid in step (5) is preferably pAAV-RC8, and the auxiliary plasmid is preferably pHelper.

[0035] The present invention also provides the application of the recombinant adeno-associated virus particles in the preparation of drugs for treating tumors.

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. It should also be noted that all raw materials used in the present invention are conventional commercially available products.

[0037] Example 1: Detection of transcriptional activity of the human telomerase reverse transcriptase core promoter (hTERTcp) in mouse tumor cells.

[0038] The pEGFP-N1 plasmid and the phTERTcp-EGFP plasmid were transfected into mouse melanoma cells B16 and human hepatocellular carcinoma cells HepG2, respectively, using Liposome 2000 (purchased from Invitrogen). The transfection procedure strictly followed the standard protocol in the reagent instructions, and plasmid incubation and co-transfection with liposomes were performed under serum-free medium conditions to minimize the interference of serum components on transfection efficiency. After transfection, the medium was replaced with complete medium, and the cells were incubated at 37°C and 5% C. After being cultured in a constant temperature incubator for 48 h, the expression of green fluorescent protein (EGFP) in the two groups of cells was qualitatively observed using a fluorescence inverted microscope. Figure 1 .

[0039] Figure 1 Fluorescence detection results showed that EGFP protein was highly expressed in both murine and human tumor cells under the regulation of the hTERTcp promoter, indicating that the transcriptional activity of the hTERTcp promoter is comparable to that of the CMV promoter. These results demonstrate that the hTERTcp promoter can effectively regulate the transcriptional expression of target genes in mouse tumor cells.

[0040] Example 2: Construction and identification of HEK293T / TetR stable cell line

[0041] Lentiviral vector construction: A DNA fragment containing the TetR protein-coding gene regulated by the CMV promoter was designed and synthesized. This fragment was then inserted into the lentiviral vector FUGW using seamless cloning technology to obtain the recombinant plasmid FUGW-CMV-TetR. Figure 2 ).

[0042] Lentiviral packaging: Recombinant plasmid FUGW-CMV-TetR (3.0 μg), packaging plasmid psPAX2 (1.9 μg) and envelope plasmid pMD2.G (1.1 μg) were co-transfected into HEK293T cells cultured to 80% confluence in 6-well plates. After 48 h of transfection, the supernatant was collected and concentrated 40 times using lentivirus concentration reagent to obtain concentrated lentivirus.

[0043] Selection of stable cell lines: HEK293T cells were seeded in 24-well plates until the confluence reached 50%, then 100 μL of the above-mentioned concentrated lentivirus was added, and the cells were cultured for another 48 hours. The cells were then passaged into 6-well plates, and puromycin was added to a final concentration of 2 μg / mL for selection. The selection was continued for 2 weeks to obtain the stable cell line HEK293T / TetR expressing the TetR protein.

[0044] Identification of stable cell lines: Total RNA was extracted from HEK293T (control group, untransfected with lentivirus) and HEK293T / TetR (stable cell line) cells, and cDNA was obtained using a reverse transcription kit. PCR amplification was performed using TetR-specific primers. PCR was used to detect whether the TetR gene expression cassette was integrated into the genomic DNA of the stable cell line HEK293T / TetR. Results are shown below. Figure 3 The stable cell line showed a specific amplification fragment of TetR at 250 bp in the stable transfected group, while no target band was detected at this position in the control group, indicating successful construction of the stable cell line. The phTERTcp-EGFP and pGL3-hTERTcp-TRE-EGFP plasmids were transiently transfected into HEK293T / TetR cells, and the transfection effect was observed after 48 h of culture. Figure 4 As shown, without the introduction of a TRE regulatory sequence (no TRE sequence is involved in regulation without the addition of doxycycline), the hTERTcp promoter can normally initiate the transcription of the EGFP gene. When a TRE regulatory sequence is inserted between the hTERTcp promoter and the EGFP gene, TetR in the cell specifically binds to TRE, inhibiting the downstream EGFP gene transcription driven by the hTERTcp promoter, thus preventing EGFP gene expression. However, after the addition of 2 μg / mL doxycycline, TetR binds to doxycycline, causing a conformational change and losing its ability to bind to the TRE sequence. The transcription process driven by the hTERTcp promoter is restored, leading to EGFP gene expression. This inducible transcriptional regulation characteristic confirms that this study successfully constructed a tetracycline-inducible expression system, which can avoid host cell death caused by continuous expression of the therapeutic gene during AAV packaging, thereby solving the problem of viral packaging failure.

[0045] Example 3 Construction and Packaging of Recombinant AAV Vectors

[0046] Artificially synthesized gene expression cassettes: These cassettes contain transcriptional regulatory elements and gene sequences (human telomerase reverse transcriptase core promoter (hTERTcp), tetracycline response element (TRE), optimized melivexin gene (with a methionine codon and the corresponding nucleic acid sequence for enterokinase recognition added sequentially to its N-terminus after sequence optimization), ribosome insertion sequence (IRES), and human enterokinase light chain-melivexin fusion gene (hEK). LAfter combining -hmel, the sequence was artificially synthesized by Nanjing GenScript Biotech Co., Ltd., resulting in hTERTcp-TRE-hmel-IRES-hEK. L -hmel gene sequence.

[0047] The nucleotide sequence of the expression cassette is shown in SEQ ID NO.1. After transcription and translation, the expression cassette yields two expression polypeptides. One is a melittin peptide with Met and enterokinase recognition sequences added to its N-terminus, and its amino acid sequence is shown in SEQ ID NO:2 (corresponding to the optimized melittin peptide gene). The other is a human enterokinase light chain-melittin peptide fusion protein with an enterokinase recognition sequence added at the junction, and its amino acid sequence is shown in SEQ ID NO:3 (corresponding to the human enterokinase light chain-melittin peptide fusion gene).

[0048] The nucleotide sequences of each transcriptional regulatory element are as follows: human telomerase reverse transcriptase core promoter (hTERTcp, SEQ ID NO:4), tetracycline response element (TRE, SEQ ID NO:5), optimized melivesin gene (melivesin gene with methionine codon and enterokinase recognition sequence corresponding nucleic acid sequence added sequentially to its N-terminus after sequence optimization, SEQ ID NO:6), ribosome insertion sequence (IRES, SEQ ID NO:7), and human enterokinase light chain-melivesin fusion gene (hEK). L -hmel, SEQ ID NO:8).

[0049] SEQ ID NO:1:

[0050] SEQ ID NO:2: MDDDDKGIGAVLKVLTTGLPALISWIKRKRQQ。

[0051] SEQ ID NO:3: MHHHHHIVGGSNAKEGAWPWVVGLYYGGRLLCGASLVSSDWLVSAAHCVYGRNLEPSKWTAILGLHMKSNLTSPQTVPRLIDEIVINPHYNRRRKDNDIAMMHLEFKVNYTDYIQPICLPEENQVFPPGRNCSIAGWGTVVYQGTTANILQEADVPLLSNERCQQQMPEYNITENMICAGYEEGGIDSCQGDSGGPLMCQENNRWFLAGVTSFGYKCALPNRPGVYARVSRFTEWIQSFLHDDDDKGIGAVLKVLTTGLPALISWIKRKRQQ。

[0052] SEQ ID NO:4: agtggattcgcgggcacagacgcccaggaccgcgcttcccacgtggcggagggactggggacccgggcacccgtcctgccccttcaccttccagctccgcctcctccgcgcggaccccgccccgtcccgacccctcccgggtccccggcccagccccctccgggccctcccagcccctccccttcctttccgcggccccgccctctcctcgcggcgcgagtttcaggcagcgctgcgtcctgctgcgcacgtgggaagccct。

[0053] SEQ ID NO:5: tccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagaga。

[0054] SEQ ID NO:6: atggatgacgatgacaaaggcatcggcgccgtgctgaaggtgctgaccaccggcctgcccgccctgatcagctggatcaagcgcaagcgccagcagtga。

[0055] SEQ ID NO:7: gagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa。

[0056] SEQ ID NO:8: .

[0057] Construction of recombinant AAV vector: The constructed gene expression cassette was inserted into the AAV vector plasmid to obtain the recombinant plasmid pAAV-hTERTcp-TRE-2mel ( Figure 2 The correct insertion sequence of the vector was verified by sequencing.

[0058] Recombinant AAV Packaging: The vector plasmid pAAV-hTERTcp-TRE-2mel, packaging plasmid pAAV-RC8, and helper plasmid pHelper were co-transfected into the constructed HEK293T / TetR stable cell line. After transfection, the cells were cultured for 72 h. Cells containing AAV virus particles were then gently scraped off with a cell scraper and collected into 15 mL centrifuge tubes. The cells were centrifuged at 150×g for 3 min to pellet the cells. The supernatant was discarded, and the cell pellet was washed once with PBS. Finally, the cells were resuspended in 300 μL of PBS. A 37°C constant temperature water bath and liquid nitrogen were prepared in advance. The centrifuge tubes containing the resuspended cells were alternately placed in liquid nitrogen and the 37°C water bath for three freeze-thaw cycles to promote complete cell lysis and release of virus particles. The cells were then centrifuged at 4°C and 2000×g for 5 min. The precipitated cell debris was discarded, and the supernatant was collected as the crude AAV virus extract. Nuclease was added to the crude viral extract at a ratio of 0.1 μL Benonase per 1 mL, and the mixture was incubated in a 37°C water bath for 1 h to degrade residual cellular genomic DNA and plasmid DNA. After the enzymatic digestion reaction, the supernatant was collected by centrifugation at 4°C and 600×g for 10 min. The AAV genome content was detected using qPCR to determine the AAV titer. AAV virus was digested using DNase I and proteinase K. The standard plasmid was diluted, and the copy gradient of the standard was set to 10-1. 5 10 6 10 7 10 8 10 9 10 10 Configure the qPCR reaction system, designing three replicates for each sample and standard, and determine the viral titer. The resulting recombinant AAV is AAV-mel, which should be frozen at -80℃ for later use.

[0059] Example 4: Application in Tumor Treatment

[0060] Animal model establishment: Fifteen 7-week-old SPF-grade C57BL / 6J mice were housed in a well-ventilated environment at a room temperature of approximately 22°C. Light was circulated every 12 hours, and ample water and food supplies were provided. Bedding was changed every 3 days to maintain a clean environment. Five mice were randomly selected as the WT group for normal feeding, while the remaining 10 mice were used to establish a hepatocellular carcinoma mouse model via high-pressure tail vein injection of plasmids. At the start of the experiment, the weight of each mouse was accurately weighed and recorded. Plasmids encoding myr-AKT1 and NRASV12 (pT3-myr-AKT-HA and pT / Caggs-NRASV12, both purchased from Addgene) were diluted 5 μg each with the Sleeping Beauty transposase expression plasmid (pCMV / SB11, purchased from Addgene) at a ratio of 25:1 in physiological saline (0.9% sodium chloride) to a final volume of 2 mL. After filtering through a 0.22 μm filter, the solutions were injected into mice via the tail vein over 5 to 7 seconds to establish the AKT / NRas group (model group). The growth status and tumor growth of the mice were dynamically monitored weekly. The trend of weight change during the feeding period is shown in the figure below. Figure 5 As shown, the body weight of the model group was close to that of the control group and showed an upward trend within 1 to 3 weeks. After the fourth week, the body weight showed a downward trend. In order to avoid the mice's condition from continuing to deteriorate or even die, they were treated with medication in the fifth week.

[0061] Treatment: Ten tumor-bearing mice were randomly divided into two groups: a treatment group (AAV-mel) and a negative control group (AAV-GFP, which, compared to the treatment group's AAV-mel, only expressed the GFP gene). Each mouse in the treatment group received a single tail vein injection of 100 μL of AAV-mel (titer: 2 × 10⁻⁶). 12 (vg / mL), each mouse in the negative control group received a single tail vein injection of 100 μL of AAV-GFP (titer: 2 × 10⁻⁶ vg / mL). 12 vg / mL). A second injection was given one week later, for a total of two injections. From the start of the drug injection: the mice in the treatment group were not significantly affected; their eating, drinking, and other activities were normal. The mice in the negative control group showed lethargy, significant abdominal swelling, reduced activity, and decreased food intake after the model was established. As time progressed, the abdominal swelling increased, their movements became more sluggish, and they gradually died, while the mice in the treatment group survived. Figure 6 Initially, the weight difference between the two groups of mice was small in the first week. As the condition of the virus-infected mice worsened, their food intake decreased, and their weight decreased, while the weight of the treated mice steadily increased, approaching that of normal mice. Figure 7 ).

[0062] Efficacy evaluation: After 30 days of continuous treatment, mice in both groups were dissected to systematically observe the overall situation of liver tumor formation (see...). Figure 8From a gross morphological perspective, the livers of mice in the negative control group exhibited significant tumorigenesis characteristics: a high tumorigenesis rate, numerous white tumor masses visible on the liver surface, irregular morphology, and a scattered distribution. Besides the main lesion area, large areas of scattered tumor infiltration were also observed in other liver lobes, and the entire liver showed significant enlargement and deformation due to tumor proliferation. Measurements showed that the average liver weight of mice in the AAV-GFP group was 6.62 g ± 0.46 g, while the average liver weight of mice in the AAV-mel treatment group was only 2.93 g ± 0.07 g. The difference in liver weight between the two groups was statistically significant. Figure 9 (Left). Significant differences were also observed in spleen tissue weight analysis: the average spleen weight in the AAV-GFP group was 0.244 g ± 0.01 g, and the average spleen weight in the AAV-mel group was 0.128 g ± 0.01 g; these differences were also statistically significant. Figure 9 (Right). This result suggests that AAV-mel-mediated meliotide treatment not only effectively inhibits the growth and proliferation of liver tumors but also improves tumor-induced abnormal proliferation of spleen tissue, further confirming the effectiveness of this treatment regimen in inhibiting tumor progression. Further HE staining results of liver tissue ( Figure 10 The results showed that in the negative control group, the liver tissue structure was severely disordered, the normal structure of the liver lobules disappeared, and neoplastic lesions were visible. The tumor cells showed atypia, increased nuclear-cytoplasmic ratio, and darkened nuclear staining. In some areas, cells were densely packed and arranged in a disordered manner. Vacuole degeneration of hepatocytes was visible in the lesion areas, and local inflammatory cell infiltration was possible. In contrast, the liver tissue in the treatment group recovered a relatively normal lobular structure. Hepatocytes were arranged neatly in a radial pattern along the central vein, with no obvious residual tumor lesions. The hepatocytes were more uniform in morphology, with relatively consistent nuclear size and staining, and no obvious atypia. Only a small amount of scattered mild cellular edema or a very small amount of inflammatory cell infiltration were observed, without severe damage such as vacuolar degeneration or necrosis.

[0063] As shown in the above embodiments, this invention provides a recombinant adeno-associated virus (AAV) particle containing a gene for targeted transcription of melivesin, its preparation method, and its application. The expression cassette containing the melivesin gene contains a human telomerase reverse transcriptase core promoter, a tetracycline-responsive element, an optimized melivesin gene, a ribosomal insertion sequence, and a human enterokinase light chain-melivesin fusion gene. This invention aims to address how to achieve precise targeted therapy for tumors, effectively killing tumor cells while minimizing damage to normal cells, overcoming the limitations of traditional treatment methods. It also solves the problem of pre-transcription of the melivesin gene leading to cell death during recombinant AAV packaging, and ensures that the melivesin expressed in tumor cells has its natural sequence and activity, providing a safer and more effective treatment option for cancer patients.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An expression cassette containing a gene that targets the transcription of melitoxin, characterized in that, The expression cassette includes a human telomerase reverse transcriptase core promoter, a tetracycline response element, an optimized melitoxin gene, a ribosome insertion sequence, and a human enterokinase light chain-melitoxin fusion gene.

2. The expression box according to claim 1, characterized in that, The gene sequence of the expression cassette is hTERTcp-TRE-hmel-IRES-hEK. L -hmel, the nucleotide sequence of the expression cassette is shown in SEQ ID NO:

1.

3. The use of the expression cassette according to any one of claims 1 to 2 in the preparation of a drug for treating tumors.

4. A recombinant adeno-associated virus particle containing a gene that targets the transcription of melitoxin, characterized in that, It is prepared from the expression cassette and recombinant adeno-associated virus plasmid vector described in claim 1.

5. The method for preparing the recombinant adeno-associated virus particles according to claim 4, characterized in that, Includes the following steps: (1) A DNA fragment containing the gene encoding the cytomegalovirus promoter and tetracycline repressor protein was synthesized and cloned into a lentiviral vector to obtain the recombinant plasmid FUGW-CMV-TetR; (2) The recombinant plasmid FUGW-CMV-TetR, packaging plasmid and envelope plasmid were co-transfected into HEK293T cells. After 48-72 h, the supernatant was collected and concentrated to obtain concentrated lentivirus. (3) HEK293T cells were infected with concentrated lentivirus and screened with puromycin for 1-2 weeks to obtain a stable cell line HEK293T / TetR that stably expresses tetracycline repressor protein for later use. (4) Insert the expression cassette described in claim 1 into the adeno-associated virus vector plasmid to obtain the recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel for later use; (5) The recombinant adeno-associated virus vector plasmid pAAV-hTERTcp-TRE-2mel, packaging plasmid and helper plasmid were co-transfected into HEK293T / TetR cells. After culturing for 48-72 h, the cells were centrifuged, the precipitated cells were collected, and the cells were repeatedly frozen and thawed and lysed to collect the supernatant. (6) Degrade the cellular DNA and plasmid DNA in the supernatant to obtain recombinant adeno-associated virus particles.

6. The preparation method according to claim 5, characterized in that, The cloning method described in step (1) is seamless cloning technology, and the lentivirus vector is FUGW.

7. The preparation method according to claim 5, characterized in that, The packaging plasmid in step (2) is psPAX2, the coating plasmid is pMD2.G, and the concentration factor is 30 to 50 times.

8. The preparation method according to claim 5, characterized in that, The concentration of puromycin in step (3) is 1~3 μg / mL.

9. The preparation method according to claim 5, characterized in that, The packaging plasmid in step (5) is pAAV-RC8, and the auxiliary plasmid is pHelper.

10. The use of the recombinant adeno-associated virus particles according to claim 4 in the preparation of a tumor-treating drug.

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