KR20230061194A - Transcription system of RNA of interest, cells comprising same, and uses and methods thereof - Google Patents

Abstract

The present invention relates to an RNA polymerase gene suitable for a cell expression system, an RNA transcription system of interest using the same, a cell containing the same, and a use thereof. In addition, the present invention relates to a cell expressing an RNA of interest obtained by transforming the above system into a host cell and a use thereof, and the production method according to the present invention is suitable for safely and efficiently mass-producing the RNA of interest.

Description

Transcription system of RNA of interest, cells comprising same, and uses and methods thereof}

The present invention relates to an RNA transcription system of interest using an RNA polymerase gene suitable for a host cell, a cell comprising the same, and uses and methods thereof. In addition, the present invention relates to a cell prepared by transforming the above system into a host cell, an RNA of interest, exosome or vesicle produced therefrom, and uses and methods thereof.

Methods of amplifying, producing, and using RNA in host cells have been reported previously. For example, ① a method using E. coli lacking ribonuclease III (RNaseIII) as a host to accumulate RNA in bacteria (Tenllado F. , et. al., Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol. 2003 Mar. 20; 3:3), ② Pseudomonas with the ability of phi 6 bacteriophage to accumulate RNA in capsid syringae as a host (Aalto A P, et. al., Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA. 2007 March; 13(3):422-9 ), ③ A method for accumulating RNA fused with a partial sequence of tRNA in bacteria using Escherichia coli as a host (Ponchon L, et. al., A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc. 2009; 4(6):947-59), ④ Method of producing siRNA using Escherichia coli as a host and at the same time allowing siRNA to form a complex with a protein that binds to siRNA (Huang L, et. al., Efficient and specific gene knockdown by small interfering RNAs generated in bacteria Nat Biotechnol.2013 April, 31(4):350-6), ⑤ RNA production method by secretory production using bacteria of the genus Rhodovulum as a host (WO2009/063969 Reference), ⑥ A method for accumulating RNA in yeast mitochondria using yeast as a host (see WO2010/084371). and ⑦ a method for efficiently producing RNA using a coryneform bacterium deficient in ribonuclease III (RNaseIII) as a host (see US Patent Application 20200024629).

The present invention relates to an RNA transcription system of interest using an RNA polymerase gene suitably for a host cell expression system, a host cell comprising the same, and a use thereof.

In addition, the present invention relates to a cell prepared by transforming the system into a host cell, an RNA of interest, an exosome or a vesicle produced therefrom, and uses and methods thereof, and the production method according to the present invention is safe and It should be suitable for efficient mass production of RNA and vesicles.

The present invention relates to vesicles, RNAs of interest and exosomes derived from host cells that amplify the RNA of interest.

The present invention relates in particular to the field of transcription systems of RNAs of interest in host cells.

As one of the present invention for achieving the above object, a system comprising at least one protein translating and transcribing component in a host cell, comprising an RNA polymerase expression unit and an RNA unit of interest,

a) the RNA polymerase expression unit comprises (i) a promoter operating in a host cell; (ii) a structure enabling intracellular translation; (iii) an open reading frame (ORF) containing RNA polymerase [DNA dependent RNA Polymerase (DdRp) and/or chimeric enzyme containing capping enzyme or RNA dependent RNA Polymerase (RdRp)]; (iv) a DNA unit comprising a transcription termination sequence; and

b) the RNA unit of interest comprises (i) a promoter driven by the DdRp; (ii) a DNAi unit capable of being transcribed by the DdRp polymerase and encoding an RNA of interest (RNAi); and (iii) a unit comprising a transcription termination sequence or (i) a promoter operating in a host cell; (ii) a DNA unit comprising DNA encoding a replicon RNA replicated by the RdRp and comprising RNAi; and (iii) a unit comprising a transcription termination sequence;

Here, c) RNA transcription systems, cells transformed therefrom, and RNAs of interest, exosomes or vesicles (Cell derived vesicels, CDV) derived therefrom are provided; .

The system can add expression units of H/ACA sRNP or eukaryotic stand-alone pseudouridine synthases (Pus enzymes) to pseudouridylate uridine in the RNA of interest.

The eukaryotic stand-alone pseudouridine synthases (Pus enzymes) may originate from any one selected from the group including Escherichia coli, yeast, and humans.

Preferably, when the host cell of the present invention is a eukaryotic cell, the RNA polymerase expression unit can translate any one selected from the group consisting of (i) DNA dependent RNA Polymerase (DdRp) ORF and variants thereof in the host cell. An mRNA construct capable of translating a chimeric enzyme comprising a catalytic unit of a capping enzyme in addition to the mRNA, or an mRNA construct; and

(ii) may include a DNA construct capable of expressing the DdRp, wherein the DNA construct comprises a promoter driven by the DdRp; 5'UTR; Any one selected from the group containing DNA dependent RNA Polymerase (DdRp) ORF and variants thereof, or any one selected from the group containing RNA dependent RNA Polymerase (RdRp) ORF or variants thereof; And a DNA structure containing a 3'UTR. Here, or the DNA construct may additionally include DNA capable of translating a chimeric enzyme including a catalytic unit of a capping enzyme.

Preferably, the DdRp may be a capping enzyme or a poly(A) polymerase and a chimeric enzyme.

Preferably, the RNA transcription system of interest includes a protein-RNA tethering system comprising an RNA binding domain binding with high affinity and a peptide binding RNA sequence, in addition to the RNA polymerase expression unit DNA encoding the RNA binding domain; and DNA encoding one or more of the peptide-binding RNA sequences in addition to the expression unit of the RNA of interest.

In the present invention, the chimeric enzyme composed of DdRp and several enzymes (or proteins) is referred to as ctDdRp.

In order to produce ctDdRp in E. coli as a host cell, various methods have been attempted to construct an expression vector suitable for E. coli and to increase the expression rate of the protein of interest in the expression vector. First, factors related to plasmid self-replication, gene copy number and stability, and second, mRNA, initiation rate of mRNA synthesis, elongation and termination of mRNA synthesis, and mRNA Factors related to the degradation rate, third, factors related to the translation initiation rate of the protein of interest, and fourth, factors related to the degradation rate of the protein of interest should be considered to be suitable for the host cell.

It is related to the transcription process and mRNA. The first step in gene expression is a transcription initiation event catalyzed by RNA polymerase. In E. coli, the rate of transcription initiation is controlled by the nucleotide sequence of the promoter, that is, regulatory proteins bind to specific nucleotide sites of the promoter to control the transcription mechanism. When mRNA transcription is initiated by the promoter, the mRNA should continue to elongate, but sometimes non-specific immature termination occurs. Non-specific nucleotide sequences that can cause this phenomenon must be excluded from mRNA, that is, a portion of the promoter transcribed into mRNA and the DNA sequence of the target gene determine this. In general, when human or animal genes are expressed in E. coli in the form of cDNA (complementary DNA), expression is insufficient in most cases, so reconstructing the gene of interest to be suitable for the E. coli expression system is the first step in constructing an expression vector. It is an essential process. Transcription termination occurs at the point where mRNA, mRNA polymerase, and DNA are separated, and in this process, the sequence of nucleotides, the secondary structure of mRNA transcripts, and host cell proteins such as ρ, NusA and NusB, etc. become involved as an influencer. As such, the efficiency of the process of transcription from DNA to mRNA depends on the ability of RNA polymerase to recognize and act on transcription initiation and termination sites. In order for RNA polymerase to perform its basic function, each promoter region and terminator region are There may be similarities in common structures, but on the contrary, there may also be differences in structures that give differences in function (efficiency and effect factor dependence).

In addition, sequences that are generally symmetrical to each other of transcription terminators form a stem-loop structure, which plays a role in terminating transcription by RNA polymerase and is located at the 3' end of the mRNA transcript. Existing U bases are known to play a role in separating transcripts from DNA, but how the length of the stem structure, the size of the loop structure, the length of the U base, and the ratio of G/C bases affect the mRNA transcription efficiency. Not sure if that will affect it. On the other hand, the transcription terminator is preferably located close to the 3' end of the target gene, which has the advantage of minimizing unnecessary transcription by RNA polymerase to increase mRNA stability and reduce energy used by cells.

This is related to the translation process. The translation process is a process in which a polypeptide is made from mRNA, and the efficiency of translation initiation is affected by various sites in the mRNA. Site, initiation codon, interval and sequence between SD site and initiation codon, and secondary structure of mRNA are important factors. The SD region has a sequence complementary to the 3' end of the 16S rRNA and is generally composed of 3 to 6 nucleotides, and the initiation codon is mainly AUG, but sometimes GUG, UUG, AUU, AUA, etc. are used.

In addition, the interval between the SD site and the initiation codon is usually composed of 9 ± 3 nucleotides, and it is effective to have many base compositions A and U. On the other hand, the spacing and sequence between the SD region and the initiation codon are closely related to the 5' end of the target gene, and play an important role in the formation of mRNA secondary structure in the initiation codon region.

Therefore, the efficiency of translation initiation can be successfully increased by destabilizing the mRNA stem-loop structure near the initiation codon by changing the gene spacing and sequence of this region to be suitable for the target gene.

The elongation step from which translation is initiated to form a polypeptide does not proceed at a constant rate. This phenomenon is because several parts of mRNA regulate this step. Since mRNA is transcribed from the sequence of the target gene, the codon usage and codon context of the target gene have a large effect on the gene expression rate. will affect In other words, one of the main causes of poor expression of cDNA-type genes in the E. coli expression system is the use of codons for tRNA that are less distributed in E. coli or the lack of compatibility between adjacent codons. In E. coli, two release factors (hereinafter referred to as 'RF') that recognize three stop codons are involved in the termination stage of the translation process.

RF1 recognizes UAA and UAG, while RF2 recognizes UAA and UGA. Although the efficiency of the three stop codons is different, it is known that UAA is mainly used for highly expressed genes. However, in order to ensure translation termination, it is ideal to design two stop codons in succession.

After all, in order to obtain a desired protein, several steps must be performed from the gene, and the efficiency of expression of the gene is determined according to the efficiency of each step. That is, all steps must be harmonized in a complex way, regardless of the quantity and stability of the gene, the transcription efficiency and stability of mRNA, and the translation efficiency. Not only the composition but also the base composition of the target gene must be compatible with each other to increase the efficiency of each step and increase the expression of the protein of interest.

On the other hand, the function and structure of microorganisms are determined according to the vast amount of genetic information they have, and they have the ability to grow while adapting to various environments. Therefore, the genetic background of the host cell plays an important role in producing a specific target product. However, although there are determinants that can superficially determine the role of the host cell, such as plasmid stability and degradation of the target product, the genetic reason why the production rate of the target product varies greatly depending on the host cell has not yet been clarified in detail. In most cases where a recombinant product is overexpressed, the host cell undergoes an enormous metabolic burden to grow, and this causes the segregation of the plasmid to occur, so selection of a host cell that stably produces the target product is a very important task.

In addition, host cells should be selected by considering the following two factors in addition to the genetic background. That is, the growth requirements of the host cell and the regulation of gene expression. Growth requirements include: 1) requirements for nutrients such as vitamins, amino acids, and metal ions; 2) sensitivity to temperature; and 3) oxygen dependence, these factors greatly affect the efficiency of the fermentation process. In addition, since the induction method of the target product varies depending on the gene expression system, the selection of host cells according to the promoter must be considered. If the induction method, such as heat, chemical inducer, or nutrient starvation, varies depending on the promoter, the response may be subtle or greatly change depending on the host cell. will be highlighted as important.

Step (a): Codon Optimization Step

The host cell containing the RNA transcription system of interest of the present invention may codon optimize the RNA polymerase nucleotide sequence to be used in the present invention to be suitable for expression in Escherichia coli in order to use E. coli.

In the present invention, the codon was converted into a codon suitable for E. coli so that RNA polymerase can be more highly expressed in E. coli, which contains an RNA transcription system. Through the codon optimization process, only the nucleotide sequence of the gene sequence is converted, not the amino acid sequence.

Nucleotide sequences used in the present invention are construed to include nucleotide sequences showing substantial identity to the nucleotide sequences in addition to the above-mentioned sequences. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned so as to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art. A nucleotide sequence exhibiting homology, more preferably at least 90% homology, most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc., and blastp, blasm, It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLAST is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A sequence homology comparison method using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Step (b): Preparation of recombinant vector

Next, the RNA polymerase expression unit and the RNA unit of interest included in the RNA transcription system of the present invention may be one or each independent replicon. A recombinant vector is prepared by inserting the RNA polymerase and the codon-optimized nucleotide sequence of the RNA of interest into an expression vector.

As used herein, the term "expression vector" is a linear or circular DNA molecule composed of a fragment encoding a polypeptide of interest operably linked to an additional fragment that serves for transcription of the expression vector. Such additional fragments include promoter and terminator sequences. Expression vectors also include one or more origins of replication, one or more selectable markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

The nucleotide sequence of the codon-optimized RNA polymerase of the present invention may be operably linked to an expression control sequence and inserted into an expression vector. As used herein, 'operably linked to' means that one nucleic acid fragment is combined with another nucleic acid fragment and its function or expression is affected by the other nucleic acid fragment. In addition, 'expression control sequence' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a specific host cell. Such regulatory sequences include promoters to initiate transcription, optional operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences to control termination of transcription and translation.

Vectors of the invention may typically be constructed as vectors for expression. When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., T7 promoter, tre promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter) promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter and trp promoter and the like), a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator regions of the E. coli tryptophan biosynthesis pathway and the leftward promoter (pL λ promoter) of phage λ can be used as control regions. there is. Preferably, the promoter used in the vector of the present invention is a T7 promoter, and the termination sequence is a T7 terminator.

A vector injected into a host cell can be expressed in the host cell, in which case a large amount of RNA polymerase is obtained. For example, when the expression vector includes the lac operon, gene expression can be induced by treating the host cell with isopropylthio-β-D-galactoside (IPTG).

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (e.g., pETDuet-1, pTrc99A, pSTV28, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9 , pHC79, pIJ61, pLAFR1, pHV14, pET22b, pGEX series, pET series, and pUCP19, etc.), phage (eg, λgt4, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.). However, preferably pETDuet-1 is used.

On the other hand, the vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes for resistance to mycin and tetracycline.

The expression vector of the present invention includes a promoter sequence and a nucleotide sequence of a gene to be expressed (structural gene), and it is preferable that the sequences are linked in the order of 5'→3'.

According to the present invention, the promoter sequence, the nucleotide sequence of the codon-optimized RNA polymerase and the transcription terminator sequence are operatively linked in the order of 5'→3'.

In the expression vector of the present invention, it is necessary to sequence and insert only the base sequence including the ribosomal binding site (RBS) and essential parts for enzyme expression so that the genes have a minimum length including the function of over-expression of the enzyme, and insert the sequence into the host cell. It is desirable in terms of reducing the metabolic burden.

The RNA unit of interest of the present invention is preferably designed to suit the genetic information of the target cell according to its use. Depending on the use, if the target cell for which the RNA is to exert efficacy is a human cell, the structure and codon optimization can be performed to suit the genetic information of the human cell.

Preferably, the RNA unit of interest of the present invention can be designed to be optimized for the genetic information of the target cell rather than the host cell.

Step (c): Preparation of transformed E. coli

In the present invention, host cells can be appropriately selected and used from prokaryotic cells, yeast, plant, animal cells, and the like.

Preferably, the recombinant vector can be transduced into E. coli.

The recombinant vector containing the nucleotide sequence of the codon-optimized RNA polymerase is then introduced into E. coli. The method of delivering the vector of the present invention into E. coli can be carried out by heat shock method, CaCl2 method, and electroporation method. And, preferably, step (c) of the present invention is carried out by a thermal shock method.

Any host cell capable of stably and continuously cloning and expressing the vector of the present invention can be used as any host cell known in the art, for example, E. coli BL21 (DE3), E. coli W3110, E. coli JM109 , E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, strains of the genus Bacillus such as Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcessons and various Pseudomonas There are enterobacteria and strains such as species.

In the present invention, the term 'Cell derived vesicles (Cell derived vesicles, CDV)' refers to vesicles artificially manufactured in nucleated cells, and in almost all types of cells, they are separated from cell membranes to form double phospholipids, which are the structure of cell membranes. It means that it has the form of a membrane. The cell-derived vesicles of the present invention may have a micrometer size, for example, 0.03 to 1 μm, and may be used interchangeably within the present specification. The cell-derived vesicles of the present invention are distinguished from naturally secreted vesicles, and the 'vesicles' of the present invention are separated from the inside and outside by a lipid bilayer composed of cell membrane components of the derived cell, and the cell membrane lipid of the cell It has cell membrane proteins, nucleic acids, and cellular components, and has a size smaller than the original cell, but is not limited thereto.

Hereinafter, the present invention will be described in more detail. Of course, the following description of the present invention is only intended to embody the present invention and is not intended to limit or limit the scope of the present invention. What can be easily inferred by an expert in the technical field to which the present invention belongs from the detailed description of the present invention is interpreted as belonging to the scope of the present invention. References cited herein are incorporated herein by reference.

I. Host Cell

The host cells of the present invention can be roughly divided into host cells including prokaryotic, yeast and plant cells having cell walls and host cells including animal cells without cell walls.

The host cell of the present invention is any one selected from the group containing animals, plants, yeasts and bacteria, wherein the bacteria are Corynebacterium glutamicum, Escherichia coli BE42, Escherichia coli HB101, Escherichia coli JM101, E. coli LE392, E. coli MC1061, E. coli Y1088 and E. coli W3110 selected from the group consisting of attenuating the expression of the gene encoding the ribonuclease III or destroying the gene to reduce the activity of ribonuclease III It is characterized by doing

Cells for preparing the cell-derived vesicles may be included without limitation as long as they are nucleated cells, but are preferably stem cells, acinar cells, myoepithelial cells, red blood cells, monocytes, dendritic cells, natural killer cells, and platelets. It may be one or more selected from. The stem cells may be any one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells, more preferably mesenchymal stem cells, still more preferably the mesenchymal stem cells It can be mesenchymal stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood.

Host cells have been developed and applied based on microorganisms with established genetic tools such as Escherichia coli and yeast, but the range of target microorganisms is gradually expanding to Bacillus subtilis, Pichia pastoris, Streptomyces avermitilis, and cyanobacteria. Corynebacterium glutamicum (Corynebacterium glutamicum) is a Gram-positive bacterium that does not form spores and is a GRAS (Generally recognized as safe) strain that is not pathogenic and is widely used industrially for producing various L-amino acids and nucleic acids. It is an industrial strain.

In addition, Corynebacterium has a great advantage not only in amino acid production but also in easy protein secretion production due to the characteristics of Gram-positive bacteria. And beyond the current production of amino acids or proteins, research is being actively conducted to produce various bio compounds through Corynebacterium.

The steps necessary to obtain an expression unit of an RNA polymerase with intracellular activity and a transcription unit of the RNA of interest are very simple on a theoretical level. The RNA of interest may also be prepared by selecting RNA polymerase and the RNA of interest, cleaning into the desired expression vector, transforming into the host of choice, and inducing.

Selection of RNA polymerase and host cell to produce the RNA of interest is the starting point of the whole process. Among microorganisms, available host systems included bacteria, yeasts, molds, and unicellular algae. All have their pros and cons, and your choice may depend on RNA polymerase and mRNA.

The advantages of using E. coli as a host organism are well known. (i) It has unparalleled rapid growth dynamics. Given optimal environmental conditions in glucose-salt medium, the doubling time is approximately 20 minutes. This means that cultures inoculated with a 1/100 dilution of a saturated starter culture can reach termination within a few hours. However, it should be noted that the expression of recombinant proteins imposes a metabolic burden on the microbe and can significantly shorten the production time. (ii) high cell density cultures were easily achieved; The theoretical density limit of E. coli was estimated at about 200 g dry cell weight/l or approximately 1 x 10 ¹³ viable bacteria/ml. However, the exponential growth of complex media leads to densities approaching that figure. In the simplest laboratory setup (i.e., batch culture of E. coli at 37 °C using LB medium), <1 × ¹⁰ cells/ml may be the upper limit, which is 0.1% of the theoretical limit. For this reason, high-density cell culture methods have been designed to promote E. coli growth even when producing recombinant proteins. Being flagship organisms, these strategies have arisen thanks to a wealth of knowledge about their physiology. (iii) rich and complex media can be made from readily available and inexpensive components; (iv) Transformation with exogenous DNA is quick and easy. Plasmid transformation of E. coli can be performed within 5 minutes.

There are dozens of potential candidates for E. coli strains suitable for use as hosts. All of them have advantages and disadvantages and are special strains used in specific situations. In the case of the first expression strain, it is BL21 (DE3) and some derivatives of the K-12 line.

BL21 is valuable for several genetic characteristics of BL21 after various transformations of the B line. Like other parental B strains, BL21 cells lack the Lon protease that degrades many foreign proteins. Another gene missing from the genome of BL21 parental cells is the gene encoding OmpT, an outer membrane protease that functions to degrade extracellular proteins. The free amino acids were then absorbed into the cells. This is problematic for the expression of the recombinant protein because OmpT can digest it after cell lysis. Plasmid loss was also prevented thanks to the hsd SB mutation already present in the parental strain that produced BL21 (B834). As a result, DNA methylation and degradation were disrupted. If the gene of interest is located under the T7 promoter, T7 RNAP should be provided. In strain BL21(DE3), the λDE3 prophage was inserted into the chromosome of BL21 and contained the T7 RNAP gene under lac.

BL21(DE3) and its derivatives are the most used strains for protein expression. Nevertheless, K-12 may be used for this purpose. AD494 and Origami TM (Novagen) strains are trx B (thioredoxin reductase) mutants, resulting in enhanced disulfide bond formation in the cytoplasm (Origami strains also lack the glutathione reductase gene). Another strain widely used in the K-12 repertoire is the recA mutant HMS174. This mutation has a positive effect on plasmid stability.

Plasmid multimer formation, an important cause of instability, is in E. coli. All three strains have λDE3-containing derivatives (available from Novagen), so the T7 RNAP system can be used. In particular, E. coli has been used as the most common host cell because genetic and physiological research has been well preceded, and it is relatively easy to manipulate genetically, and the cost of culture is low, and it has the advantage of being able to increase the amount of cells produced, so it is possible to mass-produce proteins. is most widely used in

E. coli strain HT115 (DE3) is used to utilize the isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase gene contained within a stable insert of the modified lambda prophage λ DE3 . This strain lacks RNase III, an enzyme that normally degrades most dsRNAs in bacterial cells, resulting in enhanced accumulation of extended dsRNA duplexes compared to the RNase III expressing strain, BL21(DE3).

The host cell is a cell with reduced ribonuclease III activity.

Host cells can be modified to reduce the activity of ribonuclease III (RNaseIII). Specifically, bacteria may be modified such that the activity of ribonuclease III is reduced compared to unmodified strains. The activity of ribonuclease III can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less or 0% of the activity of the unmodified strain. That is, the bacterium may be modified such that, for example, the activity of ribonuclease III is deleted (removed). By modifying the bacteria to reduce the activity of ribonuclease III, the ability of the bacteria to produce the RNA of interest can be improved, that is, the production of the RNA of interest using the bacteria can be increased.

Ribonucleases (commonly abbreviated RNase) break down RNA into smaller components by nuclease-catalyzed action. Ribonucleases can be divided into: It consists of several subclasses within the classes of endoribonucleases and exoribonucleases.

There are many RNases in all organisms, and RNA degradation is a very ancient and important process. In addition to removing cellular RNA that is no longer needed, RNases play a key role in the maturation of all RNA molecules, messenger RNA that carries the genetic material to make proteins, and noncoding RNA that functions in a variety of cellular processes. Active RNA degradation systems are also the first line of defense against RNA viruses.

Some cells also secrete large amounts of non-specific RNases such as A and T1. Therefore, RNases are very common and RNAs that are not in a protected environment have a very short lifespan. All intracellular RNAs are protected from RNase activity by several strategies, including 5' end-capping, 3'-end polyadenylation, and folding within RNA-protein complexes (ribonucleoprotein particles or RNPs).

RNase III is a type of ribonuclease that cleaves rRNAs (16s rRNA and 23s rRNA) in polycistronic RNA operons transcribed in prokaryotes. In addition, it degrades the double-stranded RNA (dsRNA)-Dicer RNAse family to cleave pre-miRNA (60-70 bp in length) at specific sites and converts it to miRNA (22-30 bp), actively involved in transcription and transcriptional regulation.

Hereinafter, ribonuclease III and the gene encoding it are described.

The term “ribonuclease III” refers to a protein having the activity of catalyzing a reaction that cleave specific RNA such as double-stranded RNA. A gene encoding ribonuclease III is also referred to as a “ribonuclease III gene”.

Examples of ribonuclease III genes include the mc gene. The protein encoded by the mc gene (ribonuclease III) is also referred to as the "Rnc protein".

The nucleotide sequence of the ribonuclease III gene, such as the mc gene, and the amino acid sequence of the ribonuclease III encoded by these genes, such as the Rnc protein, which bacteria possess can be found, for example, at NCBI (National Center for Biotechnology Information). can The expression "a gene or protein has a base or amino acid sequence" means that the gene or protein contains a base or amino acid sequence unless otherwise specified, and includes the case where the gene or protein has only a base or amino acid sequence.

Such modifications that retain the original function are also referred to as “conservative modifications”. The term "mc gene" includes the previously exemplified mc gene as well as conservative variants thereof. Similarly, the term "Rnc protein" includes the Rnc proteins exemplified above as well as conservative variants thereof. Examples of conservative variants include, for example,

The expression "original function is maintained" means that a variant of a gene or protein has a function (eg activity or property) corresponding to that of the original gene or protein. That is, the expression "the original function is maintained" used for the ribonuclease III gene means that the variant of the gene encodes a protein that maintains the original function, that is, a protein having ribonuclease III activity. Also, the expression "the original function is maintained" used for ribonuclease III means that the variant of the protein has ribonuclease III activity.

Ribonuclease III activity can be measured, for example, by incubating the enzyme with RNA serving as a substrate (eg, double-stranded RNA) and measuring enzyme-dependent cleavage of the RNA. Specifically, an example of ribonuclease III activity is a method of adding an enzyme, such as a crude extract of cells or a partially purified enzyme thereof, to a synthetic substrate. The H-labeled poly(AU) in double helix form was reacted at 35° C., the reaction mixture was treated with trichloroacetic acid, and the degree of radioactivity reduction of the precipitated fraction with reaction time was measured, and it contained high molecular weight nucleotides. That is, ribonuclease III activity can be calculated based on the degree of radioactivity reduction, which is an index of substrate cleavage. Additionally, another example is the method of adding 32 enzyme-containing reaction mixtures (30 mM Tris-HCl (pH8.0), 250 mM potassium glutamate or 160 mM NaCl, 5 p-radiolabeled double as substrates). Stranded RNA was incubated in mM spermidine, 0.1 mM EDTA and 0.1 mM DTT) at 37 °C for 5 min and MgCl 2 was added thereto to initiate the RNA cleavage reaction at a final concentration of 10 mM, and after appropriate reaction progress, EDTA and electrophoresis Equal volume mixtures of marker dyes are added, of which the EDTA concentration is to give the final concentration. Above 20 mM, the reaction is stopped. Then, ribonuclease III activity was reacted by electrophoresis using a denaturing 15% (w/v) polyacrylamide gel with TBE buffer (89 mM Tris/Tris-borate, and 2 mM EDTA) containing 7 M urea. After applying the sample, it can be detected. and applying the gel to a radiographic image analyzer to analyze the cleaved RNA fragments.

Examples of conservative variants are described below.

Homologs of the ribonuclease III gene or homologs of the ribonuclease III can be found in a BLAST search or FASTA search using, for example, the nucleotide sequence of any of the ribonuclease III genes or any amino acid sequence exemplified above. can be readily obtained from public databases by Ribonuclease III exemplified above as the query sequence, and homologs of the ribonuclease III gene, for example, PCR using bacterial chromosomes as templates, and these known ribonuclease III genes and their adjacent regions. It can be obtained by an oligonucleotide prepared based on the nucleotide sequence of

The ribonuclease III gene has the amino acid sequence of any of the ribonuclease IIIs exemplified above, but with substitutions, deletions, insertions and/or one or several amino acid residues at one or several positions so long as the original function is maintained. can be added. The number meant by the term "one or several" mentioned above may vary depending on the position of the amino acid residue or the type of amino acid residue in the three-dimensional structure of the protein, but specifically, for example, 1, 1 to 50, 1 to 40, or 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

The aforementioned substitutions, deletions, insertions and/or additions of one or more amino acid residues are conservative mutations that maintain the normal function of the protein. A typical example of a conservative mutation is a conservative substitution.

The ribonuclease III gene may be, for example, a gene encoding a protein having an amino acid sequence showing 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology, The total amino acid sequence of any one of the ribonuclease IIIs exemplified above as long as the original function is maintained. In this description "homology" means "identity".

A probe can be, for example, part of a complementary sequence of a gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and using a DNA fragment containing any of these nucleotide sequences as a template. As a probe, a DNA fragment of about 300 bp in length can be used, for example. In this case, the hybridization washing conditions may be, for example, 50°C, 2xSSC and 0.1% SDS.

In addition, since codon degeneracy is host-dependent, any codon in the ribonuclease III gene can be replaced with each equivalent codon. That is, the ribonuclease III gene may be a variant of any of the ribonuclease III genes exemplified above due to the degeneracy of the genetic code.

The percentage of sequence identity between two sequences can be determined using, for example, a mathematical algorithm. These mathematical algorithms are non-limiting. By using programs based on these mathematical algorithms, sequence comparisons (ie, alignments) can be performed to determine sequence identity. The program can properly run on a computer. Examples of such programs include, but are not limited to, the CLUSTAL of PC/Gene program (available from Intelligenetics, Mountain View, CA), the ALIGN program (version 2.0), and GAP, BESTFIT, BLAST, FASTA, and Wisconsin's TFASTA. Alignment using the Genetics Software Package, version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA) program can be performed using initial parameters, for example.

In order to obtain a nucleotide sequence homologous to the target nucleotide sequence, in particular, for example, a BLAST nucleotide sequence with a score of 100 and a word length of 12 can be obtained using the BLASTN program. For example, a search for a target protein, particularly a BLAST protein, can be performed using a BLASTX program with a score of 50 and a word length of 3. For BLAST nucleotide searches and BLAST protein searches, see ncbi.nlm.nih.gov. Additionally, Gapped BLAST (BLAST 2.0) can be used to obtain alignments that include gap(s) of comparative interest. You can also use PSI-BLAST to perform iterative searches to detect distant relationships between sequences. When using BLAST, either Gapped BLAST or PSI-BLAST, the initial parameters of each program (e.g. BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. Sorting can also be done manually.

Sequence identity between two sequences is calculated as the percentage of identical residues in the two sequences when the two sequences are aligned for maximal fit.

The foregoing descriptions of genes and variants of proteins can be similarly applied to any other proteins and variants of RNA of interest and the genes encoding them.

Hereinafter, a method for reducing the activity of a protein (enzyme) such as ribonuclease III will be described.

The expression “the activity of a protein is reduced” means that the activity of the protein is reduced compared to the unmodified strain. Specifically, the expression "the activity of the protein is reduced" means that the activity of the protein per cell is reduced compared to the unmodified strain. "Unmodified strain" refers to a control strain that has not been modified to reduce the activity of the protein of interest. Examples of unmodified strains include wild type strains and parental strains. Specific examples of unmodified strains include strains of various types of bacterial species. Specific examples of unmodified strains also include the strains exemplified above in relation to the description of bacteria. That is, in one embodiment, the activity of the protein may be reduced compared to the type strain, i.e., the type strain of the species to which the bacteria as described herein belong.

A state in which "the activity of a protein is reduced" also includes a state in which the activity of a protein completely disappears. More specifically, the expression “the activity of a protein is reduced” can mean that the number of molecules of the protein per cell is reduced and/or the function of each molecule of the protein is reduced relative to non-protein molecules. modified strains. That is, the term "activity" in the expression "the activity of a protein is reduced" is not limited to the catalytic activity of a protein, and may also mean the amount of transcription (ie, the amount of mRNA) of a gene encoding it. The amount of a protein or translation of a protein (i.e., amount of protein). The state "the number of protein molecules per cell decreases" includes a state in which no protein exists at all. The state of "the function of each protein molecule is reduced" includes a state in which the function of each protein molecule has completely disappeared. The degree of decrease in activity of the protein is not particularly limited as long as the activity is reduced compared to the unmodified strain. The activity of the protein can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less or 0% of the activity of the unmodified strain. The degree of decrease in activity of the protein is not particularly limited as long as the activity is reduced compared to the unmodified strain. The activity of the protein can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less or 0% of the activity of the unmodified strain. The degree of decrease in activity of the protein is not particularly limited as long as the activity is reduced compared to the unmodified strain. The activity of the protein can be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less or 0% of the activity of the unmodified strain.

Modifications to reduce the activity of a protein can be achieved, for example, by reducing expression of the gene encoding the protein. The expression "gene expression is reduced" means that the expression of the gene is reduced compared to the wild-type strain and the unmodified strain such as the parent strain. Specifically, the expression "gene expression is reduced" means that the expression of the gene per cell is reduced compared to the unmodified strain. More specifically, the expression “the expression of a gene is reduced” may mean that the amount of transcription of a gene (ie, the amount of mRNA) is reduced and/or the amount of translation of a gene (ie, the amount of protein expressed is reduced). there is. genes) is reduced. A state in which "expression of a gene is reduced" also includes a state in which the gene is not expressed at all. The state of "decreased expression of a gene" is also referred to as a state of "decreased expression of a gene". Expression of the gene may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less or 0% of the unmodified strain.

A decrease in gene expression can be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Expression of a gene can be reduced by modifying the expression control sequences of a gene, such as the promoter, the Shine-Dalgarno (SD) sequence (also called the ribosome binding site (RBS)), and the spacer region between the RBS. and the start codon of a gene. When an expression control sequence is modified, one or more nucleotides, two or more nucleotides, or three or more nucleotides of the expression control sequence are modified. For example, the efficiency of transcription of a gene can be reduced, for example by replacing the gene's promoter on a chromosome with a weaker promoter. The term “weak promoter” refers to a promoter that provides for attenuated transcription of a gene compared to the wild-type promoter that is constitutively present for the gene. Examples of weaker promoters included, for example, inducible promoters. That is, an inducible promoter can function as a weaker promoter in non-induced conditions, such as in the absence of a corresponding inducer. In addition, some or all of the expression control sequences may be deleted. Expression of a gene can also be reduced, for example by manipulating factors responsible for regulating expression. Examples of factors responsible for expression control include small molecules responsible for transcriptional or translational control (inducer, repressor, etc.), proteins responsible for transcriptional or translational control (transcription factors, etc.), and nucleic acids responsible for transcriptional or translational control (siRNA, etc.) etc. In addition, the expression of a gene can also be reduced, for example, by introducing a mutation that reduces the expression of the gene into the coding region of the gene. For example, expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons used less frequently in the host. Also, for example, gene expression can be reduced due to disruption of a gene as described herein. Expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons used less frequently in the host. Also, for example, gene expression can be reduced due to disruption of a gene as described herein. Expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons used less frequently in the host. Also, for example, gene expression can be reduced due to disruption of a gene as described herein.

Modifications to reduce the activity of a protein can also be accomplished, for example, by disrupting the gene encoding the protein. The expression “a gene is disrupted” means that a gene is altered so that a protein capable of functioning normally is not produced. The state of "normally functioning protein is not produced" is a state in which no protein is produced from the gene, and a state in which the function (activity or property, etc.) of the protein per molecule is reduced or removed from the gene.

Disruption of a gene can be achieved, for example, by deleting the gene on a chromosome. The term "deletion of a gene" refers to deletion of part or all of the coding region of a gene. In addition, the entire gene including sequences upstream and downstream from the coding region of the gene on the chromosome may be deleted. The region to be deleted can be any region such as an N-terminal region (region encoding the N-terminal region of the protein), an internal region or a C-terminal region (region encoding the C-terminal region of the protein). ), as long as it can reduce the activity of the protein. Deletion of longer regions can generally inactivate genes more reliably. The region to be deleted is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more or more in length, 80% of the total length of the coding region of the gene. Greater than, greater than 90%, or greater than 95%. Moreover, the reading frames of sequences upstream and downstream from the region to be deleted need not be identical. Frame movement downstream of a region may be discarded due to inconsistencies in frame reading.

Disruption of genes can also be caused by, for example, mutations to amino acid substitutions (missense mutations), stop codons (nonsense mutations), additions or deletions of one or two nucleotide residues (frameshift mutations), or coding of genes on a chromosome, etc. am. Disruption of a gene can also be achieved, for example, by inserting another nucleotide sequence into the coding region of the gene on the chromosome. The insertion site can be in any region of the gene, and insertion of longer nucleotide sequences will generally more reliably inactivate the gene. A particular example is that the reading frames of sequences upstream and downstream from the insertion site are not identical. Frame movement downstream of a region may be discarded due to inconsistencies in frame reading. Other nucleotide sequences are not particularly limited as long as a sequence that reduces or eliminates the activity of the encoded protein is selected, and includes, for example, marker genes such as antibiotic resistance genes and genes useful for their production.

In particular, disruption of the gene can be performed such that the amino acid sequence of the encoded protein is deleted. That is, modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein, specifically modifying a gene to encode a protein in which the amino acid sequence is deleted. The term “deletion of an amino acid sequence of a protein” refers to a deletion of part or an entire region of the amino acid sequence of a protein. In addition, the term "deletion of the amino acid sequence of a protein" means that the original amino acid sequence disappears from the protein, and includes a case where the original amino acid sequence is changed to another amino acid sequence. That is, a region changed to a different amino acid sequence by frame shift, for example, may be regarded as a deletion region. Deletion of the amino acid sequence of a protein generally shortens the overall length of the protein, but there may be cases where the overall length of the protein is unchanged or extended. For example, by deletion of part or all of the coding region of a gene, the region encoded by the deleted region may be deleted from the encoded protein. Also, the region encoded by the region downstream of the introduction site can be deleted from the encoded protein, for example by introducing a stop codon into the coding region of the gene. Also, for example, by frameshifting in the coding region of a gene, the region encoded by the frameshift region may be deleted from the encoded protein.

Gene modification on such a chromosome can be achieved, for example, by preparing a disrupted gene that is modified so that it cannot produce a normally functioning protein, and transforming a host with a recombinant DNA containing the disruption. - A gene that causes homologous recombination between a disrupted gene on a chromosome and a wild-type gene to replace a wild-type gene on a chromosome with a disrupted gene. In this process, experimental manipulation is facilitated by including marker genes selected according to host characteristics, such as auxotrophy, in the recombinant DNA. Examples of disruptive genes include genes in which part or all of the coding region is deleted, genes containing missense mutations, genes containing nonsense mutations, genes containing frameshift mutations, and insertions of transposons or marker genes. contains a gene that Even if the protein encoded by the disruptive gene is produced, its function is reduced or eliminated because it has a different structure from the wild-type protein. Gene disruption by gene replacement using such homologous recombination has already been established, such as a method using linear DNA.

Modifications to reduce the activity of a protein can also be accomplished, for example, by mutagenesis. Examples of mutagenesis treatment include X-ray or ultraviolet irradiation and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). that is included

These methods of reducing the activity of a protein may be used alone or in any combination.

When a protein functions as a complex having a plurality of subunits, some or all of the plurality of subunits may be modified, as long as the activity of the protein is eventually reduced. That is, for example, some or all of a plurality of genes encoding each subunit may be disrupted. Also, when there are multiple isozymes of a protein, some or all of the activities of the multiple isozymes may be reduced, as long as eventually the activity of the protein is reduced. That is, for example, some or all of a plurality of genes encoding each isoenzyme may be disrupted.

A decrease in the activity of a protein can be confirmed by measuring the activity of the protein.

A decrease in the activity of a protein can also be confirmed by confirming a decrease in the expression of a gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the amount of transcription of the gene or a decrease in the amount of protein expressed from the gene.

A decrease in the amount of gene transcription can be confirmed by comparing the amount of mRNA transcribed from the gene with the amount of the unmodified strain.

Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, and RNA-seq. The amount of mRNA (eg, the number of molecules of mRNA per cell) can be reduced, for example, by 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that.

A decrease in the amount of protein can be confirmed by Western blotting using an antibody. The amount of protein (eg, number of protein molecules per cell) can be reduced, for example, by 50% or less, 20% or less, 10% or less, 5% or less, or 0%. It is an unmodified strain.

Disruption of the gene can be confirmed by determining the nucleotide sequence of part or all of the gene, a restriction enzyme map, the full length of the gene, etc., depending on the means used.

The aforementioned method of reducing the activity of a protein can be applied not only to the reduction of ribonuclease III activity, but also to the reduction of the activity of any protein and the reduction of the expression of any gene.

II. Vectors containing expression units and transcription units

The RNA transcription system of interest of the present invention comprises (a) DNA comprising an RNA polymerase expression unit that provides RNA polymerase; And (b) it may be a DNA system characterized in that the DNA containing the RNA unit of interest encoding the RNA of interest (RNAi) transcribed by the RNA polymerase is managed by each independent replicon or one replicon. .

The DNA system additionally includes an RNA polymerase expression unit including a promoter recognizable in host cells for transcription of the RNA polymerase; and an RNA unit of interest comprising a DNA encoding a promoter to which the RNA polymerase can bind, and a DNA system characterized in that they are operably linked to each other.

Plasmids are circular DNA molecules that exist independently in bacterial cells. Plasmids almost always contain more than one gene, and these genes are often responsible for useful features displayed by the host bacterium. For example, the ability to survive normal toxic concentrations of antibiotics is due to the presence of plasmids containing antibiotic resistance genes in bacteria. In the laboratory, this antibiotic resistance is often used as a selectable marker.

All plasmids have at least one DNA sequence that can serve as a replication origin (a site recognized by the host cell's replication enzyme, and replication starts from this site), and therefore a main bacterial chromosome ( It can replicate within cells independently of the main bacterial chromosome. Some types of plasmids can replicate by inserting themselves into the bacterial chromosome. These integrating plasmids (called episomes) can stably maintain this conformation during numerous cell divisions. However, at some stage, it exists as an independent element by excision (reverse process of integration).

A selectable marker is a gene carried by a vector, and refers to a gene that enables the recognition of characteristics of cells containing the vector or recombinant DNA molecule.

Plasmids range in size from 1.0 kb for the smallest to over 250 kb for the largest. Copy number refers to the number of plasmids normally found in one bacterial cell. Although the factors controlling copy number are not well understood, each plasmid has a characteristic number ranging from 1 to 50 or more.

Although plasmids are widely distributed in bacteria, they are by no means so common in other organisms. The most studied eukaryotic plasmid is present in yeast as a 2 μm circle. The discovery of the 2 μm plasmid was very fortunate as it enabled the construction of vectors for cloning the genes of industrial organisms of great importance as hosts. However, plasmids have not been found in other eukaryotes.

In the present invention, the basic requirement for the delivery system of the RNA polymerase expression unit and the RNA unit of interest is an appropriate cloning vector, that is, transport and amplification in a host cell, which is a bacterial host cell, and transport and amplification in a host cell, which is a host cell, that serves as a vehicle when transported. It is a selection of DNA molecules. A variety of natural replicons exhibit characteristics that allow them to act as cloning vectors, but vectors must also be designed to have certain minimum qualifications to function as efficient agents for delivery, maintenance, and amplification.

An ideal vector should possess the following four properties:

(1) low molecular weight

(2) the ability to confer readily selectable phenotypic traits to host cells;

(3) Multicloning site with multiple restriction endonuclease cleavage sites for cloning of genes with desired proteins of interest

(4) the ability to replicate within the host cell so that many copies of the vector can be generated and passed on to daughter cells;

Examples of vectors naturally occurring or artificially constructed as delivery vehicles usable in the present invention include Escherichia coli plasmids, bacteriophages (λ, M13, P1), viruses (animal virus-retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia Insect viruses - Baculovirus, Plant viruses - Cauliflower mosaic virus, Potato virus X, Gemini virus, etc.), Agrobacterium tumefaciens based vectors, chimeric plasmids (cosmids, phagemids, phasmids and fosmids), artificial chromosomes (YAC, BAC, PAC, MAC and HAC) and non-E. coli vectors (eg, Bacillus and Pseudomonas vectors, etc.).

To determine the vector selection for the RNA polymerase expression unit and the RNA unit of interest in the present invention, the following various factors should be considered.

(1) Vector size: DNA unit size can vary for different vector types, ranging from 5-25 kb for plasmid vectors to >2,000 kb for HACs.

(2) Vector size: Vector sizes range from 5 kb plasmid vectors to 6-10 megabase HAC high-capacity vectors.

(3) Restriction enzyme cleavage sites: The number of restriction enzyme cleavage sites found in vectors varies greatly. Small plasmid vectors may have a few cleavage sites, but inserting multiple replication sites into the vector can increase the cleavage sites of the restriction enzyme.

(4) Copy number: Cloning vectors are maintained at different copy numbers depending on the replicon of the plasmid. High copy number vectors are preferred. Expression vectors with an origin of replication derived from the low copy number plasmid pBR322 determine the vector copy number which can range from 25-50 copies/cell or 150 to 200 copies/cell if derived from the high copy number plasmid pUC. .

(5) Cloning efficiency: The ability to clone a DNA unit inserted into a vector is determined by the vector's cloning efficiency.

(6) Ability of the vector to screen the protein of interest: In the case of recombinant selection, a specific selectable marker must be present in the vector to distinguish it from non-recombinant.

(7) The type of downstream experiments, such as evaluation of the protein of interest, should be considered.

Efficient retention and long-term expression of RNA polymerase expression units in the present invention is a major goal, but a major obstacle for many RNA transcription systems of interest. The choice of vector to use for the RNA transcription system of interest is very important as it greatly affects the level of gene expression and maintenance over time.

The most common vectors in use today are the result of multiple combinations of replicons, promoters, selectable markers, and multiple replication sites. To make an informed decision, you should carefully evaluate these features based on your individual needs.

replication origin

Genetic elements that replicate as autonomous units, such as plasmids, contain an origin of replication. An important parameter to keep in mind when choosing a suitable vector is the number of copies. Control of copy number resides in copies. Since many expression units are present in cells, it is logical to think that a higher plasmid dosage equals a higher recombinant protein yield. However, high plasmid numbers impose a metabolic burden that reduces bacterial growth rate and can lead to plasmid instability, thus reducing the number of healthy organisms for protein synthesis. For this reason, the use of high copy number plasmids for protein expression does not imply increased production yields by any means.

Commonly used vectors, such as the pET series, retain the pMB1 origin (ColE1-derivative, 15–60 copies per cell), whereas mutant versions of the pMB1 origin, such as the pUC series (500-700 copies per cell). The wild-type ColE1 origin (15-20 copies per cell) can be found in the pQE vector (Qiagen). They all belong to the same incompatibility group and cannot propagate together in the same cell because they compete with each other for the replication machinery. For dual expression of recombinant proteins using two plasmids, systems with p15A ori can be used (pACYC and pBAD plasmid series, 10-12 copies per cell). Although rare, triple expression can be achieved using the pSC101 plasmid. This plasmid is under tight control over replication and therefore exists in low copy number (<5 copies/cell). It may be advantageous to use a plasmid containing this replicon if the presence of high doses of the cloned gene or its product has detrimental effects on the cell. Alternatively, the Duet vector (Novagen) allows cloning of two genes from the same plasmid, simplifying double expression. The Duet plasmid has two multicopy sites preceded by a T7 promoter, a lac operator, and a ribosome binding site. By combining different compatible Duet vectors, up to 8 recombinant proteins can be generated from 4 expression plasmids.

promoter

A typical promoter in prokaryotes is the lac promoter, which is a key component of the lac operon. Lactose drives the system and this sugar can be used for protein production. However, induction is difficult in the presence of readily metabolizable carbon sources (e.g., glucose present in rich media). In the presence of lactose and glucose, expression of the lac promoter is not fully induced until all glucose is utilized. At this point (low glucose), cyclic adenosine monophosphate (cAMP) is produced, which is required for full activation of lac. This regulation of expression is called catabolism inhibition. Glucose also suppresses lactose absorption because lactose permase is inactive in the presence of glucose. To achieve expression in the presence of glucose, a mutant, the lac UV5 promoter, was introduced that reduces (but does not eliminate) sensitivity to catabolic regulation. However, when present in multicopy plasmids, both promoters suffer from low levels of the lac promoter repressor protein LacI, which sometimes results in unacceptably high levels of expression (so-called "leakage") in the absence of an inducer. Basic expression control in chromosomal copies of the gene (about 10 molecules per cell) can be achieved by introduction of a mutated promoter of the lacI gene, termed lacI Q, which leads to a higher level of expression (nearly 10-fold) of lacI. . The lac promoter and lac UV5 are rather weak and not very useful for recombinant protein production. Synthetic hybrids can be used that combine the strengths of other promoters with the advantages of the lac promoter. For example, the tac promoter consisted of the -35 region of the trp (tryptophan) promoter and the -10 region of the lac promoter. This construct is about 10 times more potent than lac UV5. Notable examples of commercially available plasmids that use lac or tac promoters to drive protein expression include the pUC series (lac UV5 promoter, Thermo Scientific) and the pMAL series of vectors (tac promoter, NEB).

The T7 promoter system present in the pET vector (pMB1 ori, medium copy number, Novagen) is very useful for recombinant protein expression. Targeted proteins can represent 50% of total cellular proteins if successful. In this system, the gene of interest is cloned in the region downstream of the promoter recognized by phage T7 RNA polymerase (T7 RNAP). This highly active polymerase can be provided on another plasmid or placed into the bacterial genome of a prophage (λDE3) encoding T7 RNAP, most commonly under the transcriptional control of the lac UV5 promoter. Thus, the system can be induced with lactose or isopropyl β-D-1-IPTG. Basal expression can be controlled by co-expression of lac I Q as well as T7 lysozyme. T7 lysozyme binds to T7 RNAP and inhibits transcriptional initiation from the T7 promoter. If the T7 RNAP gene is expressed and a small amount of T7 RNAP is produced, T7 lysozyme will effectively control the unintended expression of the heterologous gene under the T7 promoter. T7 lysozyme is provided by compatible plasmids (pLysS or pLysE). The amount of T7 RNAP produced after induction exceeds the polymerase level that T7 lysozyme can inhibit. Thus, "free" T7 RNAP may be involved in the transcription of recombinant genes. Another level of control inserts the lacO operator downstream of the T7 promoter to create a hybrid T7/lac promoter. All three mechanisms (tight repression of the lac-inducible T7 RNAP gene by lacIQ, T7 RNAP inhibition by T7 lysozyme and presence of a lacO operator after the T7 promoter) make the system ideal for avoiding basal expression.

Another widely used approach is to place genes under the control of regulated phage promoters. The strong left promoter (pL) of phage lambda directs the expression of early lytic genes. The promoter is tightly repressed by the λcI repressor protein, which is located in the operator sequence during lysogenic growth. When the host SOS response is triggered by DNA damage, expression of the protein RecA is stimulated, which in turn catalyzes the self-cleavage of λcI, allowing transcription of the pL control gene. This mechanism has been used in expression vectors containing the pL promoter. The SOS response (and recombinant protein expression) can be induced by adding nalidixic acid, a DNA gyrase inhibitor. Another way to activate a promoter is to place the corresponding gene under the influence of another promoter to control λcI production. This two-step control system is already a T7 promoter/T7 RNAP based vector. In the pLEX series vectors (Life Technologies), the λcI suppressor gene was integrated into the bacterial chromosome under the control of the trp promoter. In the absence of tryptophan, this promoter is always "on" and λcI is continuously produced. Addition of tryptophan results in the formation of a tryptophan-TrpR repressor complex that binds tightly to the trp operator and blocks λcI repressor synthesis. Expression of the desired gene then occurs under the pL promoter.

Transcription of all promoters was initiated by chemical agents. Systems that respond to physical signals (eg temperature or pH) can also be used. The pL promoter is one example. The mutant λcI suppressor protein (λcI 857) is temperature sensitive and unstable at temperatures higher than 37°C. E. coli host strains containing the λcI 857 protein (either chromosomally or integrated into a vector) were first grown to the desired density at 28-30 °C and then protein expression was induced by a temperature shift to 40-42 °C. The industrial advantage of this system lies in part in the fact that heat is generated during fermentation and the temperature can be easily raised in high-density cultures. On the other hand, the gene under the control of the cold-inducible promoter cspA is induced by a temperature drop to 15°C. The pCold series of plasmids have the pUC118 backbone (pUC18 derivative) with the cspA promoter and have been successfully expressed for more than 30 recombinant proteins, reaching levels of 20-40% of the total expressed proteins. However, it should be noted that in various cases the target protein was obtained in an insoluble form.

transcription termination sequence

Bacteria have transcription units for the RNA of interest. A bacterium having an expression unit of the RNA of interest can be obtained by introducing the RNA unit of interest into the bacterium.

A termination sequence for terminating gene transcription may be located downstream of the RNA gene of interest. The terminator sequence is not particularly limited as long as it functions in the host. The terminator sequence may be a terminator sequence derived from a host or a heterologous terminator sequence. The termination sequence may be the unique termination sequence of the RNA gene of interest or the termination sequence of another gene. Specific examples of the terminal sequence include, for example, the terminal sequence of bacteriophage BFK20.

Two classes of transcription termination sequences, Rho-dependent and Rho-independent, have been identified throughout the prokaryotic genome. These widely distributed sequences trigger termination of transcription upon normal completion of transcription of a gene or operon, mediate premature termination of transcription as a regulatory means as observed in transcriptional attenuation, and early termination to prevent unnecessary energy expenditure in the cell. Escape the sequence accidentally.

<Rho dependent termination sequence>

Rho-dependent transcription termination sequences require large proteins called Rho factors that exhibit RNA helicase activity to disrupt the mRNA-DNA-RNA polymerase transcription complex. Rho-dependent termination sequences have been found in bacteria and phages.

The Rho-dependent termination sequence occurs downstream of the translational stop codon, and the unstructured cytosine-rich sequence of mRNA, known as the Rho utilization site (rut) for which no consensus sequence has been identified, and the downstream transcription stop point , tsp).

rut serves as an mRNA loading site and an activator of Rho. Activation allows Rho to efficiently hydrolyze ATP and down-translocate mRNA while the mRNA maintains contact with the stereotypical site. Rho can catch up with RNA polymerase because it stops at the downstream tsp site. Several different sequences can function as tsp sites. Contact between Rho and the RNA polymerase complex stimulates dissociation of the transcription complex through a mechanism involving Rho's allosteric effect on RNA polymerase.

<Rho independent termination sequence>

The native transcription termination sequence or Rho-independent termination sequence requires the formation of a self-annealing hairpin structure in the elongated transcript, which leads to the breakdown of the mRNA-DNA-RNA polymerase ternary complex. The terminator sequence of DNA contains a 20-base pair GC-rich bipair symmetric region followed by a short poly-A track or "A stretch". This region was transcribed to form a 7-9 nucleotide "U track" with a terminal hairpin, respectively. The termination mechanism has been hypothesized to occur through a combination of allosteric effects of RNA polymerase and hairpin binding interactions and direct promotion of dissociation through “competition kinetics”. Hairpin formation causes RNA polymerase to stall and destabilize, increasing the time paused at that position and reducing the stability of the complex, making dissociation of the complex at that position more likely to occur. In addition, the elongation protein factor NusA interacts with RNA polymerase and hairpin structures to stimulate transcriptional termination.

Two classes of transcription termination sequences, Rho-dependent and Rho-independent, have been identified throughout the prokaryotic genome. These widely distributed sequences trigger termination of transcription upon normal completion of transcription of a gene or operon, mediate premature termination of transcription as a regulatory means as observed in transcriptional attenuation, and early termination to prevent unnecessary energy expenditure in the cell. Escape the sequence accidentally.

selection marker

Resistance markers can be added to the plasmid backbone to inhibit the growth of plasmid-free cells. In E. coli, antibiotic resistance genes are habitually used for this purpose. Resistance to ampicillin can be conferred by the bla gene, a periplasmic enzyme that inactivates the β-lactam ring of β-lactam antibiotics. However, as β-lactamase is continuously secreted, degradation of the antibiotic occurs and ampicillin is almost depleted within a few hours. In this situation, cells that do not carry the plasmid may increase in culture. Chloramphenicol and kanamycin resistance based on degradation may also have this problem, although this has not been experimentally verified. For this reason, tetracycline is considered to be very stable in culture. Because resistance is based on the activity of antibiotics in resistant cells.

The cost of antibiotics and the spread of antibiotic resistance are major concerns for projects with large-scale culture. We are putting a lot of effort into the development of antibiotic-free plasmid systems. These systems are based on the concept of plasmid poisoning, a phenomenon that occurs when cells without plasmids cannot grow or live. For example, essential genes can be deleted from the bacterial genome and then inserted into a plasmid. Therefore, bacteria lacking the plasmid die after cell division. Different subtypes of plasmid poisoning systems exist according to their functional principle: (i) toxin/antitoxin based systems, (ii) metabolic based systems and (iii) operator inhibitor titration systems. Although this promising technology has proven successful in large-scale fermentors, transcription systems based on plasmid poisoning have not yet become widespread.

III. Expression unit of RNA polymerase

The present invention provides an RNA transcription system capable of continuously expressing an mRNA of interest by RNA polymerase. The RNA transcription system of the present invention comprises an RNA polymerase expression unit and an RNA unit of interest.

In the present invention, host cells for the RNA transcription system may use animals, plants, yeast, bacteria, etc., wherein the bacteria are Corynebacterium glutamicum (Corynebacterium glutamicum), Escherichia coli BE42, Escherichia coli HB101, Escherichia coli JM101, Escherichia coli Host cells containing LE392, E. coli MC1061, E. coli Y1088 and E. coli W3110 in which the activity of ribonuclease III is reduced by attenuating the expression of the gene encoding ribonuclease III or disrupting the gene can be used. .

Preferably, E. coli strain HT115 (DE3) with reduced activity of ribonuclease II can be used.

Protein synthesis in heterologous expression systems such as bacteria is one of the main goals of biotechnology. Proteins are synthesized by ribosomes according to instructions encoded in the sequence of mRNA.

In order to achieve the above object, the present invention provides an RNA polymerase gene in which the expression unit of the RNA polymerase of the RNA transcription system of interest of the present invention comprises the nucleotide sequence of Formula (I) below.

Formula (I): W-X-Y-Z

In the above formula, W may include a promoter recognized by RNA polymerase in the host cell and 5*?**?*UTR including an SD region.

X represents the 5'end nucleotide sequence of the RNA polymerase gene, translation start codon ATG, and the RNA polymerase gene

(a) the DNA dependent RNA Polymerase or a variant thereof; and

(b) a capping enzyme or a variant thereof; and

(c) a linker; may also be included.

Here, the expression unit of the RNA polymerase may additionally include the RNA-binding tethering peptide. The RNA polymerase is characterized by comprising at least one RNA-binding domain of a protein-RNA tethering system, wherein the RNA-binding domain comprises a specific RNA sequence and / Or it may be an RNA structure characterized by binding specifically to the RNA element of the protein-RNA tethering system composed of the structure.

Even more preferably, the expression unit of the RNA polymerase of the RNA transcription system of interest of the present invention may include RNA dependent RNA Polymerase (RdRp) or a variant thereof.

Y represents a nucleotide sequence corresponding to the 2nd to translation stop codon of the RNA polymerase gene;

Z represents a nucleotide sequence including a transcription termination sequence below the stop codon at the 3' end of the RNA polymerase gene.

In addition, in the present invention, as a transcription termination sequence below the stop codon, the translation termination codon of TGA TAA was ligated to the 3' end of the Z region to induce translation termination to be sure.

The present invention provides an RNA polymerase amino acid sequence expressed from the RNA polymerase gene nucleotide sequence of the present invention.

In addition, the present invention provides an expression vector containing the RNA polymerase gene of the present invention.

Various methods can be considered to increase the expression rate of the RNA polymerase expression unit for the RNA transcription system of interest in the host cell E. coli. First, self-replication of plasmid [gene copy number and stability related factors, second, mRNA [mRNA synthesis initiation rate, mRNA synthesis elongation and termination (termination) and mRNA degradation rate related factors Third, factors related to the rate of initiation of translation of the protein of interest, and fourth, factors related to the degradation rate of the protein of interest should be considered to be compatible with the host cell.

DNA sequences involved in transcription

Three different DNA sequences and one multicomponent protein are involved in gene transcription: (1) promoter, (2) transcription termination sequence, (3) regulatory sequence, and (4) RNA polymerase.

RNA polymerase is made up of five different components called a, b, b', w and s. While a2bbw constitutes the core enzyme, the addition of s conferring promoter specificity constitutes the complete enzyme. The N-terminal portion is involved in dimer formation and binding to b and b', and the C-terminus linked to the N-terminus via a flexible linker is responsible for interaction with UP elements present upstream of some promoters. below) or with some transcriptional activators. The b subunit binds rNTP, contains a catalytic domain and is a target for the antibiotic rifampicin, while b' allows non-specific binding to DNA. The role of w is largely unknown, but it is assumed to play a role in RNA polymerase assembly.

All bacterial species analyzed so far contain only one gene each encoding a component of a key enzyme, but most species have genes encoding multiple s-factors. One of these factors functions as a primary or housekeeping factor and is involved in the transcription of all genes required for growth in the vegetative stage. Additional factors are called secondary or substitute factors and are only needed under certain growing conditions. E. coli encodes six replacement factors that require s ³² after a rapid rise in temperature and where s ^S replaces the housekeeping s factor s ⁷⁰ during the shutdown phase. So far, only s ⁷⁰ has been used for the production of recombinant proteins.

The W region of the formula (I) of the present invention is a promoter

a group comprising pta, aceA, aceB, adh and amyE promoters inducible with acetic acid, ethanol, pyruvic acid and the like;

a group containing the cspB, SOD and tuf promoters;

a group containing a lac promoter, a tac promoter, and a trc promoter;

A group comprising phage-derived promoters such as F1 promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter;

RNA polymerase is responsible for recognizing promoters, and each RNA polymerase recognizes a different promoter. A promoter consists of three regions, commonly referred to as -35 and -10 boxes, and a spacer region separating the two boxes. Alignment of many promoters allows the inference of a so-called consensus sequence, and the consensus sequence for s ⁷⁰ is TTGACA-N17-TATAAT. This sequence is an optimal promoter sequence with a spacer region of 17 nucleotides. There is no single promoter in E. coli. For most chromosomes that are identical to the common sequence, there is one or two deviations in both the -35 and -10 boxes. Some promoters also contain a fourth region, a UP element located upstream of the -35 box. The UP element consists of an AT-rich sequence that interacts with the C-terminal domain of the subunit to increase promoter strength. It functions as an independent promoter module and stimulates transcription when fused with other promoters such as lacUV5. Promoters directing the production of recombinant proteins do not use UP elements.

The lactose (lac) operon promoter region consists of a core promoter comprising RNAP binding sites, -35 and -10 hexamers (bold letters), -45 region (filled box) and CAP binding site (open box).

The lactose (lac) operon promoter is positively regulated by a catabolic gene activator-cyclic AMP complex (CAP) that binds to DNA located 61.5 bp upstream of the transcriptional start site. There is a 13-bp sequence (-38 to -50 [-45 region]) between the CAP binding site and the core promoter sequence.

-37C is distinguished to indicate that this position has been removed from the original definition of the -45 region. The +1 position corresponds to the P1 transcription start site. P2 transcription starts at -22, P3 transcription starts at -15, and P4 transcription starts at -33 or -34.

Prokaryotic Enhancer Sequences The base sequence of 5 prokaryotic enhancer bases is -10 in the middle of TATAAT, and this sequence was obtained from a large number of enhancer sequences. The E. coli enhancer is called a pribnow box with six sequences at -10 points, and the base sequence is TATAAT (the first TA and the last T are conserved).

Preferably, a tryptophan promoter derived from a tryptophan operon may be used as a promoter for the W region of the formula (I), and the tryptophan promoter is transcribed by the intracellular tryptophan concentration. Transcription is regulated when the intracellular tryptophan concentration is lowered or when competitive inhibitors such as indole-3-propionic acid or indole acrylic acid (3-β-indolylacrylic acid: 3-IAA) are present. It goes on.

The non-coding region of mRNA, especially the 5'-untranslated region (5'-UTR), greatly contributes to the efficiency of protein synthesis. Bacterial 5'-UTRs have many functions that affect translation. The most important and best known 5'-UTR sequence element of bacterial mRNA is the Shine-Dalgarno (SD) box complementary to the 3'-terminal region of the small subunit 16S rRNA. In E. coli, the length of the complementary region varies between 4 and 8 nucleotides (nt). The optimal distance between the SD box and the start codon for efficient gene expression is 5-9 nt.

The RNA polymerase expression unit of the RNA transcription system of interest of the present invention may include a promoter, a 5'-UTR, an RNA polymerase coding region and a transcription termination sequence.

The Shine-Dalgarno Sequence or SD sequence is the ribosome binding site of prokaryotic messenger RNA, and is generally located 8 bases ahead of the start codon AUG. This RNA sequence recruits the ribosome to the messenger RNA (mRNA) and aligns the ribosome at the start codon position to help initiate protein synthesis.

Shine-Dalgarno sequences are present in both bacteria and archaea. Also, this arrangement is present in some chloroplast and mitochondrial transcripts. The six common arrangements are AGGAGG. For example, in E. coli, it is AGGAGGU, whereas in the early stages of E. coli virus T4, the GAGG sequence predominates.

The nucleotide tract at the 3' end of E. coli 16S rRNA is rich in pyrimidine and has a specific nucleotide sequence (PyACCUCCUUA 3'OH). They recognize a complementary purine-rich sequence (AGGAGGU) of these ribosome bases. The base binding of the Shine-Dalgarno sequence to mRNA and the 3' end of 16S rRNA is important for initiation of transcription by bacterial ribosomes.

In eukaryotic cells, the 3' end of small 18S rRNA plays an important role in termination of protein synthesis by binding complementary bases with the stop codon. It is based on the complementary relationship between UUA-OH at the 3' end of and E. coli stop codon.

Mutations in the Shine-Dalgarno sequence can reduce or increase translation in prokaryotic cells. This change was demonstrated by the fact that mRNA-ribosomes use an increase or decrease in base-binding efficiency, and compensatory mutations in the 3'-terminal 16S rRNA sequence can restore translation.

The intercistronic spacer (IS) contains the S/D sequence if an Au(UAUUUUAAUUAAUUAA) rich sequence is introduced upstream of the intercistronic spacer translational efficiency. Each IS size varies from ~20ntds to 40-50ntds. This spacer contains S/D sequences for the 30s ribosomal subunit to bind to the next cistron and restart translation.

The region between the start codon and the stop codon is commonly referred to as an ORF (Open Reading Frame). The coding region of each cistron (most bacterial mRNAs are polycistronic) begins with the initiator codon AUG (mostly) or GUG (rarely) of each cistron, and then UAG (Opel), UGA (Amber), or UAA. Termination sequence ends with a codon.

This spacer region is used for ribosome dissociation and subsequent recombination with the cistron at the end of the orf and begins translation again. The size of the intercistron space varies, but is mostly very short. Often the terminator codon sequence overlaps with the next cistron containing the AUG. This will allow you to seamlessly start translating again (e.g. UGAUG ).

The transcription termination sequences of the present invention permit transcription termination, and there are two types of termination sequences, namely rho-independent and rho-dependent. The first class consists of inverted repeats in which the template DNA strand is followed by several A residues. When RNA polymerase transcribes an inverted repeat, it immediately folds into a stem-loop structure at the mRNA level, pausing the enzyme. Dissociation of the enzyme occurs because there are several U residues behind the stem-loop structure that interact weakly with the A residues of the template DNA. However, the terminator causes dissociation of each RNA polymerase molecule and does not result in readthrough-transcription to adjacent gene(s). To reduce this read-through, often two different transcription termination sequences are placed side by side in an expression vector. Two tandem transcriptional termination sequences, T1 and T2, are particularly effective, the rrnB rRNA operon of E. coli.

The rho independent terminal sequence consists of a stable hairpin and a U-rich region. Intrinsic termination mechanisms include the pausing of the transcription elongation complex (TEC) at the termination site and the formation of RNA termination sequence hairpins inside RNA polymerase (RNAP). Three nucleic acid binding sites have been characterized in TEC: double-stranded DNA binding site (DBS), RNA:DNA hybrid binding site (HBS) and single-stranded RNA binding site (RBS). Formation of stable RNA hairpins, together with dissociation of weak U-rich RNA:DNA hybrid duplexes of nascent mRNAs, leads to disruption of protein-nucleic acid interactions in HBS and RBS of RNAP, destabilization and dissociation and release of TECs.

The unique rho-independent termination sequence is a model of a typical native termination sequence consisting of an RNA hairpin followed by a 15nt 'T-region'. Hairpin and thymidine-rich regions can be separated by 'spacers' of less than 2 nt. An adenosine-rich region was also identified upstream of the hairpin, but is not included in this terminal sequence model. It also contains an 11nt region upstream of the adenosine-rich hairpin, termed the 'A-region'. The efficiency of transcription termination depends on both the stability of the RNA hairpin and RNA:DNA hybrid duplex within the TEC and the difference between the TEC's pause at the termination point.

Genes are expressed or regulated constitutively, and there are two different classes of regulators: transcriptional repressors and transcriptional activators. Repressors bind to operators located within or immediately downstream of the promoter region and in most cases prevent or act as a barrier to RNA polymerase-promoter binding. To relieve oppression, the oppressor must be separated from the operator. In some cases, an inducer is synthesized by the cell or taken up in the environment where it binds to an inhibitor and causes dissociation from the operator. The LacI repressor is the best studied example. Other types of repressors require an operator to bind to a cognate operator. As long as a large amount of co-inhibitor is present in the cell, inhibition occurs. If the corepressor is being consumed by the cell, the repressor fails to bind to its operator. The TrpR inhibitor and its key inhibitor tryptophan are the most prominent examples.

A third, though artificial possibility, is a temperature-sensitive suppressor. These repressor alleles are isolated after mutagenesis of the repressor gene and undergo amino acid replacement to synthesize temperature-sensitive proteins. At low temperatures (30-32 °C), the repressor is activated and binds the actuator. When the cells are moved to higher temperatures (40-42 °C), the repressor changes its structure and separates from the effector. This principle is used with the cI inhibitor of bacteriophage l.

These repressor alleles are isolated after mutagenesis of the repressor gene and undergo amino acid replacement to synthesize temperature-sensitive proteins. At low temperatures (30-32 °C), the repressor is activated and binds the actuator. When the cells are moved to higher temperatures (40-42 °C), the repressor changes its structure and separates from the effector. This principle is used with the cI inhibitor of bacteriophage l. These repressor alleles are isolated after mutagenesis of the repressor gene and undergo amino acid replacement to synthesize temperature-sensitive proteins. At low temperatures (30-32 °C), the repressor is activated and binds the actuator. When the cells are moved to higher temperatures (40-42 °C), the repressor changes its structure and separates from the effector. This principle is used with the cI inhibitor of bacteriophage l.

A transcriptional activator binds to a sequence designated UAS (upstream activating sequence), usually upstream of a promoter. By binding to the UAS, the activator increases the likelihood of RNA polymerase binding to the promoter and further activates transcriptional initiation by interaction with one of its subunits (the a or s subunit in most cases). Expression systems using transcriptional activators are not described.

DNA sequences involved in translation

The complexity of the process has made it difficult to decipher the determinants of protein synthesis initiation. The wide range of translation efficiencies of various mRNAs is mainly due to the structure of the 5' end of each mRNA species. Therefore, no universal sequence has been devised for efficient translation initiation, where the translation initiation region is (1) the Shine-Dalgarno sequence, (2) the start codon, (3) the spacer region between the Shine-Dalgarno sequence and the start codon, (4) It is sometimes composed of four different sequences of translational enhancers.

Shine and Dalgarno identified sequences in the ribosome-binding site (RBS) of bacteriophage mRNA and that this region interacts with the complementary 3' end of the 16S rRNA during translation initiation. In E. coli, the initiation codon AUG is predominantly (91%) used, followed by GUG (8%) and UUG (1%). This preference is consistent with AUG's predominant translational efficiency. The spacing between the Shine-Dalgarno sequence and the start codon varies from 5 to 13 nucleotides and also affects translation efficiency. To determine the optimal nucleotide sequence of the translation initiation region, (1) the Shine-Dalgarno sequence UAAGGAGG enables 3-6 fold higher protein production than AAGGA for all intervals, and (2) the optimal interval for UAAGGAGG is AAGGA It may be 4-8 nucleotides and 5-7 nucleotides for.

In addition, the secondary structure of the translation initiation site of mRNA plays an important role in the efficiency of gene expression. Closure of the Shine-Dalgarno sequence and/or start codon by the stem-loop structure prevents accessibility to the 30S ribosomal subunit and inhibits translation. There are two reported cases where this principle is used to significantly reduce the downstream reading frame namely translation, rpoH heat shock sigma factor S-encoding mRNA in Escherichia coli and Morita rhizobiae small heat-shock protein-encoding mRNA. In both cases, translation of these mRNAs is achieved under heat shock conditions leading to melting of the secondary structure. There is potential to minimize mRNA secondary structure in the translation initiation region. Enrichment of RBS with adenine and thymine residues enhanced the expression of specific genes, but mutation of specific nucleotides upstream or downstream of the Shine-Dalgarno sequence inhibited the formation of mRNA secondary structures and reduced translation efficiency. Sequences that significantly enhance the expression of recombinant genes have been identified, and these modules are called translational enhancers. One example is the U-rich region immediately upstream of the Shine-Dalgarno sequence in the E. coli atpE gene. These 30 base sequences have been successfully used to overexpress human interleukin-2 and interferon beta genes.

Expression system for E. coli

Tight expression of transcripts of recombinant genes is often desirable or necessary because tight expression can be detrimental or even lethal to cell growth. Regulated gene expression requires an inducible or repressible system, so all expression systems are based on controllable promoters. Promoters enabling constitutive expression have proven unsuitable for the production of recombinant proteins for two main reasons. First, it does not allow the production of toxic proteins and second, even non-toxic proteins produced at physiological concentrations can be harmful to the human body. cells when produced at higher levels. One prominent example is an integral membrane protein that, when overproduced, disrupts the inner membrane and results in cell death. Four regulatable promoter systems are widely used, where three are based on the already mentioned repressors (LacI, TrpR and phage 1 cI) and the fourth is based on phage RNA polymerase.

The Lac system consists of the former promoter/operator region of the Lac operon and coding for the lacI repressor, the repressor of the lacI gene. In the absence of an inducer, the Lac repressor binds to an operator immediately downstream of the promoter as a homotetramer. The wildtype lac promoter sequence contains one deviation at -35 and two deviations in the -10 box, and the spacer region contains 18 nucleotides compared to the consensus sequence. One of the many isolated promoter mutants was named lacUV5. If its DNA sequence is compared with that of the wild-type promoter, two nucleotides are exchanged, the consensus is -10 box, and the promoter strength of lacUV5 is increased by 2.5-fold. Mutations that increase the promoter strength are generally referred to as promoter-up mutations.

The promoter of the trp operon exhibits the primary -35 box and optimal spacer length, but has three deviations within the -10 box. Based on the lacUV5 and trp promoters, an artificial promoter representing the consensus sequence of the s 70-dependent promoter was constructed, which was called Ptac.

The LacI and TrpR repressors are inactivated to initiate expression of the recombinant gene. For the Plac, PlacUV5 and Ptac promoters, the repressors are inactivated by the addition of isopropyl-bD-thiogalactopyranoside (IPTG). . This compound binds to the active LacI inhibitor and causes dissociation from the effector. IPTG has two advantages over lactose. First, its uptake is not dependent on Lac permease, and second, it is cleaved by b-galactosidase and cannot prevent blockage of transcription. Lac Gene The lacI gene is part of an expression plasmid or is present in a chromosome.

Expression systems based on the trp system use synthetic media with defined tryptophan concentrations. The concentration is chosen in such a way that the system can self-induce when the tryptophan concentration in the cell falls below a threshold level. Additionally, 3-b-indole-acrylic acid can be added to inactivate the TrpR repressor and inhibit the charge of tRNA trp by tryptophanil-tRNA synthetase.

The third system uses the bacteriophage l repressor cI. A more convenient method is a temperature-sensitive version of the cI inhibitor called cI857. Therefore, cells with an E. coli-based expression system are grown at low temperature to the mid-exponential phase and then transferred to high temperature to induce recombinant gene expression.

The most widely applied expression system uses phage T7 RNA polymerase, which recognizes only the promoter present in the T7 DNA and not the promoter present in the E. coli chromosome. Thus, the expression vector contains one T7 promoter (usually a promoter present in front of the gene) to which the recombinant gene is fused. The gene encoding T7 RNA polymerase is either present in the expression vector itself or in a second compatible plasmid or integrated into the E. coli chromosome. In all three cases the gene is fused to an inducible promoter allowing transcription and translation during the expression phase. T7 RNA polymerase offers three advantages over E. coli enzymes. First, it consists of only one subunit, the second exerts a higher processivity, and the third is insensitive to rifampicin. The latter feature can be used to increase the amount of recombinant protein, especially by adding this antibiotic about 10 minutes after induction of the gene encoding T7 RNA polymerase. During that time, enough polymerase is synthesized to allow high-level expression of the recombinant gene and inhibition of E. coli. The enzyme prevents further expression of all other genes present on both the plasmid and chromosome. Because all promoter systems are leaky, low levels of expression of the gene encoding T7 RNA polymerase can be detrimental to the cell if the recombinant gene encodes a toxic protein. These polymerase molecules present during growth can be suppressed by expressing the T7 encoding gene for lysozyme. This enzyme is a dual-function protein that cleaves bonds in the cell wall of E. coli and binds and selectively inhibits T7 RNA polymerase, a feedback mechanism that ensures a controlled burst of transcription during T7 infection.

Another expression system, hitherto not widely used, is induced by cold shock. When the mid-exponential phase culture of E. coli is rapidly moved from 37 °C to the 10–15 °C temperature range, the synthesis of most cellular proteins is greatly reduced, whereas the synthesis of about 15 cold shock proteins is transiently upregulated. CspA, the major cold shock protein, is virtually undetectable at 37 °C, but more than 10% of the total protein synthesis is used for production in the first hour after the temperature drops. Transcribed into the 150 nucleotide-long 5' untranslated region of cspA mRNA, the transcript stability is 10-fold greater when cells are moved to 15-10 °C than 37 °C (conferring transcripts at t and high instability 1/2-10 s). increase in degree A vector was constructed based on the untranslated region following the cspA promoter to express the recombinant protein at low temperature. While the growth rate of E. coli strains drops off rapidly as the incubation temperature decreases to 20 °C, addition of the groESL operon from Oleispira antarctica isolated from Antarctic seawater allows 3-fold faster growth at 15 °C and 36-fold greater growth at 10 °C. grow rapidly It can also be shown that both molecular chaperones exhibited high protein folding activity in vitro at temperatures of 4–12 °C. This result suggests that these engineered E. coli strains can produce large amounts of correctly folded recombinant proteins at low temperatures.

IV. RNA polymerase

After all, in order to obtain RNA polymerase, several steps must be performed from the gene, and the efficiency of gene expression is determined by the efficiency of each step. In other words, all steps must be harmonized in a complex way, regardless of the amount and stability of the gene, the transcription efficiency and stability of mRNA, and the translation efficiency. In addition to the base composition, the base composition of the gene of interest must be compatible with each other to increase the efficiency of each step and increase the expression of RNA polymerase.

"Chimeric enzyme" refers to an enzyme that is not a natural enzyme found in nature (ie, non-natural). Thus, chimeric enzymes include catalytic domains derived from different sources (eg, from different enzymes) or from catalytic domains that are derived from the same source (eg, from the same enzyme) but are arranged in a different way than found in nature. can do. "Chimeric enzymes" include monomeric (ie single unit) enzymes as well as oligomeric (ie multi unit) enzymes, particularly heterooligomeric enzymes. More specifically, a "chimeric enzyme" refers to one (i.e., a single unit) or several (i.e., multiple units) catalytic domain(s) (one or more catalytic domains of a capping enzyme) or protein(s) (one or more capping enzymes). can be

A “catalytic domain” of an enzyme relates to a protein domain necessary and sufficient to ensure enzyme function, particularly in a three-dimensional structure. For example, the catalytic domain of RNA triphosphatase is a necessary and sufficient domain to ensure RNA triphosphatase function. A "catalytic domain" includes the catalytic domain of a wild-type or mutant enzyme.

"Protein domains" define discrete functional and/or structural building blocks and elements of proteins that fold and function independently.

In particular, chimeric enzymes according to the present invention are monomeric or oligomeric non-natural enzymes.

A "monomeric enzyme" relates to a single unit enzyme composed of only one polypeptide chain.

"Oligomer enzyme" refers to a multi-unit enzyme composed of two or more polypeptide chains linked together, either covalently or non-covalently. "Oligomer enzymes" include multi-unit enzymes, wherein two or more units of the enzyme are covalently or non-covalently linked together. "Oligomer enzymes" include homo-oligomer enzymes, which are multiunit enzymes composed only of a single type of monomer (subunit), and hetero-oligomer enzymes composed of different types of monomers (subunits).

The present inventors are particularly concerned with the catalytic domain of capping enzymes, including the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase; And a RNA-binding domain of a protein-RNA tethering system comprising an RNA-binding domain that specifically binds to an RNA element composed of a specific RNA sequence and / or structure; a monomeric or oligomeric chimeric (non-natural) enzyme comprising The capping ratio of specific mRNA produced by RNA polymerase could be improved to a very high level.

A cytoplasmic monomeric or oligomeric chimeric enzyme comprising the catalytic domain of a capping enzyme and the bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system is U1-RNA 70K (also known as SNRN P70) described in U.S. Patent No. 5,866,680 above. Compared to other RNA-binding domains of protein-RNA tethering systems such as the -U1 snRNA system, the capping ratio of specific mRNA produced by bacteriophage DNA-dependent RNA polymerase could be greatly improved.

The present inventors also found that a monomeric or oligomeric chimeric enzyme comprising a catalytic domain of a capping enzyme, an RNA-binding domain of a protein-RNA tethering system, and a catalytic domain of poly(A) polymerase is capped in the presence of poly(A) polymerase. It was surprisingly able to cooperatively increase the capping rate of specific mRNAs produced by RNA polymerase compared to a combination of enzymes. This result was unexpected because the capping enzyme and poly(A) polymerase are not physically linked in nature and do not contain known expected binding domains for specific RNA sequences.

1.ctDdRp

The present invention relates in particular to the field of transcription systems of RNAs of interest in host cells. As one of the present invention for achieving the above object, a system comprising at least one intracellular protein translating and transcribing component, comprising an RNA polymerase expression unit and an RNA unit of interest,

a) the RNA polymerase expression unit comprises (i) a promoter operating in a host cell; (ii) a structure enabling intracellular translation; (iii) an open reading frame (ORF) containing RNA polymerase [DNA dependent RNA Polymerase (DdRp) and/or chimeric enzyme containing capping enzyme or RNA dependent RNA Polymerase (RdRp)]; (iv) a DNA unit comprising a transcription termination sequence; and

b) the RNA unit of interest comprises (i) a promoter driven by the DdRp; (ii) a DNAi unit capable of being transcribed by the DdRp polymerase and encoding an RNA of interest (RNAi); and (iii) a unit containing a transcriptional termination sequence.

or (i) a promoter operative in the host cell; (ii) a DNA unit comprising DNA encoding a replicon RNA replicated by the RdRp and comprising RNAi; and (iii) a unit comprising a transcription termination sequence;

Here, c) a configuration that enables their operation; provides an RNA transcription system characterized by and a cell line transformed therewith.

Preferably, the DdRp may be a capping enzyme or a poly(A) polymerase and a chimeric enzyme.

The DNA fragment encoding the chimeric DdRp of the present invention is the ORF of NP868R-T7RNAP (SEQ ID NO: 1). A DNA fragment encoding a fusion between NP868R, the mRNA capping enzyme of African swine fever virus, and the wild-type phage DdRp of bacteriophage T7 was inserted into one plasmid. was fused to the amino terminus of

The nucleotide sequence for the ORF of NP868R-T7RNAP is shown in SEQ ID NO: 1 below.

NP868R-T7RNAP:

ATGGAATTCG CCAGCCTGGA CAACCTGGTG GCCAGATACC AGCGGTGCTT CAACGACCAG 60

AGCCTGAAGA ACAGCACCAT CGAGCTGGAA ATCCGGTTCC AGCAGATCAA CTTCCTGCTG 120

TTCAAGACCG TGTACGAGGC CCTGGTCGCC CAGGAAATCC CCAGCACCAT CAGCCACAGC 180

ATCCGGTGCA TCAAGAAGGT GCACCACGAG AACCACTGCC GGGAGAAGAT CCTGCCCAGC 240

GAGAACCTGT ACTTCAAGAA ACAGCCCCTG ATGTTCTTCA AGTTCAGCGA GCCCGCCAGC 300

CTGGGCTGTA AAGTGTCCCT GGCCATCGAG CAGCCCATCC GGAAGTTCAT CCTGGACAGC 360

AGCGTGCTGG TCCGGCTGAA GAACCGGACC ACCTTCCGGG TGTCCGAGCT GTGGAAGATC 420

GAGCTGACCA TCGTGAAGCA GCTGATGGGC AGCGAGGTGT CAGCCAAGCT GGCCGCCTTC 480

AAGACCCTGC TGTTCGACAC CCCCGAGCAG CAGACCACCA AGAACATGAT GACCCTGATC 540

AACCCCGACG ACGAGTACCT GTACGAGATC GAGATCGAGT ACACCGGCAA GCCTGAGAGC 600

CTGACAGCCG CCGACGTGAT CAAGATCAAG AACACCGTGC TGACACTGAT CAGCCCCAAC 660

CACCTGATGC TGACCGCCTA CCACCAGGCC ATCGAGTTTA TCGCCAGCCA CATCCTGAGC 720

AGCGAGATCC TGCTGGCCCG GATCAAGAGC GGCAAGTGGG GCCTGAAGAG ACTGCTGCCC 780

CAGGTCAAGT CCATGACCAA GGCCGACTAC ATGAAGTTCT ACCCCCCCGT GGGCTACTAC 840

GTGACCGACA AGGCCGACGG CATCCGGGGC ATTGCCGTGA TCCAGGACAC CCAGATCTAC 900

GTGGTGGCCG ACCAGCTGTA CAGCCTGGGC ACCACCGGCA TCGAGCCCCT GAAGCCCACC 960

ATCCTGGACG GCGAGTTCAT GCCCGAGAAG AAAGAGTTCT ACGGCTTTGA CGTGATCATG 1020

TACGAGGGCA ACCTGCTGAC CCAGCAGGGC TTCGAGACAC GGATCGAGAG CCTGAGCAAG 1080

GGCATCAAGG TGCTGCAGGC CTTCAACATC AAGGCCGAGA TGAAGCCCTT CATCAGCCTG 1140

ACCTCCGCCG ACCCCAACGT GCTGCTGAAG AATTTCGAGA GCATCTTCAA GAAGAAAACC 1200

CGGCCCTACA GCATCGACGG CATCATCCTG GTGGAGCCCG GCAACAGCTA CCTGAACACC 1260

AACACCTTCA AGTGGAAGCC CACCTGGGAC AACACCCTGG ACTTTCTGGT CCGGAAGTGC 1320

CCCGAGTCCC TGAACGTGCC CGAGTACGCC CCCAAGAAGG GCTTCAGCCT GCATCTGCTG 1380

TTCGTGGGCA TCAGCGGCGA GCTGTTTAAG AAGCTGGCCC TGAACTGGTG CCCCGGCTAC 1440

ACCAAGCTGT TCCCCGTGAC CCAGCGGAAC CAGAACTACT TCCCCGTGCA GTTCCAGCCC 1500

AGCGACTTCC CCCTGGCCTT CCTGTACTAC CACCCCGACA CCAGCAGCTT CAGCAACATC 1560

GATGGCAAGG TGCTGGAAAT GCGGTGCCTG AAGCGGGAGA TCAACTACGT GCGCTGGGAG 1620

ATCGTGAAGA TCCGGGAGGA CCGGCAGCAG GATCTGAAAA CCGGCGGCTA CTTCGGCAAC 1680

GACTTCAAGA CCGCCGAGCT GACCTGGCTG AACTACATGG ACCCCTTCAG CTTCGAGGAA 1740

CTGGCCAAGG GACCCAGCGG CATGTACTTC GCTGGCGCCA AGACCGGCAT CTACAGAGCC 1800

CAGACCGCCC TGATCAGCTT CATCAAGCAG GAAATCATCC AGAAGATCAG CCACCAGAGC 1860

TGGGTGATCG ACCTGGGCAT CGGCAAGGGC CAGGACCTGG GCAGATACCT GGACGCCGGC 1920

GTGAGACACC TGGTCGGCAT CGATAAGGAC CAGACAGCCC TGGCCGAGCT GGTGTACCGG 1980

AAGTTCTCCC ACGCCACCAC CAGACAGCAC AAGCACGCCA CCAACATCTA CGTGCTGCAC 2040

CAGGATCTGG CCGAGCCTGC CAAAGAAATC AGCGAGAAAG TGCACCAGAT CTATGGCTTC 2100

CCCAAAGAGG GCGCCAGCAG CATCGTGTCC AACCTGTTCA TCCACTACCT GATGAAGAAC 2160

ACCCAGCAGG TCGAGAACCT GGCTGTGCTG TGCCACAAGC TGCTGCAGCC TGGCGGCATG 2220

GTCTGGTTCA CCACCATGCT GGGCGAACAG GTGCTGGAAC TGCTGCACGA GAACCGGATC 2280

GAACTGAACG AAGTGTGGGA GGCCCGGGAG AACGAGGTGG TCAAGTTCGC CATCAAGCGG 2340

CTGTTCAAAG AGGACATCCT GCAGGAAACC GGCCAGGAAA TCGGCGTCCT GCTGCCCTTC 2400

AGCAACGGCG ACTTCTACAA TGAGTACCTG GTCAACACCG CCTTTCTGAT CAAGATTTTC 2460

AAGCACCATG GCTTTAGCCT CGTGCAGAAG CAGAGCTTCA AGGACTGGAT CCCCGAGTTC 2520

CAGAACTTCA GCAAGAGCCT GTACAAGATC CTGACCGAGG CCGACAAGAC CTGGACCAGC 2580

CTGTTCGGCT TCATCTGCCT GCGGAAGAAC GGGCCCGGCG GAGGCGGAAG TGGAGGCGGA 2640

GGAAGCGGAG GGGGAGGATC TGGCGGCGGA GGCAGCCTCG AGAACACCAT CAATATCGCC 2700

AAGAACGACT TCAGCGACAT CGAGCTGGCC GCCATCCCTT TCAACACCT GGCCGACCAC 2760

TATGGCGAGC GGCTGGCCAG AGAACAGCTG GCCCTGGAAC ACGAGAGCTA CGAAATGGGC 2820

GAGGCCCGGT TCCGGAAGAT GTTCGAGAGA CAGCTGAAGG CCGGCGAGGT GGCCGATAAT 2880

GCCGCCGCTA AGCCCCTGAT CACCACCCTG CTGCCCAAGA TGATCGCCCG GATCAACGAT 2940

TGGTTCGAGG AAGTGAAGGC CAAGCGGGGC AAGAGGCCCA CCGCCTTCCA GTTTCTGCAG 3000

GAAATCAAGC CCGAGGCCGT GGCCTACATC ACCATCAAGA CCACCCTGGC CTGCCTGACC 3060

AGCGCCGACA ATACCACCGT GCAGGCTGTC GCTTCTGCCA TCGGCAGAGC CATCGAGGAC 3120

GAGGCCAGAT TCGGCAGAAT CCGGGACCTG GAAGCCAAGC ACTTCAAGAA AAACGTGGAG 3180

GAACAGCTGA ACAAGCGCGT GGGCCACGTG TACAAGAAAG CCTTCATGCA GGTGGTGGAG 3240

GCCGACATGC TGAGCAAGGG CCTGCTGGGC GGAGAAGCCT GGTCCAGCTG GCACAAAGAG 3300

GACAGCATCC ACGTCGGCGT GCGGTGCATC GAGATGCTGA TCGAGTCGAC CGGCATGGTG 3360

TCCCTGCACA GGCAGAATGC CGGCGTGGTG GGCCAGGACA GCGAGACAAT CGAACTGGCC 3420

CCCGAGTATG CCGAGGCCAT TGCCACAAGA GCCGGCGCTC TGGCCGGCAT CAGCCCCATG 3480

TTCCAGCCCTGCGTGGTGCCTCCTAAGCCCTGGACAGGCATCACAGGCGG CGGATACTGG 3540

GCCAACGGCA GACGCCCTCT GGCCTCTGGTG CGGACCCACA GCAAGAAAGC CCTGATGCGC 3600

TACGAGGACG TGTACATGCC CGAGGTGTAC AAGGCCATCA ACATTGCCCA GAACACCGCC 3660

TGGAAGATCA ACAAGAAAGT GCTGGCCGTG GCCAATGTGA TCACCAAGTG GAAGCACTGC 3720

CCCGTGGAGG ACATCCCCGC CATCGAGCGG GAGGAACTGC CCATGAAGCC CGAGGACATC 3780

GACATGAACC CCGAGGCCCT GACAGCCTGG AAAAGGGCCG CTGCCGCCGT GTACCGGAAG 3840

GACAAGGCCC GGAAGTCCCG GCGGATCAGC CTGGAGTTCA TGCTGGAACA GGCCAACAAG 3900

TTCGCCAACC ACAAGGCCAT TTGGTTCCCT TACAACATGG ACTGGCGGGG CAGAGTGTAC 3960

GCCGTGAGCA TGTTCAATCC ACAAGGCAAC GACATGACCA AGGGGCTGCT GACCCTGGCC 4020

AAGGGCAAGC CCATCGGCAA AGAGGCTAC TACTGGCTGA AGATCCACGG CGCCAACTGC 4080

GCTGGCGTGG ACAAGGTGCC CTTCCCAGAG CGGATCAAGT TCATCGAGGA AAACCACGAG 4140

AACATCATGG CCTGCGCCAA GAGCCCTCTA GAAAACACTT GGTGGGCCGA GCAGGACAGC 4200

CCCTTCTGCTTCCTGGCCTT CTGCTTTGAG TACGCCGGAG TGCAGCACCA CGGCCTGAGC 4260

TACAACTGCA GCCTGCCCCT GGCCTTCGAT GGCAGCTGCA GCGGCATCCA GCACTTCAGC 4320

GCCATGCTGA GGGACGAAGT GGGCGGCAGA GCCGTGAATC TGCTGCCAAG CGAGACAGTG 4380

CAGGACATCT ACGGGATCGT GGCCAAGAAA GTGAACGAGA TCCTGCAGGC CGACGCCATC 4440

AACGGCACCG ACAACGAGGT GGTGACCGTG ACCGATGAGA ACACCGGCGA GATCAGCGAG 4500

AAAGTGAAGC TCGGCACCAA GGCCCTGGCT GGCCAGTGGC TGGCCTACGG CGTGACCAGA 4560

TCCGTGACCA AGCGGAGCGT GATGACACTG GCCTATGGCA GCAAAGAGTT CGGCTTCCGG 4620

CAGCAGGTGC TGGAAGATAC CATCCAGCCT GCCATCGACA GCGGCAAGGG GCTGATGTTC 4680

ACCCAGCCCA ACCAGGCCGC TGGCTACATG GCCAAGCTCA TATGGGAGAG CGTGTCCGTG 4740

ACAGTGTGG CCGCCGTGGA GGCCATGAAT TGGCTGAAGT CCGCCGCCAA ACTGCTGGCC 4800

GCTGAAGTGA AGGACAAAAA GACCGGCGAA ATCCTGCGGA AGAGATGCGC CGTGCACTGG 4860

GTGACCCCCG ATGGCTTCCC CGTGTGGCAG GAGTACAAGA AGCCCATCCA GACCCGGCTG 4920

AACCTGATGT TCCTGGGCCA GTTCAGACTG CAGCCCACCA TCAACACCAA CAAGGACTCC 4980

GAGATCGACG CCCACAAGCA GGAAAGCGGC ATTGCCCCCA ACTTCGTGCA CAGCCAGGAC 5040

GGCAGCCACC TGAGAAAGAC CGTGGTGTGG GCTCACGAGA AGTACGGCAT CGAGAGCTTC 5100

GCCCTGATCC ACGACAGCTT CGGCACCATT CCCGCCGACG CCGCCAACCT GTTCAAGGCC 5160

GTGCGGGAGA CAATGGTGGA CACCTACGAG AGCTGCGACG TGCTGGCCGA CTTCTACGAC 5220

CAGTTCGCCG ACCAGCTGCA CGAGAGCCAG CTGGACAAGA TGCCCGCCCT GCCCGCCAAG 5280

GGGAACCTGA ACCTGCGGGA CATCCTGGAA AGCGACTTCG CCTTCGCCTG A 5331 (SEQ ID NO: 1)

Preferably, the RNA transcription system of interest includes a protein-RNA tethering system comprising an RNA binding domain binding with high affinity and a peptide binding RNA sequence, in addition to the RNA polymerase expression unit DNA encoding the RNA binding domain; and DNA encoding one or more of the peptide-binding RNA sequences in addition to the expression unit of the RNA of interest.

In the present invention, the chimeric enzyme composed of DdRp and several enzymes (or proteins) is referred to as 'ctDdRp'.

DNA dependent RNA Polymerase

A polymerase is a molecule capable of catalyzing the synthesis of polymer molecules, generally from monomeric building blocks. An “RNA polymerase” is a molecule capable of catalyzing the synthesis of RNA molecules from ribonucleotide building blocks. A "DNA polymerase" is a molecule capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA polymerases and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of a plurality of proteins. Typically, a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, typically a DNA molecule. Typically, an RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, and the template nucleic acid is either a DNA molecule (if the RNA polymerase is a DNA-dependent RNA polymerase, DdRp) or an RNA molecule (where RNA polymerase is an RNA-dependent RNA polymerase). in the case of the polymerase RdRp).

DdRp is a synonym for RNAP (RNA Polymerase), and the DdRp in eukaryotic nuclei are RNAP I, RNAP II, and RNAP III, which are structurally very different from each other and each transcribe a different set of genes (other polymerases are in mitochondria and chloroplasts). However, all three have two interrelated large subunits and the two largest subunits of bacterial RNAP. In addition, there are several small subunits found in common in all three enzymes or only between RNAP I and RNAP III.

Eukaryotic DdRp, like bacterial RNAP, requires specialized cofactors for transcriptional initiation. Unlike bacteria, however, eukaryotic initiators recognize the promoter element independently and not as part of the polymerase holoenzyme. Many different initiators are involved, particularly in genes transcribed by RNAP II, and some are themselves very complex proteins. Purified eukaryotic RNA polymerase alone cannot selectively initiate transcription from promoters.

Holoenzymes are sometimes used to refer to eukaryotic RNAPs, but in this case they mean something similar to bacterial 'core' enzymes and cannot be transcribed from a promoter. However, unlike bacterial core enzymes, eukaryotic whole enzymes may involve many other proteins involved in the transcription or processing of RNA.

A chimeric enzyme according to the present invention further comprises one or more catalytic domains of a DNA-dependent RNA polymerase.

"DNA-dependent RNA polymerase" (RNAP) relates to a nucleotidyltransferase that synthesizes a complementary strand of RNA from a single or double stranded DNA template in the 5' - 3' direction.

Preferably, the catalytic domain of a DNA-dependent RNA polymerase has a relatively simple structure and more preferably is a catalytic domain of an enzyme characterized by genomic enzyme regulatory elements (ie promoters and transcription termination signals).

Thus, in particular, the catalytic domain of a DNA-dependent RNA polymerase may be the catalytic domain of a bacteriophage DNA-dependent RNA polymerase, a bacterial DNA-dependent RNA polymerase or a DNA-dependent RNA polymerase of various eukaryotic organelles.

The catalytic domain of DNA-dependent RNA polymerase is the catalytic domain of bacteriophage DNA-dependent RNA polymerase.

Bacteriophage DNA-dependent RNA polymerases have the advantage of optimizing transgene expression levels, in particular by having a higher processivity than eukaryotic RNA polymerases. Bacteriophage DNA-dependent RNA polymerases also have much simpler structures than most nuclear eukaryotic polymerases, consisting of multiple subunits (eg RNA polymerase II) and transcription factors. Most of the bacteriophage DNA-dependent RNA polymerases characterized to date are single subunit enzymes and do not require helper proteins for transcription initiation, elongation or termination. Some of these enzymes, named after the cloned bacteriophages, are also well-characterized for regulatory genomic elements important for transformation (eg, promoters and termination signals).

Also, there is no competition between endogenous gene transcription and transgene transcription. A chimeric enzyme according to the present invention comprising a bacteriophage DNA-dependent RNA-polymerase moiety allows production of RNA transcripts in any eukaryotic species (eg yeast, rodent and human). They are inexpensive, quick and easy to implement, making them suitable for large-scale assays and protein production. In contrast to eukaryotic RNA polymerases such as RNA polymerase II, most bacteriophage DNA-dependent RNA polymerases do not require the involvement of factors for the initiation, elongation or termination of transcription, and thus all biological systems (e.g., cell-free reaction mixtures, It allows the production of RNA transcripts in cultured cells and living organisms).

Said catalytic domain of bacteriophage DNA-dependent RNA polymerase is in particular wild-type T7 RNA polymerase (NCBI genome sequence NC_001 604; gene 1261 050; Gene 1261 050); UniProtKB/Swiss-Prot P00573), wild-type T3 RNA polymerase (NCBI genome sequence NC_003298; Gene 927437; UniProtKB/Swiss-Prot Q778M8), wild-type K1 E RNA polymerase (NCBI genome sequence AM084415.1, UnisisProtKB/Sw Prot Q2WC24) , wild-type K1-5 RNA polymerase (NCBI genome sequence AY370674.1, NCBI YP_654105.1), wild-type K11 RNA polymerase (NCBI genome K11 RNAP sequence NC_004665; Gene 12588850; Q859H5), wild-type cpA1 122 RNA polymerase (NCBI genome sequence sequence NC_004777; gene 1733944; UniProtKB/Swiss-Prot protein Q858N4), wild-type cpYeo3-12 RNA polymerase (NCBI genome sequence NC_026/Gene2) Q9T145) and wild-type gh-1 RNA poly erase (NCBI genome sequence NC_004665; gene 1258850; UniProtKB /Swiss-Prot protein Q859H5), wild-type SP6 RNA polymerase (NCBI genome sequence NC_004831; Gene 1481778; UniProtKB/Swiss-Prot protein Q7Y5R1), and mutants or derivatives thereof capable of synthesizing complementary single-stranded RNA 5' -Sequences for double-stranded template DNA in the 3' direction, more specifically wild-type T7 RNA polymerase, wild-type T3 RNA polymerase, wild-type SP6 RNA polymerase, wild-type K1-5 RNA polymerase and duplex in the 5' - 3' direction wild-type K1 E RNA polymerase capable of synthesizing single-stranded RNA sequence-complementary to strand template DNA, and mutants or derivatives thereof; and the like.

The prototype of bacteriophage RNA polymerase, namely bacteriophage T7 RNA polymerase, has the advantage that in vitro the enzyme is highly processive and extends 240-250 nucleotides/sec in the 5' - 3' direction at 37 °C. Moreover, when expressed in eukaryotic cells, bacteriophage T7 RNA polymerase mostly remains in the cytoplasm, thus active transfer of large DNA molecules (i.e., transgenes) from the cytoplasm to the nucleus of eukaryotic cells and transfer of RNA molecules from the nucleus to the cytoplasm. By preventing spillage, transgene expression levels can be optimized.

The catalytic domain of DNA-dependent RNA polymerase may be either the wild type of K1 E or K1-5 RNA polymerase, but may also be a mutant of K1 E or K1-5 RNA polymerase capable of mono-synthesis. Stranded RNA (including processability) that is sequence-complementary to the double-stranded template DNA in the 5' - 3' direction. For example, the mutation may be the K1E RNA polymerase mutations R551 S, F644A, Q649S, G645A, R627S, 181 OS, D812E and K631 M, particularly R551 S, and the like.

Preferably, said catalytic domain of the DNA-dependent RNA-polymerase of the chimeric enzyme according to the invention is from an enzyme different from that of the host cell to prevent competition between endogenous gene transcription and transcription of said DNA sequence.

Capping enzyme and Poly(A) polymerase

In eukaryotes, the precursor of messenger RNA (mRNA), or pre-mRNA, is synthesized by RNA polymerase II and then undergoes a number of post-transcriptional modifications necessary for biological activities including translation, stability, or immune response. Two of these modifications that are particularly important for mRNA metabolism and translation are the addition of a cap at the 5'-end and a polyadenylation tail at the 3'-end.

Capping is a specialized structure found at the 5'-end of almost all eukaryotic messenger RNAs. The simplest cap structure, cap-0, is the result of the addition of a guanine nucleoside methylated at N7, which is formed by a 5'-5' triphosphate attached to the end of primary RNA (i.e., m7GpppN where N is any base, m is a methyl group and p is a phosphate). Formation of cap-0 in the so-called canonical pathway involves a series of three enzymatic reactions. That is, RNA triphosphatase (RTPase) removes the γ phosphate residue at the 5' triphosphate end of the nascent pre-mRNA and converts it to diphosphate, and RNA guanylyltransferase (GTase) transfers GMP from GTP to the diphosphate 5' end of the nascent RNA end. , RNA N7-guanine methyltransferase (N7-MTase) is a reaction that forms a GpppN cap by adding a methyl residue in azode 7 of guanine.

In higher eukaryotes and some viruses, the first (i.e., cap-1 structure; m7GpppNm2'-°pN) and two separate ribose-2'-0 MTases, termed cap1- and cap2-specific MTases, respectively. The 2'-hydroxyl group of the ribose of the second (i.e., cap2 structure; m7GpppNm2' 0pNm2' 0) transcribed nucleotide can be methylated (Langberg and Moss 1981). However, cellular N7-MTase activity is exclusively in the nucleus and in contrast cap-1 ribose-2′-0 MTase activity was detected in both the cytoplasm and nucleus of HeLa cells whereas cap2 MTase activity was only found in the cytoplasm.

Formation of the 5' terminal m7GpppN cap is the first step in pre-mRNA processing. The m7GpppN cap plays an important role in mRNA stability and transport from the nucleus to the cytoplasm. In addition, the 5'-terminal m7GpppN cap is important for the translation of mRNA into protein by anchoring the eukaryotic translation initiation factor 4F (elF4F) complex, which mediates the transfer of the 16S portion of the small ribosomal subunit into mRNA. Thus, the 5'-end m7GpppN cap greatly enhances the translation of mRNA in vitro and in cells. cap-0, cap-1 and cap-2 modifications participate in innate immunity by discriminating self from non-self RNA via the RNA sensors RIG-1 and MDA5, which in turn induce interferon type-l responses.

"Capping enzyme" means any enzyme capable of adding an m7 GpppG cap to the 5'-end of mRNA and/or modifying the terminal or penultimate base of an RNA sequence. standard capping enzymes and cap-1 or cap-2 nucleoside 2' methyltransferase, N6-methyl-adenosine transferase.

"cap-0 standard capping enzyme" refers to an enzyme capable of adding a cap-0 structure to the 5' end of an RNA molecule by involving a series of three enzymatic reactions. Namely, RNA triphosphatase (RTPase) that removes the γ-phosphate residue at the 5' triphosphate end of the nascent pre-mRNA to produce diphosphate ppRNA, and RNA guanylyltransferase that transfers GMP from GTP to the nascent end of the diphosphate ppRNA RNA. (GTase), and an RNA N7-guanine methyltransferase (N7-MTase) that adds a methyl to the nitrogen 7 residue of guanine for the GpppRNA cap.

The enzymatic domains of eukaryotes and viruses involved in the canonical formation of cap-0 structures can assemble into a variable number of protein subunits.

A single subunit capping enzyme that contains all three important enzymatic domains: RTPase, GTase and N7-MTase. These enzymes include, but are not limited to: ① Acanthamoeba Polyphaga mimivirus capping enzyme R382 (NCBI APMV genomic sequence NC_006450; UniProtKB/Swiss-Prot accession number Q5UQX1), ② ORF3 capping enzyme from yeast Kluyveromyces lactis linear extrachromosomal episomal pGKL2 (NCBI Kluyveromyces 599 pGgenomic 7 sequence ) UniProtKB/Swiss-Prot accession number P05469), ③ African swine fever virus NP868R capping enzyme (NCBI ASFV genome sequence strain BA71 V NC_001659; UniProtKB/Swiss-Prot accession number P32094), VP4 Bluetongue virus capping enzyme (NCBI BTV serotype 10 genome sequence Y00421; UniProtKB/Swiss-Prot accession number P07132).

Capping enzymes composed of two subunits, including but not limited to: ① Mammalian capping enzymes composed of the RNGTT subunit having both RTPase and GTase enzyme activities (also named HCE1; human and mouse UniProtKB/Swiss-Prot Accession No. 060942 and 055236, respectively) and RNMT with N7-MTase enzyme activity (human and mouse UniProtKB/Swiss-Prot Accession Nos. Q05D80 and D3YYS7, respectively), ② consisting of the D1R gene product with RTPase, GTase and N7-MTase enzyme domains. Vaccinia capping enzyme (sequence strain Western Reserve NC_006998.1; UniProtKB/Swiss-Prot Accession No. P04298) and the D1R subunit that greatly enhances RNA N7-MTase activity (genomic sequence strain Western Reserve NC_006998 .1; Gene 3707515; UniProtKB/Swiss-Prot Accession No. P04318), etc.

Capping enzymes composed of three subunits include Saccharomyces cerevisiae CET1 with RTPase (UniProtKB/Swiss-Prot Accession No. 013297), CEG1 with GTase (UniProtKB/Swiss-Prot Accession No. Q01 159) and ABD1 with N7-MTase catalytic activity. (UniProtKB/Swiss-Prot Accession No. P32783) et al.

"cap-0 noncanonical capping enzyme" means an enzyme capable of adding a cap-0 structure to the 5' end of an RNA molecule, but in a pathway different from the normal enzymatic process. Three non-canonical 5' RNA cap synthesis mechanisms have been reported.

The term "cap-1 capping enzyme" refers to an enzyme capable of adding a cap-1 structure to the 5' end of an RNA molecule. As used herein, the term "cap-2 capping enzyme" refers to an enzyme capable of adding a cap-2 structure to the 5' end of an RNA molecule.

Two cap modifications are found in mammals, higher eukaryotes and some viruses, which are absent in yeast and plant mRNAs and are produced by methylation of the 2' hydroxy group of the ribose portion of the nucleotide. 5' end of the mRNA: cap-1 at the first mRNA nucleotide, cap-2 at the second mRNA nucleotide. Cap-1 methylation is found in almost all mammalian mRNA molecules, whereas only half of mRNAs contain a 2'-O-methylated residue at the second transcribed nucleotide.

In mammals, cap-1 and cap-2 modifications are carried out by two ribose-2'-0 methyltransferases (also called nucleoside-2'-methyltransferases or 2'-0-MTases).

First, MTR1 (cap-1 ribose-2′-0 MTase activity, also known as FTSJD2, KIAA0082 or ISG95; UniProtKB/Swiss-Prot accession number Q8N1 G2) is found only in the nucleus and is potentially involved in putative nuclear localization signals and RNA binding. It is the G-patch domain involved. Surprisingly, MRT1 is associated with the CTD of RNA polymerase II, indicating that cap-1 formation occurs early in mRNA synthesis.

Second, MTR2 (cap 2 ribose-2'-0 MTase, also named FTSJD1 or FLJ1 1 171; UniProtKB/Swiss-Prot Accession No. Q8IYT2) is a methyl group from S-adenosylmethionine to the second 2'-O-ribose convey Nucleotides of mRNA and small nuclear RNA. Although MTR2 does not require guanosine cap-0 or N7 methylation of cap-1 modifications, the presence of cap-1 increases MTR2 activity. MTR2 protein is distributed throughout the nucleus and cytoplasm, in contrast to nuclear MTR1.

Some eukaryotic viruses have their own cap-1 and/or cap-2 2'-0-MTase, including: VP39 from vaccinia virus (NCBI genome sequence NC_006998.1; UniProtKB/Swiss-Prot accession number YP_232977 with both cap-1 and cap-2 2'-O-MTase enzyme activities. Orf69 from Autographa californica Nucleopolyhedrovirus (NCBI genome sequence NC_001623.1; UniProtKB/Swiss-Prot accession number P41469) nspl 6 of coronavirus (residues 6776-7073 of polyprotein 1 ab of human SARS coronavirus NCBI genome sequence NC_004718.3; accession UniProtKB/Swiss-Prot No. P0C6X7) Reovirus with 2′-0-MTase in addition to GTase and N7-MTase enzymatic activities (e.g. mammalian orthoreovirus type 3, strain Dearing; NCBI genome sequence J03488; UniProtKB/Swiss-Prot accession number P1 1079). λ2 protein from Bujnicki and Rychlewski 2001, VP4 from blue tongue virus (NCBI BTV serotype 10 genome sequence ID Y00421; UniProtKB/Swiss-Prot accession number P07132), dengue fever virus, sequence virus, flavi including zika virus NS5 from viruses, Meaban virus, Yokose virus, St. Louis encephalitis virus, Japanese encephalitis virus, tick-borne encephalitis virus (e.g. polyprotein of dengue virus type 1 strain Nauru/West Pac/1974; NCBI genome sequence U88535; UniProtKB/ Swiss-Prot Accession No. P17763) can also methylate internal adenosine residues of mRNA.

A chimeric enzyme according to the present invention comprises or consists of one or more catalytic domains of RNA triphosphatase; one or more catalytic domains of a guanylyltransferase; one or more catalytic domains of N7-guanine methyltransferase; and at least one RNA-binding domain of a protein-RNA tethering system.

In particular, at least one of said catalytic domains is a catalytic domain of a cap-0 canonical capping enzyme, more specifically a viral cap-0 canonical capping enzyme.

"RNA triphosphatase" (RTPase) relates to an enzyme that removes the Y phosphate residue at the 5' triphosphate end of nascent pre-mRNA to diphosphate. “RNA guanylyltransferase” (GTase) refers to an enzyme that transfers GMP from GTP to the nascent diphosphate RNA terminus. "N7-guanine methyltransferase" (N7-MTase) relates to an enzyme that adds a methyl residue at azot 7 of guanine to the cap of GpppN.

The catalytic domain of RNA triphosphatase, guanylyltransferase, and N7-guanine methyltransferase may be from the same or different capping enzymes.

Preferably, the catalytic domain of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase is advantageously derived from one or several cytoplasmic enzymes with relatively simple structures and well-characterized enzymatic activities. Thus, in particular, the catalytic domain of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may be the catalytic domain of one or more viral capping enzymes, or cytoplasmic episomal capping enzymes.

The catalytic domain of RNA triphosphatase, the catalytic domain of N7-guanine methyltransferase of guanylyltransferase, in particular wild-type vaccinia virus capping enzyme, wild-type bluetongue virus capping enzyme, wild-type bamboo mosaic virus capping enzyme, wild-type African swine fever Viral capping enzyme, Lake phycodnavirus 2 (OLPV2) capping enzyme, wild-type Phaeocystis globosa virus capping enzyme, wild-type Chrysochromulina ericina virus capping enzyme, and mutants or derivatives thereof, respectively, can remove the γ phosphate residue at the 5' triphosphate terminal before initialization. there is. Diphosphate conversion of mRNA, transfer of GMP from GTP to the initial diphosphate RNA terminus, or addition of a methyl residue to azot 7 of guanine. For the GpppN cap, more specifically, a wild-type African swine fever virus capping enzyme capable of transferring γ phosphate residues at the 5' triphosphate end of the nascent pre-mRNA to diphosphate or GMP, and mutants or derivatives thereof. Add a GTP to the RNA end or add a methyl residue to azot 7 of guanine in the GpppN cap.

"Vaccinia virus capping enzyme" relates to the heterodimeric D1 R/D12L capping enzyme of vaccinia virus. The D1 R gene product (genomic sequence strain Western Reserve NC_006998.1 ; UniProtKB/Swiss-Prot accession number P04298) and the D12L gene product, which have RTPase, GTase and N7-MTase enzymatic domains, have no intrinsic enzymatic activity, but the RNA N7-D1R subunit of MTase activity (genomic sequence strain Western Reserve NC_006998.1, gene 3707515, UniProtKB/Swiss-Prot Accession No. P04318).

"Blue tongue virus capping enzyme" relates to the single unit VP4 capping enzyme of blue tongue virus (BTV), a 76 kDa protein (644 amino acids; sequence referenced, eg, NCBI BTV serotype 10 genome). sequence Y00421; gene 29431 57; UniProtKB/Swiss-Prot P07132). This capping enzyme can homodimerize through a leucine zipper located near the carboxyl terminus. VP4 catalyzes all three enzymatic steps required for the synthesis of mRNA m7GpppN capping: RTPase, GTase and N7998 MTase.

"Bamboo mosaic virus capping enzyme" relates to the bamboo mosaic virus (BMV) mRNA capping enzyme, ORF1, which is a single unit 155-kDa protein (1365-amino acid; NCBI BMV isolate BaMV-0). Genome sequence NC_001642, gene 1497253, UniProtKB/Swiss-Prot Q65005). The ORF1 protein has all the enzymatic activities necessary to generate m7GpppNm RNA capping, namely RTPase, GTase and N7-MTase.

"African swine fever virus capping enzyme" (ASFV) relates to a single unit 100 kDa protein (868 amino acids; NCBI ASFV genome sequence strain BA71V), NP868R capping enzyme (G4R). NC_001 659; Gene 1488865; UniProtKB/Swiss-Prot P32094), which has all the enzymatic activities required to generate m7GpppN mRNA capping, namely RTPase, GTase and N7-MTase.

"Gasimoeba polyphaga mimivirus capping enzyme" relates to R382 (APMV), another single unit 136.5 kDa protein (1170 amino acids; NCBI APMV genome sequence NC_006450; Gene 3162607; Gene 3162607; UniProtKB/Swiss-Prot Q5UQX1). will be.

"Organic lake picornavirus 1 (OLPV1) capping enzyme" refers to 889 single unit amino acids referenced by NCBI accession number ADX05869.1.

"Organic Lake Picodnavirus 2 (OLPV2) capping enzyme" refers to 942 single unit amino acids referenced by NCBI accession number ADX06468.1.

"Pheocystis globosa virus capping enzyme" refers to 1066 single unit amino acids referenced as UniProtKB/Swiss-Prot YP_008052553.1.

"Chrysochromurina erysina virus capping enzyme" refers to 965 single unit amino acids referenced as UniProtKB/Swiss-Prot YP_009173557.1.

The catalytic domain of N7-guanine methyltransferase, RNA triphosphatase, and guanylyltransferase are derived from cytoplasmic episomal capping enzymes such as pGKL2. In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase, and N7-guanine methyltransferase are all or part of the yeast linear extrachromosomal episomal capping enzyme of pGKL2 encoded by the ORF3 gene of Kluyveromyces lactis pGKL2. included in (NCBI Kluyveromyces lactis CB 2359 pGKL2 genome sequence NC_010187; UniProtKB/Swiss-Prot P05469) and it is a 594 amino acid protein (70.6 kDa protein).

The catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, and the catalytic domain of N7-guanine methyltransferase are contained in a monomer, i.e., one polypeptide. For example, the monomer may be a monomeric capping enzyme or a monomeric chimeric enzyme according to the present invention.

In particular, the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, and the catalytic domain of N7-guanine methyltransferase are included in the monomeric capping enzyme. In this case, the chimeric enzyme according to the present invention comprises a monomer capping enzyme comprising a catalytic domain of RNA triphosphatase, a catalytic domain of guanylyltransferase, and a catalytic domain of N7-guanine methyltransferase. The monomeric capping enzyme may be a monomeric virus capping enzyme, and is particularly selected from the group consisting of a wild-type blue tongue virus capping enzyme, a wild-type bamboo mosaic virus capping enzyme, a wild-type African swine fever virus capping enzyme, and a wild-type capping enzyme. Acanthamoeba polyphaga mimivirus capping enzyme, wild-type OLPV1 capping enzyme, wild-type OLPV2 capping enzyme, wild-type Pyocystis globosa virus capping enzyme, wild-type chrysochromulina erysina virus capping enzyme and m7GpppN capable of adding caps Mutants and derivatives thereof, and more specifically wild-type African swine fever virus capping enzymes and mutants and derivatives thereof capable of adding an m7GpppN cap to the 5'-end of RNA molecules, and even more Specifically, it is a wild-type African swine fever virus capping enzyme.

The chimeric enzyme according to the present invention also comprises at least one catalytic domain of the chimeric enzyme of the present invention, in particular at least one catalytic domain of the capping enzyme, more particularly at least the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, N7 -It is one catalytic domain selected from the group consisting of the catalytic domain of guanine methyltransferase, preferably the catalytic domain of N7-guanine methyltransferase.

For example, the domain that enhances the activity of one or more catalytic domains of the chimeric enzyme of the present invention is the vaccinia virus D12L gene (genomic sequence NC_006998.1; Gene3707515; UniProtKB/Swiss-Prot YP_232999.1), the intrinsic enzyme activity is but greatly enhances the RNA N7-guanine methyltransferase activity of the D1R subunit of the vaccinia mRNA capping enzyme.

The chimeric enzyme according to the present invention further comprises at least one catalytic domain of a 5'-end RNA processing enzyme other than cap-0, cap-1 and cap-2 capping enzymes.

"5'-terminal RNA processing enzyme other than cap-0, cap-1 and cap-2 capping enzyme" means the penultimate or penultimate base of an mRNA sequence other than cap-0, cap-1 and cap-2 capping enzyme It is about enzymes that can transform 0, cap-1 and cap-2 capping enzymes, N6-methyl-adenosine transferase and 2,2,7-trimethylguanosine (TMG) and 2,7-trimethylguanosine (DMG) cap modifications at the 5' end Other capping modifications other than cap-0, cap-1 and cap-2 modifications of appendable enzymatic RNA molecules have been characterized in eukaryotic or viral mRNAs. For example, m6A methylation by the N6-methyl-adenosine transferase of the first base is a reversible modification affecting cellular mRNA fate. The 2,2,7-trimethylguanosine (TMG) and 2,7-trimethylguanosine (DMG) cap modifications present in snRNA, telomerase RNA, trans-spliced nematode mRNA and certain viral mRNAs play a role in translational advantage can be given. TMG and DMG are carried out by specialized enzymes in viruses (e.g. L320 in DNA mimivirus, UniProtKB/Swiss-Prot accession number Q5UQR2, protozoa (e.g. Giardia lamblia trimethylguanosine synthase (NCBI accession number EAA4643)). Lower eukaryotes (e.g. : Schizosaccharomyces pombe trimethylguanosine synthase, UniProtKB/Swiss-Prot accession number Q09814), human trimethylguanosine synthase 1 UniProtKB/Swiss-Prot accession number Q96RS0.

The chimeric enzyme of the present invention further comprises one or more catalytic domains of a poly(A) polymerase.

The present inventors provide that a monomeric or oligomeric chimeric enzyme comprising at least one catalytic domain of a capping enzyme, at least one RNA-binding domain of a protein-RNA tethering system and a poly(A) polymerase is an RNA polymerase, poly(A) polymerization Compared to the combination of capping enzymes in the presence of the enzyme, it was possible to synergistically increase the capping rate of a specific mRNA produced by RNA polymerase. This result is unexpected because capping enzymes and poly(A) polymerases are not physically linked in nature and do not contain binding domains for specific RNA sequences. A person skilled in the art would expect the same expression rate because the components are the same.

"Poly(A) polymerase" refers to any enzyme capable of catalyzing the non-template addition of an adenosine residue from ATP to the 3' end of an RNA molecule.

The catalytic domain of poly(A) polymerase is the catalytic domain of standard poly(A) polymerases including mammalian (eg PAPOLA, PAPOLG), yeast (eg Saccharomyces cerevisiae PAP1, Schizosaccharomyces pombe PLA1, Candida). albicans PAP, Pneumocystis carinii PAP), protozoan, viral and bacterial standard poly(A) polymerases.

In particular, said catalytic domain of a poly(A) polymerase is a cytoplasmic canonical poly(A) polymerase, more specifically selected from the group consisting of:

mammalian cytoplasmic poly(A) polymerase, including at least in part the cytoplasmic enzyme PAPOLB (human and mouse PAPOLB, UniProtKB/Swiss-Prot Accession Nos. Q9NRJ5 and Q9WVP6); Mutants of PAPOLA (human and mouse PAPOLA, UniProtKB/Swiss-Prot Accession Nos. P51003 and Q61 183, respectively), in which mutations or deletions of nuclear localization signals can relocate nuclear enzymes to the cytoplasm, mutants of PAPOLG (respectively Mutations or deletions of nuclear localization signals in human and mouse PAPOLG, UniProtKB/Swiss-Prot accession numbers Q9BWT3 and Q6PCL9) have the potential to relocate nuclear enzymes to the cytoplasm.

Yeast or protozoan poly(A) polymerase (e.g. mutation of Saccharomyces cerevisiae PAP1, UniProtKB/Swiss-Prot accession number P29468; Schizosaccharomyces pombe PLA1, UniProtKB/Swiss-Prot accession number Q10295), Candida albicans PAP (UniProtKB/Swiss-Prot accession number Q10295) Prot accession number Q9UW26), Pneumocystis carinii PAP (also named Pneumocystis jiroveci, Z0A2 accession number A2Prot0/Sw mutation; deletion of nuclear localization signal likely relocates nuclear enzymes to the cytoplasm), as well as other pyrophilic, mesophilic , thermophilic or thermophilic yeast or protozoan strain viral poly(A) polymerase, VP55 catalytic subunit (UniProtKB/Swiss-Prot accession number strain Western Reserve P23371) and fairness factor (UniProtKB/Swiss-Prot accession number strain Western Reserve P07617), other varicella-virus poly(A) polymerases (e.g. Cowpox virus, Monkeypox virus or Camelpox virus), African swine fever virus (C475L), UniProtKB/Swiss-Prot accession number A0A0A1 E081), Acanthamoeba polyphaga mimivirus R341 (UniProtKB/Swiss-Prot accession number E3VZZ8) and Megavirus chilensis Mg561 poly(A) polymerase (NCBI accession number: A2EXum602) (NCBI accession number: Number: YP_602), Mamavirus (NCBI Accession No. AEQ60527), Cafeteria roenbergensis BV-PW1 Virus (NCBI Accession No. YP_003969918), Megavirus Iba (NCBI Accession No. AGD92490), Yellowstone Lake mimivirus (NCBI Accession No. YP_012917), YP_009173345), organic lake Picodnavirus 1 (NCBI Accession No. ADX05881), Organic Lake Picodnavirus 2 (NCBI Accession No. ADX06298), and Faustovirus (NCBI Accession No. AMN83802) have an untemplated adenosine residue from ATP to the 3' end of the RNA molecule. It is a mutant or derivative thereof capable of catalyzing uncontrolled addition.

In addition, the catalytic domain of a poly(A) polymerase is a catalytic domain of a non-standard poly(A) polymerase, in particular a catalytic domain of a cytoplasmic non-standard poly(A) polymerase, "non-standard poly(A) polymerase" refers to the N-terminus It refers to an enzyme that does not have a triple structure comprising a nucleotidyltransferase (NT) catalytic domain, a central domain and a C-terminal domain corresponding to an RNA binding domain (RBD). Irregular poly(A) polymerases exist in mammals.

Said catalytic domain of a poly(A) polymerase is in particular a catalytic domain of a yeast or protozoan poly(A) polymerase selected from the group consisting of wild type Saccharomyces cerevisiae PAP1 poly(A) polymerase. pombe PLA1, Candida albicans PAP (UniProtKB/Swiss-Prot Accession No. Q9UW26), Pneumocystis carinii PAP (UniProtKB/Swiss-Prot Accession No. A0A0W4ZDF2), cytoplasmic mutant polymerase of Saccharomyces omboscerevisiae, wherein the nuclear localization signal is non-functional or deleted) and mutants or derivatives thereof, which are capable of catalyzing the untemplated addition of adenosine residues from ATP to the 3' end of RNA molecules.

Yeast or protozoan poly(A) polymerases have the advantage of reduced molecular weight compared to standard mammalian poly(A) polymerases. "PAP1 poly(A) polymerase" means Saccharomyces cerevisiae PAP1 poly(A) polymerase (UniProtKB/Swiss-Prot Accession No. P29468). "PLA1 poly(A) polymerase" includes Schizosaccharomyces pombe PLA1 poly(A) polymerase, UniProtKB/Swiss-Prot Accession No. Q10295.

The catalytic domain of poly(A) polymerase is a catalytic domain of viral poly(A) polymerase, in particular wild-type C475L poly(A) polymerase selected from the group consisting of wild-type VP55 poly(A) polymerase, wild-type R341 poly(A) A) Polymerase and wild-type MG561 poly(A) polymerase and mutants or derivatives thereof, capable of catalyzing the non-template addition of an adenosine residue from ATP to the 3' end of an RNA molecule.

Viral poly(A) polymerase has the advantages of reduced molecular weight compared to mammalian poly(A) polymerase, strong enzymatic activity when expressed in mammalian cells, and presence in cytoplasmic compartments. In addition, some of these enzymes, such as R341 and MG561 poly(A) polymerases, do not require known accessory protein cofactors.

“VP55 poly(A) polymerase” relates to the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase (UniProtKB/Swiss-Prot accession number strain Western Reserve P23371). The second subunit, VP39, acts as a processability factor (UniProtKB/Swiss-Prot accession number strain Western Reserve P07617).

“C475L poly(A) polymerase” relates to African swine fever virus poly(A) polymerase (UniProtKB/Swiss-Prot Accession No. A0A0A1 E081).

“R341 poly(A) polymerase” relates to Acanthamoeba polyphaga mimivirus R341 poly(A) polymerase (UniProtKB/Swiss-Prot Accession No. E3VZZ8).

"MG561 poly(A) polymerase" relates to Megavirus chilensis MG561 poly(A) polymerase (NCBI accession number: YP_004894612).

Protein-RNA tethering system

"Protein-RNA tethering system" refers to a system in which a protein (or peptide) recognizes and specifically (with high affinity) binds to a specific RNA element composed of a specific RNA through its RNA-binding domain. Thus, a protein (or peptide) may be capable of binding to an RNA element having a specific sequence and/or structure. Specific binding between a protein (or peptide) and a specific RNA element through an RNA binding domain means that the protein (or peptide) and a specific RNA element interact with high affinity. Interactions with high affinity include interactions with an affinity of about 10 ⁶ M or greater, particularly at least 10 ⁷ M, at least 10 ⁸ M, at least 10 ⁹ M, more particularly at least 10 ¹⁰ M. Whether or not the RNA-binding domain specifically binds has a high affinity for a specific RNA element, in particular, by comparing the reaction of the RNA binding domain and a specific RNA element with RNAs other than the RNA binding domain and a specific RNA element. can be easily tested.

RNA-protein affinity can be determined by a variety of methods well known to those skilled in the art. These methods include, but are not limited to, steady-state fluorescence or electrophoretic measurements, RNA electrophoretic mobility shift assays.

"RNA-binding domain of a protein-RNA tethering system" refers to the domains of a protein (or peptide) necessary and sufficient to ensure recognition, especially in its three-dimensional structure. and interaction with a specific RNA element composed of a specific RNA sequence and/or structure with high affinity, thus making it possible to bind this RNA element with this protein (or peptide) through the RNA binding domain. Thus, the "RNA-binding domain" (RNA-tethering domain) binds RNA with sequence and/or structural specificity. That is, it specifically binds to an RNA element composed of a specific RNA sequence and/or structure. The term "RNA-binding domain of a protein-RNA tethering system" includes RNA-binding domains of wild-type or mutant proteins (or peptides).

The chimeric enzyme according to the present invention includes at least the RNA-binding domain, but may further include all or part of a protein (or peptide) containing the RNA-binding domain. In fact, according to the chimeric enzyme according to the present invention, the RNA-binding domain of the protein-RNA tethering system may be included in all or part of the protein (or peptide) system of protein-RNA tethering.

Some specialized protein-RNA tethering systems include bacteriophage protein-RNA tethering systems such as the MS2 coat protein-RNA tethering system, the R17 coat protein-RNA tethering system, and the Ramdoid N antitermination protein-RNA tethering system. included MS2 coat protein and R17 coat protein recognize specific RNA elements composed of stem-loop RNA structures and interact with high affinity. The lambdoid N antitermination protein-RNA tethering system recognizes and interacts with high affinity with specific RNA elements composed of boxBL and boxBR stem-loop RNA structures. Bacteriophages so far exhibit characteristics belonging to the lambdoid family (ie, λ, P22, Φ21, HK97 and 933W viruses) or MS2-related families (ie, MS2 and R17).

In the chimeric enzyme according to the present invention, the RNA-binding domain is a bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system, particularly selected from the group consisting of MS2 coat protein-RNA tethering system.

R17 envelope protein-RNA tethering system and lambdide N anti-termination protein-RNA tethering system, more specifically lambda N anti-termination protein-RNA tethering system, phi21 N anti-termination protein-RNA tethering system, HK97 N anti-termination a protein-RNA tethering system and a p22 N anti-terminating protein-RNA tethering system, more particularly a lambda N anti-terminating protein-RNA tethering system, and the like.

In particular, when the chimeric enzyme of the present invention is used to add a 5'-end cap to an RNA molecule synthesized by a bacteriophage DNA-dependent polymerase (included or not included in the chimeric enzyme), the RNA-binding domain is a bacteriophage RNA, which is the binding domain of the bacteriophage protein-RNA tethering system.

In fact, unexpectedly, we found that a cytoplasmic monomeric or oligomeric chimeric enzyme comprising the catalytic domain of a capping enzyme and the bacteriophage RNA-binding domain of a bacteriophage protein-RNA tethering system could greatly increase the capping rate of a specific mRNA produced. proved. Compared to other RNA-binding domains of protein-RNA tethering systems, including the U1-RNA 70K system described in US Patent 5,866,680, bacteriophage DNA-dependent RNA polymerase was performed.

In particular, the RNA binding domain comprises wild-type MS2 envelope protein (NCBI accession number NC_001417.2, UniProtKB/Swiss-Prot P03612), type R17 envelope protein (NCBI accession number EF108465.1, UniProtKB/Swiss-Prot P69170) and certain RNA elements. , more specifically wild-type lambda N anti-terminal protein and its mutants and derivatives capable of recognizing and interacting with high affinity wild-type lambda N anti-terminal protein (NCBI accession number NC_001416.1, complete genome sequence; UniProtKB/Swiss-Prot Accession No. P03045), wild-type phi21 N anti-termination protein (NCBI Accession No. AH007390.1, partial genome sequence, UniProtKB/Swiss-Prot Accession No. P07243), wild-type HK97 N anti-termination protein-RNA tethering system (NCBI Accession No. NC_002167. 1, complete genome sequence; NCBI protein accession number NP_037732.1) and wild-type p22 N antitermination protein (particularly NCBI sequence (NCBI accession number NC_002371.2, complete genome sequence; UniProtKB/Swiss-Prot accession number P04891), more particularly wild-type lambda N antitermination protein (NCBI accession number NC_001416.1, complete genome sequence; UniProtKB/Swiss-Prot accession number P03045) All or nearly the entire MS2/R17 protein is required for competent binding to tethered RNA. This is because several amino acid residues spread across these proteins are involved in stem-loop interactions.

Thus, the chimeric enzyme of the present invention is wild-type MS2 coat protein (NCBI accession number NC_00141 7.2, UniProtKB/Swiss-Prot P03612) or its isolate wild-type R17 coat protein (NCBI accession number EF108465.1, UniProtKB/Swiss-Prot P69170), Alternatively, mutants or derivatives thereof capable of recognizing specific RNA elements and interacting with them with high affinity may be used.

Importantly, the 18- to 22-amino acid region from the N-terminal sequence of the lambdoid N-protein binds to the cognate RNA sequence with similar affinity and specificity to that of the full-length N-protein.

In the chimeric enzyme of the present invention, the RNA-binding domain of the protein-RNA tethering system comprises or consists of a peptide consisting of amino acids at positions 1 to 22, in particular 1 to 18, of wild-type lambda N. No. NC_001416.1, complete genome sequence; UniProtKB/Swiss-Prot Accession No. P03045), or wild-type phi21 N antitermination protein (NCBI Accession No. AH007390.1, partial genome sequence; UniProtKB/Swiss-Prot Accession No. P07243), or Wild-type HK97 N anti-terminal protein-RNA tethering system (NCBI accession number NC_002167.1, complete genome sequence, NCBI protein accession number NP_037732.1) or wild-type P22 N anti-termination protein (NCBI accession number) NC_002371.2, complete genome sequence , UniProtKB/Swiss-Prot accession number P04891) or mutants or derivatives thereof capable of recognizing specific RNA elements and interacting with high affinity, in particular wild-type lambda N antitermination protein (NCBI accession number NC_001416.1, complete genome sequence; UniProtKB/Swiss -Prot Accession No. P03045) and the like.

In particular, the RNA-binding domain of the protein-RNA tethering system comprises a peptide (UniProtKB/Swiss-Prot Accession No. P03045) composed of amino acids at positions 1 to 22 of the wild-type Lambda N antitermination protein (NCBI Accession No. NC_001416.1) contains or consists of

In particular, the RNA-binding domain of the protein-RNA tethering system is not derived from the same source (eg, from the same enzyme) as the different catalytic domains of the chimeric enzyme of the invention.

An "RNA element of a protein-RNA tethering system that specifically binds to said RNA-binding domain" relates to an RNA sequence, generally a stem capable of binding with high affinity to a corresponding RNA-binding domain of a protein RNA-binding system. form a loop

In particular, said DNA sequence is operably linked to a promoter for a bacteriophage DNA-dependent RNA polymerase or to a promoter for said DNA-dependent RNA polymerase of a chimera of the present invention.

In particular, when the RNA binding domain of the protein-RNA tethering system is the RNA binding domain of the rhamdoid N antitermination protein-RNA tethering system, the element specifically binding to the RNA binding domain is boxBL and / or SEQ ID NO. 7 and the boxBR stem loop RNA structure comprising the elements encoded by SEQ ID NO: 38.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the MS2 coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain is 19 nucleotides or SEQ ID NO: 39 A 21 nucleotide stem loop sequence (nucleotides 1748-766 Enterobacteriophage MS2 isolate DL52, NCBI Accession No. JQ966307.1) comprising elements encoded by, which contains the initiation codon of the gene for viral replicase.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the R17 separate coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain may be 19 nucleotides . or a 21 nucleotide stem-loop (nucleotides 1746-764 of Enterobacteriophage R17, NCBI Accession No. EF1 08465.1) containing the initiation codon of the gene for viral replicase.

linker

The chimeric enzyme according to the present invention includes at least the catalytic domain, but may further include all or part of the enzyme containing the catalytic domain.

Indeed, according to the chimeric enzyme according to the present invention, said catalytic domain of a DNA-dependent RNA polymerase may also be included in whole or in part of a DNA-dependent RNA polymerase, preferably a monomeric DNA-dependent RNA polymerase.

A capping enzyme, preferably a monomeric capping enzyme, the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase may be included in whole or in part.

As used herein, the terms «connection» and «coupled» include covalent and noncovalent connections.

Said RNA-binding domain may in particular be linked by covalent linkage (directly or indirectly by means of a linking peptide) to one catalytic domain of a chimeric enzyme according to the invention selected from the group consisting of:

catalytic domains of capping enzymes, in particular RNA triphosphatases, guanylyltransferases, N7-guanine methyltransferases; At least one catalytic domain selected from the group consisting of the above catalytic domain of poly(A) polymerase, at least one catalytic domain of the chimeric enzyme of the present invention, preferably the catalytic domain of RNA triphosphatase and the catalytic domain of guanylyltransferase the above domains enhancing activity, the catalytic domain of N7-guanine methyltransferase, in particular the catalytic domain of N7-guanine methyltransferase; and the catalytic domain of a 5' terminal RNA processing enzyme other than cap-0, cap-1 and cap-2 capping enzymes.

Linking peptides have the advantage of minimizing steric hindrance and creating a fusion protein in which sufficient space is provided for the components of the fusion protein to maintain their original conformation.

The linking peptide of the present invention may be selected from the group consisting of:

A peptide of the formula (GlymSerP)n, wherein: m represents an integer from 0 to 12, particularly from 1 to 8, more particularly from 3 to 6, even more particularly from 4; p represents an integer from 0 to 6, particularly from 0 to 5, more particularly from 0 to 3, and still more particularly from 1; and n represents an integer from 0 to 30, particularly from 0 to 12, more particularly from 0 to 8, still more particularly from 1 to 6, inclusive; In particular a peptide of formula (GlymSerP)n, wherein: m represents 4; p represents 0 or 1; and n represents 1, 2 or 4; more particularly of the formulas Gly4, (Gly4Ser)1(Gly4Ser)2 and (Gly4Ser)4.

A flexible linker peptide of the formula (GlymSerP)n has the advantage that the glycine residue provides peptide flexibility, while the serine provides some solubility. In addition, the absence of sensitive sites for chymotrypsin I, factor Xa, papain, plasmin, thrombin and trypsin in the (GlymSerP)n linker sequence is presumed to increase the overall stability of the resulting fusion protein.

Variable length (GlymSerP)n linkers are commonly used to engineer single chain Fv fragment (sFv) antibodies. Additionally, the (GlymSerP)n linker has been used to generate a variety of fusion proteins that frequently retain the biological activity of each component.

The RNA-binding domain and the catalytic domain of the protein-RNA tethering system can also be assembled by specific protein elements such as leucine zippers. The C-terminus or N-terminus of the RNA-binding domain may be linked to the N-terminus or C-terminus of one of the catalytic domains of the chimeric enzyme of the present invention, respectively.

Preferably, the C-terminus of said RNA binding domain is covalently bonded (preferably of formula Gly4, (Gly4Ser)i(Gly4Ser)2 or (Gly4Ser)4, even more preferably of formula (Gly4Ser)i or Gly4). linked directly or indirectly by a linking peptide) to the N-terminus of one of the catalytic domains selected from the group consisting of: catalytic domains of capping enzymes, in particular RNA triphosphatases, guanylyltransferases, N7-guanine methyltransferases; It may be the catalytic domain of a poly(A) polymerase.

At least one catalytic domain of the chimeric enzyme of the present invention, preferably the domain that enhances the activity of at least one catalytic domain selected from the group consisting of the catalytic domain of RNA triphosphatase and the catalytic domain of guanylyltransferase, N7-guanine methyl catalytic domains of transferases, particularly N7-guanine methyltransferases; and the catalytic domain of a 5' terminal RNA processing enzyme other than cap-0, cap-1 and cap-2 capping enzymes.

The C-terminus of the RNA binding domain is covalently linked (preferably by a linking peptide of the formula Gly4, (Gly4Ser)i(Gly4Ser)2 or (Gly4Ser)4, even more preferably of the formula (Gly4Ser)i or Gly4). linked directly or indirectly) to the N-terminus of one of the catalytic domains selected from the group consisting of:

catalytic domains of capping enzymes, in particular RNA triphosphatases, guanylyltransferases, N7-guanine methyltransferases; and the catalytic domain of poly(A) polymerase.

The chimeric enzymes of the present invention are fusion proteins. A “fusion protein” refers to an artificial protein created through the joining of two or more proteins or protein domains that originally encode separate proteins. Translation of this fusion gene produces single or multiple polypeptides with functional properties derived from each original protein.

In the chimeric enzyme according to the present invention, by means of linking peptides (in particular linking peptides of the formula Glv4, (Gly4Ser)i, (Gly4Ser)2 or (Gly4Ser)4, more specifically of the formula (Gly4Ser)2), in particular two or more, In particular three or more, and more particularly all catalytic domains may be directly or indirectly assembled, fused or joined. In particular, it may be at least two, in particular at least three, more particularly whole catalytic domains selected from the group consisting of the catalytic domains of capping enzymes, in particular the catalytic domains of RNA triphosphatases, the catalytic domains of guanylyltransferases and It may be a catalytic domain of a DNA-dependent RNA polymerase selected from the group consisting of a catalytic domain of N7-guanine methyltransferase, and a catalytic domain of poly(A) polymerase.

In particular, the constructs include a catalytic domain of RNA triphosphatase, a catalytic domain of guanylyltransferase, a catalytic domain of N7-guanine methyltransferase, and a catalytic domain of DNA dependent RNA polymerase; linked directly or directly by a linking peptide selected from the group consisting of Gly4Ser)2 and (Gly4Ser)4, more particularly linking peptides of the formula (Gly Ser)2.

Preferably, at least said catalytic domain of the DNA-dependent RNA polymerase is linked by a linking peptide (particularly selected from the group consisting of linking peptides of the formulas Gly4, (Gly4Ser)i, (Gly4Ser)2 and (Gly4Ser)4). . In particular, at least one of the catalytic domains of the capping enzyme of formula (GlySer)2) may be one or more particularly selected from the group consisting of:

In the chimeric enzyme according to the invention, at least two of the catalytic domains can be assembled by non-covalent bonds, in particular leucine zippers.

Preferably, at least said catalytic domain of a DNA-dependent RNA polymerase or poly(A) polymerase is assembled by a non-covalent bond, in particular a leucine zipper, to one or more of the catalytic domains of a capping enzyme, preferably consisting of It may be one or more of the catalytic domains selected from the group, said catalytic domain of RNA triphosphatase; the catalytic domain of guanylyltransferase; and the catalytic domain of N7-guanine methyltransferase.

At least said catalytic domain of the poly(A) polymerase is assembled to at least one of the catalytic domains of the capping enzyme by a non-covalent bond, particularly preferably by a leucine zipper at its C-terminal end, in particular selected from the group consisting of It may be at least one of the catalytic domains, said catalytic domain of RNA triphosphatase; the catalytic domain of guanylyltransferase; and the catalytic domain of N7-guanine methyltransferase; and more specifically to said catalytic domain of RNA triphosphatase.

Leucine zippers, dimeric coil protein structures composed of two interacting amphiphilic α-helices, are commonly used to homo- or heterodimerize proteins. Each helix consists of a repeat of seven amino acids, the first amino acid (residue a) is hydrophobic, the fourth (residue d) is usually leucine, and the other residues are polar.

The leucine zippers VELCRO ACID-p1 and BASE-p1, which form a parallel heterodimer two-stranded coil structure, have a high tendency to form parallel protein heterodimers. They have been used to heterodimerize membrane proteins and several soluble proteins.

Other types of oligomerizing peptide domains also include chimeric enzymes according to the present invention, particularly ACID-a1/BASE-a1, ACID-Kg/BASE-Eg, NZ/CZ, ACID-pLL/BASE-pLL, and EE1234L and RR1234L It can be considered to create a chimeric enzyme according to the present invention to assemble at least two of the catalytic domains of a leucine zipper to form the same antiparallel heteromeric structure. The disulfide-linked version of the leucine zipper can also be used to generate interchain disulfide bridges between cysteine residues under oxidative conditions and disulfide-coil-linked heterodimeric chimeric enzymes according to the present invention.

Thus, at least two of the catalytic domains of poly(A) polymerase, capping enzymes (particularly RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase) and DNA-dependent RNA polymerase may be . Leucine zippers, especially those assembled into leucine zippers to form antiparallel heterostructures such as ACID-a1 /BASE-a1, ACID-Kg/BASE-Eg, NZ/CZ, ACID-pLL/BASE-pLL leucine zippers, disulfide coil coils bond, which is a disulfide bridge between cysteine residues.

5 comprising the RNA polymerase or a mutant or derivative thereof R551 SK1 E RNA polymerase mutant fused to the amino terminal end of wild-type T7, T3, SP6, K1-5, K1 E, fused to a viral capping enzyme; It is possible to synthesize single-stranded RNA sequence-complementary to the double-stranded template DNA in the '-3' direction, in particular via a linker, preferably of the formulas Glv4, (Gly4Ser)i, (Gly4Ser)2 and (Gly4Ser)4, It is preferably selected from the group consisting of linking peptides of formula Gly4, (Gly4Ser)i or (Gly4Ser)2.

In particular via a linker, preferably (Gly4Ser) i , (Gly4Ser)2 and (Gly4Ser)4 selected from the group consisting of connecting peptides of the formula Glv4, more preferably of the formula Gly4 or (Gly4Ser)2).

The present invention also relates to an isolated nucleic acid molecule or group of isolated nucleic acid molecules, wherein said nucleic acid molecule(s) is a nucleic acid molecule encoding a chimeric enzyme according to the present invention or an isolated nucleic acid molecule encoding a chimeric enzyme, The sequence may include a nucleic acid sequence encoding an RNA-binding domain of a protein-RNA tethering system fused in frame, particularly in sequence,

a nucleic acid sequence encoding one or more catalytic domains of a poly(A) polymerase; a nucleic acid sequence encoding one or more catalytic domains of a capping enzyme; and optionally

a nucleic acid sequence encoding at least one catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase; wherein the RNA-binding domain specifically binds to an RNA element of the protein-RNA tethering system composed of a specific RNA sequence and/or structure.

The group of isolated nucleic acid molecules encoding the chimeric enzyme according to the present invention comprises or consists of all nucleic acid molecules necessary and sufficient to obtain the chimeric enzyme according to the present invention by expression.

A "nucleic acid molecule" is any molecule composed of linked nucleotides including DNA and RNA molecules.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme according to the present invention comprises or consists of:

A nucleic acid molecule encoding said RNA binding domain of a protein-RNA tethering system, and one or more catalytic domains of a capping enzyme, in particular one or more catalytic domains of RNA triphosphatase, one or more catalytic domains of guanylyltransferase and N7-guanine nucleic acid molecules encoding one or more catalytic domains of a methyltransferase; and optionally: a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase; and/or a nucleic acid molecule encoding at least one catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme according to the invention may comprise or consist of: a nucleic acid molecule encoding said RNA binding domain of a protein-RNA tethering system, one or more catalysts of RNA triphosphatase nucleic acid molecules encoding domains, nucleic acid molecules encoding one or more catalytic domains of a guanylyltransferase, nucleic acid molecules encoding one or more catalytic domains of an N7-guanine methyltransferase; and optionally: a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase; and/or a nucleic acid molecule encoding at least one catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

An isolated nucleic acid molecule of the present invention may comprise or consist of a nucleic acid sequence encoding the RNA-binding domain of a protein-RNA tethering system fused in frame, particularly in sequence, wherein the catalyst of poly(A) polymerase A nucleic acid sequence encoding a domain, a nucleic acid sequence encoding said catalytic domain of a capping enzyme, in particular a nucleic acid sequence encoding said catalytic domain of RNA triphosphatase, a nucleic acid sequence encoding said catalytic domain of guanylyltransferase, N7 - a nucleic acid sequence encoding the catalytic domain of guanine methyltransferase, and a nucleic acid sequence encoding the catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

An isolated nucleic acid molecule according to the present invention may be a nucleic acid sequence encoding an RNA-binding domain of a protein-RNA tethering system, the sequence of which is fused in frame, in particular in sequence, comprising a catalytic domain of poly(A) polymerase. a nucleic acid sequence that encodes; a nucleic acid sequence encoding a catalytic domain of a capping enzyme, and optionally a nucleic acid sequence encoding said catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase; and a nucleic acid sequence encoding a ribosome skipping motif between a nucleic acid sequence encoding the catalytic domain of a poly(A) polymerase and a nucleic acid sequence encoding one of the catalytic domains selected from the group consisting of an enzyme, and a DNA-dependent RNA polymerase. It may include the catalytic domain of.

An isolated nucleic acid molecule according to the present invention may be a nucleic acid sequence encoding an RNA-binding domain of a protein-RNA tethering system, the sequence of which is fused in frame, in particular in sequence, comprising a catalytic domain of poly(A) polymerase. a nucleic acid sequence that encodes; A nucleic acid sequence encoding the catalytic domain of RNA triphosphatase, a nucleic acid sequence encoding the catalytic domain of guanylyltransferase, a nucleic acid sequence encoding the catalytic domain of N7-guanine methyltransferase, and optionally a DNA-dependent RNA polymerase can include

In particular, a nucleic acid sequence encoding said catalytic domain of a bacteriophage DNA-dependent RNA polymerase; and a nucleic acid sequence encoding an IS between a nucleic acid sequence encoding the catalytic domain of poly(A) polymerase and a nucleic acid sequence encoding one of the catalytic domains selected from the group consisting of the catalytic domain triphosphatase of RNA. , the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase, and the catalytic domain of DNA-dependent RNA polymerase, preferably the catalytic domain of RNA triphosphatase.

An isolated nucleic acid molecule of the invention encoding a chimeric enzyme may be a sequence thereof, comprising: a nucleic acid sequence encoding one or more catalytic domains of a poly(A) polymerase; a nucleic acid sequence encoding one or more catalytic domains of a capping enzyme; and optionally a nucleic acid sequence encoding said catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase; and a sequence further comprising a nucleic acid sequence encoding a ribosome skipping motif between a nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase and a nucleic acid sequence encoding one or more catalytic domains of a capping enzyme.

Indeed, unexpectedly, the inventors have found that such a nucleic acid molecule is framed in <a nucleic acid molecule encoding a chimeric enzyme comprising at least one domain of a capping enzyme> and <a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase>. DNA-dependent RNA polymerase associated with a nucleic acid encoding the RNA-binding domain of the RNA tethering system fused in

In a host cell, the method comprises expressing in the host cell a nucleic acid molecule or group of isolated nucleic acid molecules according to the invention, wherein the DNA sequence is covalently linked to at least one sequence encoding an RNA element. The protein-RNA tethering system that specifically binds to the RNA-binding domain.

An "RNA element of a protein-RNA tethering system that specifically binds to said RNA-binding domain" relates to an RNA sequence, generally a stem capable of binding with high affinity to a corresponding RNA-binding domain of a protein RNA-binding system. form a loop

In particular, when the RNA binding domain of the protein-RNA tethering system is the RNA binding domain of the rhamdoid N antitermination protein-RNA tethering system, the element specifically binding to the RNA binding domain is a boxBL and/or boxBR stem It is a loop RNA structure.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the MS2 coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain is a 21 nucleotide stem loop sequence ( nucleotides 1748-766 Enterobacteriophage MS2 isolate DL52, NCBI Accession No. JQ966307.1), which contains the initiation codon of the gene for viral replicase.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the R17 separate coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain may be 19 nucleotides . or a 21 nucleotide stem-loop (nucleotides 1746-764 of Enterobacteriophage R17, NCBI Accession No. EF1 08465.1) containing the initiation codon of the gene for viral replicase.

In particular, said DNA sequence is operably linked to a promoter for a bacteriophage DNA-dependent RNA polymerase or to a promoter for said DNA-dependent RNA polymerase of a chimera of the invention and is covalently linked to at least one at its 3' end. do. Preferably at least 2, at least 3, more preferably at least 4 sequences encode elements that specifically bind to said RNA-binding domain.

In particular, the transcription termination sequence may be a bacteriophage T7 phil 0 transcription termination sequence (Genbank Accession No. GU071091.1) or an E. coli RNA polymerase rrnB t1 stop (Genbank Accession No. LN832404.1).

The present invention also relates to a kit for the production of an RNA molecule having a 5'-end cap, in particular a 5'-end m7GpppN cap, comprising at least one chimeric enzyme according to the present invention as defined above and/or an isolated nucleic acid. .

a group of acid molecules and/or nucleic acid molecules according to the present invention as defined above, and/or a vector according to the present invention as defined above, or one or more catalytic domains of a chimeric enzyme, in particular RNA triphosphatase, guanylyl one or more catalytic domains of a transferase, one or more catalytic domains of an N7-guanine methyltransferase, and a DNA-dependent RNA polymerase and/or an isolated nucleic acid molecule and/or said chimeric enzyme and poly(A) polymerase, particularly a cytoplasmic poly(A) polymerase (A) A group of isolated nucleic acid molecules that encode a polymerase, comprising at least one RNA. a binding domain of a protein-RNA tethering system linked to at least one catalytic domain of said poly(A) polymerase and/or an isolated nucleic acid molecule encoding said poly(A) polymerase; and optionally a DNA sequence encoding the RNA molecule covalently linked to at least one sequence encoding an RNA element of the protein-RNA tethering system that specifically binds the RNA-binding domain.

2. RdRp

The expression unit of the present invention RNA polymerase is (i) a promoter operating in the host cell;

(ii) an RNA dependent RNA Polymerase (RdRp) ORF or variant thereof; and

(iii) a transcription termination sequence;

Preferably, the expression unit of RNA polymerase for expressing alphavirus RdRp includes an open reading frame (ORF) encoding alphavirus RdRp.

"RdRp" means RNA dependent RNA polymerase (RdRP). RdRp are generally capable of catalyzing the synthesis of polypeptides or (-)-strand RNA based on (+)-strand RNA templates, and/or those capable of catalyzing the synthesis of (+)-strand RNA based on (-)-strand RNA templates. Refers to a complex or combination of two or more identical and/or non-identical proteins.

RdRp may additionally have one or more additional functions, such as protease (for self-cleavage), helicase, terminal adenylyltransferase (for poly(A) tail addition), methyltransferase and guanylyltransferase (5'- to provide a cap to the nucleic acid), nuclear localization sites, tryphosphatase.

“Alphavirus RdRp” refers to RNA RdRp from an alphavirus, including RNA RdRp from a naturally occurring alphavirus and RNA RdRp from a variant or derivative of an alphavirus, such as an attenuated alphavirus. The terms "RdRp" and "alphavirus RdRp" are used interchangeably unless the context indicates that any particular RdRp is not an alphavirus RdRp.

The term "RdRp" refers to all variants of alphavirus RdRp expressed by alphavirus-infected cells or by cells transfected with a nucleic acid encoding for RdRp, in particular post-translational modified variants, conformations, isoforms. and homologues. Also, the term "RdRp" includes all forms of RdRp that are produced and can be produced by recombinant methods. For example, RdRp comprising tags facilitating detection and/or purification of RdRp in the laboratory, eg myc-tag, HA-tag or oligohistidine tag (His-tag) can be prepared by recombinant methods. there is.

Optionally, the RdRp additionally comprises any one or more of alphavirus conserved sequence 1 (CSE 1) or its complementary sequence, conserved sequence 2 (CSE 2) or its complementary sequence, conserved sequence 3 (CSE 3) or its complementary sequence. , it functionally means that it has the ability to bind to conserved sequence configuration 4 (CSE 4) or its complementary sequence.

Preferably, the RdRp is capable of binding to CSE 2 [i.e., on the (+) strand] and/or CSE 4 [i.e., on the (+) strand], or the complement of CSE 1 [i.e., on the (-) strand]. ] and/or the complement of CSE 3 [ie, on the (-) strand].

The origin of RdRp is not limited to a specific alphavirus. In a preferred embodiment, the alphavirus RdRp is derived from a Semliki Forest Virus, including naturally occurring Semliki Forest Virus and variants or derivatives of Semliki Forest Virus, such as attenuated Semliki Forest Virus. Also preferably, the RdRp is derived from Sindbis viruses, including naturally occurring Sindbis viruses and variants or derivatives of Sindbis viruses, such as attenuated Sindbis viruses. Also preferably, the RdRp is derived from Venezuelan encephalitis virus (VEEV), including naturally occurring VEEV and variants or derivatives of VEEV, such as attenuated VEEV.

Alphavirus RdRp typically comprise or consist of an alphaviral non-structural protein (nsP). "Non-structural protein" refers to any one or more distinct non-structural proteins (nsP1, nsP2, nsP3, nsP4) of alphavirus origin, or the polypeptide sequence of two or more non-structural proteins of alphavirus origin, for example nsP1234. refers to a poly-protein comprising "Non-structural protein" refers to nsP123. "Non-structural protein" refers to nsP1234. "Non-structural protein" refers to a complex or complex of nsP123 (synonymously P123) and nsP4. "Non-structural protein" refers to a complex or complex of nsP1, nsP2 and nsP3. "Non-structural protein" refers to a complex or complex of nsP1, nsP2, nsP3 and nsP4. "Non-structural protein" refers to any one or more complexes or complexes selected from the group consisting of nsP1, nsP2, nsP3 and nsP4.

The term "composite" or "assembly" is a functional ensemble of multiple elements. In relation to alphavirus RdRp, the term "complex or complex" refers to at least two multiple protein molecules, at least one of which is an alphaviral non-structural protein, wherein the complex or complex is RNA-dependent RNA polymerase (RdRP) indicates activity. A complex or complex may consist of many different proteins (heteromultimers) and/or multiple copies of one particular protein (homomultimers). With reference to multimers or multiples, “multi” means two or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10.

In addition, complexes or complexes may also include proteins from two or more other alphaviruses. For example, in a complex or complex according to the present invention comprising different alphavirus nonstructural proteins, not all nonstructural proteins need be of the same alphavirus origin. Heterogeneous complexes or conjugates are equally included in the present invention. For illustrative purposes only, a heterologous complex or complex may comprise one or more non-structural proteins (eg, nsP1, nsP2) from a first alphavirus (eg, Sindbis virus) and a second alphavirus (eg, Semliki Forest virus) from one or more non-structural proteins (nsP3, nsP4).

A "complex" or "assembly" refers to two or more identical or different protein molecules in spatial proximity. The proteins of the complex are preferably in direct or indirect physical or physicochemical contact with each other. A complex or complex may consist of multiple copies of a number of different proteins (heteromultimers) and/or of one particular protein (homomultimers).

"RdRp" includes each co- or post-translationally modified form, including carbohydrate-modified (eg glycosylated) and lipid-modified forms of alphavirus non-structural proteins. “RdRp” includes each and every functional fragment of alphavirus RdRp. Fragments are functional when they act as RNA-dependent RNA polymerase (RdRP). RdRp can form membranous replication complexes and/or vacuoles in cells in which RdRp is expressed.

Preferably, the expression unit of RNA polymerase comprises the coding region(s) for RdRp as defined above. Coding region(s) may consist of one or more open reading frame(s). Expression units of RNA polymerase encode all of the alphaviruses nsP1, nsP2, nsP3 and nsP4. The expression unit of RNA polymerase is encoded by a single open translational feature, encoding the alphaviruses nsP1, nsP2, nsP3 and nsP4 as a single, selectively cleavable polyprotein, nsP1234. The expression unit of RNA polymerase is encoded by a single open translational feature, encoding the alphaviruses nsP1, nsP2 and nsP3 as a single, selectively cleavable polyprotein, nsP123. nsP4 can be individually encrypted.

Preferably, the expression unit of the RNA polymerase of the present invention does not contain an alphavirus subgenomic promoter.

Preferably, the expression unit of RNA polymerase is an mRNA molecule. The mRNA molecule preferably does not contain CSE 1 and CSE 4. It is thought that such mRNAs do not compete with the replicon for binding of RdRp, so that the replicon can be replicated very efficiently by RdRp.

The RNA of the RNA polymerase expression unit is preferably non-double-stranded, preferably single-stranded, and more preferably positive-stranded RNA. RdRp is encoded by an open reading frame on the RdRp mRNA unit.

The expression unit of the RNA polymerase of the present invention is intron-free RNA, preferably intron-free mRNA. Preferably, the expression unit of the RNA polymerase is naturally intron-free RNA (mRNA). For example, intron-free RNA (mRNA) can be obtained by synthesis in vitro, eg by in vitro transcription. The expression unit of RNA polymerase includes an open reading frame encoding nsP1234 without an intron. The expression unit of RNA polymerase includes an open reading frame encoding nsP123 without an intron.

An “intron” is defined as a non-coding section of a precursor mRNA (pre-mRNA) that is removed prior to translation of the coding sequence of RNA into a polypeptide, i.e., spliced from RNA. Once the intron is spliced from the pre-mRNA, the resulting mRNA sequence is ready to be translated into a polypeptide. That is, the nucleotide sequence of an intron is typically not translated into a protein. An intron-free mRNA is an mRNA that contains a contiguous sequence of codons (base triplets) for translation into a polypeptide. An intron-free mRNA either naturally lacks an intron (e.g. can be first synthesized as an intron-free mRNA in a cell or in vitro transcription) or matures into an intron-free mRNA by splicing of an intron-containing pre-mRNA. It can be. Naturally intron-free in vitro transcribed RNAs are preferred in the present invention.

The expression unit of the RNA polymerase of the present invention differs from alphavirus genomic RNA at least in that it is not capable of self-replication and/or does not contain an open reading frame under the control of a sub-genomic promoter. When self-amplification is not possible, an expression unit of RNA polymerase may also be referred to as a "suicide construct".

Preferably, the expression unit of the RNA polymerase of the present invention is not related to alphavirus structural proteins. Preferably, the expression units of RNA polymerase are not packaged by alphaviral structural proteins. More preferably, the expression units of RNA polymerase are not packaged within viral particles. Preferably, the expression units of RNA polymerase do not associate with viral proteins (viral protein-free systems). Viral protein-free systems offer advantages over prior art, eg helper-virus based systems.

Preferably, the expression unit of RNA polymerase has at least one Conserved Sequence Configuration (CSE) required for (-) strand synthesis based on a (+) strand template and/or (+) strand synthesis based on a (-) strand template. lack. More preferably, the expression unit of RNA polymerase does not contain any alphavirus conserved sequence construct (CSE). In particular, in the four CSEs of alphavirus, any one or more of the following CSEs are preferably not present on the expression unit of RNA polymerase.

- CSE 1, which is believed to function as a promoter for synthesis of the (+) strand based on the (-) strand template in alphaviruses found in nature;

- CSE 2, which is believed to function as a promoter for negative-strand synthesis based on the positive-strand genomic RNA of alphaviruses found in nature;

- CSE 3, believed to contribute to the efficient transcription of subgenomic (+) strand RNA in alphaviruses found in nature;

- CSE 4, which is believed to act as a core-promoter for the initiation of negative-strand synthesis based on positive-strand genomic RNA in alphaviruses found in nature.

CSE 1, CSE 3 and CSE 4 do not exist and CSE 2 may or may not exist.

In particular, in the absence of any one or more of the alphavirus CSEs, the RdRp mRNA units of the present invention are more similar to typical eukaryotic mRNAs than to alphaviral genomic RNAs.

The expression unit of the RNA polymerase of the present invention is an isolated nucleic acid molecule.

3. Codon Usage

In general, degeneracy of the genetic code allows for the replacement of certain codons (base triplets encoding amino acids) present in RNA sequences by other codons (base triplets) while retaining the same coding ability (replacing a codon is encodes the same amino acid as the replaced codon).

In the present invention, at least one codon of an open reading frame included in an RNA molecule is different from each codon of each open reading frame in the species from which the open reading frame is derived. The coding sequence of an open reading frame is referred to as "adapted".

For example, when the coding sequence of an open reading frame is modified, frequently used codons can be selected. WO2009/024567 A1 describes modifications of the coding sequence of nucleic acid molecules, including substitution of more frequently used rare codons.

Because the frequency of codon usage varies depending on the cell or organism of interest, such types of modifications are appropriate to tailor the nucleic acid sequence for expression in a particular cell or organism of interest. Generally speaking, codons that are used more frequently are typically translated more efficiently in a target cell or organism, but modification of all codons in an open reading frame is not always necessary.

For example, when the coding sequence of the open reading frame is modified, the content of G (guanylate) residues and C (cytidylate) residues can be altered by selecting the codon with the most abundant GC-content for each amino acid. can It has been reported that RNA molecules with GC-rich open reading frames have the potential to reduce immune activity and improve translation and half-life of RNA.

V. RNA units of interest

Since RNA is widely used in life sciences, biotechnology, and pharmaceuticals, the RNA transcription system of interest of the present invention needs to be designed to efficiently produce proteins and/or RNAs, particularly in eukaryotic cells.

RNA of interest (RNAi) of the present invention is mRNA (messenger RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), miRNA (micro RNA), siRNA (small interfering RNA), shRNA, trans-activating crispr RNA, Circular It may be any one selected from the group containing RNA, ribozymes and RNA aptamers.

The present inventors have previously developed an artificial transcription system (ie, chimeric enzyme, ctDdRp) for efficient transformation that autonomously produces mRNA molecules in eukaryotic cells, particularly in the cytoplasm of said cells. Using this system, RNA chains were synthesized by the RNA polymerase portion of this chimeric enzyme and capped at the 5'-end by the mRNA capping enzyme portion. In addition, a poly(A) tail can be created at the 3'-end of the transcript by transcription of a polyadenosine track from a DNA template.

In the RNA transcription system of the present invention, the RNA unit of interest encoding RNAi is either a DdRp-operated RNA unit or an RdRp-operated RNA unit of interest, and the RNA unit of interest is converted by each of the two types of RNA polymerase expression units. It can be configured to be operably connected.

1. RNA units of interest operated by DdRp

In the RNA transcription system of the present invention, the RNA unit of interest encoding RNAi may be constructed by being operably linked by an RNA polymerase expression unit encoding the chimeric enzyme, ctDdRp.

The transcription unit of the RNA of interest of the present invention is

(1) a promoter to which the ctDdRp binds;

(2) 5'UTR;

(3) DNA fragments encoding RNAi; and

(4) 3'UTR; and may be configured by operably linking them.

The chimeric enzyme of the present invention, ctDdRp, can increase the expression of any one selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase to continuously express the RNA of interest for a long period of time.

In the present invention, T7 RNA polymerase has very high specificity for the T7 promoter and can only transcribe DNA downstream of the T7 promoter.

Therefore, by using the T7 RNA polymerase in the present invention, binding and transcription are performed only to the RNA unit of interest having the T7 promoter without reacting with other promoters in the cell, and the RNA of interest can be transcribed and amplified with high efficiency.

The chimeric enzyme, ctDdRp, may additionally include a capping enzyme, a tethering system, or a Poly(A) polymerase.

Preferably, the RNA unit of interest operated by DdRp of the RNA transcription system of the present invention may include a 5'UTR and a 3'UTR including a 5'cap or an IRES in the mRNA of interest for expressing the protein of interest in a eukaryotic cell. there is.

"5'cap", "cap", "capped" and ""5'cap structure" are synonyms that refer to a dinucleotide found at the 5' end of primary transcripts of some eukaryotic cells, such as precursor messenger RNA. A 5'cap is a structure in which a (optionally chemically modified) guanosine is linked to the first nucleotide of the mRNA molecule via the 5' to the 5' triphosphate linkage (or chemically modified triphosphate linkage in the case of certain cap analogs). The term may refer to a conventional cap or cap analog.

For example, providing RNA with a 5' cap can be achieved by in vitro transcription of a DNA template in the presence of the 5' cap, wherein the 5' cap is co-transcriptionally incorporated into the resulting RNA strand. Alternatively, the RNA can be generated, for example, by in vitro transcription, and the 5'cap can be attached to the RNA after transcription using a capping enzyme, for example, the capping enzyme of vaccinia virus. In capped RNA, the 3' position of the first base of a (capped) RNA molecule is linked at the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphodiester bond.

"Conventional 5'cap" means a naturally occurring 5'cap, preferably a 7-methylguanosine cap. In a 7-methylguanosine cap, the guanosine of the cap is a chemically modified guanosine, wherein The modification consists of methylation at the 7-position.

The 5' cap on the mRNA of interest is induced by the chimeric enzyme ctDdRp. Efficient production of the mRNA of interest encoded in trans can be achieved.

An mRNA of interest of the present invention may also contain an internal ribosome entry site (IRES) element. Generally, an internal ribosome entry site, abbreviated IRES, is a nucleotide sequence in the middle of an mRNA sequence that allows initiation of translation from the mRNA from a position different from the 5' end of the mRNA sequence of interest, such as a position in the middle of the mRNA sequence of interest. Located.

Newly synthesized mRNA is usually modified into a 5' cap structure when the transcript reaches a length of 20 to 30 nucleotides. First, the 5' terminal nucleotide pppN (ppp represents triphosphate and N represents any nucleoside) is 5' in cells by RNA 5' triphosphatase and capping enzyme having guanylyl transferase activity. converted to GpppN. GpppN can then be methylated in the cell by a second enzyme with (guanine-7)-methyltransferase activity to form a mono-methylated m7GpppN cap.

The 5'cap used in the present invention is a natural 5'cap.

In the present invention, the natural 5' cap dinucleotide typically comprises a non-methylated cap dinucleotide (G(5')ppp(5')N; also referred to as GpppN) and a methylated cap dinucleotide ((m7G(5')ppp (5') N; also referred to as m7GpppN).

The presence of the 5' cap also provides an unexpected synergistic effect in the trans-replication system of the present invention. It is more efficient for the capped chimeric enzyme mRNA to trigger production of the protein of interest when the capped chimeric enzyme mRNA is provided in trans to RNA. Therefore, the synergistic effect of 5' cap and replication in trans, that is, an effect superior to that of cis replication.

The capped RNA of the present invention does not depend on the capping device of the target cell. The method most frequently used to form capped RNA in vitro is the use of all four ribonucleoside triphosphates and cap dinucleotides such as m7G(5')ppp(5')G (also called m7GpppG) in bacteria in the presence of or to transcribe the DNA template with bacteriophage RNA polymerase.

RNA polymerase then initiates transcription by nucleophilic attack by the 3'OH of the guanosine moiety of m7GpppG on the α-phosphate of the template nucleoside triphosphate (pppN), resulting in the intermediate m7GpppGpN (where N is the second base of the RNA molecule). is) to form Formation of the competing GTP-initiated product pppGpN is inhibited by setting the molar ratio of cap to GTP between 5 and 10 during in vitro transcription.

A 5' cap is a 5' cap analogue. It is particularly suitable if the RNA is obtained by in vitro transcription, for example in vitro transcribed RNA (IVT-RNA). Cap analogs were initially described to facilitate large-scale synthesis of RNA transcripts by in vitro transcription.

UTR

An Untranslated region or “UTR” refers to a region that is transcribed but not translated into an amino acid sequence or the corresponding region within an RNA molecule such as an mRNA molecule. The UTR can be upstream of the open reading frame and/or downstream of the open reading frame encoding the chimeric enzyme.

Specifically, in the chimeric enzyme mRNA of the present invention, "3'UTR" refers to a region located 3' of the coding region for the chimeric enzyme, and "5'UTR" refers to a region located 5' of the coding region for the chimeric enzyme. It is about the area in which it is located.

The 3'UTR, if present, is located at the 3' end of the gene, downstream of the stop codon of the protein coding region, but the "3'UTR" preferably does not contain a poly(A) tail. Thus, the 3'UTR is upstream of the poly(A) tail, eg directly adjacent to the poly(A) tail (if present).

The 5'UTR, if present, is located at the 5' end of the gene upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5' cap, eg right next to the 5' cap (if present).

The 5' and/or 3'UTRs can be functionally linked to the open reading frame, such that these regions are associated with the open reading frame to increase stability and/or translational efficiency of the RNA comprising the open reading frame.

UTR is involved in RNA stability and translation efficiency, which are prerequisites for effective RNA delivery. By selecting specific 5' and/or 3' untranslated regions (UTRs), both, in addition to structural modifications regarding the 5' cap and/or 3' poly(A)-tail, can be improved.

Sequence elements within the UTR are generally understood to affect translation efficiency (mainly 5'UTR) and RNA stability (mainly 3'UTR). Preferably, an activated 5'UTR is present to increase the translational efficiency and/or stability of the nucleic acid sequence. Independently or additionally, it is preferred that an activated 3'UTR is present to increase translational efficiency and/or stability of the nucleic acid sequence.

Preferably, the chimeric enzyme mRNA contains a 5'UTR and/or 3'UTR that is heterologous or non-unique to the ctDdRp from which it was derived. This allows designing untranslated regions according to the desired translational efficiency and RNA stability.

The 3'UTR of the present invention is one or more that can fold to provide a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins known to increase RNA stability (eg RNA-binding proteins). May contain reverse iterations.

Two consecutive identical copies of the human beta-globin 3'UTR, in particular the human beta-globin 3'UTR, contribute to high transcriptional stability and translational efficiency.

Thus, the chimeric enzyme mRNA of the present invention contains two consecutive identical copies of the human beta-globin 3'UTR. Thus, it is (a) optionally 5'UTR in the 5'→3' direction; (b) an open reading frame encoding the chimeric enzyme; (c) 3'UTR; A 3'UTR comprising two consecutive identical copies of the human beta-globin 3'UTR, a fragment thereof, or a variant of the human beta-globin 3'UTR or a fragment thereof.

The chimeric enzyme mRNA of the present invention activates to increase translation efficiency and/or stability of the 3'UTR, but not human beta-globin 3'UTR, fragments thereof, or variants of human beta-globin 3'UTR or fragments thereof. includes

The chimeric enzyme mRNA of the present invention contains an activated 5'UTR to increase translation efficiency and/or stability.

UTR-containing RNAs according to the present invention can be prepared, for example, by in vitro transcription. These can be achieved by genetically modifying the expression of a nucleic acid molecule (eg DNA) of the invention in a manner that permits transcription of RNA having a 5'UTR and/or 3'UTR.

A 3'UTR is a portion of an mRNA that is usually located between the protein coding region of the mRNA (i.e., the open reading frame) and the poly(A) sequence. The 3'UTR of mRNA is not translated into an amino acid sequence. 3'UTRs are generally encoded by genes that are transcribed into respective mRNAs during gene expression. The genomic sequence is first transcribed into immature mRNA containing optional introns. Thereafter, the immature mRNA is further processed to become a mature mRNA through a maturation process. This maturation process involves 5' capping, immature mRNA splicing to cut an additional intro and adenylation of the immature mRNA 3' end and additional endo- or exo nucleases. Include modifications at the 3' end, such as first truncation.

In the present invention, the 3'UTR corresponds to the sequence of mature mRNA located at the 3' end of the stop codon of the protein coding region, preferably immediately adjacent to the 3' end of the stop codon of the protein coding region, and is a poly(A) sequence. , preferably to the nucleotide right next to the 5' end of the poly(A) sequence. In the present invention, the 3'UTR of a gene, such as the 3'UTR of alpha or beta globin, is a sequence corresponding to the 3'UTR of a mature mRNA derived from such a gene, i.e., obtained by transcription of the gene and maturation of the immature mRNA. It is the sequence corresponding to the mRNA. The term "3'UTR of a gene" includes the DNA sequence and RNA sequence of the 3'UTR.

A 5'UTR is generally understood to be a specific part of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Generally, the 5'UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'UTR may contain elements for regulating gene expression, also referred to as regulatory elements. Such a regulatory element may be, for example, a ribosome binding site or a 5' terminal oligopyrimidine tract (TOP). The 5'UTR can be modified post-transcriptionally, for example by the addition of a 5' cap. In the present invention, the 5'UTR corresponds to the sequence of the mature mRNA located between the 5' cap and the start codon. Preferably, the 5'UTR is from the nucleotide located 3' of the 5' cap, preferably from the nucleotide located immediately 3' of the 5' cap, 5' of the start codon of the protein coding region. It corresponds to a sequence extending up to the nucleotide located, preferably up to the nucleotide located immediately 5' to the start codon of the protein coding region. Nucleotides located immediately 3' to the 5' cap of the mature mRNA generally correspond to the start of transcription.

The 5'UTR sequence may be an RNA sequence, such as an mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to such an RNA sequence. The 5'UTR of a gene is the sequence corresponding to the 5'UTR of a mature mRNA derived from this gene, i.e., the sequence corresponding to the mRNA obtained by transcription of the gene and maturation of the immature mRNA. The 5'UTR of a gene includes the DNA sequence and RNA sequence of the 5'UTR.

A 5'TOP is generally a stretch of pyrimidine nucleotides located at the 5'terminal region of a nucleic acid molecule, such as the 5'terminal region of a specific mRNA molecule or a functional entity of a specific gene, for example, the 5'terminal region of a transcriptional region. )am. The sequence usually begins with a cytidine corresponding to the start of transcription, followed by a stretch of usually about 3 to 30 pyrimidine nucleotides. The pyrimidine stretch thus ends the 5'TOP with one nucleotide 5' of the first purine nucleotide located downstream of the TOP. Messenger RNA containing a 5' terminal oligopyrimidine tract is often referred to as TOP mRNA. Thus, genes providing such messenger RNA are referred to as TOP genes. TOP sequences are found, for example, in mRNAs and ribosomal proteins encoding genes and peptide elongation factors.

In the present invention, the TOP motif is a nucleic acid sequence corresponding to 5'TOP as defined above. Thus, in the present invention, the TOP motif is a stretch of pyrimidine nucleotides preferably having a length of 3 to 30 nucleotides. Preferably, the TOP-motif is at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 pyrimidine nucleotides nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5' end with a cytosine nucleotide. In the TOP gene and TOP mRNA, the TOP-motif preferably starts at its 5' end with the transcriptional start and ends one nucleotide 5' of the first purine residue in the gene or mRNA.

In the sense of the present invention, the TOP motif is preferably located at the 5' end of the sequence representing the 5'UTR or at the 5' end of the sequence encoding the 5'UTR. Thus, preferably, if such a stretch is from the 5' end of each sequence, such as SM_mRNAi according to the present invention, the 5'UTR element of SM_mRNAi according to the present invention, or the 5'UTR of the TOP gene as described herein A stretch of three or more pyrimidine nucleotides is called a "TOP motif" in the sense of the present invention if located in the nucleic acid sequence from which it was derived. In other words, a stretch of 3 or more pyrimidine nucleotides located anywhere within the 5'UTR or 5'UTR element that is not located at the 5' end of the 5'UTR or 5'UTR element is preferably not referred to as a "TOP motif". .

TOP genes are generally characterized by the presence of a 5' terminal oligopyrimidine tube. Moreover, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with tissue-specific translational regulation are also known. The 5'UTR of the TOP gene corresponds to the sequence of the 5'UTR of the mature mRNA derived from the TOP gene, and extends from the nucleotide located 3' of the 5' cap to the nucleotide located 5' of the start codon. The 5'UTR of the TOP gene usually does not contain any start codon, upstream AUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstream AUGs and upstream open reading frames are generally understood as AUGs and open reading frames appearing 5' of the start codon (AUG) of the open reading frame to be translated. The 5'UTR of TOP genes is usually relatively short. The 5'UTR length of a TOP gene can vary from 20 nucleotides to 500 nucleotides, and is usually less than about 200 nucleotides, less than about 150 nucleotides, and less than about 100 nucleotides. In the meaning of the present invention, a 5'UTR example of a TOP gene is a nucleic acid sequence extending from the nucleotide at position 5 to the nucleotide located immediately 5' to the start codon (eg ATG). A particularly preferred segment of the 5'UTR of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif. '5'UTR of TOP gene' preferably refers to the 5'UTR of a naturally occurring TOP gene.

poly(A) sequence

The chimeric enzyme RNA expression unit of the present invention comprises a 3' poly(A) sequence.

A poly(A) sequence is essentially at least 20, at least 26, at least 40, at least 80, at least 100, and not more than 500, not more than 400, not more than 300, not more than 200, in particular not more than 150 A nucleotides, in particular not more than Consists of or contains about 120 A nucleotides.

Depending on the number of nucleotides in the poly(A) sequence, typically at least 50%, at least 75% of the nucleotides in the poly(A) sequence are A nucleotides, but the remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (free delay nucleotide), G nucleotide (guanylate), C nucleotide (cytidylate). It means that all nucleotides in the poly(A) sequence, that is, 100% are A nucleotides according to the number of nucleotides in the poly(A) sequence. The term “A nucleotide” or “A” refers to adenylate.

The present invention is based on a DNA template comprising dT nucleotides (deoxythymidylate) repeated in the strand complementary to the coding strand, to which 3' poly(A ) gives the sequence. A DNA sequence (coding strand) that encodes a poly(A) sequence is referred to as a poly(A) unit.

The 3' poly(A) units present in the coding strand of DNA consist essentially of dA nucleotides, but are interrupted by a random sequence with an even distribution of four nucleotides (dA, dC, dG, dT). These random sequences may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 in length.

Poly(A) units consist essentially of dA nucleotides, but have an even distribution of 4 nucleotides (dA, dC, dG, dT) and are interrupted by random sequences of, for example, 5 to 50 nucleotides in length, in DNA At the RNA level, it exhibits constant proliferation of plasmid DNA in E. coli, and at the RNA level is still associated with beneficial properties with respect to supporting RNA stability and translational efficiency.

Consequently, the 3' poly(A) sequences contained in the RNA molecules described herein consist essentially of A nucleotides, but in a random sequence with an even distribution of four nucleotides (A, C, G, U). interrupted by These random sequences may be 5 to 50, 10 to 30, or 10 to 20 in length.

Polyadenylation is generally understood as the addition of a poly(A) sequence to a nucleic acid molecule such as an RNA molecule, eg immature mRNA. The adenylic acid polymerization reaction can be induced by a so-called adenylic acid polymerization reaction signal. This signal is located within the stretch of nucleotides at the 3' end of a nucleic acid molecule, such as an RNA molecule, to be adenylated. The adenylic acid polymerization signal usually includes a hexamer consisting of adenine and uracil/thymine nucleotides, the hexamer sequence AAUAAA. Other sequences, hexamer sequences, are also possible. Adenylic acid polymerization occurs during processing of pre-mRNA (also called immature-mRNA). RNA maturation (from pre-mRNA to mature mRNA) involves the step of adenylation.

Protein-RNA tethering system

A nucleic acid molecule encoding said RNA binding domain of a protein-RNA tethering system, and one or more catalytic domains of a capping enzyme, in particular one or more catalytic domains of RNA triphosphatase, one or more catalytic domains of guanylyltransferase and N7-guanine nucleic acid molecules encoding one or more catalytic domains of a methyltransferase; and optionally: a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase; and/or a nucleic acid molecule encoding at least one catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme according to the invention may comprise or consist of: a nucleic acid molecule encoding said RNA binding domain of a protein-RNA tethering system, one or more catalysts of RNA triphosphatase nucleic acid molecules encoding domains, nucleic acid molecules encoding one or more catalytic domains of a guanylyltransferase, nucleic acid molecules encoding one or more catalytic domains of an N7-guanine methyltransferase; and optionally: a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase; and/or a nucleic acid molecule encoding at least one catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

An isolated nucleic acid molecule of the present invention may comprise or consist of a nucleic acid sequence encoding the RNA-binding domain of a protein-RNA tethering system fused in frame, particularly in sequence, wherein the catalyst of poly(A) polymerase A nucleic acid sequence encoding a domain, a nucleic acid sequence encoding said catalytic domain of a capping enzyme, in particular a nucleic acid sequence encoding said catalytic domain of RNA triphosphatase, a nucleic acid sequence encoding said catalytic domain of guanylyltransferase, N7 - a nucleic acid sequence encoding the catalytic domain of guanine methyltransferase, and a nucleic acid sequence encoding the catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase.

An isolated nucleic acid molecule according to the present invention may be a nucleic acid sequence encoding an RNA-binding domain of a protein-RNA tethering system, the sequence of which is fused in frame, in particular in sequence, comprising a catalytic domain of poly(A) polymerase. a nucleic acid sequence that encodes; a nucleic acid sequence encoding a catalytic domain of a capping enzyme, and optionally a nucleic acid sequence encoding said catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase; and a nucleic acid sequence encoding a ribosome skipping motif between a nucleic acid sequence encoding the catalytic domain of a poly(A) polymerase and a nucleic acid sequence encoding one of the catalytic domains selected from the group consisting of an enzyme, and a DNA-dependent RNA polymerase. It may include the catalytic domain of.

An isolated nucleic acid molecule according to the present invention may be a nucleic acid sequence encoding an RNA-binding domain of a protein-RNA tethering system, the sequence of which is fused in frame, in particular in sequence, comprising a catalytic domain of poly(A) polymerase. a nucleic acid sequence that encodes; A nucleic acid sequence encoding the catalytic domain of RNA triphosphatase, a nucleic acid sequence encoding the catalytic domain of guanylyltransferase, a nucleic acid sequence encoding the catalytic domain of N7-guanine methyltransferase, and optionally a DNA-dependent RNA polymerase can include

In particular, a nucleic acid sequence encoding said catalytic domain of a bacteriophage DNA-dependent RNA polymerase; and a nucleic acid sequence encoding a ribosome skipping motif between a nucleic acid sequence encoding the catalytic domain of poly(A) polymerase and a nucleic acid sequence encoding one of the catalytic domains selected from the group consisting of: the catalytic domain of RNA may comprise the catalytic domain of triphosphatase, guanylyltransferase, the catalytic domain of N7-guanine methyltransferase, and the catalytic domain of DNA-dependent RNA polymerase, preferably the catalytic domain of RNA triphosphatase. there is.

An isolated nucleic acid molecule of the invention encoding a chimeric enzyme may be a sequence thereof, comprising: a nucleic acid sequence encoding one or more catalytic domains of a poly(A) polymerase; a nucleic acid sequence encoding one or more catalytic domains of a capping enzyme; and optionally a nucleic acid sequence encoding said catalytic domain of a DNA-dependent RNA polymerase, particularly a bacteriophage DNA-dependent RNA polymerase; and a sequence further comprising a nucleic acid sequence encoding a ribosome skipping motif between a nucleic acid sequence encoding a catalytic domain of a poly(A) polymerase and a nucleic acid sequence encoding one or more catalytic domains of a capping enzyme.

Indeed, unexpectedly, the inventors have found that such a nucleic acid molecule is framed in <a nucleic acid molecule encoding a chimeric enzyme comprising at least one domain of a capping enzyme> and <a nucleic acid molecule encoding one or more catalytic domains of a poly(A) polymerase>. DNA-dependent RNA polymerase associated with a nucleic acid encoding the RNA-binding domain of the RNA tethering system fused in

In a host cell, the method comprises expressing in the host cell a nucleic acid molecule or group of isolated nucleic acid molecules according to the invention, wherein the DNA sequence is covalently linked to at least one sequence encoding an RNA element. The protein-RNA tethering system that specifically binds to the RNA-binding domain.

An "RNA element of a protein-RNA tethering system that specifically binds to said RNA-binding domain" relates to an RNA sequence, generally a stem capable of binding with high affinity to a corresponding RNA-binding domain of a protein RNA-binding system. form a loop

In particular, when the RNA binding domain of the protein-RNA tethering system is the RNA binding domain of the rhamdoid N antitermination protein-RNA tethering system, the element specifically binding to the RNA binding domain is a boxBL and/or boxBR stem It is a loop RNA structure.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the MS2 coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain is a 21 nucleotide stem loop sequence ( nucleotides 1748-766 Enterobacteriophage MS2 isolate DL52, NCBI Accession No. JQ966307.1), which contains the initiation codon of the gene for viral replicase.

In particular, when the RNA-binding domain of the protein-RNA tethering system is the RNA-binding domain of the R17 separate coat protein-RNA tethering system, the element specifically binding to the RNA-binding domain may be 19 nucleotides . or a 21 nucleotide stem-loop (nucleotides 1746-764 of Enterobacteriophage R17, NCBI Accession No. EF1 08465.1) containing the initiation codon of the gene for viral replicase.

In particular, said DNA sequence is operably linked to a promoter for a bacteriophage DNA-dependent RNA polymerase or to a promoter for said DNA-dependent RNA polymerase of a chimera of the invention and is covalently linked to at least one at its 3' end. do. Preferably at least 2, at least 3, more preferably at least 4 sequences encode elements that specifically bind to said RNA-binding domain.

In particular, the transcription termination sequence may be a bacteriophage T7 phil 0 transcription termination sequence (Genbank Accession No. GU071091.1) or an E. coli RNA polymerase rrnB t1 stop (Genbank Accession No. LN832404.1).

The present invention also relates to a kit for the production of an RNA molecule having a 5'-end cap, in particular a 5'-end m7GpppN cap, comprising at least one chimeric enzyme according to the present invention as defined above and/or an isolated nucleic acid. .

a group of acid molecules and/or nucleic acid molecules according to the present invention as defined above, and/or a vector according to the present invention as defined above, or one or more catalytic domains of a chimeric enzyme, in particular RNA triphosphatase, guanylyl one or more catalytic domains of a transferase, one or more catalytic domains of an N7-guanine methyltransferase, and a DNA-dependent RNA polymerase and/or an isolated nucleic acid molecule and/or said chimeric enzyme and poly(A) polymerase, particularly a cytoplasmic poly(A) polymerase (A) A group of isolated nucleic acid molecules that encode a polymerase, comprising at least one RNA. a binding domain of a protein-RNA tethering system linked to at least one catalytic domain of said poly(A) polymerase and/or an isolated nucleic acid molecule encoding said poly(A) polymerase; and optionally a DNA sequence encoding the RNA molecule covalently linked to at least one sequence encoding an RNA element of the protein-RNA tethering system that specifically binds the RNA-binding domain.

2. RNA units of interest operated by RdRp

The RNA unit of interest operated by RdRp of the RNA transcription system of the present invention may include a 5'UTR and a 3'UTR including a 5'cap or an IRES of interest for expressing a protein of interest in a eukaryotic cell.

In the RNA transcription system of the present invention, an RNA unit of interest encoding RNAi may be operably linked by an expression unit encoding the RdRp.

The RNA unit of interest of the present invention is

(1) a promoter operating in the host cell;

(2) DNA encoding the replicon; and

(3) a transcription termination sequence that operates in the host cell; and may be configured by operably linking them.

RNA units of interest of the present invention include replicons. Nucleic acid constructs capable of being replicated by RdRp, preferably alphavirus RdRp, are called replicons. Typically, a replicon according to the present invention is an RNA molecule.

A “replicon” defines an RNA molecule capable of being replicated by an RNA-dependent RNA polymerase, producing one or multiple copies of the same or essentially identical RNA replicon without DNA intermediates. "No DNA intermediate" means that in the process of forming a replicon of an RNA replicon, a copy of deoxyribonucleic acid (DNA) of the replicon or the complement of the replicon forms a copy of the RNA replicon. is not formed, and/or the deoxyribonucleic acid (DNA) molecule is not used as a template in the process of forming a copy of the RNA replicon, or its complement.

“Can be duplicated” generally describes that one or more identical or essentially identical copies of a nucleic acid can be made. When used with the term "RdRp" in "capable of being replicated by RdRp", the term "capable of being replicated" describes the functional property of the replicon for RdRp.

These functional properties include at least one of (i) RdRp can recognize a replicon and (ii) RdRp can act as an RNA dependent RNA polymerase (RdRP).

Preferably, the RdRp is capable of (i) recognizing a replicon and (ii) acting as an RNA dependent RNA polymerase. An RNA-dependent RNA polymerase can use a replicon, its complement, or a portion thereof as a template.

The expression "recognizable" describes that RdRp is capable of physically binding to a replicon, and preferably RdRp is capable of binding to a replicon, typically in a non-covalent bond. The term “binding” means that an RdRp is any one or more of conserved sequence 1 (CSE 1) or its complementary sequence (if included in a replicon), conserved sequence 2 (CSE 2) or its complementary sequence (replicon). ), conserved sequence configuration 3 (CSE 3) or its complementary sequence (if included in a replicon), conserved sequence configuration 4 (CSE 4) or its complementary sequence (if included in a replicon) It can mean that there is a binding ability. Preferably, the RdRp is capable of binding to CSE 2 [i.e., on the (+) strand] and/or CSE 4 [i.e., on the (+) strand], or the complement of CSE 1 [i.e., on the (-) strand]. ] and/or the complement of CSE 3 [ie, on the (-) strand].

The expression “capable of acting as RdRP” includes the meaning that RdRp is capable of catalyzing the synthesis of the (−) strand complement of alphaviral genomic (+) strand RNA, wherein the (+) strand RNA has a template function and , and/or RdRp can catalyze the synthesis of (+) strand alphavirus genomic RNA, wherein the (-) strand RNA has a template function. In general, the expression "capable of acting as RdRP" also means that the RdRp is capable of catalyzing the synthesis of a positive-strand subgenomic transcript, wherein the negative-strand RNA has a template function and the (+)-strand RNA has a template function. Synthesis of a strand subgenomic transcript is typically initiated at an alphavirus subgenomic promoter.

The expressions "capable of binding" and "capable of acting as RdRP" refer to the ability under normal physiological conditions. In particular, the expression refers to an intracellular condition that expresses alphavirus RdRp or has been transfected with a nucleic acid encoding alphavirus RdRp. The cell is preferably a eukaryotic cell. The binding capacity and/or ability to act as RdRP can be tested experimentally, eg in a cell-free in vitro system or in a eukaryotic cell. Optionally, the eukaryotic cell is a cell derived from a species infected with a particular alphavirus representing the origin of the RdRp. For example, when using the alphavirus RdRp of a particular alphavirus that infects humans, normal physiological conditions are those in human cells. More preferably, the eukaryotic cells (eg human cells) are derived from the same tissue or organ infected with the particular alphavirus representing the origin of the RdRp.

In view of these functional properties, the replicon of the present invention and the RdRp mRNA unit of the present invention form a functional pair. The alphavirus RdRp may be any alphavirus RdRp according to the present invention, and the nucleotide sequence of the replicon RNA is not particularly limited as long as the replicon can be replicated by the alphavirus RdRp in trans.

When the system of the present invention is introduced into a cell, preferably a host cell, the RdRp encoded on the RdRp expression unit can be translated, thereby generating RdRp. After translation, RdRp is capable of replicating the RNA replicon of interest in trans. Accordingly, the present invention provides a system for replicating RNA in trans. Consequently, the system of the present invention is a trans-replication system. Thus, a replicon according to the present invention is a trans-replicon.

Here, trans (eg, in the context of trans-acting, trans-modulation) generally means "action from a different molecule" (ie, intermolecular). It is the opposite of cis (eg, with respect to cis-acting, cis-regulating), which generally means "acting on the same molecule" (ie, intramolecularly). In the context of RNA synthesis (including transcription and RNA replication), trans-actors include nucleic acid sequences containing genes encoding enzymes capable of RNA synthesis (RdRp).

RdRp functions in the synthesis of a second nucleic acid molecule, i.e. another molecule. Both the trans-acting RNA and the protein it encodes are referred to as "acting in trans" on the gene of interest. In the present invention, the trans-acting RNA encodes an alphaviral RNA polymerase. Therefore, alphavirus RNA polymerase is capable of replicating RNA and is therefore called RdRp.

RdRp acts in trans on a second RNA molecule (replicon). A replicon capable of being cloned in trans by RdRp according to the present invention is synonymously referred to herein as "trans-replicon" or "replicon according to the present invention".

Contrary to suggestions in the prior art, the trans-replication system of the present invention is well suited for gene expression.

First of all, the versatility of the system of the present invention comprising two separate RNA molecules allows replicons and RdRp expression units to be designed and/or manufactured at different times and/or at different sites. The RdRp expression unit is made at a first time point and the replicon is made at a later time point. For example, after its preparation, the RdRp expression unit can be stored for use at a later time point. The present invention provides increased flexibility compared to cis-replicons. When a new specific genome editing target locus(s) emerges, a new genome editing can be designed by constructing a guide nucleic acid that directs genome editing to the new genome editing tool. Pre-prepared RdRp expression units can be retrieved from the storage room. In other words, RdRp expression units can be designed and produced independently of any particular replicon. This is because preparation of a replicon without RdRp requires less effort and resources than preparation of a cis-replicon, which is characterized by the selection of a new specific genome editing target locus(s) or at least one new genome editing target. Enables rapid response to the emergence of specific genome editing target locus(s).

Second, trans-replicons according to the present invention are typically shorter nucleic acid molecules than typical cis-replicons. This allows faster cloning of replicons encoding genome editing tools, eg, genome editing tools, and provides higher yields of genome editing tools.

Preferably, the replicon can be replicated by alphavirus RdRp from Semliki Forest Virus, including naturally occurring Semliki Forest Virus and variants or derivatives of Semliki Forest Virus, such as attenuated Semliki Forest Virus.

Also preferably, the replicon can be replicated by alphavirus RdRp from Sindbis virus, including naturally occurring Sindbis virus and variants or derivatives of Sindbis virus, such as attenuated Sindbis virus. In another preferred embodiment, the replicon can be replicated by alphavirus RdRp from Venezuelan nephritic virus (VEEV), including naturally occurring VEEV and variants or derivatives of VEEV, such as attenuated VEEV.

An RNA replicon of interest according to the present invention is preferably a single-stranded RNA molecule. Generally, a single-stranded encoding nucleic acid molecule contains twice as much genetic information per unit of weight (eg μg) of nucleic acid material. Thus, the single-stranded nature represents an additional advantage compared to, for example, the double-stranded prior art DNA vectors employed by the present invention. Replicons according to the present invention are typically positive-stranded RNA molecules.

An RNA replicon of interest is an isolated nucleic acid molecule.

The trans-replication system of the present invention is suitable for expression of genome editing tools in the introduction of cells of a subject. In the present invention, the trans-replicon RNA contains a locus encoding a reporter protein (e.g., a fluorescent protein such as GFP or eGFP) as a genome editing tool, and the expression efficiency of each genome editing tool is easily determined. let it do The trans-replication system comprising two RNAs is significantly superior in expression efficiency of the target RNA compared to cis-replication.

The trans-replication system of the present invention is suitable for efficient expression, eg high level expression, of genome editing tools in humans or animals.

For example, the approximately 7400 nucleotides encoding RdRp in a typical alphavirus found in nature is burdensome to the cell as amplification of the full-length replicon would be required. A major part of this burden is removed if the non-replicating RdRp expression unit is used for RdRp expression, eg in the form of mRNA. This allows the use of trans-replicons for RNA amplification and transfer RNA expression. If the RdRp expression unit does not replicate in the cell (i.e., the only foreign construct replicated is the trans-replicon of the present invention), waste of cellular energy and resources (such as nucleotides) is avoided, which may explain the superior expression level. there is. Also, short replicon RNAs require less time for RNA synthesis. Thus, it can be considered that savings in time, cellular energy and/or resources can contribute to higher levels of replication.

Replicon conserved sequence

A replicon is, comprises, or is derived from alphaviral genomic RNA. A replicon according to the present invention contains one or more Conserved Sequence Configurations (CSEs). In particular, the replicon may include one or more conserved sequence constructs (CSEs) of alphaviruses found in nature or variants or derivatives thereof.

- CSE 1, which is believed to function as a promoter for synthesis of the (+) strand from the (-) strand template. If present, the CSE 1 is typically located at or near the 5' end of the replicon RNA.

- CSE 2, which is believed to act as a promoter or enhancer for the synthesis of the negative strand from the genomic (+) strand RNA template. If present, CSE 2 is typically located downstream of CSE 1 but upstream of CSE 3 .

- CSE 3, believed to contribute to efficient transcription of subgenomic RNA; If present, CSE 3 is typically located upstream of the coding sequence of the RNA of interest (if present), but downstream of CSE 2.

- CSE 4, which is believed to function as a core promoter for initiation of negative strand synthesis. If present, CSE 4 is typically located downstream of the coding sequence for the RNA of interest (if present). In any case, CSE 4 is typically downstream of CSE 3. Sequence details of CSE 4 (also referred to as 3' CSE) in various alphaviruses are described in the literature, and in the present invention, the sequence of CSE 4 can be selected, for example, according to the teachings of that document.

The RNA replicon of interest according to the present invention

(1) Alphavirus 5' replication recognition sequence:

(2) RNA of interest; and

(3) It contains the alphavirus 3' replication recognition sequence.

The alphavirus 5' replication recognition sequence includes alphavirus CSE 1 and/or CSE 2. In naturally occurring alphaviruses, CSE 1 and/or CSE 2 are typically included in the 5' replication recognition sequence.

The alphavirus 3' replication recognition sequence includes alphavirus CSE 4. In naturally occurring alphaviruses, CSE 4 is typically included in the 3' replication recognition sequence.

The alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence can direct replication of the RNA replicon according to the present invention in the presence of RdRp. Thus, alone or preferably together, these recognition sequences direct replication of the RNA replicon in the presence of RdRp. Without wishing to be bound by theory, it is understood that alphavirus conserved sequence constructs (CSEs) 1, 2 and 4 (which are included in the recognition sequence) direct replication of the RNA replicon in the presence of RdRp. Thus, a replicon will typically contain CSEs 1, 2 and 4.

The RdRp encoded by the RdRp expression unit is preferably an alphavirus RdRp capable of recognizing both the alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence of the replicon.

This is achieved when the alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence are unique to the alphavirus that induces RdRp. Natural means that these sequences are alphaviruses of the same natural origin. In one embodiment, CSE 1, CSE 2 and CSE 4 are unique to the alphavirus from which RdRp is induced.

If the alphavirus RdRp can recognize both the 5' duplicated recognition sequence (and/or CSE 1 and/or CSE 2) and the 3' duplicated recognition sequence (and/or CSE 4) of the replicon, the 5' duplicated recognition sequence (and/or CSE 1 and/or CSE 2) and/or alphavirus 3' replication recognition sequence (and/or CSE 4) are not silent in alphaviruses that induce RdRp. In other words, the RdRp is compatible with the 5' duplicate recognition sequence (and/or CSE 1 and/or CSE 2) and the 3' duplicate recognition sequence (and/or CSE 4). An RdRp is compatible (compatibility between viruses) if the non-native alphavirus RdRp can recognize each sequence or sequence element. Examples of interviral compatibility with non-native RdRp from different alphaviruses for (3'/5') replication recognition sequences and CSEs, respectively, are known in the art. As long as there is compatibility between viruses, each of the (3'/5') replication recognition sequence and CSE can be arbitrarily combined with the alphavirus RdRp.

Compatibility between viruses can be easily tested by those skilled in the art practicing the present invention by incubating the RdRp to be tested with RNA, which RNA is to be tested under conditions suitable for RNA replication, e. It has a 5' duplication recognition sequence. If duplication occurs, it is determined that the (3'/5') duplication recognition sequence and RdRp are compatible.

subgenomic promoter

The RNA replicon according to the present invention includes an expression control sequence. A typical expression control sequence is or comprises a promoter. An RNA replicon according to the present invention includes a subgenomic promoter. Preferably, the subgenomic promoter is an alphavirus subgenomic promoter. The nucleotide sequence of the subgenomic promoter is highly conserved among alphaviruses.

Preferably, the subgenomic promoter is a promoter for a structural protein of an alphavirus. This means that the subgenomic promoter is unique to the alphavirus and controls the transcription of one or more structural protein genes in the alphavirus.

It is preferred that the subgenomic promoter is compatible with the RdRp of the RdRp mRNA unit.

This is achieved when the subgenomic promoter is unique to the alphavirus that drives RdRp. Unique means that the natural origin of the subgenomic promoter and the natural origin of the RdRp are the same alphavirus.

The subgenomic promoter is not unique to the alphavirus inducing RdRp if the alphavirus RdRp can recognize the subgenomic promoter of the replicon. That is, RdRp is compatible with subgenomic promoters (compatibility between viruses). Combinations of any subgenomic promoter with RdRp are possible as long as compatibility between viruses exists. Compatibility between viruses can be readily tested by those skilled in the art practicing the present invention by incubating the RdRp being tested with an RNA, wherein the RNA has a subgenomic promoter to be tested under conditions suitable for RNA synthesis from the subgenomic promoter.

Once the subgenomic transcripts are prepared, the subgenomic promoter and RdRp are determined to be compatible. Various examples of compatibility between viruses are known: in some cases, non-native subgenomic promoters induce more efficient transcription than native subgenomic promoters.

Preferably, the replicon according to the present invention comprises conserved sequence configuration 3 (CSE 3) from alphavirus. CSE 3 is believed to contribute to efficient transcription of subgenomic RNA. Transcription of subgenomic RNA is known to occur very efficiently in the presence of CSE 3. Typically, CSE 3 is a polynucleotide stretch of about 24 nucleotides. In the alphavirus genome, CSE 3 is located in the junction region between the coding sequences of nonstructural and structural proteins. In the trans-replicon of the present invention, CSE 3 is usually upstream (5') of the open reading frame (ORF) under the control of a subgenomic promoter.

When the replicon according to the present invention includes one ORF under the control of a subgenomic promoter, CSE 3 is located 5' to the above-mentioned one ORF. If a replicon according to the present invention contains two or more ORFs under the control of a subgenomic promoter, CSE 3 may be located 5' to each such ORF. In one embodiment, CSE 3 is unique to the alphavirus that induces RdRp. CSE 3 is not unique to the alphavirus that induces RdRp if the alphavirus RdRp can recognize the CSE 3 of the replicon.

Random add-on of the Replicon

An RNA replicon according to the present invention comprises a 3' poly(A) sequence. The poly(A) sequence of alphavirus RNA plays an important role in the translational efficiency and RNA stability of viral non-structural proteins. In the present invention, the poly(A) sequence is believed to play an important role in the translation efficiency and replicon stability of the target RNA.

The 3' poly(A) sequence of the replicon according to the present invention may be selected from the 3' poly(A) sequence of the RdRp mRNA unit. In addition, the poly(A) sequence (if present on the replicon according to the present invention) is preceded by alphavirus conserved sequence configuration 4 (CSE 4). Preferably, it is directly adjacent to CSE 4, such that the most 3' nucleotide of CSE 4 is directly adjacent to the most 5' nucleotide of the poly(A) tail.

RNA replicons according to the present invention include a 5'-cap (including cap analogs). Cap (or cap derivative) is symbolized by the letter C. The aspect of the cap (or analogue) of the replicon according to the present invention is the cap (or analogue) of the RdRp expression unit in the present invention, except that the cap must necessarily be present in the case of the RNA replicon. The aspects described for are the same. Regardless of the fact that an RNA replicon is schematically depicted in another embodiment comprising a cap denoted by the letter C, it is equally encompassed by the present invention.

The coding sequence (if present) of the open reading frame(s) present on the replicon according to the present invention is adapted. Embodiments and preferred embodiments of codon alteration of replicons can be selected from among those disclosed herein for the case of codon alteration of RdRp expression units. The codons of the open reading frame(s) may be suitable for expression in a cell of interest or organism of interest.

heterologous nucleic acid sequence

The replicon further comprises at least one nucleic acid sequence that is not of viral origin, in particular a nucleic acid sequence that is not of alphavirus. In a preferred embodiment, an RNA replicon according to the present invention comprises a heterologous nucleic acid sequence.

The term “heterologous” refers to situations in which a nucleic acid sequence is not naturally functionally linked to an alphavirus nucleic acid sequence, such as an alphavirus expression control sequence, particularly an alphavirus subgenomic promoter. A heterologous nucleic acid sequence is included in the nucleic acid sequence of the replicon.

Preferably, the heterologous nucleic acid sequence is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter, more preferably, the heterologous nucleic acid sequence is located downstream of a subgenomic promoter. Alphavirus subgenomic promoters are highly efficient and are suitable for high-level heterologous gene expression. Preferably, a subgenomic promoter controls the production of subgenomic RNA comprising a transcript of a heterologous nucleic acid sequence or portion thereof.

Preferably, the subgenomic promoter is a promoter for a structural protein of an alphavirus. This means that a subgenomic promoter is a promoter that is unique to an alphavirus and controls transcription of the coding sequence of one or more structural proteins in said alphavirus. According to the present invention, a heterologous nucleic acid sequence may partially or completely replace a viral nucleic acid sequence, such as a nucleic acid sequence encoding an alphaviral structural protein. Preferably, the heterologous nucleic acid sequence is not derived from an alphavirus; In particular, it is preferred that the heterologous nucleic acid sequence is not derived from the same alphavirus as the alphavirus from which the subgenomic promoter was derived. In one embodiment, the replicon comprises CSE 3 and the heterologous nucleic acid sequence is heterologous to CSE 3.

V. Pseudouridine Formation

Pseudouridine formation after transcription in cells is performed by pseudouridine synthases, and related enzyme groups are largely classified into two types.

The best-known taxa are guide RNA-dependent enzymes called H/ACA sRNPs (small ribonucleoproteins) that use a guide RNA complementary to the sequence to be modified and a protein (Cbf5 in yeast, diskerin in humans) for target selection. H/ACA sRNPs are found in eukaryotes and archaea but not prokaryotes and modify many uridines, all of which enzymatically construct by multiple guide RNAs. Pseudouridines found on yeast ribosomes are all modified by H/ACA sRNPs found on the nucleolus, hence the term small nucleolar RNPs (snoRNPs). H/ACA sRNPs are also involved in the modification of U2 snRNA and mRNA, but also play a role in other cellular processes such as processing of pre-rRNA and stabilization of the mammalian telomerase RNA component (TERC).

Another taxon that undergoes pseudouridine formation is a guide-independent mechanism that acts via stand-alone enzymes. In eukaryotic cells, these enzymes are Pus (Pseudo Uridine Synthases) enzymes, which recognize sequences and/or RNA secondary structure elements containing the target. and correct it These enzymes classify pseudouridine synthase into six families (TruA, TruB, TruD, RsuA, RluA and Pus10) on the basis of bacteria. However, RsuA-type pseudouridine synthase has so far been found only in bacteria, and Pus10 enzymes exist only in a few archaea and eukaryotes.

Importantly, all pseudouridine synthetases, including Cbf5, share a conserved catalytic domain and a conserved catalytic mechanism based on a strictly conserved catalytic aspartate residue. Despite sequence diversification among pseudouridine synthetases, the conserved structure implies a common evolutionary origin for all these enzymes. In general, pseudouridylation involves cleavage of the NC glycosidic bond, rotation of the uracil ring and reattachment to the ribose sugar without requiring ATP for catalysis. The catalytic mechanism of pseudouridine synthetase occurs through deprotonation of the ribose sugar by a catalytic aspartate moiety, resulting in the cleavage of base and glycol intermediates.

While many structures have been studied for prokaryotic and archaeal pseudouridine synthase, the structures studied to date for eukaryotic Pus enzymes are those of the catalytic domain of human Pus1 and human Pus10. Pus1 and Pus10 have a fold similar to conserved catalytic domains found in prokaryotic enzymes, with a central antiparallel β-sheet flanked by helices and rings.

Eukaryotes have 10 different types of Pus enzymes, called Pus1 through Pus10, and some organisms have several analogues of a given Pus enzyme. There are nine types of Pus in S. cerevisiae, and each Pus enzyme has several specific or sometimes multi-target uridines in the cell and can pseudouridylate in the nucleus, cytoplasm or mitochondria. Besides pseudouridins in cytoplasmic and mitochondrial tRNAs, Pus enzymes also modify several small nuclear RNAs (snRNAs) and mitochondrial rRNAs. The Pus10 enzyme, which is not found in yeast and has been studied only in archaea, is responsible for the formation of pseudouridin from tRNA. In particular, pseudouridins of eukaryotic tRNAs are (also) produced by Pus4.

First, eukaryotic independent pseudouridine synthase belongs to four families of E. coli pseudouridine synthase. In yeast, the TruA *?**?* family, which includes members Pus1, Pus2 and Pus3, is responsible for modification at numerous sites on tRNAs as well as some sites on snRNAs and mRNAs. U55 of the highly conserved tRNA is modified by Pus4, the only member of the TruB family, which also modifies the mRNA. The RluA enzyme family is the largest group, including Pus5, Pus6, Pus8 and Pus9, which pseudouridine uridines in tRNA, mitochondrial 21S rRNA and mRNA. RluA enzymes sometimes contain an N-terminal domain similar to the ribosome building protein S4. Pus7 is the only member of the TruD family and modifies sites on tRNA, rRNA and snRNA.

VI. RNA of interest (RNAi)

RNA of interest (RNAi) of the present invention is mRNA (messenger RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), miRNA (micro RNA), siRNA (small interfering RNA), shRNA, trans-activating crispr RNA, Circular It may be any one or more selected from the group containing RNA, ribozymes and RNA aptamers.

An RNA unit of interest may be prepared using a plurality of RNAs selected above.

mRNA (messenger RNA messenger RNA) serves as a kind of blueprint that transfers the genetic information of DNA. Based on this, ribosomes synthesize proteins. It is divided into three parts: (Poly) cap, Polyadenyl, and translation sequence (actually translated part). rRNA (ribosomal RNA ribosomal RNA) is RNA that makes up ribosomes. tRNA (transfer RNA transfer RNA) has an anticodon corresponding to the codon of mRNA, and an amino acid corresponding to the anticodon is attached to the tail side by an enzyme that connects the tRNA and a specific amino acid according to the corresponding anticodon. miRNA (micro RNA micro RNA) is a small RNA that plays a role in controlling gene expression in organisms.

snRNA (small nuclear RNA) has the function of RNA splicing in the nucleus. snoRNA (small nucleolar RNA) causes RNA modifications in the nucleus. CircRNA (circular RNA) is generated through back-splicing from precursor mRNAs of thousands of eukaryotic genes. Although the expression level of circRNA is generally maintained at a low level, there is a specific expression level depending on the cell type and tissue. In addition, circRNAs contribute to the complexity and diversity of eukaryotic transcriptomes by regulating the expression of other genes by interacting with miRNAs (microRNAs) that regulate splicing and transcription processes. Trans-activating crispr RNAs are CRISPR systems that contain one or more guide RNAs designed to bind an effector protein and a corresponding target molecule.

aRNA (antisense RNA antisense RNA) plays a regulatory role in translation. siRNA (small interfering RNA) induces RNA interference. It interferes with gene expression by inhibiting the production of certain proteins.

1. dsRNA

Preferably, the present invention relates to double-stranded RNA (dsRNA) and RNAi constructs. The term "dsRNA" as used herein refers to a double-stranded RNA molecule capable of RNA interference (RNAi), including siRNA (see, eg, Bass, Nature 411: 428-429, 2001; Elbashir et al., Nature 411: 494-498, 2001; Kreutzer et al., PCT Publication WO 00/44895; Zernicka-Goetz et al., PCT Publication WO 01/36646; Fire, PCT Publication WO 99/32619; Plaetinck et al., PCT Publication WO 00/01846; Mello and Fire, PCT Publication WO 01/29058; Deschamps-Depaillette, PCT Publication WO 99/07409; and Li et al., PCT Publication No. WO 99/07409; see WO 00/44914). RNAi is also a term initially applied to a phenomenon observed in plants and worms in which double-stranded RNA (dsRNA) blocks gene expression in a specific post-transcriptional manner. RNAi provides a useful method for inhibiting or reducing gene expression in vitro or in vivo.

The terms "single-chain interfering RNA", "siRNA" or "single-chain interfering nucleic acid" as used herein refers to any nucleic acid capable of mediating RNAi or gene silencing when properly processed by cells. For example, the siRNA can be a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises complementarity to a target gene. The siRNA may be a single-stranded hairpin polynucleotide having a self-complementary sense region and an antisense region, wherein the antisense region includes complementarity to a target gene. The siRNA may be a circular single-stranded polynucleotide having a stem and two or more loop structures including a self-complementary sense region and an antisense region, wherein the antisense region includes complementarity to a target gene, and the circular polynucleotide Nucleotides can be engineered in vivo or in vitro to generate active siRNAs capable of mediating RNAi. The siRNA may also comprise a single-stranded polynucleotide having complementarity to a target gene, wherein the single-stranded polynucleotide has a terminal phosphate group, such as a 5'-phosphate (e.g., Martinez et al., Cell 110: 563-574, 2002], or may further include 5',3'-diphosphate. In certain embodiments, the siRNA is a non-enzymatic nucleic acid that binds to and alters the activity of the target nucleic acid. Binding and/or activity of the siRNA may be facilitated by interaction with one or more proteins or protein complexes, such as the RNA Induced Silencing Complex or RISC. In certain embodiments, the siRNA comprises a sequence complementary to the target sequence along one contiguous sequence of one strand of the siRNA molecule.

Optionally, a siRNA of the invention comprises a nucleotide sequence that hybridizes under physiological conditions (eg, in an intracellular environment) to a nucleotide sequence of at least a portion of an mRNA transcript for a gene to be inhibited (a "target" gene). include A double-stranded RNA only needs to be sufficiently similar to native RNA that it has the ability to mediate RNAi.

Therefore, the present invention has the advantage of being able to allow predictable sequence variation due to genetic mutation, strain polymorphism or evolutionary diversification. The number of tolerated nucleotide mismatches between the target sequence and the siRNA sequence is less than or equal to 1 base pair in 5 base pairs, or 1 base pair in 10 base pairs, or 1 base pair in 20 base pairs, or 1 base pair in 50 base pairs.

Mismatches in the center of the siRNA duplex are of paramount importance and can substantially disrupt cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that are complementary to the target RNA do not significantly contribute to the specificity of target recognition. Sequence identity can be determined using sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 and references cited therein) and, for example, the BESTFIT software program using default parameters. It can be optimized by calculation of the percent difference between nucleotide sequences by the Smith-Waterman algorithm (eg University of Wisconsin Genetic Computing Group) run on . Greater than 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, or even 100% sequence identity, between the siRNA and a portion of the target gene is preferred. Alternatively, the double-stranded region of the RNA is subjected to stringent conditions (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, hybridization at 50°C or 70°C for 12-16 hours followed by washing). It can be functionally defined as a nucleotide sequence capable of hybridizing with a portion of a target gene transcript.

The double-stranded structure of a dsRNA can be formed by one self-complementary RNA strand, two complementary RNA strands, or a DNA strand and a complementary RNA strand. Optionally, RNA double-strand formation can be initiated intracellularly or extracellularly.

RNA can be introduced in an amount capable of delivering more than one copy per cell. Higher doses (eg, at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may exert more effective inhibition, while lower doses may also be useful for certain applications. Inhibition is sequence specific in that the nucleotide sequence corresponding to the double-stranded region of RNA is targeted for inhibition.

When used in the present invention, the siRNA of the present invention is about 19-30 nucleotides in length, about 21-27 nucleotides in length, about 21-25 nucleotides in length, or about 21-23 nucleotides in length. contains the area It is understood that siRNAs recruit nuclease complexes to pair with specific sequences, thereby guiding the complexes to target gene transcripts. As a result, the target gene transcript is degraded by nucleases in the protein complex. In certain embodiments, the siRNA molecule includes a 3' hydroxyl group. In certain embodiments, the siRNA constructs can be generated by processing longer double-stranded RNA in situ, eg, in the presence of the enzyme dicer. In one embodiment, the Drosophila in vitro system is used. In this embodiment, dsRNA is combined with a soluble extract from Drosophila embryos to form a blend. The combination is maintained under conditions in which the dsRNA is processed into RNA molecules of about 21 to about 27 nucleotides. The siRNA molecule can be purified using a number of techniques known to those skilled in the art. For example, gel electrophoresis can be used to purify siRNA. Alternatively, non-denaturing methods such as non-denaturing column chromatography can be used to purify siRNA. In addition, chromatography (eg, size exclusion chromatography), glycerol gradient centrifugation, affinity purification using antibodies can be used to purify siRNA.

The dsRNA (eg, siRNA) of the present invention can be produced by chemical synthesis or recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell can mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. When used in the present invention, the dsRNA or siRNA molecules of the present invention need not be limited to molecules comprising only RNA, and further include chemically modified nucleotides and non-nucleotides. For example, the dsRNA may, for example, reduce susceptibility to intracellular nucleases, improve bioavailability, improve formulation properties, and/or alter other pharmacokinetic properties. To achieve this, modifications to the phosphate-sugar backbone or nucleoside may be included. For example, the phosphodiester bonds of native RNA can be modified to include one or more of nitrogen or sulfur heteroatoms. Modifications in RNA structure can be tailored to allow specific genetic inhibition while avoiding the general response to dsRNA. Similarly, bases can be modified to block the activity of adenosine deaminase. The dsRNA can be produced enzymatically or by partially/completely organic synthesis, and any modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. Methods of chemical modification of RNA molecules can be adapted to suit modification of dsRNA (see, e.g., Heidenreich et al. Nucleic Acids Res. 25:776-780, 1997; Wilson et al., J Mol Recog.7:89-98, 1994] Chen et al., Nucleic Acids Res. , 1997]). For example, the backbone of a dsRNA or siRNA can be a phosphorothioate, phosphoramidate, phosphodithioate, chimeric methylphosphonate-phosphodiester, peptide nucleic acid, 5-propynyl-pyrimidine containing oligomer or sugar modification. (eg, 2'-substituted ribonucleosides, a-configuration). In certain cases, the dsRNA of the present invention lacks 2'-hydroxy (2'-OH) containing nucleotides. In certain embodiments, the siRNA molecule comprises a phosphorothioate sense strand. In certain embodiments, the siRNA molecule comprises a phosphodiester antisense strand.

In certain embodiments, at least one strand of the siRNA molecule is about 1 to about 10 nucleotides in length, about 1 to about 5 nucleotides in length, about 1 to 3 nucleotides in length, or about 2 to 4 nucleotides in length. It has a 3' overhang of nucleotides in length. In certain embodiments, the siRNA may comprise one strand with a 3' overhang and the other strand with a blunt end at the 3' end (eg, no 3' overhang). In another embodiment, the siRNA may include 3' overhangs on both strands. The length of the protrusions may be the same or different for each strand. To further enhance the stability of the siRNA, the 3' overhang can be stabilized from degradation. In one embodiment, the RNA is stabilized by including purine nucleotides such as adenosine or guanosine nucleotides. Alternatively, substitution by modified analogs of pyrimidine nucleotides, eg, substitution of uridine nucleotide 3' overhangs by 2'-deoxytinidine, is permissible and does not affect the efficiency of RNAi. The absence of the 2' hydroxyl significantly enhances the nuclease resistance of the overhangs in tissue culture media and can be beneficial in vivo.

In another specific embodiment, the dsRNA of the invention may also be in the form of long double-stranded RNA. For example, a dsRNA is at least 25, 50, 100, 200, 300 or 400 bases. In some cases, the dsRNA is 400-800 bases in length. Optionally, the dsRNA is degraded within the cell, eg to generate siRNA sequences within the cell.

However, the use of long double-stranded RNA in vivo is not always practical, presumably because of the detrimental effects that can be caused by sequence-independent dsRNA reactions. In such embodiments, the use of topical delivery systems and/or agents that reduce the effect of interferon or PKR is preferred.

[0062] In a further specific embodiment, the dsRNA or siRNA is in the form of a hairpin structure (or hairpin RNA). The hairpin RNA can be exogenously synthesized or formed in vivo by transcription from an RNA polymerase III promoter. Examples of the preparation and use of such hairpin RNAs for gene silencing in mammalian cells are described, eg, in Paddison et al., Genes Dev. 16:948-58, 2002]; McCaffrey et al., Nature 418:38-9, 2002; McManus et al., RNA 8:842-50, 2002; See Yu et al., Proc. Natl. Acad. Sci. U. S. A. 99:6047-6052, 2002. Preferably, these hairpin RNAs are engineered in cells or animals to ensure continuous and stable repression of target genes. It is known in the art that siRNAs can be generated by processing of hairpin RNAs within cells.

PCT application WO 01/77350 describes an exemplary vector for bidirectional transcription of a transgene such that both sense RNA transcripts and antisense RNA transcripts of the same transgene are produced in eukaryotic cells. Accordingly, in certain embodiments, the present invention provides a recombinant vector having the following unique properties: It is a viral replica having two overlapping transcription units arranged in opposite directions and flanking the transgene for the dsRNA of interest. A cone, wherein the two overlapping transcription units generate both sense RNA transcripts and antisense RNA transcripts from the same transgene fragment in the host cell.

Ribozymes are catalytic RNAs that exchange genes only in the presence of special small molecules (ACS Chem. Biol. 2014, DOI: 10.1021/cb500567v). The splicing of genes can be controlled through ribozymes that can turn themselves off and on. There are group 1 introns, which are large ribozymes capable of splicing RNA from themselves. Trans splicing ribozymes replace genes at the ends of their genes and exchange them with the target gene they are targeting.

2. mRNA

The mRNAi of the present invention may be any one selected from the group consisting of antigens, antibodies, therapeutic proteins, peptides, and fusion proteins. An mRNAi contains at least one open reading frame that encodes a therapeutic protein or peptide. Antigens such as pathogenic antigens, oncogenic antigens, allergenic antigens or autoimmune antigens are encoded by at least one open reading frame. Therein, administration of mRNAi encoding an antigen is used in a genetic vaccination approach against diseases involving the antigen. An antibody is encoded by at least one open reading frame of an mRNAi according to the present invention.

mRNAi according to the present invention do not contain a reporter gene or a marker gene. Preferably, the mRNAi according to the present invention is selected from, for example, luciferase; green fluorescent protein (GFP) and its variants (eg eGFP, mRNAi or BFP); α-globin; hypoxanthine-guanine phosphoribosyltransferase (HGPRT); β-galactosidase; galactokinase; alkaline phosphatase; It does not encode secreted embryonic alkaline phosphatase (SEAP)) or resistance genes (eg neomycin, puromycin, hygromycin and zeocin). mRNAi according to the present invention do not encode luciferase. mRNAi according to the present invention do not encode GFP or variants thereof.

<Fusion protein>

A fusion protein is a protein composed of at least two or more domains encoded by separate genes that are combined to produce a single polypeptide that is transcribed and translated as a single unit. There are basically two types of fusion proteins. The first consists of two proteins or protein subunits fused end-to-end and usually connected by a linker, and the second has amino acids from two donors interspersed in the fusion protein.

The former is a heterologous protein polypeptide fused to a linker peptide; It may be any one selected from the group comprising; and a polypeptide in which a special purpose peptide and a protein are bound.

Fusion proteins can be classified according to the protein domains they have been incorporated into. Often, one fusion partner has molecular recognition functions while the other partner may have specific functions, such as improved stability and half-life, cytotoxic effects, or novel targeting and delivery pathways. Therapeutic fusion proteins can be immunotoxins, cytokine fusions, fragment crystallizable (Fc) fusions, albumin fusions and others not within these classes. Although most clinical trials of recombinant proteins currently in progress involve monoclonal antibodies, fusion proteins are expected to become important therapeutic molecules in the future.

<Pathogenic antigens>

The mRNAi according to the present invention may encode proteins or peptides including pathogenic antigens or fragments, variants or derivatives thereof. Such pathogenic antigens are derived from pathogenic organisms, in particular bacteria, viruses or protozoan (multicellular) pathogenic organisms that evoke an immune response in a subject, in particular a mammalian subject, more particularly a human. More specifically, preferably the pathogenic antigen is a surface antigen, eg a protein (or a fragment of a protein, eg an external portion of a surface antigen) located on the surface of a viral or bacterial or protozoan organism.

Pathogenic antigens are preferably selected from antigens derived from pathogens associated with infectious diseases. Particularly preferred antigens are influenza virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus aureus), Dengue virus, Chlamydia trachomatis, cytomegalovirus (CMV), hepatitis B virus, Mycobacterium tuberculosis, rabies virus and yellow fever virus am.

mRNAi according to the present invention encode rabies virus proteins or peptides or antigenic fragments thereof. Preferably, mRNAi according to the present invention is rabies virus glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein M (RAV-M) or RNA polymer Encodes an antigenic protein or peptide selected from the group consisting of lase L (RAV-L), or fragments, variants or derivatives thereof.

An mRNAi according to the present invention encodes a respiratory syncytial virus (RSV) protein or peptide or an antigenic fragment thereof. Preferably, SM_mRNAi according to the present invention is a respiratory syncytial virus (RSV) fusion protein F, glycoprotein G, short hydrophobic protein SH, matrix protein M, nucleoprotein N, large polymerase L, M2-1 protein, M2 -2 Encodes an antigenic protein or peptide selected from the group consisting of a protein, phosphoprotein P, non-structural protein NS1 or non-structural protein NS2, or a fragment, variant or derivative thereof.

<Tumor antigen>

The mRNAi according to the present invention may encode a tumor antigen, a peptide including a fragment, variant or derivative of the tumor antigen, or a protein or peptide including a protein, and is an antigen or a fragment, variant or derivative of the tumor antigen.

Cancer antigens of oncogenes (KRAS, p53, etc.) mutants, tumor antigens NY-iO-1, 5T4, MAGE-C1, MAGE-C2, Survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP are particularly preferred. The mRNAi to be administered encode proteins or peptides, including therapeutic proteins or fragments, variants or derivatives thereof.

Therapeutic proteins of the present invention are peptides or proteins that are beneficial in the treatment of any inherited or acquired disease or improve the condition of an individual. In particular, therapeutic proteins play a large role in the creation of therapeutics and, among other functions, can modify and repair genetic errors, destroy cancer cells or pathogen-infected cells, treat immune system disorders, and treat metabolic or endocrine disorders. For example, erythropoietin (EPO), a protein hormone, can be used to treat patients with red blood cell deficiency, a common cause of renal complications. Adjuvant proteins, therapeutic antibodies are also encompassed by therapeutic proteins and hormone replacement therapy, which can be used, for example, in the treatment of postmenopausal women. In a more recent approach, a patient's somatic cells are used to dedifferentiate into pluripotent stem cells replacing disputed stem cell therapy. In addition, these proteins used for dedifferentiation of somatic cells or for differentiation of stem cells are defined in the present invention as therapeutic proteins. In addition, therapeutic proteins may be used for other purposes, such as wound healing, tissue regeneration, and angiogenesis.

Thus, therapeutic proteins may be used, for example, in infectious diseases, neoplasms (eg cancer or tumor diseases), diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, respiratory system diseases, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system (if independently inherited or acquired).

Particularly preferred therapeutic proteins that can be used in the treatment of metabolic or endocrine disorders, among others, are intended to treat a subject by replacing their insufficient endogenous production of a functional protein with a sufficient amount, and are therefore understood as therapeutic proteins. Accordingly, such therapeutic proteins are generally mammalian, particularly human proteins. Therapeutic proteins can be used for the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, cancer or neoplastic diseases, infectious diseases or immunodeficiencies:

Furthermore, adjuvants or immune stimulatory proteins are also included in the term therapeutic protein. An adjuvant or immune stimulatory protein can be used to treat a particular disease or alleviate a condition in a subject by inducing, modifying or improving the immune response in the subject. An adjuvant protein is a mammalian, in particular human adjuvant protein, which generally includes any human protein or peptide capable of eliciting an innate immune response (in mammals), for example, as a binding response of a foreign TLR ligand to the TLR. can be selected from. It is selected from the group consisting of proteins such as The mammalian, particularly human adjuvant protein may also be selected from the group consisting of a heat shock protein, or any species homolog of any of such human adjuvant proteins. Mammalian, particularly human adjuvant proteins may also include proteins that induce or enhance the innate immune response.

<Therapeutic protein>

Therapeutic proteins or adjuvant proteins for the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, cancer or tumor diseases, infectious diseases or immunodeficiencies are generally proteins of mammalian origin, preferably of human origin, depending on the animal to be treated. A human subject is preferably treated with a therapeutic protein of human origin, for example.

Pathogenic adjuvant proteins generally include pathogenic adjuvant proteins capable of inducing an innate immune response (in mammals), more preferably pathogenic adjuvant proteins derived from bacteria, protozoa, viruses or fungi; For example, a bacterial (adjuvant) protein, a protozoan (adjuvant) protein (e.g., a propylin-like protein of Toxoplasma gondii), a viral (adjuvant) protein, or a fungal (adjuvant) and pathogenic adjuvant proteins selected from proteins and the like.

Bacterial (adjuvant) proteins may also include bacterial flagellin. Protozoan (adjuvant) proteins are further examples of pathogenic adjuvant proteins. Protozoan (adjuvant) proteins can be selected from any protozoan protein that exhibits adjuvant properties, and viral (adjuvant) proteins are another example of pathogenic adjuvant proteins. The viral (adjuvant) protein can be selected from any viral protein exhibiting adjuvant properties, more preferably, but not limited to, a Respiratory Syncytial Virus Fusion Glycoprotein (F-protein) , MMT virus-derived envelope protein, mouse leukemia virus protein, wild-type measles virus hemagglutinin protein, and the like. Fungal (adjuvant) proteins are further examples of pathogenic adjuvant proteins. In the present invention, the fungal (adjuvant) protein may be selected from any fungal protein exhibiting adjuvant properties, more preferably consisting of fungal immunomodulatory protein (FIP; LZ-8) and the like. can be selected from the group. Finally, the adjuvant protein may further be selected from the group consisting of keyhole-limpet hemocyanin (KLH), OspA and the like.

Therapeutic proteins may be used in hormone replacement therapy, particularly in the treatment of postmenopausal women. These therapeutic proteins are preferably selected from oitrogens, progesterone or progitins, and often testosterone.

Moreover, therapeutic proteins can be used to dedifferentiate somatic cells into pluripotent or omnipotent stem cells. For this purpose, several factors, in particular Oct-3/4, Sox gene family (Sox1, Sox2, Sox3 and Sox15), Klf family (Klf1, Klf2, Klf4 and Klf5), Myc family (c-myc, L-myc and N-myc), Nanog and LIN28 are described.

A therapeutic antibody is also defined herein as a therapeutic protein. These therapeutic antibodies include, inter alia, antibodies used in the treatment of cancer or neoplastic diseases, inter alia antibodies used in the treatment of hematological disorders, inter alia antibodies used in immunomodulation, antibodies used in the treatment of diabetes, antibodies used in the treatment of Alzheimer's disease, among others. antibodies, antibodies used to treat asthma,

The coding region of an mRNAi according to the present invention may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA carrying the coding sequence of one, two or more proteins or peptides. Coding sequences in such di-, or even multicistronic RNAs direct cleavage of polypeptides, including, for example, at least one internal ribosome entry site (IRES) or several proteins or peptides as described herein. can be separated by a signal peptide that

3. Aptamers

Aptamers are specific ligands of target compounds or molecules that have high specific binding to target molecules. Aptamers are nucleic acid molecules that exhibit a high degree of specific binding affinity to molecules through interactions other than classical Watson-Crick base pairing. An aptamer is capable of modulating the activity of a target by specifically binding to a selected target, and for example, it is possible to block the function of a target through aptamer binding. Aptamers corresponding to proteins including growth factors, transcription factors, enzymes, immunoglobulins and receptors have been generated by an in vitro selection process from a pool of random sequence oligonucleotides.

A typical aptamer is 10-15 kDa (30-45 nucleotides) in size, binds to its target with sub-nanomolar affinity, and differentiates against closely related targets (e.g., aptamers are typically of the same gene family). does not bind to other proteins belonging to).

As an aptamer selection method, the "SELEX™" (Systematic Evolution of Ligands by Exponential Enrichment) method is widely used. The SELEX method is a method for in vitro development of nucleic acid molecules with high specific binding to target molecules. U.S. Patent Application Serial No. 07/536,428 filed on June 11, 1990 (now abandoned), U.S. Patent No. 5,475,096 (titled "Nucleic Acid Ligands") and U.S. Patent No. 5,270,163 (titled "Nucleic Acid Ligands"). Nucleic Acid Ligands") and the like.

The SELEX method requires nucleic acids to have sufficient ability to form a variety of two-dimensional and three-dimensional structures and to act as ligands (i.e., form specific binding pairs) with virtually any chemical compound (whether monomeric or polymeric). It is based on having a chemical versatility available in the monomer that is sufficient for Molecules of any size or composition can be used as targets.

A series of structural studies have shown that aptamers have the same type of binding interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contact, steric exclusion) conferring affinity and highly selective binding in antibody-antigen complexes. was found to be available. Aptamers possess a number of desirable characteristics for use as therapeutic and diagnostic agents, including high selectivity and affinity, biological potency and excellent pharmacokinetic properties. In addition, aptamers have specific competitive advantages over antibodies and other biological proteins, as exemplified below.

4. Ribozyme

As used herein, the term "ribozyme" is a molecule composed of an RNA molecule that acts like an enzyme or a protein containing the RNA molecule, and is also called an RNA enzyme or catalytic RNA. An RNA molecule with a clear tertiary structure that performs chemical reactions and has catalytic or autocatalytic properties. Some ribozymes inhibit their activity by cleaving their own or other RNA molecules, and other ribozymes are known to catalyze the activity of ribosome aminotransferases.

Such ribozymes may include hammerhead ribozymes, VS ribozymes, and hairpin ribozymes.

The ribozyme according to the present invention not only exhibits a selective anticancer effect by inhibiting the activity of a cancer-specific gene through a trans-splicing reaction, but also activates a cancer therapeutic gene by being expressed in a conjugated form with a cancer therapeutic gene. there is. Therefore, it exhibits properties capable of inactivating cancer-specific genes and activating cancer therapeutic genes.

VII. RNA of interest production and purification

The RNA of interest can be produced and purified using the thus obtained host cell containing the RNA transcription system of interest.

The method is a method for producing the RNA of interest, which includes culturing the host cell and collecting the transcribed RNA of interest. By culturing the host cells in the medium, the RNA of interest can be transcribed and accumulated in the host cells. That is, specifically, the method is a method for producing the RNA of interest, comprising the steps of culturing the host cell in a medium, transcribing the RNA of interest to accumulate the RNA of interest in the host cell, and collecting the RNA of interest. way to do it

Bacteria, for example, can be cultured according to culture conditions commonly used for culturing bacteria such as bacteria. Bacteria can be cultured in a conventional medium containing, for example, a carbon source, a nitrogen source and inorganic ions. In addition, for example, organic micronutrients such as vitamins and amino acids may be added as needed.

As the carbon source, for example, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohol, and the like can be used. As a nitrogen source, ammonia gas, ammonia water, ammonium salt, etc. can be used, for example. As the inorganic ion, for example, a calcium ion, magnesium ion, phosphate ion, potassium ion, iron ion, etc. can be appropriately used as needed. Culturing may be performed for 10 to 120 hours under aerobic conditions within an appropriate range of pH 5.0 to 8.5 and 15 to 37 °C. In addition, culture conditions for L-amino acid production using bacteria such as bacteria and culture conditions for secretion production methods of proteins using bacteria such as bacteria may be referenced (WO01/23591, WO2005/103278, WO2013/065869, WO2013/065772, WO2013/118544, WO2013/062029, etc.). In addition, expression of the RNA of interest can be appropriately induced by using an inducible promoter for expression of the RNA of interest.

By culturing the bacteria under these conditions, the RNA of interest is transcribed and accumulated in the cells of the bacteria.

The expression and accumulation of the RNA of interest can be confirmed, for example, by subjecting a fraction of a cell extract as a sample to electrophoresis and detecting a band corresponding to the molecular weight of the RNA of interest.

RNA of interest can be collected from cells by any suitable method used for isolation and purification of the compound. Examples of such methods include, for example, centrifugation, salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, affinity chromatography and electrophoresis. These methods may be used alone or may be used in appropriate combination. Specifically, for example, a supernatant can be obtained by disrupting cells with ultrasonic waves or the like and removing cells from the disrupted suspension by centrifugation or the like, and the RNA of interest can be collected from the supernatant. The ion exchange resin method etc. are mentioned. The collected RNA of interest may be a free compound, a salt thereof, or a mixture thereof. In addition, the RNA of interest collected can also be complexed with high molecular weight compounds such as proteins. That is, the term RNA of interest may refer to a free form of the RNA of interest, a salt thereof, a complex with a high molecular weight compound such as a protein, or a mixture thereof, unless otherwise specified. Examples of salts include, for example, ammonium salts and sodium salts.

The collected RNA of interest may include components such as, for example, bacterial cells, media components, water, and by-product metabolites of bacteria, in addition to the RNA of interest. RNA of interest can also be purified to the desired extent. The purity of the collected RNA of interest is, for example, 30% (w/w) or more, 50% (w/w) or more, 70% (w/w) or more, 80% (w/w) or 90% (w/w) or more. w) or more or 95% (w/w) or more.

Purification after in vitro transcription of mRNA can be achieved through a series of tangential flow filtration (TFF) steps, and can be set to a purity of 90% to 99.9% and a yield of 90% to 95%. TFF can be supplemented with purification techniques such as size exclusion, ion exchange, and poly dT-based affinity chromatography. From the viewpoint of removing the template DNA described above, plasmid DNA is easier to remove through chromatography in terms of size and charge ratio than the method using a PCR product. The possibility that in vitro transcription products of mRNA include double-stranded RNA (dsRNA) forms has been mentioned above, and dsRNA, which is similar to an intermediate during replication of RNA viruses, can promote type I IFN production. Therefore, the in vitro transcription product is purified in a way that can reduce the type I IFN immune response through a process using chromatography, and the purified mRNA can also increase the translation efficiency. The resulting mRNA is made into lipid nanoparticles (LNPs). The LNP process and formulation are independent of the RNA sequence and therefore can be used in common for mRNA vaccines for different applications.

The nature of mRNA technology is to enable rapid development of almost infinite combinations of sequences for vaccines that require different sequences. This requires selecting the optimal structure, formulation and process for further development. For example, for inactivated and attenuated virus vaccines or recombinant protein vaccine candidates, product-specific manufacturing processes must be developed, optimized, validated, and cGMP production approved, which inevitably takes considerable time. On the other hand, mRNA vaccines can be developed relatively quickly by converting only the sequence (of course, it is necessary to develop an E. coli strain that produces template DNA), and it is easy to modify or verify the entire process. In addition, the productivity expressed in terms of unit volume and unit time is significantly higher in the mRNA platform than in conventional systems. The mRNA vaccine production process can be operated at 1/2 to 1/3 the scale of the existing vaccine production process in terms of facility size, and requires only 1/20 to 1/35 of the initial cost from a capital investment point of view. Therefore, it has a strong advantage in that it can be constructed more quickly and easily, and it is possible with a small facility that can widely use single-use equipment. Establishing a process using disposable equipment has the advantage of shortening the design and development schedule of the process and quality control because it is easy to convert the production facility when producing a vaccine using a different sequence later. When a rapid response is required, such as Covid-19, a strategy to respond by producing a traditional type of vaccine can be established by relocating existing large-scale facilities, but if it is difficult to meet demand or negatively affects the production of other medicines, additional facilities construction is urgently needed. If the construction of such a conventional facility would take years and tens of billions of dollars in capital, a new platform technology such as mRNA could offer significant advantages in rapid response and manufacturing. The gains in time and resources available through the mRNA platform are expected to become more apparent in the future when this platform is fully developed.

VIII. Cell-derived vesicles containing the RNA of interest

Recently, studies have been reported that cell secretome contains various bioactive factors that control cell behavior. Since it contains 'extracellular vesicles', research on its components and functions is being actively conducted.

Cells release vesicles of various membrane types into the extracellular environment, and these releasing vesicles are commonly referred to as extracellular vesicles. Extracellular vesicles are also referred to as cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they are used separately from exosomes.

Exosomes are vesicles with a size of tens to hundreds of nanometers made of a double-phospholipid membrane identical to the structure of a cell membrane, and contain proteins, mRNAs, miRNAs, etc. called exosome cargos inside. It is known that the exosome cargo contains a wide range of signaling factors, and these signaling factors are cell type-specific and differently regulated depending on the environment of the secreting cell. Exosomes are intercellular signaling mediators secreted by cells, and various cell signals transmitted through them regulate cell behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. It is known. Exosomes contain specific genetic materials and bioactive factors depending on the nature and state of the derived cell. In the case of proliferating stem cell-derived exosomes, they regulate cell behaviors such as cell migration, proliferation, and differentiation, and reflect the characteristics of stem cells related to tissue regeneration.

Host cells comprising the 'RNA transcription system of interest' of the present invention can be divided into host cell groups including prokaryotic, yeast and plant cells with cell walls and animal cells without cell walls.

The 'cell-derived vesicle containing the RNA of interest' of the present invention is divided into inside and outside by a lipid bilayer composed of cell membrane components of the derived cell, and has cell membrane lipids, cell membrane proteins, nucleic acids, and cell components. And, it means that the size is smaller than the original cell, but is not limited thereto.

1. Host cell without a cell wall

The host cell containing the RNA transcription system of interest for preparing the cell-derived vesicle may be included without limitation as long as it is a nucleated cell, but preferably stem cells, acinar cells, myoepithelial cells, red blood cells, monocytes, dendritic cells, It may be at least one selected from the group consisting of natural killer cells and platelets. The stem cells may be any one or more selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells and salivary gland stem cells, more preferably mesenchymal stem cells, still more preferably the mesenchymal stem cells It may be mesenchymal stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood.

The cell may be a mammalian nucleated cell or a transformed cell thereof, but any cell capable of producing an artificial vesicle through the manufacturing apparatus may be used. The transformed cells include, but are not limited to, cells transformed to express specific proteins, target-inducing substances, or substances required for cell membrane fusion with target cells, and transformed cells composed of a combination of two or more thereof. It is not.

Vesicles are a type of organelle with a size of 0.03 to 1 μm, and are naturally separated from cell membranes in almost all types of cells, and have a double phospholipid membrane structure, mRNA , contains intracellular components such as DNA and proteins.

These vesicles are basic tools of cells for metabolism, transport of metabolites, storage of enzymes, chemical reactions, and the like, and play a mediating role in signal transduction between cells by transferring mRNA, miRNA, and proteins between cells. For example, vesicles derived from cancer cells induce changes in normal cells by delivering cancer proteins or miRNAs to surrounding normal cells. In addition, since it is directly released from the cell membrane and retains the antigenic body of the parent cell, it is also used in research for vaccine development. In addition to this, vesicles play a role in removing misfolded proteins, cytotoxic substances, and by-products generated by intracellular metabolism.

Focusing on the cell-to-cell signaling function of vesicles, development of technologies for delivering drugs or biomaterials to cells using artificial double phospholipid membranes is being actively conducted. In the case of such artificial vesicles, delivery efficiency is high and delivery It is widely used in biological experiments because of the advantage of protecting the material to be tested from the external environment.

However, phospholipids conventionally used in the manufacture of artificial vesicles are extracted and purified from soybeans or eggs, and artificial vesicles prepared using these phospholipids do not contain membrane proteins, so they are not suitable for inducing cell fusion. there is. In addition, since surfactants and organic solvents used at this time are toxic, a process of removing the organic solvent is essential. Therefore, there is a need for a method for manufacturing artificial vesicles that is more friendly to cell experiments.

Cell-derived vesicles refer to vesicles artificially produced in nucleated cells, and are separated from cell membranes in almost all types of cells and have a double phospholipid membrane structure, which is the structure of cell membranes. The cell-derived vesicles of the present invention may have a micrometer size, for example, 0.03 to 1 μm.

Cell-derived vesicles are distinguished from naturally secreted vesicles, and may be prepared by extruding a sample containing cells into micropores, preferably having a large micropore size and a small micropore size. It may be produced by sequentially extruding, preferably a membrane filter having a micropore size of 9 to 11 μm, 2 to 4 μm and 0.6 to 0.2 μm, more preferably 10 μm, 3 μm and It may be manufactured by sequentially extruding on a 0.4 μm membrane filter.

In addition, the membrane of the artificial vesicle may further include components other than the cell membrane of the cell. Specifically, components other than the cell membrane may include a targeting molecule, a substance required for cell membrane fusion with the target cell (fusogen), cyclodextrin, polyethylene glycol, and the like, and components other than the cell membrane. Can be added by various methods, including, but not limited to, chemical modification of cell membranes.

2. Host cells with cell walls

In the present invention, host cell-derived extracellular vesicles having a cell wall containing the RNA transcription system of interest can induce various immune responses in the host or contain an exotoxin such as LPS, so that LPS contained in bacterial-derived vesicles can induce a variety of immune responses. There is a problem of toxicity that needs to be addressed. Therefore, in the present invention, in order to eliminate the toxicity problem of bacterial-derived vesicles, protoplasts made by removing the outer membrane of the vesicle and the cell wall including the peptidoglycan layer After preparing, artificially prepared extracellular vesicles were used.

Protoplast-derived artificial vesicles that can be produced in host cells including prokaryotes, yeast and plant cells having cell walls can be prepared by applying various mechanical, electrical, and chemical methods to protoplasts.

Specifically, in the present invention, extracellular vesicle surface antigen (Coagulase, CoA) derived from Escherichia coli as a representative Gram-negative bacterium and Staphylococcus aureus-derived as a representative Gram-positive bacterium Each was overexpressed in Escherichia coli and then cultured, and then treated with lysozyme to prepare protoplasts, and then extracellular vesicles were prepared by extrusion.

In the present invention, extracellular vesicles may be artificially prepared, and the method for preparing them is not particularly limited, but may be obtained by extruding through a filter, and may further include processes such as centrifugation and ultracentrifugation. Cell lysis using osmotic pressure, electroporation, sonication, homogenization, detergent treatment, freeze-thaw, extrusion, mechanical degradation, chemical treatment, etc. Methods of may be used, but the production method of the present invention is not limited thereto.

In the present invention, the method for separating extracellular vesicles is not particularly limited as long as it includes bacterial-derived extracellular vesicles. Extracellular vesicles can be separated using methods such as , capillary electrophoresis, polymer addition, and combinations thereof. In addition, processes such as washing to remove impurities and concentration of the obtained extracellular vesicles may be further included. The extracellular vesicles include naturally secreted or artificially secreted extracellular vesicles.

Protoplasts can also be broken up mechanically by placing a metal, ceramic, or sufficiently hard plastic ball in the solution containing the protoplasts and shaking them. In the case of manufacturing vesicles by extrusion, the cells may be broken into appropriate sizes by sequentially passing a solution containing cells from a filter having a large pore size to a filter having small pores. For example, a vesicle may be manufactured by sequentially using three filters having a hole size of 10 μm → 5 μm → 1 μm.

One embodiment of the manufacturing method according to the present invention includes the following steps: removing cell walls from cells to obtain protoplasts; preparing vesicles from a suspension containing the protoplasts; and separating the prepared vesicle from the suspension.

Another embodiment of the manufacturing method according to the present invention includes the following steps. Obtaining protoplasts by removing cell walls from cells containing therapeutic, diagnostic, or vaccine RNA; preparing vesicles from a suspension containing the protoplasts; and separating the prepared vesicle from the suspension.

Another embodiment of the manufacturing method according to the present invention includes the following steps: removing cell walls from cells containing RNA to obtain protoplasts; Externally loading a material for treatment, diagnosis, or vaccine into the protoplasts; preparing vesicles from a suspension containing protoplasts loaded with the material; and separating the prepared vesicle from the suspension.

Another embodiment of the manufacturing method according to the present invention includes the following steps: removing cell walls from cells containing RNA to obtain protoplasts; preparing a vesicle by adding a material for treatment, diagnosis, or vaccine to the suspension containing the protoplasts; and separating the prepared vesicle from the suspension.

Another embodiment of the manufacturing method according to the present invention includes the following steps: removing cell walls from cells containing RNA to obtain protoplasts; preparing vesicles from a suspension containing the protoplasts; Adding a material for treatment, diagnosis, or vaccine to the prepared vesicle suspension and loading the vesicle; and separating a vesicle loaded with a material for treatment, diagnosis, or vaccine from the suspension.

Another embodiment of the manufacturing method according to the present invention includes the following steps: removing cell walls from cells containing RNA to obtain protoplasts; preparing vesicles from a suspension containing the protoplasts; Separating the vesicles prepared from the suspension; and adding and loading a substance for treatment, diagnosis, or vaccine to the suspension containing the separated vesicles.

The manufacturing method may further include separating the vesicle loaded with the material for treatment, diagnosis, or vaccine from the suspension containing the vesicle.

The separation step may be performed using a method selected from the group consisting of ultracentrifugation, density gradient, filtration, dialysis, and free-flow electrophoresis. can

Density gradient is the most commonly used method for classifying materials with different densities, and since the vesicles of the present invention are distinguished from free molecules in density, they can be separated through the density gradient. As a specific example of this method, a density gradient such as Ficoll, Glycerol, Sucrose, OptiPrep™, etc. may be used, but the density screening method of the present invention is not limited thereto. Two types of vesicles can be separated by using the different densities of vesicles loaded with molecules for treatment and diagnosis and vesicles that are not loaded. Density gradients can be used in conjunction with centrifugation or electrophoresis. Gel filtration or ultrafiltration may also be used to select vesicles. Dialysis may be used instead of filtration to remove small molecules. In addition to this, a free flow electrophoresis method may also be used.

Depending on the purpose, vesicles within a certain range of sizes can be selected and used. The step of selecting the size of the vesicle may be performed before, concurrently, or later than the step of loading the vesicle with a material for treatment or diagnosis.

Materials for treatment, diagnosis, and/or vaccine loaded onto the protoplast-derived vesicle are not particularly limited. For example, it may be derived from a cell or transformed cell producing a protoplast-derived vesicle, or it may be a material that is not derived from the cell and prepared outside the cell, and the material to be loaded may be one or a combination of two or more materials. It may be possible, but is not limited thereto.

Loading the protoplast-derived vesicle with a material for treatment, diagnosis, and/or vaccine means exposure to the surface of the protoplast-derived vesicle or encapsulation thereof, but is not limited thereto.

The manufacturing method may further include modifying some of the components of the cell membrane. Modification of the components of the cell membrane can be achieved in a variety of ways. For example, a vesicle having the fusion protein exposed on the surface can be prepared by preparing a vesicle from a separately prepared mixture of the fusion protein and cells. It is possible to manufacture stealth-vesicles by binding polyethylene glycol to the surface of the vesicles. In addition, when cyclodextrin is added to vesicles, non-specific targeting can be reduced. Cyclodextrin is a substance that has water and fat solubility at the same time, and can inhibit non-specific binding between lipids by attaching to the surface of vesicles. The vesicle may be chemically modified and used. For example, after preparing a vesicle from a cell in which a portion of a protein including cysteine is exposed on the surface of a cell membrane, the vesicle can be modified by chemically binding various molecules to the thiol group of cysteine. In addition, it can be modified by binding various molecules to the amine groups present in cell membrane proteins by chemical methods.

A step of removing vesicles having a topologically modified membrane compared to the cell membrane of the protoplast from which the method originated may be further included. That is, after manufacturing vesicles, only vesicles having the same topology as the original cell membrane may be selected and used depending on the purpose. Vesicles exposed to the outside of the cytoplasmic domain can be removed by using a substance such as an antibody that recognizes the cytoplasmic domain of cell membrane proteins. In this way, vesicles whose inside and outside of the cell membrane have been changed are removed, and only cell membrane proteins exposed to the outside of the cell membrane of the original cell exist on the outside of the remaining vesicles.

Provided is a method for delivering a material for treatment, diagnosis, and/or vaccine including the protoplast-derived vesicle according to the present invention to a specific cell or tissue. Specifically, the method of delivering the material for treatment, diagnosis, and/or vaccine to a specific cell or tissue is a protoplast-derived vesicle in which a membrane is formed with protoplast-derived cell membrane components and is smaller in size than the protoplast and loaded with the material. including using If necessary, the treatment, diagnosis, and/or vaccine material may be delivered to a specific cell or tissue using a protoplast-derived vesicle loaded with a target-inducing material and/or a cell membrane fusion material.

The specific cell or tissue is not limited. For example, the specific cell may be an endothelial cell, cancer cell, inflammatory cell, or immune cell, and the specific tissue may be a blood vessel, cancer tissue, or inflammatory tissue. In addition, the material for treatment, diagnosis or vaccine is as described above.

Two or more of these substances can be delivered to specific cells or tissues. The material may be delivered using two or more vesicles selected from the group consisting of a vesicle loaded with one of the materials, a vesicle loaded with two or more materials, and a combination thereof. For example, two or more of the vesicles may be administered simultaneously.

In another embodiment of the present invention, sequentially administering two or more vesicles selected from the group consisting of vesicles loaded with one of the substances, vesicles loaded with two or more substances, and combinations thereof The material can be delivered.

More specifically, two or more materials can be delivered using a vesicle loaded with two or more materials at the same time. Alternatively, two or more materials may be delivered by using a plurality of vesicles loaded with one or two or more materials. For example, in the case of delivering three materials, first, second, and third vesicles including each material may be manufactured and used. A fourth vesicle containing the first and second materials and a fifth vesicle containing the third material may be prepared, and the three materials may be delivered using the fourth and fifth vesicles. The first, second, and third vesicles may be administered simultaneously or sequentially. The fourth and fifth vesicles may be administered simultaneously or sequentially.

Another aspect of the present invention provides a method for treating and/or diagnosing a disease, comprising delivering the material to a specific cell or tissue using a cell protoplast-derived vesicle containing RNA. That is, a substance for diagnosing or treating a disease can be delivered to a target cell, tissue or blood by using the vesicle.

Vesicles prepared from protoplasts can be easily made in various sizes like existing liposomes, and can be easily loaded with various therapeutic, diagnostic, or vaccine substances to be delivered, so that one or several substances can be delivered, so single treatment, It can be used for diagnosis-treatment (theragnosis, pharmacodiagnosis), which is used for both purposes of diagnosis and treatment, as well as combination therapy and diagnosis. At this time, various substances to be delivered may be encapsulated in the vesicle and exist inside the double membrane of the vesicle, or all or part of the substances may be embedded or embedded in the double membrane of the vesicle, such as a cell membrane protein. and may be bonded to the surface of the vesicle.

For example, when a substance is delivered to a cancer tissue through cancer blood vessels, a substance having a size of 100 nm or larger may remain in the cancer tissue for a long time due to an enhanced permeability and retention (EPR) effect. Therefore, drugs loaded in vesicles larger than 100 nm can stay in cancer tissue for a long time, which is useful for diagnosis and treatment because the drug's effect can be maximized. In addition, since only particles of a size of 1 μm or less can be delivered to the alveoli through inhalation due to the structure of lung tissue, substances such as anti-inflammatory agents for the treatment of asthma are loaded into vesicles of 1 μm or less in size to effectively penetrate the lung tissue. drugs can be delivered. In this way, the size of the vesicle may be manufactured in various sizes depending on the tissue in which a substance for diagnosis or treatment is required. Preferably, the size of the protoplast-derived vesicles of the present invention may be 10 nm or more and 10 μm or less.

Another aspect of the present invention is to prevent and/or prevent disease, including delivering a vaccine material to a specific cell or tissue using a vesicle derived from protoplasts of cells containing RNA and containing a vaccine material. provide a treatment method. The material for the vaccine is as described above.

Another aspect of the present invention is a disease diagnosis kit comprising a vesicle derived from protoplasts of cells containing RNA, having a smaller size than the protoplasts, and loaded with primers, probes, antisense nucleic acids, or antibodies necessary for diagnosis of diseases. provides

In the present invention, vesicles derived from protoplasts loaded with various material(s), including materials for treatment, diagnosis, or vaccine, and a method for producing the same are used to deliver specific materials to specific cells or tissues. It can be used in vitro and/or in vivo for therapeutic, diagnostic and/or vaccine, or experimental purposes. For example, enzymes, protoplast-derived vesicles loaded with materials for treatment, diagnosis, or vaccine, and methods for producing the same can be used for in vitro experiments, and can be treated in vivo through in vitro experiments. It can also be used for the purpose of transforming cells, but is not limited thereto.

VIIII. Exosomes containing the RNA of interest

Exosomes derived from the endocytic pathway of host cells containing the RNA transcription system of interest of the present invention can be released from MVBs containing several vesicles generated through exocytic fusion with the cell membrane. Exosome production involves four steps: budding, invasion, MVB formation and secretion.

As a form of budding, cell membrane invagination forms clathrin-coated vesicles, which then enter the cytoplasm to form early endosomes. Proteins, lipids and nucleic acids are specifically sorted and encapsulated to form multiple intraluminal vesicles (ILVs), the precursors of exosomes. After late endocytosis, several ILVs further develop into late endosomes (LEs). Then, part of the LE enters the lysosomal pathway and the rest fuses with the cell membrane.

Exosomes are evaluated as ideal vesicles for drug delivery because they can deliver substances across cell membranes. This lipid bilayer vesicle system can overcome the problem of skin penetration, making it one of the most effective strategies for delivering drugs to the dermal layer of the skin. are being evaluated

In addition, exosomes contain RNA, proteins, lipids and metabolites that reflect the cell type from which they are derived. Exosomes contain various molecular components (eg, proteins and RNA) of the cell from which they are derived. Exosome protein composition varies depending on the cell and tissue from which the exosome is derived, but most exosomes contain a common set of evolutionarily conserved protein molecules.

Exosomes can be obtained by isolating them from a culture solution obtained after culturing cells, and the size and content of exosomes can be influenced by molecular signals received by cells producing exosomes.

When intracellular Ca2+ concentration is increased, exosome secretion is promoted. monCRY2 can form a fusion protein by being fused with a protein capable of inducing Ca2+ influx into cells (eg, FGFR1, STIM1, etc.). monFGFR1 is dimerized by light to induce IP3-induced Ca2+ release from intracellular stores such as the endoplasmic reticulum (ER). monSTIM1 is dimerized by light and turns on the CRAC (Ca2+ Release Activated Ca2+) channel, resulting in the flux of Ca2+ ions into cells. The reaction by light occurs reversibly, and when light is removed, monCRY2 is separated into monomers, and eventually STIM1 is also separated into monomers, and CRAC is closed. Since these fusion proteins can dimerize only with low light irradiation, they can operate even when light is irradiated to cells in a non-invasive manner.

The specific mechanism and sequence of STIM1 can be found in the papers (Soboloff, J. et al. Nat Rev Mol Cell Biol 13, 549-565 (2012) & Yang, X. et al. Proc Natl Acad Sci USA 109, 5657-5662 (2012) )).

In the present invention, the culture solution itself can be used as a raw material without separating the exosomes, but the exosomes can also be separated and used. In addition, exosomes can be separated to check the size and physical properties of exosomes in the culture medium for quality assurance.

As a method for separating exosomes from the culture medium, centrifugation, immunocombining, filtration, and the like can be used.

Ultracentrifugation, which is the most commonly used exosome isolation method, cannot separate a large amount of exosomes at once, requires expensive equipment and takes a lot of time to separate, and strong centrifugation causes exosomes to be separated. Physical damage may occur, and in particular, there are disadvantages such as low purity of the separated exosomes. Among the methods for improving these disadvantages, a PS affinity method that increases the purity of separated exosomes by using a substance that specifically binds to phosphatidylserine (PS), a protein present in the membrane of exosomes (PS affinity method). Compared to the ultracentrifugation method, this method can separate exosomes of high purity, but has a disadvantage in that the yield is low.

The present invention provides an RNA polymerase suitable for a host cell E. coli expression system and an mRNA transcription system of interest using the RNA polymerase, thereby enabling stable and low-cost mass production of the RNA of interest in the host cell E. coli.

Figure 1 is the structure of the RNA transcription system using ctDdRp *?**?*
Figure 2 is an image of electrophoresis after digestion of pUC19 having an RNA transcription system using ctDdRp with a restriction enzyme,
Figure 3 is an image of electrophoresis after digestion of pUC19 having an RNA transcription system using RddRp with a restriction enzyme,
4 is an electrophoresis image of an mRNA of interest produced in E. coli strain HT115 (DE3) transformed with an RNA transcription system.

Hereinafter, the present invention is described in more detail with the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

Example 1. RNA transcription system of interest and host cell

The RNA transcription system of the present invention provides a role capable of continuously expressing an RNA of interest by RNA polymerase in a host cell capable of at least protein translation. The RNA transcription system of the present invention comprises an RNA polymerase expression unit and an RNA unit of interest.

As shown in FIG. 1, in the RNA transcription system of the present invention, the RNA polymerase expression unit and the RNA unit of interest are either one molecule or independent isolated DNA molecules, and a plasmid may be used. The RNA polymerase can be divided into DdRp and RdRp.

In the present invention, DdRp that can be synthesized in the RNA polymerase expression unit of the RNA transcription system, a fusion of <DdRp and capping enzyme>, a fusion of <DdRp and Poly(A) polymerase>, a fusion of <DdRp, capping enzyme and Poly(A) ) polymerase> was collectively referred to as 'ctDdRp'.

In the case of ctDdRp that can be synthesized in the RNA polymerase expression unit of the RNA transcription system, it was referred to as 'an RNA transcription system using ctDdRp'.

In the present invention, RdRp that can be synthesized in the RNA polymerase expression unit of the RNA transcription system is referred to as 'RNA transcription system using RdRp'.

The DNA encoding the mRNA of interest in the mRNA of interest unit of the RNA transcription system was referred to as 'DNAi'.

Host cells in the present invention may be animals, plants, yeasts and bacteria. Expression of a gene encoding ribonuclease III in cells, including Corynebacterium glutamicum, E. coli BE42, E. coli HB101, E. coli JM101, E. coli LE392, E. coli MC1061, E. coli Y1088 and E. coli W3110 Host cells in which the activity of ribonuclease III is reduced by attenuating or disrupting the gene can be used.

In the examples below, E. coli strain HT115 (DE3) with reduced activity of ribonuclease II was used. The genotype of HT115 (DE3) is: F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10 (DE3 lysogen: lavUV5 promoter-T7 polymerase) (RNAse III minus). E. coli HT115 (DE3) cells were transformed with pUC19 (control), pCPB-hp, or pCPB-hp+2T and grown overnight at 37°C or 25°C.

Monocytes include U937 cells (ATCC No.CRL-1593.2), macrophage Raw264.7 cells (ATCC No.TIB-71), cord blood mesenchymal stem cells (UC MSC), and PT67 cells (ATCC No. CRL-12284) and the like were used.

1.1. prokaryotic cell

In order to achieve the above object in prokaryotes, the present invention provides an RNA polymerase gene in which the expression unit of the RNA polymerase of the RNA transcription system of interest of the present invention comprises the nucleotide sequence of Formula (1) below.

Equation (1): W-X-Y-Z

In the above formula, W includes a promoter recognized by an RNA polymerase gene in a host cell and 5*?**?*UTR including an SD site.

The promoter recognized by RNA polymerase of the host cell at the W site of formula (I) of the present invention includes (a) pta, aceA, aceB, adh and amyE promoters that can be induced by acetic acid, ethanol, pyruvic acid, etc. army; (b) a group containing cspB, SOD and tuf promoters; (c) a group containing a lac promoter, a tac promoter, and a trc promoter; (d) a group containing phage-derived promoters such as F1 promoter, T7 promoter, T5 promoter, T3 promoter and SP6 promoter; any one selected from the group containing may be used.

In this example, the Lac promoter was used and SEQ ID NO: 2.

Lac Promoter (31nt): TTTACACTTTATGCTTCCGGCTCGTATGTTG (SEQ ID NO: 2)

The sequence of the 5'-UTR is SEQ ID NO: 3, the Shine-Dalgarno sequence is shown in bold blue, the start codon is shown in bold black, and the 4nt random fragment is shown in red. The Shine-Dalgarno Sequence or SD sequence is the ribosome binding site of prokaryotic messenger RNA, and is generally located 8 bases ahead of the start codon AUG.

5'-UTR: GGCACACACCGGAGCNNNNCAUAUG (SEQ ID NO: 3)

The 4nt random fragment NNNN may include any one selected from the group consisting of AAAU, AAUA, AUAA and AUAU.

X represents the 5'end nucleotide sequence of the RNA polymerase gene, translation start codon ATG, and the RNA polymerase gene

(a) the DNA dependent RNA Polymerase or a variant thereof; and

(b) a capping enzyme or a variant thereof; and

(c) a linker; may also be included.

Here, the expression unit of the RNA polymerase may additionally include the RNA-binding tethering peptide. The RNA polymerase is characterized by comprising at least one RNA-binding domain of a protein-RNA tethering system, wherein the RNA-binding domain comprises a specific RNA sequence and / Or it may be an RNA structure characterized by binding specifically to the RNA element of the protein-RNA tethering system composed of the structure.

Even more preferably, the expression unit of the RNA polymerase of the RNA transcription system of interest of the present invention may include RNA dependent RNA Polymerase (RdRp) or a variant thereof.

Y represents a nucleotide sequence corresponding to the 2nd to translation stop codon of the RNA polymerase gene;

Z represents a nucleotide sequence including a transcription termination sequence below the stop codon at the 3' end of the RNA polymerase gene.

In addition, in the present invention, as a transcription termination sequence below the termination codon, the translation termination codon of TGA TAA was ligated to the 3' end of the Z region to ensure translation termination.

In SEQ ID NO: 4, UAA is a stop codon, UUG is a start codon, and an underlined base sequence is an IS including S/D.

The transcription termination sequence of this example is SEQ ID NO: 4, which consists of inverted repeats followed by several A residues in the template DNA strand. When RNA polymerase transcribes an inverted repeat, it immediately folds into a stem-loop structure at the mRNA level, pausing the enzyme. Dissociation of the enzyme was allowed to occur because there are several U residues behind the stem-loop structure that interact weakly with the A residues of the template DNA.

TGAGATAAAAATGCCAGCCGATCGGGCTGGCATTTTGCCTTTTAG (SEQ ID NO: 4)

In the present invention, X of Formula (1) can be prepared using DdRp, ctDdRp or RdRp DNA fragments as a template.

2.1. DdRp

In the RNA transcription system of the present invention, when the RNA of interest is not mRNA, dsRNA, RNA aptamer, and ribozyme, a DdRp DNA fragment based on SEQ ID NO: 1 was used as an RNA polymerase.

2.2. ctDdRp

In the RNA transcription system of the present invention, when the RNA of interest is mRNA, the following ctDdRp DNA fragment based on SEQ ID NO: 1 was used as the RNA polymerase.

The ctDdRp expression unit of the present invention can be composed of a 5'UTR sequence upstream of the ctDdRp orf, a stop codon and a transcription termination sequence downstream.

Preferably, the structural unit of the ctDdRp orf includes DdRp and a capping enzyme, and a linker or an intercistronic spacer (IS) may be included between the structural units of the orf included in the structural units of the ctDdRp orf. . Additionally, poly(A) polymerase may be included.

The orfs of related proteins were optimized for expression in human cells with respect to the codon adaptation index using the GeneOptimizer algorithm. All gene sequences were artificially synthesized, amplified by step-by-step PCR using oligonucleotides, cloned and fully sequenced.

To prepare the ctDdRp expression unit of the present invention, first, a ctDdRp DNA fragment producing the ctDdRp expression unit was designed and prepared, and cloned into pUC19.

To prepare the ctDdRp expression unit of the present invention, the ctDdRp orf was designed as shown in Formula 2.

Formula (2): Nλ-X1-R341-X2-Nλ-X1-NP868R-X3-T7 Pol

where Nλ = tethering domain, X1 = G4, X2 = IS, X3 = IS.

Enzymes prepared in formula (2) may be Nλ-X1-R341, Nλ-X1-NP868R and T7 Pol.

The ctDdRp DNA fragment contained a 5'UTR sequence upstream of the orf of Formula (2) and a stop codon and a transcription termination sequence downstream.

The intercistronic spacer (IS) of the present invention contains the S/D sequence if an Au(UAUUUUAAUUAAUUAA) rich sequence is introduced upstream of the intercistronic spacer translation efficiency. IS sizes vary from ~20ntds to 40-50ntds. This spacer contains S/D sequences for the 30s ribosomal subunit to bind to the next cistron and restart translation. The IS used in this example is SEQ ID NO: 4.

UAA UAACGGAGUGAUC UUG (SEQ ID NO: 4)

For the design of ctDdRp DNA contained in the ctDdRp expression unit of the present invention, DdRp constituting the core structural unit ctDdRp ORF of formula (II) is a type of DdRp composed of a single subunit, T7 RNA polymerase (Gene ID: 1261050) , T7 RNA polymerase was selected from the group containing T3 RNA polymerase (Gene ID: 14675519) and SP6 RNA polymerase (Gene ID: 19685893).

Acanthamoeba polyphaga mimivirus R341 (UniProtKB/Swiss-Prot accession number E3VZZ8) was selected as the core structural unit Poly(A) Polymerase in formula (2).

Capping enzyme, the core structural unit of formula (2), was selected as African swine fever virus NP868R capping enzyme (NCBI ASFV genome sequence strain BA71 V NC_001659; UniProtKB/Swiss-Prot accession number P32094).

In the mRNA transcription system of the present invention, ctDdRp in the RNA expression unit is, as shown in equation (2), the tethering domain (Nλ) of the protein-RNA tethering system, and also the mRNA unit of interest , the tethering RNA of the protein-RNA tethering system was added.

In the tethering system, the tethering domain is 22 amino acids of antitermination N protein from lambda bacteriophage (Nλ; amino acids 1-22 of enterobacteria phage lambda nucleocapsid protein AAA32249; SEQ ID NO: 5 and SEQ ID NO: 6 corresponding to nucleotides) was selected.

ATGGATGCTC AGACCAGACG CAGAGAACGG CGGGCAGAGA AGCAGGCACA GTGGAAGGCC GCTAAT 66 (SEQ ID NO: 5)

Met Asp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn 22 (SEQ ID NO: 6)

The tethering RNA contains BoxBr of the λ virus (nucleotides 38312-38298 of the genomic sequence of enterobacterial phage lambda KT232076.1; SEQ ID NO: 7 corresponding to the nucleotide sequence of the BoxBr RNA stem-loop from λ bacteriophage) in series of four Constructed RNA sequences were selected.

GCCCTGAAAA AGGGC 15 (SEQ ID NO: 7)

In the linker, X1 in Formula (2) may include G4 or (G4S) ₂ .

The ctDdRp expression unit of interest of the present invention can be constructed using a plasmid or viral vector. The DNA fragment of interest is <Lac promoter - 5'UTR - ctDdRp DNA - transcription termination sequence>, which can be prepared by inserting it into a plasmid.

In order to achieve the above object in eukaryotic organisms, the present invention is an RNA polymerase mRNA unit that can be translated in a eukaryotic cell and / or is transcribed and translated by the RNA polymerase. It is provided as an RNA Polymerase DNA unit that becomes

2.3. RdRp

RdRp may also be used as the RNA polymerase used in formula (1) in the above section 1.1 of the present invention.

In order to prepare the mRNA transcription system of interest using RdRp proposed in the present invention, an mRNA encoding RdRp and a DNA fragment encoding the mRNA of interest were prepared.

The RdRp expression unit produces RdRp, and was prepared with a template DNA fragment encoding the Semliki Forest Virus (SFV) RdRp mRNA used in the examples.

In order to achieve the above object, the present invention provides an RNA polymerase gene in which the RNA polymerase expression unit of the RNA transcription system of interest of the present invention comprises the nucleotide sequence of formula (1).

Equation (1): W-X-Y-Z

In the above formula, W includes a promoter recognized by an RNA polymerase gene in a host cell and 5*?**?*UTR including an SD site.

X is Semliki Forest virus (SFV) RdRp mRNA. First, the reference Semliki Forest virus replicon plasmid (pSFV1; Invitrogen #18448-019) was purchased from ThermoFisher (USA), and alphavirus RdRp (without start codon) The product was obtained by amplifying an open reading frame encoding nucleotides 222-6321 of the RdRp ORF.

To generate the RdRp expression unit, according to Example 1, <promoter recognized in host cells - 5'UTR - open reading frame encoding RdRp (nucleotides 222-6321 of RdRp ORF) DNA - transcription termination sequence> were ligated in order to prepare recombinant DNA.

2.4. Pus expression unit

Pus was expressed to pseudouridine the mRNA produced by the RNA polymerase of the present invention.

Human PUS1 cDNA fragment was DNA obtained by PCR using primers HuPus1pET16bFor (5′-AACATATGGCCGGGAACGCGGAGCC, SEQ ID NO: 8) and HuPus1pET16bRev (5′-GGATCCTCGAGTCCCATCGCCTCAGTCAGTG SEQ ID NO: 9) against IMAGE consortium clone #2822709 (American Type Culture Collection, USA). A fragment, SEQ ID NO: 10, was inserted into the pUC vector between the T7 promoter and the transcription termination sequence.

PUS 1 ORF Nucleotide Sequence (Length: 1506bp) Accession no. XM_010228061.1

ATGGATGTGG AGGTGGCCT GGCAGGAGAG CGGGGACACG AACACAGCCC CTTTAGGCAG

GAGAAGTTCA CGCTGCACC CTCGAGCAGT GTCTGTGATA GGCGCCTGCC TGGCTGGGGA

ATGGTGGAAC ACAGGGAGCC AAAGCTCCCA CACAAGAGCA GTCAGCCTGC CGTGCCCGAG

GCTGCCGCGC GCCAGCAAAC GCCTCGCAGG CCCACGGATG CTTCGTGGCA TCCCACGCAT

TTGCTTGCAT GGACAGAGAG CACGTCTTGT GCTGAGTCCT GCCTGCTCTC CTGCCCCTTG

CAGGTTCTCA TCATGGCTGA AGAGGTGAGG GCAGCGGTTG CCGGCCAGCC CAAGAGACTG

ATGAGCAGCA GCGAGGAGCC AGAAAGGCTG GAGGAAAATG GGCACCTGTC CAAAAGGCTG

AAGAGCCAGG TGGATGAGGA GGACCAGACT AAGAAATTGC CCAAAAGGAA GATCGTGCTG

TTGATGGCGT ACTCAGGGAA AGGCTACCAT GGGATGCAAA GAAATGTGGG ATCTTCGCAG

TTCAAGACAA TTGAAGATGA CTTAGTATCT GCCCTTGTTC AGTCAGGCTG CATTCCAGAA

AACCATGGGG AAGATATGAA GAAAATGTCT TTCCAGAGAT GTGCTCGAAC AGATAAGGGT

GTATCTGCAG CTGGACAGAT TGTGTCGCTG AAGGTCAGAT TAATAGATGA CATCTTAGAA

AAGATCAATA GCCATCTTCC TTCTCACATC AGAATTCTGG GGCTGAAGAG AGTCACTGGA

GGATTCAACT CCAAGAACAA ATGTGATGCC AGAACCTACT CTTACATGCT GCCAACATTT

GCCTTTGTCC ATAAAGACCA CGATGTGCAA GAGGAGACTT TCCGACTGAA TAAAGAGACC

CTTGAAAAAG TGAACAAACT GCTCGCTTGT TACAAAGGAA CACACAATTT CCACAACTTT

ACGTCTCAGA AGGGGCCCAA GGACCCCAGT GCCAAGCGAT ATATAATGGA AATGTACTGT

GGAGAGCCCT TTGTAAGGGA AAGCATAGAA TTTGCAGTGA TCAAAGTAAA AGGCCAGAGC

TTCATGATGC ACCAAATAAG GAAGATGATT GGACTGGTGA TCGCAATTGT GAAAGGTTAT

GCTGCTGAGT CCATCATAGA ACGCAGCTGG GGTGAGGAGA AAGTAGATGT CCCCAAAGCT

CCAGGACTTG GGCTGGTCCT GGAGAGGGTG CACTTTGAAA AGTACAACAG GCGTTTTGGA

AATGATGGGC TGCACGAGCC GCTCGAATGG AAGGAGGAGG AGGAGAAGAT TGCTGTTTTC

AAAGAGCAGT ACATCTATCC TACTATCATC AACACGGAAA GGGAGGAGAA ATCCATGATG

AACTGGCTGA GCACCCTTTC CATTCATGAC TTCAACTCTT CTGCAGTTGA GCTGCAAGCT

AACAACAAAA CCTCAAAGAA CAGTGATTTT GAAGGCAGTG ATGGTTGTGA TGATGATTCT

GAGTGA (SEQ ID NO: 10)

Example 3. RNA unit of interest

3.1. mRNA unit of interest

The mRNA unit of interest of the present invention may include a promoter and a DNAi fragment (DNA encoding the mRNA of interest) to which the ctDdRp can bind and function.

The mRNA unit of interest may consist of a 5'UTR sequence upstream of the DNA orf of interest, a stop codon downstream, a 3'UTR sequence, a Poly(A) tail sequence, a poly(C) sequence, and a histone stem-loop.

The mRNA unit of interest includes DNA corresponding to several types of mRNAs of interest, and a linker or an intercistronic spacer may be included between these units.

Promoters corresponding to these may include the T7 promoter, the T3 promoter, and the SP6 promoter.

Specifically, the mRNA unit of interest of the present invention can be prepared to include a promoter and DNAi to which the ctDdRp can bind and function.

The mRNA unit of interest, singly or in combination, may provide a DNA segment comprising a DNAi encoding the mRNA of interest downstream of the T7 promoter.

The T7 promoter of the present invention is recognized by T7 RNA Polymerase and the downstream region is transcribed into RNA, and its nucleotide sequence is SEQ ID NO: 11.

T7 promoter: TAATACGACT CACTATAGGG (SEQ ID NO: 11)

Preferably, the 5'UTR is SEQ ID NO: 12 as hAg-Kozak

hAg-Kozak: ATTCTTCTGG TCCCCACAGA CTCAGAGAGA ACCCGCCACC (SEQ ID NO: 12)

The 5'UTR sequence may include G and Kozak consensus at the 5*?**?* end for the CAP1 structure, and its base sequence is SEQ ID NO: 13.

Kozak Consensus Sequence: ACCAUGG (SEQ ID NO: 13)

The 3'UTR may be any one selected from structures including muag-A64-C30-histone-SL and 2 BgUTR-A30LA70.

Preferably, the 3'UTR is muag<mutated UTR of (alpha-)globin, SEQ ID NO: 14>, the poly (A) tail is 64 adenine nucleotides (SEQ ID NO: 15), and the poly (C) sequence is 30 cytosines. It is an RNA sequence consisting of a nucleotide (SEQ ID NO: 16) and a histone stem-loop (SEQ ID NO: 17).

muag<mutated UTR of (alpha-)globin>; 5′-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3′ (SEQ ID NO: 14; underlined nucleotides are mutations compared to the wild-type sequence)

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA (SEQ ID NO: 15)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 16)

Histone stem-loop: 5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' (SEQ ID NO: 17)

When the protein of interest is a fusion protein, a (G4S)3-linker (SEQ ID NOs: 18 and 19) may be used as a linker.

(G4S)3-linker; GGAGGCGGTG GTAGTGGAGG TGGCGGGTCC GGTGGAGGTG GAAGC (SEQ ID NO: 18)

(G G G G S G G G G S G G G G S; SEQ ID NO: 19)

The mRNA units of interest of the present invention can be constructed using plasmids or viral vectors. The DNA fragment of interest can be prepared by inserting the T7 promoter - 5'UTR - DNA of interest - muag - A64 - C30 - (Tethering domain)4 - Histone-SL) into a plasmid.

Looking specifically at the mRNA of interest used in the present invention,

The mRNAi used in this example are TNFR2 protein, GM-CSF, and EPO, rabies virus G protein (RAV-G) and respiratory syncytial virus (RSV) long strain RSV-F protein It is an mRNA encoding the etc., but is not limited thereto.

<Therapeutic protein>

sTNFR2

The therapeutic protein of the present invention is a TNFR2 protein or an extracellular domain fragment thereof. Tumor necrosis factor (TNF) is a pleiotropic cytokine, especially TNF-α, which plays an important role in the inflammatory response and immune system. As receptors that bind to TNF, TNFR1 (molecular weight, 55 kD; TNF-R55) and TNFR2 (molecular weight, 75 kD; TNF-R75) are known, and the affinity of TNF-α for TNFR2 is the affinity for TNFR1. It is known to be several times higher than

In the present invention, the TNFR2 protein is encoded in 1386 bp DNA (NM_001066.3), and this protein or its extracellular domain fragment (sTNFR2) has a high binding affinity to TNF-α and inhibits the action of TNF-α, thereby preventing autoimmunity. It can show a particularly excellent effect in the treatment of diseases.

Preferably, the sTNFR2 may be an amino acid sequence polypeptide of SEQ ID NO: 20 including positions 1 to 256 from the N-terminus of the TNFR2 protein.

Also preferably, the sTNFR2 cDNA may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 21.

sTNFR2 : Transmembrane sequence fragment consisting of 1 to 256 amino acids of TNFR2

MAPVAVWAAL AVGLELWAAA HALPAQVAFT PYAPEPGSTC RLREYYDQTA QMCCSKCSPG QHAKVFCTKT SDTVCDSCED STYTQLWNWV PECLSCGSRC SSDQVETQAC TREQNRICTC RPGWYCALSK QEGCRLCAPL RKCRPGFGVA RPGTETSDVV CKPCAPGTFS NTTSSTDICR PHQICNVVAI PGNASMDA VC TSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTAPSTS FLLPMGPSPP AEGSTG (SEQ ID NO: 20)

sTNFR2 cDNA ; cDNA corresponding to sTNFR2 (768 bp)

ATGGCGCCC GTCGCCGTCT GGGCCGCGCT GGCCGTCGGA CTGGAGCTCT GGGCTGCGGC GCACGCCTTG CCCGCCCAGG TGGCATTTAC ACCCTACGCC CCGGAGCCCG GGAGCACATG CCGGCTCAGA GAATACTATG ACCAGACAGC TCAGATGTGC TGCAAAT GCTCGCCGGG CCAACATGCA AAAGTCTTCT GTACCAAGAC CTC GGACACC GTGTGTGACT CCTGTGAGGA CAGCACATAC ACCCAGCTCT GGAACTGGGT TCCCGAGTGC TTGAGCTGTG GCTCCCGCTG TAGCTCTGAC CAGGTGGAAA CTCAAGCCTG CACTCGGGAA CAGAACCGCA TCTGCACCTG CAGGCCCGGC TGGTACTGCG CGCTGAGCAA GCAGGAGGGG TGCCGGCTGT GCGCGCCGCT GCGCAAGTGC CGCCCGGGCT TCGGCGTGGC CAGACCAGGA ACTGAAACAT CAGACGTGGT GTGCAAGCCC TGTGCCCCGG GGACGTTCTC CAACACGACT TCATCCACGG ATATTTGCAG GCCCCACCAG ATCTGTAACG TGGTGGCCAT CCCTGGGAAT GCAAGCATGG ATGCAGTCTG CACGTCCACG TCCCCCACCC GGAGTATGGC CCCAGGGGCA GTACACTTAC CCCAGCCAGT GTCCACACGA TCCCAACACA CGCAGCCAAC TCCAGAACCC AGCACTGCTC CAAGCACCTC CTTCCT GCTC CCAATGGGCC CCAGCCCCCC AGCTGAAGGG AGCACTGGC (SEQ ID NO: 21)

EPO

For the preparation of the EPO RNA expression unit of the present invention, the nucleotide sequence of the murine EPO cDNA is SEQ ID NO: 22.

EPO cDNA

ATGGGCGTGC CCGAGCGGCC GACCCTGCTC CTGCTGCTCA GCCTGCTGCT CATCCCCCTG GGGCTGCCCG TCCTCTGCGC CCCCCCGCGC CTGATCTGCG ACTCCCGGGT GCTGGAGCGC TACATCCTCG AGGCCAAGGA GGCGGAGAAC GTGACCATGG GCTGCGCCGA GGGGCCCCGG CTGAGCGAGA ACATCACGGT CCCCGACACC AAGGTGAACT TCT ACGCCTG GAAGCGCATG GAGGTGGAGG AGCAGGCCAT CGAGGTCTGG CAGGGCCTGT CCCTCCTGAG CGAGGCCATC CTGCAGGCGC AGGCCCTCCT GGCCAACTCC AGCCAGCCCC CGGAGACACT GCAGCTCCAC ATCGACAAGG CCATCTCCGG GCTGCGGAGC CTGACCTCCC TCCTGCGCGT GCTGGGCGCG CAGAAGGAGC TCATGAGCCC GCCC GACACG ACCCCCCCGG CCCCGCTGCG GACCCTGACC GTGGACACGT TCTGCAAGCT CTTCCGCGTC TACGCCAACT TCCTGCGGGG CAAGCTGAAG CTCTACACCG GGGAGGTGTG CCGCCGGGGC GACCGCTGA (SEQ ID NO: 22)

GM CSF

For the production of the GM CSF RNA expression unit of the present invention, the base sequence of GM CSF (CSF2) (NM_000758) is SEQ ID NO: 23.

GM CSF (CSF2) cDNA (NM_000758)

ATGTGGCTGC AGAGCCTGCT GCTCTTGGGC ACTGTGGCCT GCAGCATCTC TGCACCCGCC CGCTCGCCCA GCCCCAGCAC GCAGCCCTGG GAGCATGTGA ATGCCATCCA GGAGGCCCGG CGTCTCCTGA ACCTGAGTAG AGACACTGCT GCTGAGATGA ATGAAACAGT AGAAGTCATC TCAGAAATGT TTGACCTCCA GGAGCCGACC TG CCTACAGA CCCGCCTGGA GCTGTACAAG CAGGGCCTGC GGGGCAGCCT CACCAAGCTC AAGGGCCCCT TGACCATGAT GGCCAGCCAC TACAAGCAGC ACTGCCCTCC AACCCCGGAA ACTTCCTGTG CAACCCAGAT TATCACCTTT GAAAGTTTCA AAGAGAACCT GAAGGACTTT CTGCTTGTCA TCCCCTTTGA CTGCTGGGAG CCAGTCCAGG AGTGA 23)

<Vaccinating protein>

G protein of rabies virus (RAV-G)

For the preparation of the RAV-G RNA expression unit of the present invention, the nucleotide sequence of the rabies virus G protein (RAV-G) is SEQ ID NO: 24.

RAV-G cDNA

ATGGTGCCCC AGGCCCTGCT CTTCGTCCCG CTGCTGGTGT TCCCCCTCTG CTTCGGCAAG TTCCCCATCT ACACCATCCC CGACAAGCTG GGGCCGTGGA GCCCCATCGA CATCCACCAC CTGTCCTGCC CCAACAACCT CGTGGTCGAG GACGAGGGCT GCACCAACCT GAGCGGGTTC TCCTACATGG AGCTGAAGGT GGGCTACATC AGCGCCATCA AGATGAACGG GTTCACGTGC ACCGGCGTGG TCACCGAGGC GGAGACCTAC ACGAACTTCG TGGGCTACGT GACCACCACC TTCAAGCGGA AGCACTTCCG CCCCACGCCG GACGCCTGCC GGGCCGCCTA CAACTGGAAG ATGGCCGGGG ACCCCCGCTA CGAGGAGTCC CTCCACAACC CCTACCCCGA CTACCACTGG CTGCGGACCG TCAAGACC AC CAAGGAGAGC CTGGTGATCA TCTCCCCGAG CGTGGCGGAC CTCGACCCCT ACGACCGCTC CCTGCACAGC CGGGTCTTCC CCGGCGGGAA CTGCTCCGGC GTGGCCGTGA GCTCCACGTA CTGCAGCACC AACCACGACT ACACCATCTG GATGCCCGAG AACCCGCGCC TGGGGATGTC CTGCGACATC TTCACCAACA GCCGGGGCAA GCGCGCCTCC AAGGGCAGCG AGACGTGCGG GTTCGTCGAC GAGCGGGGCC TCTACAAGTC CCTG AAGGGG GCCTGCAAGC TGAAGCTCTG CGGCGTGCTG GGCCTGCGCC TCATGGACGG GACCTGGGTG GCGATGCAGA CCAGCAACGA GACCAAGTGG TGCCCCCCCG GCCAGCTGGT CAACCTGCAC GACTTCCGGA GCGACGAGAT CGAGCACCTC GTGGTGGAGG AGCTGGTCAA GAAGCGCGAG GAGTGCCTGG ACGCCCTCGA GTCCATCAT G ACGACCAAGA GCGTGTCCTT CCGGCGCCTG AGCCACCTGC GGAAGCTCGT GCCCGGGTTC GGCAAGGCCT ACACCATCTT CAACAAGACC CTGATGGAGG CCGACGCCCA CTACAAGTCC GTCCGCACGT GGAACGAGAT CATCCCGAGC AAGGGGTGCC TGCGGGTGGG CGGCCGCTGC CACCCCCACG TCAACGGGGT GTTCTTCAAC GGCATCATCC TCGGGCCCGA CGGCAACGTG CTGATCCCCG AGATGCAGTC CAGCCTGCTC CAGCAGCACA TGGAGCTGCT GGTCTCCAGC GTGATCCCGC TCATGCACCC CCTGGCGGAC CCCTCCACCG TGTTCAAGAA CGGGGACGAG GCCGAGGACT TCGTCGAGGT GCACCTGCCC GACGTGCACG AGCGGATCAG CGGCGTCGAC CTCGGCCTGC CGAACTGGGG GAAGTACGTG CTGCTCTCCG CCGGCGCCCT GACCGCCCTG ATGCTGATCA TCTTCCTCAT GACC TGCTGG CGCCGGGTGA ACCGGAGCGA GCCCACGCAG CACAACCTGC GCGGGACCGG CCGGGAGGTC (SEQ ID NO: 24)

RSV-F protein from RSV long strains

For the preparation of the RSV-F RNA expression unit of the present invention, the nucleotide sequence of the RSV-F protein of the long strain of respiratory syncytial virus (RSV) is SEQ ID NO: 25.

RSV-F long (GC) cDNA

ATGGAGCTGC CCATCCTCAA GGCCAACGCC ATCACCACCA TCCTGGCGGC CGTGACGTTC TGCTTCGCCA GCTCCCAGAA CATCACCGAG GAGTTCTACC AGAGCACCTG CTCCGCCGTC AGCAAGGGCT ACCTGTCCGC CCTCCGGACC GGGTGGTACA CGAGCGTGAT CACCATCGAG CTGTCCAACA TCAAGGAGAA CAAGTGCAAC GG CACCGACG CGAAGGTGAA GCTGATCAAC CAGGAGCTCG ACAAGTACAA GAACGCCGTC ACCGAGCTGC AGCTGCTCAT GCAGAGCACG ACCGCCGCCA ACAACCGCGC GCGGCGCGAG CTGCCGCGGT TCATGAACTA CACCCTGAAC AACACCAAGA AGACGAACGT GACCCTCTCC AAGAAGCGCA AGCGGCGCTT CCTGGGGTTC CTGCTC GGCG TGGGGAGCGC CATCGCCTCC GGCATCGCCG TCAGCAAGGT GCTGCACCTG GAGGGCGAGG TGAACAAGAT CAAGTCCGCC CTCCTGAGCA CCAACAAGGC GGTCGTGTCC CTGAGCAACG GGGTGTCCGT CCTCACCAGC AAGGTGCTGG ACCTGAAGAA CTACATCGAC AAGCAGCTCC TGCCCATCGT GAACAAGCAG TCCTGCCGGA TCAGCAACAT CGAGACGGTC ATCGAGTTCC AGCAGAAGAA CAACCGCCTG CTCGAGATCA CCCGGGAGTT CAGCGT GAAC GCCGGCGTGA CCACCCCCGT CTCCACGTAC ATGCTGACCA ACAGCGAGCT GCTCTCCCTG ATCAACGACA TGCCCATCAC CAACGACCAG AAGAAGCTGA TGAGCAACAA CGTGCAGATC GTGCGCCAGC AGTCCTACAG CATCATGTCC ATCATCAAGG AGGAGGTCCT CGCCTACGTG GTGCAGCTGC CGCTGTACGG GGTCATCGAC ACCCCCTGCT G GAAGCTCCA CACGAGCCCC CTGTGCACCA CCAACACCAA GGAGGCTCC AACATCTGCC TGACGCGGAC CGACCGCGGG TGGTACTGCG ACAACGCCGG CAGCGTGTCC TTCTTCCCCC AGGCCGAGAC CTGCAAGGTC CAGAGCAACC GGGTGTTCTG CGACACCATG AACTCCCTCA CGCTGCCGAG CGAGGTGAAC CTGTGCAACG TCGACATCTT CAACCCCAAG TACGACTGCA AGATCATGAC CTCCAAGACC GACGTGAGCT CCAGC GTGAT CACCTCCCTC GGCGCGATCG TCAGCTGCTA CGGGAAGACG AAGTGCACCG CCAGCAACAA GAACCGCGGC ATCATCAAGA CCTTCTCCAA CGGGTGCGAC TACGTGAGCA ACAAGGGCGT GGACACCGTC TCCGTGGGCA ACACCCTGTA CTACGTGAAC AAGCAGGAGG GGAAGCCCT GTACGTCAAG GGCGAGCCCA TCATCAACTT CTACGACC CC CTCGTGTTCC CGTCCGACGA GTTCGACGCC AGCATCTCCC AGGTGAACGA GAAGATCAAC (SEQ ID NO: 25)

3.2. dsRNA

In the present invention, when the RNA of interest is dsRNA, RNA aptamer, and ribozyme, the RNA polymerase expression unit constituting the RNA transcription system of interest is DdRp in SEQ ID NO: 1 as an RNA polymerase according to Example 1 according to Formula (1) prepared as in

Another component, the RNA unit of interest, was prepared as follows using <DdRp-operating promoter-RNA-of-interest-transcription termination sequence>.

3.2. CPB dsRNA

The target sequence of the dsRNA used in the present invention, nucleotides 30 to 309 of SEQ ID NO: 26, was selected from COPI β (Coatomer subunit beta) of Colorado potato beetle (CPB).

A plasmid vector for producing CPB dsRNA was constructed using the pUC19 cloning vector containing the ampicillin resistance gene, the N-terminal fragment of the E. coli lac Z gene, multiple cloning sites and the origin of replication.

The different terminators cloned into the same expression plasmid are shown in Table 1.

Sequences of terminators used in RNA production vectors sequence number terminator order 26 PET ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttg 27 PTH1 catctgtttt 28 PTH2 ctcatgcttg ccatctgttt tcttgcaagt cagatggga 29 rrnBT1 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 30 rrnBT2 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctac 31 CJ ctgtgtccct atctgttaca gtctcctaaa gtat 32 B1002 ccccgcttcg gcggggtttt tt 33 B1002 ccccgcccct gacagggcgg ggttttttttt 34 PTH+PET catctgtttt cttgcaagat cagctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccgga

The rrn BT+PET+PTH+PET terminator construct is shown in SEQ ID NO: 35.

aagcttgctt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacctagcat 60

aaccccttgg ggcctctaaa cgggtcttga ggggtttttt ggccatctgt tttcttgcaa 120

gatcagctga gcaataacta gcataacccc ttggggcctc taaacggggtc ttgaggggtt 180

ttttgctgaa aggaggaact atatccgga 209 (SEQ ID NO: 35)

The DNA sequence (SEQ ID NO: 36) encoding a 397-mer hairpin derived from COPI β (Coatomer subunit beta) of the Colorado potato beetle (CPB) is upstream of the terminator and T7 in the pUC19-rrn BT2+PET+PTH+PET vector. It was inserted downstream of the promoter and was called pCPB.

gatgatgtcg gactttccta atttgtcttc gatgatttga ttgctagtga ttggcaccct 60

ttggccgttc accttggcga aacctctctt ataaatcagt tctctgacag atttcaagtt 120

agggtatccc caagtgatgt aaggttcgca tattctgagc atgttgatgg tagctttgtt 180

gagcttgaca aagacaccat tgttgatttg aaggagccgg aacaattgga gaaccttgcg 240

tactttagga gctactttgt tgataccctt tatacgaatt acgaatgcca acttggcttc 300

agcgggaacg taaaagtttc ctctggtttt agcttgtcgg atcaacctaa cttcatctct 360

ttctttgagc cggtattctt taacatatg ttcggccaag tactgcgatc gcgttaacgc 420

tttatcacga taccttctac cacatatcac taacaacatc aacactcatc atctagactc 480

tcgacgacat ccactcgatc actactctca cacgaccgat taactcctca tccacgcggc 540

cgcgagcggc cgaacagtat gttaaagaat accggctcaa agaaagagat gaagttaggt 600

tgatccgaca agctaaaacc agaggaaact tttacgttcc cgctgaagcc aagttggcat 660

tcgtaattcg tataaagggt atcaacaaag tagctcctaa agtacgcaag gttctccaat 720

tgttccggct ccttcaaatc aacaatggtg tctttgtcaa gctcaacaaa gctaccatca 780

acatgctcag aatatgcgaa ccttacatca cttggggata ccctaacttg aaatctgtca 840

gagaactgat ttataagaga ggtttcgcca aggtgaacgg ccaaagggtg ccaatcacta 900

gcaatcaaat catcgaagac aaattaggaa agtccgacat catc 944 (SEQ ID NO: 36)

HT115 (DE3) cells were transformed with the plasmid prepared above. Cell culture and total RNA extraction were performed as follows. After culturing for 18 hours, the strain with the expression vector containing the PTH+PET terminator reached an OD600 of 5.78 and showed an RNA yield of 44 mg/L, and the expression vector containing the rrn BT2+PET+PTH+PET terminator The strain with reached an OD600 of 5.55 and showed an RNA yield of 132 mg/L.

The results showed that the presence of an additional medium-sized hairpin structure rrn BT2+PET+PTH+PET terminator construct led to higher RNA production yields.

3.2.2. DV49 dsRNA

In the present invention, by designing dsRNA for the corn rootworm (Diabrotica virgifera) gene, anti-sense DV49 + loop + sense DV49 + PTH-terminator + T7 driving expression of pET-T7 terminator A pUC skeletonized plasmid having a promoter was constructed and called pDV49.

The nucleotide sequence of the DV49 expression construct is provided as SEQ ID NO:37.

taatacgact cactataggg atccatgata tcgtgaacat catctacatt caaattctta 60

tgagctttct taagggcatc tgcagcattt ttcatagaat ctaatacagc agtatttggg 120

ctagctcctt cgagggcttc cctctgcatt tcaatagttg taagggttcc atctatttgt 180

agttgggtct tttccaatcg tttcttcttt ttgagggctt ggagtgcaac tcttttattt 240

ttcgacgcat ttttctttgc aagtactgcg atcgcgttaa cgctttatca cgataccttc 300

taccacatat cactaacaac atcaacactc atcactctcg acgacatcca ctcgatcact 360

actctcacac gaccgattaa ctcctcatcc acgcggccgc ctgcaggagc gcaaagaaaa 420

atgcgtcgaa aaataaaaga gttgcactcc aagccctcaa aaagaagaaa cgattggaaa 480

agacccaact acaaatagat ggaaccctta caactattga aatgcagagg gaagccctcg 540

aaggagctag cacaaatact gctgattag attctatgaa aaatgctgca gatgccctta 600

agaaagctca taagaatttg aatgtagatg atgttcacga tatcatggat aagcttgcca 660

tctgttttct tgcaagatca gctgagcaat aactagcata accccttggg gcctctaaac 720

gggtcttgag gggttttttg ctgaaaggag gaactatatc cgga 764 (SEQ ID NO: 37)

Nucleotides 1 to 17 encode the T7 promoter; Nucleotides 21 to 260 encode an anti-sense sequence that is substantially complementary to the target nucleotide sequence of the corn rootworm (Diabrotica virgifera) gene; Nucleotides 261 to 410 encode a loop forming region; Nucleotides 411 to 650 encode a sense sequence that is substantially identical to the target nucleotide sequence of the corn rootworm (Diabrotica virgifera) gene; Nucleotides 659 to 668 encode a PTH-terminator; Nucleotides 681 to 764 encode the PET-T7 terminator.

E. coli HT115 (DE3) cells were transformed with two plasmids, one plasmid containing the DV49 dsRNA expression construct and the other plasmid containing T7 RNA polymerase under the control of an inducible element (IPTG-inducible T7 polymerase) do. The cells are expanded to the desired cell density and the dsRNA yield is determined.

3.3. RNA aptamer

In the present invention, when the RNA of interest is dsRNA, RNA aptamer, or ribozyme, RNA polymerase expression unit used RNA polymerase DdRp. The RNA unit of interest is <the promoter-RNA-of-interest-transcription termination sequence on which DdRp operates>.

3.4. Ribozyme

In the present invention, when the RNA of interest is dsRNA, RNA aptamer, or ribozyme, RNA polymerase expression unit used RNA polymerase DdRp. The RNA unit of interest is <the promoter-RNA-of-interest-transcription termination sequence on which DdRp operates>.

Example 4. Preparation of RNA transcription system

The RNA transcription system of the present invention is composed of an RNA polymerase expression unit and an RNA unit of interest, and the RNA of interest is described below as mRNA, which is not limited to mRNA, and the concept is dsRNA, RNA aptamer, and ribozyme. applied to.

4.1. mRNA transcription system suitable for ctDdRP

In this example, a plasmid was used to design a DNA fragment containing an RNA polymerase expression unit providing ctDdRp and an mRNA unit of interest.

The expression vector can be selected according to the host cell. First, the pUC 19 vector (Promega, USA) is digested with HindIII/EcoRI restriction enzyme treatment, and (a) <Lac promoter - 5'UTR - Tethering domain - ctDdRp orf- ctDdRp expression unit containing transcription termination sequence>, and (b) RNA unit of interest containing <T7 promoter - 5'UTR - DNA of interest - muag - A64 - C30 - (Tethering domain)4 - histone-SL> pUC 19 was prepared.

The prepared ctDdRp expression unit was amplified using forward primer SEQ ID NO: 38 and reverse primer SEQ ID NO: 39.

Forward Primer: 5'-AAGCTTTTTA CACTTTATGC TTCCGGCTCG TATGTTG-3*?**?* (SEQ ID NO: 38)

Reverse Primer: 5*?**?*-GGATCCCTAA AAGCAAAATG CCAGCCCGAT CGGCTGGCAT TTTTATCTCA-3*?**?* (SEQ ID NO: 39)

The prepared mRNA unit of interest was amplified using forward primer SEQ ID NO: 40 and reverse primer 41.

Forward Primer: GGATCCTAAT ACGACTCACT ATAGGG (SEQ ID NO: 40)

Reverse Primer: GAATCCTGGC TGGCTCTGTT TTGTGCCTTT G (SEQ ID NO: 41)

PCR products containing the amplified ctDdRp expression unit and the mRNA unit of interest were digested using HindIII, EcoRI, and BamHI enzymes, respectively, and then cloned using pUC19 vector linearized with HindIII/EcoRI enzymes and T4 DNA ligase.

The host cell is any one selected from the group including animals, plants, yeasts and bacteria, wherein the bacteria are Corynebacterium glutamicum, Escherichia coli BE42, Escherichia coli HB101, Escherichia coli JM101, Escherichia coli LE392, Characterized in that the activity of ribonuclease III is reduced by attenuating the expression of the gene encoding the ribonuclease III or destroying the gene in any one selected from the group consisting of E. coli MC1061, E. coli Y1088 and E. coli W3110 It can be used by selecting a strain rule.

In this example, E. coli strain HT115 (DE3) with reduced activity of ribonuclease II was used.

The reaction solution was transfected into Escherichia coli HT115 (DE3), and plasmids were isolated from the selected strain, treated with HindIII/EcoRI restriction enzymes, and subjected to electrophoresis (FIG. 2).

This procedure was applied to dsRNA, RNA aptamers and ribozymes.

4.2.. mRNA transcription systems suitable for RdRp

An RNA unit comprising the mRNA of the protein of interest of the present invention can be replicated by SFV RdRp and comprises the mRNA of the protein of interest.

To generate an RNA unit containing the protein mRNA of interest suitable for RdRp, the protein mRNA unit of interest is <promoter recognized by the host cell - 5' cloned recognition sequence DNA - SGP DNA - POI orf- 3' cloned recognition sequence DNA - A64 - C30 - histone-SL> were ligated in order to produce recombinant DNA in cells as a template.

The 5' replication recognition sequence (CSE 1 & 2 DNA) was prepared by amplifying the corresponding nucleotide sequence from the Semliki Forest virus replicon plasmid (pSFV1, Invitrogen # 18448-019) purchased from ThermoFisher (USA) or organically synthesized, SGP (CSE3 DNA) and 3' replication recognition sequence (CSE 4 DNA) were organically synthesized.

mComponent of the RNA unit of interest name base sequence Length (nt) 5' cloned recognition sequence
(CSE 1&2) ATGGCGGATG TGTGACATAC ACGACGCCAA AAGATTTTGT TCCAGCTCCT GCCACCTCCG CTACGCGAGA GATTAACCAC CCACGATGGC CGCCAAAGTG CATGTTGATA TTGAGGCTGA CAGCCCATTC ATCAAGTCTT TGCAGAAGGC ATTTCCGTCG TTCGAGGTGG AGTCATTGCA GGTCACACCA AATGACCATG CAAATGCCAG AGCATTTTCG CACCTGGCTA CCAAATTGA (SEQ ID NO: 42) 239 GATGGCGGAT GTGTGACATA CAAGACGCCA AAAGATTTTG TTCCAGCTCC TGCCACCTCC GCTACGCGAG AGATTAACCA CCCACGACGG CCGCCAAAGT GCTTGTTGAT ATTGAGGCTG ACAGCCCATT CATCAAGTCT TAGCAGAAGG CATTTCCGTC GTTCGAGGTG GAGTCATTGG AGGTGACACC AAATCACCAT CCAAATCCCA GAGCATTTTC GCAC CTGGGT ACCAAATTGA TCGAGCAGGA GACTGACAAA GACACACTCA TCTTGGATAT C (SEQ ID NO: 43) 281 Kozak sequence GCC(A/G)CCATGG (SEQ ID NO: 44) 10 3' cloned recognition sequence
(CSE 4) ATTTTGTTTT TAATATTTC (SEQ ID NO: 45) 19 SGP (CSE 3) ACCTCTACGG CGGTCCTAAA TAGG (SEQ ID NO: 46) 23

The mRNA transcription system using RdRp used in the present invention was prepared by inserting the DNA fragment of the RdRp expression unit and the mRNA unit of interest into plasmid pUC19 and transforming the completed construct into a host cell.

When the host cell is Escherichia coli, first, the RdRp expression unit DNA fragment is in the order of <Lac promoter - 5'UTR - RdRp ORF DNA - transcription termination sequence>, and the m interest RNA unit DNA fragment is <Lac promoter - 5' replication recognition Sequence DNA - SGP DNA - POI orf - 3' replication recognition sequence DNA - A64 - C30 - Histone-SL> sequence.

The prepared RdRp expression unit was amplified using forward primer SEQ ID NO: 38 and reverse primer SEQ ID NO: 39.

The prepared mRNA unit of interest was amplified using forward primer SEQ ID NO: 47 and reverse primer SEQ ID NO: 39.

Forward Primer: GGATCC TTTA CACTTTATGC TTCCGGCTCG TATGTTG (SEQ ID NO: 48)

PCR products containing the amplified RdRp expression unit and the mRNA unit of interest were digested using HindIII, EcoRI and BamHI enzymes, respectively, and then subjected to cloning reaction using pUC19 vector linearized with HindIII/EcoRI enzymes and T4 DNA ligase to obtain a host cells were transformed.

In this example, E. coli strain HT115 (DE3) with reduced activity of ribonuclease II was used. The reaction solution was transfected into E. coli HT115 (DE3), and plasmids were isolated from the selected strain, treated with HindIII/EcoRI restriction enzymes, and subjected to electrophoresis (FIG. 3).

This procedure was applied to dsRNA, RNA aptamers and ribozymes.

Example 5. Mass production of RNA of interest

5.1. mRNA

The mRNA transcription system of the present invention was transformed into E. coli strain HT115 (DE3) according to the description, and then mRNA synthesis of interest was analyzed at 0 and 24 hours.

The cultured E. coli was diluted in cold washing buffer (10 mM Tris-Cl pH 8.0, 150 mM NaCl) and harvested. Dissolve the harvested E. coli in the following lysis solution. Lysis solution is B-Per (Thermo Scientific™ B-PER™) containing 1 mM Mg-OAc, 0.5 mM CaCl, ~0.1 mM EDTA, 0.01-0.1 mg/mL lysozyme and RNase-free DNase, Roche DNase used I was diluted 1/1000 with B-Per with Mg and Ca to 10 units per mL final.

Cold buffer solution (50 mM bis-Tris pH 6.5 containing 400 mM NaCl, 5 mM EDTA and 1 μL/400 μL of 10 mg/mL linear polyacrylamide (LPA)) in the harvested solution after principle separation of the dissolved sample is about 200-400 uL was added. This solution chelates all Mg++, disrupts ribosomes, increases ionic strength, and provides a carrier for precipitation.

About 300-500 μL of acidic phenol/chloroform was added. Control RNA may be added at this step for quantification. The sample is mixed and left on the bench for about 5 minutes. Centrifuge for 5 minutes and transfer the supernatant to a new tube. If more clean RNA is required, the extraction can be repeated and a denaturing gel can be run.

A slightly larger than equal volume of isopropanol was added to the tube with the supernatant. Place the tube on ice for 30 minutes or overnight at -20 degrees. It was then centrifuged for 20 minutes.

After removing the supernatant by aspiration, the tube was washed with ~700 μL of 75% ethanol to remove salts, spun again, aspirated and then dehydrated by washing with 95% ethanol, spun again, aspirated and the tube dried . Make sure the pellet is dry before resuspending it.

After drying, in undenatured solution (10 mM Bis-Tris, pH 6.5, 0.1 mM EDTA) or denatured solution (95% stabilized formamide, 10 mM Bis-Tris pH 6.5, 0.1 mM EDTA, some bromophenol blue to taste). resuspend

For each mL of the harvested culture medium, about 50 μL was subjected to A260/280 irradiation and electrophoresis using polyacriamide gel (FIG. 4).

The purification method according to the mass production of the mRNA of interest includes the steps of (a) precipitating the mRNA of interest from an impure preparation; and (b) subjecting the impure preparation containing the precipitated mRNA of interest to a purification process including membrane filtration to capture the precipitated mRNA of interest into a membrane; and (c) eluting the captured precipitated mRNA of interest from the membrane by resolubilizing the mRNA of interest, thereby harvesting a purified mRNA solution of interest.

Isolation and purification to obtain mRNA is possible due to differences in size, electric charge, hydrophobicity, binding affinity to a specific ligand, and the like.

5.2.dsRNA

E. coli strain HT115 (DE3) was transformed with the RNA transcription system of interest containing the pCPB. After transformation, the E. coli cells were plated and single colonies were selected.

Single colonies were grown for 6-8 hours at 37° C. in 3 ml of LB medium containing 100 μg/ml ampicillin and 12.5 μg/ml tetracycline to produce seed cultures. To induce the expression of dsRNA, 200ul of seed culture was inoculated with 50ml of autoinduction medium (AIM) containing 100ug/ml ampicillin and 12.5ug/ml tetracycline (Studier, Protein Expression and Purification 41 (2005)). 207-234) were inoculated into 250 ml flasks and then cultured. The cells were harvested by centrifugation at 6000g for 10 minutes at 4°C.

Bacterial RNA was purified using a method adapted from Stead's SNAP protocol (Stead et al, Nucleic Acids Res. 2012 Nov 1; 40(20):el56.) while modifying the RNA extraction buffer as described below. did

1 ml of bacterial culture (~ 10 ⁸ cells) was centrifuged at 16,000 g for 30 seconds and the supernatant removed. The cell pellet was stored on dry ice until ready for extraction. The cell pellet was then resuspended in 100 μl of modified RNA extraction solution [18 mM EDTA, 0.025% SDS, 5 mM TCEP, 95% formaldehyde (RNA grade)] by vigorous vortexing. The 1% 2-mercaptoethanol used in the RNA extraction solution of Stead's SNAP protocol is equivalent to 5 mM TCEP as TCEP has much lower toxicity compared to 2-mercaptoethanol and has an equivalent potency to 2-mercaptoethanol. replaced by (data not shown).

After resuspension in the modified RNA extraction solution, the cells were lysed by incubating the samples at 95° C. in a water bath for 7 minutes. The cell lysate was pelleted by centrifuging the warm sample at 16,000g for 5 minutes at room temperature. The supernatant was carefully transferred to a new tube without disrupting the pellet.

Total RNA isolated from uninduced or induced pCPB E. coli cells was diluted with HO and run on an agarose gel. As shown in Fig. 5, a band corresponding to CPB dsRNA was observed in induced pCPB E. coli cells but not in uninduced pCPB E. coli cells.

The pCPB plasmid was linearized and used as a template for in vitro transcription of RNA. In vitro transcribed RNA was run on an agarose gel with purification of bacterially transcribed RNA and either diluted directly in HO or filtered with a 30K molecular cut membrane (Amicon) before dilution with HO to determine the size of RNA transcripts. to remove reagents such as EDTA, SDS, TCEP and formamide. The in vitro transcribed RNA was observed to be larger than bacterially transcribed RNA (FIG. 5).

The in vitro and in vivo (bacterially) transcribed RNA was incubated with RNAse A, which degrades single-stranded RNA but not double-stranded RNA. As shown in Figure 5, after RNAse A digestion, the size of RNA transcription products in vivo and in vitro is identical. This indicated that the increased size of in vitro transcribed RNA was due to the presence of single-stranded hairpin loops removed from bacterial cells by RNA processing.

Example 6. Preparation of protoplasts from Gram-negative bacteria by lysozyme treatment

The RNA transcription system of interest of the present invention was introduced into the target gram-negative bacteria.

It is a schematic diagram showing a method of loading a drug into vesicles derived from protoplasts after protoplasts are prepared by lysozyme treatment in Gram-negative bacteria into which the RNA transcription system of interest has been introduced.

First, protoplasts were prepared from Gram-negative bacteria. Specifically, Gram-negative E.coli HT115 (DE3) bacteria were grown in LB medium at O.D600 = 1.0, centrifuged at 3,000 × g for 10 minutes to settle the bacteria cells, and then bacterial cells were obtained. After resuspension in 100 ml of a previously prepared protoplasting buffer, EDTA was slowly added to a concentration of 0.01 M, followed by incubation at 37° C. for 40 minutes while shaking at 100 rpm. Centrifugation was performed again at 3,000 × g for 10 minutes to obtain settled bacterial cells, which were then suspended in 100 ml of a protoplasting solution containing 0.02 M MgCl2.

2mg/ml of lysozyme was added to the suspension solution, incubation was performed at 37°C for 2 hours while shaking at 100 rpm, and centrifugation was performed at 5,000 × g for 10 minutes to obtain protoplasts that had settled and CaCl2 and MgCl2 were 0.02 M was suspended in 3 ml of TBS (Tris buffered saline) solution. In addition, a supernatant in which the outer membrane and cell wall were removed was obtained.

To confirm that protoplasts were produced, biotin-labeled bacteria and protoplasts derived from biotin-labeled bacteria were prepared, respectively. Unbroken bacterial cells were removed by centrifugation at 5,000 × g for 10 minutes, and a protein-containing supernatant was obtained. Proteins obtained from each sample and the supernatant containing the outer membrane and cell wall that were removed when making protoplasts were finally stained with 5x loading dye (250 mM Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol), respectively. 1 x, and high-temperature treatment at 100 ° C. for 5 minutes. An 8% polyacrylamide gel was prepared and the sample was loaded. After electrophoresis at 80V for 2 hours, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane for 2 hours at 400 mA. After dissolving skim milk to 3% in PBS, the membrane was blocked in this solution for 2 hours. Peroxidase-attached biotin antibody was treated at 4° C. for 12 hours. After washing with PBS for 30 minutes, ECL (enhanced chemiluminescence, Amersham Co. No. RPN2106) substrate was identified and shown in FIG. 6 .

As shown in FIG. 2 , an antibody response to biotin was shown in the case of bacteria, but an antibody response to biotin was not shown in the case of protoplasts. In addition, an antibody response to biotin was shown in the supernatant containing the biotin-labeled outer membrane and cell wall. The above result means that when the lysozyme was treated, the outer membrane and cell wall present in the bacteria were removed, and the proteins present in the outer membrane and cell wall were removed. From the above results, it was confirmed that protoplasts in which the bacterial outer membrane and cell wall were removed by lysozyme were prepared.

Example 7. Preparation of protoplast-derived vesicles

7.1. Preparation of Gram-negative bacterial protoplast-derived vesicles by extrusion and ultrasound

Protoplast-derived vesicles were prepared using extrusion and ultrasound. The protoplasts of Gram-negative bacteria into which the RNA transcription system of interest was introduced were passed through a membrane filter with a pore size of 10 μm three times, then passed through a membrane filter with a pore size of 5 μm three times, and then passed through a membrane filter with a pore size of 1 μm. A primary protoplast-derived vesicle suspension was obtained by passing through a μm membrane filter three times.

Primary protoplast-derived vesicle suspensions were sonicated in 10 mM Tris-HCl (pH 8.0) at 4 °C using a Q55 Sonicator (20 kHz, QSonica, Newtown, CT). The amplitude of the sonicator was set to 40 and ultrasonic treatment was performed for 2 minutes using an ultrasonic probe having a diameter of 3 mm.

Unbroken cells were removed by centrifugation at 8000 x g for 5 minutes, and finally the samples were sonicated in an ultrasonic bath at 44 kHz (Grant, Cambridge, UK) for 30 minutes to obtain a secondary protoplast-derived vesicle suspension.

For separation and purification of the vesicles produced through the extrusion method, the vesicles were purified using tangential flow filtration (TFF) to have a size of 100 to 200 nm and a uniformity of PDI of 0.2 to 0.3 .

Specifically, cell-derived vesicles were separated and purified using Spectrum® KR2i TFF Systems (Repligen), hollow fiber filter (Repligen, 750 kDa, 0.01 m ² ).

Benzonase nuclease (Merck, 71206-3), 50mM Tris-HCl (Teknova, 50-843-335), 2mM chloride in proportion to the DNA amount of the vesicle suspension extracted from the cell extruder in the same manner as above Magnesium chloride (SIGMA-ALDRICH, M1028-100mL) was treated at 37°C for 90 minutes. Before separating/purifying the vesicles using the TFF method, aggregates in the vesicle suspension were removed by centrifugation at room temperature at 3000 x G for 10 minutes. The suspension was concentrated at a flow rate of 80 mL/min while maintaining a transmembrane pressure of 0.2 bar, and impurities were filtered out. In the process of ultrafiltration, the suspension was concentrated until the volume of the suspension was about 1/4, and impurities were removed through desalination and buffer exchange in the process of diafiltration. Desalting and buffer exchange were performed continuously, and a buffer solution (PBS) having a volume at least 6 times the volume of the concentrated solution was used. Finally, the ultrafiltration process was performed again, and the volume of the suspension was concentrated until it became 1/20 of the starting volume (the volume of the vesicle suspension before purification). The separated/purified cell-derived vesicles were centrifuged at 3000 x G for 10 minutes at room temperature to remove aggregates, and filtered through a 0.45um syringe filter (pal, 4614) to finally obtain cell-derived vesicles. Thereafter, the physical properties of the purified cell-derived vesicles were analyzed through DLS, NTA, and measurement, and the impurity removal rate compared to before purification was confirmed through quantification of protein, DNA, and benzonase.

Cell-derived vesicles were prepared using the extrusion method. Gram-negative bacteria introduced with the RNA transcription system of interest were passed through a membrane filter with a pore size of 10 μm three times, then passed through a membrane filter with a pore size of 5 μm three times, and then passed through a membrane filter with a pore size of 1 μm three times. A cell-derived vesicle suspension can also be obtained by passing through a membrane filter three times.

7.2. Characteristics of vesicles derived from protoplasts of Gram-negative bacteria

Vesicles derived from protoplasts of E. coli, a gram-negative bacterium into which the RNA transcription system of interest was introduced, were prepared and their characteristics were analyzed.

Specifically, each vesicle was adsorbed on a glow-discharged carbon-coated copper grid for 3 minutes. After washing the grid with distilled water, it was stained with 2% uranyl acetate (uranylacetate). It was observed with a JEM101 (Jeol, Japan) electron microscope and the results are shown in FIG. 7 .

7 is a transmission electron microscope (TEM) photograph of a vesicle derived from a gram-negative bacterium, Escherichia coli protoplast. As can be seen from the transmission electron microscopy image of FIG. 7, it was confirmed that the protoplast-derived vesicles prepared from bacteria by the extrusion method consisted of a lipid bilayer and were generally spherical. It can be seen that the protoplasts derived from Gram-negative bacteria have a size of 100 to 200 nm.

In addition, each of the vesicles was diluted in 1 ml of PBS at a concentration of 5 ug/ml. 1 ml of PBS containing each vesicle was placed in a cuvette and analyzed with a dynamic light scattering particle size analyzer, and the results are shown in FIG. 8 .

7.3. Preparation of bacterial protoplast-derived vesicle loaded with EGF fusion protein

In order to load human EGF on the surface of the protoplast, in order to bind to the nucleotide sequence encoding the peptide region of the 290th amino acid from the N-terminus of the nucleotide sequence encoding PrsA, a bacterial inner membrane protein, first stop based on the known DNA sequence of PrsA Primers were prepared as follows by artificially inserting a specific restriction enzyme except for the codon.

Forward Primer: 5'-CATATGAAGAAAATCGCAATAGCAG-3' (SEQ ID NO: 49)

Reverse Primer: 5'-TCTAGATTTAGAATTGCTTGAAGATGAAG-3' (SEQ ID NO: 50)

(Bold font indicates the recognition sequences of restriction enzymes NdeI and XbaI)

After PCR polymerization using the primers and genomic DNA obtained from the strain B. subtilis as a template, the resulting PCR band was extracted and purified using a Quiaquick gel purification kit. The purified product was treated with restriction enzymes NdeI and XbaI at 37° C. for 8 hours, then purified again with a QuickAquick PCR purification kit, and then ligated into a pHCE vector treated with the same enzymes and inserted. The prepared recombinant vector was named "pHCE-prsA".

In order to fuse the gene expressing human EGF to the pHCE-prsA vector, a specific restriction enzyme was artificially inserted based on the DNA nucleotide sequence of known human EGF, and primers were prepared as follows.

Forward Primer: 5′- GCTCTAGAAATACTGACTCTGA ATGTCCC-3′ (SEQ ID NO: 51)

Reverse Primer: 5'- CAAGCTTTCAGCGCAGTTCC CACCACTTC-3' (SEQ ID NO: 52)

(Bold font means recognition sequences of restriction enzymes XbaI and HindII)

After PCR polymerization using the primers and genomic DNA obtained from human non-small cell lung cancer A549 cells as a template, the resulting PCR band was extracted and purified using a QuickAquick gel purification kit. The purified product was treated with restriction enzymes XbaI and HindIII at 37°C for 8 hours, then purified again with the QuickAquick PCR purification kit, and then ligated with the pHCE-prsA vector treated with the same enzymes and inserted (FIG. 9). The prepared recombinant fusion vector was named "pHCE-prsA-EGF".

E. coli HT115 (DE3) into which the RNA transcription system of interest was introduced was transformed with the pHCE-prsA-EGF fusion vector by heat shock transformation, and 1 ml LB medium was added to the transformed E. coli strain and grown at 37 ° C. for 1 hour. After incubation for 16 hours at 37 ℃ in the LB agar plate mixed with ampicillin (ampicilin). Among the transformed E. coli colonies generated on the agar plate, colonies containing pHCE-prsAEGF were selected and obtained through colony-PCR and restriction enzyme mapping.

EGF fusion protein-loaded and unloaded vesicles were prepared from the previously prepared pHCE-prsA-EGF vector and from Escherichia coli, a gram-negative bacterium introduced with the RNA transcription system of interest, and from E. coli without it. After preparing 20 μg of each of the prepared vesicles, 5 x loading dye was finally added to 1 x, and treated at 100 ° C. for 5 minutes.

A 12% polyacrylamide gel was prepared and the sample was loaded. After electrophoresis at 80V for 2 hours, proteins were transferred to a PVDF membrane at 400 mA for 2 hours. After dissolving skim milk to 3% in PBS, the membrane was blocked in this solution for 2 hours. The EGF antibody was treated at 4°C for 12 hours. After washing twice with PBS, the secondary antibody attached with peroxidase was treated for 1 hour at room temperature. After washing with PBS for 30 minutes, the ECL matrix was identified and shown in FIG. 10 .

As shown in FIG. 10, it was confirmed that the EGF fusion protein-loaded vesicles were identified by the EGF antibody and appeared black. In contrast, vesicles without EGF were not identified. From the above results, it is clear that the vesicle loaded with the EGF fusion protein was properly made.

In addition, it was confirmed using trypsin to confirm that the EGF fusion protein is properly positioned toward the outside of the outer surface of the vesicle. Taking advantage of the fact that trypsin does not pass through bacterial protoplasts, vesicles were treated with trypsin to digest a region exposed to the outside of vesicles among cell membrane proteins, and an experiment was performed to determine whether EGF fusion proteins remained. First, after treatment with trypsin, treatment with high temperature to denature trypsin, dissolution of the vesicles, exposing both proteins inside and outside the membrane to the solution, and treatment with an antibody recognizing EGF, the results are shown in FIG. 11 .

In FIG. 11, '+' indicates the result of treatment with trypsin, and '-' indicates the result of not treating with trypsin.

As shown in FIG. 11, it can be deduced that all of the EGF portions of the EGF fusion protein are directed to the outside of the vesicle, since all of the EGF antibody marks disappeared when trypsinization was performed, which indicates that the vesicle having the EGF fusion protein is EGF. This means that it can be delivered specifically to cells having receptors.

7.4. Confirmation of cell-specific delivery of vesicles loaded with EGF fusion proteins

In order to confirm the cell-specific delivery of the RNA transcription system of interest and the in vitro EGF fusion protein-loaded vesicle in vitro, human non-small cell lung cancer A549 cells were inoculated in an amount of 2×10 ⁴ in a 24-well plate and then , cultured for 24 hours in a 24-well plate. A549 cells are cells expressing a large amount of the EGF receptor in the cell membrane.

Vesicles were prepared from protoplasts of E. coli introduced with the prepared EGF and the RNA transcription system of interest and protoplasts of E. coli without EGF. DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate, Invitrogen, No. V22885) staining agent was added to the prepared E. coli protoplast-derived vesicles to a concentration of 5 μM and stored at room temperature for 30 minutes. DiI staining exhibits red fluorescence.

After washing twice with PBS, 500 μl of medium was added to the plate on which the cells were laid, treated with DiI-labeled vesicles at a concentration of 10 μg/ml, and incubated for 20 minutes.

After washing with PBS, 500 μl of serum-free medium was added, and CellTracker solution was added to a concentration of 5 μM, followed by incubation for 30 minutes. After washing with PBS, 500 μl of medium containing serum was added and incubated for another 30 minutes. The cover glass was put in 500 μl of 4% paraformaldehyde and stored at room temperature for 10 minutes. Using a fluorescence microscope, cells tinged with green were observed at the FITC wavelength and vesicles tinged with red at the Rhodamine wavelength. The number of cells and the number of vesicles were measured, and the number of vesicles was divided by the number of cells to calculate the number of vesicles per cell. Results are shown in FIG. 12 .

As shown in FIG. 12, the number of vesicles derived from E. coli protoplasts without EGF bound to one A549 cell was about 1.2, and the number of vesicles derived from E. coli protoplasts loaded with EGF bound to one A549 cell was about 7.9. It is a dog.

The above result means that the EGF-loaded vesicles can enter cells expressing the EGF receptor present in the cell membrane, such as A549.

In addition, in order to examine whether this binding was caused by specific binding between EGF-EGF receptors, mouse colon cancer cells (CT26) were inoculated in an amount of 2×10 ⁶ in a 12-well plate, and then incubated in a 12-well plate for 24 hours. cultured. CT26 is a cell that expresses the mouse EGF receptor on its cell membrane, but can bind to human EGF. After washing twice with PBS, 500 μl of medium was added to the well plate, treated with 100 ng/ml of EGF protein, and incubated for 30 minutes to prevent EGF receptors from binding to EGF on the surface of CT26 cells. Then, the cells were washed with PBS to remove remaining EGF protein, treated with DiI-labeled vesicles at a concentration of 10 μg/ml, and incubated for 20 minutes.

After washing with PBS, 500 μl of a serum-containing medium was added and incubated for another 30 minutes, and then added to 500 μl of 4% paraformaldehyde and stored at room temperature for 10 minutes. Vesicles tinged with red were observed at the Rhodamine wavelength using a fluorescence microscope.

As shown in FIG. 13, when EGF protein was pre-treated to block the EGF receptor, it was confirmed that all red signals showing vesicles bound to cells disappeared compared to when EGF protein was not treated.

The above result means that the vesicle expressing the EGF fusion protein can bind to the EGF receptor present in the cell membrane and enter the cell.

The results of ELISA for the expression pattern of the mRNA of interest are shown in FIG. 14, and the protein of interest increased proportionally with time when EGF-free protoplast-derived vesicles and EGF-fused protoplast-derived vesicles were introduced into human non-small cell lung cancer A549 cells. . The latter increased significantly over time than the former.

7.5. Induction of apoptosis of colorectal cancer cells in vitro using protoplast-derived vesicles loaded with doxorubicin

A cover glass coated with 0.1% gelatin was placed in a 24-well plate, and a mouse colon cancer 26 cell line (mouse colon 26) was inoculated in an amount of 2×10 ⁴ on the cover glass, and cultured in the 24-well plate for 16 hours.

After washing twice with PBS, 500 μl of medium was added, and vesicles derived from protoplasts of Gram-positive bacteria prepared according to the methods of Examples 1 and 2 and vesicles derived from protoplasts of doxorubicin-loaded Gram-positive bacteria prepared according to the method of Example 17 were added. The cells were treated at concentrations of 0, 1.25, 2.5, and 5 μg/ml, respectively, and then cultured for 24 hours.

After washing with PBS, 500 μl of serum-free medium was added, and Cell Tracker solution was added to a concentration of 5 μM, followed by incubation for 30 minutes. After washing with PBS, 500 μl of medium containing serum was added and incubated for another 30 minutes. The cover glass was put in 500 μl of 4% paraformaldehyde and stored at room temperature for 10 minutes. A photograph was taken using a fluorescence microscope, and the number of living cells was measured and shown in FIG. 15 .

15 shows the number of living cells when protoplast-derived vesicles not loaded with doxorubicin and protoplast-derived vesicles loaded with doxorubicin were added at each concentration, % of control = {number of living cells at each concentration / (0 ug/ The number of viable cells at ml concentration) } * 100 It is obtained by the formula and shown in a table.

As shown in FIG. 15 , it can be seen that the doxorubicin-loaded protoplast-derived vesicle induces colon cancer cell death much more effectively than the protoplast-derived vesicle itself without doxorubicin-loaded.

From the above results, it is clear that doxorubicin loaded into protoplast-derived vesicles is delivered to colon cancer cells and induces apoptosis.

Example 8. Biological efficacy of dsRNA

Protoplast-derived vesicles were prepared from Escherichia coli HT115 (DE3) into which the pCPB transcription system of interest was introduced according to the above section 7.1. Bioefficacy of dsRNA protoplast-derived vesicles was tested against the Colorado potato beetle (CPB), Leptinotarsa decemlineata.

Bioassay using CPB larvae was performed in 13.2 g/L agar (Serva 11393), 140.3 g/L Bio-Serve pre-mix (F9380B), 5 ml/L KOH (18.3 % w/w), and 1.25 ml/L formalin (37%) of the artificial diet. The diet was dispensed into a 96-well plate with 200 uL of the titrant and briefly dried before sample application.

Twenty (20) uL titrations of test samples (dsRNA protoplast derived vesicles) were applied per well and sterile water was used as an untreated control (UTC). Plates were left to dry before adding larvae. One neonate CPB larva was added per well with a fine paint brush. The plate was then sealed with mylar and ventilated using insect pins.

32 larvae were tested per treatment.

The bioassay plates were incubated at 27° C., 60% relative humidity (RH) in complete darkness for 10-12 days. The plates were then scored for mortality (Table 2) and larval stunting. Data were analyzed using JMP©4 statistical software (SAS Institute, 1995) and complete factorial ANOVA was performed with Dunnet's test to observe treatment effects compared to untreated controls (P<0.05). A Tukey-Kramer post hoc test was performed to compare all pairs of treatments (P<0.05).

All dsRNA protoplast-derived vesicle preparations showed significant activity against the Colorado potato beetle. For example, 87.5% of beetle growth was inhibited at the lowest concentration of dsRNA protoplast derived vesicles tested (0.00002 mg/ml).

Example 9. mRNA transcription system of interest suitable for eukaryotic cells

In order to achieve the above object in eukaryotic organisms, the present invention is an RNA polymerase mRNA unit that can be translated in a eukaryotic cell and / or is transcribed and translated by the RNA polymerase. It is provided as an RNA Polymerase DNA unit that becomes

When the host cell is a eukaryotic cell, the RNA polymerase expression unit is a) an mRNA construct capable of translating any one selected from the group including DNA dependent RNA Polymerase (DdRp) ORF and variants thereof in the host cell, wherein , or mRNA capable of translating the catalytic unit of the capping enzyme may be added to the mRNA structure; and

(b-1) a promoter operating on the DdRp; (b-2) 5'UTR; (b-3) any one selected from the group containing DNA dependent RNA Polymerase (DdRp) ORF and variants thereof, or any one selected from the group containing RNA dependent RNA Polymerase (RdRp) ORF and variants thereof; and (b-4) 3'UTR;

9.1. Preparation of ctDdRp expression unit

In this embodiment, the RNA polymerase is ctDdRp, the RNA polymerase expression unit is a ctDdRp mRNA unit, and a ctDdRp DNA fragment can be prepared as a template.

The ctDdRp mRNA unit has a 5*?**?* cap structure and a 5*?**?*UTR sequence upstream of the ctDdRp orf, a stop codon downstream, a 3*?**?*UTR sequence and Poly(A) It can consist of a tail sequence, a poly(C) sequence, and a histone stem-loop.

The constituent units of the ctDdRp orf are DdRp, capping enzyme and poly(A) polymerase>, and a linker or a ribosome skipping domain may be included between the constituent units of the orf included in the RNA unit.

The orfs of related proteins were optimized for expression in human cells with respect to the codon adaptation index using the GeneOptimizer algorithm. All gene sequences were artificially synthesized, amplified by step-by-step PCR using oligonucleotides, cloned and fully sequenced.

To prepare the RNA unit of the present invention, first, a promoter-ctDdRp DNA fragment used as a template in the in vitro transcription process for preparing the RNA unit is designed and prepared, cloned into pUC19, and then the promoter-ctDdRp mRNA DNA fragment is PCR-reacted. In vitro transcription is performed using the product obtained by amplification as a template.

In order to prepare the RNA unit of the present invention, the ctDdRp orf was designed according to the same formula (2) as in the above section 2.2.

Formula (2): Nλ-X1-R341-X2-Nλ-X1-NP868R-X3-T7 Pol

where Nλ = tethering domain, X1 = G4, X2 = T2A, X3 = F2A.

Enzymes prepared in Formula 3 may be Nλ-X1-R341, Nλ-X1-NP868R and T7 Pol.

The promoter-ctDdRp DNA fragment places a T7 promoter and a 5*?**?*UTR sequence upstream of the orf of Formula 2, and a stop codon, a 3*?**?*UTR sequence and a Poly(A) tail downstream. sequence, poly(C) sequence and histone stem-loop.

For the design of ctDdRp DNA included in the promoter-ctDdRp DNA fragment of the present invention, DdRp constituting the core structural unit ctDdRp ORF of Formula 1 is a type of DdRp composed of a single subunit, T7 RNA polymerase (Gene ID: 1261050) , T7 RNA polymerase was selected from the group containing T3 RNA polymerase (Gene ID: 14675519) and SP6 RNA polymerase (Gene ID: 19685893).

Acanthamoeba polyphaga mimivirus R341 (UniProtKB/Swiss-Prot accession number E3VZZ8) was selected as the core structural unit Poly(A) Polymerase in formula (2).

Capping enzyme, the core structural unit of formula (2), was selected as African swine fever virus NP868R capping enzyme (NCBI ASFV genome sequence strain BA71 V NC_001659; UniProtKB/Swiss-Prot accession number P32094).

In the RNA/DNA system of the present invention, ctDdRp in the RNA unit is a tethering domain (Nλ) of the protein-RNA tethering system, as shown in equation (2), and each of the DNA units has a protein -Added tethering RNA of RNA tethering system.

In the tethering system, the tethering domain is 22 amino acids of antitermination N protein from lambda bacteriophage (Nλ; amino acids 1-22 of enterobacteria phage lambda nucleocapsid protein AAA32249; SEQ ID NO: 5 and SEQ ID NO: 6 corresponding to nucleotides) was selected.

ATGGATGCTC AGACCAGACG CAGAGAACGG CGGGCAGAGA AGCAGGCACA GTGGAAGGCC GCTAAT 66 (SEQ ID NO: 5)

Met Asp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala Gln Trp Lys Ala Ala Asn 22 (SEQ ID NO: 6)

The tethering RNA contains BoxBr of the λ virus (nucleotides 38312-38298 of the genomic sequence of enterobacterial phage lambda KT232076.1; SEQ ID NO: 7 corresponding to the nucleotide sequence of the BoxBr RNA stem-loop from λ bacteriophage) in series of four Constructed RNA sequences were selected.

GCCCTGAAAA AGGGC 15 (SEQ ID NO: 7)

In the linker, X1 is G4 or (G4S) ₂ .

F2A and T2A were selected from the 2A self-cleaving peptidis in <Table 3> and used as the ribosome skipping domain and in vivo cleavable linkers instead of the intercistronic spacer (IS) in section 2.2.

The ribosome skipping domain is residues 934-955 of foot-and-mouth disease virus aphtovirus (UniProtKB/Swiss-Prot AAT01756, also referred to as "F2A") or porcine tescovirus-1 (residues 979-979 of UniProtKB/Swiss-Prot Q9WJ28). 997, also referred to as “T2A”).

2A self-cleaving peptide designation amino acid sequence T2A (GSG) EGRGSLL TCGDVEENPGP (SEQ ID NO: 53) P2A (GSG) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 54) E2A (GSG) QCTNYalLKLAGDViNPGP (SEQ ID NO: 55) F2A (GSG) VKQTLNFDLLKLAGDViNPGP (SEQ ID NO: 56)

T2A: 5'GGAAGCGGAG AGGGCAGAGG AAGTCTGCTA ACATGCGGTG ACGTCGAGGA GAATCCTGGA CCT-3' (SEQ ID NO: 57)

P2A: 5'GGAAGCGGAG CTACTAACTT CAGCCTGCTG AAGCAGGCTG GAGACGTGGA GGAGAACCCT GGACCT-3' (SEQ ID NO: 58)

E2A: 5'GGAAGCGGAC AGTGTACTAA TTATGCTCTC TTGAAATTGG CTGGAGATGT TGAGAGCAAC CCTGGACCT-3' (SEQ ID NO: 59)

F2A: 5'GGAAGCGGAG TGAAACAGAC TTTGAATTTT GACCTTCTCA AGTTGGCGGG AGACGTGGAG TCCAACCCTG GACCT-3' (SEQ ID NO: 60)

The RNA unit has a 5*?**?* cap structure and a 5*?**?*UTR sequence upstream of the ctDdRp orf, a stop codon downstream, a 3*?**?*UTR sequence and a Poly(A) tail sequences, poly(C) sequences and histone stem-loops.

The 5*?**?*CAP structure is described in the Examples below.

5*?**?*UTR sequences contain the Kozak consensus.

Kozak consensus sequence ACCAUGG (SEQ ID NO: 13).

3*?**?*UTR sequence is muag (mutated alpha-globin-3'UTR) sequence is SEQ ID NO: 17 (underlined nucleotides are mutations compared to the wild-type sequence). The poly(A) tail consists of 64 adenine nucleotides, the poly(C) sequence consists of 30 cytosine nucleotides and the histone stem-loop is an RNA sequence according to SEQ ID NO: 17.

muag - A64 - C30 - Histone SL: GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG AGATTA ATAAAAAAAA AAAAAAAAAAA AAAAAAAAA AAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAATGCA TCCCCCCCCC CCCCCCCCCC CCCCCCCCCC CCAAAGGCTC TTTTCAGAGC CACCA (SEQ ID NO: 61):

muag: 5'-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3' (SEQ ID NO: 17)

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAAAA AAAAAAAAA AAAAAAAA (SEQ ID NO: 14)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 16)

Histone stem-loop: 5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' (SEQ ID NO: 17)

In the present invention, the ctDdRp DNA fragment was synthesized with <T7 promoter - 5'UTR -ctDdRp orf- muag - A64 - C30 - Histone-SL> (Biosynthesis, USA), and a forward primer 5'- GAATTC TAAT ACGACTCACT ATAG was used as a template. GGGAGA CCACAACGGT TTCCCTCTAG AAAGAACC AT G -3' (SEQ ID NO: 62) and reverse primer 5'- GGATCC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 63). Here the target sequence of T7pol is underlined. Both ends of the primers used contain EcoRI/BamHI site sequences.

The amplified PCR product was digested using EcoRI/BamHI enzymes, and then cloned using pUC19 vector linearized with EcoRI/BamHI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and plasmids were isolated from the selected strain, treated with EcoRI/BamHI restriction enzymes, and subjected to electrophoresis (FIG. 5).

The prepared recombinant pUC19 can synthesize mRNA by ctDdRp synthesized from ctDdRp RNA units in prokaryotes and synthesize ctDdRp by protein synthesis tools, so ctDdRp can also be used. However, in the present invention, since eukaryotic cells do not have an origin of replication, they serve as expression vectors only for a short period of time.

The RNA unit of the present invention was prepared by preparing a ctDdRp DNA fragment and in vitro transcription using the prepared ctDdRp DNA fragment as a template.

In order to prepare the ctDdRp RNA unit of the present invention, ctDdRp DNA containing <T7 promoter - 5'UTR - ctDdRp orf - muag - A64 - C30 - histone -SL> was designed and constructed in the above example and inserted into the pUC19 cloning vector. After recombination and in vitro transcription of ctDdRp DNA having a T7 promoter obtained from a recombinant pUC19 vector as a template, a 5'Cap structure was added.

In order to initiate self-transcription of ctDdRp containing T7 polymerase in the RNA/DNA system of the present invention, an initial supply of ctDdRp from the RNA unit is required. In this example, a ctDdRp RNA unit containing capped T7pol mRNA was prepared.

To synthesize the capped ctDdRp mRNA, first, the ctDdRp pUC19 vector prepared in the above example was amplified using forward primer (SEQ ID NO: 39) and reverse primer SEQ ID NO: 40 as a template. Here the target sequence of T7pol is underlined. The PCR product was transcribed in vitro as a template (FIG. 6).

The ctDdRp RNA expression unit containing the capped ctDdRp mRNA was synthesized in vitro using the T7 promoter-ctDdRp DNA template using the HighYield T7 ARCA mRNA Synthesis Kit (m5CTP/Ψ-UTP) (Jena BioScience, USA).

Specifically, a reaction solution (6 mM ARCA, 1.5 mM GTP, 7.5 mM 5-Methyl-CTP, 7.5 mM Pseudo-UTP, 7.5 mM ATP). DNA nuclease was added to remove the dsDNA template, and the remaining capped ctDdRp mRNA was purified using LiCl extraction and ethanol precipitation.

As a control, synthesis of capped T7pol mRNA by adding a 5'cap structure to the native RNA transcript in the ctDdRp vector was performed using the HiScribe ^™ T7 ARCA mRNA Kit (NEB, USA).

Capped ctDdRp mRNA can also be added with a natural cap (m7G(5')ppp(5')G; m7GpppG) to the IVT product with the ScriptCap m7G capping system (Epicentre Biotechnologii, USA).

9.2. Preparation of the mRNA unit of interest

The mRNA unit of interest in the present invention included any one selected from the group consisting of a promoter and a DNAi fragment to which ctDdRp can bind and function, a ctDdRp DNA fragment, and a (ctDdRp+DNAi) fragment.

The constituent units of the mRNA unit of interest included DdRp orf, capping enzyme orf, poly(A) polymerase orf, and DNA corresponding to the mRNA of interest, and a linker and a ribosome skipping domain were included between the constituent units.

The mRNA unit of interest, either singly or in combination, comprises ① a DNAi unit comprising DNAi encoding the mRNA of interest downstream of a T7 promoter, ② a ctDdRp DNA unit comprising DNA encoding the ctDdRp orf downstream of a T7 promoter, and the DNAi unit. An auto_DNA unit 2 containing the auto_DNA unit 1 and ③ (ctDdRp+DNAi) unit was provided.

Specifically, the mRNA unit of interest of the present invention was prepared to include a promoter capable of binding to the ctDdRp and any one selected from the group consisting of DNAi, ctDdRp and ctDdRp+DNAi.

A linker and a ribosome skipping domain may be included between the structural units of the ctDdRp DNA fragment. Here, the structural units included DdRp orf, capping enzyme orf, poly(A) polymerase orf, and DNA corresponding to the mRNA of interest.

The auto_ctDdRp DNA unit, DNAi unit, and auto_(ctDdRp+DNAi) unit of the present invention can be constructed using a plasmid or viral vector. ① The auto_DdRp DNA unit is a DdRp DNA segment (T7 promoter - 5'UTR - ctDdRp DNA - muag - (Tethering RNA)4 -A64), ② The DNAi unit is a DNAi segment (T7 promoter - 5'UTR - DNAi - muag - (Tethering RNA)4 RNA)4 -A64) and ③ auto_(DdRp+DNAi) units were prepared by inserting (DdRp+DNAi) fragments into plasmids, respectively.

The synthesized auto_DdRp DNA fragments, DNAi fragments, and auto_(DdRp+DNAi) fragments were amplified by PCR as templates to obtain large quantities of easily cloned auto_DdRp DNA fragments, DNAi fragments, and auto_(DdRp+DNAi) fragments. It was recombined into a linearized expression vector by treatment with a specific restriction enzyme.

The expression vectors used in this study were mammalian expression plasmid vector pCI-neo vector (Promega, USA), EEV Episomal Vector (US, System Bioscience, Catalog # EEV600A-1), AdEasy Adenoviral Vector System (Agilent Technologii, USA) pShuttle, pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector in ViraSafe ^™ Lentiviral Exprision System (Cell Biolabs, USA), shuttle vector pSMPUW, and binary vector (Ti plasmid) for plant expression, but are not limited thereto. In this Example, only plasmid vectors were limited and described, and viral vectors can be sufficiently prepared by referring to the Examples.

In the present invention, the DNA unit is a CAG promoter [cytomegalovirus (CMV) enhancer fused to the chicken beta*?*actin promoter] or remove human cytomegalovirus (CMV) immediate-early enhancer/promoter region and Poly(A) from the pCI-neo Mammalian Expression Vector (Promega, USA) to prepare an RNA unit The template DNA containing the T7 promoter of Example 1.3 was cloned and prepared.

EEV is a non-viral, non-integrating plasmid expression system based on Epstein-Barr Nuclear Antigen1 (oriP-EBNA1) to avoid problems with viral transduction systems. A key distinguishing feature of the EBNA system from viral or other plasmid-based approaches is its ability to replicate in sync with the host genome by attaching to host chromatin and replicating with each cell cycle division.

Over time, EEVs naturally lose these episomal plasmids at a rate of 5% per cell cycle division due to plasmid dilution, promoter silencing and vector duplication errors. Since most plasmids are lost over time, cell lines can be established without risk of genomic integration or alteration.

The EEV vector is a non-viral and non-integrating system for sustained foreign gene expression in cells, and the oriP-EBNA1 gene allows these plasmids to replicate and divide into daughter cells. Consequently, this EEV system has been optimized for expression in cell culture and in vivo animal models for up to 6 weeks. Several cloning sites are available to facilitate insertion of foreign DNA.

First, the DNA structure of Example 1.3 prepared for the purpose of preparing the RNA unit was used as a template and PCR was performed with a PCR primer pair having a SalI recognition site at the 5' end and an XhoI recognition site at the 3' end, and the amplified PCR product was SalI After digestion with /XhoI enzyme, CAGs-MCS Enhanced Episomal Vector (EEV) (Catalog Number # EEV600A-1, System Biosciences, USA) linearized with SalI/XhoI enzyme and cloned using T4 DNA ligase. The reaction solution was transformed into Escherichia coli, and a plasmid was isolated from the selected strain, treated with SalI/XhoI restriction enzymes, and subjected to electrophoresis (FIG. 16).

The SalI recognition site at the 5' end of the CAG promoter was used as a restriction enzyme recognition site in MCS to remove the CAG promoter.

Human cytomegalovirus (CMV) immediate-early enhancer/promoter region and SV40 Late Poly (A) were removed from the pCI-neo Mammalian Expression Vector (Promega, USA), and template DNA containing the T7 promoter for RNA unit production was cloned. did The vector is maintained episomally in cells expressing the SV40 large T antigen, leading to much higher levels of expression.

This vector also contains a neomycin phosphotransferase gene that is a selectable marker for stable transfection of mammalian cells. The pCI-neo Vector can be used for transient or stable expression by selecting cells transfected with the antibiotic G-418. Several cloning sites are available to facilitate insertion of foreign DNA fragments.

First, for the purpose of preparing the RNA unit, PCR was performed with a PCR primer pair having a BglII recognition site at the 5' end and an HpaI recognition site at the 3' end using the DNA structure of Example 1.3 as a template, and the amplified PCR product was BglII/ After digestion using HpaI enzyme, cloning was performed using pCI-neo Mammalian Expression Vector (Promega, USA) linearized with BglII/HpaI enzyme and T4 DNA ligase. The reaction solution was transformed into Escherichia coli, and a plasmid was isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and subjected to electrophoresis (FIG. 17).

<auto_ctDdRp DNA unit>

In this example, an auto_ctDdRp DNA unit containing a DNA fragment providing ctDdRp was designed using a plasmid.

The pCI-neo vector (Promega, USA), which can construct a stable cell line as a mammalian expression vector, was cut with BglII/HpaI restriction enzyme treatment and <T7 promoter - 5'UTR - Tethering domain - ctDdRp orf- muag - A64 - C30 - The auto_ctDdRp DNA unit was prepared by inserting a ctDdRp DNA fragment containing histone-SL>.

The ctDdRp DNA fragment synthesized in this example was synthesized using the forward primer 5'- AGATCT TAAT ACGACTCACT ATAG GGGAGA CCACAACGGT TTCCCTCTAG AAAGAACC AT G -3' (SEQ ID NO: 64) and the reverse primer 5'- GTTAAC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 64). 65) was used to amplify. Here the target sequence of T7pol is underlined. Both ends of the primers used contained the BglII/HpaI site sequence.

The PCR product containing the amplified ctDdRp DNA fragment was digested using BglII/HpaI enzymes, and then cloned using pCI-neo vector linearized with BglII/HpaI enzymes and T4 DNA ligase.

The reaction solution was transfected into Escherichia coli, and plasmids were isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and subjected to electrophoresis (FIG. 18).

<DNAi unit>

In this example, DNAi units containing DNAi encoding mRNAi were designed using plasmids. As a plasmid, pCI-neo vector (Promega, USA) capable of constructing a stable cell line as a mammalian expression vector was used. The pCI-neo vector was digested with BglII/HpaI restriction enzymes, and a DNA fragment consisting of <T7 promoter followed by 5'UTR, DNAi encoding the selected mRNAi, 3'UTR, (Tethering RNA)4, and A64> was inserted. The DNAi unit was prepared.

The constructed DNAi fragment was synthesized by chemical synthesis (Biosynthesis, USA) and amplified again by PCR as a template. At this time, BglII/HpaI site sequences were assigned to both ends using PCR primers. The amplified PCR product was treated with BglII/HpaI restriction enzymes and then inserted into the pCI-neo vector (Promega, USA) using T4 DNA ligase. The resulting DNAi units were confirmed by DNA sequencing.

PCR primers were forward primer 5'-AGATCT TAAT ACGACTCACT ATAG GGGAGA CCACAACGGT TTCCCTCTAG AAAGAACCAT G-3' (SEQ ID NO: 66) and reverse primer 5'-GTTAACTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT ATTAATCT-3' (SEQ ID NO: 67) did Here the target sequence of T7pol is underlined. Both ends of the primers used contained BglII/HpaI site sequences.

Specifically, 5 μl 10 × MgCl2 reaction buffer, 1 μl forward primer (20 μM), 1 μl reverse primer (20 μM), 1 μl dNTP (10 mM), 1 μl prepared DNA fragment (10 ng / μl), 0.5 μl enzyme PCR was performed by adding 40.5 μl dH2O to the reaction solution containing the mix (i.e., polymerase).

The amplification product was treated with BglII/HpaI restriction enzymes, and a pCI-neo vector linearized with BglII/HpaI restriction enzymes was mixed and DNA ligase was added to perform a cloning reaction. 2-3 μl of the pCI-neo reaction solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated overnight at 37°C.

Pick 6-12 colonies and inoculate each in 3 ml of LB growth medium with 50 μg/ml ampicillin. Incubate overnight on a shaker at 37°C. Recombinant pCI-neo vectors were isolated using standard procedures or the commercial Qiaprep Miniprep kit (Qiagen). The recombinant pCI-neo vector was digested with BglII/HpaI, and gel electrophoresis was performed to select DNAi expression unit clones (FIG. 19).

The mRNAi used in this example encodes the G protein (RAV-G) of rabies virus, RSV-F protein of a long strain of respiratory syncytial virus (RSV), or murine EPO is an mRNA that

The order of construction of mRNAi used in the present invention is as follows.

EPO

For the preparation of the DNAi expression unit encoding the murine EPO of the present invention, the 5'UTR following the T7 promoter in the pCI-neo vector, the GC-rich sequence encoding the RSV-F protein, and the mutated alpha The following DNA fragment was prepared to carry out the method described above so as to include the 3'UTR (muag), (Tethering RNA) 4 and A64 poly (A) sequences of globin. The sequence of the corresponding mRNA is SEQ ID NO: 22.

G protein of rabies virus (RAV-G)

For the preparation of the DNAi expression unit encoding the rabies virus G protein (RAV-G) of the present invention, the 5'UTR following the T7 promoter in the pCI-neo vector, the GC-rich sequence encoding the rabies virus G protein , The following DNA fragment was prepared to carry out the method described above so as to include the mutated alpha globin 3'UTR (muag), (Tethering RNA) 4 and A64 poly (A) sequences. The sequence of the corresponding mRNA is SEQ ID NO: 24.

RSV-F protein from RSV long strains

For the preparation of the DNAi expression unit encoding the RSV-F protein of the long strain of the respiratory syncytial virus (RSV) of the present invention, the T7 promoter in the pCI-neo vector was followed by 5'UTR, RSV -F protein-encoding GC-rich sequence, mutated alpha globin 3'UTR (muag), (Tethering RNA) 4 and A64 poly (A) sequence to be able to carry out the method described above The following DNA fragments were prepared. The sequence of the corresponding mRNA is SEQ ID NO: 25.

<auto_(DdRp+DNAi) unit>

The auto_(DdRp+DNAi) unit is a DNA fragment (T7 promoter - 5'UTR - ctDdRp DNA - - muag - A64 - C30 - histone-SL4 -T7 promoter - 5'UTR - DNAi - muag - ( Tethering RNA)4 - A64) was inserted.

For the preparation of the auto_(ctDdRp+DNAi) unit, first, PCR was performed using the auto_ctDdRp unit prepared in Example as a template to obtain an auto_ctDdRp DNA fragment (T7 promoter - 5'UTR - ctDdRp DNA - muag - A64 - C30 - Histone-SL4 ) was prepared.

Using the auto_ctDdRp unit as a template, a forward primer (5'- AGATCT TAAT ACGACTCACT ATAG GGGAGA CCACAACGGT TTCCCTCTAG AAAGAACCAT G-3', SEQ ID NO: 68) and a reverse primer (5'- GAATTC TGGTG GCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 69) were used. Thus, a large amount of PCR products having BglII/EcoRI site sequences at both ends were obtained.

DNAi fragments (T7 promoter - 5'UTR - DNAi - muag - (Tethering RNA)4 - A64) were prepared in large quantities by PCR using the DNAi expression unit prepared in Example as a template. When PCR of the DNAi expression unit, forward primer (5'- GAATTC TAAT ACGACTCACT ATAG GGGAGA CCACAACGGT TTCCCTCTAG AAAGAACCAT G-3', SEQ ID NO: 66) and reverse primer (5'- GTTAAC TTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT ATTAATCT-3' , SEQ ID NO: 67) was endowed with EcoRI/HpaI site sequences at both ends of the PCR product.

After digesting the amplified PCR products by EcoRI restriction enzyme treatment, using T4 DNA ligase, the auto_(ctDdRp+DNAi) DNA fragment with HpaI site sequences was added to the 3*?**?* end of the BglII site sequences at the 5' end. manufactured. The prepared auto_(ctDdRp+DNAi) DNA fragment was digested with BglII/HpaI restriction enzyme and inserted into pCI-neo vector linearized with BglII/HpaI restriction enzyme using T4 DNA ligase.

2-3 μl of the pCI-neo vector recombinant solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated overnight at 37°C. Pick 6-12 colonies and inoculate each in 3 ml of LB growth medium with 50 μg/ml ampicillin. Incubate overnight on a shaker at 37°C. The auto_(DdRp+DNAi) DNA fragment was isolated from recombinant pCI-neo using standard procedures or the commercial Qiaprep Miniprep kit (Qiagen). The recombinant plasmid was digested with BglII/HpaI restriction enzymes and subjected to gel electrophoresis to confirm clones containing the auto_(DdRp+DNAi) DNA expression unit (FIG. 20).

The prepared auto_(ctDdRp+DNAi) DNA fragment is placed in the pCI-neo vector, and the composition of the auto_(DdRp+DNAi) DNA fragment is <T7 promoter - 5'UTR - DdRp DNA - - muag - A64 - C30 - histone -SL4 - T7 promoter - 5'UTR - DNAi - muag - (Tethering RNA)4 - A64>.

Example 10. Introducing the mRNA transcription system of interest in monocytes

The RNA transcription system of interest of the present invention was introduced into target mononuclear cells. In order to obtain vesicles from cells transfected with the RNA transcription system of interest, target cells were transformed with the RNA transcription system of interest.

The linear ctDdRp RNA expression unit prepared in the present invention, several types of circular plasmid DNA EKSDNL, and combinations thereof were used to prepare liposome delivery systems using Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA).

1 μg of the mixture of the prepared ctDdRp RNA expression unit, plasmid DNA unit, and combinations thereof (equal molar ratio) and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 5 μl of Lipofectamin 2000 and 100 μl of serum-free medium were mixed in another 1.5 ml. After mixing in a tube, it was left at room temperature for 5 minutes. Then, the contents of the two tubes were mixed and stored at room temperature for 20 minutes to form a liposome-type complex. After 20 minutes, the tube was centrifuged for 10 seconds, treated and transfected on each cell, and then changed to a new medium after 4 hours.

The complex of the ctDdRp RNA/DNAi system including the ctDdRp RNA unit and the DNAi unit and the liposome was called *?**?*auto(-)DNAi@LS and was called "DdRp RNA/DNAi@LS". ctDdRp, which is repetitively synthesized from the capped ctDdRp mRNA, can be maintained at a high concentration in the cytoplasm for a long period of time.

In addition, *?**?*auto_DNAi@LS*?**?*, which is a complex of the ctDdRp RNA/auto_ctDdRp+DNAi system or the ctDdRp RNA/auto_(ctDdRp+DNAi) system and liposomes, is used to obtain ctDdRp RNA units, auto_ctDdRp units, DNAi The complex of unit and liposome is called "ctDdRp RNA/auto_ctDdRp+DNAi@LS or ctDdRp RNA unit, auto_(ctDdRp+DNAi) expression unit and liposome complex ctDdRp RNA/auto_(ctDdRp+DNAi)@LS". By binding to the T7 promoter of the auto_ctDdRp DNA unit or the auto_(ctDdRp+DNAi) expression unit by ctDdRp synthesized from the initially capped ctDdRp mRNA, ctDdRp can be maintained at high concentrations in the cytoplasm for a long period of time as a result of autotranscription.

As a control, DOPC liposomal Nazo particles loaded with pCI-neo_DNAi were prepared and called *?**?*pCI-neo_DNAi@LS*?**?*, which is a complex of pCI-neo_DNAi and liposomes.

Cells were cultured under antibiotic conditions with 250 μg/ml of hygromycin B (Invitrogen No.10687010). Vesicles were prepared from cells loaded with VEGF receptors according to the following method.

Example 11. Preparation of Vesicles from Mononuclear Cells

11.1. Preparation of vesicles from monocyte-derived vesicles through extrusion

Monocyte U937 cells (ATCC No.CRL-1593.2) or macrophage Raw264.7 cells (ATCC No.TIB-71) transfected with the RNA transcription system of interest were transfected with PBS (phosphate phosphate) at a concentration of 5 × 10 ⁶ cells/ml. It was suspended in 3 ml of buffered saline) solution. The suspension solution was passed through a membrane filter with a pore size of 10 μm 3 times, then passed through a membrane filter with a pore size of 5 μm 3 times, and then passed through a membrane filter with a pore size of 1 μm 3 times. to obtain a vesicle suspension.

The monocyte-derived vesicle suspension produced through the cell extruder was isolated and purified by the method of Section 7.1 above.

11.2. Preparation of vesicles from monocyte-derived vesicles through sonication

Vesicles were prepared from monocytes or macrophages transfected with the RNA transcription system of interest. Methods of sonication and density gradient were used.

Monocytes or macrophages were suspended in 3 ml of PBS solution at a concentration of 2 × 10 ⁷ cells/ml. After ultrasonic disintegration was performed 30 times with an ultrasonic generator (UP400s, Heilscher) under the conditions of Cycle 0.5 and amplitute 50, ultrasonic disintegration was performed again in a water bath sonicator for 30 minutes. 1 ml of 50% OptiPrep, 1 ml of 5% OptiPrep, and 3 ml of the sonicated cell suspension were sequentially placed in a 5 ml ultracentrifugation tube.

The cell-derived vesicle suspension disaggregated by ultrasonication was separated and purified by the purification method of Section 7.1 above.

11.3. Preparation of mesenchymal stem cell-derived vesicles

A vesicle suspension was prepared from umbilical cord blood mesenchymal stem cells (UC MSC) transfected with the RNA transcription system of interest through an extrusion method. Mesenchymal stem cells cultured in stem cell complete growth medium were washed with phosphate buffered saline (PBS), and the washed stem cells were resuspended in PBS at a concentration of 0.25 to 1 x 10 ⁶ cells/ml ( resuspension). After passing the suspension solution through a membrane filter with a micropore size of 10 μm using a cell extruder, it is passed through a membrane filter with a micropore size of 3 μm, and then through a membrane filter with a micropore size of 0.4 μm. By passing through a membrane filter, a mesenchymal stem cell-derived vesicle (Crude UCMSC-CDV) suspension was prepared.

The mesenchymal stem cell-derived vesicle (Crude UCMSC-CDV) suspension produced through the cell extruder was isolated and purified by the method of Section 7.1 above.

11.4. Characterization of monocyte-derived vesicles

Vesicles prepared from monocytes transfected with the RNA transcription system of interest were adsorbed on a glow-discharged carbon-coated copper grid for 3 minutes. After washing the grid with distilled water, it was stained with 2% uranylacetate for 1 minute. The results observed with a JEM101 (Jeol, Japan) electron microscope are shown in FIG. 21 .

As shown in the transmission electron microscope image of FIG. 2, it can be seen that the vesicles prepared by the extrusion method from monocytes transfected with the RNA transcription system of interest are composed of a lipid bilayer, have a size of 100 to 200 nm, and are generally spherical. can

Vesicles prepared from monocytes transfected with the RNA transcription system of interest were diluted in 1 ml of PBS at a concentration of 5 μg/ml. 1 ml of PBS containing vesicles was placed in a cuvette and analyzed with a dynamic light scattering particle size analyzer, and the results are shown in FIG. 3 . As shown in FIG. 22, the size of the vesicles was 200 to 300 nm, and the average size was confirmed to be 250 nm.

After preparing 5 μg of each of vesicles prepared by extrusion from monocytes transfected with the RNA transcription system of interest and vesicles prepared by ultrasonic dissociation from monocytes and monocyte cells, 0.5 μg of trypsin was treated at 37° C. for 20 minutes. . 5 x loading dye (250 mM Tris-HCl, 10% SDS, 0.5% bromophenol blue, 50% glycerol) was finally added to each of the trypsin-treated and untreated cells to 1 x, and 5 at 100 ° C. processed for minutes. An 8% polyacrylamide gel was prepared and the sample was loaded. After electrophoresis at 80V for 2 hours, the protein was transferred to a PVDF (polyvinylidene fluoride) membrane for 2 hours at 400 mA. After dissolving skim milk to 3% in PBS, the membrane was blocked in this solution for 2 hours. LFA-1 and beta-actin antibodies were treated at 4°C for 12 hours. After washing twice with PBS, each secondary antibody attached to peroxidase was treated at room temperature for 1 hour. After washing with PBS for 30 minutes, the ECL (enhanced chemiluminescence, Amersham Co. No. RPN2106) substrate was identified and shown in FIG. 23 .

'+' indicates the result of treatment with trypsin, and '-' indicates the result of not treating trypsin.

As shown in FIG. 23, it was confirmed that the extracellular region of LFA-1, a cell membrane protein, disappeared in vesicles and cells prepared by extrusion and then treated with trypsin, but the amount of beta actin did not decrease. If even part of the cell membrane is inverted so that some of the extracellular domain of LFA-1 is directed inside the vesicle, it should elicit an antibody response because it is not degraded by trypsin. However, since the LFA-1 antibody reaction did not appear in the trypsin-treated vesicles and cells, it can be seen that the extracellular region of the existing LFA-1 was facing the outside of the vesicles.

From the above results, in the case of vesicles prepared by the extrusion method, it can be inferred that all the parts of the original cell membrane facing the outside of the cell face the outside of the vesicle as well for other cell membrane proteins other than LFA-1, which indicates that the vesicle membrane is the cell's This means that it maintains the same topology as the cell membrane.

Also, as can be seen in FIG. 23 , unlike the vesicles prepared by the extrusion method, in the case of the vesicles prepared by the sonication method, the LFA-1 antibody reaction signal was shown after digestion with trypsin. From the above results, it can be seen that some vesicles in which the topology of cell membrane proteins is changed exist when vesicles are prepared by the sonication method.

11.5. VEGF binding using cell-derived vesicles loaded with VEGF receptors

Vesicles were prepared from HUVECs, vascular endothelial cells transfected with the RNA transcription system of interest and the VEGF receptor. Since HUVECs, which are vascular endothelial cells, express VEGF receptors on their surface, vesicles derived from HUVECs were also expected to have VEGF receptors.

To obtain vesicles from VEGF receptor-transfected cells, the pCEP4 vector containing the VEGF receptor cDNA and the RNA transcription system of interest were transfected into mouse PT67 cells (ATCC No. CRL-12284) using Lipofectamin. Cells were cultured under antibiotic conditions with 250 μg/ml of hygromycin B (Invitrogen No.10687010). Vesicles were prepared from cells loaded with VEGF receptors according to the method of Example 1.

A 96-well plate was prepared, and each vesicle was put into the well plate to be 0, 12.5, and 25 ng, respectively, and stored at 4° C. for 12 hours or more to coat. 100 μl of 1% BSA/PBS was added and fixed for 1 hour. After 1 hour, VEGF with biotin was added to 100 ng/ml and incubated for 2 hours. After washing with 1% BSA/PBS, incubation with streptavidin-POD for 20 minutes, and BM-POD substrate was added to induce color development.

24 and 25 show RLU values after color development. 24 is a result of vascular endothelial cell-derived vesicles, and FIG. 25 is a result of cell-derived vesicles loaded with VEGF receptors transfected thereon. The RLU value represents the relative value of VEGF attached to the vesicle. As shown in FIG. 8 , it was confirmed that the vesicle derived from vascular endothelial cells exhibited a high RLU value by binding to VEGF. In addition, as shown in FIG. 25 , it was confirmed that the cell-derived vesicle loaded by transforming the VEGF receptor also binds to VEGF and exhibits a high RLU value.

The above results suggest that the VEGF receptor can be loaded on the surface of the vesicle and bind to VEGF present in an aqueous solution, and that the VEGF receptor-loaded vesicle can be used as a VEGF function inhibitor.

11.6. Preparation of vesicles loaded with anti-cancer drugs

Vesicles were prepared from monocytes or macrophages transfected with the RNA transcription system of interest. The extrusion method and the density gradient method were used, and the drug was loaded by administering an anticancer drug in the extrusion step.

Monocytes or macrophages transfected with the RNA transcription system of interest were suspended in 3 ml of PBS solution at a concentration of 5 × 10 ⁶ cells/ml. Doxorubicin (Sigma, No.D1515), an anti-cancer drug, was added to the suspension solution at concentrations of 0, 100, 200, and 400 μg/ml, respectively, and mixed well. The solution was passed through a membrane filter with a pore size of 10 μm three times, then through a membrane filter with a pore size of 5 μm three times, and then through a membrane filter with a pore size of 1 μm three times. 1 ml of 50% OptiPrep, 1 ml of 5% OptiPrep, and 3 ml of the cell suspension passed through the membrane filter were sequentially placed in a 5 ml ultracentrifugation tube. It was then ultracentrifuged at 100,000 × g for 2 hours. Doxorubicin-loaded vesicles were obtained in the layer between 50% Optiprep and 5% Optiprep.

300 μl of doxorubicin solution diluted with PBS to concentrations of 0, 5, 10, 20, and 40 μg/ml, respectively, was prepared, and doxorubicin-loaded vesicles prepared by extrusion using different concentrations of doxorubicin were 300 μl each was prepared at a concentration of μg/ml. In a 96-well plate, 100 μl of each sample was placed in 3 wells. Values were obtained at an excitation wavelength of 488 nm and an emission wavelength of 530 nm in a Wallac 1420 VICTOR plate-reader (Perkin-Elmer Life Sciences) instrument. After drawing a standard curve using the value of the doxorubicin solution, the amount of doxorubicin entering the vesicle was quantified and shown in FIG. 26 .

26 is a graph showing the amount of doxorubicin loaded in vesicles with a protein amount of 1 μg. When using 100, 200, and 400 μg/ml of doxorubicin, it was confirmed that about 50 ng, 200 ng, and 300 ng of doxorubicin were loaded into the vesicle, respectively.

In all examples below, vesicles loaded with doxorubicin were prepared using 400 μg/ml doxorubicin.

11.7. Characteristics of vesicles loaded with anti-cancer drugs three

Vesicles prepared from monocytes transfected with the RNA transcription system of interest were adsorbed on a glow-discharged carbon-coated copper grid for 3 minutes. After washing the grid with distilled water, it was stained with 2% uranyl acetate for 1 minute. It was observed with a JEM101 electron microscope and the results are shown in FIG. 27 .

As shown in the transmission electron microscope image of FIG. 27, it can be seen that the vesicles prepared by the extrusion method from monocytes are composed of a lipid bilayer, have a size of 100 to 200 nm, and are generally spherical.

Vesicles prepared from monocytes transfected with the RNA transcription system of interest were diluted in 1 ml of PBS at a concentration of 5 μg/ml. As a result of putting 1 ml of PBS containing vesicles into a cuvette and analyzing with a dynamic light scattering particle size analyzer, the size of the vesicles was 100 to 300 nm, and the average size was confirmed to be 250 nm. From the above results, it can be seen that the anti-cancer drug is the same size as the non-loaded vesicle.

Doxorubicin-loaded and unloaded vesicles were incubated with 5 μM DiO (Invitrogen, No.V22886) for 30 minutes, respectively. DiO can attach to lipids and is a substance that exhibits green fluorescence. After adjusting the vesicles labeled with DiO to 20 μg/ml, 50 μl was spotted on a cover glass. Stored at 4° C. for 12 hours, the vesicles were coated on the cover glass. After attaching the cover glass to a slide glass, it was observed under a fluorescence microscope. Vesicles were observed with green fluorescence of DiO, and doxorubicin was observed with its own red fluorescence, and the results are shown in FIG. 28 .

28 is a fluorescence image of a vesicle not loaded with doxorubicin and a vesicle loaded with doxorubicin. As shown in FIG. 28 , the vesicles not loaded with doxorubicin were labeled with DiO and showed green fluorescence, but the red fluorescence of doxorubicin was not seen. On the other hand, it was confirmed that the doxorubicin-loaded vesicle showed not only green fluorescence but also red fluorescence of doxorubicin, and it was confirmed that doxorubicin was loaded into the vesicle through the presence of the green fluorescence and the red fluorescence at the same location.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be embodied in other specific forms without modifying its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all modifications or chemically modified forms derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (31)

An RNA transcription system of interest characterized by a vesicle derived from a host cell that amplifies the RNA of interest, a cell comprising the same and a use thereof. The method of claim 1, wherein the RNA of interest is
Includes messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), shRNA, trans-activating crispr RNA, circular RNA, ribozymes and RNA aptamers An RNA transcription system of interest characterized by any one or more selected from the group and a cell line comprising the same. The method of claim 1 to claim 2, wherein the host cell
host cells including cell-walled prokaryotes, yeast and plant cells; and
A host cell including an animal cell without a cell wall; RNA transcription system of interest characterized by any one selected from the group comprising, a cell comprising the same, and a use thereof. The method of claim 3, wherein the host cell
Any one selected from the group containing animals, plants, yeasts and bacteria,
Here, the bacteria are Corynebacterium glutamicum (Corynebacterium glutamicum), Escherichia coli BE42, Escherichia coli HB101, Escherichia coli JM101, Escherichia coli LE392, Escherichia coli MC1061, Escherichia coli Y1088 and Escherichia coli W3110. An RNA transcription system of interest characterized in that it reduces the activity of ribonuclease III by attenuating the expression of the gene encoding III or disrupting the gene, a cell comprising the same and a use thereof. The method of claim 1 to 4, wherein the host cell
An RNA transcription system of interest characterized by E. coli strain HT115 (DE3), cells comprising same and uses thereof. The method of claim 1 to 5, wherein the RNA transcription system
a) RNA Polymerase (polymerase) expression unit; and
b) a system comprising an RNA unit of interest;
Here, the RNA transcription system of interest, characterized in that it has a configuration capable of being linked to their operation, a cell comprising the same, and a use thereof. The method of claim 6, wherein in order to pseudouridine the uridine of the mRNA produced from the RNA unit of interest, the RNA transcription system
An RNA transcription system of interest characterized by adding an expression unit of H/ACA sRNP or eukaryotic stand-alone pseudouridine synthases (Pus enzymes), a cell comprising same and a use thereof. The method of claim 7, wherein the eukaryotic stand-alone pseudouridine synthases (Pus enzymes)
An RNA transcription system of interest, characterized in that it originates from any one selected from the group consisting of Escherichia coli, yeast and human, a cell comprising the same, and a use thereof. The method of claim 6 to 8, wherein the RNA polymerase expression unit; and
An RNA transcription system of interest characterized by a vector carrying any one or more selected from the group comprising; an RNA unit of interest, a cell comprising the same, and a use thereof. 10. The method of claim 9, wherein the vector is
Escherichia coli plasmid, bacteriophage (λ, M13, P1), virus (animal virus - retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, etc.; insect virus - baculovirus, plant virus - cauliflower mosaic virus , potato virus X, Gemini virus, etc.), Agrobacterium tumefaciens based vectors, chimeric plasmids (cosmids, phagemids, phsmids and fosmids), artificial chromosomes (YAC, BAC, PAC, MAC and HAC) and An RNA transcription system of interest characterized by at least one selected from the group containing vectors based on non-E. coli vectors (eg, Bacillus and Pseudomonas vectors, etc.), cells comprising the same, and uses thereof. The method of claim 6 to claim 10, wherein when the host cell is a eukaryotic cell, the RNA polymerase expression unit is
a) an mRNA structure capable of translating any one selected from the group containing DNA dependent RNA Polymerase (DdRp) ORF and variants thereof in a host cell,
Alternatively, mRNA capable of translating the catalytic unit of the capping enzyme may be added to the mRNA structure; and
(b-1) a promoter operating on the DdRp;
(b-2) 5'UTR;
(b-3) any one selected from the group containing DNA dependent RNA Polymerase (DdRp) ORF and variants thereof, or any one selected from the group containing RNA dependent RNA Polymerase (RdRp) ORF and variants thereof; and
(b-4) 3'UTR; a RNA transcription system of interest comprising a DNA structure comprising the same, a cell comprising the same, and a use thereof. The method of claim 6 to claim 11, when the host cell is a prokaryotic cell, the RNA polymerase expression unit
a) a promoter that operates in the host cell;
b) ribosome binding site (RBS)
c) any one selected from the group containing DNA dependent RNA Polymerase (DdRp) ORF and variants thereof, or any one selected from the group containing RNA dependent RNA Polymerase (RdRp) ORF or variants thereof; and
(d) a transcription termination sequence; an RNA transcription system of interest comprising the same, a cell comprising the same, and a use thereof. The method of claim 12, wherein the promoter
pta, aceA, aceB, adh and amyE promoters inducible with acetic acid, ethanol, pyruvic acid, and the like;
a group containing the cspB, SOD and tuf promoters;
a group containing a lac promoter, a tac promoter, and a trc promoter;
A group comprising phage-derived promoters such as F1 promoter, T7 promoter, T5 promoter, T3 promoter and SP6 promoter; an RNA transcription system of interest characterized in that any one selected from the group comprising, a cell comprising the same, and their Usage. The method of claim 11 to 12, wherein the DdRp is
a) DdRp or a variant thereof;
b) a capping enzyme;
c) the RNA binding domain of the tethering system; and
d) ctDdRp comprising a linker;
Here, or additionally, the RNA transcription system of interest, characterized in that it comprises a Poly (A) polymerase, a cell comprising the same and a use thereof. The method of claim 14, wherein the RNA sequence that specifically binds to the RNA binding domain of the tethering system
The RNA transcription system of interest, characterized in that located in the RNA unit of interest, a cell comprising the same and a use thereof. The method of claim 11 to 12, wherein the RdRp is
An RNA transcription system of interest characterized by alphavirus RdRp, cells comprising same and uses thereof. The method of claim 11, wherein the transcription termination sequence
An RNA transcription system of interest characterized by a Rho-dependent termination sequence or a Rho-independent termination sequence, a cell comprising the same and a use thereof. The method of claim 6, wherein the RNA unit of interest is
In the case of the RNA polymerase expression unit containing the DdRp
a) a promoter driven by the DdRp;
b) DNA encoding the RNA of interest; and
c) a transcription termination sequence; an RNA transcription system of interest characterized in that it comprises, a cell comprising the same and a use thereof. The method of claim 18, wherein the promoter
An RNA transcription system of interest characterized by at least one selected from the group consisting of a T7 promoter, a T3 promoter and an SP6 promoter, a cell comprising the same, and a use thereof. 19. The method of claim 6 or 18, wherein the RNA unit of interest is
In the case of the RNA polymerase expression unit containing the RdRp
a) a promoter that operates in the host cell;
b) DNA that is copied by said RdRp and encodes a replicon RNA comprising the RNA of interest; and
c) a transcription termination sequence; an RNA transcription system of interest comprising the same, a cell comprising the same and a use thereof. 21. The method of claim 20, wherein the replicon RNA is
An RNA transcription system of interest comprising an SGP originating from an alphavirus, a cell comprising the same and a use thereof. The method of claim 1, wherein the amplification of the RNA of interest,
a) culturing a host cell having the RNA transcription system of interest, transcribing the RNA of interest, and accumulating the RNA of interest in the host cell; and
b) collecting an RNA of interest from a host cell modified to have reduced activity of ribonuclease III compared to an unmodified strain; a RNA transcription system comprising the same, a cell comprising the same, and a use thereof . 23. The method of claim 22, wherein the method for producing a cell-derived vesicle (CDV) in a host cell containing the RNA transcription system of interest,
(a) preparing CDV from a suspension containing the cells; and
(b) separating the prepared CDV from the suspension; an RNA transcription system of interest comprising the, a cell comprising the same, and a use thereof. 24. The method of claim 23, wherein the method for producing a host cell protoplast-derived vesicle containing the RNA transcription system of interest,
(a) obtaining protoplasts by removing cell walls from host cells containing the RNA transcription system of interest;
(b) preparing a CDV from a suspension containing the protoplasts; and
(c) isolating the prepared CDV from the suspension; an RNA transcription system of interest comprising a, a cell comprising the same, and a use thereof. 25. The method of claim 23-24, wherein the separation step
An RNA transcription system of interest characterized in that it is performed using a method selected from the group consisting of density gradient, ultracentrifugation, filtration, dialysis, and free flow electrophoresis, a cell comprising the same, and a use thereof. The method of claim 23 to 25, wherein the transformed host cell
An RNA transcription system of interest, characterized in that it is a cell transformed to express a cell membrane fusion material, a cell comprising the same, and a use thereof. 27. The method of any one of claims 23 to 26, wherein the transformed host cell is
An RNA transcription system of interest, characterized in that it is equipped with a substance for treating, diagnosing or vaccinating a disease, a cell comprising the same, and a use thereof. 28. The method of claim 27, wherein the disease treatment, diagnosis or vaccine material
An RNA transcription system of interest, characterized in that at least one selected from the group consisting of anticancer agents, anti-inflammatory agents, angiogenesis inhibitors, peptides, proteins, toxins, nucleic acids, beads, microparticles, and nanoparticles, cells comprising the same, and uses thereof. The method of claim 1, wherein the RNA of interest is any one selected from the group consisting of mRNA, trans-activating crispr RNA, circular RNA, ribozyme and RNA aptamer,
The RNA transcription system of interest characterized by the selected RNA obtained by isolating and purifying the selected RNA amplified from the host cell containing the RNA transcription system of interest, a cell comprising the same and a use thereof. 30. The method of claim 29, wherein the method of purifying the RNA of interest
a) precipitating the RNA of interest from an impure preparation comprising lysed host cells;
b) subjecting the impure preparation containing the precipitated RNA of interest to a purification process including membrane filtration to capture the precipitated RNA of interest with a membrane; and
c) eluting the captured precipitated RNA of interest from the membrane by resolubilizing the RNA of interest, thereby harvesting the purified RNA of interest solution, a cell comprising the same, and their Usage. The RNA transcription system of interest according to claim 1, characterized by an exosome secreted from a host cell comprising the RNA transcription system of interest, a cell comprising the same, and a use thereof.

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