Self-replicating RNA molecule, and preparation method therefor and use thereof

Abstract

Provided are a self-replicating RNA construct, and a preparation method therefor and the use thereof. More specifically, the present invention relates to a self-replicating RNA construct derived from an alphavirus replicon, and a preparation method therefor and the use thereof. The cytotoxicity of the self-replicating RNA construct is effectively reduced by enhancing the expression of a target gene by means of a specific sequence mutation.

Description

Self-replicating RNA molecule, method of making and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of self-replicating RNA technology, in particular to the modification of self-replicating RNA replicon, more particularly to the mutation of non-structural protein of alphavirus replicon and the use thereof. BACKGROUND

[0002] Self-amplifying RNA (saRNA) is defined as an RNA molecule with the ability to self-replicate. Alphavirus is a positive-sense single-stranded RNA virus, which encodes viral non-structural proteins (nsP1-nsP4) and structural proteins (E3-E2-6K-E1), respectively. The genomic region encoding viral structural proteins is called viral subgenomic. saRNA usually replaces the structural protein gene on the alphavirus subgenomic with a heterologous or exogenous gene, while retaining other elements on the viral genome. The absence of viral structural proteins makes saRNA unable to produce infectious virus particles.

[0003] The conserved sequence elements (CSE) of the viral replicase is very important for the specific amplification of saRNA. The non-structural proteins of alphavirus are the first translated polyprotein, which forms a replication complex to achieve the basic function of saRNA replication. After saRNA is delivered to the cell, it is recognized and translated by the host ribosome to form an early replication complex, and a negative strand RNA is replicated from the saRNA template strand. Subsequently, the negative strand RNA is used as a template to further transcribe a positive strand RNA identical to the original saRNA, as well as a large number of positive strand subgenomic RNAs. Finally, the heterologous or exogenous gene on the subgenomic is expressed into the target molecule through the host protein translation mechanism. This mechanism is continuously recycled to achieve the self-replication of saRNA and the expression of the target protein. Based on this self-amplification mechanism, saRNA is believed to be able to express the desired antigen for a long time and at a high level.

[0004] Compared with traditional mRNA vaccines, saRNA vaccines can more effectively activate innate immune responses due to the production of double-stranded RNA (dsRNA) intermediates during amplification. Innate immune responses have a double-edged sword effect on RNA vaccines, although they have an adjuvant effect to promote specific immune responses, they can also promote the degradation of RNA and reduce the expression level of antigens. Excessive innate immune response is not conducive to the self-amplification of saRNA and the expression of antigens.

[0005] At present, the expression efficiency of saRNA target protein, cytotoxicity, safety, innate immune response, pre-existing immunity and many other aspects still need further research. SUMMARY

[0006] The present application provides a self-replicating RNA molecule and its preparation method and use, more particularly to a self-replicating RNA molecule against alphavirus replicon and its preparation method and use. In particular, the present application provides an infectious, replication-deficient, highly immunogenic alphavirus replicon based on a specific strain of alphavirus, TC-83 of VEEV, and its preparation method. The self-replicating RNA construct prepared by the mutation of the non-structural protein nsP2 in the present application can increase the expression level of the alphavirus replicon subgenomic protein, enhance the RNA expression efficiency, and reduce the cytotoxicity of the alphavirus replicon. The present application also provides a DNA molecule encoding the above-mentioned saRNA, an expression vector comprising the DNA, and a vaccine or a drug comprising the above-mentioned saRNA and its use. The self-replicating RNA of the present application is used for preventing or treating diseases.

[0007] The term "alphavirus" in the present application has its conventional meaning in the art and includes various species such as VEE, SFV, Sindbis, Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya, S.A. AR86, Everglades virus, Mucambo, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus and Buggy Creek virus.

[0008] The term "attenuated" in the present application means a nucleotide mutation or an amino acid encoded by a nucleotide mutation which, in the case of a live virus, results in a reduced ability of the alphavirus to cause disease in its host (i.e., loss of virulence) according to standard terminology in the art. The mutation is a substitution mutation, a deletion or an addition mutation.

[0009] The term "self-replicating RNA" in the present application, also known as "self-amplifying RNA". In the present application, "self-replicating RNA", "self-amplifying RNA", "saRNA" and "samRNA" have the same meaning, are different names of the same technical platform, and can be used interchangeably. They all refer to a genetically engineered RNA containing viral RNA replication machinery.

[0010] The term "heterologous" in the present application, also known as "exogenous", means from different species or different genetic backgrounds.

[0011] The term "neutral phospholipid" according to the present invention refers to a phospholipid molecule that is not charged, which is not a phosphoglyceride.

[0012] The term "polyethylene glycol (PEG)-lipid" according to the present invention refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

[0013] The term "lipid nanoparticle" (LNP) according to the present invention refers to a particle having at least one dimension in the nanometer range, which comprises at least one lipid.

[0014] The term "vaccine" according to the present invention refers to a composition suitable for application to an animal, including a human, which induces an immune response after administration, the strength of which is sufficient to minimally help prevent, ameliorate or treat a clinical disease or a tumor resulting from a microbial infection.

[0015] In one aspect, the present application provides a self-replicating RNA construct comprising a replicase coding unit and a protein of interest coding unit, wherein the replicase coding unit comprises a nucleic acid segment encoding a viral non-structural protein, the protein of interest coding unit comprises a multiple cloning site to be inserted into a protein of interest coding sequence and / or a nucleic acid segment encoding a protein of interest, the construct is capable of expressing the replicase in a cell of interest, the replication is only of the protein of interest coding unit, the viral non-structural protein is selected from nsPl, nsP2, nsP3 and nsP4, and the self-replicating RNA construct is derived from an Alphaviruses replicon. Preferably, the self-replicating RNA construct is derived from a Venezuelan equine encephalitis virus (VEEV).

[0016] In some embodiments, the self-replicating RNA construct according to the present application can be derived from VEEV (NCBI: NC_075022.1), VEEV TC-83 (NCBI: LO1443.1), VEEV LSE9010-15 (NCBI: MK796243.1), VEEV PE21-0029 (NCBI: MH086249.1), VEEV PE20-0043 (NCBI: MH086248.1), VEEV PE12-0123 (NCBI: MH086247.1) or VEEV ZPC738 (NCBI: MF459684.1) and attenuated strains of different VEEV strains.

[0017] In some embodiments, the self-replicating RNA construct is derived from the VEEV TC-83 attenuated strain (GenBank: LO1443.1).

[0018] The present application mutates the non-structural protein derived from the attenuated strain of VEEV TC-83 to construct a self-replicating RNA construct with more excellent performance.

[0019] Specifically, the non-structural protein nsP2 of the saRNA is mutated as follows: the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is mutated; and / or, the glutamine at position 739 of the encoded nsP2 or corresponding to the position is mutated; and / or, the proline at position 773 of the encoded nsP2 or corresponding to the position is mutated.

[0020] In some specific embodiments, the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is mutated and the glutamine at position 739 of the encoded nsP2 or corresponding to the position is mutated.

[0021] In some specific embodiments, the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is mutated, the glutamine at position 739 of the encoded nsP2 or corresponding to the position is mutated, and the proline at position 773 of the encoded nsP2 or corresponding to the position is mutated.

[0022] In some specific embodiments, the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is replaced with arginine, and / or, the glutamine at position 739 of the encoded nsP2 or corresponding to the position is replaced with leucine, and / or, the proline at position 773 or corresponding to the position is replaced with serine.

[0023] In some specific embodiments, the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is replaced with arginine and the glutamine at position 739 of the encoded nsP2 or corresponding to the position is replaced with leucine.

[0024] In some specific embodiments, the glycine at position 763 of the encoded nsP2 of the self-replicating RNA construct provided by the present application or corresponding to the position is replaced with arginine and the glutamine at position 739 of the encoded nsP2 or corresponding to the position is replaced with leucine, and the proline at position 773 of the encoded nsP2 or corresponding to the position is replaced with serine.

[0025] Specifically, the present application provides a self-replicating RNA construct derived from the replicon of the attenuated strain of VEEV TC-83, which comprises:

[0026] (A) a replicase-encoding unit comprising a nucleic acid segment encoding viral non-structural proteins nsPl, nsP2, nsP3, and nsP4, said nsP2 comprising the following mutation: glycine at position 763 or equivalent to this position is replaced with arginine, and said self-replicating RNA construct encodes nsP2 at position 739 or equivalent to this position glutamine is replaced with leucine.

[0027] (B) a heterologous RNA-encoding unit encoding at least one heterologous RNA sequence, but lacking sequences encoding alphavirus structural proteins, said heterologous RNA expressing a desired protein of interest.

[0028] The present application also provides another self-replicating RNA construct derived from the replicon of VEEV TC-83 attenuated strain, which is improved from the above-mentioned self-replicating RNA construct in that the nsP2 encoded therefrom further comprises the following mutation: proline at position 773 or equivalent to this position is replaced with serine.

[0029] In some embodiments, the self-replicating construct is derived from VEEV TC-83, and the amino acid sequence of the non-structural protein nsP2 encoded therefrom is shown in SEQ ID NO: 11. The amino acid sequence of the non-structural proteins nsPl, 2, 3, 4 encoded therefrom is shown in SEQ ID NO: 1.

[0030] In some embodiments, the amino acid sequence of the mutated non-structural protein nsP2 is shown in SEQ ID NO: 12-20.

[0031] In some embodiments, the amino acid sequence of the mutated non-structural proteins nsPl, 2, 3, 4 is shown in SEQ ID NO: 2-10.

[0032] The self-replicating RNA construct is SAMv1, SAMv2, SAMv3, SAMv4, SAMv5, SAMv6, SAMv7, SAMv8, SAMv9, SAMv10, respectively. The amino acid sequence of the nsp2 protein expressed in the above-mentioned construct corresponds to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, respectively. SEQ ID NO: 11 is the amino acid sequence of nsP2 derived from the attenuated strain VEEV TC-83.

[0033] In some embodiments, the amino acid sequence of the nsp1-4 non-structural proteins expressed by the self-replicating RNA construct SAMv1, SAMv2, SAMv3, SAMv4, SAMv5, SAMv6, SAMv7, SAMv8, SAMv9, SAMv10 corresponds to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, respectively. SEQ ID NO: 1 is the amino acid sequence of nsP1, 2, 3, 4 non-structural proteins derived from the attenuated strain VEEV TC-83.

[0034] The amino acid sequence of the self-replicating RNA construct of the present application can be an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO: 1-SEQ ID NO: 20, preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more.

[0035] According to the above-mentioned self-replicating RNA construct, the self-replicating RNA construct provided by the present application comprises, in the 5'-3' direction:

[0036] (1) a 5' cap structure, which is a natural 5' cap or a 5' cap analog, helps to protect saRNA from nuclease degradation and promotes translation initiation, and the 5' cap structure is co-transcriptionally capped;

[0037] (2) a 5' UTR sequence, which regulates the replication of RNA;

[0038] (3) a nucleotide sequence encoding the replicase;

[0039] (4) a subgenomic promoter SGP, which ensures efficient replication of the heterologous RNA sequence;

[0040] (5) a nucleotide sequence encoding a heterologous RNA, which expresses a desired target protein under the control of the subgenomic promoter;

[0041] (6) a 3'UTR sequence, which supports replication and efficient translation of the RNA;

[0042] (7) a polyA structure, which increases the stability of the RNA and facilitates translation.

[0043] Further, the 5'UTR of the self-replicating RNA construct can be modified.

[0044] In some embodiments, the 5'UTR is derived from the 5'UTR of any gene or a mutant thereof, and the 3'UTR is derived from the 3'UTR of any gene or a mutant thereof; preferably, the 3'UTR or 5'UTR is derived from the same virus as the replicase.

[0045] In some embodiments, the target protein is a reporter protein or an antigen. The present application provides a variety of self-replicating RNA constructs, with GFP protein as the target protein for convenient experimental observation. The self-replicating RNA construct of the present application can be used to express any target protein or other substance as needed.

[0046] In some embodiments, the heterologous RNA in the self-replicating RNA construct provided by the present application expresses one or more of the following substances: immunogens, antibodies, cytokines, lymphokines, chemokines, toxins, therapeutic proteins, enzymes, enzyme inhibitors, hormones, immune response modulators, reporter genes, etc. The above-mentioned substances can be derived from mammals (such as but not limited to humans, dogs, cats, monkeys, sheep, goats, horses, cattle, etc.), bacteria, viruses, fungi or parasites. In some embodiments, they are derived from humans. In some embodiments, the substances are naturally occurring, modified from the natural state, chimeric proteins containing protein fragments from at least two different proteins, engineered proteins with enhanced function or activity, engineered proteins with reduced function or activity, or targeted to specific compartments of cells, such as the cytoplasm, the membrane or the nucleus, or any combination thereof.

[0047] The gene of interest in the present application can encode a protein corresponding to all or part of a naturally occurring protein found in nature. It can also be a chimeric protein, such as a fusion of polypeptides from different sources or from a mutant with improved and / or altered biological properties. Such mutants can be obtained by conventional biological techniques, by substitution, deletion and / or addition of one or more amino acid residues.

[0048] The gene of interest used in the present application can be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional technique. Preferably, it is capable of producing a product having a therapeutic effect, and it can be a product which is homologous to the cellular host, or alternatively, a product which is heterologous. Within the scope of the present disclosure, the gene of interest can encode (1) an intracellular product or (2) a membrane product which is present on the surface of the host cell or (3) a product which is secreted outside the host cell. Thus, it can contain appropriate additional elements, such as a sequence encoding a secretion signal. Such signals are known to those skilled in the art.

[0049] Exemplary, the substance includes, but is not limited to, interleukins, interferons, etc. (FR9203120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc.; pro-lipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR9305125), dystrophin or mini-dystrophin (FR9111947), tumor suppressors: P53, Rb, Rap1A, DCC, k-rev, etc. (FR9304745), coagulation-related factors: factors VII, VIII, IX.

[0050] For example, another aspect of the present application provides a DNA molecule encoding any of the self-replicating RNA constructs described above and an expression vector comprising any of the DNA molecules described above.

[0051] Another aspect of the present application provides a vaccine or composition comprising any of the self-replicating RNA constructs described above and a nucleic acid having a therapeutic and / or prophylactic effect.

[0052] Another aspect of the present application provides a vaccine or composition comprising any of the self-replicating RNA constructs described above and a pharmaceutically acceptable carrier, such as a lipid nanoparticle (LNP), a polymeric carrier, etc.; preferably, the composition further comprises a lipid nanoparticle (LNP) in which the self-replicating RNA construct is encapsulated.

[0053] Specifically, the lipid nanoparticle comprises a cationic lipid, a neutral phospholipid, a steroidal lipid, and a polyethylene glycol (PEG)-lipid.

[0054] The vaccine or composition of the present application can further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a carrier, a diluent, an adjuvant or a coding adjuvant nucleotide sequence, a solubilizer, a binder, a lubricant, a suspending agent, a transfection promoter, etc.

[0055] In another aspect of the present application, a method for preparing any of the vaccines or compositions described above is provided. Specifically, the method for preparing the vaccines or compositions comprises encapsulating the self-replicating RNA construct into a lipid nanoparticle (LNP).

[0056] The method for preparing the vaccines or compositions comprises dissolving cationic lipid, neutral phospholipid, sterol lipid, and polyethylene glycol (PEG)-lipid into a solvent, and then mixing the solution with nucleic acid.

[0057] Preferably, the method for preparing the vaccines or compositions comprises dissolving cationic lipid, neutral phospholipid, sterol lipid, and polyethylene glycol (PEG)-lipid into ethanol, mixing the solution with diluted self-replicating RNA diluent, and then preparing the vaccines or compositions by ultrafiltration, dilution, and filtration. Preferably, the method for preparing the vaccines or compositions comprises dissolving cationic lipid, neutral phospholipid, sterol lipid, and polyethylene glycol (PEG)-lipid into ethanol, mixing the solution with diluted self-replicating RNA diluent at a certain flow rate ratio, and then preparing the vaccines or compositions by ultrafiltration, dilution, and filtration. Preferably, the ultrafiltration is tangential flow filtration. More preferably, the mixing is turbulent mixing, laminar mixing, or microfluidic mixing.

[0058] In another aspect of the present application, any of the self-replicating RNA construct, any of the DNA molecule, any of the expression vector, any of the vaccine or composition, and any of the method for preparing the vaccine or composition are used for preparing a medicament for preventing or treating a disease. The medicament can be a vaccine.

[0059] The disease is an infectious disease, a tumor, an autoimmune disease, an allergic disease, a genetic disease, a metabolic disease, or tissue repair. The disease, disorder, and / or condition includes, but is not limited to, rare diseases, infectious diseases (medicaments in the form of vaccines and therapeutic agents), cancer and proliferative diseases, genetic diseases (such as cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases (such as diabetes). In some embodiments, the infectious disease is selected from a disease caused by a coronavirus, an influenza virus, a respiratory syncytial virus, or an HIV virus; pediatric pneumonia, Rift Valley fever, yellow fever, rabies, multiple herpes, etc.

[0060] The treatment is gene therapy or immunotherapy. Preferably, the gene therapy or immunotherapy is a therapeutic vaccine. More preferably, the therapeutic vaccine can induce a high level of cellular immune response.

[0061] The feasibility of gene therapy for humans no longer needs to be demonstrated, which involves numerous therapeutic applications such as genetic diseases, infectious diseases, and cancers. Many documents in the prior art describe methods for using gene therapy. The principle of gene therapy is to deliver a functional gene, called a target gene, in which RNA or the corresponding protein will produce the desired function.

[0062] On the one hand, the insertion of genes allows the expression of complex and unstable molecules (such as RNA or proteins) to be prolonged, which are difficult or even impossible to obtain or administer directly. On the other hand, the controlled insertion of the desired gene inside the target specific cell allows the expression product to be modulated in a specific tissue. To this end, it must be possible to insert the desired therapeutic gene inside the selected cell, and therefore to have available insertion methods that allow the specific targeting of the selected cell or tissue.

[0063] In some embodiments, the pharmaceutical formulations of the present application are formulated to be suitable for rapid absorption and distribution, thereby delivering the active pharmaceutical to a mammal. The route of delivery is, for example, oral, intravenous, intramucosal (e.g., nasal, vaginal, etc.), intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous, intranasal administration, or administration by inhalation. Preferably, the vaccine or composition is a liquid formulation or a lyophilized powder; more preferably, the vaccine or composition is an oral formulation, an intramuscular injection formulation, an intravenous injection formulation, or an inhalation formulation; further preferably, the vaccine or composition is a nebulized inhalation or a dry powder inhalation.

[0064] The beneficial effects of the present application compared to the prior art are:

[0065] First, the self-replicating RNA construct described in the present application has a higher expression level of the target protein than the commonly used TC-83 attenuated strain, can efficiently express in mice in vivo, and is superior to the TC-83 control group; the expression duration of the target protein is as long as 28 days, and the safety of the mice is good;

[0066] Second, the self-replicating RNA construct described in the present application reduces the cytotoxicity of the self-replicating RNA by modifying the nsP protein of the self-replicating RNA replicon, while enhancing the expression of the target protein;

[0067] Third, the vaccine prepared from the self-replicating RNA construct described in the present application has the characteristics of low innate immune response;

[0068] Fourth, the self-replicating RNA vaccine of the present application can induce both humoral immunity and cellular immunity in the human body, and has a dual immune effect. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 is the proportion of GFP-expressing cells: flow cytometry detection after samRNA-LNP transfection of cells;

[0070] Figure 2 is the average fluorescence intensity of GFP expression: flow cytometry detection after samRNA-LNP transfection of cells;

[0071] Figure 3 is the cell viability after samRNA-LNP transfection for 24 hours;

[0072] Figure 4 shows cell viability after 48h samRNA-LNP transfection;

[0073] Figure 5 shows samRNA subgenomic (GFP) expression relative value;

[0074] Figure 6 shows samRNA genomic (nsPl) expression relative value;

[0075] Figure 7 shows Hela cell supernatant IFNβ expression level;

[0076] Figure 8 shows luciferase luminescence intensity in mice, luciferase expression in different samRNA design groups can last for 28 days, much longer than the linear mRNA group which can last for 7 days at most;

[0077] Figure 9 shows long-term weight observation results in mice, no obvious weight loss was found;

[0078] Figure 10 shows detection results after different samRNA transfection to cells for 48h;

[0079] Figure 10A shows samRNA-LNP transfection to 1x106 293T cells at the same concentration (2μg), and the results were detected by flow cytometry on the 2nd day;

[0080] Figure 10B shows 48h cell toxicity results under high (20ng) dose samRNA-LNP transfection;

[0081] Figure 10C, Figure 10D show RT-qPCR detection results of different samRNA nsP construct subgenomic and genomic replication level. DETAILED DESCRIPTION

[0082] For a better understanding of the present application, specific embodiments will be given to further illustrate the present application, however, it should be understood that the described embodiments are exemplary embodiments, and the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application, and to enable the scope of the present application to be fully conveyed to those skilled in the art.

[0083] Example 1 Different samRNA design schemes

[0084] The self-replicating RNA constructs described in this example are all derived from the VEEV TC-83 attenuated strain, and the nsP (non-structural protein) protein mutation design in the embodiments is as follows. The nsP protein mutation scheme is screened. Specifically, the nsP sequence of samRNA vl is derived from the VEEV TC83 attenuated strain, which is used as a control.

[0085] Table 1 Different nsP mutation summary table

[0086] (“ / ” in the table means no modification.)

[0087] Example 2 Construction of samRNA-LNP

[0088] The DNA vector for in vitro transcription of self-replicating RNA comprises a T7 promoter, a 5' UTR, a VEE replicon, a subgenomic promoter, a subgenomic 5' UTR, a GFP, a 3' UTR and a poly-A tail. The saRNA nsP corresponds to the amino acid sequence shown in SEQ ID NO: 1-9.

[0089] The plasmid was linearized by BspQI single enzyme digestion and purification to obtain the linearized plasmid as a template for subsequent transcription experiments. The linearized plasmid was prepared, and a T7 in vitro transcription reaction system was prepared. After transcription, DNase I digestion of the transcription template, proteinase K digestion, and purification, the samRNA stock solution was obtained. The lipid mixture solution for encapsulating samRNA was prepared according to Table 2, and the lipid encapsulated samRNA stock solution was formed by microfluidic method. Subsequently, the encapsulated solution was diluted, ultrafiltrated and concentrated using 50 mM sodium acetate buffer containing 435 mg / ml sucrose to obtain the samRNA-LNP sample. The key quality parameters are shown in Table 3.

[0090] Table 2 Lipid formulation

[0091] Table 3 samRNA-LNP key quality parameter table

[0092] Example 3 Detection of expression levels of different samRNA mutant load proteins

[0093] The same concentration (2 μg) of samRNA-LNP constructed in Example 2 was transfected into 1x10 6 293T cells, and the fluorescence intensity of different samRNA-LNP GFP was observed by fluorescence microscope. On day 1, day 3, day 5 and day 7, the proportion of GFP-expressing cells and the average fluorescence intensity (MFI) of GFP were counted by flow cytometry, and the average fluorescence intensity and the proportion of GFP-expressing cells were plotted against time, and the differences in average fluorescence intensity and proportion of GFP-expressing cells were counted.

[0094] Fig. 1 and Fig. 2 show the flow cytometry detection results of samRNA-LNP transfected cells. The results show that the nsP2 G763R mutation can enhance the expression of self-replicating vector load protein, while the nsP2 Q739L or nsP3 L121P can reduce the expression of self-replicating vector load protein, and other mutations have no obvious effect on the expression of self-replicating vector load protein.

[0095] Example 4 Cytotoxicity detection of different samRNA nsP constructs

[0096] To investigate the cytotoxicity of different samRNA nsP constructs, 293T cells were plated at 1X10^4 cells per well in a 96-well plate and incubated overnight at 37°C, 5% CO2. The culture medium was discarded, and then 100 μL of DMEM complete medium containing 1, 5 or 20 ng of different samRNA-LNP was added to the wells, and the plate was again placed in the incubator for 24 h or 48 h. The culture plate was removed, and 50 μL of culture solution was aspirated. 60 μL of DMEM complete medium containing CCK-8 reagent (10 μL) was added to each well, and the plate was incubated in the incubator for 1 h. Then, the OD value was measured at 450 nm using an enzyme-labeled instrument.

[0097] The cell viability of each group was calculated according to the following formula:

[0098] Cell viability = (average OD450 value of experimental group - average OD450 value of blank control) / (average OD450 value of negative group - average OD450 value of blank control) x 100%

[0099] As shown in Figure 3, the cytotoxicity of samRNA-LNP transfection at high (20 ng), medium (5 ng), and low (1 ng) doses had little effect on cytotoxicity within 24 h. After 48 h (Figure 4), at medium and high doses, the Q739L+G763R mutant combination had the lowest cytotoxicity at high expression of GFP, which was superior to the TC-83 attenuated strain.

[0100] Example 5 Detection of genomic and subgenomic RNA replication levels of different samRNA nsP constructs

[0101] RT-qPCR was used to detect the subgenomic and genomic replication levels of different samRNA nsP constructs. Total RNA was extracted from 293T cells transfected with the same concentration of samRNA-LNP using Trizol. RT-qPCR was detected using the HiScript IIOne Step qRT-PCR SYBR Green Kit kit method (Novozyme). The expression value of samRNA genome (nsP1) or subgenome (GFP) relative to the housekeeping gene GAPDH was calculated by the Ct difference (ΔCt), and the fold change of gene expression was calculated by the difference between ΔCt values (ΔΔCt). The RT-qPCR primers are shown in Table 4.

[0102] As shown in FIG. 5, FIG. 6, the samRNA different nsP designs in the genome (nsPl) and subgenomic (GFP) expression. The samRNA v3, samRNA v5 and samRNA v8 designs have higher genome and subgenomic RNA expression than the samRNA vl control group, and the samRNA v2, samRNA v4, samRNA v6, samRNA v7, samRNA v9 genome and subgenomic RNA expression is lower than the samRNA vl control group.

[0103] Table 4 RT-qPCR primers

[0104] Example 6 Intrinsic immune response detection

[0105] The level of innate immune activation was observed by determining the expression of IFNβ in cell supernatant using Elisa method. In a 24-well plate, Hela cells were plated at a cell amount of 2X10^5 per well. After overnight culture, 200 ng of samRNA-LNP samples were transfected, and Poly:IC treated samples were used as positive controls. After 48 h, cell supernatant was collected, and the expression of IFNβ was detected by Elisa method (Human IFN-β ELISA Kit; Elabscience).

[0106] As shown in FIG. 8, in Hela cells, the Poly I:C treatment control group effectively stimulated the production of IFNβ, but the IFNβ expression of the linear mRNA control group and the samRNA treatment group were all near the detection line, and the detection values of some groups were lower than the detection line. The results showed that the different samRNA designs had low levels of innate immune activation, and there was no significant difference.

[0107] Example 7 Luciferase expression level and time detection in mice in vivo

[0108] The GFP element in different samRNA designs was replaced with luciferase (Luciferase) to construct a samRNA-Luciferase vector. As in Example 2, samRNA (Luciferase)-LNP was constructed. 6-8 week old BALB / c mice were selected, and 1 μg of samRNA (Luciferase)-LNP was injected intramuscularly on day 0 (D0). On D1, D3, D5, D7, D10, D14, D21, D28 after injection, the luciferase luminescence intensity was detected by in vivo imaging. At the same time, the body weight of the mice was monitored on D0 and before each in vivo imaging.

[0109] As shown in FIG. 9, luciferase expression in different samRNA design groups can last for up to 28 days, which is much longer than the maximum of 7 days of linear mRNA group. The luciferase expression levels of different samRNA design groups are all higher than those of the linear mRNA group with the same dose. The luciferase expression level of samRNA v2 group is lower than that of samRNA v1 group. The luciferase expression levels of samRNA v3 and samRNA v6 groups are close, both of which are higher than those of samRNA v1 and samRNA v2 groups. The results show that the nsP2 Q739L and G763R mutations can enhance the luciferase expression level in mice, which is better than the TC-83 control. In the long-term weight observation of mice (FIG. 9), no obvious weight loss is found, indicating that the samRNA-LNP has good safety.

[0110] Example 8 samRNA mutation design scheme

[0111] Based on the samRNA v6 design, this embodiment supplements the nsP3-P773S mutation scheme, aiming to achieve a better vector effect than samRNA v6. The amino acid sequence corresponding to the nsP of samRNA is shown in SEQ ID NO: 10.

[0112] Table 5 supplementary mutation design scheme

[0113] The results show that, based on samRNA v6, the addition of P773S mutation has no obvious effect on cell toxicity, subgenomic and genomic replication levels, but the expression level of self-replicating vector load protein is improved.

[0114] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, which all belong to the protection scope of the present application.

[0115] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A self-replicating RNA construct (saRNA) derived from a replicon of the attenuated strain VEEV TC-83, characterized in that, The self-replicating RNA construct comprises: (A) a replicase-encoding unit comprising a nucleic acid segment encoding viral non-structural proteins, the viral non-structural proteins being nsP1, nsP2, nsP3 and nsP4, the nsP2 comprising the following mutation: glycine at position 763 or a position equivalent thereto is replaced by arginine, and the self-replicating RNA construct encoding nsP2 at position 739 or a position equivalent thereto is replaced by leucine. (B) a heterologous RNA-encoding unit encoding at least one heterologous RNA sequence, but lacking sequences encoding alphavirus structural proteins, the heterologous RNA expressing a desired target protein.

2. The self-replicating RNA construct derived from a replicon of the attenuated strain VEEV TC-83 according to claim 1, characterized in that, The nsP2 encoded by the self-replicating RNA construct further comprises the following mutation: proline at position 773 or a position equivalent thereto is replaced by serine.

3. The self-replicating RNA construct derived from a replicon of the attenuated strain VEEV TC-83 according to claim 1 or 2, characterized in that, The self-replicating RNA construct comprises, in the 5'-3' direction: (1) a 5' cap structure, which is a natural 5' cap or a 5' cap analog, helping to protect the saRNA from nuclease degradation and promoting translation initiation, the 5' cap structure being co-transcriptionally capped; (2) a 5' UTR sequence, regulating replication of the RNA; (3) a nucleotide sequence encoding the replicase; (4) a subgenomic promoter SGP, ensuring efficient replication of the heterologous RNA sequence; (5) a nucleotide sequence encoding the heterologous RNA expressing a desired target protein, which is controlled by the subgenomic promoter; (6) a 3' UTR sequence, supporting replication and efficient translation of the RNA; (7) a polyA structure, increasing RNA stability and promoting translation.

4. The self-replicating RNA construct derived from a replicon of the attenuated strain VEEV TC-83 according to any one of claims 1 to 3, characterized in that, The heterologous RNA expresses one or more of the following: an immunogen, an antibody, a cytokine, a lymphokine, a chemokine, a toxin, a therapeutic protein, an enzyme, an enzyme inhibitor, a hormone, an immune response modulator, a reporter gene, etc.

5. A DNA molecule encoding the self-replicating RNA construct according to any one of claims 1-4.

6. An expression vector comprising the DNA molecule according to claim 5.

7. A vaccine comprising the self-replicating RNA construct according to any one of claims 1-4.

8. Use of the self-replicating RNA construct according to any one of claims 1-4, the DNA molecule according to claim 5, the expression vector according to claim 6, or the vaccine according to claim 7, for the preparation of a medicament for the prevention or treatment of a disease.

9. Use according to claim 8, characterized in that, The disease is an infectious disease, a tumor, an autoimmune disease, an allergic disease, a genetic disease, a metabolic disease or tissue repair.

10. Use according to claim 8 or 9, characterized in that, The treatment is gene therapy or immunotherapy; preferably, the gene therapy or immunotherapy is a therapeutic vaccine; more preferably, the therapeutic vaccine can induce a high level of cellular immune response.

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