CN117070464A - Protein-coated self-replicating RNA and preparation method thereof - Google Patents

Protein-coated self-replicating RNA and preparation method thereof Download PDF

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CN117070464A
CN117070464A CN202310546809.3A CN202310546809A CN117070464A CN 117070464 A CN117070464 A CN 117070464A CN 202310546809 A CN202310546809 A CN 202310546809A CN 117070464 A CN117070464 A CN 117070464A
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protein
nucleic acid
acid molecule
self
recombinant cell
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吴可行
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Shanghai Xingshen Biotechnology Co ltd
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Shanghai Xingshen Biotechnology Co ltd
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Abstract

The application provides a protein-coated self-replicating RNA and a preparation method thereof, wherein the protein-coated self-replicating RNA is obtained by culturing recombinant cells, and the recombinant cells comprise: a first nucleic acid molecule comprising a self-replicating RNA molecule; a second nucleic acid molecule encoding a delivery vehicle protein. The delivery vehicle protein encoded by the second nucleic acid molecule is capable of self-assembling in a cell to encapsulate the self-replicating RNA molecule, whereby in vivo delivery of the self-replicating RNA molecule can be achieved.

Description

Protein-coated self-replicating RNA and preparation method thereof
Technical Field
The application relates to the field of biology, in particular to a protein-coated self-replicating RNA and a preparation method thereof.
Background
The development of mRNA-based drugs or mRNA vaccines has become one of the focus of attention in recent years. However, traditional mRNA is not very stable and degrades within a few days of the cell, resulting in an unpredictable level of protein expression. If used for long term disease treatment, patients may be required to inject large amounts of mRNA, which may increase the toxic side effects of mRNA treatment.
Self-replicating RNA molecules can replicate themselves in the cytoplasm, thus achieving protein expression levels not lower than conventional mRNA at very low doses, and long-acting expression of the protein over time, thus exhibiting a more promising technical advantage over mRNA.
However, there is currently a lack of effective delivery means for RNA molecules, which greatly limits the application of self-replicating RNA molecule technology.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent.
In one aspect of the application, the application provides a recombinant cell. According to an embodiment of the application, the recombinant cell comprises: a first nucleic acid molecule comprising a self-replicating RNA molecule; a second nucleic acid molecule encoding a delivery vehicle protein.
To achieve self-replicating RNA molecules into cells and stable in the cells, they are carried on a delivery vehicle that can carry the self-replicating RNA molecules into cells.
By providing the first nucleic acid molecule comprising the self-replicating RNA molecule and the second nucleic acid molecule encoding the delivery carrier protein in the form of recombinant cells, the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cells to encapsulate the self-replicating RNA molecule, and the delivery carrier encapsulated with the self-replicating RNA molecule produced by the cell strain can realize in vivo delivery of the self-replicating RNA molecule, is beneficial to realizing self-replication of RNA in the cells, maintains long-acting high protein level and plays a role better.
In another aspect of the application, the application provides a method of preparing a recombinant cell as described above. According to an embodiment of the application, the method comprises: allowing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, said first nucleic acid molecule and second nucleic acid molecule being as defined in the recombinant cell as described above.
In yet another aspect of the application, the application provides a method of preparing a self-replicating RNA molecule composition. According to an embodiment of the application, the self-replicating RNA molecule comprises a self-replicating RNA and a delivery carrier protein, the method comprising: culturing the recombinant cell described above; and obtaining the self-replicating RNA molecule composition.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a self-replicating RNA molecule (reRNA) according to one embodiment of the application TM ) Is a structural schematic diagram of (a);
FIG. 2 shows a schematic structural diagram of a protein-RNA complex containing a self-replicating RNA molecule according to one embodiment of the application;
FIG. 3 shows a helper plasmid as a source of coat protein for a RERNA according to one embodiment of the application TM A production schematic;
FIG. 4 shows a VSV-G helper plasmid according to an embodiment of the present application as an helper material, reRNA TM -GFP-producing cell pictures;
FIG. 5 shows an LCMV-GP helper plasmid as an adjunct reRNA according to one embodiment of the present application TM -GFP-producing cell pictures;
FIG. 6 shows a coat protein stably transfected cell line reRNA according to one embodiment of the application TM A production schematic;
FIG. 7 shows a 293 cell line and a VSV-G stably transformed 293 cell line reRNA according to an embodiment of the application TM GFP productionCell picture.
Detailed Description
Embodiments of the present application are described in detail below. The following examples are illustrative only and are not to be construed as limiting the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The term "self-replicating RNA molecule" as used herein may also be referred to as "self-amplifying RNA" and is distinguished from common mRNA by the fact that it can replicate itself using its own RNA sequence as a template. According to embodiments of the application, self-replicating RNA molecules can also be translated and replicated in the cytoplasm without entering the nucleus, and the potential risk of integration with the genome can be avoided. Typically mRNA encodes a protein to be expressed and intracellular ribosomes are used to accomplish translation and protein production. According to embodiments of the present application, the self-replicating RNA molecule carries a sequence capable of expressing an RNA polymerase (RNA-dependent RNA polymerase), which can be used as a template to produce more self-replicating RNA molecules after the RNA molecule is produced in the cytoplasm by translation to produce the RNA polymerase.
Recombinant cells
In one aspect of the application, the application provides a recombinant cell. According to an embodiment of the application, the recombinant cell comprises: a first nucleic acid molecule comprising a self-replicating RNA molecule; a second nucleic acid molecule encoding a delivery vehicle protein.
To achieve self-replicating RNA molecules into cells and stable in the cells, they are carried on a delivery vehicle that can carry the self-replicating RNA molecules into cells.
By providing the first nucleic acid molecule comprising the self-replicating RNA molecule and the second nucleic acid molecule encoding the delivery carrier protein in the form of recombinant cells, the delivery carrier protein encoded and expressed by the second nucleic acid molecule can self-assemble in the cells to encapsulate the self-replicating RNA molecule, and the delivery carrier encapsulated with the self-replicating RNA molecule produced by the cell strain can realize in vivo delivery of the self-replicating RNA molecule, is beneficial to realizing self-replication of RNA in the cells, maintains long-acting high protein level and plays a role better.
The inventors have found during the course of the study that, since self-replicating RNA molecules link at least the sequence encoding the RNA polymerase to the sequence expressing the target protein, the molecular weight of the entire mRNA molecule is much greater than that of conventional mRNA, and that excessive molecular weight may result in significant decreases in delivery efficiency, translation efficiency, and replication efficiency. In order to improve these efficiencies, the inventors have conducted intensive studies and have desired to find the shortest nucleic acid fragment that can normally perform the functions of self-replication and translation.
According to an embodiment of the application, the self-replicating RNA molecule comprises: a first RNA sequence encoding an N protein or a functional fragment thereof; a second RNA sequence encoding a P protein or a functional fragment thereof; a third RNA sequence encoding an L protein or a functional fragment thereof; and a target molecule coding region encoding at least one target molecule.
Referring to FIGS. 1 and 2, the inventors have conducted intensive studies on intracellular translation and self-amplification mechanisms of RNAs of various RNA viruses, and have found that by using RNA molecules encoding N, P and L proteins from Rhabdoviruses as core regions, self-replication and translation of RNAs in animal cells can be achieved, and the core regions act as powerful "engines" which can provide efficient transcriptional amplification and initiation of "kinetic energy" of macromolecular proteins, and can further carry "cargo regions" to replicate or translate target molecules, which cover almost all protein drugs on the market at present. According to embodiments of the present application, the "cargo region" may be designed to allow the body to produce a variety of peptides, enzymes, antibodies, channel proteins, receptor proteins, etc. in cells, thereby achieving different prophylactic or therapeutic objectives, covering tumor lines, vaccine lines, rareDisease pipeline and prospective general-purpose product pipeline. The novel self-replicating RNA molecule is named as the rRNA TM 。
The term "functional fragment" as used herein refers to a part of the full-length sequence of a protein, but can still exert a function related to the self-replication of an RNA molecule, and may be, for example, a truncated full-length sequence or a protein in which the amino acid sequence of the full-length sequence of the protein is altered, such as substitution, mutation or deletion. According to embodiments of the application, RNA molecules can be bound to functional fragments of N protein, RNA is protected from nuclease, N protein can be bound to functional fragments of P protein, L polymerase can be positioned on a template, and can also serve as a basic component of an RNA polymerase transcription and replication complex, and further the functional fragments of L protein can play the function of RNA polymerase and are related to RNA transcription and replication.
According to an embodiment of the application, at least one of the N protein, the P protein, the L protein is each independently from a rhabdoviridae virus.
The N protein, P protein, L protein may be from the vesicular stomatitis virus indiana strain, N proteins including, but not limited to Uniprot ID: sequences of P03521, P11212, Q77E03, Q8B0H4, B7UCZ 2; the P proteins include, but are not limited to Uniprot ID: sequences of P04880, Q8B0H8, P04879, P03520, B7UCZ 3; l proteins include, but are not limited to, uniprot ID: Q8B0H0, Q98776, Q8B0I0, Q8B0H5, P03523.
The N protein, the P protein and the L protein can be from vesicular stomatitis virus New Jersey strain, and the N protein comprises but is not limited to Uniprot ID: sequences of P04881, Q89034, S5TKS4, Q89036, Q89037; the P proteins include, but are not limited to Uniprot ID: sequences of P04877, Q89057, Q89052, Q89050, Q89049; l proteins include, but are not limited to, uniprot ID: p16379, P16379, I7DDL0, Q8B545, S5TC 82.
The N protein, the P protein and the L protein can also be from other vesicular virus genera (such as Qian Dipu vesicular virus, malabar vesicular virus) and rabies virus genera.
According to an embodiment of the application, the N protein has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% homology thereto, said P protein having the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 80% homology thereto, said L protein having the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least 80% homology thereto.
MSVTVKRIIDNTVVVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTALLLRSTR
ARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPT
PDMMQYAKRAVMSLQGLREKTIGKYAKSEFDK(SEQ ID NO：1)
MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSDTESEPEIEDNQGLYAPD
PEAEQVEGFIQGPLDDYADEEVDVVFTSDWKQPELESDEHGKTLRLTSPEGLSGEQKSQWLSTIKAVVQSAKYWNLAE
CTFEASGEGVIMKERQITPDVYKVTPVMNTHPSQSEAVSDVWSLSKTSMTFQPKKASLQPLTISLDELFSSRGEFISV
GGDGRMSHKEAILLGLRYKKLYNQARVKYSL(SEQ ID NO：2)
MEVHDFETDEFNDFNEDDYATREFLNPDERMTYLNHADYNLNSPLISDDIDNLIRKFNSLPIPSMWDSKNWDGVLEML
TSCQANPIPTSQMHKWMGSWLMSDNHDASQGYSFLHEVDKEAEITFDVVETFIRGWGNKPIEYIKKERWTDSFKILAY
LCQKFLDLHKLTLILNAVSEVELLNLARTFKGKVRRSSHGTNICRIRVPSLGPTFISEGWAYFKKLDILMDRNFLLMV
KDVIIGRMQTVLSMVCRIDNLFSEQDIFSLLNIYRIGDKIVERQGNFSYDLIKMVEPICNLKLMKLARESRPLVPQFP
HFENHIKTSVDEGAKIDRGIRFLHDQIMSVKTVDLTLVIYGSFRHWGHPFIDYYTGLEKLHSQVTMKKDIDVSYAKAL
ASDLARIVLFQQFNDHKKWFVNGDLLPHDHPFKSHVKENTWPTAAQVQDFGDKWHELPLIKCFEIPDLLDPSIIYSDK
SHSMNRSEVLKHVRMNPNTPIPSKKVLQTMLDTKATNWKEFLKEIDEKGLDDDDLIIGLKGKERELKLAGRFFSLMSW
KLREYFVITEYLIKTHFVPMFKGLTMADDLTAVIKKMLDSSSGQGLKSYEAICIANHIDYEKWNNHQRKLSNGPVFRV
MGQFLGYPSLIERTHEFFEKSLIYYNGRPDLMRVHNNTLINSTSQRVCWQGQEGGLEGLRQKGWSILNLLVIQREAKI
RNTAVKVLAQGDNQVICTQYKTKKSRNVVELQGALNQMVSNNEKIMTAIKIGTGKLGLLINDDETMQSADYLNYGKIP
IFRGVIRGLETKRWSRVTCVTNDQIPTCANIMSSVSTNALTVAHFAENPINAMIQYNYFGTFARLLLMMHDPALRQSL
YEVQDKIPGLHSSTFKYAMLYLDPSIGGVSGMSLSRFLIRAFPDPVTESLSFWRFIHVHARSEHLKEMSAVFGNPEIA
KFRITHIDKLVEDPTSLNIAMGMSPANLLKTEVKKCLIESRQTIRNQVIKDATIYLYHEEDRLRSFLWSINPLFPRFL
SEFKSGTFLGVADGLISLFQNSRTIRNSFKKKYHRELDDLIVRSEVSSLTHLGKLHLRRGSCKMWTCSATHADTLRYK
SWGRTVIGTTVPHPLEMLGPQHRKETPCAPCNTSGFNYVSVHCPDGIHDVFSSRGPLPAYLGSKTSESTSILQPWERE
SKVPLIKRATRLRDAISWFVEPDSKLAMTILSNIHSLTGEEWTKRQHGFKRTGSALHRFSTSRMSHGGFASQSTAALT
RLMATTDTMRDLGDQNFDFLFQATLLYAQITTTVARDGWITSCTDHYHIACKSCLRPIEEITLDSSMDYTPPDVSHVL
KTWRNGEGSWGQEIKQIYPLEGNWKNLAPAEQSYQVGRCIGFLYGDLAYRKSTHAEDSSLFPLSIQGRIRGRGFLKGL
LDGLMRASCCQVIHRRSLAHLKRPANAVYGGLIYLIDKLSVSPPFLSLTRSGPIRDELETIPHKIPTSYPTSNRDMGV
IVRNYFKYQCRLIEKGKYRSHYSQLWLFSDVLSIDFIGPFSISTTLLQILYKPFLSGKDKNELRELANLSSLLRSGEG
WEDIHVKFFTKDILLCPEEIRHACKFGIAKDNNKDMSYPPWGRESRGTITTIPVYYTTTPYPKMLEMPPRIQNPLLSG
IRLGQLPTGAHYKIRSILHGMGIHYRDFLSCGDGSGGMTAALLRENVHSRGIFNSLLELSGSVMRGASPEPPSALETL
GGDKSRCVNGETCWEYPSDLCDPRTWDYFLRLKAGLGLQIDLIVMDMEVRDSSTSLKIETNVRNYVHRILDEQGVLIY
KTYGTYICESEKNAVTILGPMFKTVDLVQTEFSSSQTSEVYMVCKGLKKLIDEPNPDWSSINESWKNLYAFQSSEQEF
ARAKKVSTYFTLTGIPSQFIPDPFVNIETMLQIFGVPTGVSHAAALKSSDRPADLLTISLFYMAIISYYNINHIRVGP
IPPNPPSDGIAQNVGIAITGISFWLSLMEKDIPLYQQCLAVIQQSFPIRWEAVSVKGGYKQKWSTRGDGLPKDTRISD
SLAPIGNWIRSLELVRNQVRLNPFNEILFNQLCRTVDNHLKWSNLRRNTGMIEWINRRISKEDRSILMLKSDLHEENSWRD(SEQ ID NO：3)
According to an embodiment of the application, the self-replicating core sequence includes:
a first RNA sequence encoding an N protein or a functional fragment thereof, said first RNA sequence having a sequence as set forth in SEQ ID NO:4, a nucleotide sequence shown in seq id no;
a second RNA sequence encoding a P protein or a functional fragment thereof, said second RNA sequence having the sequence set forth in SEQ ID NO:5, a nucleotide sequence shown in seq id no;
a third RNA sequence encoding an L protein or a functional fragment thereof, said third RNA sequence having the sequence set forth in SEQ ID NO:6, and a nucleotide sequence shown in FIG. 6.
AUGUCCGUGACCGUGAAGAGAAUCAUCGAUAACACCGUGGUGGUGCCUAAGCUGCCUGCCAACGAGGACCCUGUGGAG
UACCCCGCCGACUACUUCAGGAAGUCCAAGGAGAUCCCCCUGUACAUCAACACCACAAAGAGCCUGAGCGACCUGAGG
GGCUACGUGUACCAGGGCCUGAAGUCCGGCAACGUGUCCAUCAUCCACGUGAACUCCUACCUGUACGGCGCCCUGAAG
GAUAUCAGGGGCAAGCUGGACAAGGACUGGAGCAGCUUCGGCAUCAACAUCGGCAAGGCCGGCGAUACAAUCGGCAUC
UUCGAUCUGGUGUCCCUGAAGGCCCUGGACGGCGUGCUGCCUGACGGCGUUUCCGACGCCAGCAGGACAAGCGCCGAU
GACAAGUGGCUGCCUCUGUACCUGCUGGGCCUGUACAGAGUGGGCAGAACACAGAUGCCCGAGUACAGAAAGAAGCUG
AUGGACGGCCUGACCAACCAGUGCAAGAUGAUCAACGAGCAGUUCGAGCCCCUGGUGCCUGAGGGCAGGGACAUCUUC
GACGUGUGGGGCAACGACAGCAACUACACAAAGAUCGUGGCCGCCGUGGACAUGUUCUUCCACAUGUUCAAGAAGCAC
GAGUGCGCCUCCUUCAGAUACGGCACCAUCGUGUCCAGAUUCAAGGACUGUGCCGCCCUGGCCACCUUCGGCCACCUG
UGUAAGAUCACCGGCAUGUCCACCGAGGAUGUGACAACAUGGAUCUUGAACAGAGAGGUGGCCGAUGAGAUGGUGCAG
AUGAUGCUGCCUGGCCAGGAGAUCGAUAAGGCCGAUAGCUACAUGCCUUACCUGAUCGAUUUCGGCCUGAGCAGCAAG
UCCCCUUACAGCAGCGUGAAGAACCCUGCCUUCCACUUCUGGGGCCAGCUGACAGCCCUGCUGCUGAGGUCCACAAGA
GCCAGAAACGCCAGGCAGCCUGAUGAUAUCGAGUACACAUCCCUGACCACCGCCGGCCUGCUGUACGCCUACGCCGUG
GGAAGCAGCGCCGAUCUGGCCCAGCAGUUCUGUGUGGGCGACAACAAGUACACCCCCGAUGACAGCACCGGCGGCCUG
ACCACAAACGCCCCCCCUCAGGGCAGAGAUGUGGUGGAGUGGCUGGGCUGGUUCGAGGACCAGAACAGAAAGCCCACC
CCUGACAUGAUGCAGUACGCCAAGAGAGCCGUGAUGUCCCUGCAGGGCCUGAGAGAGAAGACCAUCGGCAAGUACGCCAAGUCCGAGUUCGAUAAGUGA(SEQ ID NO：4)
AUGGAUAACCUGACAAAGGUGAGAGAGUACCUGAAGUCCUACUCCAGACUGGACCAGGCCGUGGGCGAGAUCGACGAGAUCGAGGCCCAGAGGGCCGAGAAGUCCAACUACGAGCUGUUCCAGGAGGAUGGCGUGGAGGAGCACACAAAGCCUUCCUACUUCCAGGCCGCCGAUGAUAGCGACACCGAGUCCGAGCCUGAGAUCGAGGACAACCAGGGCCUGUACGCCCCUGAUCCCGAGGCCGAGCAGGUGGAGGGCUUCAUCCAGGGCCCCCUGGACGAUUACGCCGAUGAGGAGGUGGACGUGGU
GUUCACCUCCGACUGGAAGCAGCCUGAGCUGGAGUCCGAUGAGCACGGCAAGACCCUGAGACUGACCUCCCCCGAGGG
CCUGUCCGGCGAACAGAAGAGCCAGUGGCUGUCCACCAUCAAGGCCGUGGUGCAGAGCGCCAAGUACUGGAACCUGGC
CGAGUGCACAUUCGAGGCCUCCGGCGAGGGCGUGAUCAUGAAGGAGAGACAGAUCACCCCUGAUGUGUACAAGGUGAC
CCCUGUGAUGAACACCCACCCUAGCCAGUCCGAGGCCGUGAGCGACGUGUGGAGCCUGUCCAAGACCUCCAUGACCUU
CCAGCCUAAGAAGGCCUCCCUGCAGCCUCUGACCAUCAGCCUGGACGAGCUGUUCUCCUCCAGAGGCGAGUUCAUCUC
CGUGGGCGGCGACGGCAGGAUGUCCCACAAGGAGGCCAUCCUGCUGGGCCUGAGGUACAAGAAGCUGUACAACCAGGC
CAGGGUGAAGUACAGCCUGUGA(SEQ ID NO：5)
AUGGAGGUGCACGACUUCGAGACAGACGAGUUCAACGACUUCAACGAGGAUGAUUACGCCACCAGGGAGUUCCUGAAC
CCCGAUGAGAGGAUGACAUACCUGAACCACGCCGAUUACAACCUGAACAGCCCUCUGAUCUCCGAUGAUAUCGACAAC
CUGAUCAGGAAGUUCAACUCCCUGCCUAUCCCUAGCAUGUGGGAUUCCAAGAACUGGGAUGGCGUGCUGGAGAUGCUG
ACAUCCUGCCAGGCCAACCCUAUCCCCACCUCCCAGAUGCACAAGUGGAUGGGCAGCUGGCUGAUGAGCGACAACCAC
GAUGCCAGCCAGGGCUACUCCUUCCUGCACGAGGUGGACAAGGAGGCCGAGAUCACCUUCGAUGUGGUGGAGACAUUC
AUCAGGGGCUGGGGCAACAAGCCUAUCGAGUACAUCAAGAAGGAGAGGUGGACCGACUCCUUCAAGAUCCUGGCCUAC
CUGUGCCAGAAGUUCCUGGAUCUGCACAAGCUGACCCUGAUCCUGAACGCCGUGUCCGAGGUGGAGCUGCUGAACCUG
GCCAGAACCUUCAAGGGCAAGGUGAGAAGGAGCAGCCACGGCACCAACAUCUGUAGAAUCAGGGUGCCUUCCCUGGGC
CCUACAUUCAUCAGCGAGGGCUGGGCCUACUUCAAGAAGCUGGACAUCCUGAUGGAUAGAAACUUCCUGCUGAUGGUG
AAGGACGUGAUCAUCGGCAGGAUGCAGACAGUGCUGAGCAUGGUGUGUAGGAUCGACAACCUCUUCUCCGAGCAGGAC
AUCUUCUCCCUGCUGAACAUCUACAGAAUCGGCGAUAAGAUCGUGGAGAGACAGGGCAACUUCAGCUACGACCUGAUC
AAGAUGGUGGAGCCCAUCUGCAACCUGAAGCUGAUGAAGCUGGCCAGGGAGAGCAGACCCCUGGUGCCUCAGUUCCCU
CACUUCGAGAACCACAUCAAGACAAGCGUGGAUGAGGGCGCCAAGAUCGACAGAGGCAUCAGGUUCCUGCACGAUCAG
AUCAUGAGCGUGAAAACUGUUGAUCUGACCCUGGUCAUCUAUGGCAGCUUCAGACACUGGGGCCACCCCUUCAUCGAC
UACUACACCGGCCUGGAGAAGCUGCACUCCCAGGUGACCAUGAAGAAGGACAUCGAUGUGAGCUACGCCAAGGCCCUG
GCCUCCGAUCUGGCCAGAAUCGUGCUGUUCCAGCAGUUCAACGAUCACAAGAAGUGGUUCGUGAACGGCGACCUGCUG
CCUCACGACCACCCUUUCAAGAGCCACGUGAAGGAGAACACAUGGCCCACCGCCGCCCAGGUGCAGGAUUUCGGCGAC
AAGUGGCACGAGCUGCCUCUGAUCAAGUGCUUCGAGAUCCCUGACCUGCUGGAUCCUUCCAUCAUCUACUCCGAUAAG
UCCCACUCCAUGAACAGAAGCGAGGUGCUGAAGCACGUGAGGAUGAACCCUAACACCCCCAUCCCCUCCAAGAAGGUG
CUGCAGACCAUGCUGGACACCAAGGCCACAAACUGGAAGGAGUUCCUGAAGGAGAUCGAUGAGAAGGGCCUGGAUGAC
GACGACCUGAUCAUCGGCCUGAAGGGCAAGGAGAGGGAGCUGAAGCUGGCCGGCAGAUUCUUCAGCCUGAUGAGCUGG
AAGCUGAGAGAGUACUUCGUGAUCACCGAGUACCUGAUCAAGACACACUUCGUGCCUAUGUUCAAGGGCCUGACAAUG
GCCGACGAUCUGACAGCCGUGAUCAAGAAGAUGCUGGAUAGCUCCAGCGGCCAGGGCCUGAAGAGCUACGAGGCCAUC
UGUAUCGCCAACCACAUCGAUUACGAGAAGUGGAACAACCACCAGAGGAAGCUGUCCAACGGCCCCGUGUUCAGGGUC
AUGGGCCAGUUCCUGGGCUACCCCAGCCUGAUCGAGAGAACACACGAGUUCUUCGAGAAGAGCCUGAUCUACUACAAC
GGCAGACCUGACCUGAUGAGAGUGCACAACAACACCCUGAUCAACUCCACAUCCCAGAGAGUGUGUUGGCAGGGCCAG
GAGGGCGGCCUGGAGGGACUGAGACAGAAGGGCUGGUCCAUCCUGAACCUGCUGGUCAUUCAGAGAGAGGCCAAGAUC
AGAAACACCGCCGUGAAGGUGCUGGCCCAGGGCGACAACCAGGUCAUUUGCACACAGUACAAGACCAAGAAGAGCAGG
AACGUGGUGGAGCUGCAGGGCGCCCUGAACCAGAUGGUGAGCAACAACGAGAAGAUCAUGACCGCCAUCAAGAUCGGC
ACCGGCAAGCUGGGCCUGCUGAUCAACGAUGACGAGACAAUGCAGAGCGCCGACUACCUGAACUACGGCAAGAUCCCC
AUCUUCAGGGGCGUGAUCAGAGGCCUGGAGACAAAGAGAUGGUCCAGAGUGACAUGUGUGACCAACGACCAGAUCCCU
ACCUGCGCCAACAUCAUGUCCUCCGUGUCCACAAACGCCCUGACCGUGGCCCACUUCGCCGAGAACCCCAUCAACGCC
AUGAUCCAGUACAACUACUUCGGCACAUUCGCCAGACUGCUGCUGAUGAUGCACGACCCCGCCCUGAGGCAGAGCCUG
UACGAGGUGCAGGACAAGAUCCCUGGCCUGCACAGCAGCACCUUCAAGUACGCCAUGCUGUACCUGGACCCUAGCAUC
GGCGGCGUGAGCGGCAUGUCCCUGUCCAGGUUCCUGAUCAGAGCCUUCCCUGACCCUGUGACAGAGAGCCUGUCCUUC
UGGAGGUUCAUCCACGUGCACGCCAGGAGCGAGCACCUGAAGGAGAUGAGCGCCGUGUUCGGCAACCCCGAGAUCGCC
AAGUUCAGAAUCACCCACAUCGACAAGCUGGUGGAGGAUCCCACAAGCCUGAACAUCGCCAUGGGCAUGAGCCCUGCC
AACCUGCUGAAAACUGAAGUUAAGAAGUGCCUGAUCGAGUCCAGACAGACCAUCAGGAACCAGGUCAUUAAGGAUGCC
ACCAUCUACCUGUACCACGAGGAGGAUAGACUGAGAAGCUUCCUGUGGUCCAUCAACCCUCUGUUCCCCAGAUUCCUG
AGCGAGUUCAAGUCCGGCACAUUCCUGGGCGUGGCCGACGGCCUGAUCAGCCUGUUCCAGAACUCCAGAACCAUCAGA
AACUCCUUCAAGAAGAAGUACCACAGGGAGCUGGACGAUCUGAUCGUGAGGAGCGAGGUGUCCUCCCUGACACACCUG
GGCAAGCUGCACCUGAGAAGGGGCAGCUGUAAGAUGUGGACAUGUAGCGCCACCCACGCCGACACCCUGAGGUACAAG
AGCUGGGGCAGAACCGUGAUCGGCACCACCGUGCCUCACCCCCUGGAGAUGCUCGGCCCUCAGCACAGGAAGGAGACA
CCUUGCGCCCCUUGCAACACAAGCGGCUUCAACUACGUGAGCGUGCACUGCCCUGACGGCAUCCACGAUGUGUUCAGC
AGCAGAGGCCCCCUGCCUGCCUACCUGGGCAGCAAGACAAGCGAGUCCACAUCCAUCCUGCAGCCUUGGGAGAGAGAG
UCCAAGGUGCCCCUGAUCAAGAGGGCCACAAGGCUGAGGGACGCCAUCAGCUGGUUCGUGGAGCCCGAUUCCAAGCUG
GCCAUGACCAUCCUGAGCAACAUCCACAGCCUGACCGGCGAGGAGUGGACAAAGAGGCAGCACGGCUUCAAGAGGACA
GGCUCCGCCCUGCACAGGUUCUCCACCUCCAGAAUGAGCCACGGCGGCUUCGCCUCCCAGUCCACAGCCGCUCUGACC
AGACUGAUGGCCACCACAGACACCAUGAGAGACUUGGGCGAUCAGAACUUCGAUUUCCUGUUCCAGGCCACACUGCUG
UACGCCCAGAUCACAACCACCGUGGCCAGAGACGGCUGGAUCACAAGCUGUACCGACCACUACCACAUCGCCUGCAAG
AGCUGCCUGAGGCCCAUCGAGGAGAUCACACUGGACAGCAGCAUGGAUUACACCCCCCCCGACGUGUCCCACGUGCUG
AAAACUUGGCGUAACGGCGAGGGCAGCUGGGGCCAGGAGAUCAAGCAGAUCUACCCCCUGGAGGGCAACUGGAAGAAC
CUGGCCCCCGCCGAGCAGAGCUACCAGGUGGGAAGGUGCAUCGGCUUCCUGUACGGCGACCUGGCCUACAGAAAGUCC
ACCCACGCCGAGGAUAGCAGCCUGUUCCCUCUGUCCAUCCAGGGCAGGAUCAGAGGCAGAGGCUUCCUGAAGGGCCUG
CUGGACGGCCUGAUGAGGGCCUCCUGCUGUCAGGUCAUUCACAGGAGAUCCCUGGCUCACCUGAAGAGGCCUGCCAAC
GCCGUGUACGGCGGCCUGAUCUACCUGAUCGACAAGCUCAGCGUGAGCCCUCCCUUCCUGUCCCUGACCAGGAGCGGC
CCUAUCAGGGACGAGCUGGAGACAAUCCCUCACAAGAUCCCCACAAGCUACCCCACCAGCAACAGAGACAUGGGCGUG
AUCGUGAGAAACUACUUCAAGUACCAGUGCAGGCUGAUCGAGAAGGGCAAGUACAGAAGCCACUACUCCCAGCUGUGG
CUGUUCAGCGACGUGCUGUCCAUCGACUUCAUCGGCCCCUUCUCCAUCUCCACCACACUGCUGCAGAUCCUGUACAAG
CCUUUCCUGUCCGGCAAGGAUAAGAACGAGCUGAGGGAGCUGGCCAACCUGAGCAGCCUGCUGAGGAGCGGCGAGGGC
UGGGAGGAUAUCCACGUGAAGUUCUUCACCAAGGACAUCCUGCUGUGCCCUGAGGAGAUCAGACACGCCUGCAAGUUC
GGCAUCGCCAAGGAUAACAACAAGGACAUGAGCUACCCUCCUUGGGGCAGAGAGAGCAGAGGCACCAUCACAACAAUC
CCCGUGUACUACACCACAACCCCCUACCCUAAGAUGCUGGAGAUGCCCCCCAGGAUCCAGAACCCCCUGCUGUCCGGC
AUCAGGCUGGGCCAGCUGCCCACCGGAGCCCAUUACAAGAUCAGGAGCAUCCUGCACGGCAUGGGCAUCCACUACAGG
GACUUCCUGAGCUGUGGCGAUGGCAGCGGCGGCAUGACAGCCGCUCUCCUGAGGGAGAACGUGCACAGCAGGGGCAUC
UUCAACUCCCUCCUGGAGCUGUCCGGCUCCGUGAUGAGGGGCGCCUCCCCUGAGCCCCCUAGCGCUCUGGAGACACUG
GGCGGCGACAAGAGCAGGUGCGUGAACGGCGAGACAUGUUGGGAGUACCCCUCCGACCUGUGUGACCCCAGAACAUGG
GACUACUUCCUGAGGCUGAAGGCCGGCCUGGGCCUGCAGAUCGAUCUGAUCGUCAUGGACAUGGAGGUGAGGGAUAGC
AGCACAAGCCUGAAGAUCGAGACAAACGUGAGAAACUAUGUGCACAGAAUCCUGGACGAGCAGGGCGUGCUGAUCUAC
AAGACCUACGGCACAUACAUCUGUGAGAGCGAGAAGAACGCCGUGACCAUCCUGGGCCCUAUGUUCAAAACCGUGGAU
CUGGUGCAGACAGAGUUCUCCAGCAGCCAGACAUCCGAGGUGUACAUGGUGUGUAAGGGCCUGAAGAAGCUGAUCGAC
GAGCCUAACCCCGACUGGAGCAGCAUCAACGAGAGCUGGAAGAACCUCUACGCCUUCCAGAGCAGCGAGCAGGAGUUC
GCCAGAGCCAAGAAGGUGUCCACAUACUUCACCCUGACAGGCAUCCCCAGCCAGUUCAUCCCUGACCCUUUCGUGAAC
AUCGAGACAAUGCUGCAGAUCUUCGGCGUGCCUACAGGCGUGUCCCACGCCGCUGCCCUGAAGUCCAGCGAUAGGCCC
GCCGAUCUGCUGACCAUCUCCCUGUUCUACAUGGCCAUCAUCAGCUACUACAACAUCAACCACAUCAGGGUGGGCCCC
AUCCCCCCCAACCCCCCUUCUGAUGGCAUCGCCCAGAACGUGGGCAUCGCCAUCACCGGCAUCUCCUUCUGGCUGAGC
CUGAUGGAGAAGGACAUCCCCCUGUACCAGCAGUGCCUGGCCGUGAUCCAGCAGUCCUUCCCUAUCAGAUGGGAGGCC
GUGUCCGUGAAGGGCGGCUACAAGCAGAAGUGGAGCACAAGGGGCGACGGCCUGCCCAAGGACACCAGGAUCUCCGAC
UCCCUGGCCCCCAUCGGCAACUGGAUCAGGAGCCUGGAGCUGGUGAGGAACCAGGUGAGGCUGAACCCUUUCAACGAG
AUCCUGUUCAACCAGCUGUGUAGAACAGUGGACAACCACCUGAAGUGGUCCAACCUGAGAAGGAACACAGGCAUGAUC
GAGUGGAUCAACAGAAGAAUCUCCAAGGAGGACAGAUCCAUCCUGAUGCUGAAGUCCGAUCUGCACGAGGAGAACAGCUGGAGGGACUAA(SEQ ID NO：6)
According to an embodiment of the application, the proteins include non-human proteins and human proteins. The type of human protein is not critical in the present application, as long as it is capable of delivering a nucleic acid molecule into a cell as a delivery vehicle, including but not limited to human proteins including the SNARE family of proteins. According to an embodiment of the application, the non-human protein comprises a viral capsid protein. Compared with the humanized protein, the delivery effect of the viral capsid protein is better, and the viral capsid protein does not have viral genetic material, is easy to infect into cells, has strong specificity of targeted cells, and can realize intracellular delivery of protein and genetic material. In addition, the viral capsid protein is easy to obtain, the efficiency of preparing the delivery carrier is improved, and the cost is reduced.
According to an embodiment of the application, the virus comprises at least one of poxviruses, rabies viruses, flaviviruses, measles viruses, coronaviruses, vesicular stomatitis viruses, newcastle disease viruses, lymphocytic choriomeningitis viruses. The viruses all have capsid proteins, which can self-assemble in cells, thereby encapsulating the first nucleic acid molecule, resulting in a delivery vehicle carrying the first nucleic acid molecule.
According to an embodiment of the application, the delivery carrier protein is selected from vesicular stomatitis virus receptor and/or lymphocytic choriomeningitis virus coat protein. Specifically, vesicular stomatitis virus receptor VSV-G has the sequence set forth in SEQ ID NO:7, the specific sequence information of lymphocytic choriomeningitis virus coat protein LCMV-GP is referred to UniProtKB/Swiss-Prot: p09991. The inventor finds that the self-replicating RNA delivery efficiency of the two proteins is high through a large amount of experimental researches.
According to an embodiment of the application, the first nucleic acid molecule and the second nucleic acid molecule are each independently present in the recombinant cell in free form or integrated into the genome.
According to an embodiment of the application, the first nucleic acid molecule and the second nucleic acid molecule are each independently present in the recombinant cell in the form of a plasmid.
According to an embodiment of the application, the first nucleic acid molecule and the second nucleic acid molecule are present on the same plasmid.
The first nucleic acid molecule and the second nucleic acid molecule may be present in the recombinant cell in three ways, the first way being that both are present in episomal form, e.g. constructed on a plasmid and transferred into the recombinant cell, in episomal form. Specifically, the first nucleic acid molecule and the second nucleic acid molecule may be constructed on the same plasmid, or may be constructed on two different plasmids, respectively, and when constructed on the same plasmid, may be expressed under the same or different promoters, respectively. The second way is that both are present in the cell in a form integrated in the genome of the cell, whereby the objective of long-term stable expression of the first nucleic acid molecule and the second nucleic acid molecule can be achieved. The third way is that one of the two nucleic acid molecules is present in episomal form and the other is present in integrated genomic form, e.g. the first nucleic acid molecule is present in episomal form and the second nucleic acid molecule is present in integrated genomic form, whereby the protein can be expressed with high efficiency.
Method for preparing recombinant cells
In another aspect of the application, the application provides a method of preparing a recombinant cell. According to an embodiment of the application, the method comprises: allowing the cell to carry a first nucleic acid molecule and a second nucleic acid molecule, said first nucleic acid molecule and second nucleic acid molecule being as defined in the recombinant cell as described above. In the cell, the second nucleic acid molecule can express the delivery carrier protein and self-assemble to encapsulate the first nucleic acid molecule, thereby obtaining a composition comprising the delivery carrier protein and the first nucleic acid molecule carried thereby, which aids in delivering the first nucleic acid molecule.
According to an embodiment of the application, the method comprises: introducing at least one of the first nucleic acid molecule and the second nucleic acid molecule into the cell in plasmid form. Thus, the plasmid may be present in the cell in either episomal form or integrated into the genome of the cell.
According to an embodiment of the application, the introduction is performed by transfecting the plasmid. In particular, transfection means include calcium phosphate transfection, liposome transfection or electroporation transfection. Thus, the plasmid can be transferred into the cell rapidly.
According to an embodiment of the application, the second nucleic acid molecule is provided in the form of a lentivirus or CRISPR/Cas 9. Thus, the second nucleic acid molecule is integrated into the genome of the cell, and the purpose of stably expressing the delivery carrier protein for a long period of time is achieved.
According to an embodiment of the application, the cells are selected from HEK 293 cell lines, vero cell lines, CHO-K1 cells or NIH-3T3 cells. Therefore, a stable transgenic cell line can be constructed, and the purpose of stable expression is realized.
Those skilled in the art will appreciate that the features and advantages described above for recombinant cells are equally applicable to the method of preparing recombinant cells and are not described in detail herein.
Methods for preparing self-replicating RNA molecule compositions
In yet another aspect of the application, the application provides a method of preparing a self-replicating RNA molecule composition. According to an embodiment of the application, the self-replicating RNA molecule comprises a self-replicating RNA and a delivery carrier protein, the method comprising: culturing the recombinant cell described above; and obtaining the self-replicating RNA molecule composition.
As described above, the recombinant cell contains a first nucleic acid molecule comprising a self-replicating RNA molecule and a second nucleic acid molecule encoding a delivery carrier protein, and the second nucleic acid molecule expresses the delivery carrier protein by culturing the recombinant cell, and the delivery carrier protein self-assembles to encapsulate the first nucleic acid molecule, thereby obtaining a composition comprising the delivery carrier protein and the first nucleic acid molecule carried thereby, which aids in delivering the first nucleic acid molecule.
The method of how the self-replicating RNA molecule composition is obtained from the recombinant cells is not strictly limited, and the method can be flexibly selected according to actual needs as long as the purpose of separation can be achieved. For example, the recombinant cells in the culture broth obtained by the culture are subjected to centrifugal separation, disruption of the recombinant cells, and the like.
Those skilled in the art will appreciate that the features and advantages described above for recombinant cells are equally applicable to the method of preparing the self-replicating RNA molecule composition and will not be described in detail herein.
The scheme of the present application will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
reRNA TM The protein delivery system of (2) requires adding adjuvants to the production system during production, the production pattern is shown in FIG. 3, and the production pattern is the reRNA TM And the plasmid is taken as a raw material to be put into cells, thereby generating the rRNA TM A medicinal stock solution. The receptor of vesicular stomatitis virus (VSV virus for short) is Low Density Lipoprotein (LDLR), has wide tissue distribution, and the specific VSV-G (SEQ ID NO: 7) screened by the inventor has higher affinity,the VSV-G protein is used as a delivery protein.
The preparation method comprises the following specific preparation steps:
1. transfection reagent Lipo8000 for cultured HEK 293T cells TM Performing a reRNA TM Plasmid (carrying the reRNA on the plasmid) TM A sequence comprising an mRNA sequence, SEQ ID NO:4-6 sequence, GFP sequence) and helper plasmids (helper plasmids comprising the sequences comprising SEQ ID NOs: 4-6, a VSV-G plasmid, a T7 RNA polymerase plasmid, and the amino acid sequence of the VSV-G is shown in SEQ ID NO: 7), after the transfection, culturing the cells in an incubator for 24-48 hours, collecting the supernatant for purification to obtain the reRNA TM -GFP seed.
2. The cultured HEK 293 cells were treated with transfection reagent Lipo8000 TM Transfection of helper plasmids (carrying the nucleic acid molecule encoding VSV-G on helper plasmids) was performed and the reRNA was added after transfection TM GFP2 seed, culturing in incubator for 48 hr, collecting supernatant, purifying to obtain VSV-G coated rRNA TM -GFP。
As shown in FIG. 4, when only reRNA is dosed TM When GFP was used as a seed, only few cells showed fluorescent expression of GFP after 48 hours of cell culture, and little productivity was achieved. When VSV-G plasmid is used as an adjuvant, the rRNA can be efficiently delivered TM GFP enters the cell, GFP in the cell is obviously visible, and can be used as the reRNA under the same feeding TM The proportion of GFP-producing cells was greatly increased and the productivity was also considerable (4.83. Mu.g/ml).
MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO：7)
Example 2
The coat protein of lymphocytic choriomeningitis virus (LCMV virus for short) is LCMV-GP, and its receptor is alpha-muscular dystrophy protein, which is a ubiquitous protein and also is reRNA TM A superior alternative to delivering proteins. Reference example 1 method, with reRNA TM GFP and LCMV-GP plasmids as raw materials are put into cells to produce the reRNA TM A medicinal stock solution.
When only reRNA is fed TM GFP was used as seed, and after 48 hours, only few cells had fluorescent expression of GFP (FIG. 5), and little productivity was achieved. When LCMV-GP plasmid is used as an auxiliary material, the rRNA can be effectively delivered TM GFP enters the cell, GFP in the cell is obviously visible, and can be used as the reRNA under the same feeding TM The proportion of GFP-producing cells was greatly increased (FIG. 5), and the productivity was also considerable (3.15. Mu.g/ml).
Example 3
Referring to FIG. 6, coat protein-encapsulated reRNA was prepared from coat protein stable transgenic cell lines TM The medicine comprises the following specific steps:
1. infecting HEK 293 cells with lentivirus containing VSV-G gene (corresponding amino acid sequence shown as SEQ ID NO: 7), adding puromycin to screen positive cell strain, screening monoclonal positive cell strain, screening out cell strain with high VSV-G gene expression, and performing stable passage and database establishment.
2. After stable passage and warehouse establishment, cells are revived, and the reRNA is fed after culture TM GFP seed (preparation method reference example 1), culture was continued for 48 hours after feeding, and the supernatant was collected as a harvest for purification.
In HEK 293 cells not containing VSV-G protein, the unsteady transgenic cell line was fed with reRNA only TM GFP seed, after 48 hours, only few cells will have fluorescent expression of GFP (FIG. 7), the productivity is very low, and the stable cell line can effectively deliver the reRNA due to the VSV-G protein TM GFP was introduced into the cells, the proportion of cells which could be used as producer cells was greatly increased, almost 100%, and the productivity was also considerable (5.94. Mu.g/ml).
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.

Claims (15)

1. A recombinant cell comprising:
a first nucleic acid molecule comprising a self-replicating RNA molecule;
a second nucleic acid molecule encoding a delivery vehicle protein.
2. The recombinant cell of claim 1, wherein the self-replicating RNA molecule comprises:
a first RNA sequence encoding an N protein or a functional fragment thereof;
a second RNA sequence encoding a P protein or a functional fragment thereof;
a third RNA sequence encoding an L protein or a functional fragment thereof; and
a target molecule coding region encoding at least one target molecule.
3. The recombinant cell of claim 2, wherein at least one of the N protein, the P protein, and the L protein is each independently from a rhabdoviridae virus.
4. The recombinant cell of claim 2, wherein the N protein has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% homology thereto, said P protein having the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 80% homology thereto, said L protein having the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least 80% homology thereto.
5. The recombinant cell of claim 1, wherein the delivery vehicle protein comprises a non-human protein and a human protein;
the non-human proteins include viral capsid proteins;
the human proteins include a SNARE protein family;
optionally, the virus comprises at least one of poxviruses, rabies viruses, flaviviruses, measles viruses, coronaviruses, vesicular stomatitis viruses, newcastle disease viruses, lymphocytic choriomeningitis viruses.
6. The recombinant cell of claim 1, wherein the delivery carrier protein is selected from the group consisting of vesicular stomatitis virus receptor and/or lymphocytic choriomeningitis virus coat protein.
7. The recombinant cell of claim 1, wherein the first nucleic acid molecule and the second nucleic acid molecule are each independently present in the recombinant cell in free form or integrated into the genome.
8. The recombinant cell of claim 1, wherein the first nucleic acid molecule and the second nucleic acid molecule are each independently present in the recombinant cell in the form of a plasmid.
9. The recombinant cell of claim 8, wherein the first nucleic acid molecule and the second nucleic acid molecule are present on the same plasmid.
10. A method of producing the recombinant cell of any one of claims 1-9, comprising:
allowing the cell to carry the first nucleic acid molecule and the second nucleic acid molecule,
the first nucleic acid molecule and the second nucleic acid molecule are as defined in any one of claims 1 to 9 in a recombinant cell.
11. The method according to claim 10, comprising:
introducing at least one of the first nucleic acid molecule and the second nucleic acid molecule into the cell in plasmid form.
12. The method of claim 11, wherein said introducing is by transfecting said plasmid.
13. The method of claim 10, wherein the second nucleic acid molecule is provided in the form of a lentivirus or CRISPR/Cas 9.
14. The method of claim 10, wherein the cells are selected from HEK 293 cell line, vero cell line, CHO-K1 cells, or NIH-3T3 cells.
15. A method of preparing a composition of self-replicating RNA molecules, wherein the self-replicating RNA molecules comprise self-replicating RNA and a delivery carrier protein, the method comprising:
culturing the recombinant cell of any one of claims 1-9; and
obtaining the self-replicating RNA molecule composition.
CN202310546809.3A 2022-05-16 2023-05-15 Protein-coated self-replicating RNA and preparation method thereof Pending CN117070464A (en)

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US20030077251A1 (en) * 2001-05-23 2003-04-24 Nicolas Escriou Replicons derived from positive strand RNA virus genomes useful for the production of heterologous proteins
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