Construction and application of multifunctional self-replicating vector taking yellow virus as skeleton

Abstract

The invention provides a yellow virus replicon (sef-replicating mRNA (messenger ribonucleic acid), srRNA (srRNA)) vector with a plurality of structures aiming at functional localization of different therapeutic proteins. Recombinant sites and structural characteristics of the srRNAs and carrying target genes are explained in detail, therapeutic proteins are expressed according to functions of the therapeutic proteins and are correctly distributed inside and outside cells or embedded into cell membranes, the half-life period and stability of srRNA expression are improved, and the srRNAs are effectively applied to gene therapy of diseases.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering and biomedicine, and in particular to nucleic acid molecules for gene modification of Flavivirus self-replicating vectors and recombinant foreign genes. It also provides a variety of Flavivirus replicon vectors that express intracellular or transmembrane proteins according to the functional localization and distribution of different gene products, as tools and applications for effective nucleic acid gene therapy. Background Art

[0002] Gene vector-based nucleic acid drug therapy is an advanced medical technology that regulates or repairs gene expression by introducing specific nucleic acids (such as DNA or RNA) into cells to achieve the purpose of treating diseases. It has made significant progress in recent years and is widely used in the fields of tumor treatment, genetic diseases, infectious diseases, nervous system diseases and cardiovascular diseases. For example, the AAV 9 vector gene drug that has been approved for the treatment of spinal muscular atrophy is the development of a drug that uses gene editing tools such as CRISPR-Cas9 to precisely target tumor cells (Nat Rev Drug Discov. 2020 Jun;19(6):367-372.); mRNA vaccines have performed well in the COVID-19 pandemic (Nat Rev Drug Discov 17, 261–279 (2018)); and siRNA and miRNA have shown potential in antiviral and neurodegenerative disease drugs. At present, gene therapy uses nucleic acid delivery vectors (common lipid nanoparticles, LNPs) to deliver nucleic acid drugs into cells to complete gene modification and / or perform therapeutic functions according to the central dogma of genetics that DNA transcribes mRNA and translates proteins. Therefore, the delivery efficiency and mRNA translation efficiency directly determine the expression of the target protein. However, the delivery efficiency of the vector and its mediated efficient expression of protein drugs are still the two major bottlenecks in the development of nucleic acid drugs, which need further research and solution.

[0003] The types of non-infectious viral gene vectors are shown in Table 1, including DNA (adeno-associated virus vectors, cDNA, etc.) and RNA (mRNA, srRNA, etc.) vectors. The diversity of these vectors in chemical structure and function provides a wide range of application possibilities for gene therapy. (AAPS J 23, 78 (2021))

[0004] Table 1 Comparison of characteristics of vector-based nucleic acid drugs

[0005]

[0006] As shown in Table 1, AAV vectors can provide persistent gene expression, which is beneficial for the treatment of chronic diseases. Various serotypes of AAV can be used for targeted gene therapy for specific tissues, but they are immunogenic, have limited ability to carry long fragments or multiple genes, and replicate in the cell nucleus, with the risk of random integration into the host genome. The main AAV vector gene therapy products currently on the market include Luxturna and Zolgensma. Luxturna was developed by Spark Therapeutics for the treatment of inherited retinal diseases caused by RPE65 gene mutations, which can significantly improve patients' vision. Zolgensma was developed by AveXis (a subsidiary of Novartis) for the treatment of spinal muscular atrophy (SMA), and the provision of replacement genes through a one-time intravenous injection greatly improved the patient's motor function and survival rate. However, compared with DNA or mRNA vector nucleic acid drugs, AAV production is more complex and costly, and pre-existing immunity to AAV reduces the efficacy of gene therapy.

[0007] Compared with AAV vectors, DNA vectors transcribe mRNA in the cell nucleus, and the probability of integration into the host genome is very low. There is no risk of insertion mutagenesis, and they can be stably mass-produced, making it easy to quickly construct and produce drugs for different indications. Marketed DNA vector gene therapy products include Gendicine and Neovasculgen. Gendicine is the world's first approved gene therapy drug. It restores tumor suppressor function by carrying the p53 gene, thereby inhibiting tumor growth, and is used to treat squamous cell carcinoma of the head and neck. Neovasculgen promotes angiogenesis and improves blood circulation by carrying the VEGF gene, and is used to treat peripheral arterial disease. However, an effective delivery method is still needed to ensure that the DNA plasmid is absorbed by the target cells, and the expression cycle is usually relatively short (measured in days).

[0008] mRNA vectors are generally divided into non-replicating mRNA (nrRNA) and self-amplifying mRNA (saRNA or srRNA). nrRNA is generally used in vaccines and can be quickly designed and produced, making it an ideal choice for rapid response to infectious disease pandemics. The main mRNA vaccines currently on the market include BNT162b2 (trade name Comirnaty) developed by Pfizer and BioNTech and mRNA-1273 (trade name Spikevax) developed by Moderna. These vaccines provide protection by introducing mRNA encoding the viral spike protein (S protein) to induce the human immune system to produce an immune response against the virus. Most nrRNA nucleic acid drugs that enter cells through the transmitter system are single-copy molecules ( Figure 2), which can usually only express proteins briefly in cells, with a short half-life (measured in hours), and may require multiple doses to maintain the therapeutic effect of expressed proteins. mRNA molecules are very unstable and need to be stored and transported at extremely low temperatures. At the same time, an effective transmitter system (such as lipid nanoparticles) is also crucial for the stability and absorption of mRNA, and the production process involves high-cost raw materials and equipment, which increases the cost of use and limits its widespread application. (Biotechnology Communications, 2006, (02): 228-231)

[0009] srRNA is an emerging form of nucleic acid drug that has emerged in the past two years. It has shown great advantages in cost-effectiveness, scope of application and safety potential, and is extremely attractive in the fields of gene therapy and vaccine applications. Most of them are derived from positive-strand RNA viruses, using their genomes as vectors. They delete and replace one or more structural proteins of wild-type viruses with target proteins (therapeutic genes) and are able to replicate themselves (Cancer Gene Ther 30, 785–793). srRNA with positive-strand RNA viruses as the backbone has more advantages than mRNA: (1) It produces multiple copies of mRNA molecules in host cells ( Figure 2 ); (2) replicate in endoplasmic reticulum vesicles ( Figure 3 ), to avoid degradation; (3) more stable and efficient expression, long medicinal half-life. K. Lundstrom's research shows the therapeutic potential of srRNA. srRNAs such as alphavirus (VEEV), measles virus (MV), rhabdovirus (VSV) and flavivirus (ZIKV) are used to directly destroy cancer cells and stimulate immune responses against tumors (Int JMol Sci. 2022 Dec 9;23(24):15622.). Maine et al.'s 2021 study showed that synthetic srRNA controlled tumor growth in mice and non-human primates and induced anti-tumor T cell responses, providing a new approach for cancer immunotherapy. (Molecular Therapy. 2021 Mar 3;29(3):1186-1198) Lundstrom's 2023 study also discussed the application of srRNA in vaccine development, not only for cancer, but also for infectious diseases (Cancer Gene Ther30, 771–784). These studies highlight the versatility of srRNA in therapeutic and preventive applications, demonstrating that the main advantages of this technology include no need to integrate into the host genome, non-infectiousness, greater stability than simple mRNA, and more efficient production of therapeutic proteins. Therefore, srRNA can both synthesize more target proteins and extend the treatment cycle.

[0010] In previous studies, Charles Rice and his team successfully cloned the 3' end sequence of the HCV genome in 1996 after numerous attempts, and found that the complete RNA can replicate autonomously in animals. They then successfully developed an animal model of HCV, accelerating the development of HCV therapeutic drugs (Journal of Virology, 70(6), 3363–3371). This proved that non-infectious viruses can initiate complete replication of the viral genome after being transferred into host cells.

[0011] Following the discovery of the hepatitis C virus replicon, more researchers found that positive-strand RNA viruses of the Flavivirus genus can still replicate themselves in cells after losing some structural proteins but retaining intact non-structural proteins.

[0012] The Flavivirus genus (renamed Orthoflavivirus in 2023) is a positive-strand RNA virus in the family Flaviviridae. Viruses in this genus that are pathogenic to humans include West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus, and several other viruses that may cause encephalitis, as well as insect-specific flaviviruses (ISFs), such as cell-cell fusion agent virus (CFAV), Palm Creek virus (PCV), and Parramatta River virus (PaRV). The genome composition and arrangement of each virus in the Flavivirus genus are basically the same, with the same structural pattern (such as Figure 4 ). Its genome is a single-stranded positive-strand RNA of about 10.7 to 11 kb, containing a single open reading frame (ORF) that encodes a polyprotein precursor, which is then cleaved by the virus's own protease and host protease into three structural proteins: capsid protein (Capsid, C), membrane protein precursor (Precursormembrane, M) and envelope protein (Envelope, E), and seven non-structural proteins are NS1, NS2a, NS2B, NS3, NS4a, NS4b, and NS5. Viral proteins span the endoplasmic reticulum (ER) membrane at multiple locations. prM-M, E, NS2a, NS2b, NS4A and NS4B proteins are located in the ER cavity through hydrophobic signal sequences; NS1 is located in the ER cavity; the remaining proteins are located on the cytoplasmic side of the ER membrane, and the non-structural proteins interact with each other to form a replication complex. There are non-translated regions (NTRs) at the 5' and 3' ends on both sides of the genome, which play an important role in viral replication and translation.

[0013] All functional proteins of the genus Flavivirus are involved in viral replication. In host cells, NS1, transmembrane NS2a, NS2b, NS4a and NS4b proteins can induce the endoplasmic reticulum (ER) to form a viral RNA synthesis workshop, namely the viral vesicle, and form a replication complex with the cytoplasmic NS3 and NS5 proteins. The 12 bases at the N-terminus of the C protein form complementary base pairs with the sequence of the 3' non-coding region (NCR), which brings the 5' and 3' ends closer together, causing RNA to cyclize and become a template for the synthesis of negative-strand RNA. The viral protein NS5 interacts with the 5' stem-loop A region to synthesize a single negative strand from the 3' end of the positive strand, and releases the 3' end from the replication complex to start positive-strand RNA synthesis, and the synthesis of the positive strand continues (Viruses. 2014; 6(1):13-53). The three structural proteins and newly synthesized viral RNA are assembled into immature viral particles in the ER, and then transported to the cell membrane surface through the Golgi apparatus for maturation.

[0014] The inventor of this patent, Dr. Yu Li, and his team conducted a detailed study on West Nile virus (WNV) in the genus Flavivirus, and published their research results in 2017, which explained the role of non-structural proteins in vesicle formation during its replication. WNV's non-structural proteins, especially NS1, NS2A, NS2B, NS4A and NS4B, can use the ER to self-assemble into membrane vesicles (replication workshop) ( Figure 3 ), providing a special environment for the synthesis of viral RNA, emphasizing that vesicles contribute to the effective synthesis and replication of viral RNA in host cells, which is essential for viral replication (Virology 2017 Jun;506:130-140). In addition to the known NS3 and NS5 replicases as basic functional molecules for self-replication, these non-structural proteins also play an important role in RNA replication and are essential structural components for self-replication. This study also provides new insights into how the non-structural proteins of WNV promote the formation of replication complexes by inducing the production of vesicles in the host cell ER membrane. This discovery is crucial to understanding the replication mechanism of Flavovirus and provides valuable information for future gene therapy strategies. Compared with vaccines or gene therapy based on mRNA vectors, the stable self-replicating multi-copy molecules and long-term expression of srRNA will overcome the shortcomings of transient expression and low protein synthesis of mRNA vectors. In view of the advantages of srRNA as a drug development vector, its application field has great potential.

[0015] Since the biological characteristics of the Flavivirus, such as infectivity, antigenicity, and host tropism, are all concentrated on the E protein on the virus surface, while the virus replication function is mainly undertaken by its non-structural proteins. Therefore, the Flavivirus srRNA may not contain the three structural proteins, but must contain seven non-structural proteins that enable it to replicate itself. In addition, the structural protein prM-M of the virus and the C-terminus of the E protein ( Figure 4b) Both contain transmembrane domains (TM), with TM1 facing the cytoplasm and TM2 facing the ER cavity. This antiphase arrangement determines the direction and distribution of the proteins connected to it inside and outside the cell. For example, if the NS1 protein is connected to the C-terminus of TM2, the NS1 cleaved from the polyprotein will be distributed in the ER cavity and eventually transported outside the cell. Therefore, when constructing replicons for drug development, when the exogenous target gene is recombined with the replicon vector gene, as long as the two TMs in different directions are used well, it will not affect the expression, cutting and self-replication functions of non-structural proteins, and can also ensure the correct cutting and directional expression of the target protein. At the same time, since the structures and functions of the seven non-structural proteins of each virus in the genus Flavivirus are the same, the self-replication structure of srRNA vectors from different viruses constructed by deleting structural proteins is the same.

[0016] At the same time, since the seven non-structural proteins of each Flavivirus have the same structure and function, the structures of the srRNA vectors of each Flavivirus that do not contain structural proteins are identical. In summary, the development of srRNA vectors suitable for clinical treatment not only requires the retention of its normal self-replication function, but also requires the simultaneous expression and correct cleavage of the target gene and viral non-structural protein in the form of a polyprotein. More importantly, the expressed target protein is required to be independently distributed inside and outside the cell or embedded in the cell membrane according to its natural function and positioning to exert its therapeutic function.

[0017] So far, replicon (srRNA) drugs used in clinical applications have not been able to clearly define the intracellular and extracellular localization of the expressed products, thus losing the opportunity for widespread clinical application. The present invention studies and optimizes the structure of the srRNA of the Flavovirus, enhances its replication efficiency and gene expression level in host cells, and flexibly designs exogenous insertion gene sites according to the functional localization of the target protein, thereby providing at least four different structures of replicon vector structures to meet the distribution and expression of different therapeutic proteins inside and outside the cell, and can meet different clinical application needs and a wider range of treatment. It is precisely because the Flavovirus has the same non-structural protein that the present invention uses WNV in the Flavovirus as the srRNA to construct its four structures, but these structural models can represent the same structural srRNA from all Flavovirus sources. Summary of the invention

[0018] The purpose of the present invention is to provide four types of self-replicating nucleic acid vectors with a Flavivirus backbone capable of efficiently expressing functional proteins and methods for constructing gene drugs thereof.

[0019] To achieve the above object, the present invention adopts the following technical solution:

[0020] The present invention provides a method for constructing a plurality of self-replicating vectors with a Flavivirus as a backbone, comprising the following steps:

[0021] S1. Design and composition of basic srRNA elements: The gene of the yellow virus is used as the basic skeleton sequence, and the non-coding region (5'NCR and 3'NCR) and non-structural proteins are retained; while the viral structural protein sequence is removed to the maximum extent, the 5'-3' looped CS sequence and the necessary E-terminal TM sequence are retained; the promoter transcribed from the replicon and the 3' terminal nuclease sequence are added at the 5' end;

[0022] Preferably, since the Flavivirus genus contains more than 70 viruses, many of which are important human pathogens, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV) and yellow fever virus (YFV). These viruses have the same genome structure and similar replication mechanism. Although there may be differences in the interaction between these viruses and host proteins, or their host range and tissue tropism may be different, the genome and replication mode are conservative, among which the structural proteins determine the antigenicity and certain biological properties. Therefore, the viral RNA replicon constructed by truncating one or more structural proteins, the replication function of its non-structural protein and the self-replication mechanism are the same in the members of the Flavivirus genus. The present invention takes WNV as an example, but is applicable to the preparation of all types of self-replicating vectors with the Flavivirus genome as the skeleton.

[0023] S2. According to the functional positioning of the target protein, the target gene insertion site is designed and optimized, the structural protein gene is replaced without affecting the expression and function of the non-structural protein, and 4 replicon (srRNA) vectors are constructed. At the same time, its replication efficiency and stability are enhanced to meet the requirements of the therapeutic target function.

[0024] S3. Modification, synthesis and recombination cloning of exogenous genes: Modify or mutate the exogenous target gene to match the insertion site. For example, when TM2 is used as the signal peptide (SP), the SP of the secretory target protein should be deleted; in order to cut the expressed target protein from the polyprotein, it is necessary to add a viral protease (2a) to the C-terminus of the target protein, or replace the E protein TM1 with the transmembrane sequence (TMX) of the target protein. The modified or mutated (mutated) exogenous gene is inserted into the replicon backbone through molecular cloning technology, and the structural protein gene is replaced.

[0025] S4. Combination of srRNA and DNA plasmid: The srRNA vector is recombined in the form of cDNA on the pBR322 plasmid vector to obtain the srRNA plasmid.

[0026] The constructed replicon plasmid is transfected into suitable host cells to be amplified in Escherichia coli to produce srRNA plasmid; it is transfected into relevant animal models and a suitable DNA transmitter system is selected to transfect the srRNA plasmid for preclinical research.

[0027] The present invention also provides a method for using the Flavivirus self-replicating vector to carry different targeted genes to recombinantly construct nucleic acid drugs as gene therapy for diseases such as tissue and organ repair.

[0028] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention uses the yellow virus genome lacking structural protein genes as the skeleton, designs four insertion sites that can express therapeutic or functional proteins inside and outside the cell or across the membrane, making it an srRNA vector with the yellow virus gene as the skeleton. These vectors carrying different targeted genes can be applied to a variety of gene therapies or as nucleic acid drugs, which can meet the needs of treating various diseases or repairing tissue damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of the recombination of different target genes and srRNA

[0030] Figure 2 Schematic diagram of the structural differences between srRNA and mRNA

[0031] Figure 3 Schematic diagram of srRNA synthesis in the endoplasmic reticulum vesicles

[0032] Figure 4 The gene structure of the Flavivirus and the transmembrane pattern of its encoded protein

[0033] Figure 5 Schematic diagram of the basic structural components of Flavovirus srRNA

[0034] Figure 6 Schematic diagram of the srRNA carrier structure after deleting the M and E structural proteins

[0035] Figure 7 Structural model of srRNA vector for secretory proteins

[0036] Figure 8 Structural model of cytoplasmic protein srRNA carrier

[0037] Fig. 9 Structural model of srRNA carrier for transmembrane type I protein

[0038] Fig.10 Structural model of srRNA carrier for transmembrane type II protein

[0039] Fig.11srRNA in vitro expression verification

[0040] Fig.12 srRNA expression verification in vivo DETAILED DESCRIPTION

[0041] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. In the following examples, no specific experimental method is specified, and conventional methods and conditions are used, or selected according to the product specifications.

[0042] Example 1 Construction of WNV replicon vector

[0043] Basic components ( Figure 5 ):

[0044] The WNV gene is used as the basic skeleton sequence, lacking the nucleic acid sequences of the three structural proteins or retaining only a part of them, retaining the non-coding regions (5'NCR and 3'NCR) and non-structural proteins.

[0045] When all viral structural protein nucleic acid sequences are deleted, the 5'-3' looped CS sequence and the TM sequence at the end of the E protein are retained;

[0046] CS is an important element required for the replication complex to recognize and initiate replication. A restriction site and a start signal (ATG) are added after the 5'-CS sequence to serve as a start signal for inserting the target gene and translating the protein.

[0047] To enhance replication and delivery efficiency, the auxiliary srRNA (DNA) is transcribed into positive-strand RNA (mRNA) after entering the cell, and a promoter for its transcription, such as the classic CMV or tissue-specific promoter, is added to the 5' end of the WNV replicon vector; in order to correctly cut the srRNA transcribed by the promoter, a nuclease sequence element is added to the 3' end. This design is used for srRNA production in the form of a DNA plasmid, greatly reducing production costs. At the same time, the DNA transmitter system can be used to enter the cell to transcribe the DNA plasmid into srRNA, thereby improving the in vitro stability of srRNA.

[0048] Example 2 Construction of replicon vectors for localization of 4 protein functions

[0049] RNA virus replicons are often used in laboratories as scientific research tools. They generally retain some viral structural proteins (C and prM). The inserted gene can only be connected between the extracellular secreted prM and TM1 of the E protein, and the target protein can only be attached to the cell membrane surface in a transmembrane form ( Figure 6), which greatly limits the application of replicons. This is because each protein has different functions and is distributed inside, outside or bound to the membrane. Intracellular proteins, such as hexokinase, catalyze glycolysis inside the cell; extracellular proteins, such as insulin, are transported through the blood and bind to insulin receptors on the surface of target cells such as muscle and fat, promoting glucose uptake into the cell; membrane-bound proteins, such as the CD28 receptor on the T cell membrane, are activated after binding to the CD86 ligand, initiating a series of signaling events in the T cell and causing a T cell immune response. Each type of protein has its fixed distribution and positioning, effectively performing its specific function. Therefore, it is very necessary to construct an srRNA vector suitable for the functional positioning of the target protein.

[0050] This example aims to construct secretory, cytoplasmic and two cross-model replicons in order to expand the application range of srRNA vectors. The replicon vectors used in this example are modified based on the basic composition structure described in Example 1.

[0051] (1) Secretory replicon:

[0052] The structural diagram is as follows Figure 7 As shown, the target gene insert containing its own signal peptide (SP) is directly connected to WNV5'NCR; the 5' end of the target gene without SP is connected to TM2, using TM2 of prM as SP. A protease fragment is added to the 3' end of the insert and connected to the 5' end of TM1 of the E protein that has lost a part. The expressed target protein is released outside the cell through SP cleavage and separation of 2a and TM1.

[0053] (2) Cytoplasmic replicon:

[0054] The structural diagram is as follows Figure 8 As shown, the 5' end of the target gene insert without SP is connected to WNV5'NCR, while the protease fragment is added to the 3' end of the insert and connected to TM2 to ensure that the expressed target protein is localized in the cytoplasm after cleavage by 2a.

[0055] (3) Transmembrane type I replicon:

[0056] Transmembrane type I proteins and transmembrane type II proteins are two important types of membrane proteins. The N-terminus of transmembrane type I proteins is located outside the cell or in the endoplasmic reticulum cavity, and the C-terminus is located in the cytoplasm. The N-terminus has a signal peptide that guides it into the endoplasmic reticulum membrane, transports it, and exposes the N-terminus outside the cell. It is commonly found in signal pathway proteins on receptors and cell membranes, such as epidermal growth factor receptor (EGFR).

[0057] The structural diagram is as follows Fig. 9As shown, the homosecretionary replicon is basically constructed. SP can be the insert itself or TM2; the transmembrane sequence at the 3' end of the inserted gene can retain its own TM (TMX) or use TM1 of the E protein. Both can anchor the N-terminus of the expressed protein on the cell membrane surface.

[0058] (4) Transmembrane type II replicon:

[0059] In contrast to type I transmembrane proteins, the N terminus of type II transmembrane proteins is located in the cytoplasm and the C terminus is located outside the cell or in the endoplasmic reticulum lumen. They use endogenous signal anchor sequences to enter the endoplasmic reticulum membrane and are commonly found in cytokine receptors and activating factor ligands (such as OX40L), both of which play a key role in cell signaling, substance transport, and immune response.

[0060] Structure Fig.10 As shown, the basic framework is the same as the secretory replicon. However, the transmembrane sequence of the target gene is retained at the 5' end, so that it is located inside the membrane; 2a is added to the 3' end and connected to the 5' end of TM1, so that the C-terminus of the expressed protein is separated from TM1 and faces the outside of the cell membrane.

[0061] Example 3. In vitro expression of reporter gene replicons (3d-7d)

[0062] Experimental method: Vero cells were selected as host cells and cultured to 70-80% confluence. Cytoplasmic replicon-Luc (expressing luciferase gene) recombinant was transfected into Vero cells using P3000 transfection reagent. Anti-luciferase antibody was used to observe and check its expression under a fluorescence microscope 24 hours, 72 hours and 7 days after transfection.

[0063] Results and analysis: After the cytoplasmic replicon-Luc was transfected into cells, strong green was observed in the transfected cells under microscope, indicating that the replicon successfully expressed luciferase; and its expression lasted for more than 7 days as the cells divided and proliferated ( Fig.11 ).

[0064] Conclusion: The results of fluorescence microscopy observation showed that the cytoplasmic replicon-Luc, which does not contain WNV structural proteins, achieved high expression of exogenous indicator genes for a long time.

[0065] Example 4 Verification of Stable Expression of WNV-Luc Replicon in Nude Mice with Human Small Cell Lung Cancer (NCI-H446) Xenograft Tumor Model

[0066] The expression of jet-PEI-encapsulated cytoplasmic replicon-Luc in tumor-bearing nude mice (NCI-H446 model) was verified in vivo through small animal in vivo fluorescence imaging experiments.

[0067] (1) Experimental method: Human small cell lung cancer cells (NCI-H446) were subcutaneously inoculated into Balb / c-Nude mice to prepare a subcutaneous tumor transplant model (inoculation volume 1X10 7 / 0.1 mL / mouse), and about 16 days later, the tumor volume grew to the dosing standard (6-7 mm in diameter), and the PEI-encapsulated cytoplasmic replicon-Luc was injected intratumorally. At different time points (days) after injection, the luciferase substrate was injected intraperitoneally into the mice, and the mice were imaged in vivo.

[0068] (2) Results and analysis: Fig.12 As shown, in the Balb / c-Nude tumor-bearing mouse NCI-H446 model, the cytoplasmic replicon-Luc replicon 1 injection began to have signals from the 7th day, and the light signal values ​​were all above 100,000. The peak expression was on the 29th day (the average light signal in the group was about 490,000), and the light signal value showed a slow growth trend, with the light signal value difference within 10,000-50,000. After the peak expression, the signal values ​​began to show a downward trend, with the light signal value difference exceeding 100,000.

[0069] 4. Conclusion: The above steps verified that the expression of cytoplasmic replicon-Luc can last up to 38 days in vivo. The replicon has stronger stability and longer half-life.

[0070] This embodiment shows the construction composition of secretory, cytoplasmic and two trans-model WNV replicons, and provides a variety of structural yellow virus self-replicating vectors for different exogenous protein functional positioning. The structural characteristics and the recombination sites between each vector and the target gene are described in detail to ensure that the inserted therapeutic protein can be correctly distributed inside and outside the cell or embedded in the cell membrane according to its functional role. Through the expression verification represented by the cytoplasmic replicon-Luc, it is proved that the virus-free structural protein srRNA can efficiently express exogenous factors for a long time in vivo and in vitro, and the expressed protein has a long half-life and stability.

[0071] The above embodiments are only used to illustrate the present invention but not to limit the technical solutions described in the present invention. The understanding of this specification should be based on the technicians in the relevant technical field. Although this specification has described the present invention in detail with reference to the above embodiments, ordinary technicians in the field should understand that technicians in the relevant technical field can still modify or replace the present invention with equivalents, and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A multi-type self-replicating nucleic acid vector with a Flavivirus gene as a backbone, characterized in that: The genome of Flavivirus was used as the backbone, retaining the 5' non-coding region (5'NCR), 3' non-coding region (3'NCR) and non-structural protein gene sequences; Delete or replace the viral structural protein gene sequence, retaining the 5'-3' cyclization sequence (CS sequence) and the necessary E protein transmembrane region (TM sequence); Design and optimize the insertion site of the exogenous target gene, replace the original structural protein gene according to the function and cellular localization of the target protein, and construct various types of self-replicating RNA (srRNA) vectors.

2. The self-replicating nucleic acid vector according to claim 1, wherein the multiple types of localization distribution of each exogenous protein used for expression include intracytoplasmic, extracellular secretion type, and cell transmembrane type vectors.

3. The self-replicating nucleic acid vector according to claim 1 or 2, wherein the Flavovirus gene is the West Nile virus (WNV) genome or the genome backbone of other Flavoviruses, including but not limited to West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus and several other viruses that may cause encephalitis.

4. A method for constructing a self-replicating nucleic acid vector according to any one of claims 1 to 3, characterized in that: The genome of the Flavivirus is specifically modified, including deleting the signal peptide of the secretory protein, adding the viral protease cleavage site, replacing the transmembrane region of the E protein, etc., to make it suitable for the insertion of the target gene and the expression distribution of the exogenous protein without affecting the replication function of the self-replicating nucleic acid vector.

5. The use of a self-replicating nucleic acid vector capable of integrating an exogenous gene into any one of claims 1 to 3, making it suitable for replacing the structural protein gene of a Flavivirus; inserting it into the srRNA backbone through molecular cloning technology to construct an srRNA nucleic acid drug for expressing a specific functional gene.

6. The nucleic acid drug according to claim 5, wherein the self-replicating nucleic acid vector is capable of carrying different targeted genes to express the desired therapeutic or functional protein.

7. A nucleic acid drug comprising the self-replicating nucleic acid vector according to claim 6, characterized in that: It can express and produce specific functional proteins and can be used for gene therapy in the fields of disease treatment and prevention, tissue repair, medical beauty and health care, anti-aging, etc.

8. The method according to claim 4, wherein the srRNA plasmid is transfected into an appropriate host cell or animal model, and a suitable DNA delivery system is used for gene expression and functional verification.

9. A gene therapy method based on the self-replicating nucleic acid vector of claim 8, comprising: delivering the self-replicating nucleic acid vector to target cells or tissues; It enables efficient expression of exogenous functional proteins in cells and exerts therapeutic effects.

10. The method according to claim 9, wherein the delivery method is in vivo or in vitro transfection, using an appropriate DNA delivery system to achieve efficient gene transfer and expression.

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