Recombinant plasmid for inhibiting PLANE gene expression, preparation method thereof and recombinant adeno-associated virus

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a recombinant plasmid for inhibiting PLANE gene expression, a preparation method of the recombinant plasmid and a recombinant adeno-associated virus, and the recombinant plasmid comprises a nucleotide sequence for encoding shRNA of a targeted PLANE gene. The scAAV5.PLANE.shRNA recombinant plasmid carrying the shRNA sequence of the targeted PLANE gene is creatively constructed, the recombinant virus with infection activity is successfully packaged, in-vivo experiments prove that the recombinant virus can remarkably inhibit in-vivo growth of lung squamous carcinoma cells, and compared with other virus vectors, the recombinant virus has lower immunogenicity and higher biological safety, and can be used for preparing the lung squamous carcinoma cells. And a new choice is provided for clinical gene therapy.

Description

Technical Field

[0001] This invention relates to a recombinant plasmid for inhibiting PLANE gene expression, a method for preparing the plasmid, and a recombinant adeno-associated virus, belonging to the field of genetic engineering technology. Background Technology

[0002] In the human genome, the PLANE gene (NCBI accession number: 100507057) is a long noncoding RNA (lncRNA) located on the distal end (3q) of the long arm of chromosome 3. Existing research indicates that the copy number of this gene is significantly amplified in lung squamous cell carcinoma (LUSC) and plays a crucial role in the malignant progression of LUSC by participating in the regulation of signaling pathways closely related to tumorigenesis and development. Clinical data analysis based on the Cancer Genome Atlas database (TCGA) further confirms that, compared with normal lung tissue, the PLANE gene exhibits specific high expression characteristics in different pathological stages of LUSC—including carcinoma in situ, invasive carcinoma, and metastatic carcinoma. Clinical evidence suggests that PLANE plays a key role in the molecular pathogenesis of LUSC and may serve as a novel therapeutic target with significant research value.

[0003] Further mechanistic studies revealed that PLANE is significantly upregulated in multiple cancer types, with its expression jointly regulated by copy number increase and transcriptional activation mediated by the transcription factor E2F1. At the molecular level, PLANE, located in the intron 45 region of the Nuclear Receptor Co-Repressor 2 (NCOR2) gene, forms an RNA-RNA double-stranded structure with the precursor mRNA and simultaneously binds to heterogeneous nuclear ribonucleoprotein M (hnRNPM), thereby promoting the stable binding of the latter to this intron region. This interaction ultimately inhibits the normal alternative splicing (AS) process of the major protein-coding variant NCOR2-202. This regulatory mechanism has been confirmed as an important biological basis for PLANE's ability to promote cancer cell proliferation and tumorigenicity. These findings not only deepen our understanding of the role of PLANE in tumors at both functional and mechanistic levels but also suggest its potential as a broad-spectrum therapeutic target across cancer types, with prospects for further translational research.

[0004] Although existing research suggests that the PLANE gene may be a highly promising anti-cancer target, there is a lack of safe and efficient delivery and intervention tools for its clinical application. Current technology uses adeno-associated virus vectors to construct recombinant plasmids targeting PLANE; however, these vectors readily elicit strong immune responses in vivo, and the gene expression they mediate is only transient, failing to achieve long-term, stable therapeutic effects. This severely limits the development and application of gene therapy strategies based on the PLANE target. Summary of the Invention

[0005] To address the aforementioned deficiencies in the prior art, this invention provides a recombinant plasmid for inhibiting PLANE gene expression, a method for preparing the plasmid, and a recombinant adeno-associated virus.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: One of the objectives of this invention is to provide a recombinant plasmid for inhibiting PLANE gene expression, comprising a nucleotide sequence encoding shRNA targeting the PLANE gene.

[0007] Furthermore, the sequence of the shRNA is selected from any one of the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Furthermore, its vector is the scAAV5 vector.

[0009] Furthermore, its base sequence is shown in SEQ ID NO: 3.

[0010] A second objective of this invention is to provide a method for preparing the above-mentioned recombinant plasmid for inhibiting PLANE gene expression, comprising the following steps: S1. Prepare the target gene fragment encoding the shRNA targeting the PLANE gene; S2. Double digestion of expression vector XM19B yields linearized vector fragment; S3. Ligate the target gene fragment from step S1 with the linearized vector fragment obtained in step S2 to obtain the recombinant plasmid used to suppress PLANE gene expression, named scAAV5.PLANE.shRNA recombinant plasmid.

[0011] Furthermore, in step S2, the restriction endonucleases used for double digestion are HindIII and KpnI.

[0012] Furthermore, in step S3, the ligation reaction is carried out using the T4 DNA ligase system, and the ligation reaction is carried out at 20℃-25℃ for 3-5 hours.

[0013] A third objective of this invention is to provide a recombinant adeno-associated virus, which is produced by a viral packaging system from the recombinant plasmid described above for inhibiting PLANE gene expression.

[0014] Furthermore, the recombinant adeno-associated virus is obtained by co-transfecting 293T cells with the recombinant plasmid used to inhibit PLANE gene expression and the packaging plasmid, followed by culture, collection, and purification.

[0015] The beneficial effects of this invention are as follows: I. This invention innovatively constructs a recombinant plasmid scAAV5.PLANE.shRNA carrying a shRNA sequence targeting the PLANE gene, and successfully packages an infectious recombinant adeno-associated virus rAAV5.shPLANE.

[0016] Second, this invention utilizes the scAAV5 vector to deliver shRNA, which has lower immunogenicity and higher biosafety compared to other viral vectors, providing a new option for clinical gene therapy.

[0017] Third, in vivo experiments have demonstrated that recombinant adeno-associated virus rAAV5.shPLANE can significantly inhibit the in vivo growth of lung squamous cell carcinoma cells, indicating its great potential for application in tumor gene therapy. Attached Figure Description

[0018] Figure 1 The spectrum of the XM19B vector; Figure 2 Map of the scAAV5.PLANE.shRNA recombinant plasmid. Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] Example 1 Construction of the scAAV5.PLANE.shRNA recombinant plasmid: I. Materials and Methods 1. Vectors and strains: Expression vector XM19B, purchased from Guangzhou Paizhen Biotechnology Co., Ltd., chromatogram as shown below. Figure 1 As shown; Escherichia coli competent cells Stbl3 were purchased from Beyotime Biotechnology Co., Ltd.

[0021] 2. The main sources of reagents are shown in Table 1 below.

[0022] Table 1. Main reagents and their sources in Example 1

[0023] 3. Instruments: The instruments mainly used in this embodiment are shown in Table 2 below.

[0024] Table 2. Main instruments and their sources in Example 1

[0025] II. Experimental Procedure 1. Prepare the target gene fragment encoding the shRNA targeting the PLANE gene. The shRNA gene sequence for PLANE was designed and synthesized by Shanghai Jima Biotechnology Co., Ltd. Its sequence is shown in the nucleotide sequence of SEQ ID NO: 1 below: SEQ ID NO: 1, shPLANE.1: GACCCAAAGAGCAAGTGATTT.

[0026] 2. Double enzyme digestion of the vector (1) Incubate the bacterial culture containing the XM19B vector overnight, and extract the XM19B plasmid from 3-5 mL of fresh bacterial culture; for specific methods, refer to the high purity plasmid small-scale extraction kit (DP107).

[0027] (2) Take 1 μL of fresh plasmid and digest it in the enzyme digestion system shown in Table 3 below at 37℃ for 2 hours: Table 3 Enzyme digestion system

[0028] The enzyme digestion products were subjected to agarose gel electrophoresis. After electrophoresis, the vector fragments were recovered using an agarose gel DNA recovery kit.

[0029] 3. Ligation of the vector fragment and the target gene fragment (seamless cloning) (1) Determine the concentration of the recovered vector and the target gene fragment.

[0030] (2) Hieff Clone TM The optimal amount of cloning vector used in the recombinant reaction system is 0.03 pmol; the optimal molar ratio of cloning vector to insert fragment is 1:2, meaning the optimal amount of insert fragment used is 0.06 pmol. The DNA mass corresponding to these molar amounts can be calculated using the following formula: Optimal cloning vector usage = [0.02 × number of base pairs in the cloning vector] ng (0.03 pmol) Optimal amount of insert fragment used = [0.04 × number of base pairs in the insert fragment] ng (0.06 pmol) (3) The vector fragment and the target gene fragment were ligated in the ligation system shown in Table 4 at 22°C for 4 hours.

[0031] Table 4 Connection System

[0032] 4. Transformation (1) Place the supercompetent Stbl3 cells on ice (4°C) and allow them to thaw naturally. Then, add 10 μL of the above ligation product to the competent cells and place them on ice (4°C) for 30 min.

[0033] (2) Heat shock in a 42℃ water bath for 60 seconds. Then quickly place on ice (4℃) for 2-3 minutes.

[0034] (3) Add 500 μL of antibiotic-free LB plate and culture at 37°C with shaking at 200 rpm for 1 hour.

[0035] (4) Centrifuge at 5000 rpm for 2 min, discard the supernatant, blow the bacterial solution at the bottom of the tube to disperse it, add it to the culture plate containing the corresponding resistance (ampicillin) on the carrier, spread it evenly with a sterile spreader, and invert it in a 37℃ constant temperature incubator for overnight culture.

[0036] Single colonies were selected for sequencing verification. The correctly sequenced clones were the desired scAAV5.PLANE.shRNA recombinant plasmids, with the sequence shown in SEQ ID NO.3, and its map is shown below. Figure 2 As shown.

[0037] Example 2 Unlike Example 1, the target gene fragment encoding the shRNA targeting the PLANE gene used in this example is selected from the nucleotide sequence shown in SEQ ID NO: 2: SEQ ID NO: 2, shPLANE.2:GCTCAGAATCACTAGAATGTT.

[0038] The other steps are the same as in Example 1, and will not be repeated here.

[0039] Example 3 Production and purification of recombinant adeno-associated virus rAAV5.shPLANE 1: I. Extraction of scAAV5.PLANE.shRNA recombinant plasmid: The plasmid DNA used for viral packaging needs to be of high quality and high purity. The clone obtained in Example 1 was extracted using the endotoxin-free plasmid large extraction kit (DP120). Finally, the plasmid DNA was dissolved in TE buffer at pH 7.5.

[0040] II. 293T cell culture: 1. Revival of cryopreserved 293T cells: (1) Add 10 mL of DMEM growth medium to a 15 mL Erlenmeyer flask and set aside.

[0041] (2) Place the cryopreservation tubes stored at -80℃ into a 37℃ water bath and gently shake for 1-2 minutes to thaw them. Remove the cryopreservation tubes from the water bath and disinfect them by wiping the surface of the tubes with 75% ethanol (at room temperature); note that only the tube body should be placed in the water bath, and the cap should not be immersed in as well, otherwise the water in the water bath will be drawn into the cryopreservation tubes.

[0042] (3) Transfer the melted cell suspension from step (2) to the conical flask prepared in step (1).

[0043] (4) Centrifuge at 200g for 3 min at room temperature to precipitate cells and discard the culture medium supernatant.

[0044] (5) Add 5 mL of fresh DMEM growth medium to the conical flask and gently pipette up and down to suspend the cells. The cells are fragile at this time after revival, so it is best to suspend the cells when pipetting and avoid excessive pipetting.

[0045] (6) Transfer 5 mL of cell suspension to a 75 cm³ container. 2 Place the cell culture flask containing 10 mL of fresh DMEM growth medium in a 37°C, 5% CO2 incubator.

[0046] (7) Monitor cell density daily and passage the cells when they reach 50% confluence.

[0047] 2.293 T cell passage: (1) Preheat DMEM complete culture medium and trypsin-EDTA solution in a water bath at 37°C for later use.

[0048] (2) Remove the growth medium and wash the cells once with 10 mL of PBS buffer (phosphate-buffered saline).

[0049] (3) Digest the cells with 5 mL of trypsin-EDTA solution for 1-3 minutes. Note: The digestion time with trypsin-EDTA solution should be the shortest time required for adherent cells to detach from the culture flask. This process can be observed using an inverted microscope to avoid over-digestion that could damage or kill the cells.

[0050] (4) Dilute the cells with 5 mL of complete culture medium to inactivate the trypsin, and transfer the cell suspension to a 15 mL conical tube.

[0051] (5) Centrifuge at 200g for 3 minutes at room temperature to precipitate cells and discard the culture medium supernatant; (6) Resuspend the cell pellet in 10 mL of growth medium, and transfer 2 mL of the cell suspension to a 75 cm³ container. 2 Five cell culture flasks were prepared. Each flask contained 28 mL of DMEM growth medium. The cells were placed in a 37°C, 5% CO2 incubator. Cell density was checked daily, and cell confluence was maintained below 50%.

[0052] III. Adeno-associated virus production and packaging: 1. Preparation of 293T cells: Add 10 mL of DMEM growth medium to a 10 cm culture dish, then add 3 × 10⁻⁶ mL of DMEM. 6 293T cells were shaken well and cultured for 48 hours before being used for transfection.

[0053] Note: For high titers, the health and passage density of 293T cells are crucial factors. Passage should be performed when cell confluence reaches 50%, and large-scale cryopreservation should be carried out while cells are in low passages and growing healthily. Cell clumping should be avoided when passaged and plated for transfection, as cells may grow to a high confluence before plasmid transfection.

[0054] 2. 293T cell transfection: (1) Before transfection, check the host cells passaged two days ago. They should reach 70-80% confluence. Replace DMEM medium with 15mL Optim-MEM medium.

[0055] (2) Add 60 μg of the recombinant plasmid DNA obtained in Example 1 to an EP tube containing 1.5 mL of 150 mM NaCl and mix gently.

[0056] (3) Add 100 μL of PEI max transfection reagent to the DNA solution and vortex for 5 s; (4) The transfection system is shown in Table 5. The solution was placed in a clean bench and incubated for 15 min to form the PEI max-DNA complex.

[0057] Table 5 Transfection System

[0058] (5) Gently blow up and down the transfection system of (4) three times with a pipette.

[0059] (6) While shaking the cell culture dish, add the PEI-DNA complex dropwise into the dish to ensure that the PEI-DNA complex is evenly distributed and to prevent the local concentration from being too high.

[0060] (7) Replace with fresh DMEM medium containing 10% serum 22 hours after transfection.

[0061] 3. Purification of the rAAV5.shPLANE virus: Procedure for purifying AAV virus using column chromatography: 3.1 Preparation of AAV extract (1) Add 1 / 80 of the culture medium volume of 0.5 M EDTA (pH=8.0) to the culture medium containing AAV packaged cells, mix thoroughly, and react at room temperature for 10 min.

[0062] (2) Transfer cells from the culture dish to a centrifuge tube.

[0063] (3) Centrifuge at 2000 rpm and 4℃ for 10 min, then remove the supernatant; (4) Centrifuge at 2000 rpm and 4℃ for 1 min to completely remove the supernatant; Note: Try to remove the supernatant completely to avoid affecting virus purification due to residual supernatant.

[0064] (5) The cell precipitate is loosened by tapping the tube wall or shaking.

[0065] Note: If the cell pellet is not completely loosened, purification efficiency may be reduced. Before proceeding to the next step, ensure there are no cell clumps.

[0066] (6) Add 10 mL of AAV Extraction Solution A plus.

[0067] (7) Vortex for 15 s to fully suspend the cells. Note: Vortex until there are no cell clumps.

[0068] (8) After standing at room temperature for 5 min, vortex oscillate for 15 s; (9) Centrifuge at 2000 rpm and 4℃ for 10 min. Repeating steps (7)-(9) above can improve efficiency.

[0069] (10) Recycle the supernatant into a new sterile centrifuge tube to avoid contamination. Add 1 / 10 of the supernatant volume of AAVExtraction Solution B.

[0070] Note: The solution can be stored at -80°C at this point. If not stored, proceed to the next step immediately. If stored at -80°C, dissolve quickly in a 37°C water bath before use.

[0071] Note: AAV Extraction Solution A plus and AAV Extraction Solution B are components in the AAV purification kit.

[0072] 3.2 Purification and Concentration of AAV Virus (1) Cryonase Cold active Nuclease was added to the solution from the previous step, with a final concentration of 200 U / mL, and the reaction was carried out at 37°C for 1 hour.

[0073] (2) According to the final volume of (1): Precipitator A is 10:1, add Precipitator A, shake and mix for 10 s, react at 37℃ for 30 min, and then shake and mix for 10 s; (3) According to the final volume of (2): Precipitator B is 20:1, add Precipitator B, shake and mix rapidly for 10 s, centrifuge at 5000-9000 rpm at 4℃ for 5 min; Note: Precipitator A and Precipitator B are components in the AAV purification kit. Adding Precipitator B will produce a precipitate, which needs to be centrifuged.

[0074] (4) Filter the supernatant using a 0.45 μm filter membrane.

[0075] (5) Add the filtered AAV solution to an Amicon Ultra-15, 100 kDa filter and centrifuge at 2,000 rpm at 15°C for 5 min until the AAV solution in the filter cup is less than 1.5 mL.

[0076] (6) After removing the filtrate, add 5 mL of Suspension Buffer to the Amicon Ultra-15 filter unit, mix well by pipetting, and centrifuge at 2000 rpm at 15℃ for 5 min. Continue until the AAV solution in the filter unit is less than 1.5 mL.

[0077] (7) Repeat the previous step 4 times (5 times in total), and finally centrifuge to concentrate to 500 μL.

[0078] (8) Remove the filtrate, blow or shake for 30 s to fully suspend it, and then transfer the AAV solution in the Amicon Ultra-15, 100 kDa filter cup to a new EP tube.

[0079] Note: Suspension Buffer is a component of the AAV purification kit.

[0080] Example 4 Production and purification of recombinant adeno-associated virus rAAV5.shPLANE 2: Unlike Example 3, this example uses the endotoxin-free plasmid extraction kit (DP120) to extract plasmids from the clones obtained in Example 2.

[0081] The other steps are the same as in Example 3, and will not be repeated here.

[0082] The following specific experimental examples illustrate the beneficial effects of the scAAV5.PLANE.shRNA recombinant plasmid of this invention in inhibiting tumors: The rAAV5.shPLANE 1 and rAAV5.shPLANE 2 used in the following experimental examples were prepared in Examples 3 and 4.

[0083] Experimental Example Evaluation of the in vivo antitumor effect of rAAV5.shPLANE.

[0084] I. Establishment of a nude mouse subcutaneous xenograft model of the NCI-H226 lung squamous cell carcinoma (LUSC) cell line.

[0085] 1. Tumor cell inoculation dosage: NOD-SCID mice, each mouse was inoculated with 8 × 10⁸ tumor cells. 6 One virus, PBS medium, 160 μL cell volume, transferred on ice to keep the cells in a low metabolic state.

[0086] 2. Inoculation site: Right lower abdomen of mouse.

[0087] 3. Inoculation method: Disinfect the injection site with an alcohol swab, use a 1 mL insulin syringe, insert the needle at a 30° angle to the cut surface, slide it a short distance under the skin to the right lower abdomen, slowly inject the cells, and slowly withdraw the needle by rotating the needle; use standard operating procedures during injection to ensure that the cell suspension is evenly distributed and that each mouse receives the same number of cells.

[0088] 4. Confirmation of tumor formation: Continuously observe the texture and size of the tumor. When the tumor hardens, reaches a diameter of 5-6 mm, and a volume of 100 mm², it is considered a tumor formation. 3 They believed it to be a tumor.

[0089] II. Intratumoral injection Mice were randomly divided into four groups (n=11), injected every 6 days, and tumor volume was measured periodically.

[0090] ①PBS group: 25μL of PBS was injected into the tumor once.

[0091] ② rAAV5.shctrl group (negative virus control group): intratumoral injection of rAAV5.shctrl, each time containing 2x10 11 GC / 25μL viral particles. The rAAV5.shctrl recombinant virus was constructed using the blank expression vector XM19B, and the construction process was the same as in Example 3.

[0092] ③ rAAV5.shPLANE1 group: intratumoral injection of rAAV.shPLANE 1, each time containing 2x10 11 GC / 25μL viral particles.

[0093] ④ rAAV5.shPLANE2 group: intratumoral injection of rAAV.shPLANE 2, each time containing 2x10 11 GC / 25μL viral particles.

[0094] The tumor volume in mice was observed, measured, and recorded every 6 days. The major and minor diameters of the tumor were measured using calipers, and the tumor volume was calculated (V = 0.5 × major diameter × minor diameter). 2 The changes in tumor volume in each group are shown in Table 6 (mean ± standard deviation).

[0095] Table 6. Tumor volume changes in each group (unit: mm) 3 )

[0096] As shown in Table 6, compared with the PBS group and the negative virus control group, the tumor volume growth of the rAAV5.shPLANE 1 and rAAV5.shPLANE2 treatment groups was significantly inhibited (P<0.05), which proves that the recombinant virus of the scAAV5.PLANE.shRNA recombinant plasmid carrying the shRNA sequence targeting the PLANE gene constructed in this invention has good anti-tumor effect.

[0097] As can be seen from the data changes in Table 6 above, rAAV5.shPLANE-mediated gene silencing initially produced a strong anti-tumor effect, but this effect is temporary and incomplete. Over time, the adaptive resistance of tumor cells, the weakening of the gene silencing effect, and the presence of tumor stem cells / heterogeneity may collectively lead to tumor regeneration and growth rebound.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant plasmid for suppressing PLANE gene expression, characterized in that, It contains the nucleotide sequence of shRNA encoding the PLANE gene.

2. The recombinant plasmid for inhibiting PLANE gene expression according to claim 1, characterized in that, The shRNA sequence is selected from any one of the nucleotide sequences shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The recombinant plasmid for inhibiting PLANE gene expression according to claim 2, characterized in that, Its vector is scAAV5.

4. The recombinant plasmid for inhibiting PLANE gene expression according to claim 3, characterized in that, The scAAV5 carrier is carrier XM19B.

5. The recombinant plasmid for inhibiting PLANE gene expression according to claim 4, characterized in that, Its base sequence is shown in SEQ ID NO:

3.

6. A method for preparing a recombinant plasmid for inhibiting PLANE gene expression as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Prepare the target gene fragment encoding the shRNA targeting the PLANE gene; S2. The expression vector is double-digested to obtain a linearized vector fragment; S3. The target gene fragment from step S1 is ligated with the linearized vector fragment obtained in step S2 to obtain the recombinant plasmid used to suppress PLANE gene expression.

7. The method for preparing the recombinant plasmid for inhibiting PLANE gene expression according to claim 6, characterized in that, In step S2, the restriction endonucleases used for double digestion are HindIII and KpnI.

8. The method for preparing the recombinant plasmid for inhibiting PLANE gene expression according to claim 6, characterized in that, In step S3, the ligation reaction is carried out using the T4 DNA ligase system at 20℃-25℃ for 3-5 hours.

9. A recombinant adeno-associated virus, characterized in that, The recombinant plasmid for inhibiting PLANE gene expression as described in any one of claims 1-5 is obtained by a viral packaging system.

10. The recombinant adeno-associated virus according to claim 9, characterized in that, It was obtained by co-transfecting 293T cells with the recombinant plasmid used to inhibit PLANE gene expression and the packaging plasmid, followed by culture, collection, and purification.

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