Engineered protein, delivery system and application thereof

Abstract

The invention provides an engineered protein, a delivery system and application of the engineered protein, the engineered protein comprises RNA binding polypeptide and cell transmembrane protein, the C end of the RNA binding polypeptide is connected with the N end of the cell transmembrane protein, or the C end of the cell transmembrane protein is connected with the N end of the RNA binding polypeptide, or the C end of the RNA binding polypeptide is connected with the N end of the cell transmembrane protein. Or an RNA binding polypeptide is inserted into an intracellular region of the cell transmembrane protein to form a fusion protein, and the engineered protein can be specifically bound with alphavirus self-replicating mRNA. The engineered protein according to the embodiment of the invention can effectively recognize and combine alphavirus self-replicating mRNA, a delivery vector prepared from the engineered protein or a vector containing a nucleic acid sequence encoding the engineered protein, such as extracellular vesicles, viruses, nucleic acid protein complexes, plasmids, RNA and cells. The alphavirus self-replicating mRNA can be efficiently delivered into cells or bodies, and the alphavirus self-replicating mRNA has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to engineered proteins, delivery systems and applications thereof. Background Art

[0002] Self-replicating mRNA (saRNA) technology is a new generation of mRNA technology. The COVID-19 vaccine (ARCT-154) developed based on this technology has been approved for marketing in Japan in 2023. Generally, self-replicating mRNA (including the approved ARCT-154 vaccine) is selected from the alphavirus replicon system. The self-replicating mRNA sequence based on the alphavirus mainly includes the following structural elements: (i) 5' end cap; (ii) 5' end non-coding region nucleic acid sequence of the alphavirus; (iii) alphavirus non-structural protein nucleic acid sequence, i.e. alphavirus RNA replicase nucleic acid sequence (NS1-NS2-NS2-NS4), which can be translated and synthesized into alphavirus replicase; (iv) alphavirus 26S promoter nucleic acid sequence, which guides the transcription of subgenomic mRNA and the translation and synthesis of target protein; (v) 3' end non-coding region nucleic acid sequence of the alphavirus; (vi) 3' end PolyA tail.

[0003] In cells, self-replicating mRNA can use the RNA replicase nucleic acid sequence it carries as a template to translate and synthesize RNA replicase. On this basis, the RNA replicase uses the self-replicating mRNA sequence as a template to transcribe multiple copies of offspring self-replicating mRNA, thereby realizing a large number of amplification of the self-replicating mRNA sequence. At the same time, in the self-replicating mRNA sequence of the alphavirus replicon, the gene encoding the target protein is placed downstream of the 26S promoter. The RNA replicase can recognize the 26S promoter and transcribe a larger number of subgenomic mRNAs containing the gene encoding the target protein. The subgenomic mRNA uses the protein translation system of the host cell to translate and synthesize the target protein in large quantities. Therefore, the self-replicating mRNA system based on the alphavirus replicon has a higher number of mRNA templates, protein expression levels, and a longer expression duration than the non-replicating mRNA system, and is hailed as one of the most promising next-generation mRNA technologies.

[0004] The approved ARCT-154 coronavirus vaccine uses a liposome nanoparticle (LNP) delivery system. However, the low delivery efficiency and lack of tissue targeting of the LNP delivery system restrict its further application in the field of self-replicating mRNA biomedicine.

[0005] Extracellular vesicles (EVs) are a new type of RNA delivery vector. Since the regulatory mechanism of cellular vesicle transport won the Nobel Prize in Physiology or Medicine in 2013, EVs have gradually become a hot research topic in the global biomedical field.

[0006] Extracellular vesicles are a heterogeneous group of membrane structures secreted by cells and wrapped by a lipid bilayer, containing tiny particles secreted by cells. All cells, prokaryotes and eukaryotes, release extracellular vesicles. Extracellular vesicles can be roughly divided into three categories: exosomes are smaller vesicles with a diameter of about 50 to 150nm and have endosomal origin; microvesicles are larger vesicles with a diameter of 50 to 500nm to 1000nm, formed by the shedding of cell membranes, and can mature and separate through protrusions on the cell membrane; apoptotic bodies are larger vesicles formed during cell death, with a diameter greater than 1000nm, and when cells enter the apoptotic process, they release these vesicles.

[0007] Extracellular vesicles can carry a variety of bioactive molecules, such as nucleic acids (RNA and DNA), proteins and metabolites, and play an important role in many biological processes such as intercellular communication and signal transduction. They can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion, etc. In addition, extracellular vesicles can be artificially loaded with drugs such as small molecules, nucleic acids, and protein peptides, and can transport bioactive molecules to receptor cells, so they have strong application potential in the field of drug delivery.

[0008] Compared with synthetic nanocarriers (including LNP delivery carriers), extracellular vesicles have many advantages as delivery carriers. First, extracellular vesicles are derived from cells and are more easily metabolized after drug delivery. Second, extracellular vesicles exhibit lower immunogenicity, avoid causing unwanted immune responses, have good tolerance, and can be repeatedly injected. In addition, extracellular vesicles can cross in vivo barriers such as the blood-brain barrier that are difficult for other nanocarriers to cross. Based on the enrichment of ligand presentation on their surface, extracellular vesicles can also make the development of receptor-mediated tissue targeting possible. Ligand enrichment on engineered exosomes can also be used to induce or inhibit signaling events in receptor cells or target exosomes to specific cell types.

[0009] There are many literature reports on the loading of small RNA or non-replicating mRNA into extracellular vesicles, and some of these drugs have entered the clinical trial stage. However, there are few public reports on the active loading of self-replicating mRNA into extracellular vesicles. Active loading of self-replicating mRNA into extracellular vesicles faces more technical challenges.

[0010] Obviously, the simplest extracellular vesicle delivery system loaded with self-replicating mRNA can be achieved by the following two methods: 1) First, prepare extracellular vesicles and self-replicating mRNA separately, and then load the self-replicating mRNA into extracellular vesicles by electroporation; 2) Express self-replicating mRNA, exosome skeleton proteins and / or viral membrane proteins in cells, and cells can self-assemble to form extracellular vesicles loaded with self-replicating mRNA. Although the above two methods can theoretically load self-replicating RNA, the loading effect is not ideal in practice. One of the most important factors is that the length of the self-replicating RNA sequence is usually 3 to 4 times the length of the non-replicating mRNA sequence, which is difficult to be effectively loaded into extracellular vesicles. Therefore, the organic combination of the alphavirus replicon system and the extracellular vesicle delivery system is not as simple as imagined. It requires more in-depth research on alphavirus and alphavirus replicons, and more sophisticated design of the extracellular vesicle delivery system.

[0011] RNA binding protein (RBP) is an important class of proteins that interact with RNA by recognizing specific RNA binding domains. In RNA viruses, viral capsid protein is a type of RBP that can recognize the RNA packaging signal of viral RNA and package the viral RNA into the viral capsid. The viral capsid protein polypeptide that interacts with the viral RNA packaging signal is the RNA binding polypeptide. The TAT peptide of lentivirus is a typical RNA binding polypeptide, which is usually used to specifically package non-replicating mRNA in exosome vectors.

[0012] Alphavirus is a type of enveloped, single-stranded, positive-sense RNA virus that is spread by arthropods such as mosquitoes. It belongs to the Togaviridae family and can widely infect humans, birds, mice, horses and other animals and cause related diseases. Alphavirus has two structural proteins, capsid protein (C) and membrane protein (E). Alphavirus C protein is also an RNA binding protein that can specifically recognize and bind to the RNA of the same virus and aggregate to form a viral capsid. Alphavirus C protein includes two domains, the N-terminal domain and the C-terminal domain. The RNA binding site of alphavirus is mainly located in the N-terminal domain of the C protein, and the RNA packaging signal of alphavirus is mainly located in the non-structural protein coding region of alphavirus RNA.

[0013] Based on the fact that the self-replicating mRNA of the alphavirus replicon contains the nucleic acid sequence of the non-structural protein coding region of the alphavirus, the N-terminal domain polypeptide of the alphavirus C protein or its truncated form is fused to the cell transmembrane protein to form an engineered protein by utilizing the characteristic that the N-terminal domain polypeptide of the alphavirus C protein specifically recognizes the self-replicating mRNA. The engineered protein has the following characteristics: 1) it can specifically bind to the self-replicating mRNA and has the performance of actively packaging the self-replicating mRNA; 2) it can anchor the self-replicating mRNA on the cell membrane surface through the transmembrane region on the cell transmembrane protein and has the performance of promoting the formation of extracellular vesicles; 3) it can specifically recognize the host cell receptor through the receptor binding site on the cell transmembrane protein and has the performance of enhancing the tissue-targeted delivery of the self-replicating mRNA; 4) it can promote membrane fusion through the fusion site on the cell transmembrane protein and has the performance of enhancing the delivery efficiency of the self-replicating mRNA.

[0014] Since the above-mentioned engineered protein is a natural binding protein of the alphavirus self-replicating mRNA and has better binding ability than other RNA binding proteins, the delivery vector prepared using the above-mentioned engineered protein has a better effect of packaging the alphavirus self-replicating mRNA, and is expected to solve the problem of extracellular vesicle packaging of self-replicating mRNA. Summary of the invention

[0015] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0016] Therefore, in the first aspect of the present invention, the present invention proposes an engineered protein. According to an embodiment of the present invention, the engineered protein includes: an RNA binding polypeptide and a cell transmembrane protein, the C-terminus of the RNA binding polypeptide is connected to the N-terminus of the cell transmembrane protein, or the C-terminus of the cell transmembrane protein is connected to the N-terminus of the RNA binding polypeptide, or the RNA binding polypeptide is inserted into the intracellular region of the cell transmembrane protein to form a fusion protein, wherein the RNA binding polypeptide is selected from the N-terminal domain polypeptide of the alphavirus C protein, the truncated polypeptide of the N-terminal domain of the alphavirus C protein, and the mutant polypeptide of the N-terminal domain of the alphavirus C protein, and the cell transmembrane protein is selected from at least one of animal cell membrane proteins, exosome skeleton proteins, enveloped virus membrane proteins, and mutant polypeptides thereof.

[0017] According to an embodiment of the present invention, the above-mentioned engineered protein may further include at least one of the following additional technical features:

[0018] According to an embodiment of the present invention, the alphavirus includes at least one selected from the group consisting of SINV, VEEV and SFV.

[0019] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence of positions 1 to 124 of protein C of VEEV or a truncated polypeptide thereof.

[0020] According to an embodiment of the present invention, the RNA binding polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-3.

[0021] KKKKNGGKKKAKTGPPNPKAQSGNKKKPNKKPGKRQRMVMKLESD (SEQ ID NO: 1).

[0022] KKKKNGGKKKAKTGPPNPK (SEQ ID NO: 2).

[0023] KPGKRQRMVMKLESD (SEQ ID NO: 3).

[0024] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence of positions 1 to 113 of the C protein of SINV or a truncated polypeptide thereof.

[0025] According to an embodiment of the present invention, the RNA binding polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 4 or 5.

[0026] KPKKPKTGEKKKKGPAKPKPGKRGRMALKLEAD (SEQ ID NO: 4).

[0027] KPKKPKTGEKKKKGPAKP (SEQ ID NO: 5).

[0028] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence at positions 1 to 118 of the C protein of SFV or a truncated polypeptide thereof.

[0029] According to an embodiment of the present invention, the RNA binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0030] KKINGKTQQQKKKDKQADKKKKKPGKRERMCMKIEND (SEQ ID NO: 6).

[0031] KKINGKTQQQKKKDKQADKKKK (SEQ ID NO: 7).

[0032] According to an embodiment of the present invention, the animal cell membrane protein includes at least one selected from the following: a cell receptor protein and a cell fusion protein.

[0033] According to an embodiment of the present invention, the cell receptor protein includes at least one selected from the following: selectin E, PDGFR, CD6, CD14, CD11b, CD36, CD3, CD4, CD8 and CD28.

[0034] According to an embodiment of the present invention, the cell fusion protein includes at least one selected from the group consisting of: Myomaker, Mymerger and syncytin.

[0035] According to an embodiment of the present invention, the exosomal scaffold protein includes at least one selected from the following: LAMP2b, CD9, CD37, CD63, CD64, CD81, CD82, ARRDC1 and SPFH.

[0036] According to an embodiment of the present invention, the enveloped virus membrane protein includes at least one selected from the following: alphavirus glycoprotein E, vesicular stomatitis virus glycoprotein G, measles virus glycoprotein H / F, rubella virus glycoprotein E, mumps virus glycoprotein HA / NA, rabies virus glycoprotein G, influenza virus glycoprotein HA / NA, parainfluenza virus glycoprotein NH / F, respiratory syncytial virus glycoprotein G / F, Japanese encephalitis virus glycoprotein E and Sendai virus HN / F.

[0037] According to an embodiment of the present invention, the engineered protein includes: protein C polypeptide of VEEV and LAMP2b.

[0038] According to an embodiment of the present invention, the N-terminus of the VEEV protein C polypeptide is connected to the C-terminus of LAMP2b.

[0039] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:8.

[0040] MGLQLNITQDKVASVINPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNVS MYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECSL DDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTLKKKKNQQKKKAKTGPPNPK(SEQ ID NO: 8).

[0041] According to an embodiment of the present invention, the engineered protein includes: protein C polypeptide of VEEV and CD11b.

[0042] CD11b belongs to the CD11 antigen-like family, also known as integrin alpha M (ITGAM), which participates in various adhesion interactions of monocytes, macrophages and granulocytes, and regulates neutrophil migration, activation and apoptosis. At the same time, CD11b is associated with tumors and is a new target for tumor immunotherapy. Studies have shown that the activation protein CD11b helps inhibit tumor growth. CD11b is a surface marker of macrophages and microglia. Although CD11b protein has been detected in exosomes derived from macrophages and microglia under physiological conditions, there are few reports on the use of CD11b protein as a skeleton protein to produce engineered exosomes.

[0043] According to an embodiment of the present invention, the N-terminus of the VEEV protein C polypeptide is connected to the C-terminus of CD11b.

[0044] According to an embodiment of the present invention, the engineered protein has the amino acids shown in SEQ ID NO:9.

[0045]

[0046] According to an embodiment of the present invention, the engineered protein includes the C protein polypeptide of VEEV and SINV glycoprotein E2.

[0047] According to an embodiment of the present invention, the E2 of SINV deletes the amino acid sequence at positions 397 to 414.

[0048] According to an embodiment of the present invention, the protein C polypeptide of VEEV is inserted after the 396th amino acid of E2 of SINV.

[0049] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:10.

[0050] SVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADDNTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFLLAKCPP GDSVTVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTAGYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTR TEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTV DRDGLEYIWGNHEPVRVYAQESAPGDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECKKKKNQQKKKAKTGPPNPKLCCVRSANA(SEQ ID NO: 10).

[0051] According to an embodiment of the present invention, the engineered protein includes the C protein polypeptide of VEEV and the glycoprotein G of VSV.

[0052] According to an embodiment of the present invention, the N-terminus of the C protein polypeptide of VEEV is connected to the C-terminus of the glycoprotein G of VSV.

[0053] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:11.

[0054] CKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTGLQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQ VTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDRELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSS ISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIG HGMLDSGLHLSSKAQVFEHPHIQDAASQLPDDEILFFGDTGLSKNPIDFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLYIKLKHTKKRQIYTDIEMNRLGRKKKKNQQKKKAKTGPPNPK(SEQ IDNO: 11).

[0055] In a second aspect of the present invention, the present invention provides a delivery vector. According to an embodiment of the present invention, the delivery vector carries the engineered protein described in the first aspect and / or the nucleic acid sequence encoding the engineered protein. The delivery vector can deliver the alphavirus self-replicating mRNA and / or the nucleic acid sequence encoding the engineered protein into a cell or body.

[0056] According to an embodiment of the present invention, the above-mentioned delivery vector may further include at least one of the following additional technical features:

[0057] According to an embodiment of the present invention, the delivery vector comprises at least one selected from the group consisting of: an extracellular vesicle, a virus, a nucleic acid-protein complex, a plasmid, RNA, and a cell.

[0058] According to an embodiment of the present invention, the delivery vector is an extracellular vesicle, which further comprises a cell membrane vesicle and an alphavirus self-replicating mRNA. The extracellular vesicle can deliver the alphavirus self-replicating mRNA into a cell or in vivo.

[0059] According to an embodiment of the present invention, the cell membrane vesicles are derived from animal cells.

[0060] According to an embodiment of the present invention, the engineered protein is loaded on the cell membrane vesicle.

[0061] According to an embodiment of the present invention, the alphavirus self-replicating mRNA carries an alphavirus RNA packaging signal, which specifically binds to an RNA-binding polypeptide through the alphavirus RNA packaging signal and is packaged in the cell membrane vesicle to form the extracellular vesicle.

[0062] According to an embodiment of the present invention, the alphavirus self-replicating mRNA includes: a 5' cap, an alphavirus 5' non-coding region nucleic acid sequence, an alphavirus non-structural protein nucleic acid sequence, an alphavirus 26S promoter, a nucleic acid sequence encoding a target protein, an alphavirus 3' non-coding region nucleic acid sequence and a 3' PolyA tail.

[0063] According to an embodiment of the present invention, the extracellular vesicle comprises an amino acid sequence shown in any one of SEQ ID NOs: 8 to 11 or an amino acid sequence shown in any one of 29, 32, 35, 45, 47, 53, and 55.

[0064] According to an embodiment of the present invention, the delivery vector is a virus, and the virus also includes an alphavirus self-replicating mRNA. Thus, the virus can deliver the nucleic acid sequence encoding the engineered protein and / or the alphavirus self-replicating mRNA into a cell or body.

[0065] According to an embodiment of the present invention, the engineered protein is loaded on the envelope of the virus.

[0066] According to an embodiment of the present invention, the virus is selected from a non-replication-defective virus or a replication-defective virus.

[0067] According to an embodiment of the present invention, the virus includes at least one selected from the following: herpes simplex virus, poxvirus, adenovirus, alphavirus, flavivirus, influenza virus, coronavirus, rabies virus, vesicular stomatitis virus, poliovirus, measles virus.

[0068] According to an embodiment of the present invention, the virus is an alphavirus.

[0069] According to an embodiment of the present invention, the alphavirus is selected from VEEV.

[0070] According to an embodiment of the present invention, the VEEV genome deletes the C protein gene and / or the E protein gene.

[0071] According to an embodiment of the present invention, the virus has the amino acid sequence shown in SEQ ID NO:11.

[0072] The beneficial effects of the present invention are at least that: in the self-replicating mRNA delivery vector, the RNA binding polypeptide that recognizes the alphavirus RNA packaging signal is introduced into the intracellular region of the cell transmembrane protein, and the delivery vector is actively loaded with the self-replicating mRNA through the specific binding of the RNA binding polypeptide and the RNA packaging signal. In the extracellular vesicle delivery vector, the self-replicating mRNA active loading system can also improve the packaging efficiency of the extracellular vesicle for the self-replicating mRNA, expand the delivery targeting range of the delivery vector, and solve the specificity problem of the extracellular vesicle loading of the self-replicating mRNA.

[0073] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0075] Figure 1 A schematic diagram of an engineered protein structure according to an embodiment of the present invention, wherein:

[0076] Figure 1 A is a fusion protein formed by the RNA binding polypeptide binding to the C-terminus of the cell transmembrane protein. Figure 1 B is an RNA binding polypeptide inserted into the C-terminus of a cell transmembrane protein to form a fusion protein. The RNA binding polypeptide on the engineered protein of the present invention can specifically recognize and bind to the alphavirus self-replicating mRNA;

[0077] Figure 2 is a schematic diagram of the structure of a self-replicating mRNA according to an embodiment of the present invention, wherein the self-replicating mRNA can specifically bind to the engineered protein of the present invention;

[0078] Figure 3 This is a schematic diagram of the structure of an extracellular vesicle delivery vector according to an embodiment of the present invention, wherein the outer capsule of the extracellular vesicle is composed of a cell membrane loaded with the engineered protein of the present invention, and the interior is loaded with self-replicating mRNA;

[0079] Figure 4 This is a diagram showing the effect of an engineered extracellular vesicle delivery vector prepared according to an embodiment of the present invention delivering mRNA into cells, wherein:

[0080] Figure 4A shows the effect of engineered extracellular vesicle delivery vector delivering mRNA to BHK-21 cells.

[0081] Figure 4 B shows the effect of the engineered extracellular vesicle delivery vector delivering mRNA to VERO cells.

[0082] Figure 4 C shows the effect of cationic polymer (PEI) delivery vector delivering mRNA to VERO cells;

[0083] Figure 5 The following are photos of BHK-21 cell pathological changes inoculated with the virus-like virus VLV-VEEV / VSVG according to an embodiment of the present invention, wherein:

[0084] Figure 5 A shows a photo of BHK-21 normal control cells.

[0085] Figure 5 B shows a photo of BHK-21 cells inoculated with the first generation virus.

[0086] Figure 5 C shows a photo of BHK-21 cells inoculated with the second generation virus.

[0087] Figure 5 D shows a photograph of BHK-21 cells inoculated with the third generation virus. DETAILED DESCRIPTION

[0088] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0089] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0090] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0091] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0092] Herein, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0093] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0094] In this article, the term "recombinant cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant expression vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, BHK-21 cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0095] In this article, the term "engineered protein" generally refers to a protein modified by genetic engineering, including fusion proteins of various transmembrane proteins and RNA binding polypeptides, wherein the C-terminus of the RNA binding polypeptide is bound to the N-terminus of the cell transmembrane protein, or the C-terminus of the cell transmembrane protein is bound to the N-terminus of the RNA binding polypeptide, or the RNA binding polypeptide is inserted into the intracellular region of the cell transmembrane protein to form a fusion protein, and the engineered protein can specifically bind to the alphavirus self-replicating mRNA.

[0096] In this article, the term "transmembrane protein or cell transmembrane protein" generally refers to a type of cell membrane protein anchored on the cell membrane with a large hydrophobic region spanning the lipid bilayer, and its structure includes three parts: the extracellular region, the transmembrane region, and the intracellular region. The cell transmembrane protein includes different forms of membrane proteins such as exosome skeleton proteins, cell receptor proteins / cell ligand proteins, cell fusion proteins, and viral membrane proteins. In some embodiments, the cell transmembrane protein is selected from the exosome skeleton protein LAMP2b, or the E protein of alphavirus SINV, or the G protein of VSV, or the cell receptor / ligand protein CD11b.

[0097] In this article, the term "RNA binding polypeptide" generally refers to a polypeptide sequence that can recognize and bind to a specific RNA sequence. The RNA binding polypeptide forms a fusion protein with a cell transmembrane protein to mediate the specific assembly of mRNA in a delivery vector. The term "alphavirus RNA binding polypeptide" refers to the N-terminal domain or N-terminal domain truncated polypeptide of the alphavirus C protein.

[0098] In this article, the term "RNA packaging signal" generally refers to an RNA sequence that can specifically bind to an RNA binding polypeptide, wherein the term "alphavirus RNA packaging signal" refers to a nucleic acid sequence in the genomic RNA of an alphavirus that can specifically bind to an alphavirus capsid protein. Typically, the alphavirus RNA packaging signal is located in the alphavirus nonstructural protein coding region.

[0099] As used herein, the term "self-replicating mRNA or alphavirus self-replicating mRNA" generally refers to a self-replicating mRNA derived from an alphavirus replicon, wherein the alphavirus replicon generally employs the replicon of VEEV.

[0100] The term "delivery vector or mRNA delivery vector" used in the present disclosure is generally a vector that can deliver mRNA to cells, including extracellular vesicles, exosomes, LNPs, viruses, etc., wherein the term "extracellular vesicle or extracellular vesicle delivery vector" refers to a membrane vesicle structure with a cell capsule membrane inlaid with cell membrane proteins on the outside and mRNA packaged inside.

[0101] In a first aspect, the present invention provides an engineered protein, comprising: an RNA binding polypeptide and a cell transmembrane protein, the C-terminus of the RNA binding polypeptide is bound to the N-terminus of the cell transmembrane protein, or the C-terminus of the cell transmembrane protein is bound to the N-terminus of the RNA binding polypeptide, or the RNA binding polypeptide is inserted into the intracellular region of the cell transmembrane protein to form a fusion protein, and the engineered protein can specifically bind to alphavirus self-replicating mRNA, wherein the RNA binding polypeptide is selected from the N-terminal domain of alphavirus C protein or a truncated polypeptide thereof, and the cell transmembrane protein is selected from at least one of animal cell membrane proteins, exosome skeleton proteins, enveloped virus membrane proteins and engineered cell transmembrane proteins.

[0102] According to an embodiment of the present invention, the above-mentioned engineered protein may further include at least one of the following additional technical features:

[0103] According to an embodiment of the present invention, the RNA binding polypeptide is selected from at least one of the following: an N-terminal domain polypeptide of an alphavirus C protein, a truncated polypeptide of an alphavirus C protein, and a mutant polypeptide of a truncated N-terminal domain of an alphavirus C protein.

[0104] According to an embodiment of the present invention, the alphavirus includes at least one selected from the group consisting of SINV, VEEV and SFV.

[0105] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence at positions 1 to 124 of protein C of VEEV or a truncate thereof.

[0106] According to an embodiment of the present invention, the RNA binding polypeptide comprises the amino acid sequence shown in SEQ ID NOs: 1-3.

[0107] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence at positions 1 to 113 of the C protein of SINV or a truncate thereof.

[0108] According to an embodiment of the present invention, the RNA binding polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 4 or 5.

[0109] According to an embodiment of the present invention, the RNA binding polypeptide includes the amino acid sequence at positions 1 to 118 of the C protein of SFV or a truncate thereof.

[0110] According to an embodiment of the present invention, the RNA binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

[0111] According to an embodiment of the present invention, the transmembrane protein is selected from at least one of animal cell membrane proteins, exosome skeleton proteins, enveloped virus membrane proteins and engineered cell transmembrane proteins.

[0112] According to an embodiment of the present invention, the animal cell membrane protein includes at least one selected from the following: a cell receptor protein, a cell ligand protein and a cell fusion protein.

[0113] According to an embodiment of the present invention, the cell receptor protein / cell ligand protein includes at least one selected from the following: selectin E, PDGFR, CD6, CD14, CD11b, CD36, CD3, CD4, CD8 and CD28.

[0114] According to an embodiment of the present invention, the cell fusion protein includes at least one selected from the group consisting of: Myomaker, Mymerger and syncytin.

[0115] According to an embodiment of the present invention, the exosomal scaffold protein includes at least one selected from the following: LAMP2b, CD9, CD37, CD63, CD64, CD81, CD82, ARRDC1 and SPFH.

[0116] According to an embodiment of the present invention, the enveloped virus membrane protein includes at least one selected from the following: alphavirus glycoprotein E, vesicular stomatitis virus glycoprotein G, measles virus glycoprotein H / F, rubella virus glycoprotein E, mumps virus glycoprotein HA / NA, rabies virus glycoprotein G, influenza virus glycoprotein HA / NA, parainfluenza virus glycoprotein NH / F, respiratory syncytial virus glycoprotein G / F, Japanese encephalitis virus glycoprotein E and Sendai virus HN / F.

[0117] According to an embodiment of the present invention, the engineered protein includes: protein C polypeptide of VEEV and LAMP2b.

[0118] According to an embodiment of the present invention, the N-terminus of the VEEV protein C polypeptide is connected to the C-terminus of LAMP2b.

[0119] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:8.

[0120] According to an embodiment of the present invention, the engineered protein includes: protein C polypeptide of VEEV and CD11b.

[0121] According to an embodiment of the present invention, the N-terminus of the VEEV protein C polypeptide is connected to the C-terminus of CD11b.

[0122] According to an embodiment of the present invention, the engineered protein has the amino acids shown in SEQ ID NO:9.

[0123] According to an embodiment of the present invention, the engineered protein includes the C protein polypeptide of VEEV and SINV glycoprotein E2.

[0124] According to an embodiment of the present invention, the E2 of SINV deletes the amino acid sequence at positions 397 to 414.

[0125] According to an embodiment of the present invention, the protein C polypeptide of VEEV is inserted after the 396th amino acid of E2 of SINV.

[0126] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:10.

[0127] According to an embodiment of the present invention, the engineered protein includes the C protein polypeptide of VEEV and the glycoprotein G of VSV.

[0128] According to an embodiment of the present invention, the N-terminus of the C protein polypeptide of VEEV is connected to the C-terminus of the glycoprotein G of VSV.

[0129] According to an embodiment of the present invention, the engineered protein has the amino acid sequence shown in SEQ ID NO:11.

[0130] In a second aspect, the present invention provides a delivery vector, the delivery vector carrying the engineered protein and / or the nucleic acid sequence encoding the engineered protein described in the first aspect. The delivery vector according to an embodiment of the present invention can deliver the alphavirus self-replicating mRNA and / or the nucleic acid sequence encoding the engineered protein into a cell or body.

[0131] According to an embodiment of the present invention, the above-mentioned delivery vector may further include at least one of the following additional technical features:

[0132] According to an embodiment of the present invention, the delivery vector comprises at least one selected from the group consisting of: an extracellular vesicle, a virus, a nucleic acid-protein complex, a plasmid, RNA, and a cell.

[0133] According to an embodiment of the present invention, the delivery vector is an extracellular vesicle, and the extracellular vesicle also includes a cell membrane vesicle and an alphavirus self-replicating mRNA. According to a specific embodiment of the present invention, the extracellular vesicle has a cell membrane vesicle, the engineered protein, and an alphavirus self-replicating mRNA, thereby being able to efficiently and accurately deliver the alphavirus self-replicating mRNA into a cell or body.

[0134] According to an embodiment of the present invention, the cell membrane vesicles are derived from animal cells.

[0135] According to an embodiment of the present invention, the engineered protein is loaded on the cell membrane vesicle.

[0136] According to an embodiment of the present invention, the alphavirus self-replicating mRNA is specifically bound to the RNA binding polypeptide through the alphavirus RNA packaging signal and is packaged in the cell membrane vesicle, thereby forming the extracellular vesicle.

[0137] According to an embodiment of the present invention, the alphavirus self-replicating mRNA includes: a 5' cap, an alphavirus 5' non-coding region nucleic acid sequence, an alphavirus non-structural protein nucleic acid sequence, an alphavirus 26S promoter, a nucleic acid sequence encoding a target protein, an alphavirus 3' non-coding region nucleic acid sequence and a 3' PolyA tail.

[0138] According to an embodiment of the present invention, the extracellular vesicle comprises an amino acid sequence shown in any one of SEQ ID NOs: 8 to 11 or an amino acid sequence shown in any one of 29, 32, 35, 45, 47, 53, and 55.

[0139] According to a specific embodiment of the present invention, the extracellular vesicle is obtained by encoding a nucleic acid comprising any one of the following nucleotide sequences:

[0140] (1) SEQ ID NO:30 and SEQ ID NO:15,

[0141] (2) SEQ ID NO:33 and SEQ ID NO:15,

[0142] (3) SEQ ID NO:36 and SEQ ID NO:15,

[0143] (4) SEQ ID NO:37 and SEQ ID NO:15,

[0144] (5) SEQ ID NO:38 and SEQ ID NO:15,

[0145] (6) SEQ ID NO:39 and SEQ ID NO:15,

[0146] (7) SEQ ID NO:40 and SEQ ID NO:15,

[0147] (8) SEQ ID NO:46 and SEQ ID NO:42,

[0148] (9) SEQ ID NO:48 and SEQ ID NO:42,

[0149] (10) SEQ ID NO:54 and SEQ ID NO:50, and

[0150] (11) SEQ ID NO: 56 and SEQ ID NO: 50. The nucleic acid according to the specific embodiment of the present invention can effectively encode the extracellular vesicle delivery vector of the present invention.

[0151] According to an embodiment of the present invention, the delivery vector is a virus, and the viral genome includes a nucleic acid sequence encoding the engineered protein. The virus can deliver the nucleic acid sequence encoding the engineered protein and / or the alpha virus self-replicating mRNA into a cell or in vivo. According to a specific embodiment of the present invention, the exovesicle and the engineered protein can form a special viral structure, in which the exovesicle is the viral envelope, and the engineered protein is on the viral envelope. This viral structure includes the viral envelope and viral RNA, wherein the viral RNA includes a self-replicating mRNA structure, i.e., an RNA sequence encoding the engineered protein is added to the self-replicating mRNA structure.

[0152] According to an embodiment of the present invention, the engineered protein is loaded on the envelope of the virus.

[0153] According to an embodiment of the present invention, the virus is selected from a non-replication-defective virus or a replication-defective virus.

[0154] According to an embodiment of the present invention, the virus includes at least one selected from the following: herpes simplex virus, poxvirus, adenovirus, alphavirus, flavivirus, influenza virus, coronavirus, rabies virus, vesicular stomatitis virus, poliovirus, measles virus.

[0155] According to an embodiment of the present invention, the virus is an alphavirus.

[0156] According to an embodiment of the present invention, the alphavirus is selected from VEEV.

[0157] According to an embodiment of the present invention, the VEEV genome deletes the C protein gene and / or the E protein gene.

[0158] According to a specific embodiment of the present invention, the virus has the amino acid sequence shown in SEQ ID NO:11.

[0159] According to a specific embodiment of the present invention, the virus is encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:12.

[0160]

[0161] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0162] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), each of which is expressly incorporated herein by reference.

[0163] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0164] Example 1: Different cell transmembrane proteins form extracellular vesicles and package self-replicating mRNA

[0165] Exosome skeleton proteins can promote the formation and secretion of exosomes. Typical exosome markers include LAMP2b, CD9, CD63, CD81, etc. As cell transmembrane proteins, glycoproteins of enveloped viruses usually have the ability to drive the formation of cell microvesicles. However, the formation mechanism of cell microvesicles driven by viral glycoproteins is different from that of exosome skeleton proteins. The formation of cell microvesicles driven by viral glycoproteins usually depends on the specific binding of viral glycoproteins to the viral capsid, thereby providing the energy required for the budding of viral particles. In addition, most transmembrane proteins on the cell surface rarely spontaneously drive the formation of cell microvesicles.

[0166] In this example, the exosome skeleton protein LAMP2b, the E protein of alphavirus SINV, and the cell receptor / ligand protein CD11b were selected as representatives to compare the ability of different cell transmembrane proteins to drive extracellular vesicles and the loading effect on self-replicating mRNA.

[0167] 1. Plasmid Construction

[0168] 1.1 Plasmid expressing the RNA replicase of VEEV (Venezuelan equine encephalitis virus) (pVEEV-nsp)

[0169] The complete genome sequence of VEEV was obtained from GenBank with the sequence number NC_001449. The RNA replicase DNA fragment was synthesized with reference to the above sequence information and inserted into the pCMV-blank plasmid to construct the plasmid pVEEV-nsp.

[0170] The amino acid sequence of the RNA replicase obtained is shown in SEQ ID NO: 13:

[0171]

[0172] Compared with the pCMV-blank plasmid (original blank plasmid), the nucleic acid sequence expressing RNA replicase inserted into the plasmid pVEEV-nsp is shown in SEQ ID NO: 14:

[0173]

[0174] 1.2 VEEV replicon plasmid (pVEEV-sa)

[0175] A DNA fragment containing the 5' non-coding region of VEEV and a DNA fragment containing the 26S promoter and the 3' non-coding region are synthesized, and the 5' non-coding region DNA fragment of VEEV is inserted into the upstream of the RNA replicase nucleic acid sequence of VEEV in the plasmid pVEEV-nsp constructed in part 1.1, and the 26S promoter and the 3' non-coding region DNA fragment are inserted into the downstream of the RNA replicase nucleic acid sequence of VEEV to construct the VEEV replicon plasmid pVEEV-sa. Thus, the VEEV replicon nucleic acid sequence in the plasmid pVEEV-sa includes the following parts: (i) 5' non-coding region nucleic acid sequence, (ii) non-structural protein nucleic acid sequence, (iii) 26S promoter nucleic acid sequence, and (iv) 3' non-coding region nucleic acid sequence.

[0176] Compared with the original blank plasmid, the inserted nucleic acid sequence in the plasmid pVEEV-sa is shown in SEQ ID NO: 15:

[0177]

[0178] 1.3 VEEV replicon plasmid expressing EGFP protein (pVEEV-sa-EGFP)

[0179] Using the VEEV replicon plasmid pVEEV-sa as the backbone, various self-replicating mRNA plasmids expressing different target proteins can be constructed.

[0180] The present invention inserts the synthesized EGFP nucleic acid fragment into the plasmid pVEEV-sa constructed in part 1.2 after the start codon ATG carried by the subgenomic promoter, thereby constructing a VEEV replicon plasmid pVEEV-sa-EGFP expressing the EGFP protein.

[0181] When the plasmid pVEEV-sa-EGFP is transfected into host cells, the VEEV RNA replicase gene carried in the plasmid will translate into RNA replicase. The RNA replicase uses the VEEV replicon as a template to transcribe a large amount of progeny replicon RNA and subgenomic RNA, and translates a large amount of EGFP protein. Therefore, the plasmid pVEEV-sa-EGFP is used to evaluate the formation of extracellular vesicles and their ability to load self-replicating mRNA.

[0182] 1.4. Plasmid expressing LAMP2b protein (pLAMP2b)

[0183] The mRNA sequence of LAMP2b protein was obtained from GenBank with the sequence number AB170382.1. A DNA fragment encoding the LAMP2b protein sequence was synthesized with reference to the above sequence information and inserted into the above pCMV-blank plasmid to construct plasmid pLAMP2b.

[0184] The amino acid sequence of the obtained LAMP2b protein is shown in SEQ ID NO: 16:

[0185] MGLQLNITQDKVASVINPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNVS MYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECS LDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTL (SEQ ID NO: 16).

[0186] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid pLAMP2b is shown in SEQ ID NO: 17:

[0187] (SEQ ID NO: 17).

[0188] 1.5. Plasmid expressing SINV E protein (pSINV-E)

[0189] The complete genome sequence of SINV was obtained from GenBank with the sequence number NC_001547. A DNA fragment capable of expressing the E protein of SINV was synthesized according to the above sequence and inserted into the pCMV-blank plasmid to construct the plasmid pSINV-E.

[0190] The amino acid sequence of the SINV E protein obtained is shown in SEQ ID NO: 18:

[0191] MSAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAYDTLLNAILRCGSSGRSKRSVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADDNTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSVTVSIVSSNSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTAGYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAPGDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNAVIPTSLALLCCVRSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLPFLVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTNQEYITCKFTTVVPSPKIKCCGSLECQPAAHADYTCKVFGGVYPFMWGGAQCFCDSENSQMSEAYVELSADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPGTSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKDLIASTDIRLLKPSAKNVHVPYTQASSGFEMWKNNSGRPLQETAPFGCKIAVNPLRAVDCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGQCPVHSHSSTATLQESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKTTCNAECKPPADHIVSTPHKNDQEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR(SEQ ID NO:18)。

[0192] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid pSINV-E is shown in SEQ ID NO: 19:

[0193]

[0194] 1.6 Plasmid expressing CD11b protein (pCD11b)

[0195] The mRNA sequence of CD11b was obtained from GenBank with the sequence number NM_001145808.2. A DNA fragment encoding CD11b protein was synthesized according to the above sequence and inserted into the pCMV-blank plasmid to construct the plasmid pCD11b.

[0196] The amino acid sequence of the CD11b protein is shown in SEQ ID NO: 20:

[0197]

[0198] Compared with the pCMV - blank plasmid control, the nucleic acid sequence inserted in plasmid pCD11b is as shown in SEQ ID NO:21:

[0199] atggctctcagagtccttctgttaacagccttgaccttatgtcatgggttcaacttggacactgaaaacgcaatgaccttccaagagaacgcaaggggcttcggg

[0200] cagagcgtggtccagcttcagggatccagggtggtggttggagccccccaggagatagtggctgccaaccaaaggggcagcctctaccagtgcgactacagc

[0201] acaggctcatgcgagcccatccgcctgcaggtccccgtggaggccgtgaacatgtccctgggcctgtccctggcagccaccaccagcccccctcagctgctgg

[0202] cctgtggtcccaccgtgcaccagacttgcagtgagaacacgtatgtgaaagggctctgcttcctgtttggatccaacctacggcagcagccccagaagttcccag

[0203] aggccctccgagggtgtcctcaagaggatagtgacattgccttcttgattgatggctctggtagcatcatcccacatgactttcggcggatgaaggagtttgtctcaa

[0204] ctgtgatggagcaattaaaaaagtccaaaaccttgttctctttgatgcagtactctgaagaattccggattcactttaccttcaaagagttccagaacaaccctaaccca

[0205] agatcactggtgaagccaataacgcagctgcttgggcggacacacacggccacgggcatccgcaaagtggtacgagagctgtttaacatcaccaacggagcc

[0206] cgaaagaatgcctttaagatcctagttgtcatcacggatggagaaaagtttggcgatcccttgggatatgaggatgtcatccctgaggcagacagagagggagtc

[0207] attcgctacgtcattggggtgggagatgccttccgcagtgagaaatcccgccaagagcttaataccatcgcatccaagccgcctcgtgatcacgtgttccaggtga

[0208] ataactttgaggctctgaagaccattcagaaccagcttcgggagaagatctttgcgatcgagggtactcagacaggaagtagcagctcctttgagcatgagatgtct

[0209] caggaaggcttcagcgctgccatcacctctaatggccccttgctgagcactgtggggagctatgactgggctggtggagtctttctatatacatcaaaggagaaaa

[0210] gcaccttcatcaacatgaccagagtggattcagacatgaatgatgcttacttgggttatgctgccgccatcatcttacggaaccgggtgcaaagcctggttctgggg

[0211] gcacctcgatatcagcacatcggcctggtagcgatgttcaggcagaacactggcatgtgggagtccaacgctaatgtcaagggcacccagatcggcgcctactt

[0212] cggggcctccctctgctccgtggacgtggacagcaacggcagcaccgacctggtcctcatcggggccccccattactacgagcagacccgagggggccaggt

[0213] gtccgtgtgccccttgcccagggggcagagggctcggtggcagtgtgatgctgttctctacggggagcagggccaaccctggggccgctttggggcagcccta

[0214] acagtgctgggggacgtaaatggggacaagctgacggacgtggccattggggccccaggagaggaggacaaccggggtgctgtttacctgtttcacggaacc

[0215] tcaggatctggcatcagcccctcccatagccagcggatagcaggctccaagctctctcccaggctccagtattttggtcagtcactgagtgggggccaggacctc

[0216] acaatggatggactggtagacctgactgtaggagcccaggggcacgtgctgctgctcaggtcccagccagtactgagagtcaaggcaatcatggagttcaatcc

[0217] cagggaagtggcaaggaatgtatttgagtgtaatgatcaggtggtgaaaggcaaggaagccggagaggtcagagtctgcctccatgtccagaagagcacacgg

[0218] gatcggctaagagaaggacagatccagagtgttgtgacttatgacctggctctggactccggccgcccacattcccgcgccgtcttcaatgagacaaagaacag

[0219] cacacgcagacagacacaggtcttggggctgacccagacttgtgagaccctgaaactacagttgccgaattgcatcgaggacccagtgagccccattgtgctgc

[0220] gcctgaacttctctctggtgggaacgccattgtctgctttcgggaacctccggccagtgctggcggaggatgctcagagactcttcacagccttgtttccctttgaga

[0221] agaattgtggcaatgacaacatctgccaggatgacctcagcatcaccttcagtttcatgagcctggactgcctcgtggtgggtgggccccgggagttcaacgtgac

[0222] agtgactgtgagaaatgatggtgaggactcctacaggacacaggtcaccttcttcttcccgcttgacctgtcctaccggaaggtgtccacgctccagaaccagcgc

[0223] tcacagcgatcctggcgcctggcctgtgagtctgcctcctccaccgaagtgtctggggccttgaagagcaccagctgcagcataaaccaccccatcttcccggaa

[0224] aactcagaggtcacctttaatatcacgtttgatgtagactctaaggcttcccttggaaacaaactgctcctcaaggccaatgtgaccagtgagaacaacatgcccag

[0225] aaccaacaaaaccgaattccaactggagctgccggtgaaatatgctgtctacatggtggtcaccagccatggggtctccactaaatatctcaacttcacggcctca

[0226] gagaataccagtcgggtcatgcagcatcaatatcaggtcagcaacctggggcagaggagcctccccatcagcctggtgttcttggtgcccgtccggctgaacca

[0227] gactgtcatatgggaccgcccccaggtcaccttctccgagaacctctcgagtacgtgccacaccaaggagcgcttgccctctcactccgactttctggctgagctt

[0228] cggaaggcccccgtggtgaactgctccatcgctgtctgccagagaatccagtgtgacatcccgttctttggcatccaggaagaattcaatgctaccctcaaaggca

[0229] acctctcgtttgactggtacatcaagacctcgcataaccacctcctgatcgtgagcacagctgagatcttgtttaacgattccgtgttcaccctgctgccgggacagg

[0230] gggcgtttgtgaggtcccagacggagaccaaagtggagccgttcgaggtccccaaccccctgccgctcatcgtgggcagctctgtcgggggactgctgctcct

[0231] ggccctcatcaccgccgcgctgtacaagctcggcttcttcaagcggcaatacaaggacatgatgagtgaagggggtcccccgggggccgaaccccagtag (SEQ ID NO: 21).

[0232] 2. Plasmid Transfection

[0233] BHK-21 cells were selected as the host cells for plasmid transfection. BHK-21 cells were cultured in DMEM high-glucose medium containing 10% newborn calf serum and in an incubator with 5% CO 2 HEK 293T cells grown to a monolayer were digested with trypsin to prepare a cell suspension, which was inoculated into a 6-well cell culture plate, with 4×10 cells per well. 5 After culturing overnight, the plasmid transfection was performed when the cell confluence in the well plate reached 90%.

[0234] The plasmid transfection reagent used was TA293 transfection reagent, with a total plasmid dosage of 3.6 μg per well and a TA293 dosage of 30 μL. According to the instructions for use of the TA293 transfection reagent, the plasmid and TA293 were mixed and placed at room temperature for 10 minutes for transfection.

[0235] Before plasmid transfection, the culture medium in the well plate was removed by aspiration, and the well plate was washed twice with MEM medium. After adding the plasmid, DMEM high-glucose medium was added to each well to 2 mL. The well plate was incubated with 5% CO 2 , culture at 37°C for 48 hours, and harvest the culture supernatant containing extracellular vesicles in the well plate.

[0236] In this example, the pLAMP2b and pVEEV-sa-EGFP plasmids constructed above were co-transfected to prepare extracellular vesicles with LAMP2b as the skeleton, and the extracellular vesicles were named extracellular vesicles (LAMP2b / VEEV-sa-EGFP). The amounts of plasmids pLAMP2b and pVEEV-sa-EGFP used in the transfection system were both 3.6 μg / well; plasmids pSINV-E and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles with SINV-E as the skeleton, and the extracellular vesicles were named The extracellular vesicles are extracellular vesicles (SINV-E / VEEV-sa-EGFP), and the usage of plasmids pSINV-E and pVEEV-sa-EGFP in the transfection system are 3.6 μg / well; plasmids pCD11b and pVEEV-sa-EGFP are co-transfected to prepare extracellular vesicles with pCD11b as the skeleton, and the extracellular vesicles are named extracellular vesicles (CD11b / VEEV-sa-EGFP), and the usage of plasmids pCD11b and pVEEV-sa-EGFP in the transfection system are 3.6 μg / well.

[0237] 3. Extracellular vesicle detection

[0238] BHK-21 cells were used for the detection of extracellular vesicles. The BHK-21 cells grown to a monolayer were digested with trypsin to prepare a cell suspension, inoculated into a 96-well cell culture plate, and cultured overnight at 37°C. The extracellular vesicle samples were detected when the cell confluence in the well plate reached more than 90%. The transfection culture supernatant harvested from the above transfection system was appropriately diluted with DEME medium containing 2% newborn calf serum, added to a 96-well cell culture plate, 20 μL was added to each well, and 80 μL of DEME medium containing 2% newborn calf serum was added to each well. After culturing at 37°C for 24 hours, positive cells were counted using a fluorescence microscope, and the content of extracellular vesicles in the sample was calculated.

[0239] In this embodiment, the three extracellular vesicle culture supernatant samples harvested by testing showed that the content of extracellular vesicles (LAMP2b / VEEV-sa-EGFP) was 3.4E3 FFU / ml, the content of extracellular vesicles (SINV-E / VEEV-sa-EGFP) was 1.2E3 FFU / ml, and almost no fluorescent positive cells were observed for extracellular vesicles (CD11b / VEEV-sa-EGFP).

[0240] LAMP2b is a typical exosome skeleton protein, and overexpression of LAMP2b can promote the formation and secretion of exosomes; SINV-E is a transmembrane glycoprotein on the Sindbis virus envelope, and also has a certain ability to spontaneously form extracellular vesicles. In this embodiment, although the co-transfection plasmids pLAMP2b and pVEEV-sa-EGFP, as well as the co-transfection plasmids pSINV-E and pVEEV-sa-EGFP, observed the formation of extracellular vesicles loaded with self-replicating mRNA expressing EGFP, the content of the formed extracellular vesicles was extremely low, or the ability of the extracellular vesicles to actively load self-replicating mRNA was limited. On the other hand, the co-transfection plasmids pCD11b and pVEEV-sa-EGFP did not effectively form extracellular vesicles, indicating that the CD11b protein lacks the ability to form extracellular vesicles under natural physiological conditions.

[0241] Example 2: Promotion of extracellular vesicle formation by self-replicating mRNA transfection system

[0242] The formation of extracellular vesicles depends on the overexpression of cytoskeletal proteins on the cell membrane. Self-replicating mRNA has the ability to efficiently express proteins. Studies have reported that the expression level of self-replicating mRNA protein can reach more than 25% of the total host cell protein. Therefore, introducing the self-replicating mRNA expression system into the two-plasmid co-transfection system to prepare extracellular vesicles is expected to promote the production and secretion of extracellular vesicles.

[0243] 1. Construction of a plasmid (psaLAMP2b) containing the VEEV noncoding region expressing LAMP2b protein

[0244] The 5' non-coding region and 26S promoter nucleic acid sequence of VEEV were inserted into the upstream of the LAMP2b protein nucleic acid sequence in the plasmid pLAMP2b, and the 3' non-coding region nucleic acid sequence of VEEV was inserted into the downstream of the LAMP2b protein nucleic acid sequence in the plasmid pLAMP2b to construct plasmid psaLAMP2b.

[0245] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaLAMP2b is shown in SEQ ID NO: 22:

[0246] (SEQ ID NO:22).

[0247] 2. Construction of a plasmid containing the VEEV noncoding region expressing SINV E protein (psaSINV-E)

[0248] The 5' non-coding region and 26S promoter nucleic acid sequence of VEEV were inserted upstream of the SINV E protein nucleic acid sequence in plasmid pSINV-E, and the 3' non-coding region nucleic acid sequence of VEEV was inserted downstream of the SINV E protein nucleic acid sequence in plasmid pSINV-E to construct plasmid psaSINV-E.

[0249] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaSINV-E is shown in SEQ ID NO: 23:

[0250]

[0251] 3. Construction of a plasmid containing the VEEV noncoding region expressing CD11b protein (psaCD11b)

[0252] The 5' non-coding region and 26S promoter nucleic acid sequence of VEEV were inserted into the upstream of the CD11b protein nucleic acid sequence in the plasmid pCD11b, and the 3' non-coding region nucleic acid sequence of VEEV was inserted into the downstream of the CD11b protein nucleic acid sequence in the plasmid pCD11b to construct plasmid psaCD11b.

[0253] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaCD11b is shown in SEQ ID NO:24:

[0254]

[0255] 4. Plasmid Transfection and Detection of Extracellular Vesicles

[0256] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0257] In this embodiment, plasmids psaLAMP2b and pVEEV-sa-EGFP are co-transfected to prepare extracellular vesicles with LAMP2b as the skeleton, and the extracellular vesicles are named extracellular vesicles (saLAMP2b / VEEV-sa-EGFP); plasmids psaSINV-E and pVEEV-sa-EGFP are co-transfected to prepare extracellular vesicles with SINV-E as the skeleton, and the extracellular vesicles are named extracellular vesicles (saSINV-E / VEEV-sa-EGFP); plasmids psaCD11b and pVEEV-sa-EGFP are co-transfected to prepare extracellular vesicles with pCD11b as the skeleton, and the extracellular vesicles are named extracellular vesicles (saCD11b / VEEV-sa-EGFP).

[0258] The three extracellular vesicle culture supernatant samples harvested after testing showed that the content of extracellular vesicles (saLAMP2b / VEEV-sa-EGFP) was 4.7E4 FFU / mL, the content of extracellular vesicles (saSINV-E / VEEV-sa-EGFP) was 3.1E3 FFU / mL, and almost no fluorescent positive cells were observed for extracellular vesicles (saCD11b / VEEV-sa-EGFP).

[0259] Taking plasmid psaLAMP2b as an example, compared with plasmid pLAMP2b, plasmid psaLAMP2b adds a non-coding region gene sequence from the VEEV virus. After co-transfection with plasmid pVEEV-sa-EGFP, plasmid pVEEV-sa-EGFP translates and expresses the RNA replicase of the VEEV virus, and the RNA replicase can recognize the mRNA transcribed from plasmid psaLAMP2b and replicate a large amount of subgenomic RNA containing the LAMP2b gene, resulting in a large amount of LAMP2b protein expression. Therefore, co-transfection of plasmid psaLAMP2b and pVEEV-sa-EGFP can theoretically produce more LAMP2b protein than co-transfection of plasmid pLAMP2b and pVEEV-sa-EGFP.

[0260] In this embodiment, the double plasmid transfection system based on the self-replicating mRNA transfection system can improve the production of extracellular vesicles with LAMP2b as the skeleton and SINV-E as the skeleton, but does not promote the production of extracellular vesicles with CD11b as the skeleton. Therefore, the introduction of the self-replicating mRNA transfection system into the preparation of extracellular vesicles can effectively improve the production efficiency of extracellular vesicles.

[0261] In this example, compared with Example 1 of the present invention, the content of extracellular vesicles (saLAMP2b / VEEV-sa-EGFP) detected was higher than that of extracellular vesicles (LAMP2b / VEEV-sa-EGFP), but the content of extracellular vesicles (saLAMP2b / VEEV-sa-EGFP) only reached 4.7E4 FFU / ml, which is far lower than the theoretical content of extracellular vesicles produced by LAMP2b. One possible reason is that the plasmid pVEEV-sa-EGFP used as an indicator package transcribes a self-replicating mRNA, which is very long, about 8000 kb, and is difficult to be effectively loaded into extracellular vesicles.

[0262] Example 3: Specific binding of self-replicating mRNA to cell transmembrane proteins promotes the formation of extracellular vesicles

[0263] Usually, naturally formed extracellular vesicles are packaged with metabolites of host cells, including cell enzymes, cytokines, small fragments of nucleic acids, etc., among which mRNA is also packaged in small amounts, and these contents are passively packaged into extracellular vesicles. Adding RNA packaging signals to mRNA and adding RNA binding polypeptides to the intracellular region of cell transmembrane proteins can promote the active loading of mRNA in extracellular vesicles. This example uses boxB signal nucleic acid and N peptide to verify the effect of RNA binding polypeptides on extracellular vesicle packaging of self-replicating mRNA.

[0264] 1. Construction of VEEV replicon plasmid expressing EGFP carrying boxB signal (pVEEV-sa-EGFP-boxB)

[0265] The box B sequence was inserted upstream of the VEEV5' non-coding region sequence in the plasmid pVEEV-sa-EGFP to construct the plasmid pVEEV-sa-EGFP-boxB. In the host cell, the plasmid pVEEV-sa-EGFP-boxB can transcribe the EGFP self-replicating mRNA carrying the boxB signal, wherein the boxB signal sequence can specifically bind to the matching N-peptide sequence.

[0266] The box B nucleic acid sequence is as follows: cgggaaaaagtcccg (SEQ ID NO: 25).

[0267] 2. Construction of LAMP2b protein plasmid carrying box B sequence RNA binding N peptide (psaLAMP2bmut-N)

[0268] The nucleic acid sequence of the box B sequence RNA binding N peptide was inserted into the downstream of the LAMP2b sequence in the plasmid psaLAMP2b to construct the plasmid psaLAMP2bmut-N.

[0269] The protein expressed by plasmid psaLAMP2bmut-N is a LAMP2b fusion protein carrying an N peptide at the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 26:

[0270] MGLQLNITQDKVASVINPNTTHSTGSCRSHTALLRLNSSTIKYLDFVFAVKNENRFYLKEVNVS MYLVNGSVFSIANNNLSYWDAPLGSSYMCNKEQTVSVSGAFQINTFDLRVQPFNVTQGKYSTAQECS LDDDTILIPIIVGAGLSGLIIVIVIAYVIGRRKSYAGYQTLMDAQTRRRERRAEKQAQWKAAN (SEQ ID NO: 26).

[0271] Compared with the original empty plasmid, the nucleic acid sequence inserted into the plasmid psaLAMP2bmut-N is shown in SEQ ID NO: 27:

[0272] (SEQ ID NO:27)

[0273] 3. Plasmid transfection and detection of extracellular vesicles

[0274] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0275] In this example, plasmids psaLAMP2bmut-N and pVEEV-sa-EGFP-boxB were co-transfected to prepare extracellular vesicles, which were named extracellular vesicles (saLAMP2bmut-N / VEEV-sa-EGFP-boxB). The content of the harvested extracellular vesicles (saLAMP2bmut-N / VEEV-sa-EGFP-boxB) was 1.2E6 FFU / mL. Compared with the results of the extracellular vesicles (LAMP2b / VEEV-sa-EGFP: 3.4E3 FFU / ml) in Example 1, the binding of RNA-binding polypeptides to LAMP2b can improve the packaging efficiency of extracellular vesicles for self-replicating mRNA.

[0276] Example 4: Alphavirus RNA-binding polypeptide promotes extracellular vesicle packaging of self-replicating mRNA

[0277] The alphavirus genomic RNA can be effectively packaged in the viral capsid by specifically binding to the capsid protein (C protein). The RNA sequence that specifically binds to the alphavirus is called the packaging signal. The protein that the alphavirus packaging signal binds to is the alphavirus C protein. The binding site can be either a linear polypeptide of the C protein or a spatial conformation site. The alphavirus packaging signal does not specifically bind to the membrane protein of the virus.

[0278] In this embodiment, a polypeptide on the alphavirus C protein that specifically binds to the alphavirus packaging signal is introduced into the intracellular region of the cell transmembrane protein. It is expected that the packaging signal carried by the self-replicating mRNA itself will be used to mediate the specific binding of the self-replicating mRNA to the cell transmembrane protein, thereby promoting the effective active packaging of the self-replicating mRNA by extracellular vesicles.

[0279] 1. Construction of plasmids expressing SINV E protein carrying VEEV C protein RNA binding polypeptide (psaSINV-E1mut-VEEV-C1, psaSINV-E1mut-VEEV-C2, psaSINV-E1mut-VEEV-C3, psaSINV-E2mut-VEEV-C2)

[0280] The C protein of VEEV can specifically bind to the packaging signal on the VEEV genomic RNA. The present invention optimizes and screens a plurality of C protein polypeptides that can specifically bind to the VEEV genomic RNA.

[0281] The nucleic acid sequence of one of the polypeptides (polypeptide VEEV-C1, amino acid sequence as shown in SEQ ID NO: 1) was inserted after the E protein nucleic acid sequence in the plasmid psaSINV-E to construct plasmid psaSINV-E1mut-VEEV-C1.

[0282] The nucleic acid sequence of polypeptide VEEV-C1 is shown in SEQ ID NO: 28:

[0283] aagaagaagaagaaccaggggaagaagaaggccaagacggggccgcctaatccgaaggcacagagtggaaacaagaagaagcccaacaagaaa ccaggcaagagacagcgcatggtcatgaaattggaatctgac (SEQ ID NO: 28).

[0284] Plasmid psaSINV-E1mut-VEEV-C1 expresses a fusion protein, wherein the N-terminus of the polypeptide VEEV-C1 is bound to the C-terminus of the SINV E protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 29:

[0285]

[0286] Compared with the original blank plasmid, the nucleic acid sequence inserted in the plasmid psaSINV-E1mut-VEEV-C1 is shown in SEQ ID NO: 30:

[0287]

[0288] The nucleic acid sequence of the N-terminal truncated polypeptide of polypeptide VEEV-C1 (polypeptide VEEV-C2, the amino acid sequence is shown in SEQ ID NO: 2) was inserted into the E protein nucleic acid sequence in plasmid psaSINV-E to construct plasmid psaSINV-E1mut-VEEV-C2.

[0289] The nucleic acid sequence of polypeptide VEEV-C2 is shown in SEQ ID NO:31:

[0290] aagaagaagaagaaccaggggaagaagaaggccaagacggggccgcctaatccgaag (SEQ ID NO: 31).

[0291] Plasmid psaSINV-E1mut-VEEV-C2 expresses a fusion protein, wherein the N-terminus of the polypeptide VEEV-C2 is bound to the C-terminus of the SINV E protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO:32:

[0292]

[0293] Compared with the original blank plasmid, the nucleic acid sequence inserted in the plasmid psaSINV-E1mut-VEEV-C2 is shown in SEQ ID NO: 33:

[0294]

[0295] The nucleic acid sequence of the C-terminal truncated polypeptide of polypeptide VEEV-C1 (polypeptide VEEV-C3, the amino acid sequence is shown in SEQ ID NO: 3) was inserted into the E protein nucleic acid sequence in plasmid psaSINV-E to construct plasmid psaSINV-E1mut-VEEV-C3.

[0296] The nucleic acid sequence of polypeptide VEEV-C3 is shown in SEQ ID NO:34:

[0297] aaaccaggcaagagacagcgcatggtcatgaaattggaatctgac (SEQ ID NO: 34).

[0298] Plasmid psaSINV-E1mut-VEEV-C3 expresses a fusion protein, wherein the N-terminus of the polypeptide VEEV-C3 is combined with the C-terminus of the E protein of SINV. The amino acid sequence of the fusion protein is shown in SEQ ID NO:35.

[0299]

[0300]

[0301] The nucleic acid sequence corresponding to the L397-L414 sequence polypeptide of the E2 protein in the plasmid psaSINV-E was deleted, and the nucleic acid sequence of the polypeptide VEEV-C2 was inserted at the deleted position to construct the auxiliary plasmid psaSINV-E2mut-VEEV-C2.

[0302] Plasmid psaSINV-E2mut-VEEV-C2 expresses a fusion heterodimeric E protein, wherein a polypeptide VEEV-C2 fragment is inserted into the intracellular region of the E2 protein to form an E2 fusion protein, the amino acid sequence of which is shown in SEQ ID NO:10.

[0303] Compared with the original blank plasmid, the nucleic acid sequence inserted in the plasmid psaSINV-E2mut-VEEV-C2 is shown in SEQ ID NO: 37:

[0304]

[0305] 2. Plasmid transfection and detection of extracellular vesicles

[0306] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μL.

[0307] In this embodiment, plasmids psaSINV-E1mut-VEEV-C1 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saSINV-E1mut-VEEV-C1 / VEEV-sa-EGFP); plasmids psaSINV-E1mut-VEEV-C2 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saSINV-E1mut-VEEV-C2 / VEEV-sa-EGFP). FP); plasmids psaSINV-E1mut-VEEV-C3 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, which were named extracellular vesicles (saSINV-E1mut-VEEV-C3 / VEEV-sa-EGFP); plasmids psaSINV-E2mut-VEEV-C2 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, which were named extracellular vesicles (saSINV-E2mut-VEEV-C2 / VEEV-sa-EGFP).

[0308] After testing, four samples of extracellular vesicle culture supernatant were harvested, among which the content of extracellular vesicles (saSINV-E1mut-VEEV-C1 / VEEV-sa-EGFP) was 4.1E6 FFU / ml, the content of extracellular vesicles (saSINV-E1mut-VEEV-C2 / VEEV-sa-EGFP) was 2.8E6 FFU / ml, the content of extracellular vesicles (saSINV-E1mut-VEEV-C3 / VEEV-sa-EGFP) was 2.1E6 FFU / ml, and the content of extracellular vesicles (saSINV-E2mut-VEEV-C2 / VEEV-sa-EGFP) was 6.3E6 FFU / ml.

[0309] In this example, the alphavirus C protein RNA binding polypeptide is introduced into the extracellular vesicle packaging system for self-replicating mRNA. Compared with the results of the extracellular vesicles in Example 1 (SINV-E / VEEV-sa-EGF: 1.2E3 FFU / ml), this method increases the production of extracellular vesicles for packaging self-replicating mRNA by about 1000 times, greatly promoting the packaging of self-replicating mRNA by extracellular vesicles.

[0310] In this example, it was surprisingly found that the fusion protein formed by inserting the polypeptide VEEV-C2 into the intracellular region of the SINV E2 protein produced a higher level of extracellular vesicles than the fusion protein formed by fusing the polypeptide VEEV-C2 to the C-terminus of the SINV E1 protein, which may be related to the conformation of the heterodimeric E protein of SINV and its mechanism of forming extracellular vesicles.

[0311] The E protein of alphavirus is a heterodimeric protein composed of two subunits, E1 and E2. The E2 protein has a longer intracellular polypeptide chain and can specifically recognize and bind to the capsid of alphavirus. The binding of E2 protein to the capsid causes the conformation of E2 protein and capsid protein to change, providing energy for the budding of virus particles. The E1 protein has only two amino acid residues in the intracellular region.

[0312] When alphavirus E protein is expressed alone on the cell surface, its ability to promote the formation of extracellular vesicles is limited due to the lack of binding to the viral capsid. Binding self-replicating mRNA to the intracellular region of alphavirus E protein, especially the intracellular region of E2 protein, through RNA-binding polypeptide may help compensate for the energy required for E protein budding and promote the formation and budding of extracellular vesicles mediated by E protein.

[0313] Example 5: Promotion of the Alphavirus RNA Binding Peptide on the Formation of Extracellular Vesicles by Exosome Skeleton Proteins and Cell Receptor / Ligand Proteins

[0314] 1. Construction of LAMP2b plasmid expressing VEEV-C2 peptide (psaLAMP2bmut-VEEV-C2)

[0315] The polypeptide VEEV-C2 nucleic acid sequence was inserted into the downstream of the LAMP2b sequence in the plasmid psaLAMP2b to construct the plasmid psaLAMP2bmut-VEEV-C2. The protein expressed by the plasmid psaLAMP2bmut-VEEV-C2 is a LAMP2b fusion protein with the polypeptide VEEV-C2 added to the C-terminus, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:8.

[0316] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaLAMP2bmut-VEEV-C2 is shown in SEQ ID NO: 38:

[0317] atgggcggcgcaagagagaagcccaaaccaattacctacccaaactctctacggcggctaacctgaatggactacgacatagtctagtccgccaagatggggctgcagctgaacatcactcaggataaggttgcttcagttattaacatcaaccccaatacaactcactccacaggcagctgccgttctcacactgctctacttaggctgaatagcagcaccattaagtatcttgactttgtctttgctgtgaaaaatgaaaaccgattttatctgaaggaagtgaacgtcagcatgtatttggttaatggctccgttttcagcattgcaaataacaatctcagctactgggatgcccccctgggaagttcttatatgtgcaacaaagagcagactgtttcagtgtctggagcatttcagataaatacctttgatctaagggttcagcctttcaatgtgacacaaggaaagtattctacagcccaagagtgttcgctggatgatgacaccattctaatcccaattatagttggtgctggtctttcaggcttgattatcgttatagtgattgcttacgtaattggcagaagaaaaagttatgctggatatcagactctgaagaagaagaagaaccaggggaagaagaaggccaagacggggccgcctaatccgaagtgaacatagcagcaattggcaagctgcttatatagaacttgcggcgattggcatgccgctttaaaattttattttattttcttttcttttccgaatcggattttgtttttaatatttc(SEQ ID NO:38).

[0318] 2. Construction of the CD11b plasmid (psaCD11bmut-VEEV-C2) expressing the polypeptide VEEV-C2

[0319] The polypeptide VEEV-C2 nucleic acid sequence was inserted into the downstream of the CD11b sequence in the plasmid psaCD11b to construct the plasmid psaCD11bmut-VEEV-C2. The protein expressed by the plasmid psaCD11bmut-VEEV-C2 is a CD11b fusion protein with the polypeptide VEEV-C2 added to the C-terminus, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:9.

[0320] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaCD11bmut-VEEV-C2 is shown in SEQ ID NO: 39:

[0321]

[0322] 3. Plasmid transfection and detection of extracellular vesicles

[0323] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0324] In this embodiment, plasmid psaLAMP2bmut-VEEV-C2 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saLAMP2bmut-VEEV-C2 / VEEV-sa-EGFP); plasmid psaCD11bmut-VEEV-C2 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saCD11bmut-VEEV-C2 / VEEV-sa-EGFP). The content of the harvested extracellular vesicles (saLAMP2bmut-VEEV-C2 / VEEV-sa-EGFP) was 1.7E7 FFU / mL, and the content of the extracellular vesicles (saCD11bmut-VEEV-C2 / VEEV-sa-EGFP) was 5.2E5 FFU / mL.

[0325] In this example, the alphavirus RNA binding polypeptide is introduced into the exosome skeleton protein and the cell receptor / ligand protein. Compared with the results of Example 3 of the present invention (saLAMP2bmut-N / VEEV-sa-EGFP-boxB: 1.2E6 FFU / mL), the polypeptide VEEV-C2 derived from the C protein of the alphavirus VEEV is added to the C-terminus of the exosome skeleton protein LAMP2b, and the detectable exosome content is increased by about 10 times, which indicates that the alphavirus RNA binding polypeptide has a better promoting effect on the packaging of self-replicating mRNA in extracellular vesicles than RNA binding polypeptides from other sources. At the same time, the inventors were pleasantly surprised to find that adding the polypeptide VEEV-C2 to the C-terminus of the cell membrane protein CD11b produced detectable extracellular vesicles that packaged self-replicating mRNA.

[0326] In Examples 1 and 2 of the present invention, BHK-21 cells were used as host cells to express CD11b protein on the cell surface, but no extracellular vesicles loaded with self-replicating mRNA were detected, which at least indicates that the ability of CD11b protein as a skeleton to promote the production of extracellular vesicles is not strong, or its passive loading ability for self-replicating mRNA is extremely weak.

[0327] This example integrates the alphavirus RNA binding polypeptide into the C-terminus of the CD11b protein, and uses the RNA binding polypeptide to tightly bind the self-replicating mRNA to the intracellular region of the CD11b protein, thereby innovatively producing CD11b protein extracellular vesicles that actively package the self-replicating mRNA. This strategy for preparing extracellular vesicles greatly expands the preparation and application potential of extracellular vesicles.

[0328] Example 6: Promotion of the Alphavirus RNA Binding Peptide on the Formation of Extracellular Vesicles by Other Virus Membrane Proteins

[0329] 1. Construction of a G protein plasmid expressing VSV carrying the VEEV-C2 peptide (psaVSV-Gmut-VEEV-C2)

[0330] The G protein mRNA sequence of VSV is from GenBank, with the sequence number X03633.1. Referring to the above sequence information, a DNA fragment containing the 5' non-coding region sequence of VEEV, the 26S promoter nucleic acid sequence, the G protein nucleic acid sequence of VSV, the polypeptide VEEV-C2, and the 3' non-coding region nucleic acid sequence of VEEV was synthesized in sequence, and inserted into the plasmid pCMV-blank to construct the plasmid psaVSV-Gmut-VEEV-C2. The protein expressed by the plasmid psaVSV-Gmut-VEEV-C2 is a VSV-G fusion protein with the polypeptide VEEV-C2 bound to the C-terminus, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 11.

[0331] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid psaVSV-Gmut-VEEV-C2 is shown in SEQ ID NO: 40:

[0332]

[0333] 2. Plasmid transfection and detection of extracellular vesicles

[0334] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0335] In this example, plasmids psaVSV-Gmut-VEEV-C2 and pVEEV-sa-EGFP were co-transfected to prepare extracellular vesicles, which were named extracellular vesicles (saVSV-Gmut-VEEV-C2 / VEEV-sa-EGFP). The content of the harvested extracellular vesicles was 4.5E6 FFU / ml.

[0336] In this embodiment, the C protein polypeptide (VEEV-C2) of the alpha virus VEEV is fused to the C-terminus of the G protein inner region of VSV to form a fusion protein. The polypeptide VEEV-C2 can specifically bind to the self-replicating mRNA sequence, promote the G protein of VSV to form extracellular vesicles, and enhance the ability of extracellular vesicles to actively load self-replicating mRNA.

[0337] Example 7: Promotion of extracellular vesicles by RNA-binding polypeptides of SINV

[0338] 1. Plasmid Construction

[0339] 1.1 SINV (Sindbis virus) replicon plasmid (pSINV-sa)

[0340] The complete genome sequence of SINV is from GenBank, with the sequence number NC_001547. Referring to the above sequence information, a DNA fragment containing the 5' non-coding region, non-structural protein (RNA replicase) coding region, 26S promoter and 3' non-coding region of SINV was synthesized and inserted into the pCMV-blank plasmid to construct the SINV replicon plasmid pSINV-sa. Thus, the replicon nucleic acid sequence in the plasmid pSINV-sa includes the following parts: (i) 5' non-coding region nucleic acid sequence, (ii) non-structural protein nucleic acid sequence, (iii) 26S promoter nucleic acid sequence, (iv) 3' non-coding region nucleic acid sequence.

[0341] The amino acid sequence of the RNA replicase expressed by the plasmid pSINV-sa is shown in SEQ ID NO:41.

[0342]

[0343] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted in the plasmid pSINV-sa is shown as SEQ ID NO:42.

[0344]

[0345] 1.2 SINV replicon plasmid expressing EGFP protein (pSINV-sa-EGFP)

[0346] Using the SINV replicon plasmid pSINV-sa as the backbone, various self-replicating mRNA plasmids expressing different target proteins can be constructed.

[0347] The invention inserts the synthesized EGFP nucleic acid fragment into the start codon ATG carried by the subgenomic promoter in the plasmid pSINV-sa, and constructs a replicon plasmid pSINV-sa-EGFP expressing the EGFP protein.

[0348] When the plasmid pSINV-sa-EGFP is transfected into the host cells, the RNA replicase gene carried in the plasmid will translate into RNA replicase, which will transcribe a large amount of progeny replicon RNA and subgenomic RNA using the SINV replicon as a template, and translate a large amount of EGFP protein. Therefore, the plasmid pVEEV-sa-EGFP is used to evaluate the formation of extracellular vesicles and their ability to load self-replicating mRNA.

[0349] 1.3 Plasmids expressing VSV G protein carrying RNA-binding polypeptide (psaVSV-Gmut-SINV-C1, psaVSV-Gmut-SINV-C2)

[0350] The C protein of SINV can specifically bind to the packaging signal on the SINV genomic RNA. The present invention optimizes and screens out multiple C protein polypeptides that can specifically bind to the VEEV genomic RNA, wherein the amino acid sequence of the polypeptide SINV-C1 is shown in SEQ ID NO:4, and the nucleic acid sequence is shown in SEQ ID NO:43; the amino acid sequence of the polypeptide SINV-C2 is shown in SEQ ID NO:5, and the nucleic acid sequence is shown in SEQ ID NO:44.

[0351] The nucleic acid sequence of polypeptide SINV-C1 is as follows:

[0352] aagccgaagaaaccaaaaacgcaggagaagaagaagcaacctgcaaaacccaaacccggaaagagacagcgcatggcacttaagttggaggc cgac (SEQ ID NO: 43)

[0353] The nucleic acid sequence of polypeptide SINV-C2 is as follows:

[0354] aagccgaagaaaccaaaaacgcaggagaagaagaagaagcaacctgcaaaaccc (SEQ ID NO:44)

[0355] The G protein mRNA sequence of VSV is from GenBank, with the sequence number X03633.1. Referring to the above sequence information, a DNA fragment containing the 5' non-coding region sequence of SINV, the 26S promoter nucleic acid sequence, the G protein nucleic acid sequence of VSV, the polypeptide SINV-C1, and the 3' non-coding region nucleic acid sequence of SINV was synthesized in sequence, and inserted into the plasmid pCMV-blank to construct the plasmid psaVSV-Gmut-SINV-C1.

[0356] The protein expressed by the plasmid psaVSV-Gmut-SINV-C1 is a VSV-G fusion protein with the peptide SINV-C1 bound to the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 45:

[0357] (SEQ ID NO:45)

[0358] Compared with the original blank plasmid control, the inserted nucleic acid sequence in plasmid psaVSV-Gmut-SINV-C1 is shown in SEQ ID NO:46:

[0359] attgacggcgtagtacacactattgaatcaaacagccgaccaattgcactaccatcacaatctctacggtggtcctaaatagtcagcatagtacatttcatctgact

[0360] aatactacaacaccaccaccatgaagtgctttttgtacttagcttttttattcatcggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactgga

[0361] aaaatgttccttccaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacaggcttacaagtcaaaatgcccaagagtcacaaggc

[0362] tattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtacggaccgaagtatataacacattccatccgatccttcactccat

[0363] ctgtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccg

[0364] aagcagtgattgtccaggtgactcctcaccatgtgcttgttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaatgacatatgc

[0365] cccactgtccataactccacaacctggcattccgactataaggtcaaagggctatgtgattctaacctcatttccacggacatcaccttcttctcagaggacagagag

[0366] ctatcatccctaggaaaggagggcacagggttcagaagtaactactttgcttatgaaactggagacaaggcctgcaaaatgcagtactgcaagcattggggagtc

[0367] agactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacc

[0368] tcagtggatgtaagtctcattcaggacgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttcccatctctccagtggat

[0369] ctcagctatcttgctcctaaaaacccaggaaccggtcctgcctttaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctc

[0370] caatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactgggctccatatgaagacgtggaaattggacccaatgga

[0371] gttctgaggaccagttcaggatataagtttcctttatatatgattggacatggtatgttggactccggtcttcatcttagctcaaaggctcaggtgtttgaacatcctcaca

[0372] ttcaagacgctgcttcgcagcttcctgatgatgagattttattttttggtgatactgggctatccaaaaatccaatcgactttgtcgaaggttggttcagtagttggaaga

[0373] gctccattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatttatctttacattaaattaaagcacaccaagaaaagacagatttat

[0374] acagacatagagatgaaccgacttggaaggaagccgaagaaaccaaaaacgcaggagaagaagaagaagcaacctgcaaaacccaaacccggaaagaga

[0375] cagcgcatggcacttaagttggaggccgactaaccgctacgccccaatgatccgaccagcaaaactcgatgtacttccgaggaactgatgtgcataatgcatcag

[0376] gctggtacattagatccccgcttaccgcgggcaatatagcaacactaaaaactcgatgtacttccgaggaagcgcagtgcataatgctgcgcagtgttgccacata

[0377] accactatattaaccatttatctagcggacgccaaaaaactcaatgtatttctgaggaagcgtggtgcataatgccacgcagcgtctgcataacttttattatttcttttattaatcaacaaaattttgtttttaacatttc(SEQID NO:46)

[0378] A DNA fragment containing the 5' non-coding region sequence of SINV, the 26S promoter nucleic acid sequence, the G protein nucleic acid sequence of VSV, the polypeptide SINV-C2, and the 3' non-coding region nucleic acid sequence of SINV was synthesized and inserted into the plasmid pCMV-blank to construct the plasmid psaVSV-Gmut-SINV-C2.

[0379] The protein expressed by plasmid psaVSV-Gmut-SINV-C2 is a VSV-G fusion protein with the peptide SINV-C2 bound to the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 47:

[0380] CKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTGLQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDRELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSGLHLSSKAQVFEHPHIQDAASQLPDDEILFFGDTGLSKNPIDFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLYIKLKHTKKRQIYTDIEMNRLGRKPKKPKTGEKKKKGPAKP(SEQ ID NO:47)

[0381] Compared with the original blank plasmid control, the inserted nucleic acid sequence in plasmid psaVSV-Gmut-SINV-C2 is shown as SEQ ID NO:48:

[0382]

[0383] 2. Plasmid transfection and detection of extracellular vesicles

[0384] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0385] In this embodiment, plasmids psaVSV-Gmut-SINV-C1 and pSINV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saVSV-Gmut-SINV-C1 / SINV-sa-EGFP); plasmids psaVSV-Gmut-SINV-C2 and pSINV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saVSV-Gmut-SINV-C2 / SINV-sa-EGFP). The contents of the harvested extracellular vesicles saVSV-Gmut-SINV-C1 / SINV-sa-EGFP and saVSV-Gmut-SINV-C2 / SINV-sa-EGFP were 8.2E6 FFU / ml and 6.7E6 FFU / ml, respectively.

[0386] In this embodiment, the C protein polypeptide (SINV-C1 and SINV-C2) of the alpha virus SINV is fused to the C-terminus of the G protein inner region of VSV to form a fusion protein. The obtained fusion protein can specifically bind to the self-replicating mRNA sequence, promote the G protein of VSV to form extracellular vesicles, and enhance the ability of extracellular vesicles to actively load self-replicating mRNA.

[0387] Example 8: Promotion of extracellular vesicles by SFV RNA-binding polypeptide

[0388] 1. Plasmid Construction

[0389] 1.1SFV (Semliki Forest Encephalitis Virus) Replicon Plasmid (pSFV-sa)

[0390] The complete genome sequence of SFV is from GenBank, with the sequence number NC_003215. A DNA fragment containing the 5' non-coding region, non-structural protein (RNA replicase) coding region, 26S promoter and 3' non-coding region of SFV was synthesized with reference to the above sequence information, and inserted into the pCMV-blank plasmid to construct the SFV replicon plasmid pSFV-sa. Thus, the replicon nucleic acid sequence in the plasmid pSFV-sa includes the following parts: (i) 5' non-coding region nucleic acid sequence, (ii) non-structural protein nucleic acid sequence, (iii) 26S promoter nucleic acid sequence, and (iv) 3' non-coding region nucleic acid sequence.

[0391] The amino acid sequence of the RNA replicase expressed by the plasmid pSFV-sa is shown in SEQ ID NO:49:

[0392]

[0393] Compared with the pCMV-blank plasmid, the nucleic acid sequence inserted into the plasmid pSFV-sa is shown in SEQ ID NO:50:

[0394]

[0395] 1.2 SFV replicon plasmid expressing EGFP protein (pSFV-sa-EGFP)

[0396] Using the SFV replicon plasmid pSFV-sa as the backbone, various self-replicating mRNA plasmids expressing different target proteins can be constructed.

[0397] The invention inserts the synthesized EGFP nucleic acid fragment into the start codon ATG carried by the subgenomic promoter in the plasmid pSFV-sa, and constructs a replicon plasmid pSFV-sa-EGFP expressing the EGFP protein.

[0398] When the plasmid pSFV-sa-EGFP is transfected into the host cells, the RNA replicase gene carried in the plasmid will translate into RNA replicase, which will transcribe a large amount of progeny replicon RNA and subgenomic RNA using the SFV replicon as a template, and translate a large amount of EGFP protein. Therefore, the plasmid pVEEV-sa-EGFP is used to evaluate the formation of extracellular vesicles and their ability to load self-replicating mRNA.

[0399] 1.3 Plasmids expressing VSV G protein carrying RNA-binding polypeptide (psaVSV-Gmut-SFV-C1, psaVSV-Gmut-SFV-C2)

[0400] The C protein of SFV can specifically bind to the packaging signal on the SFV genomic RNA. The present invention optimizes and screens out multiple C protein polypeptides that can specifically bind to the VEEV genomic RNA, wherein the amino acid sequence of the polypeptide SFV-C1 is shown in SEQ ID NO:6, and the nucleic acid sequence is shown in SEQ ID NO:51, the amino acid sequence of the polypeptide SFV-C2 is shown in SEQ ID NO:7, and the nucleic acid sequence is shown in SEQ ID NO:52.

[0401] The nucleic acid sequence of polypeptide SFV-C1 is as follows:

[0402] aagaagatcaacggaaaaacgcagcagcaaaagaagaaagacaagcaagccgacaagaagaagaagaaacccggaaaaagagaaagaatgtgca tgaagattgaaaatgac (SEQ ID NO: 51)

[0403] The nucleic acid sequence of polypeptide SFV-C2 is as follows:

[0404] aagaagatcaacggaaaaacgcagcagcaaaagaagaaagacaagcaagccgacaagaagaagaag (SEQ ID NO: 52)

[0405] The G protein mRNA sequence of VSV is from GenBank, with the sequence number X03633.1. Referring to the above sequence information, a DNA fragment containing the 5' non-coding region sequence of SFV, the 26S promoter nucleic acid sequence, the G protein nucleic acid sequence of VSV, the polypeptide SFV-C1, and the 3' non-coding region nucleic acid sequence of SFV was synthesized in sequence, and inserted into the plasmid pCMV-blank to construct the plasmid psaVSV-Gmut-SFV-C1.

[0406] The protein expressed by plasmid psaVSV-Gmut-SFV-C1 is a VSV-G fusion protein with polypeptide SFV-C1 bound to the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO:53.

[0407] (SEQ ID NO:53)

[0408] Compared with the original blank plasmid, the nucleic acid sequence inserted in the plasmid psaVSV-Gmut-SFV-C1 is shown in SEQ ID NO:54:

[0409]

[0410] A DNA fragment containing the 5' non-coding region sequence of SFV, the 26S promoter nucleic acid sequence, the G protein nucleic acid sequence of VSV, the polypeptide SFV-C2, and the 3' non-coding region nucleic acid sequence of SFV was synthesized and inserted into the plasmid pCMV-blank to construct the plasmid psaVSV-Gmut-SFV-C2.

[0411] The protein expressed by plasmid psaVSV-Gmut-SFV-C2 is a VSV-G fusion protein with the polypeptide SFV-C2 bound to the C-terminus. The amino acid sequence of the fusion protein is shown in SEQ ID NO: 55:

[0412] CKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTGLQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQV TPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDRELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSIS APSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGGH MLDSGLHLSSKAQVFEHPHIQDAASQLPDDEILFFGDTGLSKNPIDFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLYIKLKHTKKRQIYTDIEMNRLGRKKINGKTQQQKKKDKQADKKKK(SEQ IDNO:55)

[0413] Compared with the original blank plasmid, the nucleic acid sequence inserted in the plasmid psaVSV-Gmut-SFV-C1 is shown in SEQ ID NO:56:

[0414]

[0415] 2. Plasmid transfection and detection of extracellular vesicles

[0416] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. In the two-plasmid transfection system, the amount of each plasmid was 1.8 μg, and the amount of TA293 was 30 μl.

[0417] In this embodiment, plasmids psaVSV-Gmut-SFV-C1 and pSFV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saVSV-Gmut-SFV-C1 / SFV-sa-EGFP); plasmids psaVSV-Gmut-SFV-C2 and pSFV-sa-EGFP were co-transfected to prepare extracellular vesicles, and the extracellular vesicles were named extracellular vesicles (saVSV-Gmut-SFV-C2 / SFV-sa-EGFP). The contents of the harvested extracellular vesicles saVSV-Gmut-SFV-C1 / SFV-sa-EGFP and saVSV-Gmut-SFV-C2 / SFV-sa-EGFP were 6.1E6 FFU / ml and 5.8E6 FFU / ml, respectively.

[0418] In this example, the C protein polypeptides (SFV-C1 and SFV-C2) of the alphavirus SFV are fused to the C-terminus of the G protein inner region of VSV to form a fusion protein. The obtained fusion protein can specifically bind to the self-replicating mRNA sequence, promote the G protein of VSV to form extracellular vesicles, and enhance the ability of extracellular vesicles to actively load self-replicating mRNA.

[0419] Example 9: Alphavirus containing engineered proteins

[0420] In addition to the extracellular vesicle delivery vector containing the engineered protein described in this patent, virus is also a good delivery vector. The present invention uses the engineered protein to creatively develop a viroid derived from alphavirus, which does not contain the original structural protein of alphavirus and only uses the engineered protein described in this patent as the structural protein of the viroid.

[0421] The engineered protein used in this example is the engineered VSV-G fusion protein described in Example 6, and the constructed viroid is named VLV-VEEV / VSVG.

[0422] 1. Construction of viral expression plasmid (pVLV-VEEV / VSVG)

[0423] Using the VEEV replicon plasmid pVEEV-sa as the backbone, the DNA fragment of the engineered VSV-G fusion protein described in Example 6 was inserted after the start codon ATG carried by the subgenomic promoter in the plasmid pVEEV-sa to construct a viral expression plasmid (pVLV-VEEV / VSVG). The nucleotide sequence carried by the plasmid is shown in SEQ ID NO:12.

[0424] 2. Plasmid transfection and virus rescue

[0425] BHK-21 cells were selected as host cells for plasmid transfection, and TA293 transfection reagent was used as transfection reagent. The amount of plasmid was 1.8 μg each, and the amount of TA293 was 30 μl. The supernatant was harvested two days after plasmid transfection and the virus content was detected.

[0426] In this example, the plasmid pVLV-VEEV / VSVG was used to successfully rescue the viroid VLV-VEEV / VSVG, which can be serially propagated and maintain biological activity.

[0427] Table 1: Virus content detection at different generations (PFU / mL)

[0428] First Generation Second Generation Third Generation Fourth Generation VLV-VEEV / VSVG 1.5E6 1.8E7 1.6E7 9.2E6

Claims

1. An engineered protein, characterized in that include: An RNA binding polypeptide and a cell transmembrane protein, wherein the C-terminus of the RNA binding polypeptide is connected to the N-terminus of the cell transmembrane protein, or the C-terminus of the cell transmembrane protein is connected to the N-terminus of the RNA binding polypeptide, or the RNA binding polypeptide is inserted into the intracellular region of the cell transmembrane protein, wherein The RNA binding polypeptide is selected from the group consisting of an alphavirus C protein N-terminal domain polypeptide, a truncated polypeptide of the alphavirus C protein N-terminal domain, and a mutant polypeptide of the alphavirus C protein N-terminal domain, and the cell transmembrane protein is selected from at least one of an animal cell membrane protein, an exosome skeleton protein, an enveloped virus membrane protein, and a mutant polypeptide thereof.

2. The engineered protein according to claim 1, characterized in that The alphavirus comprises at least one selected from the group consisting of SINV, VEEV and SFV; Optionally, the RNA binding polypeptide comprises the amino acid sequence of positions 1 to 124 of protein C of VEEV or a truncated polypeptide thereof; Optionally, the RNA binding polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3; Optionally, the RNA binding polypeptide comprises the amino acid sequence of positions 1 to 113 of the C protein of SINV or a truncated polypeptide thereof; Optionally, the RNA binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 or 5; Optionally, the RNA binding polypeptide comprises the amino acid sequence of positions 1 to 118 of the C protein of SFV or a truncated polypeptide thereof; Optionally, the RNA binding polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6 or 7.

3. The engineered protein according to claim 1, characterized in that The animal cell membrane protein comprises at least one selected from the following: a cell receptor protein and a cell fusion protein; Optionally, the cell receptor protein comprises at least one selected from the following: selectin E, PDGFR, CD6, CD14, CD11b, CD36, CD3, CD4, CD8 and CD28; Optionally, the cell fusion protein comprises at least one selected from the group consisting of: Myomaker, Mymerger and syncytin; Optionally, the exosomal scaffold protein comprises at least one selected from the following: LAMP2b, CD9, CD37, CD63, CD64, CD81, CD82, ARRDC1 and SPFH; Optionally, the enveloped virus membrane protein includes at least one selected from the following: alphavirus glycoprotein E, vesicular stomatitis virus glycoprotein G, measles virus glycoprotein H / F, rubella virus glycoprotein E, mumps virus glycoprotein HA / NA, rabies virus glycoprotein G, influenza virus glycoprotein HA / NA, parainfluenza virus glycoprotein NH / F, respiratory syncytial virus glycoprotein G / F, Japanese encephalitis virus glycoprotein E and Sendai virus HN / F.

4. The engineered protein according to claim 1, characterized in that The engineered proteins include: VEEV C protein polypeptide and LAMP2b; Optionally, the N-terminus of the VEEV protein C polypeptide is linked to the C-terminus of LAMP2b; Optionally, the engineered protein has the amino acid sequence shown in SEQ ID NO:

8.

5. The engineered protein according to claim 1, characterized in that The engineered protein includes: VEEV protein C polypeptide and CD11b; Optionally, the N-terminus of the VEEV protein C polypeptide is linked to the C-terminus of CD11b; Optionally, the engineered protein has the amino acids shown in SEQ ID NO:

9.

6. The engineered protein according to claim 1, characterized in that The engineered proteins include VEEV protein C polypeptide and SINV glycoprotein E2; Optionally, the E2 of the SINV deletes the amino acid sequence at positions 397 to 414; Optionally, the protein C polypeptide of VEEV is inserted after amino acid 396 of E2 of SINV; Optionally, the engineered protein has the amino acid sequence shown in SEQ ID NO:

10.

7. The engineered protein according to claim 1, characterized in that The engineered protein includes VEEV protein C polypeptide and VSV glycoprotein G; Optionally, the N-terminus of the C protein polypeptide of VEEV is linked to the C-terminus of the glycoprotein G of VSV; Optionally, the engineered protein has the amino acid sequence shown in SEQ ID NO:

11.

8. A delivery vector, characterized in that The delivery vector carries the engineered protein according to any one of claims 1 to 7 and / or a nucleic acid sequence encoding the engineered protein according to any one of claims 1 to 7; Optionally, the delivery vector comprises at least one selected from the group consisting of: an extracellular vesicle, a virus, a nucleic acid-protein complex, a plasmid, RNA, and a cell.

9. The delivery vector according to claim 8, characterized in that The delivery vector is an extracellular vesicle, which also includes a cell membrane vesicle and an alphavirus self-replicating mRNA; Optionally, the cell membrane vesicles are derived from animal cells; Optionally, the engineered protein is loaded on the cell membrane vesicle; Optionally, the alphavirus self-replicating mRNA carries an alphavirus RNA packaging signal; Optionally, the alphavirus self-replicating mRNA comprises, in sequence: 5' end cap, 5' end non-coding region nucleic acid sequence of alphavirus, non-structural protein nucleic acid sequence of alphavirus, 26S promoter of alphavirus, nucleic acid sequence encoding target protein, 3' end non-coding region nucleic acid sequence of alphavirus and 3' end PolyA tail.

10. The delivery vector according to claim 8, characterized in that The delivery vector is a virus, and the virus also includes an alphavirus self-replicating mRNA; Optionally, the engineered protein is loaded on the viral envelope; Optionally, the virus is selected from a non-replication-defective virus or a replication-defective virus; Optionally, the virus comprises at least one selected from the group consisting of herpes simplex virus, poxvirus, adenovirus, alphavirus, flavivirus, influenza virus, coronavirus, rabies virus, vesicular stomatitis virus, poliovirus, measles virus.

11. The delivery vector according to claim 10, characterized in that The virus is an alphavirus; Optionally, the alphavirus is selected from VEEV; Optionally, the VEEV genome has a C protein gene and / or an E protein gene deleted.

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