SaRNA, application thereof, saRNA vaccine, method and medicine composition

Abstract

The invention relates to saRNA (Ribonucleic Acid) and application thereof, a saRNA vaccine, a method and a pharmaceutical composition, the saRNA for coding HSV (Herpes Saturation Virus) ICP34.5 is constructed, the translation and expression capability of a gene can be obviously enhanced, the use of the saRNA in a gene delivery system is optimized, a more excellent application carrier is provided for vaccine and gene therapy, the antiviral innate immune response of a host can be inhibited by coding ICP34.5 and saRNA, and the anti-virus innate immune response of the host can be inhibited by coding ICP34.5 and saRNA. According to the present invention, the expression and the translation of the gene are enhanced by inhibiting PKR and IFN signal channels, particularly by inhibiting PKR and IFN signal channels and reducing translation closure, the addition of the ICP34.5 is beneficial to prolonging the expression time of the gene of interest and providing the lasting treatment effect, and the integration of the ICP34.5 ensures the same immune escape characteristic of the offspring saRNA, optimizes the gene delivery system, and improves the efficiency and the effect of the offspring saRNA. By coding ICP34.5 and saRNA, strong innate immune response can be reduced, side effects in gene therapy can be reduced, and a therapeutic window can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to saRNA and its application, saRNA vaccine, method and drug combination. Background Art

[0002] The development of self-replicating RNA (saRNA) has introduced a revolutionary approach to vaccine design and gene therapy. Currently, several vaccines based on saRNA are undergoing clinical trials, showing good safety and efficacy. saRNA is derived from viruses of the Alphaviridae family, such as Venezuelan equine encephalitis virus (VEEV), Sendbi Forest virus (SFV) and Sindbis virus, which are characterized by their ability to replicate genetic material within host cells. The key advantage of saRNA is its ability to replicate itself, which can produce high copy numbers of up to 2 x 10^5 in cells. This feature allows for effective gene delivery and the production of high titers of protective antibodies using less saRNA than traditional mRNA. The self-replication process of saRNA is initiated after transfection, and the encoded replicase is translated and interacts with the 5' and 3' ends of the genomic RNA to synthesize complementary genomic RNA copies. These copies serve as templates for the synthesis of new positive-strand, capped and polyadenylated genomic copies and subgenomic transcripts, resulting in high production of antigens in vivo. In terms of vaccine development, saRNA has been shown in preclinical models to be superior to non-replicating mRNA and DNA vaccines because it can provide very high expression levels and simultaneously induce a strong innate immune response, thereby enhancing immunity.

[0003] The unique properties of saRNA are a platform for next-generation technologies in vaccines and gene therapy because it can produce high levels of protein from tiny transfection templates, showing significant advantages in terms of efficacy and dosage requirements compared to conventional mRNA. However, the strong innate immune response triggered by saRNA, especially the activation of protein kinase R (PKR) and interferon (IFN) signaling pathways, has been a major obstacle to its full utilization. This immune response is initiated by pattern recognition receptor sensing of double-stranded RNA intermediates formed during saRNA translation, resulting in translation shutdown and hindering the expression of the target gene. Recent studies have shown that incorporating modified nucleosides such as 5-methylcytosine (5mC) into saRNA can inhibit the early interferon response and enhance the expression of saRNA. Despite some progress in this approach, inherent limitations of this approach still exist. Specifically, the progeny saRNA copies generated by the replication of modified saRNA molecules contain wild-type nucleotides because the viral RNA-dependent RNA polymerase (RdRp) only uses wild-type nucleotides for replication. Therefore, while initially modified saRNAs may exhibit properties that enhance gene expression, subsequent rounds of replication that produce wild-type saRNAs are still subject to innate immune responses that restrict antigen expression. Summary of the invention

[0004] In order to solve the defects and shortcomings of the prior art, the present invention provides a saRNA and its application, saRNA vaccine, method and drug combination.

[0005] The technical solution adopted by the present invention is: a saRNA, wherein the saRNA includes a nucleotide sequence complementary to the ICP34.5 protein gene of the herpes simplex virus (HSV).

[0006] The ICP34.5 protein gene is the ICP34.5 protein gene of herpes simplex virus type 1 (HSV-1), and its nucleotide sequence is shown in SEQ ID NO.1.

[0007] The saRNA includes a nucleotide sequence complementary to the -218 to -233 site region of the ICP34.5 protein gene promoter region of the herpes simplex virus (HSV).

[0008] A saRNA vaccine for the ICP34.5 protein gene of the HSV virus, wherein the saRNA vaccine is constructed by inserting a nucleic acid fragment encoding the ICP34.5 protein into an expression vector of the saRNA vaccine.

[0009] The nucleotide sequence of the gene encoding the ICP34.5 protein is SEQ ID NO: 1.

[0010] An application of saRNA in the preparation of gene therapy drugs.

[0011] The gene therapy drug is a drug that enhances the expression of specific genes and prolongs the antigen expression time.

[0012] The gene therapy drug is a drug that inhibits the host's anti-viral innate immune response.

[0013] A method for inhibiting a host antiviral innate immune response triggered by saRNA, wherein the method comprises inhibiting PKR and IFN signaling pathways by saRNA encoding ICP34.5 to enhance the expression of foreign genes and reduce immune-mediated side effects.

[0014] A method for optimizing gene delivery efficiency, wherein the method is to improve gene delivery efficiency and therapeutic effect by integrating the ICP34.5 coding sequence into saRNA.

[0015] A method for prolonging the antigen expression time in gene therapy, wherein the method is to promote the dephosphorylation of eIF2α and prolong the expression time of the gene in the host cell by integrating the ICP34.5 coding sequence into saRNA.

[0016] A drug combination for gene therapy, comprising the saRNA and a vector cell.

[0017] The carrier cells are mesenchymal stem cells, T cells or NK cells.

[0018] A gene delivery system, comprising saRNA capable of activating an ICP34.5 protein gene.

[0019] The beneficial effects of the present invention are as follows: the present invention provides a saRNA and its application, saRNA vaccine, method and drug combination, and the invention has the following advantages: (1) Enhanced gene expression and translation: By encoding ICP34.5, saRNA can inhibit the host's antiviral innate immune response, especially by inhibiting the PKR and IFN signaling pathways and reducing translation shutdown, thereby enhancing gene expression and translation.

[0020] (2) Prolonging the antigen expression time: The addition of ICP34.5 helps to prolong the expression time of the gene of interest, which is crucial for gene therapy and vaccine development because it can provide a longer-lasting therapeutic effect.

[0021] (3) Optimizing the gene delivery system: The integration of ICP34.5 ensures that the subsequent saRNAs have the same immune evasion properties, which may optimize the gene delivery system and improve its efficiency and effectiveness.

[0022] (4) Reduce immune response: By encoding ICP34.5, saRNA can reduce strong innate immune responses, which is of great significance for reducing side effects in gene therapy and increasing the therapeutic window. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of saRNA structure.

[0024] Figure 2 This is the T7-VEE-EGFP plasmid map.

[0025] Figure 3 This is the T7-VEE-ICP34.5 (P2A) EGFP plasmid map.

[0026] Figure 4 This is the plasmid map of T7-VEE-ΔICP34.5 (P2A) EGFP.

[0027] Figure 5 The fluorescence expression of 293T cells transfected with different saRNAs.

[0028] Figure 6 The fluorescence intensity of 293T cells transfected with different saRNAs.

[0029] Figure 7 Fluorescence expression and fluorescence intensity analysis after UC-MSC cells were transfected with different saRNAs.

[0030] Figure 8 The expression levels of eIF2α and its phosphorylated forms in host cells transfected with different saRNAs.

[0031] Fig. 9 Schematic diagram of saRNA structure.

[0032] Fig.10 The expression of type I interferon induced in host cells after transfection with different saRNAs. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and the following embodiments. It should be understood that the accompanying drawings and the following embodiments are only used to illustrate the present invention rather than to limit the present invention.

[0034] Example 1 In vitro transcription synthesis of saRNA (1) Construction of recombination template plasmid The amino acid sequence from position 218 to 233 in the ICP34.5 gene is crucial for its binding to PP1, which mediates the dephosphorylation of eIF2a. Therefore, the present invention designed a deletion mutant of the ICP34.5 gene (ΔICP34.5) lacking amino acids 218-233, thereby disabling its eIF2a dephosphorylation activity, as a control group ( Figure 1 ). It has been reported that small activating RNAs (saRNAs) modified with 5-methylcytosine (5mC) can attenuate early antiviral innate immune responses and enhance gene expression19,20. In the present invention, wild-type (WT) and 5mC-modified saRNAs ( Figure 1 These saRNAs encoded an EGFP reporter gene to assess the effect of intact ICP34.5 on protein expression ( Figure 1 ).

[0035] A. Sequence Design and Gene Synthesis ICP34.5 is present in both type I and type II HSV, with different sequences and similar functions. In the following experiment, the ICP34.5 gene sequence of type I HSV was selected for the experiment; The ICP34.5 gene sequence of HSV-1 KOS strain was searched on the NCBI website, and the codons were optimized for easy expression in mammalian cells. In order to co-express with the EGFP gene, the present invention used a P2A linker to link the ICP34.5 and EGFP genes.

[0036] The optimized ICP34.5 sequence is as follows: GCCACCATGGCCAGAAGGAGAAGGCACAGGGGCCCTAGAAGACCTAGACCTCCAGGCCCTACCGGAGCAGTGCCTACAGCTCAGTCTCAGGTCACCAGCACCCCTAATAGCGAGCCAGCCGTGAGATCTGCTCCAGCTGCAGCTCCACCTCCACCTCCAGCCGGTGGACCTCCACCTTCTTGTTCACTGCTGCTCAGACAGTGGCTGCATGTGCCAGAATCTGCCTCAGACGACGATGACGATGACGATTGGCCAGATAGCCCTCCTCCAGAGCCAGCTCCAGAAGCTAGACCTACAGCCGCAGCTCCGAGACCTAGACCTCCACCTCCAGGAGTGGGACCAGGAGGTGGAGCAGATCCATCTCACCCTCCTAGTAGACCATTCAGACTGCCTCCTAGGCTGGCTCTGAGACTGAGAGTGACCGCAGAGCATCTGGCCAGACTCAGACTGAGGAGGGCAGGAGGTGAAGGAGCTCCAGAACCTCCAGCCACACCAGCAACACCAGCGACACCTGCAACACCAGCCACACCAGCCAGAGTGCGGTTTTCTCCTCACGTGCGAGTGAGACACCTGGTCGTGTGGGCTTCAGCAGCTAGACTGGCCCGAAGAGGCAGTTGGGCTAGAGAGAGAGCCGACAGAGCCAGGTTCAGGAGACGAGTGGCCGAGGCAGAAGCCGTGATCGGACCTTGTCTGGGACCAGAAGCCAGAGCTAGAGCCCTGGCTAGAGGAGCAGGACCAGCCAATAGCGTG Compared with the amino acid sequence of the control ICP34.5 lacking amino acids at positions 218 to 233, the DNA sequence of ΔICP34.5 is as follows: ATGGCCAGAAGGAGAAGGCACAGGGGCCCTAGAAGACCTAGACCTCCAGGCCCTACCGGAGCAGTGCCTACAGCTCAGTCTCAGGTCACCAGCACCCCTAATAGCGAGCCAGCCGTGAGATCTGCTCCAGCTGCAGCTCCACCTCCACCTCCAGCCGGTGGACCTCCACCTTCT TGTTCACTGCTGCTCAGACAGTGGCTGCATGTGCCAGAATCTGCCTCAGACGACGATGACGATGACGATTGGCCAGATAGCCCTCCTCCAGAGCCAGCTCCAGAAGCTAGACCTACAGCCGCAGCTCCGAGACCTAGACCTCCACCTCCAGGAGTGGGACCAGGAGGTGGAGCA GATCCATCTCACCCTCCTAGTAGACCATTCAGACTGCCTCCTAGGCTGGCTCTGAGACTGAGAGTGACCGCAGAGCATCTGGCCAGACTCAGACTGAGGAGGGCAGGAGGTGAAGGAGCTCCAGAACCTCCAGCCACACCAGCAACACCAGCGACACCTGCAACACCAGCCACA CCAGCCAGAGTGCGGTTTTCTCCTCACGTGCGAGTGAGACACCTGGTCGTGTGGGCTTCAGCAGCTAGACTGGCCCGAAGAGGCAGTTGGGCTAGAGAGAGCCGACAGAGCCAGGTTCAGGAGACGAAGAGCTAGAGCCCTGGCTAGAGGAGCAGGACCAGCCAATAGCGTG The P2A linker sequence is as follows: GGAAGCGGAGCTACTAACTTCAGCCTGCTAAAGCAGGCTGGAGACGTGGAGGAAAACCCTGGACCT B. The above ICP34.5 or ΔICP34.5 and P2A were concatenated and sent to Shanghai Langjing Biotechnology Co., Ltd. to synthesize the ICP34.5-P2A sequence, and then constructed into the T7-VEE-GFP plasmid by seamless cloning ( Figure 2 ), thereby obtaining T7-VEE-ICP34.5 (P2A) EGFP and T7-VEE-ΔICP34.5 (P2A) EGFP recombinant plasmids ( Figure 3 and Figure 4 ); (2) Preparation of linearized template for in vitro transcription A. Plasmid linearization: Take 5ug of small amount of extracted plasmid T7-VEE-ICP34.5 (P2A) EGFP or T7-VEE-ΔICP34.5 (P2A) EGFP, add 5uL of restriction enzyme Mlu I, and incubate at 37℃ for 30 min to fully digest. The reaction system is as follows: Plasmid 5 ug 10x NEBuffer25 uL 5U H2O200uL Total volume 250 uL B. Linearized plasmid recovery: Follow the instructions of the kit (Suzhou Uni-Easy Biotechnology Co., Ltd., PCR Cleanup Kit, #UE-PCR-50), as follows: a. Add 3 volumes of Buffer PCR-A to the digested product, i.e. 750uL Buffer PCR-A, mix well and transfer to the preparation tube. Place the preparation tube in a 2 mL centrifuge tube, centrifuge at 12000g for 1 min, and discard the filtrate. b. Place the preparation tube back into a 2 mL centrifuge tube, add 700 uL Buffer W2, centrifuge at 12000 g for 1 min, and discard the filtrate; c. Place the preparation tube in a clean 1.5 mL centrifuge tube, add 30uL of Eluent to the center of the preparation tube membrane, let stand at room temperature for 1 min, and centrifuge at 12000g for 1 min to elute the DNA.

[0037] d. Measure the concentration of the linearized plasmid template and store it at 4 degrees Celsius for later use.

[0038] (3) In vitro transcription of saRNA by wild-type CTP and 5-methyl CTP Follow the instructions of the kit (HiScribe T7 High Yield RNA synthesis Kit, NEB #E2040S): a. Thawing of reagents: Centrifuge T7 RNA Polymerase Mix briefly and place on ice. Thaw 10xTranscription Buffer and ribonucleotides (ATP, CTP, GTP, N-Me-PUTP, CAP GAG), mix and centrifuge to the bottom of the tube, place 10xTranscription Buffer at room temperature, and place ribonucleotides on ice for later use.

[0039] b. Room temperature assembly transcription reaction Configure the reaction system according to the following system

[0040] c. React at 37℃ for 2h: Mix the above reaction solution, centrifuge briefly to the bottom of the centrifuge tube, and bathe at 37℃ for 2h; d. DNase I treatment: After the reaction, add 2uL DNase I to each tube and incubate at 37℃ for 15min to remove the DNA template.

[0041] (4) Purification of in vitro transcribed saRNA Follow the instructions of the kit (GeneJET RNA purification Kit, Thermo Fisher, #K0731): a. Adjust the volume of the reaction mixture in step 2) to 100 uL with RNase-free water (included in the kit). Add 300 uL of lysis buffer without β-mercaptoethanol or DTT. Mix thoroughly by vortexing.

[0042] b. Add 180 uL of anhydrous ethanol and mix by pipetting. Transfer the mixture to the GeneJET RNA Purification Column inserted into the collection tube. Centrifuge at ≥12,000 xg for 1 min. Discard the collection tube containing the flow-through. Place the GeneJET RNA Purification Column into a new 2 mL collection tube (included in the kit). Note: Seal the bag containing the GeneJET RNA Purification Column tightly after each use! c. Add 700 uL of Elution Buffer 1 (containing ethanol, see page 3 for details) to the GeneJET RNA Purification Column and centrifuge at ≥12,000 xg for 1 minute. Discard the flow-through and return the purification column to the collection tube.

[0043] d. Add 600 uL of Elution Buffer 2 (containing ethanol, see page 3 for details) to the GeneJET RNA purification column and centrifuge at ≥12,000 xg for 1 minute. Discard the flow-through and return the purification column to the collection tube.

[0044] e. Add 250 uL of Elution Buffer 2 to the GeneJET RNA Purification Column and centrifuge at ≥12,000 xg for 2 min. Optional: If there is still residual solution in the purification column, empty the collection tube and re-centrifuge the column at maximum speed for 1 min. Discard the collection tube containing the flow-through and transfer the GeneJET RNA Purification Column to a sterile 1.5 mL nuclease-free microcentrifuge tube (included in the kit).

[0045] f. Add 50 uL of nuclease-free water to the center of the GeneJET RNA Purification Column membrane. Centrifuge at ≥12,000 xg for 1 min to elute the RNA. Note: To obtain maximum RNA yield, repeat the elution step with an additional 50 mL of nuclease-free water.

[0046] g. Discard the purification column. The purified saRNA can be used directly in downstream experiments or stored in a -80°C refrigerator for later use.

[0047] According to the above experimental steps, the present invention obtained purified WT-ΔICP34.5 / EGFP saRNA, 5mC-ΔICP34.5 / EGFP saRNA and 5mC-ICP34.5 / EGFP saRNA.

[0048] Example 2 saRNA Function Verification Since HEK293 cells lack STAT2 and IRF9, they cannot form a functional ISGF3 complex, resulting in a weakened response to type I interferon, thereby promoting high-level protein expression from saRNA27. Therefore, the above-mentioned 1. synthesized WT-ΔICP34.5 / EGFP saRNA, 5mC-ΔICP34.5 / EGFP saRNA and 5mC-ICP34.5 / EGFP saRNA were transfected into 293T cells respectively. The specific method is as follows: Cell transfection a. 293T cells were measured at 7.5x10 4 / well were inoculated into 24-well plates and incubated overnight in a 37°C, 5% CO2 incubator; b. Perform transfection according to the instructions of CALNP™ mRNA in vitro transfection reagent (Beijing Dona Pharmaceutical Technology Co., Ltd., #DN002-05); Note: saRNA is transfected at 200 ng / well; 1. Preparation of saRNA transfection complex solution: Reagent A and Reagent B are restored to room temperature in advance and vortexed. Strictly mix and prepare saRNA transfection complex solution in the order.

[0049] Add 200 ng saRNA solution to a clean RNase-free EP tube; add 7 uL Reagent A to the above step i and mix; add 2 uL Reagent B to the above step ii, mix thoroughly, and incubate at room temperature for 10 min; add 90 uL culture medium to the above step iii, mix thoroughly to obtain saRNA transfection complex solution; 2. Cell dosing: add the prepared saRNA transfection complex solution to each cell well, shake the culture plate, and mix gently. 3. Place the cells in a 37°C, 5% CO2 incubator and continue incubating.

[0050] 24 hours after transfection, photos were taken to record the fluorescence expression, and the EGFP expression level was detected by flow cytometry.

[0051] Results analysis: The present invention found that all three types of saRNA can express high levels of EGFP in 293T cells ( Figure 5 ). Flow cytometry revealed that the fluorescence intensity of 293T cells transfected with 5mC-ICP34.5 / EGFP saRNA was significantly higher than that of WT-ΔICP34.5 / EGFP saRNA and 5mC-ΔICP34.5 / EGFP saRNA transfection groups, and the levels in all samples were comparable ( Figure 6 A and 6B). These findings indicate that WT and 5mC-modified saRNAs encoding wild-type ICP34.5 or deletion mutant ICP34.5 and EGFP synthesized by in vitro transcription were successfully prepared, and saRNA encoding fully functional ICP34.5 was able to significantly enhance gene expression.

[0052] In order to confirm that ICP34.5 can also enhance gene expression in primary cells, the present invention transfected WT-ΔICP34.5 / EGFP saRNA, 5mC-ΔICP34.5 / EGFP saRNA and 5mC-ICP34.5 / EGFP saRNA into umbilical cord-derived mesenchymal stem cells (UC-MSCs) respectively. The specific method is as follows: Cell transfection a. UC-MSC cells were collected at 4x10 4 / well were inoculated into 24-well plates and incubated overnight in a 37°C, 5% CO2 incubator; b. Perform transfection according to the instructions of CALNP™ mRNA in vitro transfection reagent (Beijing Dona Pharmaceutical Technology Co., Ltd., #DN002-05); Note: saRNA is transfected at 200ng / well. The method is the same as above; c. Take photos and record the fluorescence expression 24, 48 and 72 hours after transfection. Collect cells 96 hours after transfection for flow cytometry detection of fluorescence expression and fluorescence intensity analysis.

[0053] Consistent with the results in 1) above, saRNA encoding functionally intact ICP34.5 expressed higher levels of EGFP in UC-MSC cells compared with the deletion mutation group ( Figure 7 These results again demonstrate that genes encoding ICP34.5 are able to enhance expression of saRNA vector-delivered genes.

[0054] Example 3 ICP34.5 mediates eIF2α dephosphorylation During HSV-1 infection, ICP34.5 protein binds to PP1 and eIF2α, mediates the dephosphorylation of eIF2α, and promotes protein synthesis in host cells. In order to evaluate the effect of saRNA encoding ICP34.5 on the level of phosphorylated eIF2α, the present invention transfected three different saRNAs into UC-MSCs with a complete interferon signaling pathway, and the control group cells were not transfected with any RNA. WB analysis was performed 24 hours after transfection to evaluate the expression level of eIF2α and its phosphorylated form. The specific method is as follows: 1) Cell transfection and preparation of cell lysates a. UC-MSC cells were collected at 4x10 4 / well were inoculated into 24-well plates and incubated overnight in a 37°C, 5% CO2 incubator; b. Perform transfection according to the instructions of CALNP™ mRNA in vitro transfection reagent (Beijing Dona Pharmaceutical Technology Co., Ltd., #DN002-05); Note: saRNA is transfected at 200 ng / well; the method is the same as above; c. Remove the culture supernatant 24 hours after transfection and wash twice with PBS d. Add 1 mL of trypsin to digest the cells, terminate the digestion with 4 ml of complete medium, centrifuge at 1000 rpm for 5 min, remove the supernatant, and wash twice with PBS; e. Add 1 mL Western and IP cell lysis buffer (Beyotime, #P0013) containing 10 uL PMSF (Beyotime, #ST506); Note: The final concentration of PMSF is 1 mM; f. According to 1.5x10 6 Add 120uL of the above lysis solution to each cell and incubate on ice for 15 min; g. After sufficient lysis, centrifuge at 10,000 g for 5 minutes and carefully aspirate the supernatant; h. Add appropriate amount of 5x Loading buffer to the supernatant of step G and boil at 100℃ for 10 min. i. Store in -80℃ refrigerator for future use; 2) Western Blot a. Take 20 μL of each of the four cell lysates mentioned above, load them onto 10% SDS-PAGE, and perform electrophoresis at 120 V for 90 min. b. Transfer the proteins in SDS-PAGE to PVDF membrane by constant current of 350 mA for 120 min; c. Place the transferred PVDF membrane in a blocking solution containing 5% skim milk at 4°C overnight; d. Cut the blocked PVDF membrane into two parts, and add 10 ml of eIF2α Rabbit mAb (ABclone, #A21221) and Phospho-eIF2α-S51 Rabbit mAb (ABclone, #AP0692) and 10 ml of GAPDH antibody (Cat. No. 39-8600, Thermo Fisher) into each part, and incubate at room temperature with shaking for 1 hour. e. Wash the PVDF membrane incubated with primary antibody 4 times with TBST, 5 minutes each time f. Place the washed PVDF membrane in 10 mL of horseradish peroxidase-labeled Goat Anti Rabbit IgG H+L secondary antibody (Abcam, #ab6702) and incubate at room temperature for 50 min. g, TBST washing 3 times, 5 min each time; h. After adding ECL ultrasensitive colorimetric solution, take pictures using Tianneng imaging system (Shanghai Tianneng Technology Co., Ltd., #Tanon-4600SF); Result analysis: The present invention observed that the expression of eIF2α in all transfection groups was consistent ( Figure 8 A). 5mC-modified saRNA induced a slight decrease in eIF2α phosphorylation compared to wild-type saRNA ( Figure 8 A and 8B). This result indicates that 5mC-modified saRNA inhibits the early antiviral immune response, resulting in a decrease in the phosphorylation level of eIF2α. The saRNA transfection group encoding the complete ICP34.5 protein had the lowest eIF2α phosphorylation level in host cells ( Figure 8 A and 8B). These findings indicate that saRNA encoding ICP34.5 can promote the dephosphorylation of eIF2α, thereby indicating that saRNA encoding ICP34.5 can prolong the expression time of genes in host cells.

[0055] Example 4 saRNA encoding ICP34.5 induces low-level innate immune response Previous studies have shown that ICP34.5 inhibits interferon production by binding to and sequestering TANK-binding kinase 1 (TBK-1). Therefore, the enhanced green fluorescence caused by saRNA encoding ICP34.5 can be attributed, at least in part, to its inhibition of interferon expression in host cells. In order to evaluate the effect of ICP34.5 on saRNA-induced type I interferon expression, the present invention constructed saRNA encoding or not encoding ICP34.5 ( Fig. 9) and transfected into 293T or UC-MSCs. Cells were collected 24 hours after transfection and RT-PCR was performed to evaluate the mRNA levels of IFNa2 and IFNb in host cells. The specific method is as follows: 1. The transfection method and conditions are the same as steps 2 and 3 above. Extraction of total cellular RNA Before you begin: Add β-mercaptoethanol to the desired lysis buffer. Add 20uL of 14.3 M β-mercaptoethanol per 1mL of lysis buffer.

[0056] A. Rinse cells once with PBS to remove residual media. Remove and discard PBS. Detach cells from culture plate by trypsinization. Transfer cells to microcentrifuge tubes and centrifuge at 250 x g for 5 min, discarding supernatant. Note: Pelleted cells can be used directly for RNA isolation or stored at -70°C until use.

[0057] B. Resuspend the cells collected in step A in 600 uL of lysis buffer containing β-mercaptoethanol. Vortex for 10 seconds to mix thoroughly. Note: If the mixture becomes thick and viscous after adding the lysis buffer, homogenize the sample. Homogenize the lysate for 30 seconds using a rotary-stator homogenizer or pipette the lysate through a blunt 20-gauge needle mounted on a nuclease-free syringe multiple times. Incomplete homogenization will result in significantly reduced RNA yield.

[0058] C. Add 360uL of ethanol (96-100%) and mix the sample by pipetting.

[0059] D. Transfer approximately 700 uL of the lysate to a GeneJET RNA purification column inserted into a collection tube and centrifuge the column at ≥12,000 x g for 1 min. Discard the flow-through and place the purification column back into the collection tube. Repeat this step until all the lysate has been transferred to the column and centrifuged.

[0060] E. Discard the collection tube containing the flow-through and place the GeneJET RNA Purification Column into a new 2 mL collection tube. Note: Seal the bag containing the GeneJET RNA Purification Column tightly after each use! F. Add 700uL of Elution Buffer 1 (containing ethanol) to the GeneJET RNA Purification Column and centrifuge at ≥12,000 xg for 1 minute. Discard the flow-through and return the purification column to the collection tube.

[0061] G. Add 600 uL of Elution Buffer 2 (containing ethanol) to the GeneJET RNA Purification Column and centrifuge at ≥12,000 xg for 1 minute. Discard the flow-through and return the purification column to the collection tube.

[0062] H. Add 250 uL of Elution Buffer 2 to the GeneJET RNA Purification Column and centrifuge at ≥12,000 xg for 2 min. If there is still residual solution in the purification column, empty the collection tube and re-centrifuge the column at maximum speed for 1 min. Discard the collection tube containing the flow-through and transfer the GeneJET RNA Purification Column to a sterile 1.5 mL RNase-free microcentrifuge tube.

[0063] I. Add 50 uL of RNase-free double-distilled water to the center of the GeneJET RNA purification column membrane. Centrifuge at ≥12,000 xg for 1 min to elute the RNA. Discard the purification column and use the extracted RNA directly for downstream experiments or store in a -80°C refrigerator for later use.

[0064] RNA reverse transcription to cDNA Follow the instructions of PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA, #RR047A) for the following specific steps: a. Removal of genomic DNA Prepare the reaction mixture on ice according to the following ingredients. To ensure the accuracy of the reaction mixture preparation, when performing each reaction, prepare the Master Mix according to the amount of reaction number + 2, then dispense it into each reaction tube, and finally add the RNA sample.

[0065]

[0066] React at 42°C for 2 min, then transfer to 4°C; b. Reverse transcription reaction The reaction solution was prepared on ice. To ensure the accuracy of the reaction solution, the Master Mix should be prepared according to the amount of reaction number + 2 for each reaction, and then 10 μl should be dispensed into each reaction tube. After gentle mixing, reverse transcription reaction was immediately performed. The reaction system is as follows:

[0067] React at 37℃ for 15 min, incubate in 85℃ metal bath for 5 sec to inactivate the enzyme; finally transfer to -20℃ for storage; c. RT-PCR detection 500 ng of cDNA was taken and real-time quantitative PCR was performed using the Applied Bio Systems 7500 Sequence Detection System (AppliedBiosystems). The reaction procedure was: 95°C 30 s; followed by 95°C 5 s and 60°C 34 s for a total of 40 cycles; the melting curve reaction conditions were: 95°C 15 s; 60°C 1 min; 95°C 15 s; the primer sequences used for RT-PCR were as follows:

[0068] Results: The present invention found that compared with the wild-type saRNA treatment group, 5mC-modified saRNA reduced the mRNA level of IFNα2 in UC-MSC transfected cells ( Fig.10 However, we did not observe such differences in 293T cells ( Fig.10 A). At the same time, the present invention observed that in both cell types, IFNb mRNA levels were significantly reduced after transfection with 5mC-modified saRNA ( Fig.10 A and 10B). The addition of 5mC-modified saRNA encoding ICP34.5 further alleviated IFNβ in both 293T and UC-MSCs, whereas whether 5mC-modified saRNA contained ICP34.5 did not affect the transcription level of IFNα2 ( Fig.10 A and 10B). Overall, these results suggest that saRNA containing ICP34.5 induces lower innate immune responses in host cells and is safer for use in vaccines.

[0069] Technical personnel should note that although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention, and any modification using the inventive concept will be included in the scope of protection of this patent.

[0070] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A saRNA, characterized in that The saRNA includes a nucleotide sequence complementary to the ICP34.5 protein gene of the herpes simplex virus (HSV).

2. The saRNA according to claim 1, characterized in that The ICP34.5 protein gene is the ICP34.5 protein gene of herpes simplex virus type 1 (HSV-1), and its nucleotide sequence is shown in SEQ ID NO.

1.

3. The saRNA according to claim 1, characterized in that The saRNA includes a nucleotide sequence complementary to the -218 to -233 site region of the ICP34.5 protein gene promoter region of the herpes simplex virus (HSV).

4. A saRNA vaccine of the HSV virus ICP34.5 protein gene, characterized in that: The saRNA vaccine is constructed by inserting a nucleic acid fragment encoding the ICP34.5 protein into an expression vector of the saRNA vaccine.

5. The saRNA vaccine according to claim 4, characterized in that The nucleotide sequence of the gene encoding the ICP34.5 protein is SEQ ID NO:

1.

6. Use of the saRNA according to claim 1 in the preparation of gene therapy drugs.

7. The use according to claim 6, characterized in that: The gene therapy drug is a drug that enhances the expression of specific genes and prolongs the antigen expression time.

8. The use according to claim 6, characterized in that: The gene therapy drug is a drug that inhibits the host's anti-viral innate immune response.

9. A method for inhibiting saRNA-triggered host antiviral innate immune response, characterized in that: The method described is to inhibit PKR and IFN signaling pathways by saRNA encoding ICP34.5 to enhance the expression of foreign genes and reduce immune-mediated side effects.

10. A method for optimizing gene delivery efficiency, characterized in that The method described is to improve gene delivery efficiency and therapeutic effect by integrating the ICP34.5 coding sequence into saRNA.

11. A method for prolonging the antigen expression time in gene therapy, characterized in that: The method described is to integrate the ICP34.5 coding sequence into saRNA to promote the dephosphorylation of eIF2α and prolong the expression time of the gene in the host cell.

12. A drug combination for gene therapy, characterized in that: The drug combination comprises the saRNA according to claim 1 and a carrier cell.

13. The pharmaceutical combination according to claim 12, characterized in that The carrier cells are mesenchymal stem cells, T cells or NK cells.

14. A gene delivery system, characterized in that The gene delivery system includes saRNA that can activate the ICP34.5 protein gene.

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