STING protein mutant, nucleic acid for coding STING protein mutant and application of STING protein mutant

Abstract

The present application provides a STING mutant protein, nucleic acids and vectors encoding the protein, compositions or vaccines comprising the protein or nucleic acids or vectors, and uses thereof to stimulate immune responses or treat cancer.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, specifically to using STING mutants to change the tumor microenvironment, thereby activating the immune surveillance of the body's immune system against tumor cells; this application also relates to the application of using STING mutants to treat tumors. Background Art

[0002] The cGAS-STING signaling pathway is an important signaling pathway for monitoring abnormal cytoplasmic DNA in cells. When host cells are invaded by pathogens such as viruses, pathogen DNA in the cytoplasm will be recognized by cGAS. Activated cGAS will catalyze the formation of cGAMP using ATP and GTP as substrates. cGAMP is a cyclic dinucleotide that can bind to STING located on the endoplasmic reticulum and activate STING. Activated STING will transfer to the Golgi apparatus, and during this process, recruit and activate TBK1, further leading to the phosphorylation and nuclear translocation of IRF3 and NF-κB. The activation of IRF3 will induce the expression of type I interferon (Type I IFN) and inflammatory factors, activating the immune response.

[0003] The cGAS-STING signaling pathway can not only recognize exogenous pathogen DNA, but also recognize the abnormal increase of its own cytoplasmic DNA. For example, in tumor cells, due to the functional defects of DNA repair proteins, tumor cells show genetic instability, resulting in an increase in cytoplasmic DNA content and activating the cGAS-STING signaling pathway. In order to avoid the immune surveillance caused by the activation of the endogenous cGAS-STING in tumor cells, many cancers have evolved defects in the STING signaling pathway. Currently, it is believed that tumors inhibit the STING signaling pathway through epigenetic modifications such as DNA methylation.

[0004] Therefore, reactivation of the STING signaling pathway in tumors may reactivate the immune surveillance of the body's immune system against tumor cells. Activating STING in antigen-presenting cells such as DC cells may change the tumor microenvironment by releasing type I interferon (Type IIFN) and inflammatory factors, thereby activating the immune surveillance of the body's immune system against tumor cells. Summary of the Invention

[0006] Based on the constitutive mutation design of STING (STimulator of INterferon Genes), this application has obtained a series of human STING protein mutants with excellent STING pathway activation activity. The amino acid position numbers of the human STING protein mutants described in this application are defined as the corresponding amino acid positions shown in SEQ ID NO:1. Brief Description of the Drawings

[0007] Figure 1 It shows the detection results of the expression of the downstream pathway activated by the gain-of-function mutation of the STING function independently designed by the present invention in Example 2. Detailed Description of the Invention

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with a general definition of many of the terms used in this invention: Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2 nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5 th ED., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

[0010] Each publication, patent application, patent, and other reference cited herein is hereby incorporated by reference in its entirety to the extent that it does not conflict with the present disclosure.

[0011] STING: The term "Stimulator of Interferon Genes" or "STING" as used herein includes, but is not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide strands, complementary sequences, peptides, polypeptides, proteins, homologous and / or orthologous STING molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and active fragments thereof.

[0012] Gain-of-function mutants of STING: Refers to STING protein mutants that result in constitutive activity of STING. Gain-of-function mutants of STING are also STING variant polynucleotides with mutations in the wild-type STING protein. Exemplary constitutive activity mutations of the present application include, but are not limited to, the V155M, V155M-K289R-K338R, L170E, A233E, I235E, D237R, D237K, S275Q, V155W, N188V, or L190V mutations of SEQ ID NO:1. Among them, the STING V155M mutation is a mutation in the prior art, and the other mutations are STING mutants independently designed and prepared in the present application.

[0013] Coding sequence: Can be used to refer to the ribonucleotide sequence in mature mRNA that can be translated into a protein, or can also refer to the complementary sequence of the deoxyribonucleotide (DNA) sequence used as a template to transcribe the ribonucleotide (RNA) sequence. In addition, the "coding sequence" of the present application can further include polynucleotide sequences encoding functional nucleic acids, such as miRNA, shRNA, dsRNA, etc.

[0014] The term "5' cap" is located at the 5' most end of mRNA and contains methylated guanosine, which is linked to the 5' end of mRNA via pyrophosphate and forms a 5',5'-triphosphate linkage with the adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are respectively called type O, type I, and type II. Type O means that the ribose of the terminal nucleotide is not methylated, type I means that the ribose of the terminal one nucleotide is methylated, and type II means that the riboses of the terminal two nucleotides are both methylated. In this article, "CleanCap AG" is used to refer to the m7G(5’)ppp(5’)(2’-OMeA)pG cap.

[0015] The term "PolyA tail" or "PolyA sequence" refers to an uninterrupted or interrupted sequence of adenosine residues that is typically located at the 3'-end of an RNA molecule. The Poly-A tail or Poly-A sequence is known to those skilled in the art and can be selected according to actual needs. In mRNA, in the presence of a 3'-UTR, the Poly-A sequence is linked to the 3’-end of the 3'-UTR. An uninterrupted poly-A tail is characterized by having consecutive adenosine residues. The Poly-A tail can be of any length. In some embodiments, the Poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 adenosines (A), particularly about 120 A. Generally, the vast majority of nucleotides in the PolyA tail are adenosines, and the vast majority means at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridine), G (guanosine), or C (cytidine).

[0016] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or nucleotide sequences, as determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, after making two sequences have the same residues at as many positions as possible by introducing gaps and the like, the proportion of the number of positions with the same bases or amino acid residues to the total number of positions is determined. The percentage of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0017] As used herein, "delivery vehicle" refers to a structure formed by packaging or encapsulating larger biomolecules such as polynucleotides and polypeptides to assist their entry into cells, which has a higher affinity for cell membranes and is more prone to transmembrane transport from the extracellular to the intracellular space. Delivery vehicles and their preparation methods are known in the art and include, but are not limited to, liposomes (such as lipid nanoparticles (LNPs)), viruses (such as AAV, lentivirus), and quantum dots. The preparation methods of LNPs are known in the art, such as those disclosed in CN114901360A and CN113941011A. In some embodiments, the LNP comprises a PEGylated lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof.

[0018] As used herein, the term "adjuvant" refers to an exogenous substance that can be added to a pharmaceutical composition or formulation to enhance the antigen response of an individual's immune system, including but not limited to chemical adjuvants and bacterial antigens.

[0019] As used herein, an "open reading frame (ORF)" is a continuous DNA or RNA segment that starts with a start codon (such as the methionine codon (ATG or AUG)) and ends with a stop codon (such as TAA, TAG, or TGA, or UAA, UAG, or UGA). An ORF typically encodes a protein.

[0020] A "variant" refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant can be from the same or different species, or can be a synthetic sequence based on a natural or existing molecule. In the present application, "variant" can be used to refer to variants of proteins, polypeptides, or amino acid sequences, and can also be used to refer to variants of nucleic acid molecules or polynucleotide sequences.

[0021] In some embodiments, a "variant" of the amino acid sequence differs from the amino acid sequence by at least one amino acid, such as having at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitution" can be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys), and arginine (R, Arg)); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp), and methionine (M, Met). In some other embodiments, the variant may also include non-conservative substitutions. In some embodiments, a "variant" of the amino acid sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared with the amino acid sequence, the "variant" of the amino acid sequence may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or the range formed by any two of the foregoing values of activity. As used herein, a "conservative substitution variant" of a certain protein, polypeptide, or amino acid sequence refers to one or more amino acid residues being substituted by amino acids without changing the overall conformation and function of the protein or enzyme, which includes but is not limited to substituting the amino acids in the amino acid sequence of the parental protein in the manner described by the foregoing "conservative substitution". Therefore, the similarity between two proteins or amino acid sequences with similar functions may be different. For example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes determined by BLAST or FASTA algorithms to have more than 60% amino acid identity, preferably more than 75%, even better more than 85%, and optimally more than 90%, and having the same or substantially similar properties or functions compared to the native or parental protein or enzyme.

[0022] Therefore, the STING protein mutants described in this application should cover the above variants.

[0023] Those skilled in the art should be aware that variants of nucleic acid molecules or polynucleotide sequences encoding proteins include "synonymous mutants", which refer to nucleic acid molecules or polynucleotide sequences obtained after one or more codons in the nucleic acid molecule or polynucleotide sequence are replaced by other codons encoding the same amino acid as the replaced codon.

[0024] The term "at least comprising" means that the polynucleotide sequence may consist of the coding sequence of the above STING protein mutant, or may further comprise other polynucleotide sequences on the basis of comprising the coding sequence of the above STING protein mutant.

[0025] For example, sequences regulating the expression of the above STING protein mutant, sequences making the polynucleotide more stable.

[0026] The polynucleotide sequence may be a DNA sequence, an RNA sequence, or a hybrid of a DNA sequence and an RNA sequence.

[0027] As used herein, the term "treatment" refers to obtaining the desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of the occurrence or onset of a disease or its symptoms, partial or complete alleviation of the disease and / or its symptoms, and / or partial or complete cure of the disease and / or its symptoms, including: (a) preventing the occurrence or onset of a disease in a subject who may have a predisposition to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., blocking its formation; and (c) alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to subside or disappear.

[0028] The term "subject" in this application refers to mammals, including but not limited to mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (such as horses, cows, sheep, pigs, goats), etc.

[0029] "Therapeutically effective amount" or "effective amount" refers to an amount sufficient to achieve the prevention and / or treatment of a disease when administered to a mammal or other subject for treating the disease. The "therapeutically effective amount" will vary depending on the drug used, the disease of the subject to be treated and / or the severity of its symptoms, as well as age, body weight, etc. Those skilled in the art can easily determine the appropriate therapeutically effective amount and dosing frequency of the protein or composition of the present invention based on various parameters, especially based on the age, body weight and condition of the subject to be treated, the severity of the disease or condition, and the route of administration. The routes of administration include, but are not limited to, enteral, topical, suppository, inhalation, and parenteral administration, such as subcutaneous, intramuscular or intravenous injection. Detailed implementation manners

[0030] This application provides a STING mutant protein for reactivating the STING signaling pathway in tumors and a nucleic acid encoding the same, a delivery body, a cell, a pharmaceutical composition or a pharmaceutical product comprising the STING mutant protein or its encoding nucleic acid, and the use of the nucleic acid or the protein.

[0031] Specifically, in the first aspect of this application, an engineered human STING protein mutant is provided, wherein the mutation comprises (1) V155W; (2) V155M-K289R-K388R combination; (3) S275Q; (4) L170E; (5) A233E; (6) I235E; (7) D237R; (8) D237K; (9) L190V or (10) N188V; the amino acid position numbers are defined as the corresponding amino acid positions shown in SEQ ID NO:1.

[0032] In some embodiments, the amino acid sequence of the STING protein mutant comprises or consists of the following amino acid sequences: 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, SEQ ID NO:11 or SEQ ID NO:12, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto.

[0033] In the second aspect of this application, a nucleic acid molecule encoding any of the aforementioned STING protein mutants is provided; preferably, the nucleic acid molecule is DNA or RNA.

[0034] In some embodiments, the nucleic acid molecule comprises a 5' UTR structure; preferably, the 5' UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 13, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 13.

[0035] In some embodiments, the nucleic acid molecule further comprises a 3' UTR structure; preferably, the 3' UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 14, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 14.

[0036] In some embodiments, the nucleic acid molecule is mRNA.

[0037] In some embodiments, the open reading frame (ORF) sequence of the mRNA is selected from any one of the sequences shown in SEQ ID NOs: 18 - 27, or a sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity therewith.

[0038] In some embodiments, some or all of the uridines of the mRNA are chemically modified uridines; preferably, the chemically modified uridine is pseudouridine or N1 - methyl - pseudouridine.

[0039] In some embodiments, the mRNA further comprises a 5' cap structure; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0040] In some embodiments, the mRNA further comprises a polyA tail; preferably, the polyA tail comprises at least 50, at least 60 or at least 100 A nucleotides; more preferably, the polyA tail comprises at least the polynucleotide sequence shown in SEQ ID NO: 15, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 15.

[0041] In some embodiments, the sequence of the nucleic acid molecule is as shown in any one of SEQ ID NOs: 30 - 39.

[0042] This application also relates to a nucleic acid molecule complementary to the aforementioned nucleic acid molecule.

[0043] In a third aspect of the present application, there is provided an expression vector comprising any of the nucleic acid molecules described above; wherein the vector is a viral vector or a plasmid vector.

[0044] In some embodiments, the viral vector is selected from vesicular stomatitis virus (VSV), lentivirus, adenovirus, adeno-associated virus, vaccinia virus, and modified vaccinia virus Ankara.

[0045] In a fourth aspect of the present application, there is provided a delivery vehicle comprising any of the protein mutants or nucleic acid molecules described above.

[0046] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) or a lipid complex (LPX).

[0047] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-lipid.

[0048] In a fifth aspect of the present application, there is provided an isolated cell comprising any of the protein mutants, nucleic acid molecules, or vectors described above;

[0049] Preferably, the cell is selected from insect cells, mammalian cells, avian cells, bacteria, and yeast cells; preferably the cell is an Escherichia coli cell.

[0050] In a sixth aspect of the present application, there is provided a pharmaceutical composition comprising any of the protein mutants, nucleic acid molecules, vectors, delivery vehicles, or cells described above, and a pharmaceutically acceptable excipient.

[0051] In some embodiments, the pharmaceutical composition is an mRNA vaccine.

[0052] In a seventh aspect of the present application, there is provided a method of activating STING in a subject in need thereof, comprising administering the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery vehicle described in the fourth aspect, the isolated cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, which induces STING signaling.

[0053] In some embodiments, the present application provides a method of stimulating an immune response in a subject in need thereof, comprising administering the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery vehicle described in the fourth aspect, the isolated cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, which induces STING signaling.

[0054] In some embodiments, the subject has cancer or a microbial infection.

[0055] In some embodiments, the present application provides a method for treating cancer in a subject, comprising administering the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery vehicle described in the fourth aspect, the isolated cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, which induces STING signaling.

[0056] In some embodiments, the cancer is ovarian cancer, colon cancer, melanoma, breast cancer or lung cancer.

[0057] In some embodiments, the composition is administered intratumorally, intravenously, intraarterially, intraperitoneally, intranasally, intramuscularly, intradermally or subcutaneously.

[0058] In some embodiments, the STING protein mutant or the composition induces infiltration of immune cells into the tumor.

[0059] In the eighth aspect of the present application, an adjuvant is provided, which comprises the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery vehicle described in the fourth aspect, the isolated cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, which has the effect of inducing STING signaling.

[0060] In some embodiments, the present application provides a method for enhancing the immune response to a vaccine in a subject receiving the vaccine, the method comprising administering the adjuvant as described above.

[0061] In the ninth aspect of the present application, the use of the STING protein mutant, nucleic acid molecule, vector, delivery vehicle, cell or pharmaceutical composition as described above in the preparation of a drug for the treatment and / or prevention of cancer or microbial infection is provided.

[0062] It should be understood that the present application encompasses the various aspects, embodiments, and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements, and modifications within the scope of the present application will be apparent to those skilled in the art to which the present application pertains. Therefore, those of ordinary skill in the art should recognize that the scope of the present application also includes such improvements and modifications to the aspects and embodiments.

[0063] Examples

[0064] Example 1: Construction and Preparation of mRNA Sequences Encoding STING Mutants

[0065] 1. Synthesis of 1STING mRNA sequence and construction of recombinant vector

[0066] From the 5' end in sequence: The coding nucleotide sequence with a T7 promoter with XbaI at the 5' end, 5'UTR, tPA-SP, Flt3L, STING mutant ORF, 3'UTR and / or poly A tail is digested with XbaI and NotI, and ligated with the vector backbone fragment of pUC57-GW-Kan (Genewiz) digested with XbaI and NotI to construct a recombinant plasmid.

[0067] 1.2 mRNA preparation

[0068] 1.2.1 Plasmid linearization

[0069] The recombinant plasmid constructed in step 1 has a SapⅠ restriction site behind the last A of the polyA tail sequence. The plasmid containing the target gene is linearized with the restriction enzyme SapⅠ. The reaction system is shown in Table 1 and digested at 37°C for 3 h.

[0070] Table 1. Enzyme digestion system for plasmid linearization

[0071] 10× Cutsmart buffer 5 μL SapI enzyme (10000 U / mL) 1 μL Plasmid 10 μg ddH2O Make up to 50 μL

[0072] Take 2 μL of the enzyme digestion product for 1% agarose gel electrophoresis to detect the linearization of the plasmid. Purify the linearized plasmid using a PCR product recovery kit (ComWin Biotech).

[0073] (2) In vitro transcription and purification

[0074] Using the linearized recombinant plasmid obtained in step (1) as a template for in vitro transcription, and performing in vitro transcription using a high-yield T7 RNA transcription kit. The high-yield T7 RNA transcription kit, with the product name High Yield T7 RNA Synthesis Kit, is from Shanghai Zhaowei Technology Development Co., Ltd., and the product catalog number is ON-040; 5×Reaction Buffer, 100 mM ATP Solution, 100 mM CTP Solution, 100 mM GTP Solution, Enzyme mix, DNase I, Ammonium Acetate Stop Solution, Lithium Chloride (LiCl) Precipitation Solution are all components in the high-yield T7 RNA transcription kit. 100 mM ΨUTP Solution (pseudouridine triphosphate), with the full name N1-Me-pUTP, 100 mM, is from Shanghai Zhaowei Technology Development Co., Ltd., and the product catalog number is R5-027. Add each component according to the following system (Table 2) (taking a 20 μL reaction system as an example), mix well and react at 37 °C for 3 h.

[0075] Table 2. In vitro transcription system

[0076]

[0077]

[0078] Among them, CleanCap AG is m7G(5’)ppp(5’)(2’-OMeA)pG, with the product number ON-134, from Shanghai Zhaowei.

[0079] After the transcription reaction is completed, add 1 μL of DNase I, react at 37 °C for 15 min, add 15 μL of Ammonium Acetate Stop Solution, and mix well. Then add 1 / 3 volume of 7.5 M Lithium Chloride (LiCl) Precipitation Solution (to make the final concentration 2.5 M), place at -20 °C for 30 min. Centrifuge at 12000 g for 15 min, the RNA precipitate is at the bottom, discard the supernatant. Add 1 mL of 70% ethanol to wash the RNA, centrifuge at 12000 g for 5 min, and discard the supernatant. After air-drying, add 50 μL of RNase-free water to dissolve the precipitate, and perform mRNA quantification using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0080] Example 2. Detecting the activity of the STING gain-of-function mutants designed independently in the present invention by detecting the activation of the STING downstream pathway

[0081] Seed THP-1 cells at a density of 2×10^5 cells per well in a 12-well cell culture dish and incubate overnight in a 37°C, 5% CO 2 incubator. Transfect 200 ng of STING mRNA per well using Lipofectamine TM 3000. After 6 h, extract total RNA using the FastPure Cell / Tissue Total RNA Isolation Kit V2, synthesize cDNA using the Bio-Rad iScriptTM cDNA synthesis kit, and perform qPCR experiments using the ChamQ Universal SYBBR qPCR Master Mix according to the instructions to detect the transcriptional level of IFN-β in each group.

[0082] According to Figure 1 it can be seen that a certain level of IFN-β was shown in each group, indicating that each STING mutant has the activity to activate the downstream pathway. Among them, the V155W mutant, the V155M-K289R-K338R combined mutant, V155M, and the S275Q group showed the best performance. Among the four groups, there was a very significant difference in the IFN-β transcriptional level between the V155M-K289R-K338R combined mutant group and the V155M mutant group (p < 0.01), and there was an extremely significant difference in the IFN-β transcriptional level between the V155W mutant group and the V155M mutant group (p < 0.001). The above results indicate that the STING mutants independently designed in the present invention exhibit a STING pathway activation level comparable to or significantly superior to the prior art.

[0083] The sequences used in the above embodiments of the present application are shown in the following sequence listing. It should be understood that the following sequences are only exemplary sequences of the embodiments of the present application and do not impose any limitation on the present application. The nucleic acid sequences in the following sequence listing can represent DNA sequences or RNA sequences. When it represents an RNA sequence, "T" therein represents uridine.

[0084] Sequence Listing

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

Claims

1. An engineered human STING protein mutant, wherein the mutation comprises (1) V155W; (2) a combination of V155M-K289R-K388R; (3) S275Q; (4) L170E; (5) A233E; (6) I235E; (7) D237R; (8) D237K; (9) L190V or (10) N188V, wherein the amino acid position numbers are defined as the corresponding amino acid positions shown in SEQ ID NO:

1.

2. The STING protein mutant according to claim 1, wherein the amino acid sequence of the mutant comprises or consists of the following amino acid sequence: 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, SEQ ID NO:11 or SEQ ID NO:12, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

3. A nucleic acid molecule encoding the STING protein mutant according to claim 1 or 2, preferably, the nucleic acid molecule is DNA or RNA.

4. The nucleic acid molecule according to claim 3, comprising a 5'UTR structure, Preferably, the 5'UTR structure comprises at least the polynucleotide sequence as shown in SEQ ID NO: 13, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 13, And it further comprises a 3'UTR structure, Preferably, the 3'UTR structure comprises at least the polynucleotide sequence as shown in SEQ ID NO:14, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

14. The nucleic acid molecule according to claim 3 or 4, which is mRNA.

6. The nucleic acid molecule according to any one of claims 3 to 5, wherein the open reading frame (ORF) sequence is selected from any one of the sequences shown in SEQ ID NO: 18 to 27, or a sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

7. The nucleic acid molecule according to any one of claims 3 to 6, wherein some or all of the uridines in the mRNA are chemically modified uridines; Preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine; The mRNA further comprises a 5' cap structure; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG. The mRNA further comprises a polyA tail; preferably, the polyA tail comprises at least 50, at least 60 or at least 100 A nucleotides; More preferably, the polyA tail comprises at least the polynucleotide sequence as shown in SEQ ID NO:15, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

15.

8. The nucleic acid molecule according to claim 3, wherein the sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 30-39.

9. A nucleic acid molecule complementary to the nucleic acid molecule of any one of claims 3 to 8.

10. An expression vector comprising the nucleic acid molecule according to any one of claims 3 to 9; wherein the vector is a viral vector or a plasmid vector; Preferably, the viral vector is selected from the group consisting of vesicular stomatitis virus (VSV), lentivirus, adenovirus, adeno-associated virus, vaccinia virus and modified vaccinia Ankara virus.

11. A delivery vehicle comprising a protein mutant according to claim 1 or 2 or a nucleic acid molecule according to any one of claims 3-9; preferably, the delivery vehicle is a lipid nanoparticle (LNP) or a lipid complex (LPX), wherein the LNP comprises ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids; preferably, in the LNP, the molar ratio of ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids is (40-55): (10-15): (35-45): (0.5-2.5).

12. An isolated cell comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3 to 9, or the vector according to claim 11; Preferably, the cell is selected from the group consisting of insect cells, mammalian cells, avian cells, bacteria and yeast cells; preferably the cell is an E. coli cell.

13. A pharmaceutical composition comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-9, the vector according to claim 10, the delivery body according to claim 11 or the cell according to claim 12, and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, which is an mRNA vaccine.

15. An adjuvant comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-9, the vector according to claim 10, the delivery body according to claim 11, the cell according to claim 12 or the pharmaceutical composition according to claim 13 or 14, which has the function of making the STING protein constitutively active.

16. Use of the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-9, the vector according to claim 10, the delivery body according to claim 11, the cell according to claim 12 or the pharmaceutical composition according to claim 13 or 14 in the preparation of a drug for the treatment and / or prevention of cancer or microbial infection.

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