Gene combination, expression vector and use thereof

Abstract

The present invention belongs to the field of gene therapy or biological gene technology, and particularly relates to a gene combination and its expression vector and use. The gene combination is used to exist and express in nasal cells; including: U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets tumor suppressor gene; super hTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene; SPLUNC1 promoter and downstream artificial miRNA gene; wherein, U6 promoter, super hTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 in sequence. The sgRNA initiated by U6 promoter and the dCas9-VP64 initiated by super hTERT are tandemly activated to activate downstream tumor suppressor genes, such as P53, etc., and finally cause apoptosis of nasopharyngeal carcinoma cells, thereby achieving treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy or biological gene technology, and in particular relates to a gene combination and an expression vector and use thereof. Background Art

[0002] With the gradual development of targeted therapy strategies for specific molecules of tumors and cancers, the treatment effect can be improved to the greatest extent. Compared with radiotherapy and chemotherapy, targeted therapy for tumors has the characteristics of strong specificity and few side effects. However, compared with other common malignant tumors such as liver cancer and lung cancer, the research on targeted therapy for nasopharyngeal carcinoma started late.

[0003] Currently, drugs targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) have shown clinical efficacy in patients with NPC resistant to platinum drugs. However, there is still a lack of effective targeted treatment strategies for NPC in clinical practice. Summary of the invention

[0004] The present application provides a gene combination and its expression vector and use, so as to provide an effective and precise targeted treatment method for nasopharyngeal carcinoma.

[0005] In a first aspect, the present application provides a gene combination, the gene combination is used to exist and express in nasal cells; comprising:

[0006] U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0007] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0008] SPLUNC1 promoter and downstream artificial miRNA genes;

[0009] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0010] Optionally, the tumor suppressor gene is selected from P53, P21, BAX, Caspas3, and Caspas8.

[0011] In a second aspect, the present application provides an expression vector comprising the above-mentioned gene combination.

[0012] Optionally, the expression vector includes at least one of pGPU6 plasmid, GFP plasmid, and Neo plasmid.

[0013] In a third aspect, the present application provides a use of a gene combination in the preparation of an anti-nasopharyngeal carcinoma drug, wherein the gene combination is used to exist and express in nasal cells; comprising:

[0014] U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0015] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0016] SPLUNC1 promoter and downstream artificial miRNA genes;

[0017] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0018] Optionally, there is at least a 2-fold difference in expression of tumor suppressor genes.

[0019] Optionally, a p-value indicating differential adjustment is less than 0.05.

[0020] In a fourth aspect, the present application provides a use of an expression vector in the preparation of an anti-nasopharyngeal carcinoma drug, wherein the expression vector comprises the following gene combination, and the gene combination is used to exist and express in nasal cells; including:

[0021] U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0022] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0023] SPLUNC1 promoter and downstream artificial miRNA genes;

[0024] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0025] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0026] The gene combination provided in the embodiments of the present application is differentially expressed in normal nasal cells and nasopharyngeal carcinoma cells, achieving apoptosis of nasopharyngeal carcinoma cells, wherein the superhTERT promoter can drive nasopharyngeal carcinoma cell sgRNA so that it can only be expressed in nasopharyngeal carcinoma cells. In normal cells, the U6 promoter belongs to the promoter of the housekeeping gene, which can be expressed in most cells, and the sgRNA with the tumor suppressor gene as the target gene is inserted downstream thereof. The artificial miRNA gene in series with the SPLUNC1 promoter is expressed in nasopharyngeal epithelial cells; the superhTERT promoter with cancer cell specificity and its downstream genes dCas9 gene and VP64 gene are not expressed; therefore, it has no effect on normal cells. In nasopharyngeal carcinoma cells, the U6 promoter belongs to the promoter of the housekeeping gene, which can be expressed in most cells, and the sgRNA with P53 as the target gene is inserted downstream thereof. The superhTERT promoter, which is specific for cancer cells, the U6 promoter, the sgRNA targeting P53, the superhTERT promoter, the dCas9 gene and / or the VP64 gene in series, are transfected into nasopharyngeal cancer cells to cause apoptosis of nasopharyngeal cancer cells. The miRNA element in series with the SPLUNC1 promoter is not expressed in nasopharyngeal cancer cells. The sgRNA driven by the U6 promoter and the dCas9-VP64 driven by superhTERT in series activate downstream tumor suppressor genes, such as P53, and ultimately cause apoptosis of nasopharyngeal cancer cells, thereby achieving the treatment of nasopharyngeal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0029] Figure 1 A schematic diagram showing the arrangement of the gene combination of the embodiment of the present application is shown;

[0030] Figure 2 A diagram showing the working principle of the gene combination containing the SPLUNC1-TERT dual-specific promoter in the embodiment of the present application in normal nasopharyngeal epithelial cells and nasopharyngeal carcinoma cells;

[0031] Figure 3 The expression levels of SPLUNC1 in different nasopharyngeal carcinoma cells and normal cells are shown;

[0032] Figure 4 The flow cytometric results of apoptosis in nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells expressed by the expression vector containing the SPLUNC1-TERT dual-specific promoter are shown. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, the present specification takes precedence. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. For example, room temperature can refer to a temperature within the range of 10 to 35°C.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0036] Nasopharyngeal carcinoma is a malignant tumor originating from nasopharyngeal epithelial cells, and its occurrence has obvious regional and ethnic clustering. Most nasopharyngeal carcinoma patients are young and middle-aged people aged 30 to 50, which brings a heavy burden to social and economic development. Nasopharyngeal carcinoma is sensitive to radiotherapy. Currently, the most common and effective treatment for nasopharyngeal carcinoma is mainly radiotherapy, supplemented by chemotherapy. Although early-stage (stage I and II) patients can obtain better therapeutic effects, among which the 5-year survival rate can exceed 80%, but due to the hidden anatomical location of the nasopharyngeal cavity, early nasopharyngeal carcinoma has no obvious symptoms. Therefore, most nasopharyngeal carcinoma patients are in the late stage when they seek medical treatment (stage III and IV account for about 85%), and the treatment effect and survival rate are low.

[0037] Although radiotherapy equipment and technology are constantly updated, and treatment plans such as induction chemotherapy and synchronous chemoradiotherapy are constantly improved, the highly invasive and metastatic characteristics of nasopharyngeal carcinoma make the patient's prognosis poor once metastasis occurs. The 5-year survival rate of nasopharyngeal carcinoma has been stagnant for nearly 10 to 20 years, and the 5-year survival rate of advanced patients is only 15% to 40%. Studies have found that the occurrence of nasopharyngeal carcinoma is closely related to environmental and genetic factors and the disruption of the intracellular molecular balance caused by the Epstein-Barr virus.

[0038] At present, there is no effective targeted therapy for NPC in clinical practice, and there has been no significant progress in targeted therapy and immunotherapy for NPC. The molecular mechanisms of NPC occurrence and metastasis and the search for targeted therapy for NPC are crucial for early clinical diagnosis, treatment and prognosis of NPC, and will also bring significant benefits to society.

[0039] The inventors have studied and provided a new gene-targeted drug, which provides an optional solution for the early treatment of nasopharyngeal carcinoma.

[0040] After further research by the inventors, they designed a related sgRNA gene for the nasopharyngeal epithelial cell target SPLUNC1, replaced the original promoter with the cancer cell-specific promoter super hTERT promoter to drive sgRNA expression, and combined with miniSaCas9 or dCas9 genes, it can reversely target nasopharyngeal cancer cells, solving the current problem of non-targeted treatment of nasopharyngeal carcinoma, further reducing off-target effects, and better killing tumor cells.

[0041] In view of this, according to the first aspect of the present application, a gene combination is provided, wherein the gene combination is used to exist and express in nasal cells; Figure 1 As shown, including:

[0042] U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0043] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0044] SPLUNC1 promoter and downstream artificial miRNA genes;

[0045] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0046] In this embodiment, if Figure 2As shown in the figure, in nasopharyngeal carcinoma cells, the U6 promoter belongs to the promoter of the housekeeping gene and can be expressed in most cells. sgRNA with P53 as the target gene is inserted downstream. The superhTERT promoter with cancer cell specificity, the U6 promoter, the sgRNA with P53 as the target gene, the superhTERT promoter, the dCas9 gene and / or the VP64 gene in series are transfected into nasopharyngeal carcinoma cells to cause apoptosis of nasopharyngeal carcinoma. The miRNA element in series with the SPLUNC1 promoter is lowly expressed or not expressed in nasopharyngeal carcinoma cells. The sgRNA initiated by the U6 promoter and the dCas9-VP64 initiated by super hTERT in series activate downstream tumor suppressor genes, such as P53, and finally cause apoptosis of nasopharyngeal carcinoma cells, thereby achieving the treatment of nasopharyngeal carcinoma.

[0047] In this embodiment, if Figure 2 As shown, in nasopharyngeal epithelial cells with high expression of SPLUNC1, the expression element of the SPLUNC1-artificial miRNA sequence is activated, and can form base complementary pairing with U6promoter-sgRNA, thereby silencing the function of U6 promoter-sgRNA. In this way, normal cells with only hTERT-dCas9-VP64 biological elements will not activate the effect of the target gene P53, and thus have no effect on normal cells. In nasopharyngeal carcinoma cells without the expression element of the SPLUNC1-artificialmiRNA sequence, due to the lack of the inhibitory effect of SPLUNC1 miRNA, the two modules of U6promoter-sgRNA and superhTERT are tandemly activated to activate downstream target genes such as P53, achieving the effect of killing tumors.

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[0056] In some embodiments, the gene combination further includes: GFP fluorescent marker, U6 promoter, downstream sgRNA gene, tandem GFP fluorescent marker and tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene.

[0057] In some embodiments, the sequence of the sgRNA gene is shown as SEQ ID NO.8.

[0058] In some embodiments, the tumor suppressor gene is selected from P53, P21, BAX, Caspas3, and Caspas8.

[0059] In the second aspect, the present application provides an expression vector comprising the above gene combination. The above gene combination in the expression vector specifically kills nasopharyngeal cancer cells, has high efficiency, low off-target, stability, and can accurately and stably target and treat nasopharyngeal cancer.

[0060] In some embodiments, the expression vector comprises at least one of a pGPU6 plasmid, a GFP plasmid, and a Neo plasmid.

[0061] In a third aspect, the present application provides a use of a gene combination in the preparation of an anti-nasopharyngeal carcinoma drug, wherein the gene combination is used to exist and express in nasal cells; comprising: a U6 promoter and a downstream sgRNA gene and a tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0062] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0063] SPLUNC1 promoter and downstream artificial miRNA genes;

[0064] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0065] In the examples of this application, the gene combination can target nasopharyngeal cancer cells, activate tumor suppressor genes in nasopharyngeal cancer cells, and specifically kill nasopharyngeal cancer cells. This project uses genetic engineering methods to innovatively solve the problem of no specific markers in nasopharyngeal cancer cells, providing a new direction for the treatment of nasopharyngeal cancer.

[0066] In some embodiments, there is at least a 2-fold difference in expression of the tumor suppressor gene.

[0067] like Figure 4As shown, in the left figure, C2+C4=3.7%, and in the right figure, C2+C4=47.5%, indicating that the expression level of P53 tumor suppressor gene in nasopharyngeal carcinoma cells is much higher than that in normal cells.

[0068] In some embodiments, the p-value indicating differential regulation is less than 0.05.

[0069] In a fourth aspect, the present application provides a use of an expression vector in the preparation of an anti-nasopharyngeal carcinoma drug, wherein the expression vector comprises the following gene combination: a U6 promoter and a downstream sgRNA gene and a tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene;

[0070] superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene;

[0071] SPLUNC1 promoter and downstream artificial miRNA genes;

[0072] Among them, U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively.

[0073] The method of the present invention will be described in detail below with reference to embodiments, comparative examples and experimental data.

[0074] Example 1 Expression level of SPLUNC1 in nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells The expression level of SPLUNC1 promoter, as shown in SEQ ID NO.6, in different nasopharyngeal carcinoma cells, nasopharyngeal carcinoma cell lines 5-8f, CNE1, CNE2, 6-10B, SUNE1, SUNE2 and normal epithelial cell line NP69 were obtained. The above nasopharyngeal carcinoma cell line and normal epithelial cell line NP69 were derived from atcc-China Cell Resource Center and then subcultured. The RNA of the above nasopharyngeal carcinoma cell line and normal rhinitis epithelial cell line was reverse transcribed into cDNA. The expression level of SPLUNC1 in each cell line was detected by primer probe. Figure 3 As shown, the experimental results showed that SPLUNC1 was highly expressed in nasopharyngeal epithelial cells NP69 cells, and was lowly expressed or not expressed in various nasopharyngeal carcinoma cell lines.

[0075] Example 2 Construction of target expression vector

[0076] The sequence information of U6, sgRNA, hTERT, dCas9, VP64, SPLUNC1, and artificial miRNA was downloaded from the Pubmed website, and the U6-sgRNA, hTERT-dCas9-VP64, and SPLUNC1-artificial miRNA sequences were designed. The vectors containing the above sequences were synthesized by BGI according to the sequences, and then linked to the AAV-miniSaCas9-gRNA vector plasmid through the restriction site. The AAV virus was packaged and purified, and the AAV adeno-associated virus vector was used to improve the transduction ability to facilitate tumor killing in vivo. A new SPLUNC1 / TERT dual-specific promoter switch synthetic biological device that targets nasopharyngeal carcinoma cells was obtained, and the effect of the gene circuit on tumor growth, invasion and metastasis in vivo was further verified in the carcinoma in situ model.

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[0084] Verification: Through sequencing analysis, it was verified that the AAV-miniSaCas9-gRNA vector plasmid was introduced into the above-mentioned AAV virus.

[0085] Example 3 Effects of the expression vector containing the SPLUNC1-TERT dual-specific promoter in normal nasopharyngeal epithelial tissue and nasopharyngeal carcinoma cells

[0086] Normal nasopharyngeal epithelial tissue NP69 and nasopharyngeal carcinoma cell SUNE1 were cultured to 8*10 6 The expression vector with SPLUNC1-TERT dual-specific promoter was transfected into normal nasopharyngeal epithelial tissue NP69 and nasopharyngeal carcinoma cell SUNE1 using Lipofectamine2000 transfection reagent, and cell apoptosis was detected using Beckman CytoFLEX flow cytometer 48 hours later. The results confirmed that the apoptosis of normal cells was significantly lower than that of nasopharyngeal carcinoma cells. Figure 4As shown, the horizontal axis is Annexin V dye, and the vertical axis is PI dye. The cells of nasopharyngeal epithelial cells in the left figure are mainly concentrated in the third quadrant, indicating that the cell activity is good, and the transfected artificial module has a significant killing effect on normal nasopharyngeal epithelial cells; the flow cytometry results of nasopharyngeal carcinoma cells in the right figure are mainly concentrated in the first and fourth quadrants. It shows that most of the treated cells are in early apoptosis or late apoptosis. The results show that the constructed SPLUNC1-TERT dual-specific promoter expression vector can specifically target and kill nasopharyngeal carcinoma cells.

[0087] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the existence of other identical elements in the process, method, article or equipment including the elements.

[0088] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A gene combination for being present and expressed in nasal cells; comprising: U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene P53, wherein the sgRNA gene targets the tumor suppressor gene; superhTERT promoter and downstream dCas9 gene and / or miniSaCas9 gene, and tandem VP64 gene; SPLUNC1 promoter and downstream artificial miRNA genes; Among them, the U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively, and the sequence of the sgRNA gene is shown in SEQ ID NO.

8.

2. An expression vector comprising the gene combination according to claim 1.

3. The expression vector according to claim 2, characterized in that The expression vector includes at least one of pGPU6 plasmid, GFP plasmid and Neo plasmid.

4. Use of a gene combination in the preparation of an anti-nasopharyngeal carcinoma drug, wherein the gene combination is used to exist and express in nasal cells; comprising: U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene; The super hTERT promoter and the downstream dCas9 gene and / or miniSaCas9 gene, and the tandem VP64 gene; SPLUNC1 promoter and downstream artificial miRNA genes; Among them, the U6 promoter, superh TERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively, and the sequence of the sgRNA gene is limited to the sequence shown in SEQ ID NO.

8.

5. Use of an expression vector in the preparation of an anti-nasopharyngeal carcinoma drug, the expression vector comprising the following gene combination: the gene combination is used to exist and express in nasal cells; including: U6 promoter and downstream sgRNA gene and tandem tumor suppressor gene, wherein the sgRNA gene targets the tumor suppressor gene, super hTERT promoter and downstream dCas9 gene and / or mini SaCas9 gene, and tandem VP64 gene; SPLUNC1 promoter and downstream artificial miRNA genes; Among them, the U6 promoter, superhTERT promoter, dCas9 gene, miniSaCas9 gene, VP64 gene, SPLUNC1 promoter and artificial miRNA gene are shown in SEQ ID NO.1-7 respectively, and the sequence of the sgRNA gene is limited to the sequence shown in SEQ ID NO.8.

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