WO2022166959A1 - 顺式复制子构建体 - Google Patents

顺式复制子构建体 Download PDF

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WO2022166959A1
WO2022166959A1 PCT/CN2022/075384 CN2022075384W WO2022166959A1 WO 2022166959 A1 WO2022166959 A1 WO 2022166959A1 CN 2022075384 W CN2022075384 W CN 2022075384W WO 2022166959 A1 WO2022166959 A1 WO 2022166959A1
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virus
rna
construct
protein
coding unit
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French (fr)
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张守涛
马强
田庆南
王田
黄永辉
刘国涛
田震
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郑州大学
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Priority to EP22749238.6A priority Critical patent/EP4289956A1/en
Priority to CN202280023888.3A priority patent/CN117157402A/zh
Priority to US18/275,773 priority patent/US20240123049A1/en
Publication of WO2022166959A1 publication Critical patent/WO2022166959A1/zh

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    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
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    • C12N2770/00011—Details
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    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a cis-replicon RNA construct capable of efficiently expressing a target protein.
  • Nucleic acid molecules can be delivered to target cells or organisms by various technical methods. deliver.
  • the delivery of mRNA molecules has received increasing attention, and in vitro transcribed (IVT) mRNA has recently become the focus of attention as a potential new drug for transmitting genetic information.
  • IVTT in vitro transcribed
  • the use of mRNA has some outstanding advantages compared to that of DNA. First, higher safety: Since mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis.
  • mRNA is degraded by normal cellular processes, and its in vivo half-life can be modulated by using various modification and delivery methods. Moreover, the inherent immunogenicity of mRNA can be downregulated to further improve safety. Second, higher efficiency: various modifications make mRNA more stable and highly translatable, and in vivo administration can form an effective carrier molecule from mRNA, allowing it to be rapidly taken up and expressed in the cytoplasm.
  • mRNA production has the potential to be fast, cheap, and amplifiable in large quantities, mainly due to the high yield of in vitro transcription reactions, which is important for infectious disease pandemic situations and for patient-specific cancer-related or The possibility of cancer vaccine preparation of mutant antigens is very important (Norbert Pardi et al., 2018, Nat Rev Drug Discov., Vol. 17, pp. 261-279).
  • RNA Alphavirus belongs to togavirus family (togavirus family), contains 11-12kb non-segmented single-stranded RNA genome, 5' end is type 0 cap (N7mGppp), 3' end is poly( A) Tail, these features make the alphavirus genome equivalent to an mRNA that is translated immediately upon entry into the host cytoplasm and capable of controlling the production of all viral proteins required to initiate viral replication.
  • the alphavirus family includes about 30 members, mainly divided into Sindbis virus (SINV), Semliki forest virus (SFV), Venezuelan equine encephalomyelitis virus (Venezuelan equine encephalomyelitis virus, VEEV) and Chikungunya virus (CHIKV) et al. (Jennifer L. Hyde et al., Virus Research, 2015, Vol. 206, pp. 99-107).
  • the genome of alphavirus has two open reading frames in total, and the first 2/3 of the 5' end encodes four non-structural proteins (nsP1-nsP4), which are called non-structural regions.
  • the structural region which encodes structural proteins translated from subgenomic mRNAs, including capsid protein C, membrane glycoproteins E1, E2, E3, and 6K proteins.
  • the 5'-terminal nonstructural proteins of alphaviruses are first translated during infection of cells to generate the replicase required for RNA replication and transcription. Under the action of replicase, the transcription of subgenomic mRNA is initiated, followed by translation and cleavage into capsid proteins and membrane glycoproteins under the combined action of virus and host-encoded proteases. These structural proteins are assembled with viral genomic RNA into nucleocapsids Then it is released by budding.
  • Subgenomic RNA is neither used as a template for RNA synthesis nor packaged into virus particles, and structural genes can reach a very high copy number during virus replication, so subgenomic promoters can be used to promote high-efficiency expression of exogenous genes.
  • mRNAs for in vivo delivery can be either non-amplified mRNA molecules (about 2 kb) encoding the protein of interest or larger, self-amplifying mRNA molecules based on viral replicons (>10000 bp) (Piotr S. Kowalski et al. , Mol Ther., 2019, vol. 27, pp. 710-728; Giulietta Maruggi et al., Mol Ther., 2019, vol. 27, pp. 757-772).
  • SAM self-amplifying mRNA
  • the products of the first round of translation of the viral genomic RNA assemble into a replicase that replicates the negative strand (cRNA) complementary to the genome, serving as a template for the synthesis of other mRNA molecules. Due to the intracellular replication of antigen-encoding RNAs, the SAM platform enables the mass production of antigens from extremely small doses of vaccines (Norbert Pardi et al., 2018, Nat Rev Drug Discov., Vol. 17, pp. 261-279).
  • Alphavirus replicons have the following advantages: First, self-amplifying mRNA can increase the expression of foreign proteins and activate a stronger immune response in the body. This is because the production of exogenous proteins in SAM-transfected cells is based on the principle of virus replication. Under the action of RNA replicase, the RNA replicon replicates in large numbers in the cytoplasm, and the exogenous genes are efficiently expressed, which is a continuous B. Cell stimulation and antibody production provide ideal conformations.
  • RNA-dependent protein kinase PLR
  • TLR Toll-like receptor
  • OFA 2'-5'-oligoadenylate synthesis Enzymes
  • the alphavirus replicon does not enter the nucleus, but only briefly replicates, transcribes, and expresses the target protein in the cytoplasm. After that, the cell undergoes apoptosis and is eliminated under the action of the antiviral signal activated by dsRNA. Therefore, compared with traditional DNA vaccines, it will not be integrated into the host genome to cause cancer risk, nor will it exist in the body for a long time (Annette B. Vogel et al., Mol Ther., 2018, Vol. 26, No. 446- 455 pages). Third, it has a wide range of applications.
  • Alphavirus replicons can be efficiently applied to vaccine research of various pathogens, such as bovine viral diarrhea virus (BVDV), cytomegalovirus (CMV), swine fever virus (CSFV), dengue fever (Dengue), Ebola virus (Ebola), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis E (HeV), HIV (HIV), Human Papilloma Virus (HPV), Herpes Simplex Virus (HSV), Infection Bursal disease virus (IBDV), Influenza virus (Influenza), Infectious salmon anemia virus (ISAV), Japanese encephalitis virus (JEV), Lassa virus (Lassa), Leaping disease virus (LIV), Marburg virus (MBGV), Measles (Measles), Murray Valley encephalitis (MVE), Nipah virus (NiV), Norwalk-like virus (NLV), Rabies virus (RV), Respiratory syncytial virus (RS
  • Non-viral vectors can carry higher genetic loads, improve safety and induce lower immune responses.
  • Non-viral carriers used to deliver mRNA are cationic polymers (Polyplexes, PP), lipoplexes (lipoplex, LP), liposome nanoparticles (lipid nanoparticle, LNP) and cationic nanoemulsion (cationic nanoemulsion, CNE) Among them, lipid carriers are the most widely used (S Guan & J Rosenecker, Gene Therapy, 2017, Vol. 24, pp. 133-143; Piotr S. Kowalski et al., Mol Ther., 2019, Vol. 27; 710-728; Kristie Bloom et al., Gene Ther., 2020, pp. 1-13).
  • the object of the present invention is to provide a cis-replicon RNA construct for efficiently expressing the target protein, so as to solve the problems existing in the above-mentioned prior art, and construct an RNA replicon through the transformation of the alphavirus genome, which only drives the efficient replication of the target gene. and expression, to achieve the purpose of minimizing the expression of non-target proteins and increasing the expression of target proteins.
  • the present invention provides following scheme:
  • the present invention provides a cis-replicon RNA construct for efficiently expressing a target protein, the construct comprising:
  • a 5'UTR comprising one or more nucleotide sequences encoding replicase
  • the 5' UTR comprises a 5' cap
  • heterologous nucleotide sequence it is under the control of the subgenomic promoter
  • the 3'UTR contains a 3'end recognition sequence and 3'polyA that guide the replication of the RNA replicon.
  • the present invention provides a cis-replicon RNA construct for efficiently expressing a target protein, characterized in that, in the 5'-3' direction, the construct comprises:
  • a gene of interest or a heterologous nucleotide sequence it is controlled by the subgenomic promoter;
  • the replicase is derived from an alphavirus.
  • the alphavirus is Semliki Forest virus, Venezuelan equine encephalitis virus, Sindbis virus, and Chikungunya virus.
  • the 5' and 3' recognition sequences direct replication of the RNA replicon in the presence of the replicase; the 5' and 3' recognition sequences comprise CSE1 and CSE2.
  • the 5' and 3' recognition sequences of alphaviruses are preferred for the replicase source, and the 5'UTR is a eukaryotic 5'UTR;
  • the 5' cap structure is a natural 5'-cap or a 5'-cap analog
  • the 3'UTR is the 3'UTR of the viral mRNA.
  • the nucleotide sequence encoding the replicase comprises an open reading frame; the open reading frame encoding the replicase comprises a coding region for a nonstructural protein required for RNA replication.
  • the RNA construct used to express the alphavirus replicase and/or the RNA replicon does not comprise an open reading frame encoding the complete alphavirus structural protein.
  • the heterologous nucleotide sequence encodes a protein of interest, and expression of the open reading frame encoding the protein of interest is under the control of a subgenomic promoter.
  • the open reading frame encoding the protein of interest is non-native to the alphavirus from which the replicase is derived;
  • the subgenomic promoter is native to the alphavirus from which the replicase is derived.
  • the present invention provides recombinant viral vectors, plasmid DNA, linear DNA, in vitro transcribed RNA, or extracted intracellular transcribed RNA comprising the cis-replicon construct.
  • the present invention also provides a DNA recombinant vector comprising the cis-replicon RNA construct, which cannot drive the replication of replicase and efficiently drives the replication and expression of the target gene.
  • the present invention also provides nucleotide sequences encoding said cis-replicon RNA constructs.
  • the present invention also provides a method for producing a protein using an organism, comprising obtaining the construct or the recombinant vector for expressing a target protein, and transfecting the construct or the recombinant vector into an organism in a step.
  • the organisms include vertebrates and cells.
  • the present invention provides an expression construct as a cis-replicon comprising an RNA polymerase coding unit and a protein coding unit of interest, wherein
  • the RNA polymerase coding unit comprises a nucleic acid segment encoding RNA polymerase, and the target protein coding unit comprises an acceptor site (such as a multiple cloning site) to be inserted into a target protein coding sequence and/or a nucleic acid region encoding the target protein part,
  • the construct is capable of expressing the RNA polymerase in the target cell, and relying on the RNA polymerase to replicate, thereby providing a template for further expression of the target protein; preferably, the replication is efficient for the target protein unit For the RNA polymerase coding unit; more preferably, the duplication is to duplicate only the target protein coding unit.
  • the construct is DNA or RNA, preferably RNA, including single-stranded RNA or double-stranded RNA, eg, single-stranded RNA, in particular the single-stranded RNA is positive-stranded (+) RNA.
  • the construct is linear or circular
  • the construct is a linear RNA with a 5' cap or IRES sequence present at its 5' end that drives translation of a downstream coding sequence; or the construct is a circular RNA, present upstream of the RNA polymerase coding unit The IRES sequence that drives translation of the RNA polymerase coding unit.
  • replication promoter elements upstream and downstream of the protein-coding unit of interest that drive replication dependent on the RNA polymerase, thereby achieving replication of the protein-coding unit of interest; preferably the RNA polymerase
  • the coding unit is not between the upstream replication promoter element and the downstream replication promoter element.
  • the upstream replication initiation element comprises CSE1 and/or CSE3 sequences, preferably CSE1 and CSE3 sequences, more preferably CSE1, CSE2 and CSE3 sequences; and/or the downstream replication initiation element comprises CSE4 sequence.
  • the protein coding unit of interest is located downstream or upstream of the RNA polymerase coding unit.
  • the protein of interest coding unit is located upstream of the RNA polymerase coding unit, the protein of interest and the RNA polymerase are expressed as a fusion protein, and a linker sequence exists between the protein of interest and the RNA polymerase in the fusion protein , the linking sequence comprises a segment encoding a protease recognition site, and the RNA polymerase is provided after the fusion protein is recognized and cleaved by a protease, preferably, the protease recognition site is a 2A peptide sequence.
  • the segment encoding the protease recognition site may be located upstream or downstream of a replication initiation element (e.g., CSE4).
  • the protein-coding unit of interest is located upstream of the RNA polymerase-coding unit, and there is an IRES between the protein-of-interest-coding unit and the RNA polymerase-coding unit that drives translation of the RNA polymerase-coding unit sequence.
  • the IRES sequence can be located upstream or downstream of a replication promoter element (eg, CSE4).
  • the protein coding unit of interest is located upstream of the RNA polymerase coding unit, and there is a self-cleaving HDV-like ribozyme (HDV-like Ribozymes) between the protein coding unit of interest and the RNA polymerase coding unit ).
  • the self-cleaving HDV-like ribozyme can be located upstream or downstream of a replication initiation element (eg, CSE4).
  • the protein-of-interest coding unit is located downstream of the RNA polymerase-coding unit, wherein the construct comprises in the 5'-3' orientation:
  • a 5' cap or IRES sequence that drives translation of a downstream coding sequence, the RNA polymerase coding unit, the upstream replication initiation element, the protein-of-interest coding unit, and the downstream replication initiation element.
  • the construct comprises in the 5'-3' orientation:
  • the construct is DNA wherein the subgenomic promoter SGP is present upstream of the RNA polymerase coding unit.
  • the RNA polymerase is a viral replicase, particularly the viral replicase is an alphavirus replicase,
  • the alphavirus is selected from the group consisting of Semliki forest virus, Barmah forest virus, Chikungunya virus, O'nyong virus -nyong virus), Ross river virus (Ross river virus), Bebaru virus (Bebaru virus), Getah virus (Getah virus), Sagiyama virus (Sagiyama virus), Mayaro virus (Mayaro virus) and its sub type Una virus; Venezuelan equine encephalitis complex including Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus), Cabassou virus (Cabassou virus), Everglades virus (Everglades virus), Mucambu virus ( Mucambo virus, Tonate virus, Pixuna virus, Mosso das pedras virus, Rio Negro virus ); Western equine encephalitis complex includes Western equine encephalitis virus, Sindbis virus, Aura virus, Whataroa virus, Highland J virus ( Highlands J virus), Fort
  • the alphavirus is selected from the group consisting of Semliki Forest virus, Venezuelan equine encephalitis virus, Sindbis virus and Chikungunya virus.
  • the 5' cap structure is a native 5' cap or a 5' cap analog.
  • the construct comprises a 5'UTR derived from the 5'UTR of any gene or a mutant thereof, preferably the 5'UTR is derived from the same virus as the RNA polymerase source .
  • the replication initiation element is capable of being recognized by the RNA polymerase and directs its replication
  • the replication initiation element and the RNA polymerase are derived from the same virus or different viruses, more preferably from the same virus.
  • the construct comprises a 3'UTR derived from the 3'UTR of any gene or a mutant thereof, preferably the 3'UTR is derived from the same virus as the RNA polymerase.
  • the construct comprises 3' poly(A).
  • the subgenomic promoter is derived from a viral structural protein promoter, preferably, the subgenomic promoter and the RNA polymerase are derived from the same virus.
  • the present invention provides a vector comprising or encoding a construct as described in the first aspect.
  • the present invention provides a vaccine composition
  • a vaccine composition comprising the construct of the first aspect or the vector of the second aspect, wherein the protein of interest is capable of eliciting a protective immune response , eg, the antigens are derived from humans, animals, plants, viruses, bacteria and/or parasites.
  • the present invention provides a pharmaceutical composition comprising the construct as described in the first aspect or the vector as described in the second aspect, wherein the protein of interest is a therapeutic protein.
  • the present invention provides a method for expressing a protein of interest, comprising introducing the construct of the first aspect into a cell of interest that can rely on the construct to express the protein of interest.
  • the present invention provides a method of immunization comprising vaccinating a subject in need of the construct of the first aspect.
  • the cis-replicon RNA construct disclosed in the present invention by modifying various elements of the alphavirus genome, the constructed replicon cannot drive the replication of the replicase, but can only drive the efficient replication and expression of the target gene, which can minimize the The expression of the target protein, and increase the expression of the target protein by amplifying the target RNA greatly.
  • the present invention can realize the high-efficiency expression of the target protein in cells or organisms without culturing the alphavirus, and is suitable for effectively and safely producing the target protein in the organism, such as therapeutic protein or antigenic protein, which also It provides the basis and method for medical application.
  • Figure 1 shows the structure of the replicon in the prior art.
  • Figure 1A shows the structure of the alphavirus genome.
  • the alphavirus genome is a single-stranded (+) stranded RNA encoding two open reading frames (ORFs).
  • ORFs open reading frames
  • the first 2/3 part of the 5' end encodes 4 nonstructural proteins (nsP1-nsP4), which are translated and processed into replicase; the latter 1/3 near the 3' end is called the structural region and is affected by the subgenomic promoter ( SGP), which encodes structural proteins such as capsid protein C, membrane glycoproteins E1, E2, E3, and 6K proteins.
  • SGP subgenomic promoter
  • Figure 1B shows the structure of a conventional cis-replicon in which the region encoding the structural protein is replaced by the coding region of the gene of interest.
  • Figure 1C shows a replicon (also referred to herein as a trans-replicon) structure in which the gene of interest and the replicase are located in two separate constructs.
  • C 5' cap
  • SGP subgenomic promoter
  • CSE conserved sequence element.
  • Figure 2 shows a replicon structure in some embodiments of the invention, wherein the replicase coding unit is located upstream of the gene coding unit of interest.
  • Fig. 2A shows that the upstream replication initiation element comprises CSE1, CSE2 and SGP, and the downstream replication initiation element comprises CSE4;
  • Fig. 2B shows that the upstream replication initiation element comprises CSE1 and CSE2, and the downstream replication initiation element comprises CSE4;
  • Fig. 2C shows that the upstream replication initiation element comprises CSE1, The downstream replication promoter element contains CSE4;
  • Figure 2D shows that the upstream replication promoter element contains SGP, and the downstream replication promoter element contains CSE4.
  • Figure 3 shows a cis-replicon structure in some embodiments of the invention, wherein the replicase coding unit is located downstream of the gene coding unit of interest.
  • Figure 3A shows that there is a 2A peptide sequence between the replicase coding unit and the target gene coding unit;
  • Figure 3B shows that there is an IRES sequence between the replicase coding unit and the target gene coding unit;
  • Figure 3C shows the relationship between the replicase coding unit and the target gene coding unit There is an HDV-like ribozyme (HDVr) sequence between them;
  • Figure 3D shows that there is no self-cleavage sequence or IRES sequence between the replicase coding unit and the target gene coding unit.
  • HDVr HDV-like ribozyme
  • Figure 4 shows the fluorescence images of cells when different replicons express exogenous protein (fluorescent protein EGFP).
  • Traditional plasmid (A) traditional cis-replicon with the structure shown in FIG. 1B (B), trans-replicon with the structure shown in FIG. 1C (C), and the cis-replicon of the present invention with the structure shown in FIG. 2A (D) Comparison of fluorescent signals of EGFP expressed.
  • Figure 5 shows the detection results of the reporter gene signal when different replicons express exogenous proteins (Luc as the reporter gene), which are the traditional plasmid, the cis-replicon with the structure shown in Figure 1B, and the trans-replicon with the structure shown in Figure 1C.
  • the cis-replicon of the present invention with the structure shown in Figure 2A.
  • Figure 6 shows the WB detection results of different replicons expressing foreign proteins (new coronavirus RBD proteins), which are the traditional plasmid and the cis-replicon of the present invention with the structure shown in Figure 2A, respectively.
  • Figure 7 shows the determination of anti-SARS-CoV-2-RBD IgG titers in the serum of immunized mice by ELISA.
  • Figure 8 shows the neutralization titer (IC 50 ) of anti-SARS-CoV-2-RBD antibodies in the serum of immunized mice detected by pseudovirus neutralization assay.
  • replicas refers to a unit capable of independent replication, including one or more elements required for replication, which may be DNA or RNA.
  • “Cis-replicon” means that the elements required for replication are located on the same polynucleotide chain, which can be linear or circular.
  • “Trans-replicon” means that elements required for replication are located on different polynucleotide strands.
  • protein-encoding unit of interest in the present invention refers to a polynucleotide segment comprising a protein-encoding sequence of interest, and/or an acceptor site (eg, a multiple cloning site) to be inserted into a protein-encoding sequence of interest.
  • acceptor site eg, a multiple cloning site
  • RNA polymerase coding unit refers to a polynucleotide segment comprising an RNA polymerase coding sequence.
  • replication in the present invention refers to the use of RNA polymerase to generate a polynucleotide chain identical to the full length or segment of a template polynucleotide chain.
  • the duplication is duplication of a sequence comprising an upstream replication initiation element and a downstream replication initiation element, resulting in multiple copies of the same sequence.
  • replication initiation element in the present invention refers to a polynucleotide segment capable of being recognized by an RNA polymerase (eg, viral replicase) and initiating replication.
  • an RNA polymerase eg, viral replicase
  • upstream/downstream defines upstream and downstream in the 5'-3' direction for a particular polynucleotide sequence.
  • alphavirus is to be understood broadly and includes any viral particle having characteristics of an alphavirus.
  • Alphaviruses are characterized by the presence of (+) stranded RNA that encodes genetic information suitable for replication in host cells, including replicases and the like. Further characterization of many alphaviruses is described, for example, in Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124; Jonathan Rupp et al., 2015, J. Gen. Virology, vol. 96, pp.
  • alphavirus includes alphaviruses found in nature and any variants or derivatives thereof.
  • Alphaviruses found in nature are preferably selected from the group consisting of: Semliki forest virus complex including Semliki forest virus, Barmah forest virus, Chikungunya virus virus), O'nyong-nyong virus, Ross river virus, Bebaru virus, Getah virus, Sagiyama virus, Mayaro virus and its subtype Una virus; Venezuelan equine encephalitis complex including Venezuelan equine encephalitis virus, Cabassou virus, swampy Everglades virus, Mucambo virus, Tonate virus, Pixuna virus, Mosso das pedras virus ), Rio Negro virus; Western equine encephalitis complex including Western equine encephalitis virus, Sindbis virus, Aura virus, Vata Roa virus (Whataroa), Highlands J virus (Highlands J virus), Fort Morgan virus (Fort Morgan virus) and its subtype Boji River virus (Creek virus); Eastern equine encephalitis virus (Eastern equine encepha
  • the alphavirus is selected from the group consisting of the Semiriki Forest virus complex (including the virus types as described above, including the Semliki Forest virus), the Western Equine Encephalitis complex (including the virus types as described above, including Sindbis virus), Venezuelan equine encephalitis complex (including the virus types described above, including Venezuelan equine encephalitis virus).
  • the Semiriki Forest virus complex including the virus types as described above, including the Semliki Forest virus
  • the Western Equine Encephalitis complex including the virus types as described above, including Sindbis virus
  • Venezuelan equine encephalitis complex including the virus types described above, including Venezuelan equine encephalitis virus.
  • the alphavirus is not an alphavirus found in nature, such as an alphavirus modified by genetic engineering.
  • alphaviruses not found in nature are variants or derivatives of alphaviruses found in nature, which differ from alphaviruses found in nature by the presence of at least one mutation in the nucleotide sequence (ie, genomic RNA).
  • Mutations in the nucleotide sequence can be selected from insertions, substitutions or deletions of one or more nucleotides compared to alphaviruses found in nature.
  • a mutation in a nucleotide sequence may or may not be related to a mutation in the polypeptide or protein encoded by the nucleotide sequence. For example, Erkuden Casales et al.
  • nsP nonstructural protein
  • BHK baby hamster kidney
  • CHO Chinese hamster ovary
  • nsP1-4 non-structural proteins
  • non-structural regions which are non-structural proteins at the 5' end during cell infection
  • the cis RNA construct of the present invention contains an open reading frame (ORF) encoding an alphavirus replicase.
  • ORF open reading frame
  • a "non-structural protein” is any one or more individual non-structural proteins (nsP1, nsP2, nsP3, nsP4) of alphavirus origin, or comprising more than one alphavirus-derived non-structural protein Polyproteins of polypeptide sequences, such as nsP1234.
  • “nonstructural protein” refers to nsP123.
  • nsP1 is a guanine-7-methyltransferase (also known as viral capase) with methyltransferase and guanylyltransferase activities, as well as membrane anchoring functions for the replication complex. Its methyltransferase domain catalyzes the addition of the methyl group of S-adenosylmethionine to the GTP molecule to form a covalent m7GMP-nsP1 intermediate, and nsP2 removes the 5'- ⁇ - After phosphorylation, the m7GMP moiety is transferred to the RNA molecule through the guanosyltransferase activity of nsP1 to form a type 0 cap structure, namely m7GpppA (Leevi & Tero Ahola, 2002, Vol.
  • nsP2 is the largest replicase protein and an RNA-binding protein with NTPase, RNA helicase and protease activities, which can open RNA double strands and assist nsP1 protein to complete the capping process.
  • the protease activity of nsP2 also plays an important role in replicase breakdown into nonstructural proteins (Ellen G. Strauss et al., Virology, 1992, vol. 191, pp. 932-940; M Gomez de Cedrón et al., FEBS Lett, 1999, Volume 448, pp. 19-22; Leevi & Tero Ahola, 2002, Vol. 71, pp. 187-222; Maija K. et al., 2017, Vol. 234, pp. 44-57).
  • nsP3 is a phosphorylated protein composed of three domains: an N-terminal conserved domain with phosphatase activity and nucleic acid binding ability, an alphavirus unique domain (AUD), and a C-terminal hypervariable domain (HVD) ( Farhana Abu Bakar et al., Viruses., 2018, Vol. 10, p. 71).
  • AUD alphavirus unique domain
  • HVD C-terminal hypervariable domain
  • nsP3 has been found to interact with several host proteins, including stress granule-associated proteins, DEAD-box family proteins, heat shock proteins, and kinases, and may regulate protein-protein poly/mono-ADP ribosylation (Tyler Lark et al. , Front Microbiol, 2017, Vol. 8, p. 2652; Maija K. et al., 2017, vol. 234, pp. 44-57; Benjamin et al., Viruses, 2018, Vol. 10, p. 105).
  • nsP3 may also be involved in neurovirulence with Old World alphaviruses such as SFV (Benjamin et al., Viruses, 2018, Vol. 10, p. 105).
  • nsP4 contains an RNA-dependent RNA polymerase (RdRp) domain at the C-terminus and is the most conserved protein among alphaviruses.
  • RdRp RNA-dependent RNA polymerase
  • nsP4 forms a replication complex (RC) together with host proteins and other nsPs (such as nsP123+nsP4) to synthesize (-) strand RNA using the viral (+) strand as a template.
  • nsP123 After nsP123 is cleaved into nsP1, nsP2 and nsP3, it can form a tetramer with nsP4, which no longer has the function of synthesizing negative-strand RNA, but it can use the negative-strand RNA as a template to participate in the replication of genomic RNA and transcription of 26S subgenomic RNA.
  • Purified nsP4 also possesses terminal adenylate transferase activity, possibly generating a poly(A) tail at the 3' end of the genome (Maija K. et al., 2017, vol. 234, pp. 44-57; Farhana Abu Bakar et al., Viruses., 2018, vol. 10, pp. 71).
  • the source of replicase is not limited to any particular alphavirus.
  • the alphavirus replicase is from a Semliki Forest virus (SFV), including a naturally occurring Semliki Forest virus or variant.
  • the replicase is from Sindbis virus (SINV), including naturally occurring Sindbis viruses or variants.
  • the replicase is from Venezuelan Equine Encephalitis Virus (VEEV), including naturally occurring VEEV or variants.
  • RNA molecule e.g, an mRNA molecule
  • the 5'UTR and 3'UTR of alphaviruses contain distinct core promoter elements for negative- and positive-strand RNA synthesis.
  • the alphavirus-capped 5'UTR contains promoter elements, translational regulatory sequences, and structures of innate immune defense (Jennifer L. Hyde et al., Virus Research, 2015, vol. 206, pp. 99-107).
  • the alphavirus 5'UTR contains core promoter elements that act in conjunction with other cis-acting elements downstream and within the 3'UTR to regulate the synthesis of positive and negative strands (Kulasegaran Shylini et al., 2009, J Virol. , pp. 8327-8339).
  • the adenylated 3'UTR of alphaviruses contains highly conserved viral replication sequence elements, elements that determine cell tropism, and conserved binding regions that affect viral RNA stability (Jennifer L.Hyde et al., Virus Res, 2015, pp. 99-107 Page).
  • CSE common conserved sequence
  • the cis-RNA replicons of the present invention contain a 5'UTR of non-viral origin; in particular, non-alphavirus origin, such as the human ⁇ -globin 5'UTR, can improve translation efficiency while making the replicase not expandable Increases the RNA encoding the replicase region.
  • the RNA comprises a 5'UTR derived from a eukaryotic organism.
  • a 5'UTR according to the invention may comprise any combination of more than one nucleic acid sequence, optionally separated by a linker.
  • the cis-RNA replicon of the present invention comprises the 3'UTR of alphavirus, and the 3'UTR is active and can ensure the stability of RNA and the effective replication of the target gene.
  • the cis-RNA construct described in the present invention contains a 3'poly(A) sequence.
  • the replication complex and the poly(A) tail interact through the poly(A) binding protein (PABP), so at least 11-12 residues of the poly(A) tail are required to efficiently generate negative strand RNA.
  • Negative-strand synthesis replicates with comparable efficiency on RNA templates containing 25 or 34 adenylate residues in the poly(A) tail; however, when the tail size is reduced to 20 residues, the efficiency of negative-strand synthesis decreases significantly (>70 %). When the size of the tail was reduced to 10 residues, the efficiency dropped again. Deletion of the 3'poly(A) tail severely inhibited negative-strand RNA production.
  • RNA synthesis of a template RNA without a 3' poly(A) tail is only 4% that of a template RNA containing 25 3' terminal adenylate residues (Hardy and Rice, 2005, J Virol, vol. 79 , pp. 4630-4639).
  • poly(A) sequences also affect the function of RNA stability and protein translation in transfected eukaryotic cells. It has been reported in the literature that a 3' poly(A) sequence of approximately 120 A nucleotides has beneficial effects on RNA levels in transfected eukaryotic cells as well as on protein levels translated from open reading frames upstream of the 3' poly(A) sequence (Nadine Bangel-Ruland, J Gene Med, 2013, Vol. 15, pp. 414-426). Furthermore, the use of segmented poly(A), such as poly(A)2 ⁇ 60_G or poly(A)2 ⁇ 60_T, can significantly reduce plasmid recombination in E. coli without any negative effects on mRNA half-life and protein expression influences.
  • This poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosine nucleotides separated by spacer elements of varying lengths open.
  • segmented poly(A) tails Compared to traditional homogeneous poly(A) tails, segmented poly(A) tails have a higher potential for recombinant plasmids and the performance of the resulting mRNAs (half-life and translational efficiency) (Zeljka Trepotec et al.; RNA, 2019 25, pp. 507-518).
  • the 3' poly(A) sequence comprises or consists essentially of or consists of at least 11, preferably at least 25, preferably at least 34, preferably at least 69, preferably at least 100, and preferably at most 500, preferably at most 400, preferably at most 300, preferably at most 200, especially at most 150 A nucleotides, especially about 120 A nucleotides.
  • At least 50% of the poly(A) sequence are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotide (guanylic acid), C nucleotide (cytidylic acid), in order to form a segmented poly(A) tail.
  • 3-letter codons for mRNA means that 64 possible codons encode 20 amino acids and a translation stop signal.
  • the degeneracy of the genetic code means that in the genetic code of several triplets encoding the same amino acid, the first and second bases are mostly the same, but the third position is different. There are multiple codons for the same amino acid, and since tRNAs randomly recognize codons within the A site of the ribosome, the codons can be defined as optimal according to the efficiency of selecting suitable cognate tRNAs from the cytoplasmic pool of tRNAs. Codon frequencies are used differently across the biological world, and codon bias correlates with tRNA levels in Escherichia coli, Saccharomyces cerevisiae, C.
  • a major strategy for codon optimization is to increase translation elongation by overcoming limitations associated with species-specific differences in codon utilization and tRNA abundance. Utilize favored codons, avoid rare codons with low utilization rate, simplify the secondary structure of mRNA after gene transcription, remove motifs that are not conducive to high-efficiency expression, add motifs that are conducive to high-efficiency expression, and adjust GC content and other methods to redesign the genetic code. In addition to codon preference, the combined efficiency of codon usage also needs to be considered.
  • the codons of the open reading frame contained by the RNA molecule may be different from the corresponding codons from which the open reading frame originates, and may be optimized for different species, referred to as "codon optimization".
  • codon optimization can be performed on the open reading frames encoding the replicase of the present invention and the expressed exogenous protein, respectively.
  • conserved sequence element refers to a nucleotide sequence found in alphavirus RNA. These sequence elements are referred to as “conserved” because orthologs are present in the genomes of different alphaviruses, and orthologous CSEs of different alphaviruses preferably share a high percentage of sequence and/or similar secondary or tertiary structure.
  • conserved sequence elements CSEs
  • the replicons according to the invention comprise one or more conserved sequence elements (CSEs) (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp.
  • the first 44 nucleotides of the positive-strand 5'UTR form a conserved stem-loop structure, and its complementary sequence is located at the 3' end of the negative-strand, which initiates the synthesis of positive-strand RNA using the negative-strand RNA as a template.
  • CSE2 is a conserved stem-loop structure formed by the front 51 nucleotides of the nsP1 protein coding sequence.
  • CSE3 is a 24 nucleotides located at the terminus of nsP4 protein and is a promoter for transcribing subgenomic mRNA.
  • CSE4 is 19 nucleotides at the 3' end of RNA, and is a promoter for synthesizing negative-strand RNA using positive-strand RNA as a template (Hardy and Rice, J. Virol., 2005, Vol. 79, pp. 4630-4639).
  • the alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence are capable of directing the replication of the RNA replicon according to the invention in the presence of a replicase.
  • these recognition sequences direct RNA replicon replication in the presence of a replicase.
  • alphavirus conserved sequence elements are recognition sequences that direct replication of the RNA replicon in the presence of a replicase.
  • the replicon recognition sequences are generally referred to as CSEs 1 and 2.
  • the replicase and conserved sequence elements may be from different alphavirus genera.
  • the CSE-1, CSE-2, CSE-3 and CSE-4 described herein include variants thereof that retain their ability to initiate replication.
  • SGP subgenomic promoter
  • the term "subgenomic promoter” or “SGP” refers to a nucleic acid sequence 5' upstream of a nucleic acid sequence (eg, a coding sequence), which controls the nucleotide sequence by providing a recognition and binding site for replicase. Transcribe.
  • the RNA replicon according to the invention comprises a subgenomic promoter.
  • the SGPs described herein include variants thereof that retain their ability to initiate transcription of downstream genes.
  • the replicase encoded by the present replicon is an alphavirus replicase capable of recognizing subgenomic promoters.
  • the subgenomic promoter according to the present invention is a promoter of an alphavirus structural protein, including CSE3, which is highly conserved among alphaviruses, that is, at least 24 nucleotides at the end of the non-structural protein nsP4 (taking Sindbis virus as an example) ), which can efficiently initiate transcription of subgenomic RNAs.
  • the SGP is the same as or overlaps with or comprises CSE3.
  • the cis-replicon RNA comprises at least one nucleic acid sequence not derived from an alphavirus.
  • the term “heterologous” or “foreign” refers to the situation where the nucleic acid sequence is not homologous to the alphavirus genomic nucleic acid sequence.
  • the heterologous nucleic acid sequence is under the control of an alphavirus subgenomic promoter. More preferably, the heterologous nucleic acid sequence is located downstream of a subgenomic promoter.
  • Alphavirus subgenomic promoters are very efficient and therefore suitable for high-level heterologous gene expression (Kenneth Lundstrom, Molecules, 2018, Vol. 23, p. 3310).
  • the "target protein” referred to herein is the protein encoded by the target gene coding unit.
  • the RNA replicon according to the invention comprises an open reading frame encoding a peptide or protein of interest.
  • the protein of interest is encoded by a heterologous nucleic acid sequence.
  • the gene of interest is under the control of a subgenomic promoter. More preferably, the gene of interest is located downstream of a subgenomic promoter.
  • internal ribosome entry site refers to a sequence capable of recruiting ribosomes to initiate translation of downstream genes. When present between two coding sequences, an internal ribosome can mediate translation. start.
  • the replicons provided by the present invention can encode single or multiple proteins. Multiple proteins can be encoded as fusion-expressed proteins or separately expressed proteins. For separately expressed proteins, one or more of these proteins can be provided with an upstream IRES, or the open reading frames of multiple proteins can be constructed in the replicon, keeping each open reading frame under the control of a subgenomic promoter . Alternatively, autocatalytic proteases (eg, the foot-and-mouth disease virus 2A protein) can be added to these proteins for isolation (Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562).
  • autocatalytic proteases eg, the foot-and-mouth disease virus 2A protein
  • the term "2A peptide" (2A peptide, or 2A self-cleaving peptide) is a segment of about 18-22 amino acids in length, which can induce the self-cleavage of recombinant proteins containing 2A peptide in cells.
  • the nucleic acid segment encoding the 2A peptide is present between the gene encoding unit of interest and the RNA polymerase encoding unit.
  • HDV-like ribozyme (HDV-like ribozyme), or Hepatitis Delta Virus (HDV) ribozyme (HDVr) refers to a class of ribozymes with self-cleaving function.
  • HDV ribozymes catalyze the cleavage of the phosphodiester bond between the substrate nucleotide or oligonucleotide and the 5'-hydroxyl group of the ribozyme, thereby cleaving the polynucleotide chain where it is located from the cleavage site.
  • the HDV-like ribozyme is present between the coding unit of the gene of interest and the coding unit of the RNA polymerase.
  • the proteins of interest encoded by the replicons of the present invention may also be immunogenic compounds or epitopes or cytokines for therapeutic or protective purposes.
  • the term "antigen” or “immunogen” refers to a substance that elicits the production of antibodies, and is any substance that induces an immune response.
  • Antigens are preferably derived from the products of naturally occurring antigens, such as from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, as well as tumor antigens.
  • cytokine refers to the synthesis of immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation , secreted a class of small molecule proteins with a wide range of biological activities, such as interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc.
  • immune cells such as monocytes, macrophages, T cells, B cells, NK cells, etc.
  • non-immune cells endothelial cells, epidermal cells, fibroblasts, etc.
  • a biological entity eg, replicase, virus, subgenomic promoter SGP
  • a biological entity eg, replicase, virus, subgenomic promoter SGP
  • it is meant to include not only its wild-type form, but also variant forms that retain the function of the biological entity.
  • the system of the present invention does not contain any alphavirus structural proteins, such as: capsid protein C, membrane glycoproteins E1, E2, E3 and 6K proteins and the like.
  • the host cell does not produce viral particles, eg, progeny viral particles.
  • the replicon cannot drive the replication of the replicase itself, but can only drive the replication of the RNA of the target gene.
  • the present invention provides DNA encoding the replicons provided by the present invention.
  • the DNA provided by the present invention is a plasmid.
  • plasmid refers to DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeast, and actinomycetes, which have the ability to replicate autonomously and express the genetic information they carry, which are closed circular of double-stranded DNA molecules.
  • RNA molecule according to the invention can be obtained by in vitro transcription.
  • IVT-RNA in vitro transcribed RNA
  • the 5' cap can be obtained by adding a cap-analog to an in vitro transcription reaction, and the poly(A) tail is encoded by a poly(dT) sequence on the DNA template.
  • capping and poly(A) tailing can also be achieved by an enzymatic reaction after the transcript is obtained.
  • In vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.
  • the immunostimulatory properties of mRNA can be enhanced by the addition of adjuvants to enhance the efficacy of mRNA vaccines.
  • adjuvants include traditional adjuvants as well as novel approaches that exploit the inherent immunogenicity of mRNA or its encoded immunomodulatory proteins.
  • the compositions of the present invention may contain one or more adjuvants.
  • Adjuvants can be added to vaccines to stimulate the immune system's response.
  • exemplary adjuvants include, but are not limited to, the following: biological adjuvants such as Mycobacterium (M. tuberculosis, BCG), Corynebacterium pumilus, B.
  • pertussis Gram-negative bacilli endotoxin, Interleukin-1, Interleukin-2 , interleukin-12, interferon- ⁇ , etc.
  • inorganic adjuvants such as aluminum hydroxide, alum, aluminum phosphate, etc.
  • synthetic adjuvants such as double-chain polyinosinic acid, cytidine acid, double-chain polyadenosine acid, etc.
  • oily agents such as peanut oil emulsifying adjuvant, mineral oil, vegetable oil, lanolin, etc.
  • immune activating proteins such as CD70, CD40L and TLR4, etc.
  • RNA must cross the cell membrane to reach the cytoplasm, and the negative potential across the cell membrane creates a strong barrier for highly negatively charged RNA molecules. In addition to cell membrane barriers, RNA also faces the problem of degradation of extracellular RNA that is abundant in skin and blood. Naked RNA molecules are rapidly degraded in the organism, do not accumulate in the target tissue after systemic administration, and even if they reach the target tissue, they cannot penetrate the target cells, so a delivery system is required.
  • the RNA pharmaceutical compositions of the present invention are contained in at least one cation-containing complex.
  • the pharmaceutical composition according to the present invention can be encapsulated in cationic polymers (Polyplexes, PP), lipoplex (lipoplex, LP), lipid nanoparticle (LNP), cationic nanoemulsion (cationic nanoemulsion, CNE).
  • polymers such as polyethyleneimine (PEI)
  • PEI polyethyleneimine
  • PP cationic polymers
  • PEIs are a group of linear or branched polyethyleneimine polymers with strong affinity for nucleic acids and a proton sponge effect that promotes endosomal escape and protects nucleic acids from enzymatic degradation (Thomas Démoulins et al., Nanomedicine, vol. 2016, Vol. 12, pp. 711-722).
  • negatively charged mRNA and positively charged cationic liposomes are electrostatically aggregated to form a multilamellar vesicle complex, ie, a liposome complex (LP), in which the complex contains
  • a liposome complex LP
  • the encapsulated mRNA is not easily degraded by RNase and can be successfully delivered into the cell.
  • DOTAP (1,2-diallyl-3-trimethylammonium propane
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOTMA N-[1-(2, 3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • Cationic lipids are commonly used in mRNA vaccination because they can not only encapsulate mRNA but also act as immunogens themselves and are considered adjuvants to vaccines (Yasmin Granot et al., Seminars in Immunology, 2017, vol. 34, p. 68-77).
  • the mRNA is present in liposome nanoparticles (LNPs), the liposomes generally comprising the following: (1) ionizable or cationic lipid or polymeric material with tertiary Amines or quaternary amines, which can promote self-assembly into virus-sized ( ⁇ 100nm) particles, which are used to encapsulate polyanionic mRNA and allow endosomes to release mRNA into the cytoplasm; (2) auxiliary phospholipids generally use zwitterionic lipids, such as DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) or POPE (1-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine).
  • LNPs liposome nanoparticles
  • Auxiliary phospholipids serve as the backbone of liposomes, similar to lipids in cell membranes, assist in lipid bilayer formation and disruption, and facilitate endosomal escape.
  • Some phospholipids have polymorphic characteristics, which can be transformed from lamellar to hexagonal phase when entering endosomes, which promotes the release of mRNA from liposomes; (3) cholesterol, to stabilize the lipid bilayer of LNP and improve particle stability, Enhances the stability of particles and promotes membrane fusion; (4)
  • Lipid-linked polyethylene glycol (PEG) enables nanoparticles to have a hydrated layer, improves colloidal stability, can reduce non-specific interactions with serum proteins, and Absorption through the reticuloendothelial system and prolongation of the half-life of formulations (Norbert Pardi et al., 2018, Nat Rev Drug Discov., vol. 17, pp. 261-279; Piotr S. Kowalski et al., Mol Ther, 2019, pp. 27
  • the mRNA is present in a compound consisting of squalene, a nonionic sorbitan ester surfactant, sorbitan trioleate (Span 85) or sorbitan monostearate (Span 60), cationic lipid DOTAP, etc. in the cationic nanoemulsion (CNE), which can be used for the preparation of vaccines (Jesse H. Erasmus et al., Mol Ther., 2018, Vol. 26, pp. 2507-2522 ).
  • the present invention provides a method of producing a protein in a cell comprising the steps of obtaining an RNA construct for expressing an exogenous protein comprising a 5' cap for driving translation of the replicase and a 3' cap for enhancing mRNA stability 'poly(A), comprising the open reading frame encoding the foreign protein, and transfecting the RNA construct into the cell.
  • RNA can be used in the form of pharmaceutical compositions, or as naked RNA, eg, for electroporation or lipofection, etc.
  • the present invention provides a method of producing a protein in an organism comprising the steps of: obtaining an RNA construct for expressing a foreign protein comprising a 5' cap for driving translation of the replicase and a mRNA stability enhancing 3'poly(A), comprising the open reading frame encoding the foreign protein, and administering the RNA construct to the subject.
  • the medicaments of the present invention are useful in prophylactic as well as therapeutic methods of treating a subject.
  • the terms “immunization” or “vaccination” generally refer to the process of treating a subject for therapeutic or prophylactic reasons.
  • Treatment, particularly prophylactic treatment is preferably or is intended to include treatment aimed at inducing or enhancing an immune response in a subject against one or more antigens.
  • an immune response can be induced or enhanced by RNA.
  • the present invention provides prophylactic treatment, which preferably is or includes vaccination of the subject.
  • the present invention is particularly suitable for vaccination, wherein the replicon encodes a pharmaceutically active peptide or protein as the protein of interest, which is an immunologically active compound or antigen.
  • the medicaments of the present invention can be administered to a subject, eg, for the treatment of the subject, including vaccination of the subject.
  • subject relates to vertebrates, particularly mammals including humans.
  • the medicament according to the present invention may be administered to a subject by any suitable route.
  • the drug can be administered systemically, and administration can be accomplished in a variety of ways, such as intravenous, subcutaneous, intradermal, intramuscular, or inhalation.
  • Alternatives to administration to muscle tissue or skin include, but are not limited to, intradermal, intranasal, intraocular, intraperitoneal, intravenous, interstitial, buccal, transdermal or sublingual administration, and the like.
  • CSE1 (Semriki Forest virus SFV from Alphavirus):
  • CSE2 (Semriki Forest virus SFV from Alphavirus):
  • CSE4 (Semriki Forest virus SFV from Alphavirus):
  • 3'UTR (Semriki Forest virus SFV from Alphavirus):
  • Replicase (derived from Alphavirus genus Semliki Forest virus SFV), its amino acid sequence is as follows:
  • DNA sequences of each segment required for constructing the replicon in the present invention are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and inserted into the mammalian expression vector pVAX1 (purchased from Invitrogen) by homologous recombination to construct pVAX1 -cis-SFV-neo cis-replicon plasmid (the structure is shown in Figure 2A, where the target gene position is the multiple cloning site).
  • the pVAX1-cis-SFV-neo obtained after construction contains the following sequence:
  • MCS Multiple Cloning Site
  • the sub-vector pVAX1-trans-SFV and the cis-replicon vector pVAX1-cis-SFV-neo of the present invention have multiple cloning site regions at the 3' end of the subgenomic promoter, respectively constructed into pVAX1-EGFP/Luc, pVAX1-cis-SFV -EGFP/Luc, pVAX1-trans-SFV-EGFP/Luc, pVAX1-cis-SFV-neo-EGFP/Luc.
  • RNA obtained by in vitro transcription was transfected with RNA liposomes using the transfection reagent Lipofectamine Messenger MAX (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions.
  • RNA replicons To evaluate the level of exogenous protein (green fluorescent protein) expressed by different RNA replicons, pVAX1-EGFP (without replicon, as a control), pVAX1-cis-SFV-EGFP, pVAX1 -The RNA obtained by in vitro transcription of trans-SFV-EGFP and pVAX1-cis-SFV-neo-EGFP were respectively transfected into hamster kidney fibroblasts (BHK-21) in 96-well plates in equal amounts (Cell Bank of Type Culture Collection, Chinese Academy of Sciences) purchased), after 48 hours, the green fluorescence intensity was observed under the microscope and the image was recorded.
  • BHK-21 hamster kidney fibroblasts
  • RNA replicons To evaluate the level of exogenous protein (luciferase) expressed by different RNA replicons, pVAX1-Luc (without replicon, as a control), pVAX1-cis-SFV-Luc, pVAX1 -Trans-SFV-Luc, pVAX1-cis-SFV-neo-Luc in vitro transcribed RNAs were transfected into BHK-21 cells in 96-well plates in equal amounts, and fireflies were detected by Bright-Glo (Promega, Madison, USA) detection reagent Luciferase, bioluminescence was measured using an EnSpire multi-plate reader (PerkinElmer, MA, USA). Data are expressed in relative luciferase units [RLU], minus background signal (reads from untransfected cells).
  • RLU relative luciferase units
  • the RNAs obtained by in vitro transcription of pVAX1-RBD and pVAX1-cis-SFV-neo-RBD were transfected into BHK-21 cells in 24-well plates in equal amounts, and the cells were collected 48 hours later.
  • the expression of the new coronavirus RBD was detected and analyzed by Western Blot, and the antibody used the new coronavirus RBD antibody produced by Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • a pseudotyped lentiviral luciferase reporter system was used to evaluate the neutralization ability of vaccinated animal sera against SARS-CoV-2.
  • the SARS-CoV-2 pseudotyped virus is composed of a lentiviral packaging plasmid pCMV delta R8.2, an expression plasmid pCDH-CMV-luc encoding a luciferase reporter gene, and a plasmid pcDNA3.1- Spike was produced by co-transfection of 293T cells at a treatment ratio of 1:1.5:1 for transfection.
  • the virus-containing medium was collected by centrifugation, filtered through a 0.45 ⁇ m membrane, and then stored at -80°C until use.
  • mice aged 6-8 weeks were purchased from Henan Provincial Laboratory Animal Center and raised in an SPF animal laboratory, and all experiments were approved by the Life Science Ethics Review Committee of Zhengzhou University.
  • the liposome-encapsulated pVAX1-RBD (control) and the RNA obtained by in vitro transcription of pVAX1-cis-SFV-neo-RBD (experimental group) were injected into mice intramuscularly, and were used on the 14th and 28th days after the primary immunization. doses of RNA for enhanced immunity. Serum samples were collected 35 days after the primary immunization, heat inactivated at 56°C for 30 min, and stored at -80°C.
  • the control group of immunized mice was injected with PBS, or RNA obtained by in vitro transcription of liposome-encapsulated pVAX1 plasmid. There were 5 mice in both the experimental group and the control group, and the experimental results were expressed as mean ⁇ SEM. The comparison between groups was performed using the Mann-Whitney U test, and statistical analysis was performed by GraphPad Prism (version 8.4) software.
  • the expression vector pVAX1-EGFP, the traditional cis-replicon vector pVAX1-cis-SFV-EGFP, the trans-replicon vector pVAX1-trans-SFV-EGFP (containing the corresponding replicase), the cis-replicon vector pVAX1 of the present invention - cis-SFV-neo-EGFP in vitro transcribed RNA was transfected into BHK-21 cells in a 96-well plate for 48 hours by Lipofectamine MessengerMAX TM and other substances. ability.
  • Fig. 4 The results are shown in Fig. 4, the traditional cis-replicon (Fig. 4B), the trans-replicon (Fig. 4C) and the cis-replicon of the present invention (Fig. 4D) express green fluorescent protein far better than the traditional expression vector
  • Fig. 4A The resulting RNA was transcribed (FIG. 4A).
  • RNAs transcribed from the traditional cis-replicon, the trans-replicon (including the matching replicase), and the cis-replicon vector of the present invention are far superior to that obtained from the transcription of the traditional expression vector.
  • the RNAs transcribed from the cis-replicon and the trans-replicon (containing a matching replicase) vector of the present invention have the same ability to express luciferase, and are significantly better than the traditional cis-replicon (P ⁇ 0.01).
  • RNA obtained by in vitro transcription of pVAX1-SFV-RBD and pVAX1-cis-SFV-neo-RBD was encapsulated in liposomes to immunize mice, and enhanced with an equal dose of RNA on the 14th and 28th days after the primary immunization
  • serum samples were collected 35 days after the primary immunization, and the anti-RBD IgG titer in the serum was determined by ELISA (the coating antigen was recombinant RBD protein).
  • the control group was immunized with PBS or pVAX1 mRNA transcribed in vitro.
  • RNA obtained by in vitro transcription of pVAX1-SFV-RBD and pVAX1-RBD was encapsulated in liposomes and immunized mice, and the same dose of RNA was used for enhanced immunization on the 14th and 28th days after the initial immunization. Serum samples were collected every day, and serum neutralization titers were determined by pseudovirus neutralization assay. The control group was immunized with PBS or pVAX1 RNA transcribed in vitro. The experimental results were compared with the mean ⁇ SEM of three independent experiments, and the Mann-Whitney U test was used for comparison between groups. In neutralization experiments, if undiluted serum did not show any neutralizing effect, its titer was assigned as 1 for statistical analysis.

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Abstract

提供了一种用于高效表达目的蛋白的新型顺式复制子构建体，其包含RNA聚合酶编码单元和目的蛋白编码单元。该复制子构建体能够驱动目的蛋白编码单元的复制，并减少或避免RNA聚合酶的复制和表达，从而有效提高目的蛋白的表达，减少非目的蛋白的表达。

Description

顺式复制子构建体
本申请要求于2021年02月03日提交中国国家知识产权局、申请号为202110151575.3的中国专利申请和2021年10月09日提交中国国家知识产权局、申请号为202111178173.9的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，特别是涉及一种高效表达目的蛋白的顺式复制子RNA构建体。
背景技术
为了预防和治疗目的而引入一种或更多种外源多肽/蛋白在生物医学治疗中已经越来越广泛，可以通过多种技术方法将核酸分子(DNA或者mRNA)向靶细胞或生物体实现递送。近年来，mRNA分子的递送受到越来越多的关注，体外转录(IVT)mRNA最近已成为一种潜在的传递遗传信息的新药，成为人们关注的焦点。与DNA的相比，mRNA的使用具有一些突出的优点。第一，较高的安全性：由于mRNA是一个非感染性、非整合的平台，因此不存在感染或插入突变的潜在风险。此外，mRNA被正常的细胞过程降解，其体内半衰期可以通过使用各种修饰和传递方法来调节。而且，mRNA固有的免疫原性可以下调以进一步提高安全性。第二，较高的效率：各种修饰使mRNA更加稳定，具有高度的可翻译性，体内给药可通过将mRNA形成有效的载体分子，使其在细胞质中快速摄取和表达。第三，快速生产时效：制备mRNA具有快速、廉价和可大量扩增的潜力，这主要得益于体外转录反应的高产量， 而这对于传染病的大流行情况以及针对患者特定的癌症相关或突变抗原的癌症疫苗制备的可能性非常重要(Norbert Pardi等人，2018年，Nat Rev Drug Discov.，第17卷，261-279页)。
正链RNA病毒的基因组RNA甲病毒(alphavirus)属于披膜病毒科(togavirus family)，含有11-12kb的非分段单链RNA基因组，5’端为0型cap(N7mGppp)，3’端为poly(A)尾，这些特征使得甲病毒基因组相当于一种mRNA，在进入宿主细胞质后立即翻译，且能够控制启动病毒复制所需的所有病毒蛋白的产生。甲病毒家族包括了约30个成员，主要分为辛德毕斯病毒(Sindbis virus,SINV)、塞姆利基森林病毒(Semliki forest virus,SFV)、委内瑞拉马脑炎病毒(Venezuelan equine encephalomyelitis virus,VEEV)和基孔肯雅病毒(Chikungunya virus，CHIKV)等(Jennifer L.Hyde等，Virus Research，2015，第206卷，第99-107页)。甲病毒的基因组共有2个开放阅读框，5’末端的前2/3部分编码4种非结构蛋白(nsP1-nsP4)，称为非结构区。靠近3’末端的后1/3称为结构区，编码由亚基因组mRNA翻译的结构蛋白，包括衣壳蛋白C、膜糖蛋白E1、E2、E3以及6K蛋白等。甲病毒在感染细胞过程中5’末端的非结构蛋白首先翻译，产生RNA复制和转录所需要的复制酶。在复制酶的作用下，启动亚基因组mRNA的转录，随后经过翻译，在病毒和宿主编码蛋白酶的联合作用下裂解为衣壳蛋白和膜糖蛋白，这些结构蛋白与病毒基因组RNA装配成核衣壳后以出芽方式释放。亚基因组RNA既不作为RNA合成的模板，又不被包装入病毒颗粒，且在病毒复制时结构基因可以达到非常高的拷贝数，因此可以利用亚基因组的启动子启动外源基因的高效表达。
用于体内递送的mRNA既可以是编码目的蛋白的非扩增mRNA分子(约 2kb)，也可以是较大的、基于病毒复制子的自扩增mRNA分子(>10000bp)(Piotr S.Kowalski等，Mol Ther.，2019年，第27卷，第710-728页；Giulietta Maruggi等，Mol Ther.，2019年，第27卷，第757-772页)。当前最常用的自扩增mRNA(SAM)疫苗是基于甲型病毒基因组，其中编码RNA复制的基因是完整的，但是编码结构蛋白的基因被目的蛋白的基因所取代。病毒基因组RNA的第一轮翻译产物组装成复制酶，该复制酶复制出与基因组互补的负链(cRNA)，作为合成其他mRNA分子的模板。由于抗原编码的RNA在细胞内复制，SAM平台能够从极小剂量的疫苗中大量生产抗原(Norbert Pardi等，2018年，Nat Rev Drug Discov.，第17卷，第261-279页)。
甲病毒复制子具有以下几个优点：第一，自扩增mRNA可提高外源蛋白的表达量，激活体内更强的免疫反应。这是因为，SAM转染的细胞中产生外源蛋白是基于病毒复制的原理，在RNA复制酶的作用下，RNA复制子在细胞质中大量地复制，高效地表达外源基因，为持续的B细胞刺激和抗体产生提供了理想的构象。此外，宿主细胞通过多种模式识别受体识别转录的双链RNA中间体，如RNA依赖性蛋白激酶(PKR)、Toll样受体(TLR)19和2’-5’-寡腺苷酸合成酶(OAS)，从而会导致局部炎症，从而提供额外的免疫刺激，因此可以诱导较高的体液免疫和细胞免疫应答(Khromykh A等，2000年，Curr Opin Mol Ther，第2卷，第555-569页；Annette B.Vogel等，Mol Ther.，2018年，第26卷，第446-455页)。第二，自扩增mRNA安全性好。甲病毒复制子不进入细胞核，仅在细胞质中进行短暂的复制转录和目的蛋白的表达，之后便在dsRNA的激活的抗病毒信号作用下细胞发生凋亡而被清除。因此和传统DNA疫苗相比，其不会被整合进宿主基因组而引发致癌风险，也不会长期在 机体内存在(Annette B.Vogel等，Mol Ther.，2018年，第26卷，第446-455页)。第三，应用范围广。甲病毒复制子可以高效地应用到多种病原体的疫苗研究中，如牛病毒性腹泻病毒(BVDV)、巨细胞病毒(CMV)、猪瘟病毒(CSFV)、登革热(Dengue)、埃博拉病毒(Ebola)、、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、戊型肝炎(HeV)、艾滋病毒(HIV)、人乳头瘤病毒(HPV)、单纯疱疹病毒(HSV)、传染性法氏囊病病毒(IBDV)、流感病毒(Influenza)、传染性鲑鱼贫血病毒(ISAV)、流行性乙型脑炎病毒(JEV)、拉沙病毒(Lassa)、跳跃病病毒(LIV)、马尔堡病毒(MBGV)、麻疹(Measles)、默里谷脑炎(MVE)、尼帕病毒(NiV)、诺瓦克样病毒(NLV)、狂犬病毒(RV)、呼吸道合胞病毒(RSV)、裂谷热病毒(RVFV)、非典型肺炎病毒(SARS-CoV)、痘苗病毒(vaccinia virus)、朊病毒(Prion)、炭疽病(B.anthracis)、布鲁氏菌(B.abortus)、肉毒梭菌(C.botulinum)、疟疾(Malaria)、结核分枝杆菌(M.tuberculosis)、恶性疟原虫(P.falciparum)、葡萄球菌(Staphylococcus)等。(Kenneth Lundstrom，Viruses，2014年,第6卷，第2392-2415页；Kenneth Lundstrom，Vaccines，2019年，第7卷，第29页；Cuiling Zhang等，Front Immunol.，2019年，第10卷，第594页)。
此外，一个安全、有效和可控制的基因传递系统是基因递送的关键步骤，目前主要有病毒载体和非病毒载体。非病毒载体可以承载更高的遗传负荷，更高的安全性并诱导更低的免疫反应。用于递送mRNA的非病毒载体有阳离子聚合物(Polyplexes,PP)、脂质体复合物(lipoplex，LP)、脂质体纳米粒(lipid nanoparticle，LNP)和阳离子纳米乳(cationic nanoemulsion，CNE)等，其中以脂质载体的应用最为广泛(S Guan&J Rosenecker，Gene Therapy，2017年， 第24卷，第133-143页；Piotr S.Kowalski等，Mol Ther.，2019年，第27卷；第710-728页；Kristie Bloom等，Gene Ther.，2020年，第1-13页)。
因此，需要开发一种用于高效表达目的蛋白的复制子RNA构建体的改善方法，以实现最大限度的减少非目标蛋白的表达，大幅度扩增目标RNA而增加目标蛋白的表达的目的。
发明内容
本发明的目的是提供一种高效表达目的蛋白的顺式复制子RNA构建体，以解决上述现有技术存在的问题，通过对甲病毒基因组的改造构建RNA复制子，只驱动目的基因的高效复制和表达，实现了最大限度的减少非目的蛋白的表达，而增加目标蛋白的表达的目的。
为实现上述目的，本发明提供了如下方案：
本发明提供一种高效表达目的蛋白的顺式复制子RNA构建体，所述构建体包括：
包含编码复制酶的一种或多种核苷酸序列的5’UTR；
所述5’UTR包含5’帽；
指导RNA复制子复制的5’端识别序列和亚基因组启动子；
异源核苷酸序列：其受所述亚基因组启动子控制；
多克隆位点；以及
保证RNA的稳定性以及目的基因有效复制的3’UTR；
所述3’UTR包含指导RNA复制子复制的3’端识别序列和3’polyA。
在一些实施方案中，本发明提供一种高效表达目的蛋白的顺式复制子RNA构建体，其特征在于，按5’-3’方向，所述构建体包括：
(1)5’UTR序列，包含5’帽结构；
(2)编码复制酶的一种或多种核苷酸序列；
(3)指导RNA复制子复制的5’端识别序列和亚基因组启动子SGP；
(4)多克隆位点；
(5)目的基因或异源核苷酸序列：其受所述亚基因组启动子控制；
(6)指导RNA复制子复制的3’端识别序列；
(7)保证RNA的稳定性以及目的基因有效复制的3’UTR和3’polyA。
优选的是，所述复制酶源自甲病毒。
优选的是，所述甲病毒为塞姆利基森林病毒、委内瑞拉马脑炎病毒、辛德毕斯病毒、基孔肯雅病毒。
在一些实施方案中，5’端识别序列和3’端识别序列在所述复制酶的存在下指导所述RNA复制子的复制；所述5’端识别序列和3’端识别序列包含CSE1和CSE2。
在一些实施方案中，甲病毒的5’端识别序列和3’端识别序列对于所述复制酶源优选的是，5’UTR为真核生物的5’UTR；
5’帽结构为天然5’-帽或5’-帽类似物；
3’UTR为病毒mRNA的3’UTR。
在一些实施方案中，编码复制酶的核苷酸序列包含开放阅读框；编码复制酶的开放阅读框包含RNA复制所需的非结构蛋白的编码区。
在一些实施方案中，用于表达甲病毒复制酶的RNA构建体和/或所述RNA复制子不包含编码完整甲病毒结构蛋白的开放阅读框。
在一些实施方案中，所述异源核苷酸序列编码目的蛋白，编码所述目的蛋 白的开放阅读框的表达受到亚基因组启动子的控制。
在一些实施方案中，编码目的蛋白的开放阅读框对于所述复制酶源自的甲病毒是非天然的；
所述亚基因组启动子对于所述复制酶源自的甲病毒是天然的。
在一些实施方案中，本发明提供包含所述顺式复制子构建体的重组病毒载体、质粒DNA、线性DNA、体外转录RNA或提取的细胞内转录RNA。
本发明还提供包含所述的顺式复制子RNA构建体的DNA重组载体，所述重组载体不能驱动复制酶的复制，并且高效驱动目的基因的复制和表达。
本发明还提供编码所述的顺式复制子RNA构建体的核苷酸序列。
本发明还提供一种利用生物体生产蛋白质的方法，包括获取用于表达目的蛋白的所述构建体或所述的重组载体，并将所述构建体或所述重组载体转染生物体中的步骤。
进一步地。所述生物体包括脊椎动物和细胞。
本发明还提供以下技术方案：
在第一个方面，本发明提供一种作为顺式复制子的表达构建体，其包含RNA聚合酶编码单元和目的蛋白编码单元，其中
所述RNA聚合酶编码单元包含编码RNA聚合酶的核酸区段，所述目的蛋白编码单元包含待插入目的蛋白编码序列的接受位点(如多克隆位点)和/或编码目的蛋白的核酸区段，
所述构建体能够在目的细胞中表达所述RNA聚合酶，并依赖所述RNA聚合酶进行复制，从而提供进一步表达目的蛋白的模板；优选地，所述复制对所述目的蛋白单元的效率高于对所述RNA聚合酶编码单元；更优选地，所述 复制为仅复制所述目的蛋白编码单元。
在一些实施方案中，所述的构建体是DNA或RNA，优选为RNA，包括单链RNA或双链RNA，例如是单链RNA，特别地所述单链RNA是正链(+)RNA。
在一些实施方案中，所述的构建体是线性的或环状的；
特别地所述构建体是线性RNA，在其5’端存在驱动下游编码序列翻译的5’帽结构或IRES序列；或者所述构建体是环状RNA，在所述RNA聚合酶编码单元上游存在驱动所述RNA聚合酶编码单元翻译的IRES序列。
在一些实施方案中，所述目的蛋白编码单元上游和下游都存在驱动依赖于所述RNA聚合酶之复制的复制启动元件，从而实现所述目的蛋白编码单元的复制；优选地所述RNA聚合酶编码单元不在所述上游复制启动元件和下游复制启动元件之间。
在一些实施方案中，所述上游复制启动元件包含CSE1和/或CSE3序列，优选地包含CSE1和CSE3序列，更优选地包含CSE1、CSE2和CSE3序列；和/或所述下游复制启动元件包含CSE4序列。
在一些实施方案中，所述目的蛋白编码单元位于所述RNA聚合酶编码单元的下游或上游。
在一些实施方案中，所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，目的蛋白和RNA聚合酶作为融合蛋白表达，所述融合蛋白中目的蛋白和RNA聚合酶之间存在连接序列，所述连接序列包含编码蛋白酶识别位点的区段，在所述融合蛋白经蛋白酶识别并切割后提供所述RNA聚合酶，优选地，所述蛋白酶识别位点为2A肽序列。所述编码蛋白酶识别位点的区段可 以位于复制启动元件(例如CSE4)的上游或下游。
在一些实施方案中，所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，在所述目的蛋白编码单元和RNA聚合酶编码单元之间存在驱动所述RNA聚合酶编码单元翻译的IRES序列。所述IRES序列可以位于复制启动元件(例如CSE4)的上游或下游。
在一些实施方案中，所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，在所述目的蛋白编码单元和RNA聚合酶编码单元之间存在自裂解HDV样核酶(HDV-like Ribozymes)。所述自裂解HDV样核酶可以位于复制启动元件(例如CSE4)的上游或下游。
在一些实施方案中，所述目的蛋白编码单元位于所述RNA聚合酶编码单元的下游，其中所述构建体按5’-3’方向包含：
驱动下游编码序列翻译的5’帽结构或IRES序列、所述RNA聚合酶编码单元、所述上游复制启动元件、所述目的蛋白编码单元和所述下游复制启动元件。
在一些实施方案中，所述构建体按5’-3’方向包含：
(1)5’UTR序列，包含5’帽结构；
(2)编码RNA聚合酶的一种或多种核苷酸序列；
(3)指导RNA复制子复制的5’端识别序列和亚基因组启动子SGP；
(4)多克隆位点和/或目的基因或异源核苷酸序列：其受所述亚基因组启动子控制；
(5)指导RNA复制子复制的3’端识别序列；以及
(6)保证RNA的稳定性以及目的基因有效复制的3’UTR和3’polyA。
在一些实施方案中，所述目的蛋白编码单元的上游存在亚基因组启动子SGP，所述亚基因组启动子控制所述目的基因或异源核苷酸序列。
在一些实施方案中，所述的构建体为DNA，其中所述RNA聚合酶编码单元上游存在亚基因组启动子SGP。
在一些实施方案中，所述RNA聚合酶是病毒复制酶，特别地所述病毒复制酶为甲病毒的复制酶，
优选地，所述甲病毒选自塞姆利基森林病毒(Semliki forest virus)、巴马森林病毒(Barmah forest virus)、基孔肯尼雅病毒(Chikungunya virus)、欧尼恩病毒(O’nyong-nyong virus)、罗斯河病毒(Ross river virus)、贝巴鲁病毒(Bebaru virus)、盖他病毒(Getah virus)、鹭山病毒(Sagiyama virus)、马雅罗病毒(Mayaro virus)及其亚型乌纳(Una virus)病毒；委内瑞拉马脑炎复合体包括委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus)、卡巴斯欧病毒(Cabassou virus)、沼泽地病毒(Everglades virus)、穆坎布病毒(Mucambo virus)、图那特病毒(Tonate virus)、皮克苏纳病毒(Pixuna virus)、莫索·达斯·佩德拉斯病毒(Mosso das pedras virus)、内格罗河病毒(Rio Negro virus)；西部马脑炎复合体包括西部马脑炎病毒(Western equine encephalitis virus)、辛德毕斯病毒(Sindbis virus)、奥拉病毒(Aura virus)、瓦塔罗阿病毒(Whataroa)、高地J病毒(Highlands J virus)、摩根堡病毒(Fort Morgan virus)及其亚型博吉河病毒(Creek virus)；东部马脑炎病毒(Eastern equine encephalitis virus)、米德尔堡病毒(Middelburg virus)、图那特病毒(Trocara virus)、孜拉加奇病毒(Kyzylagach virus)、恩杜姆病毒(Ndumu virus)、巴班肯病毒(Babanki virus)、挪威鲑鱼甲病毒(Norwegian salmonid alphavirus)、鲑鱼胰腺病病毒(Salmon  pancreatic disease virus)、睡病病毒(Sleeping disease virus)和南方象海豹病毒(Southern elephant seal virus)，
更优选地，所述甲病毒选自塞姆利基森林病毒、委内瑞拉马脑炎病毒、辛德毕斯病毒和基孔肯雅病毒。
在一些实施方案中，所述5’帽结构是天然5’帽或5’帽类似物。
在一些实施方案中，所述的构建体包含5’UTR，所述5’UTR来源于任一基因的5’UTR或其突变体，优选地5’UTR来源于与RNA聚合酶来源相同的病毒。
在一些实施方案中，所述复制启动元件能够被所述RNA聚合酶识别并指导其复制，
优选地，所述复制启动元件与所述RNA聚合酶来源于相同病毒或不同病毒，更优选为来源于相同病毒。
在一些实施方案中，所述的构建体包含3’UTR，所述3’UTR来源于任一基因的3’UTR或其突变体，优选地3’UTR来源于RNA聚合酶相同病毒。
在一些实施方案中，所述的构建体包含3’poly(A)。
在一些实施方案中，所述亚基因组启动子来源于病毒结构蛋白的启动子，优选地，所述亚基因组启动子与所述RNA聚合酶来源于相同病毒。
在第二个方面，本发明提供一种载体，其包含或编码如第一个方面所述的构建体。
在第三个方面，本发明提供一种疫苗组合物，其包含如第一个方面所述的构建体或如第二个方面所述的载体，其中所述目的蛋白是能引发保护性免疫反应的抗原，例如所述抗原源自人、动物、植物、病毒、细菌和/或寄生虫。
在第四个方面，本发明提供一种药物组合物，其包含如第一个方面所述的构建体或如第二个方面所述的载体，其中所述目的蛋白是治疗性蛋白。
在第五个方面，本发明提供一种表达目的蛋白的方法，其包括将第一个方面所述构建体引入能依赖于所述构建体来表达目的蛋白的目的细胞。
在第六个方面，本发明提供一种免疫接种方法，其包括将第一个方面所述的构建体接种到有此需要的对象。
本发明公开了以下技术效果：
本发明公开的顺式复制子RNA构建体，通过对甲病毒基因组多种元件进行改造，构建的复制子不能驱动复制酶的复制，只能驱动目的基因的高效复制和表达，可以最大限度的减少目的蛋白的表达，并且通过大幅度扩增目标RNA而增加目的蛋白的表达。本发明不需要对甲病毒进行培养，就能实现目的蛋白在细胞或生物体中的高效表达，适合用于在生物体中有效和安全地产生目的蛋白，如治疗性蛋白或抗原蛋白，这也为医学上应用提供了依据和方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1示现有技术中复制子的结构。图1A示甲病毒基因组结构，甲病毒的基因组是单股(+)链RNA，编码两个开放阅读框(ORF)。5’末端的前2/3部 分编码4种非结构蛋白(nsP1-nsP4)，其被翻译并加工成复制酶；靠近3’末端的后1/3称为结构区，受到亚基因组启动子(SGP)的转录控制，编码衣壳蛋白C、膜糖蛋白E1、E2、E3以及6K蛋白等结构蛋白。图1B示一种传统顺式复制子的结构，其中编码结构蛋白的区域被目的基因编码区替代。图1C示一种复制子(本文中也称反式复制子，trans-replicon)结构，其中目的基因与复制酶位于两个独立构建体中。缩写：C：5’帽；SGP：亚基因组启动子；CSE：保守序列元件。
图2示本发明一些实施方案中的复制子结构，其中复制酶编码单元位于目的基因编码单元上游。图2A示上游复制启动元件包含CSE1、CSE2和SGP，下游复制启动元件包含CSE4；图2B示上游复制启动元件包含CSE1和CSE2，下游复制启动元件包含CSE4；图2C示上游复制启动元件包含CSE1，下游复制启动元件包含CSE4；图2D示上游复制启动元件包含SGP，下游复制启动元件包含CSE4。
图3示本发明一些实施方案中的顺式复制子结构，其中复制酶编码单元位于目的基因编码单元下游。图3A示复制酶编码单元与目的基因编码单元之间存在2A肽序列；图3B示复制酶编码单元与目的基因编码单元之间存在IRES序列；图3C示复制酶编码单元与目的基因编码单元之间存在HDV样核酶(HDVr)序列；图3D示复制酶编码单元与目的基因编码单元之间无自裂解序列或IRES序列。
图4示不同复制子表达外源蛋白(荧光蛋白EGFP)时的细胞荧光图像。传统质粒(A)、图1B所示结构的传统顺式复制子(B)、图1C所示结构的反式复制子(C)、图2A所示结构的本发明顺式复制子(D)表达EGFP的荧光 信号比较图。
图5示不同复制子表达外源蛋白(Luc作为报告基因)时的报告基因信号检测结果，分别为传统质粒、图1B所示结构的顺式复制子、图1C所示结构的反式复制子、图2A所示结构的本发明顺式复制子。
图6示不同复制子表达外源蛋白(新冠病毒RBD蛋白)的WB检测结果，分别为传统质粒和图2A所示结构的本发明顺式复制子。
图7示ELISA法测定免疫小鼠血清中抗SARS-CoV-2-RBD的IgG滴度。
图8示假病毒中和实验检测免疫小鼠血清中抗SARS-CoV-2-RBD抗体的中和效价(IC 50)。
具体实施方式
术语
本发明中术语“复制子”(replicon)是指能够独立进行复制的单位，包含一个或多个复制需要的元件，可以是DNA，也可以是RNA。“顺式复制子”(cis-replicon)是指复制需要的元件位于同一条多核苷酸链上，可以是线性，也可以是环状。“反式复制子”(trans-replicon)是指复制需要的元件位于不同的多核苷酸链上。
本发明中术语“目的蛋白编码单元”是指包含目的蛋白编码序列的多核苷酸区段，和/或包含待插入目的蛋白编码序列的接受位点(例如，多克隆位点)。“RNA聚合酶编码单元”是指包含RNA聚合酶编码序列的多核苷酸区段。
本发明中术语“复制”是指利用RNA聚合酶产生与模板多核苷酸链的全长或区段相同的多核苷酸链。在一些实施方案中，所述复制是对包含上游复制启 动元件与下游复制启动元件之间的序列进行复制，产生多个序列相同的拷贝。
本发明中术语“复制启动元件”是指能够被RNA聚合酶(例如，病毒复制酶)识别并启动复制的多核苷酸区段。
本文所述“位于…上游/下游”，针对特定多核苷酸序列，以5’-3’方向来定义上下游。
甲病毒
本发明中，术语“甲病毒”应广泛地理解，并包括具有甲病毒特征的任何病毒颗粒。甲病毒的特征包括存在(+)链RNA，其编码适于在宿主细胞中复制的遗传信息，包括复制酶等。许多甲病毒的更多特征描述于例如Strauss&Strauss，Microbiol.Rev.，1994，第58卷，第491-562页；Gould等，2010，Antiviral Res.，第87卷，第111-124页；Jonathan Rupp等，2015，J.Gen.Virology，第96卷，第2483-2500页；Kathryn Carpentier等，Curr Opin Virol，2018年，第28卷，第53-60页；Luis Carrasco等，Viruses，2018年，第10卷，第70页；Rebecca Brown等，Viruses，2018年，第10卷，第89页；Autumn Holmes等，PLoS Pathog，2020年，第16卷，e1008876等文献中。术语“甲病毒”包括自然界发现的甲病毒及其任何变体或衍生物。
在自然界中发现的甲病毒优选选自：赛姆利基森林病毒复合体包括塞姆利基森林病毒(Semliki forest virus)、巴马森林病毒(Barmah forest virus)、基孔肯尼雅病毒(Chikungunya virus)、欧尼恩病毒(O’nyong-nyong virus)、罗斯河病毒(Ross river virus)、贝巴鲁病毒(Bebaru virus)、盖他病毒(Getah virus)、鹭山病毒(Sagiyama virus)、马雅罗病毒(Mayaro virus)及其亚型乌纳(Una virus)病毒；委内瑞拉马脑炎复合体包括委内瑞拉马脑炎病毒(Venezuelan  equine encephalitis virus)、卡巴斯欧病毒(Cabassou virus)、沼泽地病毒(Everglades virus)、穆坎布病毒(Mucambo virus)、图那特病毒(Tonate virus)、皮克苏纳病毒(Pixuna virus)、莫索·达斯·佩德拉斯病毒(Mosso das pedras virus)、内格罗河病毒(Rio Negro virus)；西部马脑炎复合体包括西部马脑炎病毒(Western equine encephalitis virus)、辛德毕斯病毒(Sindbis virus)、奥拉病毒(Aura virus)、瓦塔罗阿病毒(Whataroa)、高地J病毒(Highlands J virus)、摩根堡病毒(Fort Morgan virus)及其亚型博吉河病毒(Creek virus)；东部马脑炎病毒(Eastern equine encephalitis virus)、米德尔堡病毒(Middelburg virus)、图那特病毒(Trocara virus)、孜拉加奇病毒(Kyzylagach virus)、恩杜姆病毒(Ndumu virus)、巴班肯病毒(Babanki virus)、挪威鲑鱼甲病毒(Norwegian salmonid alphavirus)、鲑鱼胰腺病病毒(Salmon pancreatic disease virus)、睡病病毒(Sleeping disease virus)和南方象海豹病毒(Southern elephant seal virus)等。
更优选地，甲病毒选自赛姆利基森林病毒复合体(包括如上所述的病毒类型，包括赛姆利基森林病毒)、西部马脑炎复合体(包括如上所述的病毒类型，包括辛德毕斯病毒)、委内瑞拉马脑炎复合体(包括如上所述的病毒类型，包括委内瑞拉马脑炎病毒)。
在本发明实施例中，甲病毒不是自然界中发现的甲病毒，例如：通过基因工程改造的甲病毒。通常，在自然界中未发现的甲病毒是自然界中发现的甲病毒的变体或衍生物，其与自然界中发现的甲病毒的区别在于核苷酸序列(即基因组RNA)至少存在一个突变。与自然界中发现的甲病毒相比，核苷酸序列中的突变可以选自一个或更多个核苷酸的插入、替换或缺失。核苷酸序列中的突 变可以与核苷酸序列编码的多肽或蛋白质中的突变相关或不相关。例如，Erkuden Casales等人利用嘌呤霉素筛选到SFV表达载体复制酶R649H/P718T双突变体，其表达的β-半乳糖水平与亲本载体相似，且能够在至少10代传代过程中保持稳定的高表达水平(Erkuden Casales等，Virology，2008年，第376卷，第242-251页)。Toey Nivitchanyong等人利用非结构蛋白(nsP)P726S点突变，构建了两株Sindbis病毒株TE和633的非细胞病变且具有复制活性病毒，感染幼仓鼠肾(BHK)细胞和中国仓鼠卵巢(CHO)细胞后可持续地表达异源基因(Toey Nivitchanyong等，Virus Res.，2009年，第141卷，第1-12页)。
复制酶构建体的表征
甲病毒的基因组共有2个开放阅读框，5’末端的前2/3部分编码4种非结构蛋白(nsP1-4)，称为非结构区，在感染细胞过程中5’末端的非结构蛋白首先翻译成一种大的多聚蛋白，产生RNA复制和转录所需要的复制酶复合体，利用基因组RNA作为模板合成负链，该过程发生在细胞感染后的3-4小时内，之后将只会合成正链RNA。
本发明中的顺式RNA构建体中包含编码甲病毒复制酶的开放阅读框(ORF)。在本发明中，“非结构蛋白”是指甲病毒来源的任意一种或更多种单独的非结构蛋白(nsP1、nsP2、nsP3、nsP4)，或包含多于一种甲病毒来源的非结构蛋白的多肽序列的多聚蛋白，例如nsP1234。在实施例中，“非结构蛋白”是指nsP123。
nsP1是一种鸟嘌呤-7-甲基转移酶(也被称为病毒帽子酶)，具有甲基转移酶和鸟苷酰基转移酶活性，以及复制复合物的膜锚定功能。其甲基转移酶结构 域催化S-腺苷甲硫氨酸的甲基基团添加到GTP分子中，形成共价m7GMP-nsP1中间产物，待nsP2去除正链RNA分子上的5’-γ-磷酸后，通过nsP1的鸟苷基转移酶活性将m7GMP部分转移到RNA分子上，形成0型帽状结构，即m7GpppA(Leevi
Figure PCTCN2022075384-appb-000001
&Tero Ahola，2002年，第71卷，第187-222页；Maija K.
Figure PCTCN2022075384-appb-000002
等，2017年,第234卷，第44-57页；Autumn T.LaPointe等，mBio.，2018年，第9卷，第e02342-18页)。
nsP2是最大的复制酶蛋白，是RNA结合蛋白，具有核酸水解酶(NTPase)、RNA解旋酶(RNA helicase)与蛋白酶活性，能打开RNA双链，辅助nsP1蛋白完成加帽的过程。nsP2的蛋白酶活性在复制酶分解为非结构蛋白过程中也发挥重要作用(Ellen G.Strauss等，Virology，1992，第191卷，第932-940页；M Gomez de Cedrón等，FEBS Lett，1999，第448卷，第19-22页；Leevi
Figure PCTCN2022075384-appb-000003
&Tero Ahola，2002年，第71卷，第187-222页；Maija K.
Figure PCTCN2022075384-appb-000004
等，2017年,第234卷，第44-57页)。
nsP3是一种磷酸化蛋白，该蛋白由三个结构域组成：具有磷酸酶活性和核酸结合能力的N端保守结构域、甲病毒独特结构域(AUD)和C端高变域(HVD)(Farhana Abu Bakar等，Viruses.，2018年，第10卷，第71页)。nsP3的功能了解较少，被称为nsPs中比较神秘的一个。近年来发现，nsP3与几种宿主蛋白质相互作用，包括应激颗粒相关蛋白、DEAD-box家族蛋白、热休克蛋白和激酶，并可能调节蛋白质蛋白质多聚/单-ADP核糖基化(Tyler Lark等，Front Microbiol，2017年，第8卷，第2652页；Maija K.
Figure PCTCN2022075384-appb-000005
等，2017年,第234卷，第44-57页；Benjamin
Figure PCTCN2022075384-appb-000006
等，Viruses，2018年，第10卷，第105页)。此外，nsP3还可能与与旧大陆甲病毒(如SFV)的神经毒力有关(Benjamin
Figure PCTCN2022075384-appb-000007
等，Viruses，2018年，第10卷，第105页)。
nsP4在C末端包含RNA依赖性RNA聚合酶(RdRp)结构域，是甲病毒中最保守的蛋白质。nsP4作为甲病毒复制复合体的主要催化核心，与宿主蛋白和其他nsP(如nsP123+nsP4)一起形成复制复合体(RC)，以病毒(+)链为模板合成(-)链RNA。nsP123裂解为nsP1、nsP2和nsP3后，可与nsP4形成四聚体，其不再具有合成负链RNA的功能，但它可利用负链RNA为模板参与复制基因组RNA和转录26S亚基因组RNA。纯化的nsP4还具有末端腺苷酸转移酶活性，可能在基因组3’端产生poly(A)尾(Maija K.
Figure PCTCN2022075384-appb-000008
等，2017年,第234卷，第44-57页；Farhana Abu Bakar等，Viruses.，2018年，第10卷，第71页)。
复制酶的来源不限于任何特定的甲病毒。在一个优选的实施方案中，甲病毒复制酶来自赛姆利基森林病毒(SFV)，包括天然存在的赛姆利基森林病毒或变异体。在一个作为替代的优选实施方案中，复制酶来自辛德毕斯病毒(SINV)，包括天然存在的辛德毕斯病毒或变异体。在一个作为替代的优选实施方案中，复制酶来自委内瑞拉马脑炎病毒(VEEV)，包括天然存在的VEEV或变异体。
UTR
术语“非翻译区”或“UTR”涉及转录但不翻译成氨基酸序列的区域，或RNA分子(例如mRNA分子)中的相应区域。甲病毒属的5’UTR和3’UTR含有不同的核心启动子元件，用于负链和正链RNA合成。甲病毒属加帽的5’UTR包含启动子元件、翻译调节序列及先天性免疫防御的结构(Jennifer L.Hyde等，Virus Research，2015，第206卷，第99-107页)。
甲病毒5’UTR包含核心启动子元件，这些元件与下游和3’UTR内的其他顺式作用元件一起作用，以调节正链和负链的合成(Kulasegaran Shylini等人，2009年，J Virol.，第8327-8339页)。甲病毒属的腺苷酸化3’UTR含有高度保守的病毒复制序列元件、决定细胞趋向性元件及影响病毒RNA稳定性保守结合区域(Jennifer L.Hyde等，Virus Res，2015年，第99-107页)。其中，聚poly(A)末端的19个核苷酸的共同保守序列(CSE)是负链合成的启动子(Kuhn等人，J Virol，1990年，第1465-1476页)，聚poly(A)尾与CSE共同支持负链合成和高效翻译，是病毒复制的必要步骤。
优选地，本发明的顺式RNA复制子中包含非病毒来源的5’UTR；特别地，不是甲病毒来源，比如人β-珠蛋白5’UTR，可提高翻译效率，同时使得复制酶不扩增编码复制酶区域的RNA。在一个实施方案中，RNA包含来源于真核生物的5’UTR。根据本发明的5’UTR可包含多于一个核酸序列的任何组合，任选地通过接头分开。
优选地，本发明的顺式RNA复制子中包含甲病毒的3’UTR，该3’UTR具有活性，可以保证RNA的稳定性及目的基因的有效复制。
Poly(A)序列
本发明中所述的顺式RNA构建体中包含3’poly(A)序列。在甲病毒复制过程中，复制复合物和poly(A)尾之间通过poly(A)结合蛋白(PABP)相互作用，因此poly(A)尾部至少需要11-12个残基才能有效地产生负链RNA。负链合成在poly(A)尾部含有25或34个腺苷酸残基的RNA模板上复制效率相当；但是，当尾部尺寸减小到20个残基时，负链合成效率显著降低(>70％)。当尾部的尺寸减少到10个残基时，效率再次下降。3’poly(A)尾的缺失严重 抑制了负链RNA的产生。一个没有3’poly(A)尾的模板RNA的负链RNA合成仅为含有25个3’末端腺苷酸残基的模板RNA的4％(Hardy and Rice，2005年，J Virol，第79卷，第4630-4639页)。
此外，poly(A)序列还会影响转染的真核细胞中RNA稳定性和蛋白质翻译的功能。据文献报道，约120个A核苷酸的3’poly(A)序列对转染的真核细胞中的RNA水平以及3’poly(A)序列上游的开放阅读框翻译的蛋白质水平具有有益影响(Nadine Bangel-Ruland，J Gene Med，2013，第15卷，第414-426页)。此外，采用分段poly(A)，如poly(A)2×60_G或poly(A)2×60_T，可以显著减少质粒在大肠杆菌中的重组，而不会对mRNA半衰期和蛋白质表达产生任何负面影响。这种poly(A)尾的特征在于，它由至少两个含A的元素组成，每个元素被定义为由40-60个腺苷酸组成的核苷酸序列，由不同长度的间隔元件隔开。与传统的同质poly(A)尾相比，分段化poly(A)尾在重组质粒和产生的mRNA性能(半衰期和翻译效率)方面具有更高的潜力(Zeljka Trepotec等人；RNA，2019年，第25卷，第507-518页)。
根据本发明，在一个实施方案中，3’poly(A)序列包含以下或基本上由以下或由以下组成：至少11个、优选至少25个、优选至少34个、优选至少69个、优选至少100个，且优选至多500个、优选至多400个、优选至多300个、优选至多200个、特别是至多150个A核苷酸，特别是约120个A核苷酸。在本发明中，poly(A)序列中至少50％，优选至少75％，是A核苷酸，但允许剩余的核苷酸是除A核苷酸外的核苷酸，例如U核苷酸(尿苷酸)、G核苷酸(鸟苷酸)、C核苷酸(胞苷酸)，以便形成分段化poly(A)尾。
密码子优化
mRNA使用3个字母的密码子意味着64个可能的密码子编码20个氨基酸和翻译停止信号。遗传密码的简并性是指编码同一氨基酸的几个三联体遗传密码中，一、二位碱基大多是相同的，只是第三位不同。同一氨基酸有多个密码子，由于tRNAs对核糖体A位点内的密码子进行随机识别，根据从tRNAs的细胞质池中选择合适的同源tRNA的效率，密码子可以被定义为最优。生物界中使用的密码子频率是不一样的，密码子偏差与大肠埃希菌、酿酒酵母、秀丽隐杆线虫、果蝇和真核生物中的tRNA水平相关(Gavin Hanson等，Nat Rev Mol Cell Biol.，2018年，第19卷，第20-30页)。密码子优化的主要策略是通过克服与密码子利用率和tRNA丰度的物种特异性差异相关的限制来提高翻译延伸率。利用偏爱密码子，避开利用率低的稀有密码子，同时简化基因转录后mRNA的二级结构，去除不利于高效表达的基序(Motif)，加入有利于高效表达的基序，调整GC含量等这些方法重新设计基因编码。除了密码子的偏好性外，还需要考虑密码子的组合使用效率。在大部分情况下，仅用高频密码子的效果并不会最好，考虑到高频和次高频密码子的组合使用效果会更佳(Vincent P Mauro等，BioDrugs.，2018年，第32卷，第69-81页)。在本发明的一些实施方案中，由RNA分子包含的开放阅读框的密码子与开放阅读框起源的相应密码子可能不同，会根据不同的物种进行优化，称为“密码子优化”。综上，可以分别对编码本发明的复制酶、表达的外源蛋白的开放阅读框进行密码子的优化。
保守序列元件(conserved sequence element,CSE)
本发明中，术语“保守序列元件”或“CSE”是指在甲病毒RNA中发现的核苷酸序列。这些序列元件称为“保守的”，因为直系同源物存在于不同甲病毒的 基因组中，并且不同甲病毒的直系同源CSE优选地共享高百分比的序列和/或相似的二级或三级结构。在甲病毒基因组中发现了四个保守序列元件(CSE)，这些元件在病毒RNA复制过程中执行特定的功能。在一个实施方案中，根据本发明的复制子包含一种或更多种保守序列元件(CSE)(Strauss&Strauss，Microbiol.Rev.，1994，第58卷，第491-562页)。例如在辛德毕斯病毒中，正链5’UTR前44个核苷酸形成一个保守的茎环结构，其互补序列位于负链的3’端，是以负链RNA为模板合成正链RNA的启动子。CSE2是nsPl蛋白编码序列前端51个核苷酸形成的保守茎环结构，是以正链RNA为模板合成负链RNA的共启动子。CSE3是位于nsP4蛋白末端的24个核苷酸，是转录亚基因组mRNA的启动子。CSE4是RNA 3’末端的19个核苷酸，是以正链RNA为模板合成负链RNA的启动子(Hardy和Rice，J.Virol.，2005，第79卷，第4630-4639页)。
在一个实施方案中，甲病毒5’复制识别序列和甲病毒3’复制识别序列能够在复制酶的存在下指导根据本发明的RNA复制子的复制。因此，当单独存在或优选地共同存在时，这些识别序列在复制酶的存在下指导RNA复制子复制。不希望受到任何特定理论的束缚，应理解甲病毒保守序列元件是在复制酶的存在下指导RNA复制子复制的识别序列。在该实施方案中，复制子识别序列通常指CSE 1和2。此外，在一个作为替选的实施方案中，复制酶和保守序列元件可以来自不同的甲病毒属。
本文所述的CSE-1、CSE-2，CSE-3和CSE-4包括其变体，所述变体保留其启动复制的能力。
亚基因组启动子(subgenomic promoter,SGP)
本发明中，术语“亚基因组启动子”或“SGP”是指核酸序列(例如：编码序列)上游5’的核酸序列，其通过提供复制酶的识别和结合位点来控制所述核酸序列的转录。在特定实施方案中，根据本发明的RNA复制子包含亚基因组启动子。本文所述的SGP包括其变体，所述变体保留其启动下游基因转录的能力。
优选地，本复制子编码的复制酶是能够识别亚基因组启动子的甲病毒复制酶。
优选地，根据本发明的亚基因组启动子是甲病毒结构蛋白的启动子，包含甲病毒中高度保守的CSE3，即最少包含非结构蛋白nsP4末端的24个核苷酸(以辛德毕斯病毒为例)，可以有效启动亚基因组RNA的转录。在一些实施方案中，SGP与CSE 3相同或与CSE3重叠或包含CSE 3。
异源核酸序列
在一个实施方案中，顺式复制子RNA包含至少一种不来源于甲病毒的核酸序列。根据本发明，术语“异源”或“外源”是指核酸序列不与甲病毒基因组核酸序列同源的情况。优选地，异源核酸序列受到甲病毒亚基因组启动子的控制。更优选地，异源核酸序列位于亚基因组启动子的下游。甲病毒亚基因组启动子非常有效，因此适合于高水平的异源基因表达(Kenneth Lundstrom，Molecules，2018年，第23卷，第3310页)。
目的蛋白
本文所指“目的蛋白”是由目的基因编码单元编码的蛋白。在一个实施方案中，根据本发明的RNA复制子包含编码目的肽或目的蛋白的开放阅读框。优选地，目的蛋白由异源核酸序列编码。优选地，目的基因受到亚基因组启动子 的控制。更优选地，目的基因位于亚基因组启动子的下游。
本发明中术语“内部核糖体进入位点”(internal ribosome entry site,IRES)是指能够募集核糖体从而启动下游基因翻译的序列，当存在于两个编码序列之间时能够介导翻译的内部起始。
本发明提供的复制子可以编码单个或多个蛋白。多个蛋白可以编码为融合表达的蛋白或者分开表达的蛋白。对于分开表达的蛋白，则可以向这些蛋白中的一个或更多个提供上游IRES，或在复制子中构建多个蛋白的开放阅读框，保持每个开放阅读框都受到亚基因组启动子的控制。另外，也可以向这些蛋白中加入自催化蛋白酶(例如：口蹄疫病毒2A蛋白)来隔开(Strauss&Strauss，Microbiol.Rev.，1994，第58卷，第491-562页)。
本发明中术语“2A肽”(2A peptide,or 2A self-cleaving peptide)是一类长约18-22个氨基酸的区段，能诱导细胞内含有2A肽的重组蛋白自剪切。在本发明的一些实施方案中，编码2A肽的核酸区段存在于目的基因编码单元与RNA聚合酶编码单元之间。
本发明中术语“HDV样核酶”(HDV-like ribozyme)，或称Hepatitis Delta Virus(HDV)ribozyme(HDVr)是指一类具有自剪切功能的核酶。HDV核酶催化底物核苷酸或寡核苷酸与核酶的5'-羟基之间的磷酸二酯键的裂解，从而将所在的多核苷酸链从剪切位点断开。在本发明的一些实施方案中，HDV样核酶存在于目的基因编码单元与RNA聚合酶编码单元之间。
本发明的复制子编码的目的蛋白也可以是免疫原性化合物或抗原表位或细胞因子，作为治疗性或保护性的目的。术语“抗原”或“免疫原”指能引起抗体生成的物质，是任何可诱发免疫反应的物质。抗原优选来源于天然存在的抗原 的产物，例如来源于变应原、病毒、细菌、真菌、寄生虫和其他感染因子和病原体以及肿瘤抗原。术语“细胞因子”指由免疫细胞(如单核、巨噬细胞、T细胞、B细胞、NK细胞等)和某些非免疫细胞(内皮细胞、表皮细胞、纤维母细胞等)经刺激而合成、分泌的一类具有广泛生物学活性的小分子蛋白质，例如白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。
本文中当使用一种生物学实体(如复制酶、病毒、亚基因组启动子SGP)时，其含义不仅包括其野生型存在形式，也包括保留该生物学实体之功能的变体形式。
本发明实施例的安全特征
优选地，本发明的系统不包含任何甲病毒结构蛋白，例如：衣壳蛋白C、膜糖蛋白E1、E2、E3以及6K蛋白等。
优选地，在用本发明的顺式复制子RNA接种宿主细胞后，宿主细胞不产生病毒颗粒，例如：子代病毒颗粒。
优选地，复制子不能驱动复制酶自身的复制，而只能驱动目的基因RNA的复制。
本发明的DNA
本发明提供了DNA，其编码本发明提供的复制子。在一个优选的实施方案中，本发明提供的DNA是质粒。如本文所用，术语“质粒”指的是细菌、酵母菌和放线菌等生物中染色体(或拟核)以外的DNA分子，具有自主复制能力，并表达所携带的遗传信息，是闭合环状的双链DNA分子。
制备RNA的方法
根据本发明的任何RNA分子，都可以通过体外转录获得。体外转录RNA(IVT-RNA)可通过从核酸分子(特别是DNA分子)在体外转录获得。5’帽可以由帽-类似物添加到体外转录反应中获得，poly(A)尾部则由DNA模板上的聚(dT)序列编码。或者，也可以在获得转录产物后，通过酶促反应实现加帽和加poly(A)尾。体外转录方法是本领域技术人员已知的，各种体外转录试剂盒均是商品化的试剂盒。
疫苗佐剂
在一些实施方案中，mRNA的免疫刺激特性可以通过加入佐剂来提高mRNA疫苗的效力。这些方法包括传统的佐剂以及利用mRNA固有的免疫原性或其编码免疫调节蛋白的新方法。本发明的组合物可包含一种或更多种佐剂。
可以在疫苗中加入佐剂以刺激免疫系统的反应。示例性佐剂包括但不限于以下：生物性佐剂如分枝杆菌(结核杆菌、卡介苗)、短小棒状杆菌、百日咳杆菌、革兰阴性杆菌内毒素、白细胞介素-1、白细胞介素-2、白细胞介素-12、干扰素-γ等；无机佐剂如氢氧化铝、明矾、磷酸铝等；人工合成佐剂如双链多聚肌苷酸、胞苷酸、双链多聚腺苷酸等；油剂如花生油乳化佐剂、矿物油、植物油、羊毛脂等；免疫激活蛋白如CD70、CD40L和TLR4等。
RNA药物递送系统
细胞膜主要由两性离子和带负电荷的磷脂的脂质双层组成，其中磷脂的极性头指向水性环境，疏水尾形成疏水核。各种离子泵和离子通道有助于在细胞膜上维持负电势(-40至-80mV)以控制大多数必需的金属离子(例如K +，Na +，Ca 2+和Mg 2+)的平衡。RNA必须穿过细胞膜才能到达细胞质，而跨细胞膜的负电位给高度带负电荷的RNA分子创建了强大的屏障。除了细胞膜屏障，RNA 还面临着皮肤和血液中大量存在的细胞外核糖核酸降解的问题。裸露的RNA分子在生物体中迅速降解，在全身性给药后不会积聚在目标组织中，即使它们到达目标组织也无法穿透目标细胞，因此需要借助递送系统。
在一个实施方案中，本发明的RNA药物组合物包含至少一种含阳离子的复合物中。根据本发明的药物组合物可以包封在阳离子聚合物(Polyplexes,PP)、脂质体复合物(lipoplex，LP)、脂质体纳米粒(lipid nanoparticle，LNP)、阳离子纳米乳(cationic nanoemulsion，CNE)中。
在本发明的一个优选实施方案中，聚合物(Polymers)，例如聚乙烯亚胺(PEI)与带负电核酸的静电相互作用形成的阳离子聚合物(PP)。PEI是一组线性或支链的聚乙烯亚胺聚合物，对核酸有很强的亲和力，并具有促进内体逃逸的质子海绵效应，保护核酸不受酶降解的影响(Thomas Démoulins等，Nanomedicine，2016年，第12卷，第711-722页)。
在本发明的一个优选实施方案中，带负电荷的mRNA与带正电荷的阳离子脂质体经静电作用聚集形成多层囊状复合物即脂质体复合物(LP)中，复合物中包封的mRNA不易被RNase降解，能被成功递送至细胞内。DOTAP(1，2-二烯丙基-3-三甲基铵丙烷)、DOPE(1，2-二油酰-sn-甘油-3-磷酸乙醇胺)和DOTMA(N-[1-(2，3-二油酰氧基)丙基]-N，N，N-三甲基氯化铵)是已被证实有效的递送载体。阳离子脂质通常用于mRNA疫苗接种，因为它们不仅可以包裹mRNA，而且本身也可以用作免疫原，被认为是疫苗的佐剂(Yasmin Granot等，Seminars in Immunology，2017年，第34卷，第68-77页)。
在本发明的一个优选实施方案中，mRNA存在于脂质体纳米粒(LNP)中， 所述脂质体通常包含以下：(1)可电离的或阳离子的脂质或聚合材料，带有叔胺或季胺，可促进自组装成病毒大小(～100nm)的颗粒，用来封装聚阴离子mRNA，并允许内体释放mRNA到细胞质中；(2)辅助磷脂一般采用两性离子脂质，如DSPC(1，2-二硬脂酰-sn-甘油-3-磷酸胆碱)，DOPE(1，2-二油酰-sn-甘油-3-磷酸乙醇胺)或POPE(1-棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺)。辅助磷脂作为脂质体的骨架，类似于细胞膜中的脂质，有助于脂质双层形成和破坏，促进内体逃逸。一些磷脂具有多态性特征，在进入内涵体时能由层状转变为六角相，促进mRNA从脂质体中释放；(3)胆固醇，以稳定LNP的脂质双层,提高颗粒稳定性，增强了颗粒的稳定性，促进膜融合；(4)脂质连接的聚乙二醇(PEG)，使纳米颗粒具有水合层，提高胶体稳定性，可减少与血清蛋白的非特异性相互作用，绕过网状内皮系统的吸收并延长制剂的半衰期(Norbert Pardi等，2018年，Nat Rev Drug Discov.，第17卷，第261-279页；Piotr S.Kowalski等，Mol Ther，2019年，第27卷，第710-728页)。
在本发明的一个优选实施方案中，mRNA存在于一种由角鲨烯、非离子山梨醇酐酯表面活性剂、山梨醇酐三油酸酯(Span 85)或山梨醇酐单硬脂酸盐(Span 60)、阳离子脂质DOTAP等制成的阳离子纳米乳液(CNE)中，可以用于疫苗的制备(Jesse H.Erasmus等，Mol Ther.，2018年，第26卷，第2507-2522页)。
产生蛋白质的方法
本发明提供了在细胞中产生蛋白质的方法，其包括以下步骤：获得用于表达外源蛋白的RNA构建体，其包含用于驱动所述复制酶翻译的5’帽和增强mRNA稳定性的3’poly(A)，包含编码所述外源蛋白质的开放阅读框，将所述 RNA构建体转染到所述细胞中。
在本发明的方法中，可以使用根据本发明的任何系统，或根据本发明的试剂盒，或根据本发明的药物组合物。RNA可以以药物组合物的形式使用，或者以裸RNA使用，例如：用于电穿孔或者脂质体转染等。
本发明提供了在生物体中产生蛋白质的方法，其包括以下步骤：获得用于表达外源蛋白的RNA构建体，其包含用于驱动所述复制酶翻译的5’帽和增强mRNA稳定性的3’poly(A)，包含编码所述外源蛋白质的开放阅读框，向所述对象施用该RNA构建体。本发明的药物可用于治疗对象的预防性以及治疗性方法。
疫苗接种
术语“免疫接种”或“疫苗接种”通常是指出于治疗或预防原因而治疗对象的过程。治疗，特别是预防性治疗，优选是或旨在包括旨在诱导或增强对象的针对一种或更多种抗原的免疫应答的治疗。根据本发明，如果期望通过使用如本文所述的RNA诱导或增强免疫应答，则可以通过RNA引发或增强免疫应答。在一个实施方案中，本发明提供预防性治疗，其优选地是或包括对对象进行疫苗接种。本发明的一个实施方案特别适用于疫苗接种，其中复制子编码作为目的蛋白的药学活性肽或蛋白质，它们是免疫活性化合物或抗原。本发明的药物可以施用于对象，例如，用于治疗对象，包括对象的疫苗接种。术语“对象”涉及脊椎动物，特别是哺乳动物包括人。
施用方式
根据本发明的药物可以以任何合适的途径施用于对象。
例如：药物可以全身性施用，可以以多种方式实现给药，例如静脉内、皮 下、皮内、肌肉或吸入等。施用于肌肉组织或皮肤的替代方案包括但不限于：皮内、鼻内、眼内、腹膜内、静脉内、间质、经颊、透皮或舌下施用等。
实施例
一、材料与方法
(1)构建携带反式复制子、传统顺式复制子和本发明的一种顺式复制子模板的DNA载体
实施例中使用的部分元件序列如下：
5’帽结构：7-甲基鸟苷帽结构(m7Gppp,Cap 0)
5’UTR(来源于人α珠蛋白):actcttctggtccccacagactcagagagaacccacc(SEQ ID NO:1)
CSE1(来源于甲病毒属的赛姆利基森林病毒SFV)：
Figure PCTCN2022075384-appb-000009
CSE2(来源于甲病毒属的赛姆利基森林病毒SFV)：
Figure PCTCN2022075384-appb-000010
SGP(CSE3)(来源于甲病毒属的赛姆利基森林病毒SFV)：
Figure PCTCN2022075384-appb-000011
CSE4(来源于甲病毒属的赛姆利基森林病毒SFV)：
Figure PCTCN2022075384-appb-000012
3’UTR(来源于甲病毒属的赛姆利基森林病毒SFV)：
Figure PCTCN2022075384-appb-000013
Figure PCTCN2022075384-appb-000014
复制酶(来源于甲病毒属的赛姆利基森林病毒SFV)，其氨基酸序列如下：
Figure PCTCN2022075384-appb-000015
Figure PCTCN2022075384-appb-000016
Figure PCTCN2022075384-appb-000017
(1)本发明中构建复制子所需的各区段DNA序列由生工生物工程(上海)股份有限公司合成后，利用同源重组插入哺乳动物表达载体pVAX1(购自Invitrogen)中，构建成pVAX1-cis-SFV-neo顺式复制子质粒(结构如图2A所示，其中目的基因位置为多克隆位点)。携带相同聚合酶的传统顺式复制子(结构如图1B所示，其中目的基因位置为多克隆位点)及反式复制子系统(结构如图1C所示，其中目的基因位置为多克隆位点)按照发明专利公开CN 109328233 A中的描述合成构建，利用同源重组插入哺乳动物表达载体pVAX1，分别命名为pVAX1-trans-SFV及pVAX1-cis-SFV。
构建后获得的pVAX1-cis-SFV-neo包含如下序列：
Figure PCTCN2022075384-appb-000018
Figure PCTCN2022075384-appb-000019
Figure PCTCN2022075384-appb-000020
Figure PCTCN2022075384-appb-000021
Figure PCTCN2022075384-appb-000022
Figure PCTCN2022075384-appb-000023
多克隆位点(MCS)：atcgatatgcatttataaggatcccctaggcgatcg(SEQ ID NO:16)
Figure PCTCN2022075384-appb-000024
(2)将增强型绿色荧光蛋白(EGFP)编码区或者荧光素酶报告基因(Luc)分别同源重组到改造后的表达载体pVAX1、传统顺式复制子载体pVAX1-cis-SFV、反式复制子载体pVAX1-trans-SFV和本发明顺式复制子载体pVAX1-cis-SFV-neo的亚基因组启动子3’端的多克隆位点区域，分别构建成pVAX1-EGFP/Luc、pVAX1-cis-SFV-EGFP/Luc、pVAX1-trans-SFV-EGFP/Luc、pVAX1-cis-SFV-neo-EGFP/Luc。
(3)将新冠病毒(SARS-CoV-2)刺突蛋白(Spike)中的受体结合域(RBD)分别同源重组到改造后的表达载体pVAX1及顺式复制子pVAX1-cis-SFV-neo 载体中，插入亚基因组启动子3’端的多克隆位点区域，构建成pVAX1-RBD和pVAX1-cis-SFV-neo-RBD。
(4)将(1)-(3)所述构建获得的载体使用NEB公司的HiScribe TM T7 ARCA mRNA Kit(with Tailing)(New England Biolabs，MA，USA)，按照说明书进行体外转录后，收集RNA，通过Nanodrop 2000超微量分光光度计(Thermo Fisher Scientific,MA,USA)检测RNA浓度，制备好的RNA储存于-80度冰箱中备用。
(5)RNA转染
本实施例中所有细胞培养基、胎牛血清(FBS)、抗生素和其他补充剂均由美国Thermo Fisher Scientific公司提供。体外转录获得的RNA使用转染试剂Lipofectamine Messenger MAX(Thermo Fisher Scientific,MA,USA)，按照制造商的说明书进行RNA脂质体转染。
(6)检测绿色荧光蛋白信号
为了评价不同RNA复制子表达外源蛋白(绿色荧光蛋白)的水平，根据(5)中描述的方法，将pVAX1-EGFP(不含复制子，作为对照)、pVAX1-cis-SFV-EGFP、pVAX1-trans-SFV-EGFP、pVAX1-cis-SFV-neo-EGFP体外转录获得的RNA分别等量转染96孔板的仓鼠肾成纤维细胞(BHK-21)(中国科学院典型培养物保藏委员会细胞库购得)，48小时后，显微镜下观察绿色荧光强度并记录图像。
(7)萤光素酶活性测定
为了评价不同RNA复制子表达外源蛋白(荧光素酶)的水平，根据(5)中描述的方法，将pVAX1-Luc(不含复制子，作为对照)、pVAX1-cis-SFV-Luc、 pVAX1-trans-SFV-Luc、pVAX1-cis-SFV-neo-Luc体外转录获得的RNA分别等量转染96孔板的BHK-21细胞，用Bright-Glo(Promega，Madison，USA)检测试剂检测萤火虫萤光素酶，使用EnSpire多功能酶标仪(PerkinElmer，MA，USA)测量生物发光。数据以相对萤光素酶单位[RLU]表示，减去背景信号(未转染细胞的读数)。
(8)蛋白表达检测
根据(5)中描述的方法，将pVAX1-RBD和pVAX1-cis-SFV-neo-RBD体外转录获得的RNA分别等量转染24孔板的BHK-21细胞，48小时后收集细胞。新冠病毒RBD的表达情况通过Western Blot检测分析，抗体采用北京义翘神州科技有限公司生产的新冠病毒RBD抗体。
(9)假病毒中和实验
采用假型慢病毒荧光素酶报告系统，评价接种动物血清对SARS-CoV-2的中和能力。
具体地，SARS-CoV-2假型病毒是由慢病毒包装质粒pCMV delta R8.2、编码荧光素酶报告基因的表达质粒pCDH-CMV-luc以及一个表达新冠病毒Spike蛋白的质粒pcDNA3.1-Spike共转染293T细胞而产生，用于转染的治疗比例为1：1.5：1。通过离心法收集含病毒培养基，通过0.45μm的膜过滤，随后-80℃储存备用。
对于中和试验，首先对待测热灭活血清进行连续稀释，然后与SARS-CoV-2假型病毒一起孵育1小时，然后将血清-病毒混合物转移到预先接种于细胞培养板的293T-hACE2细胞。48小时后裂解细胞，用Bright-Glo(Promega，Madison，USA)检测试剂测定荧光素酶活性。然后使用GraphPad Prism(version 8.4) 计算IC 50中和滴度。
(10)小鼠免疫
6-8周龄的BALB/c小鼠购买自河南省实验动物中心并在SPF级别动物实验室中饲养，所有实验均由郑州大学生命科学伦理审查委员会批准。
将脂质体包裹的pVAX1-RBD(对照)及pVAX1-cis-SFV-neo-RBD体外转录获得的RNA(实验组)对小鼠进行肌肉注射，并在初次免疫后第14、28天用等剂量的RNA进行增强免疫。初次免疫后35天收集血清标本，56℃热灭活30min，-80℃保存。免疫小鼠的对照组注射PBS，或使用脂质体包裹的pVAX1质粒体外转录获得的RNA。实验组和对照组均采用5只小鼠，实验结果用平均值±SEM表示，组间比较采用Mann-Whitney U检验，通过GraphPad Prism(version 8.4)软件进行统计分析。
二、结果与分析
1、将表达载体pVAX1-EGFP、传统顺式复制子载体pVAX1-cis-SFV-EGFP、反式复制子载体pVAX1-trans-SFV-EGFP(含相应复制酶)、本发明顺式复制子载体pVAX1-cis-SFV-neo-EGFP体外转录所得的RNA通过Lipofectamine MessengerMAX TM等物质的量转染到96孔板的BHK-21细胞中48小时后，比较各种复制子表达绿色荧光蛋白(EGFP)的能力。
结果如图4所示，传统顺式复制子(图4B)、反式复制子(图4C)与本发明顺式复制子(图4D)表达绿色荧光蛋白的效果，均远优于传统表达载体转录所得的RNA(图4A)。
2、将传统表达载体pVAX1-Luc、传统顺式复制子pVAX1-cis-SFV-Luc、反式复制子pVAX1-trans-SFV-Luc(含相应复制酶)、本发明顺式复制子 pVAX1-cis-SFV-neo-Luc转录所得的RNA，通过Lipofectamine MessengerMAX TM等物质的量转染到96孔板的BHK-21细胞中48小时后，用Bright-Glo荧光素酶测定系统检测荧光素酶活性，比较各种复制子表达荧光素酶(Luc)的能力。实验结果用平均值±SEM表示，组间比较采用Mann-Whitney U检验。
结果如图5所示，传统顺式复制子、反式复制子(含配套复制酶)、本发明顺式复制子载体转录所得的RNA表达荧光素酶的能力，均远超传统表达载体转录所得的RNA(P＜0.01)。本发明顺式复制子和反式复制子(含配套复制酶)载体转录所得的RNA表达荧光素酶的能力相当，且显著优于传统顺式复制子(P＜0.01)。
3、使用转染试剂Lipofectamine Messenger MAX，将体外转录的pVAX1-RBD/pVAX1-cis-SFV-neo-RBD的RNA转染BHK-21细胞48小时后检测表达量。Western Blot结果如图6所示，与传统表达载体转录所得的RNA相比，本发明顺式复制子表达外源蛋白RBD的量更高。
4、pVAX1-SFV-RBD及pVAX1-cis-SFV-neo-RBD体外转录获得的100ng RNA经脂质体包裹后免疫小鼠，并在初次免疫后第14、28天用等剂量的RNA进行增强免疫，初免后35天收集血清标本，用ELISA法测定血清中抗RBD的IgG滴度(包被抗原为重组RBD蛋白)。对照组采用PBS或体外转录的pVAX1 mRNA免疫小鼠。实验结果用三次独立实验的平均值±SEM进行比较，组间比较采用Mann-Whitney U检验。ELISA实验中，对于没有任何反应的抗血清，其滴度指定为1，以便用于统计学分析。
结果如图7所示，本发明顺式复制子载体pVAX1-cis-SFV-neo-RBD及传 统表达载体pVAX1-RBD转录所得的RNA免疫小鼠后，均产生了针对新冠病毒受体结合域(RBD)蛋白的IgG抗体，且本发明顺式复制子载体体外转录的RNA复制子组产生的抗体滴度明显高于传统表达载体转录所得的RNA组(P＜0.05)。
5、pVAX1-SFV-RBD及pVAX1-RBD体外转录获得的100ng RNA经脂质体包裹后免疫小鼠，并在初次免疫后第14、28天用等剂量的RNA进行增强免疫，初免后35天收集血清标本，用假病毒中和实验来测定血清中和效价。对照组采用PBS或pVAX1体外转录的RNA免疫小鼠。实验结果用三次独立实验的平均值±SEM进行比较，组间比较采用Mann-Whitney U检验。中和实验中，如果未经稀释的血清仍未显示任何中和效应，其滴度指定为1用于统计学分析。
结果如图8所示，在体外假病毒中和实验中，本发明顺式复制子载体pVAX1-cis-SFV-neo-RBD及表达载体pVAX1-RBD转录所得的RNA免疫小鼠后，血清中均产生了中和抗体，提供了对细胞的保护。同时，本发明顺式RNA复制子组的抗体效价明显高于传统表达载体pVAX1-RBD转录的RNA组(P＜0.05)。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (24)

  1. 一种作为顺式复制子的表达构建体，其包含RNA聚合酶编码单元和目的蛋白编码单元，其中
    所述RNA聚合酶编码单元包含编码RNA聚合酶的核酸区段，所述目的蛋白编码单元包含待插入目的蛋白编码序列的接受位点(如多克隆位点)和/或编码目的蛋白的核酸区段，
    所述构建体能够在目的细胞中表达所述RNA聚合酶，并依赖所述RNA聚合酶进行复制，从而提供进一步表达目的蛋白的模板；优选地，所述复制对所述目的蛋白单元的效率高于对所述RNA聚合酶编码单元；更优选地，所述复制为仅复制所述目的蛋白编码单元。
  2. 如权利要求1所述的构建体，其是DNA或RNA，优选为RNA，包括单链RNA或双链RNA，例如是单链RNA，特别地所述单链RNA是正链(+)。
  3. 如权利要求1或2所述的构建体，其是线性的或环状的；
    特别地所述构建体是线性RNA，在其5’端存在驱动下游编码序列翻译的5’帽结构或IRES序列；或者所述构建体是环状RNA，在所述RNA聚合酶编码单元上游存在驱动所述RNA聚合酶编码单元翻译的IRES序列。
  4. 如权利要求1至3任一项所述的构建体，其中在所述目的蛋白编码单元上游和下游都存在驱动依赖于所述RNA聚合酶之复制的复制启动元件，从而实现所述目的蛋白编码单元的复制；优选地所述RNA聚合酶编码单元不在所述上游复制启动元件和下游复制启动元件之间。
  5. 如权利要求4所述的构建体，其中所述上游复制启动元件包含CSE1 和/或CSE3序列，优选地包含CSE1和CSE3序列，更优选地包含CSE1、CSE2和CSE3序列；和/或所述下游复制启动元件包含CSE4序列。
  6. 如权利要求1至5任一项所述的构建体，其中所述目的蛋白编码单元位于所述RNA聚合酶编码单元的下游或上游。
  7. 如权利要求1至6任一项所述的构建体，其中所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，目的蛋白和RNA聚合酶作为融合蛋白表达，所述融合蛋白中目的蛋白和RNA聚合酶之间存在连接序列，所述连接序列包含编码蛋白酶识别位点的区段，在所述融合蛋白经蛋白酶识别并切割后提供所述RNA聚合酶，优选地，所述蛋白酶识别位点为2A肽序列。
  8. 如权利要求1至6任一项所述的构建体，其中所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，在所述目的蛋白编码单元和RNA聚合酶编码单元之间存在驱动所述RNA聚合酶编码单元翻译的IRES序列。
  9. 如权利要求1至6任一项所述的构建体，其中所述目的蛋白编码单元位于所述RNA聚合酶编码单元的上游，在所述目的蛋白编码单元和RNA聚合酶编码单元之间存在自裂解HDV样核酶(HDV-like Ribozymes)。
  10. 如权利要求1至6任一项所述的构建体，其中所述目的蛋白编码单元位于所述RNA聚合酶编码单元的下游，其中所述构建体按5’-3’方向包含：驱动下游编码序列翻译的5’帽结构或IRES序列、所述RNA聚合酶编码单元、所述上游复制启动元件、所述目的蛋白编码单元和所述下游复制启动元件。
  11. 如权利要求10所述构建体，其中所述构建体按5’-3’方向包含：
    (1)5’UTR序列，包含5’帽结构；
    (2)编码RNA聚合酶的一种或多种核苷酸序列；
    (3)指导RNA复制子复制的5’端识别序列和亚基因组启动子SGP；
    (4)多克隆位点和/或目的基因或异源核苷酸序列：其受所述亚基因组启动子控制；
    (5)指导RNA复制子复制的3’端识别序列；以及
    (6)保证RNA的稳定性以及目的基因有效复制的3’UTR和3’polyA。
  12. 如权利要求1至11任一所述的构建体，其为DNA，其中所述RNA聚合酶编码单元上游存在亚基因组启动子SGP。
  13. 如权利要求1至12任一所述的构建体，其中所述RNA聚合酶是病毒复制酶，特别地所述病毒复制酶为甲病毒的复制酶，
    优选地，所述甲病毒选自塞姆利基森林病毒(Semliki forest virus)、巴马森林病毒(Barmah forest virus)、基孔肯尼雅病毒(Chikungunya virus)、欧尼恩病毒(O’nyong-nyong virus)、罗斯河病毒(Ross river virus)、贝巴鲁病毒(Bebaru virus)、盖他病毒(Getah virus)、鹭山病毒(Sagiyama virus)、马雅罗病毒(Mayaro virus)及其亚型乌纳(Una virus)病毒；委内瑞拉马脑炎复合体包括委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus)、卡巴斯欧病毒(Cabassou virus)、沼泽地病毒(Everglades virus)、穆坎布病毒(Mucambo virus)、图那特病毒(Tonate virus)、皮克苏纳病毒(Pixuna virus)、莫索·达斯·佩德拉斯病毒(Mosso das pedras virus)、内格罗河病毒(Rio Negro virus)；西部马脑炎复合体包括西部马脑炎病毒(Western equine encephalitis virus)、辛德毕斯病毒(Sindbis virus)、奥拉病毒(Aura virus)、瓦塔罗阿病毒(Whataroa)、高地J病毒(Highlands J virus)、摩根堡病毒(Fort Morgan virus)及其亚型博吉河病毒(Creek virus)；东部马脑炎病毒(Eastern equine encephalitis virus)、 米德尔堡病毒(Middelburg virus)、图那特病毒(Trocara virus)、孜拉加奇病毒(Kyzylagach virus)、恩杜姆病毒(Ndumu virus)、巴班肯病毒(Babanki virus)、挪威鲑鱼甲病毒(Norwegian salmonid alphavirus)、鲑鱼胰腺病病毒(Salmon pancreatic disease virus)、睡病病毒(Sleeping disease virus)和南方象海豹病毒(Southern elephant seal virus)，
    更优选地，所述甲病毒选自塞姆利基森林病毒、委内瑞拉马脑炎病毒、辛德毕斯病毒和基孔肯雅病毒。
  14. 如权利要求10至13任一项所述的构建体，其中，所述5’帽结构是天然5’帽或5’帽类似物。
  15. 如权利要求1至14任一项所述的构建体，其包含5’UTR，所述5’UTR来源于任一基因的5’UTR或其突变体，优选地5’UTR来源于与RNA聚合酶来源相同的病毒。
  16. 如权利要求4至15任一项所述的构建体，其中，所述复制启动元件能够被所述RNA聚合酶识别并指导其复制，
    优选地，所述复制启动元件与所述RNA聚合酶来源于相同病毒或不同病毒，更优选为来源于相同病毒。
  17. 如权利要求1至16任一项所述的构建体，其包含3’UTR，所述3’UTR来源于任一基因的3’UTR或其突变体，优选地3’UTR来源于RNA聚合酶相同病毒。
  18. 如权利要求1至17任一项所述的构建体，其包含3’poly(A)。
  19. 如权利要求11至18任一项所述的构建体，其中所述亚基因组启动子来源于病毒结构蛋白的启动子，优选地，所述亚基因组启动子与所述RNA聚 合酶来源于相同病毒。
  20. 一种载体，其包含或编码如权利要求1至19任一项所述的构建体。
  21. 一种药物组合物，其包含如权利要求1至19任一项所述的构建体或如权利要求20所述的载体，其中所述目的蛋白是治疗性蛋白。
  22. 一种疫苗组合物，其包含如权利要求1至19任一项所述的构建体或如权利要求20所述的载体，其中所述目的蛋白是能引发保护性免疫反应的抗原，例如所述抗原源自人、动物、植物、病毒、细菌和/或寄生虫。
  23. 一种表达目的蛋白的方法，其包括将权利要求1至19任一项所述构建体引入能依赖于所述构建体来表达目的蛋白的目的细胞。
  24. 一种免疫接种方法，其包括将权利要求1至19任一项所述的构建体接种到有此需要的对象。
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