RNA replicon for gene expression and use thereof

Abstract

Provided are an RNA replicon for gene expression and the use thereof. Specifically provided are an RNA replicon for transgene expression, a system containing the RNA replicon, and the use thereof.

Description

RNA replicons for gene expression and their applications Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to trans RNA replicons for trans gene expression, systems containing said trans RNA replicons, and their applications. Background Technology

[0002] Gene therapy involves introducing exogenous genes into target cells to achieve therapeutic goals. Among these, mRNA drugs have attracted the attention of researchers both domestically and internationally due to their relative safety. However, traditional mRNA drugs suffer from instability and easy degradation, leading to non-continuous downstream protein expression. Long-term treatment may require injections of large amounts of mRNA, potentially increasing the toxic side effects. Self-amplifying RNA (saRNA) refers to nucleic acid sequences that replicate themselves using their own RNA sequence (RNA replicon) as a template, aided by their carried RNA polymerase (RdRP). saRNA can achieve protein expression levels similar to large doses of traditional mRNA formulations at relatively low doses and can continuously produce the target protein. This characteristic can reduce the injection dosage and frequency in RNA therapy while prolonging the therapeutic effect and reducing potential toxic side effects from RNA and the drug delivery vehicle.

[0003] Alphaviruses are positive-sense RNA viruses. Alphaviruses host a variety of organisms, including insects, fish, and mammals. The alphavirus genome encodes four non-structural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (forming viral particles). The genome typically contains two open reading frames (ORFs). The four non-structural proteins (nsP1–nsP4) are usually encoded by the first ORF, which begins near the 5' end of the genome, while the alphavirus structural proteins are encoded by the second ORF, downstream of the first ORF and located near the 3' end of the genome.

[0004] In alphavirus-infected cells, only the nucleic acid sequence encoding non-structural proteins is translated from genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts of RNA molecules similar to eukaryotic messenger RNA. Post-infection, the (+) strand of genomic RNA is used for direct translation of the open reading frame encoding the non-structural protein (nsP1234). Subsequently, nsP1234 is hydrolyzed into peptides nsP123 and nsP4. nsP4 acts as an RNA-dependent RNA polymerase (RdRp) and can be used for the synthesis of the (-) strand, but its efficiency for the production of (+) strand RNA is low. Peptides nsP123 and nsP4 form a (-) strand replicase complex, used for transcription of (-) strand RNA using (+) strand genomic RNA as a template. Typically, in later stages, peptide nsP123 is further cleaved into individual proteins nsP1, nsP2, and nsP3. nsP1, nsP2, nsP3, and nsP4 together form a (+) strand replicase complex, which uses the complementary sequence of the (-) strand of the genomic RNA as a template to synthesize a new (+) strand genome.

[0005] The synthesis of alphavirus RNA is regulated by four conserved sequence elements (CSEs) that act in cis. The 5' replication recognition sequence of the alphavirus genome shows low homology among different alphaviruses but possesses a conserved secondary structure. The 5' replication recognition sequence also contains two conserved sequence elements involved in viral RNA synthesis, CSE1 and CSE2. The secondary structure is considered more important for the function of CSE1 and CSE2 than for the linear sequence. The 3' terminal sequence of the alphavirus genome (the sequence immediately upstream of the poly(A) sequence) possesses a conserved primary structure (i.e., the nucleotide sequence). CSE4, in particular, also known as the "19-nt conserved sequence," is crucial for the initiation of (-) chain synthesis.

[0006] Aviral structural proteins (core nucleocapsid protein C, envelope protein E2, and envelope protein E1, all components of the viral particle) are typically encoded by a single open reading frame under the control of a subgenomic promoter (SGP). The subgenomic promoter is recognized by the cis-acting alphavirus replicase complex. The alphavirus replicase complex uses the complementary sequence of the (-) strand of the genomic RNA as a template to synthesize a (+) strand of the subgenomic transcript. The (+) strand of the subgenomic transcript encodes the alphavirus structural proteins. Avirus-derived vectors can be used to deliver exogenous genetic information to target cells or organisms.

[0007] However, further improvements are still needed to alphavirus-derived vectors to efficiently deliver gene drugs and / or express target proteins. Summary of the Invention

[0008] This disclosure provides an improved transRNA replicon and method for the efficient expression of a target protein (e.g., an antigen), applicable to gene therapy for the prevention and treatment of diseases.

[0009] According to a first aspect of this disclosure, an RNA replicon is provided comprising a modified 5' replication recognition sequence, which, compared to the natural alphavirus 5' replication recognition sequence, includes at least the removal of the first and fourth start codons in the nsP1 coding sequence.

[0010] The nsP1 coding sequence within the nsP1234 open reading frame overlaps with the 5' replication recognition sequence of the trans RNA replicon. The translation start codon AUG present in the nsP1 coding sequence leads to the synthesis of the nsP fragment. However, the nsP fragment is generally unnecessary in gene drug delivery, and the translation of nsP also places an unnecessary burden on the host cell. Through screening, the inventors discovered that only when both the first start codon AUG and the fourth start codon in the nsP1 coding sequence within the 5' replication recognition sequence are simultaneously removed can efficient expression of the target protein carried by the replicon be achieved while simultaneously preventing nsP fragment synthesis; that is, translation of the target protein is initiated at the first start codon of the target protein open reading frame.

[0011] In some embodiments, the natural alphavirus 5' replication recognition sequence includes a sequence homologous to the 200-500 nt of the alphavirus 5' end. In some embodiments, the modified 5' replication recognition sequence includes a sequence homologous to the 200-300 nt of the alphavirus 5' end. In some embodiments, the modified 5' replication recognition sequence includes a sequence homologous to approximately 200 nt, 210 nt, 220 nt, 230 nt, 240 nt, 250 nt, 260 nt, 270 nt, 280 nt, 290 nt, or 300 nt of the alphavirus 5' end.

[0012] In some embodiments, the natural alphavirus 5' replication recognition sequence has a nucleotide sequence as shown in SEQ ID NO:12, or a homologous sequence thereof, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0013] In some implementations, the modified 5' replication recognition sequence, compared to the natural alphavirus 5' replication recognition sequence, further includes the removal of one or more of the second, third, and fifth start codons in the nsP1 coding sequence.

[0014] In some embodiments, the removal of the first, second, third, fourth, and / or fifth start codons in the nsP1 coding sequence may refer to the alteration, deletion, or insertion of one or more nucleotides in the start codon AUG. In some embodiments, the removal of the first, second, third, fourth, and / or fifth start codons in the nsP1 coding sequence may refer to the replacement of the start codon AUG with, for example, but not limited to, ACG, UUG, or AUC.

[0015] In some embodiments, the modified 5' replication recognition sequence includes sequences homologous to conserved sequence elements (CSE)1 and / or CSE2 of the alphavirus.

[0016] In some embodiments, the secondary structure of the modified 5' replication recognition sequence has one or more stem-loop structures. These stem-loop structures provide the function of being recognized by the replicase. In some embodiments, one or more of the 1st, 2nd, 3rd, 4th, and 5th stem-loop structures of the modified 5' replication recognition sequence are not destroyed or missing. That is, one or more of the 1st, 2nd, 3rd, 4th, and 5th stem-loop structures of the modified 5' replication recognition sequence may be identical or similar to the 5' replication recognition sequence of the natural alphavirus.

[0017] In some implementations, in addition to start codon removal, the modified 5' replication recognition sequence may also include one or more nucleotide changes to compensate for the disruption or loss of one or more stem-loop structures introduced by start codon removal.

[0018] In some embodiments, the modified 5' replication recognition sequence does not include an open reading frame encoding an alphavirus nonstructural protein or a truncated form thereof.

[0019] In some embodiments, the modified 5' replication recognition sequence has a nucleotide sequence as shown in any one or more of SEQ ID NO:13-20 and 31, or a homologous sequence thereof, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0020] In some embodiments, the RNA replicon includes a 3' replication recognition sequence not derived from an alphavirus. In some embodiments, the RNA replicon does not include a conserved sequence element (CSE)4.

[0021] CSE4 is known to function as a core promoter for the synthesis of the alphavirus initiating (-) RNA strand. Its deletion results in the ineffective synthesis of the (-) strand. The conserved sequence of CSE4 is AUUUUGUUUUUAAUAUUUC (SEQ ID NO: 30; 19nt conserved sequence).

[0022] This disclosure unexpectedly found that replacing the 3' replication recognition sequence of the RNA replicon of this disclosure with a non-alphavirus-derived 3' replication recognition sequence, which does not include CSE4, can also effectively initiate the synthesis of the (-) RNA chain and the expression of the target protein.

[0023] In some embodiments, the non-alphavirus-derived 3' replication recognition sequence may be derived from, for example, but not limited to, primates, mice, rats, dogs, rabbits, goats, sheep, alpacas, camels, guinea pigs, viruses, bacteria, fungi, etc.

[0024] In some embodiments, the non-A virus-derived 3' replication recognition sequence may be derived from, for example, but not limited to, human, mouse, dengue virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, etc.

[0025] In some embodiments, the non-alpha virus-derived 3' replication recognition sequence may have a nucleotide sequence as shown in any one or more of SEQ ID NO:1 to 10, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0026] In some embodiments, the RNA replicon further includes an open reading frame encoding one or more target proteins. In some embodiments, the expression of the target protein can be performed using the RNA replicon as a template. Those skilled in the art will understand that this disclosure does not impose any particular limitation on the one or more target proteins, and they can be any protein. In non-limiting embodiments, the target protein can be selected from, for example, but not limited to, reporter proteins, pharmaceutically active peptides, or proteins.

[0027] In some embodiments, the reporter protein can be any conventionally used reporter protein that can be used for identification or detection. Examples of reporter proteins may include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein), luciferase, β-galactosidase, etc.

[0028] In some embodiments, the pharmaceutically active peptide or protein can treat or alleviate a disease or symptom, delay its onset, or reduce its severity. In some embodiments, the pharmaceutically active peptide or protein includes antigens that can elicit an immune response in a subject.

[0029] In some embodiments, the open reading frame encoding the functional alphavirus nonstructural protein does not overlap with the 5' replication recognition sequence.

[0030] In some embodiments, the RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein. In such embodiments, the functional alphavirus nonstructural protein for RNA replicon replication may be provided as in the trans form.

[0031] In some embodiments, the RNA replicon includes a 5'-cap structure.

[0032] In some embodiments, the RNA replicon includes a poly(A) tail downstream of the 3'-replication recognition sequence. In some embodiments, the poly(A) tail may include 20 to 500 consecutive adenosine nucleotides (A), for example, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive A nucleotides.

[0033] According to a second aspect of this disclosure, a system is provided that includes:

[0034] RNA constructs for expressing functional alphavirus non-structural proteins; and

[0035] The RNA replicon described in this disclosure is capable of trans replication via the functional alphavirus nonstructural protein.

[0036] In some embodiments, the RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein.

[0037] In some embodiments, the RNA construct for expressing functional alphavirus nonstructural proteins includes a 5' cap structure. In some embodiments, the RNA replicon includes a 5'-cap structure.

[0038] The 5'-cap structure is well known in the art and can typically be formed from modified nucleotides, particularly derivatives of guanine nucleotides. Preferably, the 5'-cap is attached to the 5'-terminus via a 5'-5'-triphosphate bond. In some embodiments, the 5'-cap may be methylated, for example, m7GpppN, where N is the 5'-terminal nucleotide carrying the 5'-cap, typically the 5'-terminus of RNA.

[0039] The capped RNA disclosed herein can be prepared in vitro and therefore does not depend on the capping mechanism in the host cell.

[0040] Other examples of 5'-cap structures include glycerol groups, reverse deoxygenated base residues (partially), 4',5' methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentafuranosyl nucleotides, and acyclic 3',4'-seco Nucleotide), acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-reverse nucleotide moiety, 3'-3'-reverse baseless moiety, 3'-2'-reverse nucleotide moiety, 3'-2'-reverse baseless moiety, 1,4-butanediol phosphate, 3'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, or bridged or non-bridged methylphosphonate moiety.

[0041] RNA can also be stabilized by adding a 5'-cap structure to prevent degradation by RNases. In some implementations, G(5')ppp(5')G or m7G(5')ppp(5')N is used as the 5' cap structure (N is A, G, C or U).

[0042] In some embodiments, the RNA construct for expressing functional alphavirus nonstructural proteins includes a 5'-untranslated region (UTR), an open reading frame, and a 3'-UTR. In some embodiments, the 5'-UTR and / or the 3'-UTR is natural, heterologous, or non-natural for the alphavirus from which the functional alphavirus nonstructural protein is obtained.

[0043] In some embodiments, the RNA construct for expressing functional alphavirus nonstructural proteins further includes a poly(A) tail. In some embodiments, the poly(A) tail may comprise 20 to 500 consecutive adenosine (A) nucleotides, for example, about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive A nucleotides.

[0044] The alphavirus disclosed herein may be an alphavirus that exists in nature. For example, naturally occurring alphaviruses can be selected from: Chikungunya virus (CHIKV); Nyong virus and its subtype Igbo-Ora virus, Ross River virus and its subtype Bebaru virus, Getah virus, Sagiyama virus, Semliki forest virus (SFV) and its subtype (Me Tri virus); Venezuelan equine encephalitis complex, including Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocara virus and its subtype Bijou Bridge virus, and Venezuelan equine encephalitis virus. The Western equine encephalitis virus (WEEV) complex includes Auravirus, Babanki virus, Kyzylagach virus, Sindbis virus (SIN or SINV), Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, and Westernequine encephalitis virus; as well as some unclassified viruses, including Salmonpancreatic disease virus; Sleeping Disease virus; Southern elephant seal virus; and Tonate virus. More preferably, the alphavirus may be selected from: Semlikie Forest virus, Sindbis virus, and Chikungunya virus.

[0045] According to a third aspect of this disclosure, a nucleic acid molecule is provided, comprising a nucleic acid sequence encoding the RNA replicon described above in this disclosure.

[0046] According to a fourth aspect of this disclosure, a method for producing a target protein is provided, the method comprising the following steps:

[0047] (1) Obtaining the RNA replicon of the present disclosure, wherein the RNA replicon comprises an open reading frame encoding a functional alphavirus nonstructural protein and an open reading frame encoding a target protein, and the RNA replicon is capable of replication via the functional alphavirus nonstructural protein, and

[0048] (2) The RNA replicon is transferred into cells or administered to a subject.

[0049] In some embodiments, the cell may be an in vitro cell or part of an organism.

[0050] According to a fifth aspect of this disclosure, a method for producing a target protein is provided, the method comprising the following steps:

[0051] (1) Obtain RNA constructs for expressing functional alphavirus non-structural proteins;

[0052] (2) Obtain the RNA replicon of the present disclosure, said RNA replicon being capable of replication via the RNA construct obtained in (1), and comprising an open reading frame encoding a target protein; and

[0053] (3) The RNA construct obtained in (1) and the RNA replicon obtained in (2) are transferred into cells or administered to the subject together.

[0054] In this embodiment, the RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein.

[0055] In some embodiments, the cell may be an in vitro cell or part of an organism.

[0056] According to a sixth aspect of this disclosure, a cell is provided that includes an RNA replicon of the first aspect of this disclosure or a system of the second aspect. In some embodiments, the cell may be an in vitro cell or part of an organism.

[0057] According to a seventh aspect of this disclosure, a pharmaceutical composition is provided comprising an RNA replicon of the first aspect of this disclosure, a system of the second aspect, a nucleic acid molecule of the third aspect, or a cell of the sixth aspect; and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier, transporter, excipient, or diluent known in the art. In some embodiments, the pharmaceutically acceptable carrier can be cationic lipids, lipid nanoparticles (LNPs), microvesicles, or exosomes.

[0059] According to the eighth aspect of this disclosure, the use of the RNA replicon of the first aspect, the system of the second aspect, or the cell of the sixth aspect of this disclosure for delivering a target gene to a cell or a subject is provided. In some embodiments, the target gene may be used, for example, but not limited to, immunization, gene editing, gene therapy, etc. Attached Figure Description

[0060] Figure 1 shows a schematic diagram of the structure of linear mRNA, RNA replicon, and replicase according to embodiments of the present disclosure. Specifically, Figure 1A shows a schematic diagram of the structure of linear mRNA; Figure 1B shows a schematic diagram of the structure of mRNA carrying an RNA replicon that recognizes alphavirus 5'CSE and 3'CSE, and an RNA replicon where 3'CSE is replaced by 3'UTR; Figure 1C shows a schematic diagram of the structure of mRNA carrying Semlikie Forest Virus (SFV) replicase and Chikungunya Virus (CHIKV) replicase.

[0061] Figure 2 shows schematic diagrams of the structures of linear mRNA, RNA replicon mutant, and replicase according to embodiments of the present disclosure. Specifically, Figure 2A shows a schematic diagram of the structure of linear mRNA; Figure 2B shows a schematic diagram of the structure of mRNA in which the alphavirus recognizes an RNA replicon in which the ATG in the 5'CSE is completely or partially removed, or the 3'CSE is replaced by the 3'UTR; and Figure 2C shows a schematic diagram of the structure of mRNA carrying the chikungunya virus (CHIKV) replicase.

[0062] Figure 3 shows schematic diagrams of the linear mRNA, RNA replicon mutant, and replicase according to embodiments of the present disclosure. Specifically, Figure 3A shows a schematic diagram of the linear mRNA; Figure 3B shows a schematic diagram of the mRNA that recognizes the alphavirus RNA replicon with simultaneous mutations of ATG1 and ATG4 in the 5'CSE and with the 3'CSE replaced by the 3'UTR; and Figure 3C shows a schematic diagram of the mRNA carrying the chikungunya virus (CHIKV) replicase.

[0063] Figure 4 illustrates the expression of open reading frames (i.e., eGFP) in the trans-replicon of alphavirus after the 3'CSE has been replaced with the universal 3'UTR according to an embodiment of this disclosure.

[0064] Figure 5 illustrates the expression of open reading frames (i.e., eGFP) in the trans replicon after partial or complete removal of ATG in the 5'UTR of alphavirus according to embodiments of the present disclosure.

[0065] Figure 6 illustrates the expression of open reading frames (i.e., eGFP) in the trans replicon after partial or complete removal of ATG in the 5'UTR of alphavirus according to embodiments of the present disclosure and replacement of 3'CSE with the universal 3'UTR. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0067] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0068] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0069] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0070] As used herein, the term "nucleic acid" includes deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In this disclosure, nucleic acids may include genomic DNA, cDNA, mRNA, viral RNA, recombinant or chemically synthesized nucleic acid molecules. In some embodiments, nucleic acids may be in the form of single-stranded, double-stranded, or linear or covalently closed circular molecules.

[0071] As used herein, the term "RNA" or "RNA molecule" refers to a molecule containing ribonucleotide residues, preferably composed entirely or substantially of ribonucleotide residues. The term "ribonucleotide" includes nucleotides having a hydroxyl group at the 2' position of the β-D-furanose group. RNA or RNA molecules can include double-stranded RNA, single-stranded RNA, isolated RNA, synthetic RNA, and recombinant RNA. RNA can differ from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides. RNA molecules may also contain non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs.

[0072] Single-stranded RNA (SRNA) generally refers to an RNA molecule that is not associated with any complementary nucleic acid molecule (typically, there is no complementary RNA molecule). SRNA may contain its own complementary sequence, which allows partial RNA folding and the formation of secondary structures, including but not limited to base pairs, stems, stem-loops, and protrusions. SRNA can exist as a negative strand [(-) strand] or a positive strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. This genetic information can be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can be directly used as a template for translation (protein synthesis). The (-) strand is the complementary strand of the (+) strand. In the case of double-stranded RNA, the (+) and (-) strands are two separate RNA molecules that bind together to form double-stranded RNA.

[0073] "nt" is an abbreviation for nucleotide, preferably referring to a series of nucleotides in a nucleic acid molecule.

[0074] As used in this article, the term "codon" refers to the base triplet encoding nucleic acids. The start codon is the first codon in an RNA molecule that is translated by ribosomes. Such codons typically encode methionine in eukaryotes and modified methionine in prokaryotes. The most common start codon in both eukaryotes and prokaryotes is AUG. Unless otherwise specified, "start codon" as used in this article refers to codon AUG. If the start codon of messenger RNA (mRNA) is AUG, then the base triplet encoding AUG is ATG. RNA molecules may contain codons AUG that are not used by ribosomes to initiate translation; these codons are not considered functional start codons.

[0075] As used herein, the term "mRNA" or "messenger RNA" refers to a transcript that is produced using a DNA template and encodes a protein. mRNA typically contains a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. In some embodiments, mRNA can be modified by stabilization modifications and capping.

[0076] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a continuous sequence of adenosine residues, typically located at the 3' end of an RNA molecule. The poly(A) sequence is usually added to the free 3' end of RNA post-transcriptionally via a template-independent RNA polymerase. However, it should be understood that this disclosure also includes poly(A) sequences encoded by DNA. In specific embodiments, the RNA replicons of this disclosure and / or RNA constructs for expressing functional alphavirus nonstructural proteins include a poly(A) tail, for example, a poly(A) tail. 90 )tail.

[0077] As used herein, the term "5' replication recognition sequence" refers to a continuous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical or homologous to the 5' segment of the alphavirus genome and is recognized by a replicase (e.g., alphavirus replicase). The 5' replication recognition sequence herein includes naturally occurring 5' replication recognition sequences and their functional equivalents. In one embodiment, the 5' replication recognition sequence comprises a conserved sequence element 1 (CSE 1) or a variant thereof from the alphavirus genome, and a conserved sequence element 2 (CSE2) or a variant thereof from the alphavirus genome. The 5' replication recognition sequence is typically capable of forming four stem-loops (SLs) starting from the 5' end: SL1, SL2, SL3, and SL4.

[0078] As used in this article, the term "conserved sequence element" or "CSE" refers to a nucleotide sequence present in alphavirus RNA. These sequence elements are called "conserved" because orthologs exist in the genomes of different alphaviruses, and orthologous CSEs of different alphaviruses preferably have a high percentage of sequence identity and / or similar secondary or tertiary structures. The alphavirus genome includes four CSEs: CSE1, CSE2, CSE3, and CSE4. In this article, 5'CSE refers to the conserved sequence element at the 5' end of the alphavirus RNA; the 5'CSE of alphavirus contains CSE1, CSE2, and CSE3. Similarly, 3'CSE refers to the conserved sequence element at the 3' end of the alphavirus RNA; the 3'CSE of alphavirus contains CSE4.

[0079] As used herein, the term "identity %" refers specifically to the percentage of identical nucleotides in an optimal alignment between two sequences to be compared. This percentage is purely statistical, and the differences between the two sequences may be randomly distributed across the entire length of the sequences. The sequences to be compared may include additions or deletions compared to a reference sequence to obtain an optimal alignment between the two sequences. The comparison of two sequences is typically performed by comparing the sequences relative to fragments or a "comparison window" after optimal alignment to identify local regions of the respective sequences. Sequence alignment can be performed using procedures conventionally used in the art, such as, but not limited to, GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA.

[0080] As used herein, the terms “stem-loop” or “hairpin” or “hairpin loop” are used interchangeably to refer to a specific secondary structure formed by a nucleic acid molecule (typically a single-stranded nucleic acid molecule, such as single-stranded RNA). A specific secondary structure represented by a stem-loop consists of a continuous nucleic acid sequence comprising a stem and a (terminal) loop, also known as a hairpin loop, where the stem is formed by two adjacent, fully or partially complementary sequence elements; they are separated by a short sequence (e.g., 3 to 10 nucleotides) forming the loop of the stem-loop structure. Two adjacent, fully or partially complementary sequences can be defined, for example, stem-loop elements stem 1 and stem 2. A stem-loop is formed when these two adjacent, fully or partially anticomplementary sequences (e.g., stem-loop elements stem 1 and stem 2) form base pairs with each other, resulting in a double-stranded nucleic acid sequence comprising an unpaired loop formed at its end by a short sequence located between stem-loop elements stem 1 and stem 2. Thus, a stem-loop comprises two stems (stem 1 and stem 2) that form base pairs with each other at the secondary structure level of the nucleic acid molecule and are separated at the primary structure level of the nucleic acid molecule by a short sequence that is not part of stem 1 or stem 2. Stem loops are typically formed by single-stranded RNA molecules.

[0081] As used herein, the terms "compensating for the disruption or deletion of secondary structure" or "compensating for the disruption or deletion of stem-loop structure" refer to situations where a change in one or more first nucleotides, in the absence of a change in one or more second nucleotides, results in the disruption or deletion of the secondary structure of a nucleic acid sequence, while the simultaneous presence of one or more first nucleotide changes and one or more second nucleotide changes does not result in the disruption or deletion of the secondary structure of the nucleic acid. Typically, one or more first nucleotide changes and one or more second nucleotide changes coexist in the same nucleic acid molecule. In embodiments of this disclosure, "compensating for the disruption or deletion of secondary structure" refers to compensating for the disruption or deletion of stem-loop structure. The one or more nucleotide changes can be the deletion, addition, substitution, and / or insertion of one or more nucleotides.

[0082] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0083] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0084] Materials and methods:

[0085] The following materials and methods are used in the embodiments described below:

[0086] The source of the DNA plasmids used for preparation suitable for in vitro transcription: pUC57 is a commonly used plasmid cloning vector in E. coli. The pUC57-Kan-BsmBl-free plasmid backbone used in the examples was purchased from GenScript. The phage polymerase was T7, and the poly(A) box was replaced with 90 adenosine residues (poly(A)). 90 The BSPQ1 restriction site was placed immediately downstream of the poly(A) box. The pETR-V5 plasmid backbone used, as described in CN202311741177.2, contains the T7 promoter, 5'-UTR, 3'-UTR, and polyA sequence.

[0087] Linear mRNA plasmid construction: The coding nucleic acid sequence of enhanced green fluorescent protein (eGFP) (SEQ ID NO:27) was cloned into the 3' end of the 5'UTR of the pETR-V5 plasmid backbone (Figure 1 A, Figure 2 A and Figure 3 A).

[0088] In vitro transcription (IVT) of plasmids and purification of RNA: In vitro transcription was performed using the plasmids constructed in Examples 1 and 2 and T7 RNA polymerase. To generate an IVT template, the plasmids were purified by precipitation with 5M sodium chloride / anhydrous ethanol after linearization. The linearized plasmid DNA was transcribed in vitro using T7 RNA polymerase (Kaika, T7P-EE1MP-C1) and purified using... Reagent AU (Trilink, N-7114) or Capping Reagent AG (Trilink, N-7113).

[0089] Following in vitro transcription, capped polyadenylated RNA was purified using a two-step precipitation process involving 8M lithium chloride and 5M sodium chloride / anhydrous ethanol. The quality of the purified RNA was evaluated spectrophotometrically and analyzed on a 5200 BioAnalyzer (Agilent, Santa Clara, USA). The purified IVT-RNA used in this example was purified.

[0090] Cell culture: BHK21 cells (ATCC, CBP60642) were grown at 37°C in an incubator equilibrated to 5% CO2 in MEM medium (Gibco, 11095080) supplemented with 10% fetal bovine serum (FCS) (Hyclone, SV30087.03), 1% non-essential amino acids (Gibco, 11140050), and 1 mM sodium pyruvate (Gibco, 11360070). Transfection was performed when cell confluence reached approximately 70%–90%. Twenty-four hours post-transfection, images were taken using an inverted fluorescence microscope (Olympus, CKX53).

[0091] Cell transfection: RNA-lipid transfection was performed using Lipofectamine Messenger MAX (Invitrogen, catalog number LMRNA001) according to the manufacturer's instructions. Cells were transfected at a rate of approximately 45,000 cells / cm². 2 The growth zone was plated, and the transfection amount of linear mRNA and RNA replicon was 20 ng / well (96-well plate, Beyotime, catalog number FULA965); the transfection amount of replicase was 50 ng / well (96-well plate, Beyotime, catalog number FULA965).

[0092] Example 1. Construction of alphavirus replicase plasmid

[0093] In this embodiment, the open reading frames (ORFs) of Semlikee Forest Virus (SFV) replicase (SEQ ID NO:25) and Chikungunya Virus (CHIKV) replicase (SEQ ID NO:26) were obtained according to information from the GeneBank database to construct the alphavirus replicase sequence. This alphavirus replicase sequence was cloned into the pUC57-Kan-BsmBl-free vector to construct alphavirus replicase plasmids, named pUC57-SFV-NSP and pUC57-CHIKV-NSP, respectively. The constructed alphavirus replicase plasmids contain the T7 promoter (SEQ ID NO:29), 5'UTR, replicase ORF, 3'UTR, and poly(A) 90(Downstream of which is immediately adjacent to the BSPQ1 restriction site) tandem (C in Figure 1, C in Figure 2 and C in Figure 3), wherein the 5'UTR (SEQ ID NO:28) originates from the fibrinogen β chain (FGB) gene and the 3'UTR (SEQ ID NO:3) originates from the α-1 antitrypsin (AAT) gene.

[0094] Example 2. Construction of RNA replicon plasmid

[0095] 2.1 Construction of the alphavirus replicon plasmid (including the original 5' replication recognition region and the alphavirus 3' replication recognition region)

[0096] In this embodiment, enhanced green fluorescent protein (eGFP) was cloned into the 3' end of the 5' replication recognition sequence of the RNA replicon (Figure 1, B) to obtain the replicon sequence. This replicon sequence was cloned into the pUC57-Kan-BsmBl-free vector to construct the alphavirus replicon plasmid pUC57-eGFP-3'CSE. The vector contains a T7 promoter, 5'CSE, eGFP ORF, a 3' replication recognition sequence (3'CSE(SFV), SEQ ID NO:11), and poly(A)... 90 (It is connected in series with the BSPQ1 restriction site downstream of it.)

[0097] 2.2 Construction of a universal 3'UTR alphavirus replicon plasmid

[0098] Construction method: As shown in Figure 1B, the 3' replication recognition sequence in the alphavirus replicon vector pUC57-eGFP-3'CSE constructed in 2.1 was replaced with a universal 3'UTR. The universal 3'UTR is derived from hAG (SEQ ID NO:1), Rps27a (SEQ ID NO:2), AAT (SEQ ID NO:3), human hemoglobin β subunit (hHBB, SEQ ID NO:4), chromium mosaic virus (BMV, SEQ ID NO:5), dengue virus type 2 polymerase (DEN2, SEQ ID NO:6), transferrin (TF, SEQ ID NO:7), hepatitis C virus (HCV, SEQ ID NO:8), human cytochrome B-245α chain (Human CYBA, SEQ ID NO:9), MOD1 (SEQ ID NO:8), and other viruses. NO:10), and the resulting replicon plasmids were named pUC57-eGFP-hAG, pUC57-eGFP-Rps27a, pUC57-eGFP-AAT, pUC57-eGFP-hHBB, pUC57-eGFP-BMV, pUC57-eGFP-DEN2, pUC57-eGFP-TF, pUC57-eGFP-HCV, pUC57-eGFP-Human CYBA, and pUC57-eGFP-MOD1, respectively.

[0099] 2.3 Construction of mutants of the 5' replication recognition region of the alphavirus replicon

[0100] Construction method: As shown in Figure 2B, the five start codons ATG in the 5' replication recognition sequence of the alphavirus replicon vector pUC57-eGFP-3'CSE constructed in 2.1 were subjected to single nucleotide mutations (or substitutions) or combination mutations (or substitutions) to obtain the corresponding mutant plasmids. The five start codons ATG in the 5' replication recognition sequence are the underlined and bolded ATG bases in the nucleotide sequence shown in SEQ ID NO:12 below.

[0101] The folding pattern of the replicon RNA encoded by the plasmid was predicted using the RNA secondary structure prediction web server Mfold (http: / / www.mfold.org / ). If the secondary structure of the 5' replication recognition sequence with the start codon ATG mutation differed from that of the unmutated 5' replication recognition sequence shown in SEQ ID NO:12, one or more additional nucleotide mutations were performed until the secondary structure was identical to that of the unmutated 5' replication recognition sequence shown in SEQ ID NO:12. The resulting 5' replication recognition sequence mutant sequences are shown in Table 1 below.

[0102] Table 1: Mutants of the 5' replication recognition sequence.

[0103] 2.4 Construction of an alphavirus replicon plasmid containing both a 5' replication recognition sequence mutation and a universal 3' UTR

[0104] Construction method: As shown in Figure 3B, the ATG-mut9 (SEQ ID NO:31) plasmid with simultaneous mutations in start codons 1 and 4 was obtained using a method similar to 2.3. Then, the 3' replication recognition sequence (3'CSE, SEQ ID NO:11) on the plasmid was replaced with universal 3'UTRs derived from hAG (SEQ ID NO:1), Rps27a (SEQ ID NO:2), AAT (SEQ ID NO:3), and hHBB (SEQ ID NO:4), respectively, to construct an alphavirus replicon that simultaneously contains the 5'CSE (replication recognition region) mutation and the universal 3'UTR. The combinations of the 5'CSE mutation and the universal 3'UTR are shown in Table 2 below.

[0105] Table 2: The following table shows the alphavirus replicons that simultaneously contain a 5' replication recognition region and a universal 3' UTR.

[0106] Example 3. Effect of replacing the 3' replication recognition sequence with a universal 3' UTR on trans-replicon RNA expression.

[0107] BHK21 cells were transfected with mRNAs of the replicase SFV-NSP or CHIKV-NSP (encoded by plasmids constructed in Example 1), respectively, in combination with mRNAs of the replicons eGFP-3'CSE, eGFP-hAG, eGFP-Rps27a, eGFP-AAT, eGFP-hHBB, eGFP-BMV, eGFP-DEN2, eGFP-TF, eGFP-HCV, eGFP-Human CYBA, and eGFP-MOD1 (encoded by plasmids constructed in 2.1 and 2.2 of Example 2). Twenty-four hours post-transfection, images were taken using an inverted fluorescence microscope (Olympus, catalog number CKX53), and the results are shown in Figure 4. Positive controls are samples transfected with linear mRNA, and blank controls are samples without transfected mRNA.

[0108] As shown in Figure 4, after replacing the SFV-recognized 3'UTR (3'CSE) with the aforementioned universal 3'UTR, the expression of the open reading frame (i.e., eGFP) in the trans replicon was achieved, and the expression was significantly higher than that of the positive control sequence.

[0109] Example 4. Effect of mutations in the 5' replication recognition region of the replicon on trans-replicon RNA expression.

[0110] The mRNA of the replicase CHIKV-NSP (encoded by the plasmid constructed in Example 1) was combined with the mRNAs of the 5'CSE mutants ATG-mut1, ATG-mut2, ATG-mut3, ATG-mut4, ATG-mut5, ATG-mut6, ATG-mut7, and ATG-mut8 (encoded by the plasmid constructed in section 2.3 of Example 2), respectively, and transfected into BHK21 cells. Twenty-four hours post-transfection, images were taken using an inverted fluorescence microscope (Olympus, catalog number CKX53), and the results are shown in Figure 5. The positive control is the sample transfected with linear mRNA, and the blank control is the sample without transfected mRNA.

[0111] As shown in Figure 5, trans replication can still occur in the alphavirus 5'CSE even without complete removal of ATG. Furthermore, replication of the replicon and expression of eGFP in the open reading frame are only possible when both start codons 1 (ATG1) and 4 (ATG4) in the 5'CSE are mutated. In other words, ATG1 and ATG4 are the more important start codons in the alphavirus 5'CSE. Other start codons besides ATG1 and ATG4 (such as ATG2, ATG3, and ATG5), regardless of mutation, have no significant impact on eGFP expression in the open reading frame of the replicon.

[0112] The observation results in Figure 5 are summarized in Table 3 below.

[0113] Table 3: Effect of 5'CSE mutation in replicon on replicon expression.

[0114] Example 5. Effect of replicons containing both 5'UTR mutations and universal 3'UTR substitutions on trans-replicon RNA expression.

[0115] BHK21 cells were co-transfected with mRNA containing the replicase pUC57-CHIKV-NSP (encoded by the plasmid constructed in Example 1) and a replicon mutant mRNA containing a combination of a 5' replication recognition region mutation and a universal 3' UTR (encoded by the plasmid constructed in section 2.4 of Example 2). Twenty-four hours post-transfection, images were taken using an inverted fluorescence microscope (Olympus, catalog number CKX53), and the results are shown in Figure 6. Positive controls are samples transfected with linear mRNA, and blank controls are samples without transfected mRNA.

[0116] As shown in Figure 6, even if only the ATG of start codon 1 (ATG1) and start codon 4 (ATG4) in the alphavirus 5'CSE is mutated, and the alphavirus 3'CSE is replaced with the universal 3'UTR, eGFP expression in the trans replicon can still be achieved. The specific expression details are shown in Table 4.

[0117] Table 4: Effects of both 5'UTR mutations and universal 3'UTR substitutions in replicons on trans-replicon RNA expression.

[0118] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An RNA replicon, characterized in that, The RNA replicon includes a modified 5' replication recognition sequence, which, compared to the natural alphavirus 5' replication recognition sequence, includes at least the removal of the first and fourth start codons in the nsP1 coding sequence.

2. The RNA replicon according to claim 1, characterized in that, Compared to the natural alphavirus 5' replication recognition sequence, the modified 5' replication recognition sequence further includes the removal of one or more of the second, third, and fifth start codons in the nsP1 coding sequence, and / or The natural alphavirus 5' replication recognition sequence includes a sequence homologous to the 200-500 nt, preferably 200-300 nt, end of the alphavirus 5' terminal, and / or The 5' replication recognition sequence of the natural alphavirus has a nucleotide sequence as shown in SEQ ID NO:12, or a homologous sequence thereof, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

3. The RNA replicon according to claim 1, characterized in that, The modified 5' replication recognition sequence includes sequences homologous to conserved sequence elements (CSE)1 and / or CSE2 of alphavirus, and / or The secondary structure of the modified 5' replication recognition sequence has one or more stem-loop structures. Preferably, one or more of the 1st, 2nd, 3rd, 4th, and 5th stem-loop structures of the modified 5' replication recognition sequence are not destroyed or missing, and / or In addition to start codon removal, the modified 5' replication recognition sequence may also include one or more nucleotide changes to compensate for the disruption or deletion of one or more stem-loop structures introduced by start codon removal, and / or The modified 5' replication recognition sequence does not include an open reading frame encoding an alphavirus nonstructural protein or a truncated form thereof.

4. The RNA replicon according to claim 1, characterized in that, The RNA replicon includes a 3' replication recognition sequence not derived from an alpha virus, and the RNA replicon does not include the conserved sequence element (CSE)4. Preferably, the non-alphavirus-derived 3' replication recognition sequence originates from primates, mice, rats, dogs, rabbits, goats, sheep, alpacas, camels, guinea pigs, viruses, bacteria, or fungi. Preferably, the non-hepatitis A virus-derived 3' replication recognition sequence is derived from human, mouse, dengue virus, chromosome virus, hepatitis A virus, hepatitis B virus, or hepatitis C virus. Preferably, the non-alphavirus-derived 3' replication recognition sequence includes nucleotide sequences as shown in any one or more of SEQ ID NO:1 to 10, or nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

5. The RNA replicon according to claim 1, characterized in that, The RNA replicon also includes open reading frames encoding one or more target proteins, and the expression of the target proteins is performed using the RNA replicon as a template. Preferably, the target protein includes a reporter protein, a pharmaceutically active peptide, or a protein.

6. The RNA replicon according to claim 1, characterized in that, The RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein.

7. The RNA replicon according to any one of claims 1 to 6, characterized in that, The RNA replicon includes a 5'-cap structure, and / or The RNA replicon includes a poly(A) tail downstream of the 3'-replication recognition sequence, the poly(A) tail preferably comprising 20 to 500 consecutive adenosine nucleotides, and / or The virus is selected from: Chikungunya virus (CHIKV), Bama forest virus complex and its subtype Ibo-Aura virus, Ross River virus and its subtype Bibaru virus, Geta virus, Heron Mountain virus, Semliki forest virus and its subtype, Kabaso virus, Swamp virus, Mostas Pedras virus, Mukamb virus, Paramana virus, Napishuna virus, Rio Negro virus, Trokala virus and its subtype Bijubridge virus, Aura virus, Babanken virus, Zilagach virus, Sindbis virus, Oklbu virus, Wodarlo River virus, Bogd River virus, Morganburg virus, Highland J virus, Western equine encephalitis virus, salmon pancreatic disease virus, sleep disorder virus, Southern elephant seal virus, and Thunat virus; preferably selected from Semliki forest virus, Sindbis virus, and Chikungunya virus.

8. The system, characterized in that, The system includes: RNA constructs for expressing functional alphavirus non-structural proteins; and The RNA replicon according to any one of claims 1 to 7 is capable of trans replication via the functional alphavirus nonstructural protein.

9. The system according to claim 8, characterized in that, The RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein, and / or The RNA construct for expressing functional alphavirus nonstructural proteins includes a 5' cap structure, and / or The RNA replicon includes a 5'-cap structure, and / or The virus is selected from: Chikungunya virus (CHIKV), Bama forest virus complex and its subtype Ibo-Aura virus, Ross River virus and its subtype Bibaru virus, Geta virus, Heron Mountain virus, Semliki forest virus and its subtype, Kabaso virus, Swamp virus, Mostas Pedras virus, Mukamb virus, Paramana virus, Napishuna virus, Rio Negro virus, Trokala virus and its subtype Bijubridge virus, Aura virus, Babanken virus, Zilagach virus, Sindbis virus, Oklbu virus, Wodarlo River virus, Bogd River virus, Morganburg virus, Highland J virus, Western equine encephalitis virus, salmon pancreatic disease virus, sleep disorder virus, Southern elephant seal virus, and Thunat virus; preferably selected from Semliki forest virus, Sindbis virus, and Chikungunya virus.

10. The system according to claim 8, characterized in that, The RNA construct for expressing functional alphavirus non-structural proteins includes: 5' - Untranslated region (UTR) Open reading box, and 3'-UTR, Wherein, the 5'-UTR and / or 3'-UTR are natural, heterologous, or non-natural for the alphavirus from which the functional alphavirus non-structural protein is obtained. Preferably, the RNA construct for expressing functional alphavirus nonstructural proteins further includes a poly(A) tail, wherein the poly(A) tail preferably comprises 20 to 500 consecutive adenosine nucleotides.

11. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleic acid sequence encoding an RNA replicon as described in any one of claims 1 to 7.

12. A method for producing a target protein, characterized in that, The method includes the following steps: (1) Obtaining an RNA replicon according to any one of claims 1 to 7, said RNA replicon comprising an open reading frame encoding a functional alphavirus nonstructural protein and an open reading frame encoding a target protein, and said RNA replicon being capable of replication via said functional alphavirus nonstructural protein, and (2) The RNA replicon is transferred into cells or administered to a subject. Preferably, the cell is an in vitro cell or part of an organism.

13. A method for producing a target protein, characterized in that, The method includes the following steps: (1) Obtain RNA constructs for expressing functional alphavirus non-structural proteins; (2) Obtaining an RNA replicon according to any one of claims 1 to 7, said RNA replicon being capable of replication by the RNA construct obtained in (1) and comprising an open reading frame encoding a target protein; and (3) The RNA construct obtained in (1) and the RNA replicon obtained in (2) are jointly transferred into cells or jointly administered to the subject. The RNA replicon does not include an open reading frame encoding a functional alphavirus nonstructural protein. Preferably, the cell can be an in vitro cell or part of an organism.

14. A cell, characterized in that, The cell comprises an RNA replicon according to any one of claims 1 to 7 or a system according to any one of claims 8 to 10.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an RNA replicon according to any one of claims 1 to 7, a system according to any one of claims 8 to 10, a nucleic acid molecule according to claim 11, or a cell according to claim 14; and a pharmaceutically acceptable carrier.

16. The use of the RNA replicon of any one of claims 1 to 7, the system of any one of claims 8 to 10, and the nucleic acid molecule of claim 11 in delivering a target gene to a cell or a subject. Preferably, the target gene is used for immunization, gene editing, or gene therapy.

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