KR20220018410A - Self-transcribing RNA/DNA system that provides Genome editing in the cytoplasm - Google Patents

Abstract

The present invention provides a genome editing RNA/DNA system which comprises a DdRp RNA expression cassette and a genome editing tool DNA (DNAge) expression cassette that can be transcribed by DdRp, and has a genome editing tool capable of self-transcription with the genome editing RNA/DNA system constructed by operatively connecting the same, and a composition thereof.

Description

A RNA/DNA system capable of self-transcription in the cytoplasm and providing genome editing

The present invention provides a system having at least one endogenous genome editing and self-transcriptional component in a cell, wherein the system comprises a DdRp RNA comprising DNA dependent RNA Polymerase (DdRp) mRNA having a structure that enables intracellular translation. expression cassette; and a DNAge expression cassette encoding a genome editing tool amplified by the DdRp; an auto_DdRp expression cassette containing a DNA encoding the DdRp in addition to the self-transcribed RNA/DNA system or the self-transcribed RNA/DNA system comprising a It is a self-transcription RNA/DNA system that increases the expression of DdRp in the cytoplasm to continuously express a genome editing tool for a long period of time.

Genome editing has provided a powerful means and unprecedented opportunity to study gene function and combat disease by introducing targeted genomic sequence changes at specific loci in living cells or organisms. Zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) nucleases. Clase is being used effectively by many laboratories.

Single-component class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), and RNA-guided endonucleases, such as multicomponent class 1 CRISPR, such as multiprotein Cas3, are relatively easy to manipulate, resulting in more attracting attention CRISPR provides a simple method for targeted gene cleavage and targeted gene insertion.

The main factors for gene editing are the Cas9 protein containing the nuclease domain and the guide RNA (sgRNA) that provides sequence specificity for the target RNA. The first 20 nucleotide sequences at the 5' end of sgRNA are complementary to the target sequence, providing specificity to the CRISPR/Cas9 system. The 3' region of the sgRNA forms the secondary structure essential for Cas9 activity. When sgRNA brings Cas9 nuclease to a specific target, Cas9 cleaves the double-stranded DNA of the target at a protospacer-adjacent motif (PAM) site. Non-homologous end-joint repair of this truncated double strand often results in deletions or small indels that perturb the target gene.

The application of CRISPR-Cas9 is largely limited due to the high resistance to guide-target mismatch, the relatively easy to degrade RNA-directed secondary structure, and the requirement of a protospacer adjacent motif (PAM).

Similar to the above Cas9 and Cpf1, algonuts play an important role in host defense and gene expression repression against exogenous nucleic acids. Cas9 and Cpf1 are found naturally only in prokaryotes, whereas members of the algonut superfamily exist in many species (from bacteria to mammals). Although most algonautes associate with single-stranded RNA and play a pivotal role in RNA silencing, some algonuts bind to ssDNA and cleave target DNA.

Recently, genome editing was performed in human cells by using Natronobacterium gregoryi Argonaute (NgAgo) together with guide DNA oligos. Argonaute is an endonuclease that uses a 5' phosphorylated short single-stranded nucleic acid as a guide to cleave a target. DNA-guided algonut binding does not appear to have special requirements for sequence or secondary structure. Since the algonut protein contains nucleic acid-induced nuclease activity, a specialized genome editing system that exhibits strong genome editing ability is currently being developed.

Recently, a fusion linking a template RNA to the 3' end of an existing guide RNA and linking a prime editing guide RNA (pegRNA) and a reverse transcriptase to Cas9 nickase, which can cut only a single strand The protein was manufactured and the Prime editing correction technology was completed.

Existing genome editing methods include CRISPR plasmid method, mRNA method encoding Cas protein and sgRNA, and ribonucleoprotein (RNP) complex method consisting of Cas protein and sgRNA. CRISPR/Cas9 plasmid containing DNA fragment encoding Cas protein and sgRNA In the genome editing method, the CRISPR/Cas9 plasmid encoding the genome editing tool is nuclear-dependent and transcribed using a tool located in the nucleus. must move to However, since less than 1% of the CRISPR/Cas9 plasmid introduced through the cell membrane migrates to the nucleus, the genome editing RNA/DNA system of the present invention intends to overcome this problem by constructing and using a transcription tool in the cytoplasm.

The DdRp RNA expression cassette constituting the genome editing RNA/DNA system of the present invention synthesizes DdRp through a tool necessary for protein synthesis from RNA in the cytoplasm. The synthesized DdRp binds to the promoter in the DNAge expression cassette of the present invention to transcribe and synthesize tools necessary for genome editing in the cytoplasm. The nuclear-independent synthesized genome editing tool forms a complex capable of genome editing and moves to the nucleus, enabling efficient genome editing. Accordingly, genome editing in a target cell or target organism is made possible by the genome editing RNA/DNA system of the present invention.

The present invention provides an RNA expression cassette for expressing DNA-dependent RNA polymerase (DdRp) in order to develop a new tool for genome editing in order to edit genomic DNA accurately and efficiently; and a DNAge expression cassette encoding a genome editing tool capable of being transcribed by DdRp; to provide a novel technology suitable for a genome editing system capable of intracytoplasmic self-transcription, including.

An object to be solved by the present invention is to provide a characterized genome editing system that exhibits a strong targeted genome editing ability, to study gene function and combat disease by introducing a targeted genome sequence change into a specific locus of a living cell or organism. We want to implement a powerful means for

In order to develop a new tool for genome editing to accurately and efficiently edit genomic DNA, the present invention provides a DNA dependent RNA Polymerase comprising a chemically modified nucleotide (CM) with a structure that enables intracellular translation. (DdRp) DdRp RNA expression cassette comprising mRNA; and a DNAge expression cassette encoding a genome editing tool capable of being transcribed by DdRp; to provide a novel technology suitable for a genome editing RNA/DNA system capable of self-transcription, including.

Additionally, the present invention relates to a method of providing a 5'Cap structure at the 5' end of a transcript for stability of an entire transcriptome generated by DdRp, a product of the auto_DdRp expression cassette of the present invention, and for translation efficiency.

In particular, it is an object of the present invention to increase the expression of a genome editing tool encoded in a DNAge expression cassette by a self-transcribed RNA/DNA system administered to a cell or tissue.

The fundamental object of the invention is solved by the claimed subject matter.

DdRp RNA comprising DNA dependent RNA Polymerase (DdRp) mRNA (capped CM_DdRp mRNA) with a possible structure that enables intracellular translation, in order to be able to develop new tools of genome editing to accurately edit genomic DNA. expression cassette; and a DNAge expression cassette encoding a genome editing tool capable of being transcribed by DdRp; to provide a novel technology suitable for a self-transcribed RNA/DNA system constituting a genome editing system comprising a.

The present invention provides a method for providing a 5'Cap structure to a transcript for stability and translation efficiency of an entire transcript produced by DdRp, a product of the auto_DdRp expression cassette of the present invention.

The present invention relates to a DNAge ("SM_DNAge") encoding a genome editing tool comprising at least one sequence modification (SM) that increases the expression of at least one genome editing tool for medical use ("SM_DNAge") and a DNAge expression cassette comprising the same provides

In the RNA/DNA system of the present invention, a pharmaceutical composition and kit of parts for a DNAge expression cassette comprising the SM_DNAge fragment encoding the genome editing tool are provided, preferably for gene therapy and/or genetic vaccination. for use in the field.

The RNA/DNA system of the present invention provides a method of increasing the expression of a genome editing tool with a DNAge expression cassette comprising a capped CM_DdRp mRNA or a capped CM/SM_DdRp mRNA expression cassette and a DNAge fragment encoding a genome editing tool.

The object of the present invention is solved by the provision of a self-transcribed RNA/DNA system, wherein the expression of a genome editing tool in a target tissue or target cell can be further increased.

The following definitions are provided for clarity and readability. Any technical feature recited for these definitions may be understood in each and all of the present invention. Further definitions and explanations may be provided in particular in these. Where nucleic acid sequences are reported herein, these sequences generally include both a specific RNA or DNA sequence as well as its corresponding DNA or RNA counterpart, respectively. For example, where a DNA sequence is provided, the skilled person knows that the corresponding RNA sequence is obtained by thymine exchange with uracil residues and vice versa.

Cells : Cells include cells from any organism suitable for genome editing. Exemplary organisms include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats; poultry such as chickens, ducks and geese; plants such as monocotyledonous plants and dicotyledonous plants such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis, and the like.

Genome : Genome encompasses genomic DNA present in the nucleus as well as organ DNA present in cell sub-components (eg mitochondria, plastids). The genome of a cell refers to the genetic material of a cell or organism. The genetic material of a cell or organism often includes one or more genes. In certain embodiments, a gene comprises or consists of one or more nucleic acids. A gene refers to a segment of DNA involved in generating a polypeptide chain, and includes a coding region (eg, an exon), the transcription/translation of a gene product, and the regions before and after the coding region (leaders and trailers) involved in the regulation of transcription/translation. ) and intervening sequences (introns) between individual coding segments (exons). A gene may not necessarily produce a peptide, or it may produce a truncated or non-functional protein due to genetic differences in the gene sequence (eg, mutations in the coding and non-coding portions of the gene). For example, the non-functional gene may be a pseudogene. A gene, whether functional or non-functional, can often be identified by homology to a gene in a reference genome. For example, any particular gene of a subject (eg, a gene of interest, counterpart gene, pseudogene, etc.) can be identified in another subject genome or in a reference genome by one of ordinary skill in the art. In diploid subjects, a gene often contains a pair of alleles (eg, two alleles). Accordingly, the methods, systems or processes in the present invention can be applied to alleles of one or both genes. In some embodiments, a method, system or process of the invention is applied to each allele of a gene.

Gene therapy : Gene therapy generally refers to the treatment of a patient's body or an isolated element of the patient's body, eg, isolated tissues/cells, by means of a nucleic acid encoding a peptide or protein. This generally involves a) administering nucleic acids, preferably RNA and DNA molecules as defined herein - by any route of administration - directly to the patient or in vivo/ex vivo or in vitro. administration to isolated cells/tissues of a patient in vitro that results in transformation of the patient's cells in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of the isolated, transformed cells to the patient if the nucleic acid was not directly administered to the patient.

Expression Cassette : Expression cassette refers to a vector suitable for expressing a target nucleotide sequence in an organism, such as a recombinant vector, and may be referred to as a "construct" for convenience in the present invention. Expression refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription (eg, transcription to produce mRNA or functional RNA) of the nucleotide sequence and/or translation of the RNA into a protein precursor or mature protein. Expression cassette refers to a vector suitable for expressing a nucleotide sequence of interest in an organism, such as a recombinant vector. "Expression" means the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription (eg, transcription to produce mRNA or functional RNA) of the nucleotide sequence and/or translation of the RNA into a protein precursor or mature protein. Expression cassettes include a variety of forms or combinations thereof, including linear nucleic acid fragments, circular plasmids, viral vectors or translatable RNA (eg, mRNA) and ribonuclear protein (RNP) complexes. Viral vectors have been commercialized for expression of lentiviruses and adeno-associated viruses. An expression cassette may comprise a subject nucleotide sequence and a regulatory sequence, which may be derived from different sources, or a subject nucleotide sequence and a regulatory sequence, which are derived from the same source but aligned in a manner different from that normally found in nature.

Upstream: Upstream refers to the relative position of a first element of a nucleic acid molecule to a second element of the nucleic acid molecule, wherein the two nucleic acid molecules are comprised in the same nucleic acid molecule, wherein the first element is 5' more than the second element of the nucleic acid molecule is located in The second element is then referred to as being downstream of the first element of the nucleic acid molecule in question. An element located upstream of a second element may be synonymous with being 5' of the second element. For double-stranded nucleic acid molecules, indications such as upstream and downstream are given for the (+) strand.

Regulatory sequences: Regulatory sequences or regulatory elements are used interchangeably and are upstream (5' non-coding sequences), within or downstream of a coding sequence, affecting the transcription, RNA processing or stability or translation of the combined coding sequence. (3' non-coding sequence) refers to a nucleotide sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

Promoter: A promoter refers to a nucleic acid fragment capable of controlling the transcription of other nucleic acid fragments. In the present invention, a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not derived from the cell. The promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally-regulated promoter or an inducible promoter. The core promoter refers to a nucleic acid sequence included in the promoter. A core promoter is typically the smallest portion of a promoter necessary to properly initiate transcription. A core promoter typically includes a transcription initiation site and a binding site for RNA polymerase.

polymerase; Polymerases generally refer to molecular entities capable of catalyzing the synthesis of polymer molecules from monomer building blocks. An “RNA polymerase” is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. A “DNA polymerase” is a molecular entity 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 aggregate 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, RNA polymerase synthesizes an RNA molecule based on a template nucleic acid, the template nucleic acid being either a DNA molecule (where the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (where the RNA polymerase is an RNA-dependent RNA) for the polymerase DdRp).

DNA dependent RNA Polymerase (DdRp): DdRp is a synonym for RNA Polymerase (RNAP), and the DdRp in the nucleus of eukaryotes are RNAP I, RNAP II and RNAP III and they are structurally mutually exclusive. They are very different and each transcribe a different set of genes (different polymerases are present in mitochondria and chloroplasts). However, all three have two large subunits related to each other and the two largest subunits of bacterial RNAP. There are also several small subunits found in common in all three of these enzymes or only between RNAP I and RNAP III.

Eukaryotic DdRp, like bacterial RNAP, requires specialized cofactors for transcription initiation. However, unlike in the case of bacteria, eukaryotic initiators recognize promoter elements independently and not as part of the polymerase holoenzyme. Many different initiators are involved, particularly in genes transcribed by RNAP II, some of which are themselves very complex proteins. Purified eukaryotic RNA polymerase alone cannot initiate transcription selectively at the promoter. The term 'holoenzyme' is sometimes used to refer to eukaryotic RNAP, but in this case it refers to something similar to a bacterial 'core' enzyme and cannot be transcribed from a promoter. However, unlike bacterial core enzymes, eukaryotic whole enzymes can contain many other proteins involved in the transcription or processing of RNA.

RNAP I is found in the nucleolus (phosphorus) and transcribes only the gene encoding the large ribosomal RNA from which most cellular RNA is synthesized. In yeast, the enzyme has 13 subunits (and a mass of nearly 600 kDa). Five of the smaller subunits are also found in yeast RNAP II and III and the remaining two in yeast RNAP III.

RNAP II transcribes the gene that encodes a protein, most of the genes in the cell. It also transcribes the genes that code for most small nuclear RNAs (snRNAs). Most organisms appear to have a 12-subunit RNAP II (mass about 550 kDa). However, several other proteins are required for full activity, and RNAP holoenzymes can have a mass of 4000 kDa.

RNAP III mainly transcribes genes encoding transfer RNA and 5S RNA, but also transcribes some genes encoding other small RNAs. RNAP III has at least 14 distinct subunits with a mass of nearly 700 kDa. The promoters for RNAP I and RNAP II are mostly upstream of the transcription start site (as is the case with prokaryotic promoters), but some promoters for RNAP III are downstream of the start site.

The entire kidney complex formed by these enzymes resembles bacterial RNAP. The mechanism by which these enzymes find a promoter is quite different from that used by bacteria, but the overall mechanism of transcription initiation is very similar. However, little is known about the termination of eukaryotes.

DdRp range in subunit complexity from single subunit polymerases of bacteriophages (bacterial viruses) to complexes of 12-15 subunits in eukaryotes (Table 1).

Many phages utilize the host cell's polymerase and a self-encoding RNA polymerase, as is the case with phage encoding smaller single-subunit RNA polymerases (eg T7). Some of these polymerases are currently biotechnological because they are relatively easy to mass-produce in bacterial cells, do not require additional protein cofactors to recognize the promoter region, and synthesize transcripts at least twice as fast as bacterial host RNA polymerases. used as the main reagent for Interestingly, single-subunit bacteriophage RNA polymerase autologous recognizes the correct gene and purified DNA, whereas multi-subunit eukaryotic nuclear polymerase requires a very large number of helper protein factors to find the proper location of the template for transcription. You can start at the right place in

Bacterial RNA polymerase contains four subunits of three non-identical proteins in the catalytic core of the enzyme, and this core complex recognizes specific sequences that promote transcription and adds one that reduces binding to non-promoter sites. It requires a cofactor (called sigma). Each species of bacteria has several sigma factors.

Subunit of DNA-dependent RNA polymerase Source Number of subunits in core Sizes (kDa) Bacteriophage T3, T7, <SP6 ^a One ~100 Bacteriophage N4 ^a,b One 320 3 40, 30, 15 Eubacteria, for example, Escherichia coli 4 150, 145, 45 Archaebacteria, for example, Sulfolobus acidocaldarius 13 122, 101, 44, 30, 27, 13.8, 12, 11.8, 10, 9.7, 9.7, 7.5, 5.5 Saccharomyces cerevisiae RNAP I 14 190, 135, 49, 43, 40, 34.5, 27, 23, 19, 14.5, 14, 12.2, 10, 10 Saccharomyces cerevisiae RNAP II 12 191, 140, 35, 25.4, 25, 19, 17.9, 16.5, 14.2, 13, 8.3, 7.7 Saccharomyces cerevisiae RNAP III 13-17 160, 128, 82, 53, 40, 37, 34, 31, 27, 25, 23, 19, 17, 14.5, 11, 10, 10 Human and yeast mitochondria One 140 Spinach and mustard chloroplast ^c One 110 4 154, 120, 78, 38

13

13 141, 110, 36, 26, and others not yet fully characterized Vaccinia virus 8 147, 132, 35, 30, 22, 19, 18, 7 Baculovirus ^a 4 98, 55, 53, 46

a, Viruses use host RNA polymerase as well as self-encoding RNA polymerase for temporal regulation of transcription.

b, The virus encodes one or more RNA polymerases.

c, One or more RNA polymerases are used.

The genomes of some bacteriophages, such as Bacillus subtilis phage SP01 and coliphage T4, encode autologous sigma factors that redirect the bacterial RNA polymerase catalytic core for transcription of viral DNA templates. The catalytic core of bacterial enzymes is used equally for all these transcriptions. In addition, many bacteriophages utilize other viral-coding and host-coding regulatory proteins to modify subsequent steps of the transcription cycle after promoter recognition.

Bacteriophage N4 uses both a host polymerase and the other two polymerases encoded by its own genome (Table 1). One of the viral polymerases is a very large single-subunit enzyme, which, unlike the smaller single-subunit bacteriophage-encoding polymerases, requires a single-stranded DNA binding protein, the host cofactor ssb. The ssb protein allows this 320 kDa polymerase to recognize a specific hairpin structure in viral DNA. This allows the synthesis of additional viral RNA, enabling the replication and assembly of new viral particles.

Like prokaryotic viruses, eukaryotic viruses use a full spectrum of transcriptional strategies, from coding their own polymerases (eg, vaccinia, baculovirus) to using host cell machinery. Many eukaryotic viruses also encode RNA-dependent RNA polymerase (RdRp) (eg hepatitis C virus, polio virus). Virus-encoded protein factors also regulate host cell transcription at various locations in the transcriptional cycle.

Eukaryotic DdRp can be found in the nucleus and organelles. A polymerase found in mitochondria and one polymerase found in chloroplasts resemble small single-subunit bacteriophage RNA polymerases. There are two other RNA polymerases in chloroplasts, one of which is very similar to bacterial enzymes (Table 1). Nuclear RNA polymerase is provided as three distinct biochemically isolated complexes, each originally classified according to the type of gene it transcribes. More recently, it has been defined by subunits and accessory protein factors that act to regulate RNA synthesis. Termed RNA polymerases I, II and III (or A, B and C, respectively) each have at least 12 subunits (Table 1). RNA polymerase I transcribes a gene encoding structural RNA for a subunit of the ribosome. RNA polymerase II transcribes genes encoding a subset of proteins and small RNAs. RNA polymerase III transcribes genes encoding a subset of ribosomal 5S RNA, tRNA, and other small RNAs.

Transcription by DdRp: DNA-dependent RNA polymerases of the bacteriophage T3, T7 or SP6 are a family of relatively small (~100 kDa) single-subunit RNA polymerases that are responsible for all stages of transcription, i.e. initiation, elongation or termination. No additional protein factors are required. These polymerases are easily overexpressed in E. coli. They are very active, terminate less frequently (compared to E. coli RNA polymerase) and transcribe very tightly in their promoters. These properties make these polymerases a very convenient tool for in vitro RNA synthesis using various experimental strategies. RNA polymerase from the bacteriophage T7 (T7 RNAP) is perhaps the most commonly used and commercially available, but more economical to purify in-house for large-scale RNA synthesis.

As in <Figure 1>, bacteriophage RNAP initiates and terminates RNA transcription at a specific sequence (with a specific efficiency). However, the simplest experimental setup for the production of ready RNA in vitro is the so-called efflux transcription, where RNA synthesis begins at a specific T7 promoter and ends when RNAP detaches from the physical end of the DNA template. This setup allows for convenient preparation of chemically synthesized DNA promoter and template sequences. Moreover, it has been found that the DNA template need not be perfectly base paired. Most or all of the coding strand may remain single stranded. The minimum base pair region spanning positions -15 to -3 (+1 indicates the start of the transcription residue) is still sufficient for fully efficient transcription using T7 RNAP. This is consistent with the notion of forming a transcriptional bubble as part of the promoter recognition process. In the crystal structure of T7 RNAP with an open promoter, the template and non-template strands are unwound downstream starting at position -4. This property makes it possible to prepare a single universal DNA top strand for transcription of any RNA sequence. Only the bottom coding DNA strand needs to be redesigned each time.

As in Figure 1, the native T7 RNAP promoter sequence is strongly conserved from position -17 to position +6. Nevertheless, RNA is transcribed even in promoters chemically modified at positions +1 to +6, although yields may be reduced. In general, the most efficient yields are achieved with RNA sequences beginning with GG or GA. To improve the yield, the transcription reaction conditions should be optimized for each RNA sequence. These include varying MgCl2 concentrations and relative amounts of T7 RNAP, template and NTP. In addition to the correct RNA fragment and several shorter stop products, in vitro transcription, T7 RNAP, often incorporates a non-template nucleotide at the 3' end of the main transcript to generate the so-called 'n+1 product'. The desired RNA product must then be isolated from mis-sized transcripts, DNA templates, and unused NTPs and RNAPs.

As shown in <Fig. 1>, RNA of almost any length and sequence can be prepared through in vitro transcription using T7 RNAP. However, for larger RNAs, the use of chemically synthesized DNA templates is not practical because the yield of templates with oligonucleotide length decreases exponentially. For templates larger than 50-100 nt, an alternative approach is to use a fully double-stranded DNA template designed within a linearized high-copy DNA plasmid (eg pUC18.31). Another potential complication of preparing large RNAs is RNA denaturation. This is done during PAGE purification. RNA must be folded back into its original form after such purification, which can sometimes be problematic.

DdRp promoter: DdRp encoded by bacteriophages T7, T3 and SP6 is particularly suitable for polymerase-promoter interaction studies. Each phage enzyme consists of a single species of protein = 100 kDa capable of carrying out all steps of the transcription process in the absence of additional protein factors. Bacteriophage T7 is a prototype of a group of bacterial viruses, each encoding a single subunit DdRp. Other members of this class include coliphages coliphages T3 and BA14, Salmonella phage Salmonella phage SP6, Pseudomonasphage Pseudomonas phage gh-1 and Klebsiella phage phage K11. Phage DdRp is useful in a variety of applications, including large-scale RNA synthesis, nucleic acid amplification, and has proven to be the basis of efficient expression systems in both prokaryotic and eukaryotic cells.

As in <Fig. 2>, despite the high level of structural similarity (judging by the conservation of amino acid sequence), phage DdRp acts precisely and specifically for its promoter sequence. Each phage promoter is associated with a consensus 23-base pair consensus sequence that extends from -17 to +6 (see Figure 1). The core nucleotide sequence, extending from -7 to +1, is highly conserved across the three promoter types, suggesting that these regions interact in a common manner with the shared features of polymerases. Promoter sequences differ significantly in the region from -8 to -12, suggesting that base pairing in this region is important for specific promoter recognition. Studies using synthetic phage promoters have shown that the key determinants of promoter specificity for T3 and T7 enzymes include the base pairs at positions -10 and -11. Substitution of the T3 residue at these two positions in the T7 promoter sequence prevents recognition by T7 polymerase and enables transcription by T3 RNA polymerase. The domains of the T3 and T7 RNA polymerases responsible for the recognition of these determinants were localized, and preliminary crystallographic data suggest that this region of the polymerase is located within the putative DNA binding cleft.

Nucleic Acid Molecules : A nucleic acid molecule is a molecule comprising a nucleic acid component. Nucleic acid molecule refers to DNA or RNA. Used synonymously with "polynucleotide". Nucleic acid molecules are polymers comprising or consisting of nucleotide monomers and are covalently linked to each other by phosphodiester bonds of sugar/phosphate-backbone.

Nucleic acids include DNA (eg, complementary DNA (cDNA), genomic DNA (gDNA), etc.), RNA (eg, message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA), tRNA, microRNA, and/or or one or more nucleic acids of any composition from DNA or RNA analogues (eg containing base analogues, sugar analogues and/or non-natural backbones, etc.), RNA/DNA hybrids and polyamide nucleic acids (PNAs) (eg a set of nucleic acids) or a subset), all of which may be in single- or double-stranded form and, unless otherwise limited, may include known analogs of natural nucleotides that may function in a manner similar to naturally occurring nucleotides.

nucleic acids, including deoxyribonucleotides, ribonucleotides, and known analogues of natural nucleotides. Nucleic acids include derivatives or variants thereof, suitable analogs of RNA or DNA synthesized from nucleotide analogs as equivalents, single-stranded (“sense” or “antisense”, “plus” or “minus” strands, “forward” reading frame or “ back" reading frame) and double-stranded polynucleotides. Nucleic acids may be single or double stranded. Nucleic acids may be of any length of 2 or more, 3 or more, 4 or more, or 5 or more contiguous nucleotides. A nucleic acid may comprise a specific 5' to 3' order of nucleotides known in the art as a sequence (eg, a nucleic acid sequence, such as a sequence).

Nucleic acids may be naturally occurring and/or may be synthetic or altered. For example, the nucleic acid may be an amplicon. The nucleic acid may be from a nucleic acid library, such as a gDNA, cDNA or RNA library. Nucleic acids can be synthesized (eg, chemically synthesized) or generated (eg, by polymerase extension in vitro, eg, by amplification, eg, by PCR). A nucleic acid is or is from a plasmid, phage, virus, self-replicating sequence (ARS), centromere, artificial genome, genome, or other nucleic acid capable of being transcribed in vitro or in a host cell, cell, cell nucleus, or cytoplasm of a cell. may be of

Nucleic acids provided for a process or method described herein may comprise nucleic acids from 1 to 1000, 1 to 500, 1 to 200, 1 to 100, 1 to 50, 1 to 20, or 1 to 10 samples. have. Oligonucleotides are relatively short nucleic acids. Oligonucleotides may be from about 2-150, 2-100, 2-50, or 2 and about 35 nucleic acids in length. Oligonucleotides are 18 to 30, 20 to 28 or 21 to 26 nucleotides in length. Oligonucleotides are single-stranded. Oligonucleotides are primers. Primers are often configured to hybridize to a selected complementary nucleic acid and to be extended by a polymerase after hybridization.

DNA: DNA is a generic abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, ie a polymer made up of nucleotides. These nucleotides usually consist of - by themselves - a sugar moiety, a base component and a phosphate component: deoxyadenosine-monophosphate, deoxythymidine-monophosphate, deoxyguanosine-monophosphate and between deoxy It is a tidine-monophosphate monomer and is polymerized by a unique backbone structure. The backbone structure is generally formed by a phosphodiester bond between the sugar moiety of the first nucleotide proximal to the monomer, i.e., deoxyribose and the second phosphate moiety. The specific order of the monomers, ie the order of the bases linked to the sugar/phosphate-backbone, is referred to as the DNA-sequence. DNA can be single-stranded or double-stranded. In the double stranded form, the nucleotides of the first strand generally hybridize with the nucleotides of the second strand, eg, A/T-base pairs and G/C-base pairs.

RNA, mRNA: RNA is a general abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer made up of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers linked together along the so-called backbone. The backbone is formed by a phosphodiester linkage between the first sugar proximal to the monomer, namely ribose, and the second phosphate component. A specific chain of monomers is called an RNA-sequence.

RNA relates to molecules comprising ribonucleotide residues, and to molecules consisting entirely or largely of ribonucleotide residues. The term "ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. "RNA" means double-stranded RNA, single-stranded RNA, isolated RNA, such as partially or fully purified RNA, essentially pure RNA, synthetic RNA, and recombinantly generated RNA, such as additions, deletions, modified RNA that differs from naturally-occurring RNA by substitution and/or alteration. Such alterations may include, for example, adding a non-nucleotide material, at one or more nucleotides of the RNA, at the end(s) of the RNA, or internally. Nucleotides of RNA molecules may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs, in particular analogs of naturally occurring RNA.

RNA may be single-stranded or double-stranded. In the present invention, single-stranded RNA is preferred. The term "single-stranded RNA" generally refers to embodiments in which a complementary nucleic acid strand (typically a complementary RNA strand; ie, no complementary RNA molecule) is associated with an RNA molecule.

Ordinary RNA can be synthesized by transcription of a DNA-sequence, for example inside a cell. In an advancing cell, transcription is usually carried out inside the nucleus or mitochondria. In vivo, transcription of DNA usually results in so-called premature RNA, which must be processed into so-called messenger-RNA, usually abbreviated as mRNA. For example, processing of immature RNA in eukaryotic organisms involves a variety of different post-transcriptional modifications, including splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. includes The sum of these processes is called the maturation of the RNA.

Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Generally, a mature mRNA comprises a 5' cap, optionally a 5'UTR, an open reading frame, optionally a 3'UTR and a poly(A) sequence. In addition to messenger RNA, several non-coding types of RNA exist, which may be involved in the regulation of transcription and/or translation.

"Variant" with respect to nucleic acid and amino acid sequences includes any variant, in particular mutants, virus strains, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, especially those naturally occurring do. Allelic variants relate to alterations in the normal sequence of a gene, the importance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. In the context of a nucleic acid molecule, "variant" includes a degenerate nucleic acid sequence, wherein a degenerate nucleic acid according to the invention is a nucleic acid that differs in codon sequence from a reference nucleic acid because of the degeneracy of the genetic code. A species homologue is a nucleic acid or amino acid sequence that has a species origin that differs from a given nucleic acid or amino acid sequence. A viral homologue is a nucleic acid or amino acid sequence that has a viral origin that differs from a given nucleic acid or amino acid sequence.

Nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, and/or insertions compared to a reference nucleic acid. Deletion includes removing one or more nucleotides from a reference nucleic acid.

Additional variants include 5' and/or 3' terminal fusions of one or more nucleotides, such as 1, 2, 3, 5, 10, 20, 30, 50 or more nucleotides. Mutations may include, but are not limited to substitutions, wherein at least one nucleotide in the sequence is removed and at least one other nucleotide is inserted in its place (eg, conversions and transitions). Mutations include monobasic sites, bridging sites, and chemically altered or modified bases. Insertion involves adding one or more nucleotides within a reference nucleic acid.

A variant of a particular nucleic acid sequence has one or more functional properties of the particular sequence and is functionally equivalent to a nucleic acid sequence that exhibits the same or similar properties as the particular sequence, eg, a particular nucleic acid sequence.

Derivatives include any chemical derivatization of nucleic acids on nucleotide bases, sugars or phosphates. "Derivatives" also encompass nucleic acids containing non-naturally occurring nucleotides and nucleotide analogs. Derivatization of a nucleic acid preferably increases its stability.

A nucleic acid sequence derived from a nucleic acid sequence refers to a nucleic acid that is a variant of the nucleic acid from which it is derived. Preferably, when replacing a specific sequence in an RNA molecule, a sequence that is a variant for that specific sequence maintains RNA stability and/or translation efficiency.

"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are interchangeably termed single- or double-stranded RNA or DNA, optionally comprising synthetic, non-natural or modified nucleotide bases. It is used to refer to a polymer that has been The sequence of a nucleic acid molecule is generally understood to be a specific and discrete order, ie, a sequence of nucleotides thereof. The sequence of a protein or peptide is generally understood to be a sequence, ie a sequence of its amino acids.

Sequence homology: Two or more sequences are identical (there are homology) if they exhibit the same length and order of nucleotides or amino acids. Percentage of homology generally describes the extent to which two sequences are identical, ie, it generally describes the percentage of nucleotides whose sequence positions correspond to nucleotides in a reference sequence. For purposes of determining the degree of identity, the sequences being compared are considered to represent the same length, ie, the length of the longest sequence of the sequences being compared. This means that the first sequence of 8 nucleotides is 80% identical to the second sequence of 10 nucleotides comprising the first sequence. In other words, in the present invention, the identity of a sequence preferably relates to the percentage of nucleotides of the sequence having the same position in two or more sequences having the same length. Gaps are usually considered non-co-located, regardless of their actual position in alignment.

Stabilized Nucleic Acid Molecules: A stabilized nucleic acid molecule is a nucleic acid molecule, preferably degraded by an enzymatic digest such as, for example, an environmental factor or an exo- or endonuclease, rather than an unmodified nucleic acid molecule. A DNA or RNA molecule that has been chemically modified so that it is more stable to disintegration or degradation. A stabilized nucleic acid molecule is one that is stabilized in cells such as prokaryotic or eukaryotic cells, mammalian cells such as human cells. The stabilizing effect can also be exerted outside the cell, for example, in a buffer solution and the like, for example, in a process for preparing a pharmaceutical composition comprising a stabilized nucleic acid molecule.

Heterologous sequence: In general, two sequences are understood to be 'heterologous' unless they can be derived from the same gene. That is, heterologous sequences may be derived from the same organism, but they do not occur naturally (in nature) in the same nucleic acid as in the same mRNA.

Expression : includes the production of RNA or the production of RNA and proteins. This also includes partial expression of nucleic acids. In addition, expression may be transient or stable. In the context of RNA, the term "expression" or "translation" relates to the process by which one strand of messenger RNA directs the assembly of amino acid sequences within the liposome of a cell to make a peptide or protein. A gene refers to a specific nucleic acid sequence for producing one or more cellular products and/or for achieving one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid section (typically DNA, but RNA in the case of RNA viruses) comprising a nucleic acid encoding a particular protein or functional or structural RNA molecule. mRNA refers to messenger-RNA and typically relates to a transcript produced using a DNA template and encoding a peptide or protein. Typically, an mRNA comprises a 5'UTR, a protein coding region, a 3'UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are well known to those skilled in the art. For example, a variety of in vitro transcription kits are available on the market. According to the present invention, mRNA can also be modified by stabilizing modification and capping.

Open reading frame: may be a sequence of several nucleotide triplets that can generally be translated into peptides or proteins. The open reading frame is preferably a start codon, i.e. a combination of the following three nucleotides (ATG or AUG), usually encoding the amino acid methionine, at its 5' end and a subsequent region, usually multiple 3 nucleotides in length. contains The ORF is preferably terminated by a stop-codon (eg TAA, TAG, TGA). In general, this is only the stop-codon of the open reading frame. Accordingly, in the present invention the open reading frame preferably begins with a start codon (eg ATG or AUG) and preferably with a stop codon (eg TAA, TGA, or TAG or UAA, UAG, UGA, respectively) ) is a nucleotide sequence consisting of a number of nucleotides that can be divided by three. The open reading frame may be isolated or may be incorporated into a longer nucleic acid sequence, such as a vector or mRNA. The open reading frame may also be referred to as a “protein coding region”.

Bicistronic RNA, multicistronic RNA: Bicistronic or multicistronic RNA typically has two (bicistronic) or more (multicistronic) open reading frames (ORFs). RNA, preferably mRNA. An open reading frame is a sequence of codons that can be translated into a peptide or protein.

The term "poly(A) sequence" or "poly(A) tail" refers to a sequence of contiguous or interrupted adenylate residues, typically located at the 3' end of an RNA molecule. Contiguous sequences are characterized by consecutive adenylate residues. In fact, a contiguous poly(A) sequence is typical. The poly(A) sequence is attached during eukaryotic transcription in the cell nucleus to the free 3' end of the RNA by template-independent RNA polymerase after transcription, but is usually not encoded in eukaryotic DNA, whereas the present invention relates to DNA and the encoded poly(A) sequence.

A nucleic acid such as RNA, for example mRNA, may encode a peptide or protein. Thus, a transcribable nucleic acid sequence or transcript thereof may comprise an open reading frame (ORF) encoding a peptide or protein.

G/C Chemical Modification: A G/C-chemically modified nucleic acid may be an RNA molecule, based on a chemically modified wild-type sequence comprising an increased number of guanosine and/or cytosine nucleotides compared to the wild-type sequence. This increased number can be produced by substitution of codons containing adenosine or thymidine nucleotides with codons containing guanosine or cytosine nucleotides. When abundant G/C content occurs in the coding region of DNA or RNA, this exploits the degeneracy of the genetic code. Thus, codon substitutions preferably do not modify the encoded amino acid residues, but only increase the G/C content of the nucleic acid molecule.

Unmodified reference RNA: RNA that corresponds to a wild-type RNA sequence as it exists in nature. For example, a non-chemically modified reference mRNA corresponds to an mRNA sequence comprising naturally occurring nucleotides and comprises nucleotides comprising a naturally occurring modification, and/or a naturally occurring untranslated region, such as , a naturally occurring 5' cap (CAP) structure m7GpppN, optionally comprising a naturally occurring 5'UTR, if present in the naturally occurring mRNA sequence, and if present in the naturally occurring mRNA sequence and approximately 30 adenosine The wild-type open reading frame, if present in the poly A sequence with

Chimeric Enzyme : Chimeric enzyme refers to an enzyme that is not a natural enzyme found in nature. Thus, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, different enzymes) or catalytic domains derived from the same source (eg, the same enzyme), arranged in a manner different from that found in nature. Chimeric enzymes include monomeric (ie single unit) enzymes as well as oligomeric (ie multiple unit) enzymes, particularly hetero-oligomeric enzymes. Monomeric enzymes refer to single-unit enzymes consisting of only one polypeptide chain.

Protein: In general, a protein comprises one or more peptides or polypeptides. In general, proteins fold into three-dimensional shapes that may be required for proteins to exert biological functions.

"Polypeptide", "peptide", "amino acid sequence" and "protein" are used herein interchangeably to refer to a polymer of amino acid residues. These terms apply to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acid. The terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” also include, but are not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-sulfation. Modifications such as ribosylation are included.

Peptide: Generally, a peptide or polypeptide is a polymer of amino acid monomers linked by peptide bonds. It generally contains less than 50 monomer units. However, the term peptide is not a waiver for molecules having more than 50 monomer units. Long peptides, also called polypeptides, generally have 50 to 600 monomer units.

By “isolated molecule” is meant a molecule that is substantially free of other molecules, such as other cellular material. The term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) e.g. cleaved and nucleic acids purified by gel-electroporation fractionation, or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that can be manipulated by recombinant techniques.

"Indel" refers to an insertion/deletion mutation consisting of a plurality of nucleotides inserted or deleted at a double strand break (DSB) site in a nucleic acid.

"Knockdown" refers to a decrease in expression of a particular gene product (eg, protein, mRNA, or both). Knockdown of a protein can be measured by detecting the protein secreted by the tissue or cell population (eg, in serum or cell medium) or by detecting the total cellular amount of the protein from the tissue or cell population of interest. Methods for measuring knockdown of mRNA are known and include sequencing of mRNA isolated from a tissue or cell population of interest. "Knockdown" is a loss of some expression of a particular gene product, e.g., a decrease in the amount of transcribed mRNA or a decrease in the amount of a protein expressed or secreted by a cell population (including an in vivo population such as that found in tissues). can refer to

Knockout refers to loss of expression of a specific protein in a cell. Knockout can be measured by detecting the amount of protein secretion from a tissue or cell population (eg, in serum or cell medium) or by detecting the total cell mass of the protein tissue or cell population. The method of the present disclosure "knocks out" one or more cells of the target protein (e.g., in a cell population comprising an in vivo population such as found in a tissue). In some embodiments, the knockout is, for example, It is not the formation of a mutant of the target protein produced by the indel, but a complete loss of target protein expression in the cell.

Transformation: The term "transfection" refers to the introduction of a nucleic acid molecule, DNA or RNA (eg mRNA) molecule into a cell, preferably a eukaryotic cell. In the present invention, the term "transformation" includes any method known to a person skilled in the art for introducing a nucleic acid molecule into a cell, preferably a eukaryotic cell such as a mammalian cell. Such methods include, for example, electroporation, for example cationic lipid and/or liposome-based lipofection, calcium phosphate precipitation, nanoparticle-based transformations, virus-based transformations, or transformations based on cationic polymers such as DEAE-dextran or polyethyleneimine. Preferably, the introduction is non-viral.

Vector: Used in its most general sense, it encompasses, for example, any intermediate carrier capable of introducing said nucleic acid into a prokaryotic and/or eukaryotic subject cell and, where appropriate, integrating it into the genome. Such vectors are preferably transcribed and/or expressed in cells. Vectors include plasmids, phagemids, viral genomes and fragments thereof. By “introducing” a nucleic acid molecule (eg, a plasmid, a linear nucleic acid fragment, RNA, etc.) or protein into an organism, it is meant using the nucleic acid or protein to transform the cells of the organism so that the nucleic acid or protein functions in the cell. In the present invention, "transformation" includes both stable transformation and transient transformation. "Stable transformation" means introducing an exogenous nucleotide sequence into a genome to achieve stable inheritance of a foreign gene. When stably transformed, the exogenous nucleic acid sequence is stably inserted into the genome of the organism and its successive generations. "Transient transformation" means that a nucleic acid molecule or protein is introduced into a cell so that an exogenous gene performs its function without being stably inherited. In the case of transient transformation, no exogenous nucleic acid sequence is inserted into the genome.

Carrier/Polymer Carrier: In the present invention, the carrier may generally be a compound that facilitates transport or complexation of another compound (cargo). A polymeric carrier is generally a carrier from which a polymer is formed. A carrier may be associated with its cargo by covalent or non-covalent interactions. A carrier is capable of transporting a nucleic acid, eg, RNA or DNA, into a target cell. The carrier may be a cationic component.

Cationic component: The term “cationic component” refers to a molecule that is generally charged, which is generally 1 to 9, preferably less than 9 (eg 5 to 9), or less than 8 (eg for example 5 to 8), or less than 7 (eg 5 to 7), most preferably positively charged (cation) at a physiological pH value of eg 7.3 to 7.4. Thus, the cationic component may be any positively charged compound or polymer, preferably a cationic peptide or protein, which is positively charged under physiological conditions, in particular in vivo physiological conditions. A 'cationic peptide or protein' may contain at least one positively charged amino acid or one or more positively charged amino acids, for example selected from Arg, His, Lys or Orn. Thus, 'polycationic' compounds are also within the range that exhibit more than one positive charge under the given conditions.

Vehicle: A vehicle is generally understood to be a material suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound. For example, it may be a physiologically acceptable lipid suitable for storage, transport and/or administration of a pharmaceutically active compound.

Pharmaceutically effective amount : In the present invention, a pharmaceutically effective amount is generally used to replace a pathological level of an expressed peptide or protein, or to replace a missing gene product, e.g., a pathology. It is understood as an amount sufficient to induce a pharmaceutical effect, such as an immune response, in the case of adverse circumstances.

The carrier of the genome editing RNA/DNA system described herein is fused or associated with one or more polypeptides (eg, toxins, ligands, receptors, cytokines, antibodies, etc. or combinations thereof). The delivery system of the genome editing system described herein may include a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (eg, glycosylation of antigen binding proteins) and dextran.

The genome editing strategy of the present invention is a promising method for studying gene function and as a powerful means to combat disease, for example by introducing targeted genomic sequence changes to specific loci in living cells or organisms. Currently used genome editing includes zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), clustered, spaced-distributed short palindromic repeats (CRISPR)-associated (Cas) nucleases. First, methods using a fusion protein of algonut endonuclease and M-MLV reverse transcriptase and Cas9 H840A nickase have been effectively used by many laboratories.

The genome editing strategy of the present invention is a promising method for studying gene function and as a powerful means to combat disease, for example by introducing targeted genomic sequence changes to specific loci in living cells or organisms.

The introduction of genome editing tools that encode one or more polypeptides for genome editing purposes has been a goal of biomedical research for many years. While the prior art discloses approaches to the delivery of genome editing tools to target cells or organisms, the types of nucleic acid molecules and/or delivery systems are different. Ribonucleic acid (RNA) molecules have received attention in recent years, influenced by the safety concerns associated with the use of deoxyribonucleic acid (DNA) molecules. Various approaches have been proposed, including administration of single-stranded RNA or double-stranded RNA in the form of naked RNA, or in complex or packaged form, for example, to a non-viral or viral carrier.

In viruses and viral delivery vehicles, genome editing tools are typically encapsulated by proteins and/or lipids (viral particles). For example, engineered RNA virus particles from RNA viruses have been proposed as delivery vehicles for genome editing.

Other embodiments and advantages of the gene editing RNA/DNA system of the present invention are described below.

The genome editing RNA/DNA system of the present invention is a genome editing RNA/DNA system (FIG. 3) having at least one endogenous genome editing and transcription-capable component in a cell,

DdRp RNA expression cassette comprising DNA dependent RNA Polymerase (DdRp) mRNA (capped CM_DdRp mRNA) having a structure capable of intracellular translation and comprising chemically modified nucleotides (CM); and

It provides an RNA / DNA system comprising a; DNAge expression cassette encoding a genome editing tool that can be transcribed in trans by DdRp,

Here, the DNAge expression cassette capable of being transcribed by DdRp may be composed of a DNAen expression cassette encoding a targeting endonuclease that performs genome editing and a DNAsg expression cassette encoding a guide nucleic acid.

The genome editing RNA/DNA system of the present invention comprises at least two nucleic acid expression cassettes. Thus, it may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more nucleic acid molecules.

As another aspect of the present invention for achieving the above target,

The RNA / DNA system additionally comprises an auto_DdRp expression cassette comprising a promoter capable of binding to DdRp for self-transcription of the DdRp mRNA and DNA encoding DdRp; It provides an RNA/DNA system that does this.

As another aspect of the present invention for achieving the above target,

DdRp RNA expression cassette comprising capped CM_DdRp mRNA; and

For self-transcription of DdRp mRNA, auto_DdRp containing a promoter capable of binding to DdRp and DNA encoding DdRp, and an auto_(DdRp+DNAge) expression cassette containing a DNAge encoding a genome editing tool transcribed by the DdRp; It provides an expression RNA / DNA system comprising a, characterized in that they are configured to be operably linked.

Here, the capped CM_DdRp mRNA, auto_DdRp expression cassette, DNAge expression cassette, or auto_(DdRp+DNAge) expression cassette may include at least one nucleotide sequence modification (SM).

In order to utter the object of the present invention, the DdRp encoded by the bacteriophages T7, T3 and SP6 that is composed of one subunit and does not require other auxiliary elements for enzymatic activity in DdRp of <Table 1> was selected.

DdRp of the auto_DdRp expression cassette of the present invention is DdRp itself, or the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, N7-guanine methyltransferase, and It may be a chimeric enzyme comprising a catalytic domain of DNA-dependent RNA polymerase (DdRp), and a method for synthesizing and providing an RNA molecule having a 5'-terminal m7GpppN cap highly translatable by a eukaryotic translation apparatus may be provided.

In the present invention, T7 RNA polymerase (polymerase) is an RNA polymerase that recognizes a phage-specific promoter sequence consisting of about 20 base pairs derived from bacteriophage T7, and catalyzes the synthesis of RNA from DNA in the 5' to 3' end direction. is an enzyme Specifically, unlike eukaryotic RNA polymerase, which requires various intracellular cofactors, T7 RNA polymerase has the advantage of being able to induce transcription without additional intracellular cofactors. In addition, since there is no other RNA polymerase specific for the T7 promoter in the cell of eukaryotes, the transcription system using the T7 promoter/T7 RNA polymerase does not receive interference from other transcription systems in the cell as well as other transcription systems. It does not interfere with the system. On the other hand, other promoters belonging to the same RNA polymerase III type as the T7 promoter, for example, the U1 promoter or the H1 promoter, do not have the property of not causing such interference of the T7 promoter because they already exist in the cell, and the transcription of the present invention The system is not suitable for controlling transcription. More specifically, the T7 RNA polymerase mRNA fragment of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

In addition, the DdRp RNA expression cassette containing the T7 RNA polymerase mRNA of the present invention may further include a 5'cap structure. The 5'cap structure refers to 7-methylguanosine present at the 5' end of mRNA of most eukaryotic cells and viruses, and mRNA is produced by ribonuclease (RNase). It prevents degradation, and in the cytoplasm, it can bind to a translation initiator and act as a ribosome binding site. In general, the cytoplasmic transcript has a problem in that the translation efficiency by the ribosome is lowered because there is no 5'cap structure essential to the nuclear transcript. Accordingly, the present invention uses a T7 RNA polymerase mRNA fragment having a 5'cap structure to not only increase intracellular stability and intracytoplasmic translation efficiency, but also lower the expression cassette DNA previously used for nuclear-dependent mRNA expression. It is characterized in that it can effectively provide early T7 RNA polymerase in the cytoplasm by overcoming the limitation of nuclear membrane permeation.

In the present invention, in order to synthesize a DdRp RNA expression cassette containing a T7 RNA polymerase mRNA containing a 5′-cap structure and a CM, a dsDNA fragment having a T7 promoter-T7 polymerase sequence is first synthesized by chemical synthesis. (Bioneer), T7 polymerase mRNA having a 5'cap structure and CM-containing T7 promoter-T7 polymerase DNA fragment was synthesized in vitro. T7 polymerase mRNA including the 5'cap structure and CM can exist for a long time by securing in vivo stability.

The DdRp RNA expression cassette encoding the RNA transcribed by DdRp of the present invention comprises a T7 promoter, a DNA encoding the RNA, a poly adenine tail (poly A tail) and a T7 termination sequence, and these are operably linked it could be

In the present invention, by the product of the RNA expression cassette containing the T7 RNA polymerase mRNA, the auto_DdRp DNA expression cassette encoding the T7 RNA polymerase mRNA having the T7 promoter or the DNAge expression cassette having the T7 promoter are also transcribed and amplified. , may be included without limitation in the expression of a specific RNA. In the present invention, T7 RNA polymerase has very high specificity for the T7 promoter, and can transcribe only DNA downstream of the T7 promoter.

Therefore, by using the T7 RNA polymerase in the present invention, it does not react with other promoters in the cell and binds and transcribes the DNA encoding the mRNA having only the T7 promoter, and RNA can be transcribed and amplified with high efficiency. .

Existing expression cassettes, plasmids, or viral systems are nuclear-dependent, requiring transcription using a tool located in the nucleus, so that the existing expression cassette must be transported to the nucleus. However, there is a disadvantage in that less than 1% of the expression cassette introduced through the cell membrane moves to the nucleus.

The expression expression RNA/DNA system of the present invention seeks to overcome the disadvantage of efficiency of transfer to the nucleus by constructing and using a transcription tool in the cytoplasm as shown in <Fig. 3>.

As shown in <Fig. 4>, the genome editing RNA / DNA system of the present invention can design and manufacture various types of expression cassettes by combining various components,

The present invention provides two nucleic acid expression cassettes, a DdRp RNA expression cassette; and a DNAge expression cassette encoding a genome editing tool.

The present invention may also provide a system comprising a DNAge expression cassette comprising three nucleic acid expression cassettes, each separately comprising a DdRp RNA expression cassette, a DNAen expression cassette encoding a targeting endonuclease, and a DNAsg expression cassette encoding an sgRNA. have.

The DdRp RNA expression cassette constituting the genome editing RNA/DNA system of the present invention synthesizes DdRp through a tool necessary for protein synthesis from RNA in the cytoplasm. The synthesized DdRp binds to the promoter in the DNAge expression cassette of the present invention to transcribe and synthesize tools necessary for genome editing in the cytoplasm. The nuclear-independent synthesized genome editing tool forms a complex capable of genome editing and moves to the nucleus, enabling efficient genome editing. Accordingly, genome editing in a target cell or target organism is made possible by the genome editing RNA/DNA system of the present invention.

Existing genome editing methods include CRISPR plasmid method, mRNA method encoding Cas protein and sgRNA, and ribonucleoprotein (RNP) complex method consisting of Cas protein and sgRNA. CRISPR/Cas9 plasmid containing DNA fragment encoding Cas protein and sgRNA In the genome editing method, the CRISPR/Cas9 plasmid encoding the genome editing tool is nuclear-dependent and transcribed using a tool located in the nucleus. must move to However, there is a disadvantage that less than 1% of the CRISPR/Cas9 plasmid introduced through the cell membrane moves to the nucleus,

As shown in <Fig. 5>, the genome editing RNA/DNA system of the present invention attempts to overcome this problem by constructing and using a transcription tool in the cytoplasm.

The present invention provides a DNA comprising a nucleic acid sequence encoding a DdRp RNA expression cassette and a DNAge expression cassette, or both, according to the present invention.

Here, the DdRp RNA expression cassette is present in the RNA strand, and the DNAge expression cassette is present in different DNA strands, and the DNAge expression cassette may be transcribed in trans action by DdRp.

Preferably, the DNAge expression cassette capable of being transcribed by the DdRp is at least one selected from the group comprising DNAen encoding a targeting endonuclease and DNAsg encoding RNA related to a specific targeting nucleotide sequence of a selected genome.

Here, in the DNAge expression cassette, together with the DNA related to the specific targeting sequence of the selected genome, RNA related to the specific targeting sequence of the selected genome artificially synthesized from the outside may also be injected into the cell, or instead externally DNA related to a specific targeting nucleotide sequence of a selected genome artificially synthesized in a cell may be injected into the cell.

The present invention provides an auto-DdRp expression cassette comprising a DNA fragment encoding DdRp transcribed by the DdRp in addition to the genome editing RNA/DNA system comprising the DdRp RNA expression cassette and the DNAge expression cassette according to the present invention. can The auto-DdRp expression cassette is capable of executing a DdRp expression loop in which the DdRp DNA fragment downstream of the promoter to which the DdRp binds is repeatedly self-transcribed by DdRp synthesized from the DdRp RNA expression cassette.

The present invention provides a DNA comprising a nucleic acid sequence encoding a DdRp RNA expression cassette, an auto-DdRp expression cassette and a DNAge expression cassette, or both, according to the present invention.

In addition, the DdRp included in the auto-DdRp expression cassette may be itself or a DdRp chimerase having a function of catalyzing the formation of a 5*?**?*Cap structure.

In the following, the DdRp RNA expression cassette and the DNAge expression cassette may be separately prepared and co-transfected, and in this case, the auto_(DdRp+DNAge) expression cassette may be used instead.

The present invention provides a method for genome editing by a self-transcribed RNA/DNA system in a cell comprising the following steps.

(a) obtaining a DdRp RNA expression cassette;

(b) obtaining a DNAge expression cassette capable of being trans-transcribed by DdRp, and

(c) co-inoculating the DdRp RNA expression cassette and the DNAge expression cassette into the cell;

Here, the DdRp RNA expression cassette includes a 5' cap for inducing translation of DdRp, and additionally, the step of obtaining an auto_DdRp expression cassette may be performed and inoculated together in step (c).

In this method, the DdRp RNA expression cassette and/or the DNAge expression cassette are as defined above for the self-transcribed RNA/DNA system of the present invention.

The present invention provides a cell comprising the self-transcribed RNA/DNA system of the present invention. Cells are seeded according to the method according to the invention. Cells are obtainable by the method of the present invention. A cell may be part of an organism.

The present invention provides a method for genome editing by a self-transcribed RNA/DNA system in a subject, comprising the following steps.

(a) obtaining a DdRp RNA expression cassette;

(b) obtaining a DNAge expression cassette capable of being trans-transcribed by DdRp, and

(c) administering to the subject a genome editing RNA/DNA system consisting of a DdRp RNA expression cassette and a DNAge expression cassette;

Here, the DdRp RNA expression cassette includes a 5' cap for inducing translation of DdRp, and additionally, the step of obtaining an auto_DdRp expression cassette may be performed and inoculated together in step (c).

In the method, the DdRp RNA expression cassette and/or the DNAge expression cassette are as defined above for the genome editing RNA/DNA system of the present invention.

I. DdRp RNA Expression Cassette

The self-transcribed RNA/DNA system of the present invention comprises an RNA expression cassette comprising DNA dependent RNA Polymerase (DdRp) mRNA; and

A DNAge expression cassette comprising a DNA fragment encoding a genome editing tool transcribed by the DdRp; includes, and is configured to be operably linked.

The DdRp RNA expression cassette is

(1) 5' UTR;

(2) an open reading frame coding for DdRp, and

(3) contains 3'UTR.

Nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be cytoplasmic or hybrid DdRp with NLS can be enzymatically activated in the nucleus to selectively start transcription at the T7 promoter placed in chromosomal or plasmid DNA and stop at specific T7 terminator sequences.

Structures that can be translated into cells

The DdRp RNA expression cassette for expressing DdRp comprises a 5' cap.

The terms "5'cap", "cap", "capped" and ""5'cap structure" are synonymous to refer to a dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as precursor messenger RNA. 5'cap is a structure in which (optionally chemically modified) guanosine is attached to the first nucleotide of an mRNA molecule through 5' in a 5' triphosphate bond (or a chemically modified triphosphate bond 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 accomplished by in vitro transcription of a DNA template in the presence of said 5' cap, wherein said 5' cap is co-transcriptionally incorporated into the resulting RNA strand. Alternatively, the RNA may be generated, for example, by in vitro transcription, and the 5'cap may be attached to the RNA after transcription using a capping enzyme, for example, a capping enzyme of a papillomavirus. In capped RNA, the 3' position of the first base of the (capped) RNA molecule is linked at the 5' position of the subsequent base ("second base") of the RNA molecule via a phosphate diester bond.

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

Translation of DdRp is driven by a 5' cap on DdRp mRNA. The 5' cap on DdRp mRNA has a very positive effect on the expression of DdRp as well as the overall performance of the system. Efficient production of trans-encoded target RNA can be achieved.

The DdRp mRNA of the present invention does not contain an internal ribosome entry site (IRES) element. In general, the internal ribosome entry site, abbreviated IRES, is a nucleotide sequence that enables translation initiation from messenger RNA (mRNA) from a location different from the 5' end of the DdRp mRNA sequence, such as in the center of the DdRp mRNA sequence. located in the middle of the sequence.

Substitution of the IRES with the 5' cap in the present invention is a specific modification of the RNA molecule that does not affect the sequence of the polypeptide encoded by the DdRp mRNA molecule (non-polypeptide-sequence modifying modification). For example, in the case of eukaryotic messenger RNA, non-polypeptide-sequence modifying modifications include selection of specific untranslated regions, introduction of introns (eg, rabbit beta-globin intron II sequence), or silent modifications of the coding sequence. , eg, by adaptation to preferential codon usage in a subject cell or subject organism without alteration (silent modification) of the encoded polypeptide sequence.

Production of the target protein encoded by according to the present invention is efficient when the cap is present on the DdRp mRNA.

The presence of a 5' cap in eukaryotic mRNA is thought to play an important role, inter alia, in regulating the intranuclear release of mRNA and facilitating processing, particularly 5' proximal intron excision. The DdRp mRNA of the present invention typically does not need to be exported or processed from the nucleus. Nevertheless, the presence of a 5' cap on the DdRp mRNA is advantageous.

For eukaryotic mRNA, it is generally described that the 5' cap is also usually involved in efficient translation of the mRNA: in general, in eukaryotic cells, translation is initiated only at the 5' end of the messenger RNA (mRNA) molecule unless there is an IRES. .

Eukaryotic cells can present RNA with a 5' cap during intranuclear transcription. Newly synthesized mRNA is usually transformed 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, N represents any nucleoside) is 5' in the cell by a capping enzyme having RNA 5' triphosphatase and guanylyltransferase 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, natural 5' cap dinucleotides are typically non-methylated cap dinucleotides (G(5')ppp(5')N; also referred to as GpppN) and methylated cap dinucleotides ((m7G(5')ppp) (5')N; also called m7GpppN).

The DdRp mRNA of the present invention does not depend on the capping device of the target cell. When transfected into a subject cell, it is believed that the DdRp mRNA of the present invention is typically not limited to the cell nucleus, ie, the site where capping occurs in a typical eukaryotic cell.

Similar to the IRES in the prior art, the 5' cap on DdRp mRNA is useful for triggering translation initiation of DdRp. The eukaryotic translation initiation factor elF4E may be thought to be involved in the binding of capped mRNAs to ribosomes. The presence of the 5' cap significantly increases performance. Accordingly, the DdRp mRNA of the present invention includes a 5' cap.

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

Since the capped RNA of the present invention can be prepared in vitro, it does not depend on the capping device of the target cell. The most frequently used method for forming capped RNAs in vitro is bacteria in the presence of all four ribonucleoside triphosphates and cap dinucleotides such as m7G(5')ppp(5')G (also called m7GpppG). Or to transcribe the DNA template with bacteriophage RNA polymerase.

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

A 5' cap is a 5' cap analog. These embodiments are particularly suitable when 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.

In contrast to the engineered replication system described previously, the DdRp mRNA of the present invention preferably comprises a cap analog. Therefore, the translation of DdRp in the system of the present invention is preferably induced by a cap analog.

Ideally, cap analogs associated with higher translational efficiency and/or increased resistance to degradation in vivo and/or increased resistance to degradation in vitro are selected. Accordingly, the present invention does not contain any modifications and provides a natural cap [(m7G(5')ppp(5')G; also called m7GpppG, commercially available ScriptCap m7G capping system (which may be added by Epicentre Biotechnologies)] The expression containing the is presented.

UTR

An “Untranslated Region” or “UTR” relates to a region that is transcribed but not translated into an amino acid sequence or to the corresponding region in an RNA molecule, such as an mRNA molecule.

In the DdRp RNA expression cassette of the present invention, the term "3'UTR" relates to a region located 3' of the DdRp coding region for DdRp, and the term "5'UTR" refers to a region located 5' of the DdRp coding region. is about

The 3'UTR, if present, is located at the 3'end of the gene, downstream of the stop codon of the protein coding region, although the term "3'UTR" preferably does not include the 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 immediately next to the 5' cap (if present).

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

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

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

Preferably, the DdRp mRNA comprises a 5'UTR and/or a 3'UTR that is not heterologous or native to that from which the DdRp is derived. This allows the design of untranslated regions according to the desired translation efficiency and RNA stability.

The 3'UTRs of the present invention have one or more foldables to provide a stem-loop structure that interacts with proteins (such as RNA-binding proteins) known to act as barriers for exoribonucleases or to increase RNA stability. Reverse iterations may be included.

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

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

The DdRp mRNA of the present invention activates to increase the translation efficiency and/or stability of but not human beta-globin 3'UTR, a fragment thereof, or a variant of human beta-globin 3'UTR or a fragment thereof, 3'UTR include

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

The UTR-containing RNA according to the 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 allows transcription of RNA with 5'UTR and/or 3'UTR.

A 3'UTR is a portion of an mRNA that is usually located between the protein coding region (ie, open reading frame) of the mRNA and the poly(A) sequence. The 3'UTR of the mRNA is not translated into the amino acid sequence. The 3'UTR is usually encoded by a gene that is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into immature mRNA containing selective introns. Thereafter, the immature mRNA is further processed to undergo a maturation process to become a mature mRNA. This maturation process includes 5' capping, splicing of immature mRNA to cut additional intro, and polymerization of adenylate at the 3' end of immature mRNA and additional endo- or exo nucleases. Modifications at the 3' end, such as truncation, are included. In the present invention, the 3'UTR corresponds to the sequence of a mature mRNA located 3' to the stop codon of the protein coding region, preferably 3' immediately next to the stop codon of the protein coding region, and 5 of the poly(A) sequence 'to the nucleotide immediately adjacent to the 'flank, preferably 5' of the poly(A) sequence. In the present invention, the term "3'UTR of a gene", such as "3'UTR of alpha or beta globin", refers to a sequence corresponding to the 3'UTR of a mature mRNA derived from such a gene, i.e., the transcription of the gene and the expression of the immature mRNA. It is the sequence corresponding to the mRNA obtained by maturation. The term "3'UTR of a gene" includes DNA sequences and RNA sequences of 3'UTRs.

A 5'UTR is generally understood as a specific section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. Generally, the 5'UTR starts with a transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'UTR may include elements for regulating gene expression, also called regulatory elements. Such a regulatory element may be a ribosome binding site or a 5'-terminal oligopyrimidine tube (Oligopyrimidine Tract).

The 5'UTR can be modified post-transcriptionally 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. 5'UTR is from the nucleotide located 3' of the 5' cap, from the nucleotide located immediately 3' to the 5' cap, to the nucleotide located 5' of the start codon of the protein coding region. Corresponds to a sequence extending up to the nucleotide located immediately 5' of the start codon of the region. The nucleotide located immediately 3' of the 5' cap of the mature mRNA generally corresponds to the start of transcription. The 5'UTR sequence may be an RNA sequence, such as an mRNA sequence used to define a 5'UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the present invention, the 5'UTR of a gene is a sequence corresponding to the 5'UTR of a mature mRNA derived from this gene, that is, a sequence corresponding to an mRNA obtained by transcription of the gene and maturation of an immature mRNA. The 5'UTR of a gene includes the DNA sequence and the RNA sequence of the 5'UTR.

The 5' terminal oligopyrimidine tube (Oligopyrimidine Tract, TOP) is generally a 5' terminal region of a nucleic acid molecule, such as a 5' terminal region of a specific mRNA molecule or a functional entity of a specific gene, for example, the 5' end of a transcription region. A stretch of pyrimidine nucleotides located in the region. The sequence usually begins with a cytidine corresponding to the beginning of transcription, usually followed by a stretch of about 3 to 30 pyrimidine nucleotides. Pyrimidine Stretch The 5' TOP is thus terminated with one nucleotide 5' of the first purine nucleotide located downstream of the TOP. A messenger RNA containing a 5'-terminal oligopyrimidine tube is often referred to as TOP mRNA. Thus, a gene providing such messenger RNA is referred to as a TOP gene. TOP sequences are found, for example, in mRNA and ribosomal proteins encoding genes and peptide elongation factors.

In the present invention, the TOP motif is a nucleic acid sequence corresponding to the 5'TOP as defined above. Therefore, in the present invention, the TOP motif is preferably a stretch of pyrimidine nucleotides having a length of 3 to 30 nucleotides. The TOP-motif consists of at least 3 pyrimidine nucleotides, at least 4 pyrimidine nucleotides, at least 5 pyrimidine nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 pyrimidine nucleotides, wherein the The stretch begins at its 5' end with a cytosine nucleotide.

In TOP genes and TOP mRNAs, the TOP-motif preferably starts at its 5' end with the start of transcription and ends with one nucleotide 5' of the first purine residue in said gene or mRNA. In the sense of the present invention, the TOP motif is 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, if this stretch is at the 5' end of each sequence, such as a nucleic acid sequence derived from the SM_mRNAi according to the invention, the 5'UTR element of the SM_mRNAi according to the invention, or the 5'UTR of the TOP gene as described herein. A stretch of three or more pyrimidine nucleotides is referred to as a "TOP motif" if located in In other words, a stretch of 3 or more pyrimidine nucleotides located anywhere within a 5'UTR or 5'UTR element that is not located at the 5'end of the 5'UTR or 5'UTR element is 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. As defined above, the 5'UTR of the TOP gene corresponds to the sequence of the 5'UTR of the mature mRNA derived from the TOP gene, preferably 5' of the start codon from the nucleotide located 3' of the 5'cap (CAP). to the nucleotide located in The 5'UTR of a TOP gene generally does not contain any start codons, 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 that appear 5' of the start codon (AUG) of the open reading frame to be translated.

The 5'UTR of TOP genes is generally relatively short. The 5'UTR length of a TOP gene can vary from 20 nucleotides to 500 nucleotides, and is generally less than about 200 nucleotides, less than about 150 nucleotides, and less than about 100 nucleotides. In the sense of the present invention, an example of a 5'UTR of a TOP gene is a nucleic acid sequence extending from the nucleotide at position 5 to the nucleotide located immediately 5' of the start codon (eg, ATG). In particular, the 5'UTR fragment of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif. The '5'UTR of the TOP gene' represents the 5'UTR of the naturally occurring TOP gene.

poly(A) sequence

The DdRp RNA expression cassette of the present invention may comprise a 3' poly(A) sequence.

The poly(A) sequence is essentially at least 20, at least 26, at least 40, at least 80, at least 100, no more than 500, no more than 400, preferably no more than 300, no more than 200, in particular no more than 150 of A nucleotides, in particular consisting of or comprising about 120 A nucleotides.

Depending on the number of nucleotides in the poly(A) sequence, typically at least 50%, at least 75% of the majority 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). ), G nucleotides (guanylate), C nucleotides (cytidylates). All nucleotides in the poly(A) sequence, i.e. 100% according to the number of nucleotides in the poly(A) sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate.

The genome editing tool DNA expression cassette, and auto_DdRp DNA expression cassette of the present invention may include a DNA sequence (coding strand) encoding a poly(A) sequence.

The present invention is based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand, a 3' poly(A) attached during RNA transcription, ie during the production of transcribed RNA in vitro. ) provides the sequence. The DNA sequence (coding strand) encoding the poly(A) sequence is referred to as the poly(A) cassette.

The 3' poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence with an even distribution of 4 nucleotides (dA, dC, dG, dT). Such random sequences may be 5 to 50, preferably 10 to 30, more preferably 10 to 20 in length.

Poly(A) consisting essentially of dA nucleotides, but having an even distribution of 4 nucleotides (dA, dC, dG, dT) and interrupted by a random sequence that is, for example, 5-50 nucleotides in length Cassettes exhibit constant proliferation of plasmid DNA in E. coli at the DNA level and are still associated with beneficial properties with respect to supporting RNA stability and translation efficiency at the RNA level.

As a result, the 3' poly(A) sequence contained in the RNA molecules described herein consists essentially of A nucleotides, but by a random sequence with an even distribution of 4 nucleotides (A, C, G, U). is stopped Such random sequences may be 5 to 50, preferably 10 to 30, more preferably 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 a stretch of nucleotides at the 3' end of a nucleic acid molecule, such as an RNA molecule, to be subjected to adenylic acid polymerization. The adenylic acid polymerization signal generally comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, hexamer sequences, are possible. Adenylic acid polymerization usually occurs during processing of pre-mRNA (also called immature-mRNA). In general, RNA maturation (from pre-mRNA to mature mRNA) involves the step of adenylic acid polymerization.

Codon usage

In general, the degeneracy of the genetic code makes it possible to replace certain codons (base triplets encoding amino acids) present in RNA sequences by other codons (base triplets) while maintaining the same coding capacity (replacement of a codon is encodes the same amino acid as the codon replaced).

In the present invention, at least one codon of the open reading frame included in the RNA molecule is different from each codon of each open reading frame in the species from which the open reading frame is derived. A coding sequence in an open reading frame is said to be "altered".

For example, when the coding sequence of the open reading frame is modified, frequently used codons may be selected. Modifications in 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 subject cell or subject organism, this type of modification is appropriate to tailor the nucleic acid sequence to expression in a particular subject cell or subject organism. Generally speaking, more frequently used codons are typically translated more efficiently in a subject cell or subject organism, but modification of all codons in the open reading frame is not always necessary.

For example, if the coding sequence of the open reading frame is modified, the content of G (guanylate) residues and C (cytodylate) residues can be modified 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 decrease immune activity and enhance translation and half-life of RNA.

Each of the open reading frames coding for DdRp according to the present invention can be adapted.

II. DNA expression cassette

The genome editing RNA/DNA system of the present invention comprises, in addition to the DdRp RNA expression cassette, a DNAge expression cassette each preferably encoding at least one genome editing tool.

The genome editing system of the present invention can be delivered to cells in various forms, including fragments of polynucleotides, plasmids, lentiviruses, adeno-associated viruses, and ribonucleoprotein (RNP) complexes, or combinations thereof.

A representative genome editing CRISPR/Cas9 system is the CRISPR/Cas9 ribonucleoprotein (RNP) system, which consists of a Cas9 protein and a single guide RNA (sgRNA) or CRISPR RNA (crRNA):trans-activating crRNA (tracrRNA) duplex.

The DNAge expression cassette capable of being transcribed by the DdRp constituting the genome editing RNA/DNA system of the present invention is a DNAen expression cassette encoding a targeting endonuclease (Cas9 protein) and a single target sequence related to a specific targeting sequence of the selected genome. Guide RNA (sgRNA) or CRISPR RNA (crRNA): may include a DNAsg expression cassette encoding a trans-activating crRNA (tracrRNA) duplex.

The targeting endonuclease is zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), CRISPR-associated Cas nuclease, algonut endonuclease and M-MLV reverse transcriptase. and Cas9 H840A nickase is one selected from the group consisting of endonuclease containing a fusion protein.

The DNAen expression cassette of the present invention is

(1) a promoter to which DdRp binds;

(2) 5*?**?*UTR;

(3) a DNA fragment encoding DNAen; and

(4) 3'UTR may be included.

The composition of the single guide RNA (sgRNA) or CRISPR RNA (crRNA):trans-activating crRNA (tracrRNA) duplex is as follows.

crRNA is a 20 nt RNA oligonucleotide designed to be complementary to a genomic target. The crRNA sequence design utilizes standard gRNA design principles and can be easily selected from the gRNA database or customized using gRNA design tools. tracrRNA is a 67nt RNA oligonucleotide that forms an activated CRISPR/Cas9 RNP complex with crRNA and Cas9 nuclease.

The sgRNA is a single RNA oligo containing both a crRNA sequence capable of identifying a target genomic sequence and a tracrRNA sequence capable of forming a complex with a Cas9 protein. When the sgRNA or crRNA:tracrRNA duplexes form a complex with the Cas9 protein, they together form a protospacer adjacent motif (PAM) sequence 3-4 bp upstream from the (5'NGG-3'), 20 nt guide RNA sequence can create a double-stranded break at a locus complementary to

The DNAsg expression cassette of the present invention is

(1) a promoter to which DdRp binds (or RNA polymerase III (Pol III) promoter);

(2) a DNA fragment encoding DNAsg; and

(3) 3'UTR may be included.

In the present invention, DdRp synthesized from the DdRp RNA expression cassette can act on each DNAge expression cassette to induce transcription and production of a genome editing tool. For example, each DNAge expression cassette may encode a targeting endonuclease or guide nucleic acid for use in genome editing. This is advantageous when genome editing of several different loci is required for the subject.

The genome editing tool of the DNAge expression cassette of the present invention may include a targeting endonuclease that performs genome editing (FIG. 5). In particular, in such cases, including when the DNAge expression cassette uses a targeting endonuclease, a guide nucleic acid that is part of a genome editing tool can be introduced through a delivery system from the outside. Exceptionally, the guide nucleic acid may be RNA or DNA.

Preferably, the genome editing tool constituting the DNAge expression cassette and capable of being transcribed by DdRp may include a guide nucleic acid used for genome editing (FIG. 6). Exceptionally in this case, the targeting endonuclease that is part of the genome editing tool may be introduced via a delivery system from the outside.

Preferably, the genome editing tool constituting the DNAge expression cassette and capable of being transcribed by DdRp may include a targeting endonuclease used for genome editing and two types of guide nucleic acids (FIG. 7). In these cases involving priming, the two types of guide nucleic acids may be in one or both DNAs.

The amplification genome editing system of the present invention is not limited to only functional elements, but is not limited thereto.

A DNAge expression cassette encoding a genome editing tool is present along with expression control sequences. The DNAge encoding the genome editing tool is under the control of the promoter to which DdRp binds. The DNAge encoding the genome editing tool is located downstream of the promoter to which DdRp binds. The genome editing tool is encoded by a heterologous nucleic acid. The downstream position of the promoter to which DdRp binds contains a DNA fragment encoding a genome editing tool.

The DNA (DNAen) encoding the targeting endonuclease constituting the DNAge expression cassette of the present invention is a unit for transcription of the targeting endonuclease, and includes a start codon (base triplet), typically AUG (RNA molecule in) or (in each DNA molecule) an open reading frame containing ATG.

Preferably, when the DNAen expression cassette according to the present invention comprises at least one open reading frame (protein coding region), the expression cassette is a transcription unit of a genome editing tool molecule.

The DNAen expression cassette according to the present invention may encode a single targeting endonuclease or multiple targeting endonucleases. Multiple targeting endonucleases may be encoded as single targeting endonucleases (fusion targeting endonucleases) or as separate targeting endonucleases.

A DNAen expression cassette in which the targeting endonuclease is encoded as a separate targeting endonuclease, one or more of these may be provided with an upstream IRES or additional viral promoter elements. Preferably, the DNAen expression cassette according to the present invention may comprise two or more open reading frames, each of which may be under the control of a promoter to which DdRp binds.

Thus, a DNAen expression cassette configured to express a polypeptide of interest comprises a coding region encoding a targeting endonuclease, one or more suitable promoters operably linked to the coding region, a translation initiation sequence, a start codon, a stop codon, a polyA signal sequence, leader sequence, NLS, and the like.

The DNAen expression cassette contains a sequence encoding a nuclear localization sequence (NLS). Any suitable NLS sequence may be used. The DNAen expression cassette is configured to express a targeting endonuclease or functional fragment thereof.

Preferably, the open reading frame encoding the genome editing tool is not unique to the virus that induces DdRp. The open reading frame under the control of the promoter to which DdRp binds does not encode any viral proteins. The open reading frame under the control of the promoter to which DdRp binds does not encode any full-length viral non-structural proteins or fragments thereof and/or does not encode any full-length viral structural proteins or fragments thereof.

The promoter to which DdRp binds can direct transcription of the DNAge expression cassette coating the genome editing tool in the presence of DdRp. Thus, for a gene editing tool to express, when the DdRp RNA expression cassette is present together with a DNAge expression cassette, these promoters direct transcription of the DNA expression cassette coating the genome editing tool.

The promoter to which DdRp binds is unique to the virus that induces DdRp.

DNAge expression cassettes contain heterologous nucleic acids.

The DNAen constituting the DNAge expression cassette of the present invention includes an open reading frame encoding a targeting endonuclease. The open reading frame encoding the targeting endonuclease is not unique to viruses that induce DdRp. The open reading frame encoding the targeting endonuclease does not encode a viral structural protein. The gene encoding the targeting endonuclease is under the control of a promoter to which DdRp binds. This allows expression of the open reading frame encoding the targeting endonuclease under the control of the promoter to which DdRp binds.

The transcript of the DNAen expression cassette contains a 5' cap.

5'UTR and/or 3'UTR may be present in the DNAen expression cassette of the present invention. The 3'UTR of the DNAen expression cassette contains the 3' poly(A) sequence.

The DNAen expression cassette of the present invention consists of a G/C content, codon optimization, UTR modification, a poly(A) tail with more than 30 adenosine nucleotides, a poly(C) sequence, and a histone-stem-loop sequence. It may include a base sequence modification (SM).

The DNAsg expression cassette of the present invention may include RNA or sgRNA related to a specific targeting nucleotide sequence of a selected genome. The DNAsg expression cassette of the present invention may include a promoter to which DdRp binds, or an RNA polymerase III (Pol III) promoter may be used instead of a promoter to which DdRp binds.

The promoter to which DdRp binds is unique to the virus that induces DdRp. The promoter to which DdRp binds is a promoter for structural proteins of the virus. This means that the promoter to which DdRp binds is unique to the virus and controls gene transcription of one or more structural proteins in the virus.

The genome editing RNA/DNA system of the present invention may include a DdRp RNA expression cassette, a DNAge expression cassette comprising a DNAge encoding a genome editing tool, and an auto_DdRp expression cassette comprising DNA encoding DdRp.

The expression cassette comprising the DNA fragment encoding the DdRp or genome editing tool may have a promoter to which DdRp binds in each upstream direction or a promoter to which one DdRp binds in an upstream direction.

For DdRp of the present invention, DdRp composed of one subunit was selected.

The DdRp RNA expression cassette of the present invention increases the expression of any one selected from the group comprising T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase, which can be nuclear-independently transcribed in the cytoplasm. Target RNA can be expressed continuously for a long period of time.

In the present invention, T7 RNA polymerase has very high specificity for the T7 promoter, and can transcribe only DNA downstream of the T7 promoter. Therefore, by using T7 RNA polymerase in the present invention, binding and transcription to the RNA expression cassette and auto_DdRp expression cassette having only the T7 promoter without reacting with other promoters in the cell are performed, and T7 RNA polymerase itself is produced with high efficiency. It can be self-transcribed and amplified by transcription of RNA.

The auto_DdRp expression cassette is characterized in that it self-transcribes the T7 RNA polymerase in the cytoplasm by repeating the T7 RNA polymerase expression loop.

Specifically, the auto_DdRp expression cassette may include a T7 promoter, an internal ribosome entry site (IRES) region, a DNA fragment encoding T7 RNA polymerase, a poly A tail, and a T7 termination sequence. , they may be operatively connected and configured. The IRES region included in the auto_DdRp expression cassette may be a viral IRES, and by generating an IRES-fused T7 polymerase transcript, not only increases the transcriptional stability of DdRp mRNA, but also increases the expression efficiency of T7 polymerase in the cytoplasm. can keep

The self-transcription may be performed by the DdRp expression loop by the auto_DdRp expression cassette for self-transcription of DdRp synthesized by the DdRp RNA expression cassette constituting the composition of the present invention.

Specifically, the DdRp mRNA or capped CM/SM DdRp mRNA is translated by ribosomes in the cytoplasm to generate DdRp, and the resulting DdRp recognizes a promoter present in the auto_DdRp expression cassette and performs transcription of DdRp. After that, the generated DdRp mRNA is translated by ribosomes in the cytoplasm again to repeat the production of DdRp, whereby DdRp self-transcription is made. Accordingly, the composition of the present invention is characterized in that it can maintain DdRp self-transcribed by the DdRp expression loop at a high concentration for a long period of time in the cytoplasm.

Accordingly, the present invention provides a DdRp RNA expression cassette; and a DdRp expression loop carried by an auto_DdRp expression cassette comprising DNA encoding DdRp for DdRp self-transcription to maintain high concentration of DdRp mRNA in the cytoplasm for a long period of time in a nuclear-independent manner, and thus also DdRp continuously for a long period of time. It is characterized by expression.

Capping is a special structure found at the 5' end of almost all eukaryotic messenger RNAs. The simplest cap structure, cap0, is the result of guanine nucleoside addition methylated at N7 bound by a gamma phosphate at the 5'5' triphosphate end bound to the end of the primary RNA (i.e., m ⁷ GpppN, where N is any base, p represents phosphate and m is a methyl group). The formation of cap0 involves a series of three enzymatic reactions: RNA triphosphatase (RTPase) removes the gamma phosphate residue at the 5' triphosphate terminus of the resulting pre-mRNA for diphosphate, and RNA guanylyltransferase ( GTase) transfers GMP from GTP to the diphosphate-generating RNA terminus, and RNA N7-guanine methyltransferase (N7-MTase) adds a methyl residue on nitrogen 7 of guanine to the GpppN cap.

In more advanced eukaryotes and some viruses, primary (ie, cap1 construct; m ⁷ GpppNm ^2'-o pN) and secondary (ie, cap2 externa; m ⁷ GpppNm ^2'-o pNm ^2'-o ) transcription The 2'-hydroxyl group of the ribose of a given nucleotide can be methylated by two distinct ribose-2'-O MTases, termed cap1- and cap2-specific MTases, respectively.

However, in contrast to cellular N7-MTase activity, which is only present in the nucleus, cap1 ribose-2'-O MTase activity was detected both in the cytoplasm and nucleus of HeLa cells, whereas cap2 MTase activity was found only in their cytoplasm. became

Formation of the 5'-end m ⁷ GpppN cap is the first step in pre-mRNA processing. The m ⁷ GpppN cap plays an important role in mRNA stability and its transport from the nucleus to the cytoplasm.

Furthermore, the 5'-terminal m ⁷ GpppN cap is important for the translation of mRNA into protein by anchoring the eukaryotic translation initiation factor 4F (eIF4F) complex, which mediates the recruitment of the 16S portion of the small ribosomal subunit into mRNA. . Thus, the 5'-terminal m ⁷ GpppN cap significantly enhances the translation of mRNA both in vitro and in vivo.

DdRp of the auto_DdRp expression cassette of the present invention is DdRp itself, or the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, N7-guanine methyltransferase, and A report that a chimeric enzyme comprising a catalytic domain of DNA-dependent RNA polymerase (DdRp) synthesizes an RNA molecule with a 5'-terminal m7GpppN cap highly translatable by a eukaryotic translation apparatus without induction of cytotoxicity and apoptosis (Korea) , was prepared using a chimeric enzyme based on Patent Publication No. 10-2013-0069632).

The present invention relates to at least one, in particular catalytic domain of RNA triphosphatase, at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular catalytic domain of N7-guanine methyltransferase, and at least one, in particular It relates to a chimeric enzyme comprising the catalytic domain of DdRp.

In particular, the chimeric enzyme according to the present invention is capable of synthesizing RNA molecules having a 5'-terminal m7 GpppN cap.

A chimeric enzyme refers to an enzyme that is not a natural enzyme found in nature. Thus, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, different enzymes) or catalytic domains derived from the same source (eg, the same enzyme), arranged in a manner different from that found in nature. Chimeric enzymes include monomeric (ie single unit) enzymes as well as oligomeric (ie multiple unit) enzymes, particularly hetero-oligomeric enzymes. Monomeric enzymes refer to single-unit enzymes consisting of only one polypeptide chain.

In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are covalently and/or non-covalently linked together.

In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are operatively linked together to synthesize an RNA molecule with a 5'-terminal m7 GpppN cap.

In particular, at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular catalytic domain of N7-guanine methyltransferase and at least one, in particular DdRp catalytic domains, wherein at least two of said catalytic domains are linked together covalently or non-covalently, preferably at their termini (N or C terminus), more particularly said at least one, in particular catalytic domain of RNA triphosphatase , said at least one, in particular catalytic domain of guanylyltransferase, at least one, in particular at least one catalytic domain selected from the group consisting of catalytic domain of N7-guanine methyltransferase, preferably at its terminus (N or C The chimeric enzyme of the present invention comprising a covalently or non-covalently linked terminal) is linked with said at least one, in particular, the catalytic domain of DdRp, preferably at its terminus (N or C terminus).

In particular, the present invention relates to at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular a catalytic domain of guanylyltransferase, at least one, in particular a catalytic domain of N7-guanine methyltransferase, and at least one, It relates in particular to the chimeric enzyme of the present invention comprising the catalytic domain of DdRp, provided that the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the nuclear eukaryotic DdRpI, only catalytic domains of II and/or III; and more particularly, chimeric enzymes comprising the only catalytic domain(s) of DdRpII.

In particular, the present invention relates to at least one, in particular a catalytic domain of RNA triphosphatase, at least one, in particular a catalytic domain of guanylyltransferase, at least one, in particular a catalytic domain of N7-guanine methyltransferase, and at least one, It relates in particular to the chimeric enzyme of the present invention comprising the catalytic domain of DdRp, provided that the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the nuclear eukaryotic DdRpI, catalytic domains of II and/or III; and more particularly, chimeric enzymes comprising at least one catalytic domain of DdRpII.

In particular, when expressed in a eukaryotic host cell, the chimeric enzyme according to the present invention is capable of synthesizing an RNA molecule having a 5' terminal m7 GpppN cap, preferably translatable by a eukaryotic translation apparatus.

In particular, when expressed in eukaryotic host cells, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase bind the m7 GpppN cap at the 5' end of the RNA molecule synthesized by the catalytic domain of DdRp. The RNA molecule, which can be added, preferably having a 5'-terminal m7 GpppN cap, can be translated by a eukaryotic translation apparatus.

In particular, when expressed in eukaryotic host cells, when the catalytic domain of DdRp is the catalytic domain of bacteriophage DdRp, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are at their 5' ends m7GpppN cap can be added to the 5' end of the RNA molecule having a guanosine ribonucleotide in

In particular, when expressed in a eukaryotic host cell, when the catalytic domain of DdRp is the catalytic domain of a bacterial DNA-dependent RNA polymerase, the catalytic domain of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase is An m7GpppN cap can be added at the 5' end of RNA molecules having guanosine or adenosine ribonucleotides at their 5' end.

In particular, when expressed in eukaryotic host cells, when the catalytic domain of DdRp is the catalytic domain of human or mouse mitochondrial DdRp, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are An m7GpppN cap can be added at the 5' end of the RNA molecule with adenosine or thymidine ribonucleotides at the 5' end.

The catalytic domain of an enzyme relates to a domain that is necessary and sufficient to ensure enzyme function, especially in its three-dimensional structure. For example, the catalytic domain of RNA triphosphatase is a necessary and sufficient domain to ensure RNA triphosphatase function. The term “catalytic domain” includes the catalytic domain of a wild-type or mutant enzyme.

The chimeric enzyme of the present invention includes at least the catalytic domain, but may further include all or a part of the enzyme containing the catalytic domain. Indeed, according to one aspect of the chimeric enzyme according to the invention, said catalytic domain of DdRp may be comprised in all or part of a DdRp, preferably a monomeric DdRp. Said catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase and said catalytic domain of N7-guanine methyltransferase may also be included in all or part of a capping enzyme, preferably a monomeric capping enzyme.

The chimeric enzyme of the present invention may be a nuclear enzyme, a subcellular compartment enzyme or a cytoplasmic enzyme. Thus, the chimeric enzyme according to the present invention may comprise a signal peptide or marker-signal known to one of ordinary skill in the art that induces delivery of the enzyme in a cell. For example, the chimeric enzyme according to the present invention may include a nuclear localization signal (NLS) that directs the enzyme to the nucleus. These NLSs are often units of five basic positively charged amino acids. The NLS can be located anywhere on the peptide chain.

A chimeric enzyme does not contain a signal peptide or marker-signal that induces the transfer of the enzyme, except to the cytoplasm, when it is a cytoplasmic chimeric enzyme.

Cytoplasmic localization of the chimeric enzyme of the present invention optimizes transgene expression levels by avoiding the vigorous movement of large DNA molecules (i.e., transgenes) from the cytoplasm to the nucleus of eukaryotic cells and export of RNA molecules from the nucleus to the cytoplasm. have an advantage

Accordingly, these cytoplasmic chimeric enzymes of the present invention may be useful for generating host-independent, eukaryotic gene expression systems capable of operating in the cytoplasm where significantly higher amounts of transfected DNA are normally found compared to the nucleus.

These cytoplasmic chimeric enzymes of the present invention are capable of synthesizing RNA molecules with a 5'-terminal m7GpppN cap, highly translatable by the eukaryotic cytoplasmic translation apparatus, without cytotoxicity and induction of apoptosis.

Since endogenous gene transcription occurs in the nucleus of eukaryotic cells, as opposed to transgene transcription that occurs in the cytoplasm, there is no competition between endogenous gene residues and transgene transcription.

Therefore, the cytoplasmic chimeric enzyme of the present invention is particularly suitable for large-scale analysis and protein production.

In the chimeric enzyme of the present invention, the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase are contained in a monomer, ie, 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 comprised in a monomeric capping enzyme. In this case, the chimeric enzyme of the present invention comprises a monomeric capping enzyme, wherein the enzyme comprises the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase. do. The monomeric capping enzyme is capable of adding an m7GpppN cap at the 5' end of the RNA molecule, in particular a wild-type blue eye virus capping enzyme, a wild-type bamboo mosaic virus capping enzyme, a wild-type African swine fever virus capping enzyme, a wild-type Acanthamoeba poly a monomeric viral capping enzyme selected from the group consisting of an acanthamoeba polyphaga mimivirus capping enzyme and mutants and derivatives thereof, and more particularly a wild-type African swine fever virus capping enzyme.

In particular, said catalytic domain of DdRp may also be comprised in a monomer, ie in one polypeptide.

For example, the monomer may be a monomeric DdRp or a monomeric chimeric enzyme according to the present invention.

In particular, said catalytic domain of DdRp is comprised in monomeric DdRp. In this case, the chimeric enzyme of the present invention contains a monomeric DdRp comprising the catalytic domain of DdRp. The monomeric DdRp may be a monomeric phage DdRp, in particular T7 RNA polymerase, T3 RNA polymerase and SP6, capable of synthesizing single-stranded RNA with sequence complementarity to double-stranded template DNA in the 5' to 3' direction. T7 RNA polymerase, which may be selected from the group consisting of RNA polymerase and mutants or derivatives thereof, and more particularly, capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5' to 3' direction; and It may be selected from the group consisting of mutants or derivatives thereof.

At least two selected from the group consisting of the catalytic domain of DdRp and the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase, and more preferably the entire catalytic domain is may be included in the monomer.

The chimeric enzymes of the present invention may be monomeric or oligomeric. Indeed, the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp may be included in one or several polypeptides.

Preferably, the chimeric enzyme of the present invention may be monomeric.

Indeed, the present invention relates to a monomeric chimeric enzyme comprising the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the catalytic domain of DdRp, without inducing cytotoxicity and apoptosis. , confirmed the report that it is possible to synthesize an RNA molecule with a 5'-terminal m7GpppN cap, which is highly translatable by a eukaryotic translation apparatus.

Due to steric hindrances and components and enzymes, which must remain in their native structure, it is not self-evident that capping of transcripts occurs with monomeric enzymes. Indeed, capping of the T7 transcript cannot be achieved by the fusion enzyme CTD-T7 RNA polymerase, even assuming that it causes an m7GpppN cap at the 5' end of the developing RNA molecule.

The monomeric chimeric enzymes of the present invention have the advantage of being particularly inexpensive, rapid and easy to implement, making them highly suitable for large-scale assays and protein production. Indeed, the production of monomeric enzymes is easier than oligomeric enzymes. Also, since only single-units exist, there is no problem of unit assembly. Monomeric enzymes are also easier to manipulate than multimeric enzymes.

DdRp (RNAP) relates to a nucleotidyl transferase that synthesizes RNA with sequence complementarity to double-stranded template DNA in the 5'→3' direction.

Preferably, said catalytic domain of DdRp is the catalytic domain of an enzyme, which has a relatively simple structure and more preferably characterizes genomic enzyme regulatory elements (ie promoter, transcription termination signal and chain splicing). Thus, in particular, the catalytic domain of DdRp may be the catalytic domain of a bacteriophage DdRp, a bacterial DdRp or a DdRp of various eukaryotic organelles (eg, mitochondria, chloroplasts and prochromosomes).

In the present invention, the catalytic domain of DdRp is the catalytic domain of bacteriophage DdRp. Bacteriophage DdRp has a significant advantage in optimizing transgene expression levels, especially by having a higher processing capacity than eukaryotic RNA polymerase. Bacteriophage DdRp also has a much simpler structure than most nuclear eukaryotic polymerases, which have a complex structure with multiple subunits (eg RNA polymerase II). Most bacteriophage DdRp characterized to date are single-subunit enzymes, which do not require helper proteins for initiation, elongation or termination of transcription. Several of these enzymes named for cloned bacteriophages also have well-characterized regulatory genomic elements (ie, promoters, termination signals, transcriptional stop sequences), which are important for transduction.

Also, there is no competition between endogenous gene transcription and transgene transcription. The chimeric enzyme according to the invention comprising a bacteriophage DNA dependent RNA-polymerase residue enables the production of RNA transcripts in any eukaryotic species (eg yeast, rodent and human). They are inexpensive, rapid, and easy to perform, making them suitable for large-scale analysis and protein production, and in contrast to eukaryotic RNA polymerases such as RNA polymerase II, most bacteriophage DdRp do not contain relevant factors for initiation, elongation or termination of transcription. Because it is not required, it enables the production of RNA transcripts in any biological system (eg, cellular reaction mixtures, cultured cells and living organisms).

Said catalytic domain of bacteriophage DdRp is the catalytic domain of bacteriophage DdRp, in particular wild type of T7 RNA polymerase, wild type of T3 RNA polymerase (NCBI genomic sequence ID# NC_003298; GeneID# 927437; UniProtKB/Swiss-Prot ID# Q778M8), K11 Wild type of RNA polymerase (NCBI genomic K11 RNAP sequence ID# NC_004665; GeneID# 1258850; UniProtKB/Swiss-Prot ID# Q859H5), wild type of ψA1122 RNA polymerase (NCBI genome sequence ID# NC_004777; GeneID# 1733944; UniProtKB/Swiss) -Prot protein ID# Q858N4), wild-type of ψYeo3-12 RNA polymerase (NCBI genomic sequence ID# NC_001271; GeneID# 1262422; UniProtKB/Swiss-Prot ID# Q9T145) and wild-type of gh-1 RNA polymerase (NCBI genomic sequence) ID# NC 004665; GeneID# 1258850; UniProtKB/Swiss-Prot protein ID#Q859H5), wild type of K1-5 RNAP RNA polymerase (NCBI genome sequence ID# NC_008125; GeneID# 5075932 UniProtKB/Swiss-Prot protein ID# Q8SCG8) and wild-type SP6 RNA polymerase (NCBI genome sequence ID# NC_004831; GeneID# 1481778; UniProtKB/Swiss-Prot protein ID# Q7Y5R1) and mutants or derivatives thereof, which may be a catalytic domain selected from the group consisting of 5'→ Single-stranded RNA having sequence complementarity to the double-stranded template DNA in the 3' direction can be synthesized, and more particularly, it may be a catalytic domain of T7 RNA polymerase.

T7 RNA polymerase relates to the bacteriophage T7 DdRp. Preferably, the T7 RNA polymerase has the amino acid sequence of SEQ ID NO: 1 (NCBI genomic sequence ID# NC_001604; GeneID# 1261050; UniProtKB/Swiss-Prot ID# P00573) and is an 883 amino acid protein with a molecular weight of 98.8 kDa .

T7 RNA polymerase is particularly capable of processing this enzyme in vitro, elongating from 240 to 250 nucleotides/s at 37° C. in the 5′→3′ direction. Moreover, when expressed in eukaryotic cells, T7 RNA polymerase is mostly retained in the cytoplasm [Elroy-Stein and Moss 1990; Gao and Huang 1993; Brisson, He et al. 1999], thus optimizing transgene expression levels by avoiding the vigorous migration of large DNA molecules (ie, transgenes) from the cytoplasm to the eukaryotic cell nucleus and export of RNA molecules from the nucleus to the cytoplasm.

The catalytic domain of DdRp can be one of the wild-type T7 RNA polymerase as well as the mutant of T7 RNA polymerase, which is single-stranded with sequence complementarity to the double-stranded template DNA in the 5'→3' direction even at reduced throughput. RNA can be synthesized. For example, the mutant may be selected from the group consisting of R551S, F644A, Q649S, G645A, R627S, I810S and D812E.

In the chimeric enzyme of the present invention, the catalytic domain of DdRp is located C-terminal to the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

Indeed, when the catalytic domain of DdRp is the catalytic domain of a bacteriophage DdRp, in particular that of a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, said catalytic domain preferably preserves its native carboxyl terminus . In particular, the C-terminus of said catalytic domain of DdRp, in particular of said catalytic domain of bacteriophage DdRp, corresponds to the C-terminus of said chimeric enzyme. In particular, when the chimeric enzyme is a whole bacteriophage DdRp, in particular a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerases, said polymerase preferably preserves its native carboxyl-terminus. In particular, the C-terminus of said catalytic domain of DdRp, in particular of said catalytic domain of bacteriophage DdRp, corresponds to the C-terminus of said chimeric enzyme. In particular, said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, is included in all or part of bacteriophage DdRp, wherein the C-terminus of said bacteriophage DdRp is said Corresponds to the C-terminus of the chimeric enzyme.

In the chimeric enzyme of the present invention, said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, is said catalytic domain of RNA triphosphatase , located C-terminal to the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

The catalytic domain of DdRp is the catalytic domain of bacterial DdRp.

Preferably, the bacterial DdRp has a suitable structural complexity.

For example, the bacterial DdRp may be E. coli DdRp (NCBI genomic sequence of K-12 substrate DH10B ID# NC_010473), which has four different subunits (α subunit: rpoA GeneID# 6060938, UniProtKB). /Swiss-Prot ID# B1X6E7;β subunit: rpoB GeneID#6058462, UniProtKB/Swiss-Prot ID#B1XBY9;β' subunit: rpoC GeneID#6058956, UniProtKB/Swiss-Prot ID#B1XBZ0;σ subunit: rpoE GeneID# 6060683, UniProtKB/Swiss-Prot ID# B1XBQ0), assembled in a complex of five ααββ′σ subunits. this. Genomic elements involved in the regulation of enzymatic activity, including E. coli RNA polymerase promoters, rho-dependent and -independent terminators and transcriptional stop sequences, have been well characterized.

The catalytic domain of DdRp is the catalytic domain of DdRp of eukaryotic cell phases, such as mitochondria, chloroplasts and proplasts. Indeed, these polymerases may also have a relatively simple structure.

The catalytic domain of DdRp may be the catalytic domain of mammalian mouse mitochondrial RNA polymerase, which is a single unit 120 kDa protein (GeneID# 216151, UniProtKB/Swiss-Prot ID# Q8BKF1) and shares homology with bacteriophage RNA polymerase. do. Several transcription factors are required for transcription initiation, elongation or termination: TFB1M (mitochondrial transcription factor Bl; mouse GeneID#224481, UniProtKB/Swiss-Prot ID# Q8JZM0) or TFB2M (mitochondrial transcription factor B2; mouse GeneID#) for transcriptional termination. 15278, UniProtKB/Swiss-Prot ID# Q3TL26), TFAM (mitochondrial transcription factor A; mouse GeneID# 21780, UniProtKB/Swiss-Prot ID# P40630), and mTERF (mitochondrial transcription terminator; mouse GeneID# 545725, UniProtKB/Swiss) -Prot ID# Q8CHZ9). The genomic elements involved in the regulation of the enzymatic activity of mitochondrial RNA polymerase, including transcription termination signals as well as two promoters in the light and heavy chains of the mitochondrial genome, have been well characterized.

RNA triphosphatase (RTPase) is an enzyme that removes the gamma phosphate residue at the 5' triphosphate terminus of the developing pre-mRNA to diphosphate. RNA guanylyltransferase (GTase) refers to an enzyme that transfers GMP from GTP to the diphosphate-generating RNA terminus. N7-guanine methyltransferase (N7-MTase) relates to an enzyme that adds a methyl residue on nitrogen 7 of guanine to the GpppN cap.

The catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may be of the same or different capping enzymes. If the catalytic domains are from the same enzyme, then the catalytic domains of DdRp are from different enzymes.

Preferably, said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase originate from one or several cytoplasmic enzymes, which advantageously have a relatively simple structure and well-characterized enzymatic activity. Thus, in particular, the catalytic domain of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may be the catalytic domain of one or several viral capping enzymes, or capping enzymes of cytoplasmic episomes.

Said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase may comprise one or several virus capping enzymes, in particular wild-type blue snow virus capping enzyme, wild-type bamboo mosaic virus capping enzyme, wild-type African swine fever virus capping. The enzyme, wild-type Acanthamoeba polyphagase, is of an enzyme selected from the group consisting of a mimivirus capping enzyme and a mutant or derivative thereof, each of which is converted to diphosphate by removing the gamma phosphate residue at the 5' triphosphate terminus of the resulting pre-mRNA. or transfer of GMP from GTP to the diphosphate generating RNA terminus, or adding a methyl residue on nitrogen 7 of guanine to the GpppN cap, more particularly that of wild-type African swine fever virus capping enzyme.

The blue tongue virus capping enzyme relates to the single-unit VP4 capping enzyme of blue tongue virus (BTV), which is a 74 kDa protein (644 amino acids; for sequence, e.g., NCBI BTV serotype 10 genomic sequence ID# Y00421; GeneID# 2943157; UniProtKB/Swiss-Prot ID# P07132, D0UZ45, Q5J7C0, Q65751, Q8BA65, P33428, P33429, P33427, C3TUP7, Q8BAD5, C5IWW0, B4E551, Q3KVQ3) QK3KVQ3) Q6Q73 Q6VP2, Q3 Q3KVQ3) . This capping enzyme can homodimerize via a leucine zipper located adjacent to its carboxyl terminus. VP4 catalyzes all three enzymatic steps required for mRNA m7GpppN capping synthesis: RTPase, GTase and N7-MTase.

Bamboo mosaic virus enzyme relates to ORF1, bamboo mosaic virus (BMV) mRNA capping enzyme, which is a single-unit 155-kDa protein (1365-amino acid; NCBI BMV isolate BaMV-0 genome sequence ID# NC 001642; GeneID# 1497253; UniProtKB/Swiss-Prot ID# Q65005). ORF1 protein has all the enzymatic activities required to produce m7GpppN mRNA capping, ie RTPase, GTase and N7-MTase. Furthermore, ORF1 has RNA-dependent RNA-polymerase activity, which is not governed by the chimeric enzyme activity of the present invention, and can be disrupted by deletion of the Asp1229 Asp1230 residue of the mRNA capping enzyme. As used herein, the term "African swine fever virus capping enzyme" relates to NP868R capping enzyme (G4R), (ASFV), which is a single-unit 100 kDa protein (868 amino acids; NCBI ASFV genome sequence strain BA71V ID). # NC_001659; GeneID# 1488865; UniProtKB/Swiss-Prot ID# P32094).

The Acanthamoeba polyphaga mimivirus capping enzyme is directed to another single unit 136.5 kDa protein, R382 (APMV) (1170 amino acids; NCBI APMV genomic sequence ID# NC_006450; GeneID#3162607; UniProtKB/Swiss-Prot ID# Q5UQX1 ).

The catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase are of one or several capping enzymes of the cytoplasmic episome, such as pGKL2. In particular, the catalytic domains of RNA triphosphatase, guanylyltransferase and N7-guanine methyltransferase are included in all or part of the capping enzyme of the yeast linear extrachromosomal episomal pGKL2.

Cytoplasmic linear episomes are double-stranded DNA structures, which replicate stably in the cytoplasm of various yeast strains [Cong, Yarrow et al. 1994]. One prototype of a yeast linear extrachromosomal episome, pgKL2 (13,457 bp; also termed pGK12), is Kluyveromyces lactis CB 2359 and Saccharomyces cerevisiae F102- was completely sequenced from various yeast strains, including 2 [Tommasino, Ricci et al. 1988]. The capping enzyme encoded by the ORF3 gene of Kluyveromyces lactis pGKL2 (NCBI Kluyveromyces CB 2359 pGKL2 genomic sequence ID# NC_010187; UniProtKB/Swiss-Prot ID# P05469) is a 594 amino acid protein (70.6 kDa protein) )to be.

In one embodiment of the chimeric enzyme of the invention, at least two, in particular at least three and more particularly the entire catalytic domain may be assembled, fused or linked directly or indirectly by a linking peptide.

In particular, at least two, in particular at least three and more particularly the entire catalytic domain selected from the group consisting of the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and DdRp are linked directly or by a linking peptide.

Linking peptides have the advantage of minimizing steric hindrance and providing sufficient space for the components of the fusion protein to create a fusion protein that maintains their native conformation.

Preferably, at least said catalytic domain of DdRp comprises said catalytic domain of RNA triphosphatase; the catalytic domain of guanylyltransferase; and the catalytic domain of N7-guanine methyltransferase is bound by a linking peptide to at least one catalytic domain selected from the group consisting of.

In particular, the linking peptide may be located N-terminal to said catalytic domain of DdRp, in particular said catalytic domain of bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, said catalytic domain of RNA triphosphatase , C-terminal to the catalytic domain of guanylyltransferase and the catalytic domain of N7-guanine methyltransferase.

In particular, the catalytic domain of DdRp, in particular the N-terminus of the catalytic domain of a bacteriophage DdRp selected from the group consisting of T7, T3 and SP6-RNA polymerase, comprises the catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase connected to the C-terminus of one catalytic domain selected from the group consisting of a catalytic domain and said catalytic domain of N7-guanine methyltransferase by a covalent bond, in particular a linking peptide.

The linking peptide is a peptide of the formula (GlymSerp)n, wherein m is an integer from 0 to 12, in particular an integer from 1 to 8 and more particularly an integer from 3 to 6 and more particularly 4, and p is an integer from 0 to 6 , in particular an integer from 0 to 5, more particularly an integer from 0 to 3 and especially 1, n is an integer from 0 to 30, especially an integer from 0 to 12, more particularly an integer from 0 to 8 and even more particularly an integer from 1 to 6),

SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 and a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 22.

The flexible linker peptide of formula (GlymSerp)n has the advantage that the glycine residue provides peptide flexibility and serine provides some solubility. Additionally, the absence of sensitive sites for kyrotrypsin I, factor Xa, papain, plasmin, thrombin and trypsin in the formula (GlymSerp)n linker sequence is thought to increase the overall stability of the resulting fusion protein.

Variable length formula (GlymSerp)n linkers are commonly used to construct single-stranded Fv fragment (sFv) antibodies. In addition, formula (GlymSerp)n linkers have been used to generate a variety of fusion proteins, which often retain the biological activity of each of their components.

Other types of peptide linkers are also chimeric enzymes according to the present invention, for example GGGGIAPSMVGGGGS (SEQ ID NO: 6), SPNGASNSGSAPDTSSAPGSQ (SEQ ID NO: 7), EGKSSGSGSESKSTE (SEQ ID NO: 8), EGKSSGSGSESKEF (SEQ ID NO: 9), GGGSGGGSGGGTGGGSGGG (SEQ ID NO: 9) 10)], GSTSGSGKSSEGKG (SEQ ID NO: 11), YPRSIYIRRRHPSPSLTT (SEQ ID NO: 12), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 13), GSTSGSGKPGSGEGS (SEQ ID NO: 14), SSADDAKKDAAKKDDAKKKDDAKKDAKKDDAKKKDDDALKDDAKKKGS SEQ ID NO: LSDADKKKDAAKKGS SEQ ID NO: l5), GSADDAKK SEQ ID NO: l5) 17), AEAAAKEAAAKEAAAKA (SEQ ID NO: 18), GSHSGSGKP (SEQ ID NO: 19), GSTSGSGKPGSGEGSTGAGGAGSTSGSGKPSGEG (SEQ ID NO: 20), LSLEVAEEIARLEAEV (SEQ ID NO: 21), and GTPTPTPTPTGEF (SEQ ID NO: 22).

Other types of covalent bonds include, but are not limited to, disulfide bonds, transglutamination and protein trans-binding by chemical and/or physical agents, such as tris(bipyridine)ruthenium(II)-dichloride and UV light irradiation.

Said catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp can also be assembled by specific protein elements, for example leucine zippers, for example chimeric according to the invention In enzymes, the biotinylation domain can be assembled into one of the catalytic domains (eg, Avi-tag II or PFBtag) and the biotin binding domain into one of the other catalytic domains (eg, strep-tagII or nono-tag).

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

Preferably, at least said catalytic domain of DdRp is by a non-covalent bond, in particular a leucine zipper, the group consisting of said catalytic domain of RNA triphosphatase, said catalytic domain of guanylyltransferase and said catalytic domain of N7-guanine methyltransferase. It may be assembled into at least one of the catalytic domains selected from

Leucine zippers, a dimeric helical-coil protein structure composed of two amphoteric α-helices interacting with each other, are commonly used to homo- or hetero-dimerize proteins. Each helix consists of a repeat of 7 amino acids, wherein the first amino acid (residue a) is hydrophobic, the fourth amino acid (residue d) is usually leucine, and the other residues are polar. The leucine zippers VELCRO ACID-p1 and BASE-p1, which form parallel heterodimeric double-stranded helical coil structures, have a high tendency to form parallel protein hetero-dimers. They have been used to heterodimerize several soluble proteins] as well as member proteins.

Other types of oligomerized peptide domains also produce chimeric enzymes according to the invention and form at least two of said domains of the chimeric enzymes according to the invention, in particular antiparallel heterodimeric structures, such as leucine zippers, for example ACID- It is thought to assemble al/BASE-al, ACID-Kg/BASE-Eg, NZ/CZ, ACID-pLL/BASE-pLL and EE1234L and RR1234L leucine zippers. The disulfide-linked version of the leucine zipper can also be used to generate interchain disulfide bridges between cysteine residues under reducing conditions as well as in the disulfide helical coil-linked heterodimeric chimeric enzymes according to the present invention.

Thus, at least two of the catalytic domains of RNA triphosphatase, guanylyltransferase, N7-guanine methyltransferase and DdRp are leucine zippers, in particular leucine zippers that form antiparallel heterodimeric structures, e.g. ACID- al/BASE-al, ACID-Kg/BASE-Eg, NZ/CZ and ACID-pLL/BASE-pLL leucine zippers, disulfide helical coil-bonded zippers, and disulfide bridges between cysteine residues.

The chimeric enzyme of the present invention is, in particular, a wild-type T7 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction via a linker and more particularly a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of T7 RNA polymerase; or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is a wild-type T3 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction, particularly through a linker and more particularly through a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of T3 RNA polymerase, or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is, in particular, a wild-type SP6 RNA polymerase capable of synthesizing single-stranded RNA having sequence complementarity to double-stranded template DNA in the 5'→3' direction via a linker and more particularly a linker of formula (Gly3Ser)4 or a wild-type mRNA capping enzyme of African swine fever virus or a mutant thereof, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, fused to the amino terminus of a mutant or derivative thereof, in particular to the wild type of SP6 RNA polymerase, or derivatives, particularly wild-type African swine fever virus capping enzymes.

The chimeric enzyme of the present invention is a wild-type mRNA capping enzyme of African swine fever virus or a mutant or derivative thereof, in particular wild-type African swine fever, capable of adding an m7GpppN cap at the 5' end of the RNA molecule, assembled by a leucine zipper. viral capping enzyme and amino terminus of wild-type T7 RNA polymerase or a mutant or derivative thereof, in particular T7 RNA polymerase, capable of synthesizing single-stranded RNA with sequence complementarity to double-stranded template DNA in the 5'→3' direction. Includes wild type.

The chimeric enzyme in the present invention comprises at least one catalytic domain of the chimeric enzyme of the present invention, in particular the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N7-guanine methyltransferase and the catalytic domain of DdRp. It further comprises a domain enhancing the activity of at least one catalytic domain selected from the group consisting of.

For example, the domain that enhances the activity of at least one catalytic domain of the chimeric enzyme of the present invention has no intrinsic enzymatic activity, but significantly inhibits the RNA N7-guanine methyltransferase activity of the D1R subunit of the vaccinia mRNA capping enzyme. It may be a 31-kDa subunit encoded by the vaccinia virus D12L gene, which enhances (genomic sequence ID#NC_006998.1; GeneID#3707515; UniProtKB/Swiss-Prot ID#YP_232999.1).

The chimeric enzymes of the present invention are, in particular, RNA-triphosphatase, RNA guanylyltransferase and RNA-guanine methyltransferase, assembled in whole or in part by a linker and more particularly by a linker of formula (Gly3Ser)4 and/or a leucine zipper. at least one catalytic domain of a vaccinia mRNA capping enzyme, having a enzyme activity, in particular a 95 kDa subunit encoded by the vaccinia virus D1R gene (genomic sequence ID# NC 006998.1; GeneID# 3707562; UniProtKB/Swiss-Prot ID # YP 232988.1); - comprises at least one catalytic domain of DdRp, in particular a polymerase selected from the group consisting of T7, T3 and SP6-RNA polymerase and more particularly of T7 RNA polymerase and a 31-kDa encoded by the vaccinia virus D12L gene .

The invention also relates to an isolated nucleic acid molecule or group of isolated nucleic acid molecules, said nucleic acid molecule(s) encoding a chimeric enzyme according to the invention.

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

Said group of isolated nucleic acid molecules encoding a chimeric enzyme of the invention comprises at least one catalytic domain of RNA triphosphatase, at least one catalytic domain of guanylyltransferase and at least one catalytic domain of N7-guanine methyltransferase. It comprises or consists of a nucleic acid molecule encoding a nucleic acid molecule and a nucleic acid molecule encoding at least one catalytic domain of DdRp.

Said group of isolated nucleic acid molecules encoding a chimeric enzyme of the invention comprises a nucleic acid molecule encoding at least one catalytic domain of RNA triphosphatase, a nucleic acid molecule encoding at least one catalytic domain of guanylyltransferase, N7- comprises or consists of a nucleic acid molecule encoding at least one catalytic domain of guanine methyltransferase and a nucleic acid molecule encoding at least one catalytic domain of DdRp.

The nucleic acid molecule of the present invention may be functionally linked to at least one, preferably the entire promoter(s) selected from the group consisting of a promoter for eukaryotic DdRp, preferably RNA polymerase II, and a promoter for said catalytic domain of DdRp. can

The ligation of the nucleic acid to the promoter for eukaryotic DdRp, preferably RNA polymerase II, is remarkable, when the chimeric enzyme of the present invention is expressed in a eukaryotic host cell, the expression of the chimeric enzyme is eukaryotic RNA polymerase, preferably It has the advantage of being induced by RNA polymerase II. These chimeric enzymes can also initiate transcription of a transgene. When a tissue-specific RNA polymerase II promoter is used, the chimeric enzyme of the present invention can be selectively expressed in a target tissue/cell.

The promoter may be a constitutive promoter or an inducible promoter known to those of ordinary skill in the art. The promoter may be developmentally regulated and may be inducible or tissue specific.

The present invention also relates to a vector comprising a nucleic acid molecule of the present invention. The vector may be suitable for semi-stable or stable expression.

The invention also relates to a group of vectors comprising said group of isolated nucleic acid molecules of the invention.

In particular, the vector of the present invention is a cloning or expression vector.

The vector may be a viral vector such as a bacteriophage or a non-viral vector such as a plasmid vector.

Said vector of the invention is, in particular, a bicistronic vector comprising a nucleic acid molecule of the invention and said catalytic domain of DdRp and/or a promoter for at least one DNA sequence of interest, said DNA sequence is operably linked to the promoter for the catalytic domain of DdRp.

The vector of the present invention may also include a promoter for the catalytic domain of DdRp.

The present invention also relates to an isolated nucleic acid molecule of the invention or a group of isolated nucleic acid molecules of the invention or a vector of the invention or a host cell comprising the group of vectors of the invention.

The host cells of the present invention may be useful for large-scale protein production.

Preferably, said catalytic domain of the DdRp chimeric enzyme of the invention is of an enzyme different from that of the host cell to prevent competition between endogenous gene transcription and transgene transcription.

The present invention also relates to genetically engineered non-human eukaryotes expressing the chimeric enzymes of the present invention.

The non-human eukaryote may be any non-human animal and plant.

The present invention also relates to a chimeric enzyme according to the invention or an isolated nucleic acid molecule according to the invention or a group of isolated nucleic acid molecules according to the invention, in particular in vitro or It relates to use ex vivo.

The invention also relates to a chimeric enzyme according to the invention, in particular for the production of a protein for therapeutic purposes, such as a protein, in particular an antibody, in a eukaryotic system, for example in vitro synthesized protein assay or in cultured cells. to the in vitro or ex vivo use of an isolated nucleic acid molecule according to the present invention or a group of isolated nucleic acid molecules according to the present invention.

The present invention also relates to a particularly in vitro or ex vivo method for producing in a host cell an RNA molecule having a 5' terminal m7 GpppN cap encoded by a DNA sequence, said method comprising in a host cell a nucleic acid molecule according to the invention or the present invention expressing a group of isolated nucleic acid molecules according to the invention, wherein said DNA sequence is operably linked to a promoter for said catalytic domain of DdRp, in particular said promoter is effective in said host cell, in vitro or It relates to an ex vivo method.

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

Ⅲ. Modification of RNA/DNA systems

The DdRp RNA expression cassette constituting the RNA/DNA system of the present invention may include at least one chemical modification (CM). In addition, the DdRp RNA expression cassette, auto_DdRp expression cassette or DNAge expression cassette of the present invention may include at least one nucleotide sequence modification (SM).

The RNA/DNA system of the present invention is composed of RNA and DNA. The DdRp RNA expression cassette is a DdRp mRNA comprising a DdRp open reading frame (ORF), 5'UTR and 3'UTR, and may include a 5'cap structure, chemical modification (CM) and sequence modification (SM), If these symptoms were lacking in SM, they were called capped CM_DdRp mRNA, and if they were all included, it was called capped CM/SM_DdRp mRNA.

The "RNA expression cassette comprising capped CM/SM_DdRp mRNA" of the present invention is an expression cassette in which CM/SM_DdRp mRNA is prepared by including chemically modified nucleotides in SM_DdRp mRNA and a 5'cap structure is added. The amount of protein or peptide produced from the capped CM/SM_mRNA is relatively increased, as capped CM/SM_mRNA has higher stability than SM_DdRp mRNA lacking CM (“capped SM_DdRp mRNA”).

In the present invention, DdRp mRNA comprises at least one modification in the modification consisting of CM and SM that increase DdRp expression. The DdRp expression is further increased by administration of CM/SM_DdRp mRNA containing CM and SM. An increase in protein production along at least one of CM/SM in capped CM/SM_DdRp mRNA is referred to herein as “capped CM/SM_DdRp mRNA-associated protein expression”.

The present invention provides an RNA expression cassette comprising capped CM/SM_DdRp mRNA comprising elements managing stability, replication, transcription and translation of the DdRp mRNA. If the capped CM/SM_DdRp mRNA method of the present invention is compared with an expression cassette with a conventional nuclear-dependent promoter, the increase in protein production of the capped CM/SM_DdRp mRNA method is referred to herein as “capped CM/SM_DdRp mRNA-associated increase”. can be mentioned

"Capped CM/SM_DdRp mRNA-associated protein expression" of the present invention means that the protein produced from the capped CM/SM_DdRp mRNA measured for a specific period of time is increased compared to the total amount of protein produced from a reference DdRp mRNA lacking CM/SM. situation, or a situation in which the protein level produced from the capped CM/SM_DdRp mRNA measured at a specific time point is increased compared to the protein level produced from a reference DdRp mRNA lacking CM/SM.

In the present invention, the DNAge expression cassette may include at least one targeting endonuclease-encoding DNA fragment, sgRNA-encoding DNA fragment, 5'UTR and 3'UTR. The DNAge expression cassette may include a nucleotide sequence modification (SM), which was referred to as SM_DNAge. The DNAge expression cassette may include SM_DNAge.

In addition to this, the DdRp DNA of the auto_DdRp DNA expression cassette may be included, and a nucleotide sequence modification (SM) may be included, and was referred to as auto_SM DdRp DNA.

In the present invention, the amount of protein or RNA produced from the SM_DNAge expression cassette measured at a specific time point in the SM_DNAge expression cassette with a nucleotide sequence modification (SM) also increases compared to the case of the DNAge expression cassette lacking SM.

In the concept of the genome editing RNA/DNA system of the present invention, when a DNAge expression cassette containing SM_DNAge in addition to a DdRp RNA expression cassette containing the capped CM/SM_DdRp mRNA of the present invention is injected into cells/tissues, the SM_DNAge expression cassette The expression of a targeting endonuclease that encodes or sgRNA is increased. The SM_DNAge expression cassette includes at least one SM. The expression of the encoded targeting endonuclease or sgRNA is increased by administration of the SM_DNAge expression cassette. The increase in targeted endonuclease production along the SM of a DNA expression cassette is referred to herein as "SM_DNA-associated protein expression".

Accordingly, the RNA expression cassette comprising the capped CM/SM_DdRp mRNA of the present invention together with the DNAge expression cassette comprising SM_DNAge or reference DNAge is injected into the target tissue, for example, preferably into the skin, intramuscularly or subcutaneously. are injected

According to the present invention, the DNAge expression cassette can be SM to enhance the expression of the encoded protein (“SM_DNAge-associated increase”). Therein, SM is not limited to any particular structure as much as expression of the encoded protein and is increased compared to a reference DNAge expression cassette as defined herein. Several DNA variants are known in the art and can be applied with a given DNAge in the present invention.

The conventional mRNA expression method in the nucleus using a DNA plasmid having a nuclear-dependent promoter depends on nuclear membrane penetration, which has an efficiency of only 1% compared to cell membrane penetration. very low In addition, the mRNA expressed in the nucleus can be synthesized only when it is released into the cytoplasm, but the exportin-5 transporter, which is involved in the ejection into the cytoplasm, is transfected into the cell due to the problem of being saturated by other RNAs at high concentrations. There is a problem in interfering with the cytoplasmic ejection of the introduced RNA, so that only a very small amount of RNA is transferred to the cytoplasm.

The RNA/DNA system, which is a composition for continuous genome editing tool expression of the present invention, is nuclear-independent of a selected DdRp synthesized in the cytoplasm from an RNA expression cassette comprising the capped CM/SM_DdRp mRNA, for example, T7 RNA polymerase. By increasing the expression, the expression of DNAge located downstream of the DdRp promoter (T7 promoter) in the DNAge expression cassette may be continuously expressed for a long period of time. Specifically, the continuous expression of the transcript encoded by the DNAge can continuously generate DNAge from the composition in the cytoplasm for a long period of time. can overcome

1. Modification of the 5' end (5' cap) of the modified RNA

The DdRp RNA expression cassette, CM_DdRp mRNA, SM_DdRp mRNA and CM/SM_DdRp mRNA expression cassette of the present invention can be modified by the addition of a so-called "5' cap (CAP)" structure.

A 5' cap is usually a modifying entity that "caps" the 5' end of an entity, usually a mature mRNA. The 5' cap can be formed in general by modifications, in particular derivatives of guanine nucleotides. Preferably, the 5' cap is connected to the 5' end via a 5'5' triphosphate bond. The 5' cap may be methylated, for example, m7GpppN, where N is the 5' cap, usually the terminal 5' nucleotide of the nucleic acid carrying the 5' end of the RNA. m7GpppN is a naturally occurring 5' cap (CAP) structure in mRNA transcribed by polymerase II, and therefore is not considered as a SM included in CM/SM_mRNA according to the present invention. This means that the CM/SM_mRNA according to the present invention may comprise m7GpppN as the 5' cap (CAP), but additionally the SM_mRNAi comprises at least one additional SM as defined herein.

Examples of additional 5' cap structures include glyceryl, inverted deoxy abasic residues (components), 4',5' methylene nucleotides, 1-(beta-D-erythrofuranosyl)nucleotides, 4 '-Thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, chemically modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3' ,4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'3' inverted nucleotide component, 3'3' inverted free base component, 3'2'-reversed nucleotide component, 3'2'-reversed base free component, 1,4-butanediol phosphate, 3' phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'phosphate, 3' phosphorothioate , phosphorodithioate, or a bridging or non-bridging methylphosphonate component. These chemically modified 5' cap (CAP) structures are considered to be at least one SM included in the SM_mRNAi according to the present invention.

Particularly preferred chemically modified 5' cap (CAP) structures are CAP1 (methylation of ribose adjacent nucleotide of m7G), CAP2 (methylation of ribose of second nucleotide downstream of m7G), CAP3 (ribose of ribose 3 nucleotide downstream of m7G). methylation of ), CAP4 (methylation of ribose downstream of the fourth nucleotide of m7G), ARCA (anti-reverse cap (CAP) analog, chemically modified ARCA (eg phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

2. Chemical modifications (CM): Chemical modifications

The term “RNA chemical modification” may refer to chemical modifications including sugar modifications or base modifications as well as backbone modifications.

The chemically modified RNA expression cassettes of the present invention may contain nucleotide analogues/variants, such as backbone modifications, sugar modifications or base modifications. A backbone modification in the context of the present invention is a modification in which the phosphate of the backbone of a nucleotide contained in a nucleic acid molecule as defined herein is chemically modified. A sugar modification in the context of the present invention is a modification of a sugar of a nucleotide of a nucleic acid molecule as defined herein. Moreover, a base modification in the context of the present invention is a modification of the nucleotide base component of a nucleic acid molecule of a nucleic acid molecule. Nucleotide analogues or variants are preferably selected from nucleotide analogues applicable to transcription and/or translation.

Sugar Modifications:

Chemically modified nucleosides and nucleotides that may be incorporated into the modified RNA of the present invention may be chemically modified in the sugar component. For example, the 2' hydroxyl group (OH) may be SM or substituted with a different number of "oxy" or "deoxy" substituents. Examples of "oxy"-2'hydroxyl group chemical modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -0(CH2CH2o)nCH2CH2OR; "locked" nucleic acids (LNAs) in which the 2' hydroxyl is linked to the 4' carbon of the same ribose sugar by, for example, a methylene bridge; An amino group (-O-amino, wherein the amino group, for example, NRR can be alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino ) or aminoalkoxy.

"Deoxy" variants include hydrogen, amino (eg, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or may be attached to the sugar via a linker, wherein the linker comprises an amino group comprising an atom, one or more of C, N and O.

A sugar group may also contain one or more carbons having the opposite stereochemical configuration to the corresponding carbon in ribose. Thus, SM_mRNA may comprise, for example, nucleotides containing arabinose as a sugar.

Backbone Modifications:

The phosphate backbone can also be modified with modified nucleosides and nucleotides that can be incorporated into modified RNA, as described herein. The phosphate group of the backbone can be modified by substituting one or more oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides may include total substitution of a non-chemically modified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates. and phosphotriesters. Phosphorothioates have all unlinked oxygens substituted with sulfur. Phosphate linkers can also be SMed by substitution of nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene-phosphonate) in the linked oxygen.

base modifications:

As described herein, chemically modified nucleosides and nucleotides that can be incorporated into a modified RNA can be further SMed at a nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. For example, nucleosides and nucleotides as described herein can be chemically SM in the major groove face. The chemical modification may include an amino group, a thiol group, an alkyl group, or a halo group.

In the present invention, the nucleotide analogs/variants are selected from base variants, which are preferably 2-amino-6-chloropurineriboside-5'triphosphate, 2-aminopurine-riboside-5'triphosphate; 2-Aminoadenosine-5'triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'triphosphate, 2-thiouridine-5'triphosphate, 2' -Fluorothymidine-5'triphosphate, 2'-O-methyl inosine-5'triphosphate 4-thiouridine-5'triphosphate, 5-aminoallylcytidine-5'triphosphate, 5-aminoallyl Uridine-5'triphosphate, 5-bromocytidine-5'triphosphate, 5-bromouridine-5'triphosphate, 5-bromo-2'-deoxycytidine-5'triphosphate, 5-Bromo-2'-deoxyuridine-5'triphosphate, 5-iodocytidine-5'triphosphate, 5-iodo-2'-deoxycytidine-5'triphosphate, 5- iodouridine-5'triphosphate, 5-iodo-2'-deoxyuridine-5'triphosphate, 5-methylcytidine-5'triphosphate, 5-methyluridine-5'triphosphate, 5-propynyl-2'-deoxycytidine-5' triphosphate, 5-propynyl-2'-deoxyuridine-5' triphosphate, 6-azacytidine-5' triphosphate, 6-aza Uridine-5'triphosphate, 6-chloropurineriboside-5'triphosphate, 7-deazadenosine-5'triphosphate, 7-deazaguanosine-5'triphosphate, 8-azaadenosine-5' Triphosphate, 8-azidoadenosine-5'triphosphate, benzimidazole-riboside-5'triphosphate, N1-methyladenosine-5'triphosphate, N1-methylguanosine-5'triphosphate, N6-methyl adenosine-5'triphosphate, O6-methylguanosine-5'triphosphate, pseudouridine-5'triphosphate, or puromycin-5'triphosphate, xanthosine-5'triphosphate. of the base-CM consisting of 5-methylcytidine-5'triphosphate, 7-deazaguanosine-5'triphosphate, 5-bromocytidine-5'triphosphate, and pseudouridine-5'triphosphate Nucleotides for base SM selected from the group are particularly preferred.

Modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- Thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudo Uridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl -uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2 -Thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

Modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethyl Tidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine Dean, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebrarin, 5-aza-zevurarin, 5-methyl-zeburarine, 5-aza-2-thio-zeburarine, 2-thio-zeburarine, 2-methoxy-cytidine, 2-methoxy-5-methyl-between tidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

The modified nucleosides are 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-de Aza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine , N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

Modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio -guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine , 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

The nucleotide may be SM and may comprise replacing the hydrogen at C-5 of uracil with a methyl group or a halo group.

Modified nucleosides are 5'0-(l-thiophosphate)-adenosine, 5'0-(1-thiophosphate)-cytidine, 5'0-(1-thiophosphate)-guanosine, 5'0 -(1-thiophosphate)-uridine or 5'0-(1-thiophosphate)-pseudouridine.

SM_mRNAi is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl- Pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5- Methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1 -Methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, a nucleoside chemical modification selected from the group consisting of:

Lipid modification:

The modified RNAs of the invention may contain lipid modifications. Such lipid-modified RNAs generally include RNAs as defined herein. Lipid-modified RNA molecules of the invention generally further comprise at least one linker covalently linked to the RNA molecule, and at least one lipid covalently linked to each linker. Instead, the lipid-modifying RNA molecule comprises at least one RNA molecule as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) to the RNA molecule. According to a third alternative, the lipid-chemically modifying RNA molecule is an RNA molecule as defined herein, at least one linker covalently linked to the RNA molecule, and at least one lipid covalently linked to each linker, and also an RNA molecule and at least one (bifunctional) lipid covalently linked (without a linker). It is particularly preferred that the lipid modification is at the terminal end of the linear RNA sequence.

3. Modification of the base sequence (SM)

Variation of open reading frame: Variation of G/C content:

SM_mRNAi of the present invention encodes at least one peptide or protein, wherein the G/C content of the coding region is modified and is particularly increased when compared to the G/C content of its specific wild-type coding region, i.e., the unmodified coding region. . Preferably, the amino acid sequence of the coding region is not modified as compared to the encoded amino acid sequence of the particular wild-type coding region.

The modification of the G/C-content of the SM_mRNAi coding region in the present invention is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of the mRNA. Thus, the composition and sequence of the various nucleotides is important. In particular, an mRNA sequence having an increased content of G (guanosine)/C (cytosine) is more stable than an mRNA sequence having an increased content of A (adenosine)/U (uracil). According to the present invention, the codon of the coding region that is continuously being translated into the amino acid sequence is different from the codon of its wild-type coding region in that the amount of G/C nucleotides in the codon is increased. In light of the fact that several codons encode one and the same amino acid (the so-called degeneration of the genetic code), the codon most favorable for stability can be determined (the so-called alternative codon usage).

Depending on the amino acid to be encoded by the coding region of SM_mRNAi in the present invention, various possibilities exist for modification of the RNA sequence, eg, the coding region, when compared to its wild-type coding region. For amino acids encoded by codons containing only G or C nucleotides, no codon modifications are necessary. Therefore, since neither A nor U is present, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) do not require modification.

In contrast, codons containing A and/or U nucleotides may be modified by substitution of other codons encoding the same amino acid but not containing A and/or U, examples of which are:

The codon for Pro may be modified from CCU or CCA to CCC or CCG;

The codon for Arg may be modified from CGU or CGA or AGA or AGG to CGC or CGG;

The codon for Ala can be modified from GCU or GCA to GCC or GCG;

The codon for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be removed from the codon, the A and U content can be reduced by using codons containing lower amounts of A and/or U nucleotides. Examples of these are:

The codon for Phe can be modified from UUU to UUC;

The codon for Leu may be modified from UUA, UUG, CUU or CUA to CUC or CUG;

The codon for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC;

The codon for Tyr can be modified from UAU to UAC;

The codon for Cys can be modified from UGU to UGC;

The codon for His can be modified from CAU to CAC;

The codon for Gln can be modified from CAA to CAG;

The codon for Ile can be modified from AUU or AUA to AUC;

The codon for Thr can be modified from ACU or ACA to ACC or ACG;

The codon for Asn can be modified from AAU to AAC;

The codon for Lys can be modified from AAA to AAG;

The codon for Val can be modified from GUU or GUA to GUC or GUG;

The codon for Asp can be modified from GAU to GAC;

The codon for Glu can be modified from GAA to GAG;

The stop codon UAA may be modified to UAG or UGA.

On the other hand, in the case of codons for Met(AUG) and Trp(UGG), there is no possibility of sequence modification.

In order to increase the G/C content of the coding region of SM_mRNAi in the present invention compared to the G/C content of a specific wild-type coding region (ie the original sequence), the substitutions listed above can be applied individually or in any possible combination. can be applied. Thus, for example, all codons for Thr occurring in the wild-type sequence can be modified to ACC (or ACG).

Preferably, in the present invention, the G/C content of the coding region of SM_mRNAi is increased by 7% or more, more preferably 15% or more, particularly preferably 20% or more, compared to the G/C content of the wild-type coding region. 5%, 10%, 20%, 30%, 40%, 50% of codons substitutable in the coding region encoding one or more peptides or proteins, including pathogenic antibodies or fragments, variants or derivatives thereof, 60% or more, more preferably 70% or more, even more preferably 80% or more, and most preferably 90% or more, 95% or more or even 100% are substituted, whereby the G/C content of said coding region is substituted. this will increase

Particularly preferably, in the present invention, the G/C content of the coding region of SM_mRNAi may increase by a maximum value (ie, 100% of substitutable codons) compared to the G/C content of the wild-type coding region.

Codon optimization:

A further preferred modification of the coding region encoding at least one peptide or protein of SM_mRNAi in the present invention is based on the finding that translation efficiency is also determined by different frequencies of appearance of tRNA in cells. Thus, if so-called "rare codons" are present to an increased degree in the coding region of a wild-type RNA sequence, they are translated to a significantly lower degree than if codons encoding a relatively "frequent" tRNA are present.

The coding region of the SM_mRNAi is preferably modified compared to the corresponding wild-type coding region so that the codons of the wild-type sequence encoding a tRNA, which are relatively rare in at least one cell, encode a tRNA that is relatively frequent in the cell and the relative is substituted with a codon carrying the same amino acid as the rare tRNA. By this modification, the coding region of the SM_mRNAi of the present invention is modified, and as a result, a codon for using a frequently occurring tRNA is inserted. In other words, according to the present invention, by this modification all codons of the wild-type coding region encoding tRNAs that are relatively rare in cells can in each case be replaced with codons encoding tRNAs that are relatively frequent in cells, and , which in each case may carry amino acids as relatively rare tRNAs.

It is known to the person skilled in the art which tRNAs occur relatively frequently in cells and, in contrast, which tRNAs occur relatively rarely. Particular preference is given to the Gly codon using the most frequently occurring tRNA for a particular amino acid, for example the most frequently occurring tRNA in (human) cells.

It is particularly preferred to associate a maximally increased sequence G/C content in the coding region of the SM_mRNAi of the present invention with “frequent” codons in which the amino acid sequence of the peptide or protein encoded by the coding region of the RNA sequence is not modified. . Particularly efficiently translated and stabilized (chemically modified) RNA sequences are provided.

Variants of UTR:

Preferably, the SM_mRNAi of the present invention has at least one chemically modified 5' and/or 3' UTR sequence. Modifications in these 5' and/or 3' UTRs may have the effect of increasing the half-life of the RNA in the cell matrix or may increase the translation efficiency and thus enhance the expression of the encoded protein or peptide. These UTR sequences may have 100% sequence homology to naturally occurring sequences occurring in viruses, bacteria and eukaryotes, but may also be partially or fully synthesized.

The SM_mRNAi according to the present invention is preferably a 5' and/or 3' UTR element, in particular a 5'UTR element comprising or consisting of a nucleic acid derived from the 5'UTR of the TOP gene or from a fragment, homologue or variant thereof, or a stable mRNA 5' and/or 3'UTR elements, which may be derived from a gene providing a histone-stem-loop structure, preferably a histone-stem-loop in its 3' untranslated region; 5' cap (CAP) structure; poly-A tail; or at least one structural element from the group comprising a poly(C) sequence.

SM_mRNAi according to the present invention comprises at least one 5' or 3'UTR element. A UTR element comprises or consists of a nucleic acid sequence derived from any naturally occurring 5' or 3'UTR, or derived from a fragment, homologue or variant of a 5' or 3'UTR of a gene. Preferably, the 5' or 3'UTR element used according to the invention is heterologous to the coding region of the SM_mRNAi according to the invention. Although 5' or 3'UTR elements derived from naturally occurring genes are preferred, synthetically engineered UTR elements may also be used in the present invention.

The sequence of SM_mRNAi according to the present invention comprises or consists of at least one 5' untranslated region element (5 'UTR element).

The nucleic acid sequence of the 5'UTR element derived from the 5'UTR of the TOP gene is 1, 2, 3, 4, 5, 6, upstream of the start codon (eg, A(U/T)G) of the gene or mRNA. It terminates at its 3' end with a nucleotide located at position 7, 8, 9 or 10. Thus, the 5'UTR element does not include any portion of the protein coding region. Thus, preferably, only the protein coding portion of the SM_mRNAi according to the present invention is provided by the coding region.

The nucleic acid sequence derived from the 5'UTR of the TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a It is preferably derived from a mammalian TOP gene, such as a human TOP gene.

SM_mRNAi according to the present invention is derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or a chordate gene, preferably a vertebrate gene, More preferably it further comprises at least one 3'UTR element comprising or consisting of a nucleic acid sequence derived from a variant of the 3'UTR of a mammalian gene, most preferably a human gene.

The term '3'UTR element' refers to a nucleic acid sequence comprising or consisting of a nucleic acid sequence derived from a 3'UTR or derived from a variant of the 3'UTR. In the sense of the present invention, a 3'UTR element may represent the 3'UTR of an mRNA. Thus, in the sense of the present invention, preferably, the 3'UTR element may be the 3'UTR of an mRNA, preferably of an artificial mRNA, which may also be an electron template for the 3'UTR of an mRNA. Thus, the 3'UTR element is preferably a nucleic acid sequence corresponding to the 3'UTR of an mRNA, preferably an artificial mRNA, such as a 3'UTR of an mRNA obtained by transcription of a genetically engineered vector plasmid. Preferably, the 3'UTR element realizes the function of the 3'UTR or encodes a sequence that realizes the function of the 3'UTR.

A novel mRNA comprises a 3'UTR element that can be derived from a gene associated with an mRNA with improved half-life (providing a stable mRNA), for example a 3'UTR element as defined and described below.

The 3'UTR element is a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(I) gene. from the 3'UTR of or from the group consisting of albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, and collagen alpha gene, such as collagen alpha 1 (I) gene. It comprises or consists of a nucleic acid sequence derived from a variant of the 3'UTR of the gene of choice. The 3'UTR element may be an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene,

Most preferably it comprises or consists of a nucleic acid sequence derived from the 3'UTR of the human albumin gene, SEQ ID NO:6.

Human Albumin 3'UTR: CATCACATTT AAAAGCATCT CAGCCTACCA TGAGAATAAG AGAAAGAAAA TGAAGATCAA AAGCTTATTC ATCTGTTTTT CTTTTTCGTT GGTGTAAAGC CAACACCCTG TCTAAAAAAC ATAAATTTCT TTAATCATTT TGCCTCTTTT CTCTGTGCTT SEQ ID NO: 6)

It is particularly preferred that the SM_mRNAi according to the present invention comprises a 3'UTR element comprising a corresponding RNA derived from another nucleic acid or a fragment, homologue or variant thereof.

Most preferably, the 3'UTR element comprises a nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID NO:6, SEQ ID NO:7.

Albumin 3'UTR: CATCACATTT AAAAGCATCT CAGCCTACC ATGAGAATAA GAGAAAGAAA ATGAAGATCA ATAGCTTATT CATCTCTTTT TCTTTTTCGT TGGTGTAAAG CCAACACCCT GTCTAAAAAA CATAAATTTC TTAA AGGTAATCATT TTGCCTCTTT GAACCT (SEQ ID NO: 7 TCTCTG TGCT TCAAT).

XRMGL preferably, the 3'UTR element of the inventive mRNA comprises or consists of the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO:6.

The 3'UTR element is an α-globin gene, preferably a vertebrate α- or β-globin gene, more preferably a mammalian α- or β-globin gene, most preferably a human α according to SEQ ID NOs: 6 to 7 - or comprises or consists of a nucleic acid sequence derived from the 3'UTR of the β-globin gene.

The 3'UTR element comprises or consists of nucleic acid sequences SEQ ID NOs:8-10 derived from the 3'UTR of the Homo sapiens hemoglobin, alpha 1 (HBA1), alpha 2 (HBA2) and beta (HBB) genes.

3'UTR of Homo sapiens hemoglobin, alpha 1 (HBA1): GCTGGAGCCT CGGTGGCCAT GCTTCTTGCC CCTTGGGCCT CCCCCCAGCC CCTCCTCCCC TTCCTGCACC CGTACCCCCG TGGTCTTTGA ATAAAGTCTG AGTGGGCGGC (SEQ ID NO: 8)

3'UTR of Homo sapiens hemoglobin, alpha 2 (HBA2): GCTGGAGCCT CGGTAGCCGT TCCTCCTGCC CGCTGGGCCT CCCAACGGGC CCTCCTCCCC TCCTTGCACC GGCCCTTCCT GGTCTTTGAA TAAAGTCTGA GTGGGCAG (SEQ ID NO: 9)

3'UTR of Homo sapiens hemoglobin, beta (HBB): GCTCGCTTTC TTGCTGTCCA ATTTCTATTA AAGGTTCCTT TGTTCCCTAA GTCCAACTAC TAAACTGGGG GATATTATGA AGGGCCTTGA GCATCTGGAT TCTGCCTAAT AAAAAACATT TATTTTCATT GC (SEQ ID NO:10)

For example, the 3'UTR element may preferably comprise or consist of a central, α-complex-binding portion of the 3'UTR of an α-globin gene, such as a human α-globin gene according to SEQ ID NO:7:

central, the α-complex-binding portion of the 3′UTR of the α-globin gene (also termed herein as “muag”); GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG (SEQ ID NO: 11).

It is particularly preferred that the 3'UTR element of SM_mRNAi according to the present invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 7 or a homologue, fragment or variant thereof.

Preferably, the at least one 5'UTR element and the at least one 3'UTR element play a synergistic role in increasing the production of the SM_mRNAi derived protein according to the invention as described above. More preferably, the at least one 5'UTR element and/or the at least one 3'UTR element plays a synergistic role with the injection of SM_mRNAi to increase protein production.

SM_mRNAi according to the present invention may further comprise - by adding at least one modification - an internal ribosome entry site (IRES) sequence or an IRES-motif, for example, if SM_mRNAi is two or more Encoding peptides or proteins can be separated into several open reading frames. The IRES-sequence can be particularly helpful if the mRNA is a bi- or multicistronic RNA.

As used herein, the term “RNA” is not limited and refers to any ribonucleic acid. Thus, the term RNA applies equally to ribonucleic acids that function as coding RNAs, for example as viral RNA, (replicon) or mRNA, or to artificial RNA expression cassettes of any type.

According to the present invention, mRNAi comprises at least one modification that increases the expression of the peptide or protein encoded by the coding region of the RNA. The mRNAi according to the invention comprises at least one type of modification as defined herein. Instead, or in combination with other types of modifications, certain modifications as defined herein, such as 3'UTR, histone stem-loop or poly C sequence, may also be present in one or more copies in the SM_mRNAi according to the invention. can This is especially true for bi- or multicistronic RNAs. SM_mRNAi according to the present invention may comprise a combination of several distinct modifications, eg, any, eg 5'UTR, poly C sequence, poly A sequence, 3'UTR or histone stem-loop and GC- Modifications that combine GC-enrichment, wherein each distinct modification may exist in the form of a single copy per RNA or multiple copies per RNA.

Modification of the 3' end of RNA:

Moreover, for SM_mRNAi comprising at least one open reading frame to be administered, the 3' region of the coding region may be modified, for example, by insertion or addition of multiple repeat stretches of adenine or cytosine residues. The 3' end of the RNA molecule is chemically modified by the addition of a series of adenine nucleotides ("poly(A) tail").

The length of the poly(A) tail has a major influence on the translation efficiency of each constituting the plasmid. To be efficiently translated, the poly(A) tail of an exogenously delivered mRNA must consist of at least 20 A residues. Furthermore, it has been demonstrated that mRNA expression positively correlates with the length of the poly(A) tail. It shows improved mRNA stability and translation efficiency compared to the shorter poly(A) tail measured with 120 nucleotides. The SM_mRNAi of the present invention comprise (optionally in combination with other modifications) a poly(A) tail, wherein the poly(A) tail has at least 30, preferably greater than 50, more preferably 100 poly(A) tails. more than, even more preferably more than 200 adenosine nucleotides. Most preferably, the RNA comprises a poly(A) tail consisting of 64 adenosine nucleotides.

If the poly(A) tail is longer than 30 adenosines, preferably longer than 50 adenosines, more preferably longer than 100 adenosines, and even more preferably longer than 200 adenosines, the poly(A) tail is only It is considered as at least one modification included in SM_mRNAi according to the present invention.

Modification of the poly(A) tail is not considered to be at least one modification of SM_mRNAi according to the present invention. This means that the SM_mRNAi according to the present invention may comprise a poly(A) tail as defined above, but may comprise at least one further modification of the present invention.

SM_mRNAi comprising at least one open reading frame comprises (optionally in combination with other modifications) a poly(C) sequence in region 3' of the coding region of the RNA. The poly(C) sequence generally comprises a plurality of cytosine nucleotides, generally between about 10 and about 200 cytidine nucleotides, preferably between about 10 and about 100 cytidine nucleotides, more preferably between about 10 and about 70 cytosine nucleotides. a stretch of tidine nucleotides or even more preferably from about 20 to about 50 or even from about 20 to about 30 cytidine nucleotides. The poly(C) sequence may preferably be located 3' of the coding region by the nucleic acid. The poly(C) sequence of the present invention is located 3' to the poly(A) sequence.

SM_mRNAi according to the present invention comprises or encodes a histone stem-loop at its 3' end as at least one modification.

A particularly preferred embodiment for a histone stem-loop sequence is the sequence according to SEQ ID NO: 12 (CAAAGGCTCTTTTCAGAGCCACCA) or the corresponding RNA sequence according to SEQ ID NO: 13 (CAAAGGCUCUUUUUCAGAGCCACCA).

heterologous nucleic acid sequence

The expression cassette comprised in the genome editing RNA/DNA system of the present invention further comprises at least one non-viral nucleic acid sequence, in particular a non-viral nucleic acid sequence. In a preferred embodiment, the expression cassette according to the invention comprises a heterologous nucleic acid sequence.

The term “heterologous” refers to a situation in which the nucleic acid sequence is not natively functionally bound to a viral nucleic acid sequence, such as a promoter to which viral expression control sequences, in particular viral DdRp, bind. The heterologous nucleic acid sequence is comprised in the nucleic acid sequence of the replicon.

Preferably, the heterologous nucleic acid sequence is under the control of a promoter to which DdRp binds, preferably a promoter to which a viral DdRp binds, more preferably, the heterologous nucleic acid sequence is located downstream of the promoter to which DdRp binds. The promoter to which the viral DdRp binds is highly efficient, making it suitable for high-level expression of heterologous genes. Preferably, the promoter to which DdRp binds controls the production of a transcript comprising a heterologous nucleic acid sequence or a transcript of a portion thereof.

Preferably, the promoter to which DdRp binds is a promoter for a structural protein of the virus. This means that the promoter to which DdRp binds is a promoter that is native to the virus and controls the transcription of the coding sequences of one or more structural proteins in the virus. According to the present invention, the heterologous nucleic acid sequence may partially or completely replace a viral nucleic acid sequence, such as a nucleic acid sequence encoding a viral structural protein. Preferably, the heterologous nucleic acid sequence is not derived from a virus; In particular, the heterologous nucleic acid sequence is preferably not derived from the same virus as the virus from which the promoter to which DdRp binds is derived.

IV. Genome editing tools

In the present invention, the DNAge expression cassette includes a DNA fragment encoding a genome editing tool. The genome editing tools are a targeting endonuclease and a guide nucleic acid. The DNAge expression cassette of the present invention may include at least one of a DNAen expression cassette encoding a targeting endonuclease and a DNAsg expression cassette encoding a guide nucleic acid.

In the present invention, the DNA encoding the genome editing tool is synonymously referred to as "DNAge". The DNA is preferably present on the expression cassette according to the invention under the control of a promoter to which DdRp binds.

The existing genome editing CRISPR/Cas9 system can be delivered to cells in a variety of forms, including plasmids, lentiviruses, adeno-associated viruses, and ribonucleoprotein (RNP) complexes. The CRISPR/Cas9 ribonucleoprotein (RNP) system consists of a Cas9 protein and a single guide RNA (sgRNA) or CRISPR RNA (crRNA):trans-activating crRNA (tracrRNA) duplex.

crRNA is a 20 nt RNA oligonucleotide designed to be complementary to a genomic target. The crRNA sequence design utilizes standard gRNA design principles and can be easily selected from the gRNA database or customized using gRNA design tools.

tracrRNA is a 67nt RNA oligonucleotide that forms an activated CRISPR/Cas9 RNP complex with crRNA and Cas9 nuclease. The sgRNA is a single RNA oligo containing both a crRNA sequence capable of identifying a target genomic sequence and a tracrRNA sequence capable of forming a complex with a Cas9 protein.

When the sgRNA or crRNA:tracrRNA duplexes form a complex with the Cas9 protein, they together form a protospacer adjacent motif (PAM) sequence 3-4 bp upstream from the (5'NGG-3'), 20 nt guide RNA sequence can create a double-stranded break at a locus complementary to

DNA (DNAgn) encoding a guide nucleic acid constituting the DNAge expression cassette of the present invention is a unit for transcription of a guide nucleic acid molecule. guide nucleic acids, such as gRNA. "Guide RNA" and "gRNA" may be used interchangeably in the present invention, typically CRISPR RNA (crRNA) and trans-coding CRISPR RNA (tracrRNA, scaffold RNA) that form a complex via some complementary moiety. is an RNA chimeric molecule composed of, crRNA is sufficiently complementary to the target sequence for hybridization; contains a sequence that directs the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to a target sequence. This sequence is 19-22 nucleotides in length, eg 20 consecutive nucleotides complementary to the target, and is typically located at the 5' end of the sgRNA molecule. A crRNA may be 100% complementary to a target sequence, but the present invention also contemplates at least 80%, 85%, 90% and 95% total complementarity with the target sequence. The tracrRNA is 100-300 nucleotides in length, and as a binding site for a nuclease, for example, the Cas9 protein allows it to form a CRISPR/Cas9 complex. Preferably, the tracrRNA is a Scaffold Template-specific sequence that is 21 nucleotides in length and forms a complex with Cas9.

However, it is known in the art that a single guide RNA (sgRNA) comprising both crRNA and tracrRNA characteristics can be designed.

The DNAsg expression cassette according to the present invention may encode a single guide nucleic acid or multiple guide nucleic acids. Multiple guide nucleic acids may be encoded as a single guide nucleic acid (fusion table guide nucleic acid) or as separate guide nucleic acids. Where the DNAge expression cassettes are encoded as separate multiple guide nucleic acids, one or more of them may be provided with Csy4 ribonuclease sequences and a Cys4 ribonucleae system or additional viral promoter elements.

In the present invention, the DNAsg expression cassette may include two or more guide nucleic acids, each of which is under the control of a promoter to which DdRp binds. In this multiple guide nucleic acid DNAsg expression cassette, multiple guide nucleic acid sgRNAs will be produced, and there are multiple gRNA expression cassettes initiated by the promoter to which each autologous DdRp binds. There is a polycistronic tRNA-gRNA expression cassette.

An advantage of the system of the present invention is that it does not require the presence or administration of a helper virus encoding a viral structural protein.

The genome editing tool includes meganuclease; zinc-finger nucleases (ZFNs); TAL-effector nucleases (TALENs); RNA-guided endonuclease; DNA-guided endonuclease; and a fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase.

1. RNA-guided endonuclease

When the genome editing tool in the present invention is an RNA-guided endonuclease, the targeting endonuclease is "Cas9 nuclease" and "Cas9", which are used interchangeably in the present invention, and include a Cas protein or a fragment thereof ( eg, a protein comprising the DNA cleavage domain of an active form of Cas9 and/or the gRNA binding domain of Cas9). Cas9 targets and cleaves a DNA target sequence under the guidance of a guide RNA to form a DNA double-stranded break (DSB), constituting the CRISPR/Cas (regularly repeating short palindromic repeat cluster and its subsystems) genome editing system is an ingredient

“Guide RNA” and “gRNA” may be used interchangeably in the present invention, and are typically composed of CRISPR RNA (crRNA) and trans-coded CRISPR RNA (tracrRNA) that form a complex through some complementary moiety. is a chimeric molecule, wherein the crRNA is sufficiently complementary to the target sequence for hybridization; contains a sequence that directs the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to a target sequence. This sequence is 19-22 nucleotides in length, eg 20 consecutive nucleotides complementary to the target, and is typically located at the 5' end of the sgRNA molecule. A crRNA may be 100% complementary to a target sequence, but the present invention also contemplates at least 80%, 85%, 90% and 95% total complementarity with the target sequence. The tracrRNA is 100-300 nucleotides in length, and as a binding site for a nuclease, for example, the Cas9 protein forms the CRISPR/Cas9 complex.

However, it is known in the art that a single guide RNA (sgRNA) comprising both crRNA and tracrRNA characteristics can be designed.

The DNA sequence encoding the sgRNA can be designed as follows (sense strand marking):

5' GAAATTAATACGACTCACTATA GGN ₁₈ GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT 3' (SEQ ID NO: 14).

The initial 22 bp (indicated in italics) is the promoter of T7 RNA polymerase used for in vitro transcription. This is followed by the GGN ₁₈ sequence (underlined), representing 20 bp consistent with the target (spacer) sequence. Cleavage results in 3 bases within the 3' end of this sequence on both strands of the target DNA. The initial GG dinucleotide is not required to achieve cleavage, but is included to optimize in vitro transcription by T7 RNA polymerase. In these first two nucleotides, slight variations can be tolerated, reducing transcriptional efficiency.

The target DNA strand, which has the same semantics as the sgRNA, should immediately carry the 20 bp 3' end consistent with the spacer into the triplet NGG. NAG is also tolerated by S. pyogenes Cas9 but is less efficient. Thus, the target has the sequence GGN18NGG. The remaining 80 bp (bold type) encodes a chimeric fusion of crRNA and tracrRNA. Shorter sgRNA segments were effective in vitro and in vivo, but appear to be less efficient. Further testing will reveal a variant with improved efficiency, but this segment is already working well.

The target sequence is endogenous to the target genome. That is, the target sequence exists with the same copy number and genomic location as the wild-type genome. or the target sequence is exogenous to the target genome. Also referred to herein as heterogeneous. In this case, it may be a gene present in the genome but at a different location, or it may be a completely foreign gene, that is, a gene that is not present in the genome containing the target sequence.

A plurality of gRNAs complementary to various target nucleic acid sequences are provided to the plant cell, and a nuclease, such as a Cas9 enzyme, cleaves the various target nucleic acid sequences in a site-specific manner. A plurality of gRNAs may be encoded from one or several species of the TRV vectors described herein. The use of multiple sgRNAs enables multiplexing.

A plurality of sgRNAs for one target gene (eg, vector B2) or a plurality of target genes are present in the expression cassette. A plurality of these (eg 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, eg 2-5, 2-4, 2-3) sgRNAs are located under one promoter. The sgRNAs may or may not be spaced apart from each other by a spacer.

Such a multiplex configuration, that is, a multiplex configuration under a promoter to which one DdRp binds, can be applied to any genome editing method using the CRISPR/Cas9 system. Accordingly, a nucleic acid expression cassette comprising two or more sgRNAs under one promoter is provided.

Such nucleic acid expression cassettes can be for any cell in prokaryotic or eukaryotic organisms, such as mammals (eg, humans), plants, insects and yeast.

The nucleic acid sequence encoding the sgRNA has ribozyme sequences located on both sides to form a ribozyme-sgRNA-ribozyme (RGR) sequence. It is shown that the primary transcript of RGR is gig-catalytically cleaved and the sgRNA is dissociated. In addition, the use of a promoter to which TRV DdRp binds is thought to expand the range of promoters that can be used for sgRNA transcription with the RGR arrangement, although it has been shown to efficiently perform transcription of sgRNA even without a flanking ribozyme. . The RGR strategy is that if the entire viral replicon is fully transcribed, any target expression product (eg, reporter, agriculturally useful trait, eg, stress tolerance, etc.) is expressed in the inoculated tissue, and the guide RNA is also in the same tissue. to ensure that it is formed in

A guide RNA is a single guide RNA (sgRNA). Methods for constructing suitable sgRNAs according to a given target sequence are known in the art.

In the present invention, the terms "tRNA" and "transfer RNA" are used interchangeably in the present invention to refer to a small molecule RNA having a function of transporting and transporting amino acids. A tRNA molecule consists of a single chain of about 70-90 nucleotides that is usually folded into a clover shape. In the case of eukaryotes, the tRNA gene in the genome is transcribed into a tRNA precursor, and then additional sequences of 5' and 3' are cleaved by RNase P and RNase Z to be processed into mature tRNA.

In the present invention, the term "ribozyme" refers to an RNA molecule having a catalytic function involved in the cleavage and processing of RNA by catalyzing the hydrolysis reaction of transphosphate and phosphodiester bonds.

In Korean Patent Publication No. 10-2019-0120287, Cas9 nuclease mutant eSpCas9 (1.0) (K810A / K1003A / R1060A), eSpCas9 (1.1) (K848A / K1003A / R1060A), and Cas9 nuclease mutant SpCas9-HF1 (N497A) /R661A/Q695A/Q926A) can significantly lower the off-target ratio during genome editing, that is, it has been reported to have high specificity.

However, these three Cas9 nuclease variants, while having high specificity, have much lower gene editing efficiency compared to wild-type Cas9. By fusing the tRNA to the 5' end of the guide RNA, the editing efficiency of the high-specificity Cas9 nuclease mutant can be increased even to the wild-type level while maintaining high specificity.

The low editing efficiency of the high-specificity Cas9 nuclease variant correlates with whether transcription of the guide RNA can be accurately initiated.

In the art, promoters commonly used to produce guide RNAs in vivo include, for example, U6 or U3 snRNA promoters whose transcription is driven by RNA polymerase III. The U6 promoter must initiate transcription at G, so for a target sequence where the first nucleotide is A, C or T, an additional G will be provided at the 5' end of the sgRNA to be transcribed. The U3 promoter initiates transcription at A, so for a target sequence where the first nucleotide is G, C or T, an additional A will be provided at the 5' end of the transcribed sgRNA. The editing efficiency of the high-specificity Cas9 nuclease variant is reduced when an additional nucleotide is present at the 5' end of the sgRNA.

In the present invention, by fusion transcription of gRNA and tRNA, due to the precise processing mechanism of tRNA (additional sequences 5' and 3' of the tRNA precursor are accurately removed to form a mature tRNA), an additional nucleotide at the 5' end is An absent sgRNA can even be easily obtained using SGP, U6 or U3 promoters, without taking into account the type of the first nucleotide of the target sequence.

Thereby, the editing efficiency of the high specificity Cas9 nuclease mutant can be improved, and the selectable range of the target sequence can be broadened. In addition, it is possible to increase the expression level of sgRNA by fusion with tRNA, which may also contribute to improving the editing efficiency of high-specificity Cas9 nuclease variants.

The tRNA and the cell to be modified are from the same species. tRNA is encoded by the following sequence. 5'aacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgca-3' (SEQ ID NO: 15).

The design of tRNA-guided RNA fusions is within the ability of one of ordinary skill in the art.

The invention also contemplates fusions of a guide RNA and a ribozyme. In the present invention, based on the fact that the editing efficiency of the high-specificity Cas9 nuclease variant is related to the precise transcription initiation of sgRNA, by using the ability of the ribozyme to cleave RNA at a specific site, a fusion of RNA and ribozyme can be rationally used. sgRNA can be produced without additional nucleotides at the 5' end by designing as a

The 3' end of the guide RNA is joined with the 5' end of the second ribozyme, and the second ribozyme cleaves the fusion at the 3' end of the guide RNA, so that the guide RNA carrying no additional nucleotides at the 3' end is is formed

The design of the first ribozyme or the second ribozyme is within the ability of one of ordinary skill in the art.

The first ribozyme is encoded by the sequence: 5'(N)6CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC-3' (SEQ ID NO: 16), wherein N is independently selected from A, G, C and T, and (N)6 is the It is a sequence complementary to the first 6 nucleotides at the 5' end position in reverse order.

The second ribozyme is encoded by the sequence: 5'GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGAC-3' (SEQ ID NO: 17).

In the present invention, the Cas9 nuclease variant with higher specificity compared to the wild-type Cas9 nuclease can be obtained from Cas9 of various species, for example, from Cas9 of Streptococcus pyogenes (SpCas9, nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: indicated amino acid sequence).

The Cas9 nuclease variant of the present invention further comprises a nuclear localization sequence (NLS). In general, for Cas9 nuclease variants, one squared NLS should have sufficient strength to allow Cas9 nuclease variants to accumulate in the nucleus of the cell in sufficient quantities for genome editing functions. In general, the strength of nuclear localization activity is determined by the number and location of NLSs, one or more specific NLSs used in the Cas9 nuclease variant, or a combination thereof.

In the Cas9 nuclease variant of the present invention, the NLS may be located at the N-terminus and/or at the C-terminus. Cas9 nuclease variants contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. Cas9 nuclease variants contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus. Cas9 nuclease variants contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus.

Cas9 nuclease variants include combinations thereof, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. If there is more than one NLS, each NLS can be selected independently of the other NLSs. Preferably, the Cas9 nuclease variant comprises two NLSs, eg, the two NLSs are located at the N-terminus and C-terminus respectively.

Generally, NLSs consist of one or more short sequences of positively charged lysine or arginine exposed on the surface of a protein, although other types of NLS are known in the art. Non-limiting examples for NLS include KKRKV (SEQ ID NO: 18), 5'AAGAAGAGAAAGGTC-3' (SEQ ID NO: 19), PKKKRKV (SEQ ID NO: 20), 5'CCCAAGAAGAAGAGGAAGGTG-3' (SEQ ID NO: 21) or CCAAAGAAGAAGAGGAAGGTT (SEQ ID NO: 21) No. 22), or SGGSPKKKRKV (SEQ ID NO: 22), 5' TCGGGGGGGAGCCCAAAGAAGAAGCGGAAGGTG -3' (SEQ ID NO: 23), and the like.

The N-terminus of the Cas9 nuclease variant contains an NLS having the amino acid sequence represented by PKKKRKV (SEQ ID NO: 24). The C-terminus of the Cas9 nuclease variant contains an NLS having an amino acid sequence represented by SGGSPKKKRKV (SEQ ID NO: 25).

In addition, the Cas9 nuclease variants of the present invention may also contain other localization sequences such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, etc. depending on the location of the DNA to be edited.

To achieve effective expression at the target sequence, the nucleotide sequence encoding the Cas9 nuclease variant is codon-optimized for the organism from which the cell to be genome-edited is derived.

Codon-optimization is one or more codons of the native sequence (e.g., about 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more codons) . do. Different species exhibit particular biases for some codons of specific amino acids. Codon bias (differences in codon usage between organisms) is largely dependent on the translation efficiency and inertia of messenger RNA (mRNA), which appears to depend in particular on the nature of the codon being translated and the availability of specific transfer RNA (tRNA) molecules.

The preference of the tRNA selected in the cell generally reflects the most frequently used codons in peptide synthesis. That is, genes can be tailored to optimize gene expression in a given organism that underlies codon optimization. Codon usage tables are readily available, for example, in the "Codon usage database" available at www.kazusa.orjp/codon/, and such tables can be modified in a number of ways.

The codon-optimized nucleotide sequence encoding the Cas9 nuclease variant is represented by SEQ ID NO: 7 (eSpCas9(1.0)), SEQ ID NO: 8 (eSpCas9(1.1)) or SEQ ID NO: 9 (SpCas9-HF1).

The nucleotide sequence encoding the Cas9 nuclease variant and/or the nucleotide sequence encoding the guide RNA fusion is operably linked to an expression control element such as a promoter.

Cas9 nuclease is basically excellent in the ability to find, recognize, and bind to a target DNA complementary to a guide RNA. When Cas9 binds to the target DNA, an R-loop form is formed. During this process, the DNA temporarily stays in a single-stranded state. Taking this into consideration, each independently developed cytosine base-correcting gene scissors. To prevent DNA double helix breakage from occurring, a mutation capable of cleaving only a single strand (Cas9 nickase; nCas9) or a mutant capable of cleaving only a single strand was used for base-correcting gene scissors. And by linking a cytidine deamination enzyme that can operate to a single strand here, deamination can be directly induced to the target base, cytosine.

When cytosine is deamination, it is changed to uracil, which is later recognized as thymine in the DNA transcription process and is transcribed. As a result, the cytosine base editing gene scissors can substitute thymine for cytosine, a single base. The researchers performed two additional tasks to maximize the efficiency of cytosine base correction.

Since uracil generated in the middle temporarily mismatches with the opposite DNA base, the intracellular base excision repair (BER) mechanism works.

At this time, a mutant repair enzyme (uracil-DNA glycosylase; UNG) removes uracil from DNA. to maximize substitution.

In addition, cytosine base-correcting gene scissors were produced to minimize removal of uracil by inducing a nick in the DNA strand opposite to the single strand where cytosine deamination occurred.

On the other hand, the adenine base editing gene scissors was developed in a form similar to the cytosine base editing gene scissors by the Liu group. That is, instead of linking cytidine deamination enzyme to Cas9, which has lost the double helix cleavage ability, an adenosine deamination enzyme was attached to replace adenine with guanine.

At that time, since an adenosine deamination enzyme acting on DNA was not found in nature, the researchers modified the adenosine deamination enzyme acting on RNA using a selective evolution method using bacteria and implemented it to work on single-stranded DNA. As a result, in the case of adenosine, deamination becomes inosine, which is later substituted with guanosine through DNA transcription or the like.

Since base-correcting gene scissors do not cause DNA double helix breaks, the problems caused by them (genetic structural change, mass deletion, DNA damage reaction caused by p53, etc.) can be basically avoided. In the meantime, it is reported that, unlike the gene editing method through the homozygous repair mechanism, the homozygous gene is not required as a template, and the efficiency is usually high, about 30-50%. In addition, it has the advantage that it can be applied to cells that hardly divide, so it has been used not only for the treatment of human diseases, but also for many research fields such as animals and plants and various biological species.

However, nucleotide-correction gene editing also has clear limitations. Because it basically works based on deamination enzymes, there is no function to add or remove bases, only substitutions are possible. Also, among the base substitutions, transversion (substitution with a base having a different number of rings) for changing cytosine to adenine is impossible. In addition, the problem of substituting cytosine or adenine in the vicinity in the target range other than the target base without distinction (bystander mutation) is also well known. In addition, in the case of adenine base-correcting gene scissors, there is a problem of substituting cytosine only for the TC motif in addition to the target adenine. This is presumed to be a problem acquired in the process of artificially improving the adenosine deamination enzyme.

In addition, since deamination enzymes naturally operate on DNA and RNA in cells, there is a possibility of inducing random mutations regardless of base-correcting gene editing, that is, even in the absence of guide RNA. It has been found that random cytosine substitutions occur throughout the genome by actual cytosine base editing gene editing. Also, it has been reported that cytosine or adenine nucleotides are randomly substituted for cytosine or adenine nucleotides in RNA transcripts, respectively.

Organisms from which cells that can be genome edited by the system of the present invention are derived include mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cattle and cats; poultry such as chickens, ducks and geese; Plants such as monocotyledonous and dicotyledonous plants include, but are not limited to, rice, corn, wheat, sorghum, barley, soybean, peanut and Arabidopsis thaliana.

2. DNA-guided endonuclease

In the present invention, when the genome editing tool is a DNA-guided endonuclease, the targeting endonuclease is Argonaute. The Argonaute polypeptide family is an essential component of the RNA-induced silencing complex (RISC) and plays a central role in the RNA silencing process. RISC is responsible for a phenomenon of gene silencing known as RNAi (RNA interference). Algonut polypeptides bind to different classes of small non-coding RNAs, including micro RNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs). Small RNAs guide algonut polypeptides through sequence complementarity (base pairing) to their specific target, resulting in mRNA cleavage or translational inhibition.

Argonaute polypeptides are well conserved in all living things. Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertion or deletion in Pasteurella multocida and E. coli with an efficiency of 80-100%. In addition, the effect is homologous arms dependent but in a DNA- and potential enzyme activity-independent manner. This effect was also observed with other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo).

The underlying mechanism of the NgAgo system is a positive selection process through a PIWI-like domain that interacts with recombinase A (recA) to enhance recA-mediated DNA strand exchange.

NgAgo is an abbreviation for Natronobacterium gregoryi Argonaute as a single-stranded DNA (ssDNA)-guided Argonaute endonuclease. NgAgo binds to the 5′ phosphorylated ssDNA of ~24 nucleotides (gDNA) and guides it to the target site and performs DNA double-strand repair at the gDNA site. Like the CRISPR/Cas system, NgAgo was reported to be suitable for genome editing, but it was not transcribed. Unlike Cas9, the NgAgo-gDNA system does not require a protospacer adjacent motif (PAM).

NgAgo has been proposed to be useful for genome editing by minimizing off-target effects due to the accuracy and efficiency of the system. The specificity of gDNA is very high because cleavage efficiency is compromised by single nucleotide mismatches between guide and target molecules. The use of 5' phosphorylated ssDNA as guide molecules reduces the likelihood of cellular oligonucleotides misleading NgAgo. Guide molecules can only be attached to NgAgo during protein expression. Once the guide is loaded, NgAgo cannot replace free floating ssDNA for gDNA. It is easier to design, synthesize and adjust the concentration of ssDNA compared to systems using sgRNA. The required amount of ssDNA is less than that of the sgRNA expression plasmid.

Argonaute polypeptides are DNA-guided endonucleases capable of editing nucleic acids in a cell or subject (eg, within the subject's genome) in a target-specific manner. An algonut polypeptide or functional fragment thereof retains the ability to edit a target nucleic acid sequence (eg, RNA, DNA, gene, promoter, etc.) within a cell (eg, a cell of a subject) in a target-specific manner. The ability to edit a target sequence in a subject's genome refers to the ability to insert, remove, and/or replace one or more specific nucleotides within a target sequence of a cell (eg, a human cell).

Algonut polypeptides or functional fragments thereof include polypeptides encoded by a portion of a nucleotide sequence, wherein the encoded polypeptide retains the ability to edit a target nucleic acid sequence in a cell when expressed in the cell.

The algonut nucleic acid or synthetic nucleic acid described herein comprises a nucleic acid sequence of 100 to 3000 nucleotides in length with 80% to 100% identity to the nucleic acid sequence of the algonut. A nucleic acid or synthetic nucleic acid described herein comprises a first nucleic acid sequence of at least 100, at least 500, at least 750, at least 1000, at least 1500, at least 1750 or at least 2000 nucleotides in length, wherein the first nucleic acid comprises SEQ ID NO: 1 at least 80%, at least 81%, at least 82%, at least 85%, at least 90% or at least 95% identity to the nucleic acid sequence of

Algonut nucleic acids are configured to express polypeptides in mammalian cells. A nucleic acid configured to express a polypeptide (eg, an algonut polypeptide) includes one or more nucleic acid regulatory sequences that direct expression of the polypeptide in a cell.

Thus, a nucleic acid configured to express a polypeptide of interest comprises a coding region encoding a protein of interest, one or more suitable promoters operably linked to the coding region, a translation initiation sequence, an initiation codon, a stop codon, a polyA signal sequence, a leader sequence , NLS, and the like. Nucleic acids include sequences encoding nuclear localization sequences (NLSs). Any suitable NLS sequence may be used. The nucleic acid is configured to express an algonut polypeptide or a functional fragment thereof.

Also, the nucleic acid is a guide oligonucleotide (oligonucleotide guide). The guide oligonucleotides comprise RNA or comprise DNA. Guide oligonucleotides are nucleic acids that are sometimes between 18 and 30 nt in length. Guide oligonucleotides are 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

Guide oligonucleotides often contain 80% to 100% of a target nucleic acid sequence (target site) or portion thereof located at a specific location within the genome of an organism or cell. 85% to 100%, 90% to 100%, 95% to 100% or 100% identical nucleic acid sequence.

The guide oligonucleotide comprises a target sequence that is 18 to 30, 18 to 28, 18 to 25, 18 to 26, 19 to 26, 19 to 25, 20 to 30, 20 to 25, 20 to 24 or 21 to 24 nucleotides in length; i.e. 100% identical to a part of a gene, intron or exon.

A target sequence refers to a specific location (a specific nucleic acid sequence) in the genome of an organism or cell to be modified using the composition or method described herein. A target sequence is a nucleic acid located within the genome of a cell or organism. The target sequence includes RNA. The target sequence includes DNA.

The target sequence may be 10 to 100, 18 to 50, 18 to 30, 18 to 28, 18 to 25, 18 to 26, 19 to 26, 19 to 25, 20 to 30, 20 to 25, 20 to 24 or 21 to 24. It is several nucleotides in length and can be located within a gene, exon, intron, or any suitable portion of the genome.

Any nucleotide within the target sequence or any portion of the target sequence may be modified by the methods described herein. Any number of nucleotides in the target sequence may be deleted, mutated, or substituted, such as by a sequence of interest (eg, an insertion sequence of a donor sequence). One or more nucleotides or sequences of interest are inserted into the target sequence by the methods described herein.

The target sequence provides a nucleic acid sequence that is complementary to or identical to a guide oligonucleotide, or a portion thereof.

The target sequence provides a nucleic acid sequence that is complementary or identical to the 5' and/or 3' flanking regions of the donor sequence. For example, a guide oligonucleotide of 18 to 28 nucleotides in length may be 85% to 100%, 90% to 100%, or 95% to 100% identical to the target sequence. Alternatively, the guide oligonucleotide comprises 1 to 2, 1 to 3, or 1, 2, 3 or 4 mismatches with respect to the target sequence in the genome. or the guide oligonucleotide is 100% identical to the target sequence.

A guide oligonucleotide comprises or consists of an exact match for a target site (ie, a target sequence) in the genome. Guide oligonucleotides are phosphorylated. Guide oligonucleotides are 5' phosphorylated (ie phosphorylated on the 5' hydroxyl terminus).

A functional algonut polypeptide uses a guide oligonucleotide to cleave the genomic DNA of an organism or cell at a specific target sequence defined by the sequence of the guide oligonucleotide. Algonut polypeptides cleave the target nucleic acid sequence anywhere within the sequence defined by the guide oligonucleotide.

Algonut polypeptides cleave the target site at a position defined by any of the first 10 nucleotides (5' nucleotides) of the guide oligonucleotide.

When both a guide oligonucleotide and a donor nucleic acid are present, the algonut polypeptide will proceed to insert and slice the donor nucleic acid into the genome of the cell at the site of the target sequence defined by the guide oligonucleotide.

In the absence of a donor sequence, an algonut polypeptide loaded with a guide oligonucleotide will often cleave the target site defined by the guide oligonucleotide sequence. Such processes often result in the introduction of one or more single nucleotide mutations introduced at the target site.

A donor sequence (donor fragment) is a nucleic acid comprising three portions: a 5' flanking sequence, a desired sequence and a 3' flanking sequence. Donor sequences include RNA. Donor sequences include DNA. The donor sequence is single-stranded. In some embodiments, the donor sequence is double stranded.

The 5' flanking sequence and the 3' flanking sequence may be the same sequence or different sequences. The 5' flanking sequence and the 3' flanking sequence are different sequences that do not share more than 10% identity. The 5' flanking sequence and the 3' flanking sequence are located on opposite sides of the desired sequence. The 5' flanking sequence and/or the 3' flanking sequence of the donor sequence is about 10-100, 10-50, 10-75, 10-25, or 20-25 nucleotides (nt) or base pairs (bp) in length .

A desired sequence refers to an algonut polypeptide and/or a nucleic acid to be inserted into a target sequence by the method described in the present invention. The term “sequence of interest” is used synonymously with the terms “nucleic acid of interest” and “nucleic acid sequence of interest”. For clarity, a “sequence of interest” may sometimes be referred to as an “insertion sequence”. A sequence of interest includes RNA. A sequence of interest includes DNA.

The sequence of interest may be any suitable sequence of any suitable length. The sequence of interest is 1 to 20,000, 1 to 5,000, 1 to 1000, 1 to 500, 10 to 5,000, 10 to 1000 or 10 to 500 nucleotides in length.

each donor sequence independently comprises a 5' flanking sequence and a 3' flanking sequence that are 80% to 100% identical to the target site, the flanking sequence also comprising a region of sequence identical to or complementary to a portion of the guide oligonucleotide do. The donor sequence comprises a sequence that is 80% to 100% identical to the guide oligonucleotide and/or 80% to 100% identical to the target site.

The donor sequence may comprise a flanking sequence that is identical to the guide oligonucleotide or overlaps a portion of the guide oligonucleotide. The donor sequence comprises (i) a sequence of interest, (ii) a 5' flanking sequence that is at least 80% to 100% identical to the guide oligonucleotide or portion thereof and/or 80% to 100% identical to the target site, and a guide oligo a 3' flanking sequence that is at least 80%-100% identical to the nucleotide or portion thereof and/or is 80%-100% identical to the target site.

The guide oligonucleotide and functional algonut polypeptide are sufficient to insert the desired sequence into a target site in the genome, wherein the donor nucleic acid (i.e., donor sequence, donor nucleic acid sequence) is a portion of the guide oligonucleotide (e.g., at least 5 to 10 canine nucleotides) and flanked on both sides (5' and 3') by the same sequence.

Two guide oligonucleotides are used to insert the desired sequence into a target site of the genome, wherein the donor nucleic acid (donor sequence) is a sequence of a first guide oligonucleotide on one side and a sequence of a second guide oligonucleotide on the other side. flanking sequences of interest.

Accordingly, provided herein is a method for editing the genome of an organism or cell. An organism is an object. The subject is a human. The cell can be any suitable cell, non-limiting examples of which include prokaryotic cells, plant cells, eukaryotic cells, mammalian cells or human cells. Methods of editing a genome include removal of a target sequence from the genome, disruption of the target sequence in the genome, and/or insertion of a desired sequence into the genome. The sequence of interest can be any suitable nucleic acid sequence, non-limiting examples of which include heterologous nucleic acids (eg, from different species), modified heterologous nucleic acids, homologous nucleic acids (eg, from the same species), synthetic nucleic acids, genes or portions thereof (eg, introns, exons, regulatory sequences, etc.), modified genes, markers, toxins, single nucleic acids, two or more nucleic acids, etc. or combinations thereof. The nucleic acid of interest encodes a chimeric antigen receptor (CAR).

The desired nucleic acid (sequence of interest) or gene may be any suitable mammalian gene, a portion thereof, or a modified form thereof.

A method of editing the genome of an organism or cell comprises contacting one or more cells with one or more nucleic acids or compositions described herein. A method of editing the genome of an organism or cell is a method of modifying a target sequence in the genome of a cell, organism or subject. A method of editing the genome of an organism or cell comprises introducing one or more nucleic acids described herein into one or more cells. The one or more nucleic acids may be introduced into one or more cells by any suitable method.

3. Prime editing

When the genome editing tool in the present invention is Prime editing, Cas9 nuclease has a mechanism of DNA-RNA hybridization between the guide RNA and the target DNA when it binds to the target DNA. Focusing on this part, a new single-stranded DNA can be produced using the guide RNA as a template using reverse transcriptase.

Since the target DNA is in the form of a double helix, when one strand binds to the guide RNA, the other single strand remains unbound and alone. If a specific template RNA additionally undergoes DNA-RNA hybridization with the remaining single strand, the reverse transcriptase can use the RNA as a template to instantly synthesize a desired DNA sequence. Prime editing guide RNA (pegRNA) can be prepared by linking the template RNA to the 3*?**?* end of the existing guide RNA.

By linking the reverse transcriptase to Cas9 nickase, which can cut only single strands, the prime editing gene editing technology was completed.

Prime editing includes three main components:

(a) a prime editing guide RNA (pegRNA) capable of identifying a target nucleotide sequence to be edited and encoding new genetic information replacing the targeted sequence: pegRNA contains a primer binding site (PBS) and a reverse transcriptase (RT) template sequence It consists of an extended single guide RNA (sgRNA) containing During genome editing, the primer binding site allows the 3' end of the nick DNA strand to hybridize to the pegRNA, whereas the RT template serves as a template for the synthesis of the edited genetic information.

(b) a fusion protein consisting of Cas9 H840A nickase fused to Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase: Cas9 H840A nickase is a Cas9 enzyme that can cleave a DNA sequence with two nuclease domains , contains a RuvC domain that cleaves the non-target strand and an HNH domain that cleaves the target strand. Introduction of the H840A substitution in Cas9 in which the histidine at amino acid position 840 is replaced with alanine inactivates the HNH domain. With only the RuvC functional domain, a catalytically damaged Cas9 can introduce a single strand nick. M-MLV reverse transcriptase is an enzyme that synthesizes DNA from a single-stranded RNA template.

(c) A single guide RNA (sgRNA) directing the Cas9 H840A nickase portion of the fusion protein to nick to the unedited DNA strand.

After the prime editing protein and pegRNA complex bind to the target DNA, the opposite single strand is first cleaved by Cas9 nickase. The cleaved DNA strand can be combined with the complementary portion of the 3*?**?* end of the pegRNA, and when this portion is combined, the reverse transcriptase uses the pegRNA as a template (RT template portion) to create a new DNA strand. Afterwards, the target DNA is restored to a double-stranded form by the cell's repair mechanism.

The first developed prime editing (PE1 version) was confirmed to work, but the efficiency was very low. To overcome this, an improved version was created by artificially improving the reverse transcriptase part in consideration of thermodynamic stability, binding to DNA-RNA structure, and inhibition of RNA-degrading enzymes (PE2 version). However, there was a limit to increasing the efficiency just by improving the prime editing self like this.

This is because, after the prime editing operation, the DNA strand directly attached to the pegRNA is intact during the DNA repair process, so there is a much higher possibility of repair based on it. That is, it is more likely to be restored based on the original DNA strand rather than the newly created DNA strand by reverse transcriptase, and as a result, the original sequence is restored. If pegRNA creates an additional nick in the directly attached strand, the efficiency of the prime editing operation can be maximized. A prime editing version (PE3 version) that additionally introduced sgRNA to create another DNA break at a location a little far from the editing site was prepared. As a result, as expected, by further cutting the opposite strand, the efficiency of the prime editing correction was further increased.

Genome editing is performed by introducing pegRNA and fusion proteins into cells. Transduction can often be accomplished by introducing a vector into a cell. Once internalized, the fusion protein cleaves the target DNA sequence and exposes a 3' hydroxyl group that can be used to initiate reverse transcription (prime) of the RT template portion of the pegRNA. The result is a branched intermediate comprising two DNA flaps, a 3' flap containing the newly synthesized (edited) sequence and a 5' flap containing a dispensable, unedited DNA sequence. The 5' flap is then cleaved with a structure-specific endonuclease or 5' exonuclease. This process allows for 3' flap ligation and produces DNA consisting of one edited strand and one unedited strand. The reannealed double-stranded DNA contains a nucleotide mismatch at the location where the edit occurred. To correct the discrepancy, the cell utilizes an intrinsic discrepancy repair mechanism with two possible outcomes. (i) information from the edited strand is copied to the complementary strand, making the edit permanent; (ii) the original nucleotide is re-integrated into the edited strand with the exception of the edit.

Prime-editing gene editing technology has a great advantage in that it does not cause double helix breaks, like base-editing gene scissors. And in that it can induce insertions or deletions as well as all kinds of base substitutions, it can be said that it is more scalable than the existing base-correcting gene scissors.

However, the process of designing the prime editing guide RNA has not been optimized yet, so in order to produce an optimal RNA, the length of the PBS (primer binding site) that the RNA can bind to the cut strand and the RT including the part to be corrected You should try various lengths of the template (template of reverse transcriptase). And in the case of base substitution, the efficiency is somewhat lower than that of the existing base-editing gene scissors, and when the final version (PE3) is used, some unwanted DNA deletions or insertions occur, etc., should be further improved in the future.

During the development of this technology, we made some modifications to the components to make them more effective.

(a) Prime Editor 1 (PE1) ; In the first system, wild-type Moloney leukemia virus (M-MLV) reverse transcriptase was fused to the C-terminus of the Cas9 H840A nickase. A detectable editing efficiency was observed.

(b) Prime Editor 2 (PE2); To improve DNA-RNA affinity, enzymatic processability and thermal stability, five amino acid substitutions were included in M-MLV reverse transcriptase. Mutant M-MLV RT was then incorporated into PE1 to give rise to (Cas9(H840A)-M-MLV RT(D200N/L603W/T330P/T306K/W313F)). An improvement in efficiency compared to PE1 was observed.

(c) Prime Editor 3 (PE3): Despite the increased efficacy, edits inserted by PE2 can still be eliminated due to DNA mismatch repair of the edited strands. To avoid this problem during DNA heteroduplex digestion, an additional single guide RNA (sgRNA) can be introduced. This sgRNA was originally designed to match the edited sequence introduced by the pegRNA and not the allele. It directs the Cas9 nickase portion of the fusion protein to nick the unedited strand at a nearby position opposite to the original nick. Selecting the unedited strand allows the cell's natural repair system to copy information from the edited strand to the complementary strand, permanently fixing the edit.

Prime editing tools offer many advantages over traditional gene editing techniques. CRISPR/Cas9 edits use Non-homoousous End Joining (NHEJ) or homology-directed repair (HDR) to fix DNA breaks, whereas the prime editing system uses DNA mismatch repair.

DNA repair mechanisms such as NHEJ and HDR produce unwanted random insertion or deletion (INDEL) byproducts in cells performing correct editing.

The Prime system introduces single-stranded DNA breaks instead of the double-stranded DNA breaks observed in other editing tools such as base editors and base editors. Base editing and prime editing Base editing and prime editing collectively provide complementary strengths and weaknesses to create targeted transition mutations. When the desired edits are transition point mutations targeted transition mutations and the PAM sequence is approximately 15 bases from the target site, the base editor provides higher editing efficiency and fewer INDEL by-products. However, since prime editing techniques require precisely positioned PAM sequences to target nucleotide sequences, they provide more flexibility and editing precision. Surprisingly, the Prime Editor allows you to insert all types of substitutions, transitions, and transformations into the target sequence.

The prime system has a total of three independent DNA bonds: (i) between the guide sequence and the target DNA, (ii) between the primer binding site and the target DNA, (iii) the 3' end of the nicked DNA strand and pegRNA, so it is better than CRISPR / Cas9. It has been suggested that there are few undesirable off-target effects.

There is considerable interest in applying gene editing methods to the treatment of diseases using genetic components. However, there are several problems with this method. Effective treatment requires editing of large numbers of target cells, which requires effective delivery methods and tissue specificity.

Currently, while prime editing looks promising for a small number of genetic modifications, more studies are needed to evaluate whether techniques for making larger changes, such as targeted insertions and deletions, are efficient. Larger genetic alterations require longer RT templates, which may interfere with efficient delivery of pegRNAs to target cells. In addition, pegRNAs containing long RT templates may be susceptible to damage caused by cellular enzymes.

Although further research is needed to improve the efficiency of prime editing, this technique offers a promising scientific improvement over other gene editing tools. Prime editing technology has the potential to correct the majority of pathogenic alleles that cause genetic diseases because it can repair insertions, deletions and nucleotide substitutions.

Much research is needed to use prime editing to correct pathogenic alleles in human disease.

The base editor used for prime editing must deliver proteins and RNA molecules into living cells. Introduction of exogenous gene editing techniques into living organisms is a major challenge. One possible way to introduce a base editor in animals and plants is to wrap the base editor in a viral capsid.

The target organism can then be transduced by the virus to synthesize the base editor in vivo. As common laboratory vectors of transduction, such as lentiviruses, elicit an immune response in humans, proposed human therapies revolve around adeno-associated viruses (AAVs) because AAV infections are often asymptomatic. Unfortunately, the effective packaging capacity of AAV vectors is small, about 4.4 kb not including inverse terminal repeats. The SpCas9-reverse transcriptase fusion protein is 6.3 kb and does not account for the elongated guide RNA required to target and prime the site of interest.

Ⅴ. The versatility of the system of the present invention

Another advantage of the system of the present invention includes the presence of a DNA expression cassette encoding a DdRp RNA expression cassette, an auto-DdRp expression cassette and a genome editing tool that provides independence from nuclear transcription and unprecedented design freedom. Considering the multi-purpose factors that can be combined with each other, the present invention can optimize DdRp expression for a desired level of DdRp synthesis, a desired target organism, a desired level of genome editing tool production, etc. in a DdRp RNA expression cassette and an auto-DdRp expression cassette. let there be Expression cassettes that can be trans-transcribed by DdRp can be designed independently.

The present invention relates to an RNA expression cassette comprising a DdRp RNA expression cassette providing the original DdRp and a DNA expression comprising an auto-DdRp expression cassette and a DNA expression cassette encoding a genome editing tool as long as the expression cassette contains a promoter for DdRp It is a genome editing RNA/DNA system composed of cassettes and has various functions depending on the type of guide RNA among genome editing tools.

For example, the following elements may be individually selected, designed and/or adapted based on the present invention. capping of RNA expression cassettes (especially selection of specific caps); chemically modified nucleotides in the RNA expression cassette; 5'UTR of the RNA expression cassette; the coding sequence of the ORF encoding DdRp (codon-optimized); 3'UTR of the RNA expression cassette; poly(A) tail of the RNA expression cassette; 5'UTR of the DNA expression cassette; the promoter sequence of the DNA expression cassette; 5'UTR of the transcript generated from the DNA expression cassette; the coding sequence of the ORF encoding DdRp and genome editing tools (codon-optimized); 3'UTR of the transcript generated from the DNA expression cassette; poly(A) tails of the DNA expression cassette and/or transcripts generated from the expression cassette.

In addition, the present invention can co-transfect an optimal amount of a genome editing tool DNA expression cassette, auto-DdRp expression cassette and DdRp RNA expression cassette for any given cell type that is quiescent or cell cycle in vitro or in vivo. have.

DdRp RNA Expression Cassette for Expression of DdRp

The present invention provides an RNA expression cassette (DdRp RNA expression cassette) for expressing DdRp comprising a 5' cap for inducing translation of DdRp. In the present invention, the DdRp RNA expression cassette of the present invention may be provided independently of the DNA expression cassette of the present invention as described herein. A DdRp RNA expression cassette may be independently characterized by any one or more properties of a DdRp RNA expression cassette of the invention. The RNA expression cassette for expressing DdRp is an isolated nucleic acid molecule. This includes embodiments that are isolated, ie, essentially free, from other nucleic acid molecules.

The RNA expression cassette according to the invention is suitable for combination with a suitable DNA expression cassette, for example in the form of a system or kit.

Any DdRp RNA expression cassette according to the present invention, which may or may not be part of the system of the present invention, may be obtained by in vitro transcription. The in vitro transcribed DdRp RNA expression cassette (IVT-RNA) is of particular interest in the present invention. IVT-RNA can be obtained by transcription from a nucleic acid molecule (especially a DNA molecule). The auto_DdRp expression cassette(s) of the present invention are suitable for this purpose, especially if they contain a promoter that can be recognized by DdRp.

The RNA according to the invention can be synthesized in vitro. This may allow the addition of cap-analogs to in vitro transcription reactions. Typically, the poly(A) tail is encoded by the poly-(dT) sequence on the auto_DdRp expression cassette template. Alternatively, capping and poly(A) tail addition can be accomplished enzymatically after transcription.

In vitro transcription methodologies are known to those skilled in the art. A variety of in vitro transcription kits are commercially available.

DNA expression cassette

The present invention provides a DNA expression cassette comprising a nucleic acid sequence encoding an RNA expression cassette (DdRp RNA expression cassette) for expressing DdRp according to the present invention, an RNA expression cassette according to the present invention, or both.

The DNA expression cassette according to the present invention encodes the genome editing tool of the system according to the present invention. The genome editing tool DNA (DNAge expression cassette is composed of a DNAen expression cassette encoding a targeting endonuclease and a DNAsg expression cassette encoding a guide nucleic acid and sgRNA, which may be in one expression cassette or an independent expression cassette, and the present invention provides such a DNA expression cassette.

The DNA expression cassette according to the invention encodes the DdRp RNA expression cassette of the system according to the invention. The auto_DdRp expression cassette encodes one RNA element (DdRp RNA expression cassette) of the system according to the invention.

Preferably, the DNA expression cassette according to the present invention is a plasmid. As used herein, the term “plasmid” generally relates to an expression cassette of extrachromosomal genetic material, usually a circular DNA duplex, capable of transcription independently of chromosomal DNA.

The auto_DdRp expression cassette of the present invention may include a promoter that can be recognized by DdRp. This allows for transcription of the encoded RNA, for example the RNA of the present invention, in vivo or in vitro. IVT vectors can use a standardized manner as a template for in vitro transcription. Examples of preferred promoters according to the invention are the promoters for SP6, T3 or T7 polymerase.

The auto_DdRp expression cassette of the present invention is an isolated nucleic acid molecule.

The present invention also provides a kit comprising a genome editing RNA/DNA system according to the present invention or an RNA expression cassette for expressing DdRp according to the present invention.

The components of the kit exist as separate entities. For example, one nucleic acid molecule in the kit may be in one entity, and the other nucleic acid molecule in the kit may be in a separate entity. For example, open containers or closed containers are suitable entities. A closed container is preferred. The container used should preferably be RNase-free or essentially RNase-free.

The kit of the present invention comprises a genome editing RNA/DNA system for inoculation of cells and/or administration to a human or animal subject.

The kit according to the invention optionally comprises a label or other form of information element, for example an electronic data carrier medium. The label or information element preferably contains instructions for use, for example printed written instructions or optionally in printable electronic form. Instructions for use may refer to at least one suitable and possible use of the kit.

Ⅵ. Expression cassettes and transporters

The self-transcription RNA/DNA system of the present invention includes the contents of the novel expression cassette expressed by DdRp, and in more detail, the developed linear DdRp RNA expression cassette, auto_DdRp expression cassette, DdRp expression system expressed by auto-loop , a DNAge expression cassette encoding a genome editing tool transcribed by DdRp, an auto_(DdRp+DNAge) expression cassette, an antibiotic resistance gene, an origin of replication and a recombinant expression cassette comprising the cloning site sequence and the expression It relates to a cell transformed with the cassette, and the like.

Restriction endonuclease is present in almost all bacteria and is an enzyme that recognizes a specific nucleotide sequence present in DNA and cuts the recognized sequence site or a specific site near it. These restriction enzymes are originally possessed by bacteria as a defense mechanism to remove foreign DNA such as viruses that invade their cells. are doing By using the properties of these restriction enzymes, a desired gene can be inserted into the vector.

In particular, there is a multicloning site (MCS) in the expression cassette, which artificially collects the recognition sites of various restriction enzymes and determines the restriction enzyme of the insert DNA for cloning. Plasmid vectors are commercially available by constructing MCS mainly from restriction enzymes commonly used. In MCS using such general-purpose restriction enzymes, there are genes in which it is difficult or impossible to clone all genes due to their high frequency.

The recombinant expression cassette of the present invention is a cassette capable of expressing a target protein or target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert. The expression cassette includes an expression control sequence and, if necessary, a signal sequence or leader sequence for membrane targeting or secretion, and may be prepared in various ways depending on the purpose. The expression control sequence (expression control sequence) refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include promoters to effect transcription, optional operator sequences to control transcription, 5'Cap structures, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. In addition, the expression cassette may include a selection marker for selecting a host cell containing the cassette, and in the case of a replicable expression cassette, it may include an origin of replication.

The recombinant expression cassette of the present invention may include a nucleotide sequence encoding a variety of known proteins, and the nucleotide sequence encoding the target protein is operably linked to a promoter. To be operably linked means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.

The expression cassette according to the present invention can be prepared by inserting the MCS polynucleotide of the present invention as a basic backbone to an existing cassette used for protein expression so as to be operatively linked by a promoter to which a DdRp selected for a structural gene binds. have. Preferably, the expression cassette of the present invention may include an origin of replication, a promoter, a fluorescent protein gene, MCS, and a selection marker, and may further include a polyA signal sequence for protein expression.

In the present invention, the selectable marker (selective marker) may be an antibiotic resistance gene, and many genes that can be such an antibiotic resistance gene are currently known (eg, kanamycin, neomycin, ampicillin, tetracycline, etc.) . Accordingly, those skilled in the art can select and use desired genes from the known antibiotic resistance genes, and the specific types of these genes do not constitute the essence of the present invention. In addition, since the introduction of the antibiotic resistance gene is for selecting transformants, any gene that can be used for selection may be introduced. Therefore, it is not limited only to the antibiotic resistance gene. Preferably, the antibiotic resistance gene of the present invention may be a kanamycin or neomycin resistance gene.

The expression cassette of the present invention includes expression cassettes in which the nucleotide sequence is slightly changed according to methods known to those skilled in the art, such as codon degeneracy, as long as the basic structure of the expression cassette of the present invention is the same or similar. In addition, it is apparent to those skilled in the art that the N-terminus and the C-terminus of the sequence listing form a circle.

On the other hand, the present invention provides a cell transformed with the recombinant cassette of the present invention.

The recombinant expression cassette of the present invention can be introduced into a host using methods known in the art. Examples include, but are not limited to, calcium chloride and heat shock methods, particle gun bombardment, silicon carbide whiskers, sonication, electroporaion, PEG- Medium fusion method, microinjection method, microinjection method, liposome-mediated method, vacuum infiltration method, etc. can be used.

The host cell may be cells of animals, plants, yeast and bacteria, preferably, in the case of other than bacteria, it can well accept a vector introduced from the outside, and the boundary between intracellular organelles such as the cytoplasm and the nucleus is clearly distinguished. Cells are preferred. More preferably, CHO-k1 (ATCC CCL-61, Cricetulus griseus, hamster, Chinese), HEK293 (ATCC CRL-1573, Homo sapiens, human), HeLa (ATCC CCL-2, Homo sapiens, human), SH-SY5Y (ATCC CRL-2266, Homo sapiens, human), Swiss 3T3 (ATCC CCL-92, Mus musculus, mouse), 3T3-L1 (ATCC CL-173, Mus musculus, mouse), NIH/3T3 (ATCC CRL-1658, Mus musculus, mouse), L-929 (ATCC CCL-1, Mus musculus, mouse), Rat2 (ATCC CRL-1764, Rattus norvegicus, rat), RBL-2H3 (ATCC CRL-2256, Rattus norvegicus, rat), MDCK (ATCC CCL-34, Canis familiaris). In addition, it may be various other stem cells, cells extracted from various tissues, and artificially made similar cell membrane structures.

Meanwhile, standard recombinant DNA and molecular cloning techniques used in the present invention are well known in the art, and are described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual) , 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984) and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience (1987)).

The present invention provides an improved expression cassette comprising the above-described nuclear-independent gene expression control sequence by DdRp in the cytoplasm.

In the present invention, the expression cassette of the present invention is a plasmid vector such as E. coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid and Ti plasmid, cosmid vector, adenovirus (Lockett LJ, et al., Clin. Cancer Res. 3:2075-2080 (1997)), Adeno-associated viruses (AAV) (Lashford LS., et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager (1999)), Retro Virus (Gunzburg WH, et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, (1999)), Lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55- 62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92:1411-1415 (1995)), vaccinia virus (Puhlmann M. et al., Human Gene) Therapy 10:649-657 (1999)), reovirus, poxvirus (GCE, NJL, Krupa M, Esteban M., The poxvirus vectors MVA and NYVAC as gene delivery systems for vaccination against infectious diseases and cancer Curr Gene Ther 8 ( 2):97-120(2008)), Semliki Forester Virus, Polymer (Hwang et al., In vitro and In vivo Transfection Efficiency of Human Osteoprotegerin Gene using Non-Viral Polymer Carriers., Korean J. Bone Metab. 13 (2):119-128 (2006)), liposomes (Methods in Molecular Biology, Vol 199, S.C. Basu and M. Basu (Eds.), Human Press 2002), can be applied to nanomaterials or niosomes.

1. Plasmids

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 characteristics exhibited by the host bacteria. For example, the ability to survive at normal toxic concentrations of antibiotics is due to the presence of plasmids with 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 an origin of replication (a site recognizable by the replication enzyme of the host cell, from which replication begins), and thus the main bacterial chromosome ( It can replicate in 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) are able to maintain their conformation stably during numerous cell divisions. However, at some stage, it exists as an independent element by excision (the reverse process of the process of being integrated).

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

The size of the plasmid ranges from 1.0 kb for the smallest to more than 250 kb for the largest. Copy number refers to the number of plasmids normally found in one bacterial cell. The factors controlling the copy number are not well understood, but each plasmid has a characteristic number of 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 exists in yeast as a 2 μm circle. The discovery of the 2 μm plasmid was very fortunate as it made possible the preparation of vectors for cloning the genes of industrial organisms of great importance as hosts. However, no plasmid has been found in other eukaryotes.

2. Adenovirus

Adenovirus has been widely used as a gene delivery vector because of its medium genome size, ease of manipulation, high titer, wide range of target cells, and excellent infectivity. Both ends of the genome contain 100-200 bp inverted terminal repeat (ITR), which is an essential cis element for DNA replication and packaging. The E1 regions of the genome (E1A and E1B) encode proteins that regulate transcription and transcription of host cell genes. The E2 region (E2A and E2B) encodes proteins involved in viral DNA replication. Among the currently developed adenovirus vectors, replication-deficient adenoviruses lacking the E1 region are widely used. On the other hand, the E3 region is removed from a conventional adenoviral vector to provide a site for insertion of a foreign gene (Thimmappaya, B. et al., Cell, 31:543-551 (1982); and Riordan, JR et al., Science, 245:1066-1073 (1989)). Therefore, the gene expression control sequence of the present invention can be inserted into the promoter sequence position of the E1A gene to regulate the expression of the E1A gene. In addition, the gene expression control sequence-expression gene cassette of the present invention is preferably inserted into the deleted E1 region (E1A region and/or E1B region, preferably E1B region) or E3 region, more preferably the deleted E1 region inserted into the area. In addition, the gene expression control sequence-expression gene cassette of the present invention can be inserted into the deleted E4 region. As used herein, the term "deletion" in relation to a viral genome sequence has a meaning including a partial deletion as well as a complete deletion of the corresponding sequence.

In addition, since adenovirus can pack up to about 105% of the wild-type genome, it can additionally package about 2 kb (Ghosh-Choudhury et al., EMBO J., 6:1733-1739 (1987)). Accordingly, the above-described foreign sequence inserted into the adenovirus may additionally bind to the genome of the adenovirus.

The foreign gene carried by the adenovirus is replicated in the same manner as the episome, and thus has very low genetic toxicity to the host cell. Therefore, it is judged that gene therapy using the adenovirus gene delivery system of the present invention is very safe.

3. Retrovirus

Retroviruses are widely used as gene delivery vectors because they insert their genes into the host genome, can transport a large amount of foreign genetic material, and have a wide spectrum of cells that can infect.

In order to construct a retroviral vector, the target nucleotide sequence to be transported is inserted into the retrovirus genome instead of the retrovirus sequence to produce a replication-incompetent virus. To produce the virion, a packaging cell line containing the gag, pol and env genes but lacking the long terminal repeat (LTR) and Ψ sequences was constructed. When a recombinant plasmid containing the target nucleotide sequence to be transported, LTR and Ψ sequence is introduced into the cell line, the Ψ sequence enables production of an RNA transcript of the recombinant plasmid, which is packaged into a virus, and the virus is discharged into the medium. The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.

Gene transfer using a second-generation retroviral vector has been published. A mutant of Moloney murine leukemia virus was prepared, and an EPO (erythropoietin) sequence was inserted into the envelope region to produce a chimeric protein having novel binding properties. The gene delivery system of the present invention can also be prepared according to the construction strategy of the second-generation retroviral vector.

4. AAV Vectors

Adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it can infect non-dividing cells and has the ability to infect various types of cells. Detailed descriptions of the preparation and use of AAV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368. A study of AAV as a gene delivery system is described in LaFace et al, Viology, 162:483486 (1988), Zhou et al., Exp. Hematol. (NY), 21:928-933 (1993), Walsh et al, J. Clin. Invest., 94:1440-1448 (1994) and Flotte et al., Gene Therapy, 2:29-37 (1995). Recently, AAV vectors are being conducted clinically I as a therapeutic agent for cystic fibrosis.

Typically, the AAV virus is a plasmid (McLaughlin et al., J. Virol., 62:1963) containing the target gene sequence (relaxin gene and the target nucleotide sequence to be transported) flanked by two AAV terminal repeats. 1973 (1988); and Samulski et al., J. Virol., 63:3822-3828 (1989)) and an expression plasmid (McCarty et al., J. Virol. , 65:2936-2945 (1991)).

5. Other Virus Vectors

Other viral vectors can also be used in the gene delivery system of the present invention. Vectors derived from basinia virus, lentivirus or herpes simplex virus, reovirus, poxvirus, and Semliki forester virus can also be used as a delivery system capable of delivering a target nucleotide sequence into a cell.

6. Polymer

As non-viral gene delivery systems, polymer-based delivery systems widely used include gelatin, chitosan, poly-LLysine (PLL) and polyethyleneamine (PEI). The advantages of polymer-based gene carriers are that the expression rate of immune response and acute toxicity is low, the preparation method is simple, and mass production is possible.

7. Liposomes and niosomes

Liposomes are automatically formed by phospholipids dispersed in an aqueous phase. Examples of successful intracellular delivery of foreign DNA molecules as liposomes are described by Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982) and Nicolau et al., Methods Enzymol., 149:157-176 (1987). Meanwhile, Lipofectamine (Gibco BRL) is the most widely used reagent for transfection of animal cells using liposomes. Liposomes containing the target nucleotide sequence to be transported interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to deliver the target nucleotide sequence to be transported into the cell.

While liposomes are made using phospholipids, niosomes are used and polar and non-polar substances can be encapsulated with a double membrane transporter composed of a mixture of nonionic surfactant and cholesterol. , osmotically active, more stable than liposomes, which are carriers of lipid particles by phospholipids, etc., and also have the advantage that the surfactant used in the preparation does not require special conditions for storage and handling.

8. Nanomaterials

As used herein, the term nanomaterial refers to a biocompatible polymer material having a size of several to hundreds of nanometers, for example, polyethylene glycol (polyetheleneglycol, PEG), polylactide (PLA), polyglycol Lead (polyglycolide, PGA), polylactide-co-glycolide (PLGA), polycaprolactone (poly-ε-carprolactone, PCL), hyaluronic acid (HA), chitosan (chitosan) ), or serum albumin.

Meanwhile, the method of introducing the gene delivery system of the present invention into a cell as described above can be carried out through various methods known in the art.

In the present invention, when the gene delivery system is constructed based on a viral vector, it is carried out according to a viral infection method known in the art. Infection of host cells with viral vectors is described in the references cited above.

When the gene delivery system in the present invention is a naked recombinant DNA molecule or plasmid, the microtransformation method (Capecchi, MR, Cell, 22:479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-1099 (1985)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), Electroporation (Neumann, E. et al., EMBO J., 1:841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6:716-718 (1986)), liposome-mediated Transfection (Wong, TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); and Nicolau et al., Methods Enzymol., 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci) ., 87:9568-9572 (1990)), the gene can be transfected into cells.

VII. pharmaceutical composition

The genome editing RNA/DNA systems described herein may be in the form of a pharmaceutical composition. The pharmaceutical composition according to the present invention may comprise one or more nucleic acid molecules according to the present invention, preferably a genome editing RNA/DNA system.

A pharmaceutical composition according to the present invention comprises a pharmaceutically acceptable diluent and/or a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable carrier. The choice of a pharmaceutically acceptable carrier, carrier, excipient or diluent is not particularly limited. Any suitable pharmaceutically acceptable carrier, vehicle, excipient or diluent known in the art may be used.

In the present invention, the pharmaceutical composition may further comprise a solvent such as an aqueous solvent or any solvent that makes it possible to preserve the integrity of the RNA. In one preferred embodiment, the pharmaceutical composition is an aqueous solution comprising RNA.

The aqueous solution may optionally contain a solute, for example, physiological saline.

In the present invention, the pharmaceutical composition is in the form of a freeze-dried composition. A freeze-dried composition can be obtained by freeze-drying each aqueous composition.

The pharmaceutical composition comprises at least one cationic entity. In general, cationic lipids, cationic polymers, and other substances having a positive charge can form complexes with negatively charged nucleic acids. It is possible to stabilize the RNA according to the invention by complexation with a cationic compound, preferably a polycationic compound such as a cationic or polycationic peptide or protein. In one embodiment, the pharmaceutical composition according to the present invention comprises at least one cationic molecule selected from the group consisting of protamine, polyethylene imine, poly-L-lysine, poly-L-arginine, histones or cationic lipids.

According to the present invention, cationic lipids are cationic amphiphilic molecules, for example molecules comprising one or more hydrophilic and lipophilic moieties. Cationic lipids may be monocationic or polycationic. Cationic lipids typically have lipophilic moieties such as sterol, acyl or diacyl chains and have an overall net positive charge. The head group of a lipid is usually positively charged. The cationic lipid preferably has a positive charge of 1 to 10 valences, more preferably 1 to 3 valences, and even more preferably monovalent positive charges. Examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); dimethyldioctadecylammonium (DDAB); 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-Diacyloxy-3-dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propane; Dioctadecyldimethyl ammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethyl ammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(s) Fermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA). Cationic lipids also include lipids having tertiary amine groups, including 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA).

Cationic lipids are suitable for formulating RNA in lipid formulations such as liposomes, emulsions and lipoplexes, as described herein. Typically the positive charge is contributed by the at least one cationic lipid and the negative charge is contributed by the RNA. In one embodiment, the pharmaceutical composition comprises at least one helper lipid in addition to the cationic lipid. Helper lipids may be neutral or anionic lipids. A helper lipid may be a natural lipid, such as a phospholipid, or an analog of a natural lipid, or a fully synthetic lipid that does not resemble a natural lipid, or a lipid-like molecule. When the pharmaceutical composition includes both a cationic lipid and a helper lipid, the molar ratio of the cationic lipid to the neutral lipid may be appropriately determined from the viewpoint of stability of the formulation and the like.

The pharmaceutical composition according to the present invention comprises protamine. According to the present invention, protamine is useful as a cationic carrier agent. The term “protamine” refers to a variety of relatively low molecular weight strongly basic proteins that are rich in arginine and have been found to be particularly related to DNA instead of somatic histones in sperm cells of animals such as fish. In particular, the term "protamine" refers to a protein found in fish sperm, which is strongly basic, soluble in water, not thermally coagulated, and comprising a number of arginine monomers. According to the present invention, the term "protamine" as used herein includes any protamine amino acid sequence obtained or derived from a natural or biological source, including multimeric forms of said amino acid sequence or fragments thereof and fragments thereof. means that The term also includes (synthetic) polypeptides that are artificial and specially designed for a particular purpose and cannot be isolated from natural or biological sources.

The compositions of the present invention may include one or more adjuvants.

The pharmaceutical composition according to the present invention may be buffered (eg buffered with acetate buffer, citrate buffer, succinate buffer, Tris buffer, phosphate buffer).

Genome Editing RNA/DNA System-Containing Particles

The genome editing RNA/DNA system of the present invention is composed of RNA and DNA, wherein the former is a single-stranded RNA fragment and the latter is a circular plasmid. Because of the instability of unprotected RNA, it is advantageous to provide the genome editing RNA/DNA system of the present invention in complex or encapsulated form. Each pharmaceutical composition is provided herein.

In particular, the pharmaceutical composition of the present invention comprises a nucleic acid-containing particle, preferably a genome editing RNA/DNA system-containing particle. Each pharmaceutical composition is referred to as a particulate formulation. In the particulate formulation according to the invention, the particles comprise a nucleic acid according to the invention and a pharmaceutically acceptable carrier or a pharmaceutically acceptable carrier suitable for delivery of the nucleic acid.

The genome editing RNA/DNA system-containing particles of the present invention may be, for example, in the form of protein particles or in the form of lipid-containing particles. Suitable proteins or lipids are referred to as particle forming agents.

When the RNA/DNA system according to the present invention is formulated in a particulate formulation, each RNA species (DdRp RNA expression cassette, and any additional RNA species such as RNA encoding a protein suitable for genome editing) and DNA species (auto -DdRp expression constructs and any additional DNA species such as genome editing tools DNA expression cassettes) can be formulated as particulate formulations. The RNA species and DNA species may be formulated in their respective individual particulate formulations, or the genome editing RNA/DNA system may be formulated in one particulate formulation.

The individual particulate formulations may exist as separate entities, for example in separate containers. Such formulations can be obtained by providing each nucleic acid individually (typically in the form of a solution containing each genome editing RNA/DNA system) with a particle forming agent, thereby allowing the formation of particles. Each particle will exclusively contain the particular nucleic acid provided when the particle is formed (individual particulate formulation).

A pharmaceutical composition according to the present invention comprises two or more individual particle formulations. Each pharmaceutical composition is referred to as a mixed particulate formulation. The mixed particulate formulation according to the present invention can be obtained by mixing the individual particulate formulations and then separately forming the individual particulate formulations as described above.

By the mixing step, one formulation comprising a mixed population of genome editing RNA/DNA system-containing particles is obtainable (eg a first population of particles may contain a DNA expression cassette according to the present invention, the particles The second formulation of may contain an RNA expression cassette according to the present invention). A population of particulates may be brought together in a container containing a mixed population of individual particulate formulations.

Alternatively, all genome editing RNA/DNA systems of a pharmaceutical composition may be formulated together as a combined particulate formulation. Such formulations can be obtained by providing a combined formulation (typically a combined solution) of all genome editing RNA/DNA systems together with a particle-forming agent to allow for the formation of particles.

In contrast to mixed particulate formulations, combined particulate formulations will typically include particles comprising a genome editing RNA/DNA system. In a combined particulate composition, the genome editing RNA/DNA system is typically present together in a single particle.

The particulate formulation of the present invention is a nanoparticulate formulation. In this embodiment, the composition according to the invention comprises a nucleic acid according to the invention in the form of nanoparticles. Nanoparticulate formulations can be obtained with a variety of protocols and with a variety of complex compounds. Lipids, polymers, oligomers or amphiphiles are typical components of nanoparticulate formulations.

The term “nanoparticle” refers to any particle having a diameter that forms a particle suitable for systemic administration, particularly parenteral administration, of a nucleic acid, generally having a diameter of 1000 nanometers (nm) or less. In one embodiment, the nanoparticles have an average diameter of from about 50 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, preferably from about 100 nm to about 300 nm, such as from about 150 nm to about 200 nm. have a range In one embodiment, the nanoparticles have a diameter in the range of from about 200 to about 700 nm, from about 200 to about 600 nm, preferably from about 250 to about 550 nm, in particular from about 300 to about 500 nm or from about 200 to about 400 nm. has

The polydispersity index (PI) of the nanoparticles described herein, as measured by dynamic light scattering, is 0.5 or less, preferably 0.4 or less or even more preferably 0.3 or less. "Polydispersity Index" (PI) is a measure of the homogeneous or heterogeneous size distribution of individual particles (eg, liposomes) in a particle mixture and represents the breadth of the particle distribution within the mixture. The PI can be determined, for example, as described in WO 2013/143555 A1.

A “nanoparticulate formulation” or similar term refers to any particulate formulation containing at least one nanoparticle. A nanoparticulate composition is a uniform aggregate of nanoparticles. Nanoparticulate compositions are lipid-containing pharmaceutical formulations or emulsions, such as liposome formulations.

Lipid-Containing Pharmaceutical Compositions

The pharmaceutical composition of the present invention comprises at least one lipid. Preferably, the at least one lipid is a cationic lipid. The lipid-containing pharmaceutical composition comprises a nucleic acid according to the present invention. The pharmaceutical composition according to the present invention comprises a genome editing RNA/DNA system encapsulated in a vesicle, for example a liposome. The pharmaceutical composition according to the present invention comprises a genome editing RNA/DNA system in the form of an emulsion. The pharmaceutical composition according to the present invention comprises RNA in a complex with a cationic compound, thereby forming, for example, a so-called lipoplex or polyplex. Encapsulation of genome editing RNA/DNA systems in vesicles such as liposomes differs from, for example, lipid/genome editing RNA/DNA system complexes. For example, a lipid/genome editing RNA/DNA system complex is obtained when the genome editing RNA/DNA system is mixed with, for example, preformed liposomes.

The pharmaceutical composition according to the present invention comprises a genome editing RNA/DNA system encapsulated in a vesicle. Such formulations are specific particulate formulations according to the invention. Vesicles are lipid bilayers surrounded by a spherical shell, enclosing a small space and separating it from the space outside the vesicle. Typically, the space within the vesicle contains the aqueous space, ie, water.

Typically, the space outside the vesicle contains the aqueous space, ie, water. The lipid bilayer is formed by one or more lipids (vesicle-forming lipids). The membrane surrounding the vesicle has a lamellar shape similar to the plasma membrane.

Vesicles according to the invention may be multilamellar vesicles, unilamellar vesicles or mixtures thereof. When encapsulated within a vesicle, the genome editing RNA/DNA system is typically isolated from any external medium. Thus, it exists in a protected form that is functionally equivalent to the protected form of the native virus. Suitable vesicles are particles as described herein, in particular nanoparticles.

For example, a genome editing RNA/DNA system can be encapsulated in liposomes. In this embodiment, the pharmaceutical composition is or comprises a liposomal formulation. Encapsulation in liposomes will typically protect the genome editing RNA/DNA system from RNase degradation. A liposome may contain some exogenous genome editing RNA/DNA system, but at least half (ideally all) of the genome editing RNA/DNA system is encapsulated within the core of the liposome.

Liposomes are often microscopic lipid vesicles with one or more bilayers of vesicle-forming lipids such as phospholipids and may encapsulate drugs, such as genome editing RNA/DNA systems. multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), sterically stabilized liposomes (SSL), polyvesicular vesicles (MV) and large polyvesicular vesicles (LMV) as well as those in the art Other types of liposomes, including other bilayer morphologies known in , may be employed, but are not limited thereto. The size and layering of the liposome will vary depending on the method of preparation. There are several other forms of supramolecular configuration in which lipids can exist in an aqueous medium, including lamellar shapes, hexagonal and inverted hexagonal shapes, cubic shapes, micelles, and inverted micelles composed of monolayers. In addition, these shapes can be obtained in combination with DNA or RNA, and the interaction of RNA and DNA can substantially affect the shape state. Such a shape may be present in the formulation of the nanoparticulate genome editing RNA/DNA system of the present invention.

Liposomes can be formed using standard known methods. Each method includes a reverse evaporation method, an ethanol injection method, a dehydration-rehydration method, sonication or other suitable method. After liposome formation, the size of the liposomes can be altered to obtain a population of liposomes having a substantially homogeneous size range.

The genome editing RNA/DNA system is present in liposomes comprising at least one cationic lipid. Each liposome may be formed from a single lipid or a mixture of lipids, when at least one cationic lipid is employed. Preferred cationic lipids have a nitrogen atom capable of being protonated; Preferably, such cationic lipids are lipids having tertiary amine groups. A particularly suitable lipid having a tertiary amine group is 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA). In one embodiment, the RNA according to the invention is in a liposomal formulation: a liposome having a lipid bilayer encapsulating an aqueous core comprising RNA, wherein the lipid bilayer has a pKa in the range of 5.0 to 7.6, preferably tertiary amine groups. lipids with A preferred cationic lipid having a tertiary amine group includes DLinDMA (pKa 5.8).

Liposomes comprising each compound are particularly suitable for encapsulation of genome editing RNA/DNA systems, and thus are suitable for liposome delivery of genome editing RNA/DNA systems. The genome editing RNA/DNA system according to the present invention is present in a liposome formulation, wherein the liposome comprises at least one cationic lipid, and the head group comprises at least one protonatable nitrogen atom (N), Liposomes and RNA have an N:P ratio of 1:1 to 20:1. According to the present invention, "N:P ratio" refers to the molar ratio of a nitrogen atom (N) in a cationic lipid to a phosphorus atom (P) in a genome editing RNA/DNA system contained in a lipid-containing particle (eg, a liposome). refers to The N:P ratio between 1:1 and 20:1 suggests that the net charge of liposomes is related to the efficiency of genome editing RNA/DNA system delivery into vertebrate cells.

A genome editing RNA/DNA system according to the present invention is present in a liposome formulation comprising at least one lipid comprising a polyethylene glycol (PEG) moiety, wherein the genome editing RNA/DNA system is encapsulated in a PEGylated liposome such that the PEG moiety is liposome exists outside of

The genome editing RNA/DNA system according to the present invention is present in a liposome formulation, and the liposome has a diameter in the range of 60-180 nm.

The genome editing RNA/DNA system according to the present invention is present in a liposome formulation, and the genome editing RNA/DNA system-containing liposome has a net charge of zero or close to negative.

The genome editing RNA/DNA system according to the present invention is in the form of an emulsion. Emulsions have conventionally been described as being used to deliver nucleic acid molecules, such as RNA molecules, to cells. Preferred in the present invention are oil-in-water emulsions. Each emulsion particle comprises an oil core and a cationic lipid. More preferred is a cationic oil-in-water emulsion in which the genome editing RNA/DNA system according to the present invention is complexed with emulsion particles. The emulsion particles comprise an oil core and a cationic lipid.

The cationic lipid interacts with the negatively charged genome editing RNA/DNA system to immobilize the genome editing RNA/DNA system in the emulsion particles. In oil-in-water emulsions, the emulsion particles are dispersed in an aqueous continuous phase. For example, the average diameter of the emulsion particles may typically be between about 80 nm and 180 nm.

The pharmaceutical composition of the present invention is a cationic oil-in-water emulsion, wherein the emulsion particles comprise an oil core and a cationic lipid. The genome editing RNA/DNA system according to the present invention may be in the form of an emulsion comprising a cationic lipid, wherein the emulsion has an N:P ratio of at least 4:1.

The genome editing RNA/DNA system according to the present invention may be in the form of a cationic lipid emulsion. In particular, the composition may comprise a genome editing RNA/DNA system complexed with particles of a cationic oil-in-water emulsion, wherein the oil/lipid ratio is at least about 8:1 (mol:mol).

The pharmaceutical composition according to the present invention includes a genome editing RNA/DNA system in the form of a lipoplex. The term “lipoplex” or “genomic editing RNA/DNA system lipoplex” refers to a complex of nucleic acids, such as lipids and genome editing RNA/DNA systems. Lipoplexes can be formed of cationic (positively charged) liposomes and anionic (negatively charged) nucleic acids. Cationic liposomes may also contain neutral “helper” lipids. In the simplest case, lipoplexes are formed spontaneously by mixing nucleic acids with liposomes in a specific mixing protocol, although a variety of other protocols can be applied. between positively charged liposomes and negatively charged nucleic acids

It can be understood that the electrostatic interaction is the driving force for lipoplex formation. In the present invention, the net charge of the genome editing RNA/DNA system lipoplex particle is close to zero or negative. Electrically-neutral or negatively charged lipoplexes of genome editing RNA/DNA systems and liposomes can induce substantial genome editing RNA/DNA system expression in cells after systemic administration.

Accordingly, in the present invention, the pharmaceutical composition according to the present invention comprises a genome editing RNA/DNA system in the form of nanoparticles, preferably lipoplex nanoparticles, wherein (i) the number of positive charges in the nanoparticles is the number of negative charges in the nanoparticles. and/or (ii) the nanoparticles are neutral or net negatively charged, and/or (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less, and/or (iv) ) The zeta potential of nanoparticles is 0 or less.

Zeta potential is the scientific term for the electrodynamic potential in colloidal systems. In the present invention, (a) the zeta potential and (b) the charge ratio of cationic lipids to genome editing RNA/DNA systems in nanoparticles can be calculated. In summary, pharmaceutical compositions that are nanoparticulate lipoplex formulations having a defined particle size in which the net charge of the particles is zero or near negative or negative are preferred pharmaceutical compositions in the context of the present invention.

An object to be solved in the present invention is to provide a characterized self-transcribed RNA/DNA system that exhibits strong genome editing ability, and the RNA/DNA system adding self-transcription and/or 5' cap structure of the present invention is a living cell or organism By introducing targeted genomic sequence changes at specific loci in

1 shows the design of a promoter and template DNA sequence for in vitro transcription using T7 RNAP, wherein the double-stranded portion of the promoter is numbered relative to the transcription start site (+1);
Figure 2 is a diagram comparing the promoter nucleotide sequence to which DdRp encoded by the bacteriophage T7, T3, SP6 and K11 binds,
3 is a basic unit of a genome editing RNA / DNA system,
4 is a type of various systems constituted by the basic unit of a genome editing RNA/DNA system.
5 is a diagram illustrating a genome editing RNA/DNA system that performs nuclear-independent self-transcription in the cytoplasm;
6 is a sequence logo showing the most conserved bases around the start codon of more than 10,000 human mRNAs. Large letters indicate higher frequencies, and larger sizes of A and G at position 8 (-3, Kozak position) and Kozak There is a G at the 14th position corresponding to the (+4) position of the sequence,
7 shows the <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL> PCR product was cloned into the pUC19 vector, and then the plasmid was separated from the selected strain and treated with EcoRI / BamHI restriction enzyme. As a result of electrophoresis,
8 is a result of in vitro transcription of the PCR product of the prepared SM_DdRp pUC19 vector having a T7 promoter-T7 polymerase sequence as a template as a template;
9 shows the prepared capped SM_T7pol mRNA (Native RNA), the capped CM/SM_T7pol mRNA (modified RNA), and the capped CM/SM_T7pol mRNA (modified RNA-choliterol) with choliterol added to the 3' end of the prepared RNA in a cell culture medium. It is the result of confirming in vitro stability by RT-PCR after treatment for various times,
Figure 10 is the result of electrophoresis after cloning the PCR product containing the SM_DdRp DNA fragment containing T7 RANP into the pCI-neo vector, separating the plasmid from the selected strain and treating with BglII / HpaI restriction enzyme;
Figure 11 is the result of electrophoresis after cloning the PCR product containing the SM_DdRp DNA fragment containing NP868R-T7RNAP into the pCI-neo vector, and then separating the plasmid from the selected strain and treating with BglII / HpaI restriction enzyme;
12 is a DNAge expression cassette clone as an image obtained by cloning a DNAge fragment encoding a genome editing tool of interest into a pCI-neo vector, digesting it with a recombinant pCI-neo vector BglII/HpaI isolated from a selected strain, and gel electrophoresis. is the result of selecting
13 is a DNAsg configuration for the target sequence of the target gene of the DNAge,
14 shows that each and every RNA used to cut several capture target nucleic acid sequences using the CRISPR complex can be designed to recognize 18 bp in front of the base PAM sequence of the region to be captured, and these are multiple gRNA expression cassettes (a) , can be prepared as a polycistronic tRNA-gRNA DNA fragment (b),
15 is an image obtained by cloning the PCR amplification product of auto_DdRp and DNAge fragment into pShuttle vector, cutting the plasmid in the selected clone with XhoI/SalI, and performing gel electrophoresis.
Figure 16 is the result of co-transformation of the linearized recombinant auto_DdRp or DNAge expression cassette with pAdEasy-1, a supercoil virus DNA plasmid, into BJ5183 bacteria, isolating the recombinant plasmid from the selected strain, treating it with Pac I, and electrophoresis;
17 is an image obtained by cloning the PCR amplification product of auto_DdRp and DNAge fragment into pSMPUW vector, cutting the plasmid with XhoI/SalI in the selected clone and performing gel electrophoresis, and is the result of selecting auto_DdRp or DNAge expression cassette clone;
18 is a schematic diagram of the PE3 strategy used for modification of the StALS1 and StALS2 genes as a prime editing strategy for correct modification of the potato ALS gene, with PAM in green, target Proline in red, and target bases identified by black asterisks, respectively. The cleavage sites of pegRNA-StALS and sgRNA StALS were marked with blue or purple lightning, respectively. PBS: primer binding site RT: reverse transcriptase,
Figure 19 is an image of auto_(DdRp + DNAge) fragment cloned into a binary vector (Ti plAsmid), separated from the selected strain, cut with EcoRI / SalI, and gel electrophoresed, auto_ (DdRp + DNAge) It is the result of selecting the Ti expression cassette clone,
Figure 20 is an image obtained by cloning the DNAge of interest into the pCI neo vector as a control, separating the plasmid from the selected strain, cutting with a restriction enzyme, and electrophoresis;
Figure 21 is the result of analyzing the expression of T7 polymerase mRNA in DdRp of auto_DdRp transformed cells using T7RNAP, there is no expression of T7 polymerase mRNA in the control pCI-neo_RSV-F@LS, DdRp RNA/RSV-F DNAi@ In LS transformed cells, it was confirmed that the transfected 5'Cap structure and the stabilized DdRp RNA were slowly degraded in the cell, DdRp RNA/auto_DdRp/RSV-F DNAi@LS DdRp RNA/auto_(DdRp/RSV- F DNAi)@LS; In cells transformed with the auto(+) DdRp expression system including the auto_(DdRp/RSV-F DNAi) adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system, the first It was confirmed that T7 polymerase binds to the T7 promoter of the auto_DdRp expression cassette and stably synthesizes DdRp through the action of the self-transcription loop synthesizing DdRp in the downstream region.
22 is a result of analyzing the expression of T7 polymerase in DdRp of auto_DdRp transformed cells using T7RNAP. As a result, DdRp RNA/RSV-F DNAi@LS auto(-) DdRp expression system and DdRp RNA/auto_DdRp/ RSV-F DNAi@LS DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; T7 polymerase synthesized in cells transformed with an auto_(DdRp/RSV-F DNAi) adeno expression system or an auto(+) DdRp expression system including an auto_(DdRp/RSV-F DNAi) lenti expression system is at least in the cytoplasm. It is maintained at a high concentration for more than 72 hours,
23 shows the results of T7E1 analysis of the genome of a cell into which the genome editing RNA/DNA system of the present invention is introduced, (a) RNA-guided endonuclease genome editing, (b) DNA-guided endonuclease genome editing ( c), The contents of genome editing by fusion protein example of M-MLV reverse transcriptase and Cas9 H840A nickase.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples only.

Example 1. Design and manufacture of self-transcribed RNA/DNA system

In order to prepare the genome editing system proposed in the present invention, as shown in <FIG. 3>, a coding RNA expression cassette and a DNAge expression cassette transcribed by the DdRp and encoding a genome editing tool were prepared. The DNAge expression cassette is composed of a DNAen expression cassette encoding a targeting endonuclease and a DNAsg expression cassette encoding a guide nucleic acid and sgRNA, which may be present on a single plasmid or on separate plasmids.

In addition, an auto_DdRp expression cassette into which DdRP is transcribed by the pT7/T7 polymerase system was added here.

The genome editing RNA/DNA system of the present invention is a DdRp composed of a single subunit in the DdRp shown in <Table 1>, T7 RNA polymerase (Gene ID: 1261050), T3 RNA polymerase (Gene ID: 14675519) and SP6 RNA polymer An RNA/DNA system capable of nuclear-independent continuous genome editing tool cytoplasmic expression using Rase (Gene ID: 19685893) was prepared as follows. The promoter sequences corresponding to them are shown in <Fig. 2>.

As in <Fig. 4>, genome editing RNA/DNA system 1 composed of a DdRp RNA expression cassette and a DNAge expression cassette capped in the present invention; genome editing RNA/DNA system 2 consisting of a capped DdRp RNA expression cassette, an auto_DdRp expression cassette and a DNAge expression cassette; and a genome editing RNA/DNA system 3 consisting of a capped DdRp RNA expression cassette and an auto_DdRp+DNAge expression cassette.

Auto(-)_DNAge@LS, a complex of RNA/DNA system 1 and liposomes of the present invention, is a T7 polymerase repeatedly synthesized from a DdRp RNA expression cassette containing a stabilized capped DdRp mRNA, and is maintained at a high concentration for a long time in the cytoplasm. By repeatedly binding to the T7 promoter of the DdRp RNA expression cassette, the expression of the genome editing tool can be maintained continuously for a long period of time.

The DdRp self-transcription loop system composed of the RNA/DNA system 2 and the RNA/DNA system 3 of the present invention was referred to as "pT7/T7 polymerase system".

<As shown in FIG. 5, auto_DNAge@LS, a complex of RNA/DNA system 2 or RNA/DNA system 3 of the present invention and a liposome, was initially synthesized from a DdRp RNA expression cassette containing a capped DdRp mRNA stabilized by T7 polymerase. By binding to the T7 promoter of the auto_DdRp expression cassette or the auto_(DdRp+DNAge) expression cassette, T7 polymerase is maintained at a high concentration in the cytoplasm for a long time as a result of autotranscription, and the T7 polymerase repeatedly binds to the T7 promoter of the DNAge expression cassette for genome editing. Tool expression can be maintained continuously for a long time.

In the case of using a viral system for the DNA expression cassette in the self-transcription RNA/DNA system of the present invention, the viral expression cassette encoding the DdRp-binding promoter and the expression RNA and the DdRp RNA expression cassette can be divided and produced, and can be independently transfected. . First, the lentivirus expression cassette is injected, stable transformed cells can be selected, and the DdRp RNA expression cassette can be transfected. Alternatively, the adenovirus expression cassette may be injected and the DdRp RNA expression cassette may be transfected.

Even when the self-transcribed RNA/DNA system of the present invention uses a viral expression cassette, the first T7 polymerase synthesized auto_DdRp expression cassette or auto_(DdRp+ By binding to the T7 promoter of the DNAge) expression cassette, T7 polymerase is maintained at a high concentration for a long time in the cytoplasm as a result of autotranscription, and the T7 polymerase repeatedly binds to the T7 promoter of the DNAge expression cassette, thereby maintaining stable target mRNA expression for a long time. .

1-1. Preparation of DdRp RNA Expression Cassettes

In order to prepare the DdRp RNA expression cassette of the present invention, a DdRp DNA having a T7 promoter capable of producing a T7 polymerase ORF was designed and manufactured, and the DdRp DNA having a T7 promoter obtained from the recombinant pUC19 vector was recombined into a pUC19 cloning vector. After in vitro transcription with a template, a 5'Cap structure was added.

<DdRp DNA Design and Manufacturing>

The DdRp DNA of the present invention is a DNA fragment capable of efficiently synthesizing DdRp mRNA, and a DNA fragment consisting of <T7 promoter followed by 5'UTR, DdRp-encoding DNA fragment, 3'UTR> was designed.

In the SM_DdRp DNA of the present invention, the T7 polymerase gene is located downstream of the T7 promoter and 5'UTR in the pUC19 vector, and at the same time muag (mutated alpha-globin-3'UTR), A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) upstream of the sequence. The SM_DdRp DNA structure may be <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL>.

Preferably, nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be activated enzymatically in the cytoplasm or hybrid DdRp with NLS in the nucleus to selectively start transcription at the T7 promoter placed in chromosomal or plasmid DNA and stop at a specific 3'UTR.

The base sequence of the unit elements constituting the SM_DdRp DNA fragment is as follows.

The 5*?**?*UTR includes the Kozak consensus.

Kozak consensus sequence ACCAUGG (SEQ ID NO: 31).

Figure 6 is a sequence logo showing the most conserved bases around the start codon of more than 10,000 human mRNAs. Large letters indicate a higher frequency, with a larger size of A and G at position 8 (-3, Kozak position) and a G at position 14 corresponding to the (+4) position of the Kozak sequence.

For the purposes of the present invention, NLS (SV40 NLS, PKKKRKVEDPYC) may be added to the N and C-terminus of Bacteriophage T7 RNA Polymerase (T7Pol). The N-terminal addition placed the NLS sequence after the start codon AUG, and the C-terminal addition placed the NLS sequence before the stop codon UAA.

SV40 NLS sequence: 5′-CCG AAG AAG AAG CGA AAG GTC-3′ (SEQ ID NO: 32),

The nucleotide sequence of the ORF of Bacteriophage T7 RNA Polymerase (T7Pol), a type of DdRP selected in the present invention, is as shown in SEQ ID NO: 33.

Bacteriophage T7 RNA Polymerase (T7Pol):

ATGAACACGA TTAACATCGC TAAGAACGAC TTCTCTGACA TCGAACTGGC TGCTATCCCG 60

TTCAACACTC TGGCTGACCA TTACGGTGAG CGTTTAGCTC GCGAACAGTT GGCCCTTGAG 120

CATGAGTCTT ACGAGATGGG TGAAGCACGC TTCCGCAAGA TGTTTGAGCG TCAACTTAAA 180

GCTGGTGAGG TTGCGGATAA CGCTGCCGCC AAGCCTCTCA TCACTACCCT ACTCCCTAAG 240

ATGATTGCAC GCATCAACGA CTGGTTTGAG GAAGTGAAAG CTAAGCGCGG CAAGCGCCCGA 300

CAGCCTTCCA GTTCCTGCAA GAAATCAAGC CGGAAGCCGT AGCGTACAT CACCATTAAG 360

ACCACTCTGG CTTGCCTAAC CAGTGCTGAC AATACAACCG TTCAGGCTGT AGCAAGCGCA 420

ATCGGTCGGG CCATTGAGGA CGAGGCTCGC TTCGGTCGTA TCCGTGACCT TGAAGCTAAG 480

CACTTCAAGA AAAACGTTGA GGAACAACTC AACAAGCGCG TAGGGCACGT CTACAAGAAA 540

AAGTTGTCGA GGCTGACATG CTCTCTAAGG GTCTACTCGG TGGCGAGGCG TGGCGAGGCG 600

TGGTCTTCGT GGCATAAGGA AGACTCTATT CATGTAGGAG TACGCTGCAT CGAGATGCTC 660

ATTGAGTCAA CCGGAATGGT TAGCTTACAC CGCCAAAATG CTGGCGTAGT AGGTCAAGAC 720

TCTGAGACTA TCGAACTCGC ACCTGATACG CTGAGGCTA TCGCAACCCG TGCAGGTGCG 780

CTGGCTGGCA TCTCTCCGAT GTTCCAACCT TGCGTAGTTC CTCCTAAGCC GTGGACTGGC 840

ATTACTGGTG GTGGCTATTG GGCTAACGGT CGTCGTCCTC TGGCGCTGGT GCGTACTCAC 900

AGTAAGAAAG CACTGATGCG CTACGAGACG TTTACATGC CTGAGGTGTA CAAAGCGATT 960

AACATTGCGC AAAACACCGC ATGGAAAATC AACAAGAAAG TCCTAGCGGT CGCCAACGTA 1020

ATCACCAAGT GGAAGCATTG TCCGGTCGAG GACATCCCTG CGATTGAGCG TGAAGAACTC 1080

CCGATGAAAC CGGAAGACAT CGACATGAAT CCTGAGGCTC TCACCGCGTG GAAACGTGCT 1140

GCCGCTGCTG TGTACCGCAA GGACAAGGCT CGCAAGTCTC GCCGTATCAG CCTTGAGTTC 1200

ATGCTTGAGC AAGCCAATAA GTTTGCTAAC CATAAGGCCA TCTGGTTCCC TTACAACATG 1260

GACTGGCGCG GTCGTGTTTA CGCTGTGTCA ATGTTCAACC CGCAAGGTAA CGATATGACC 1320

AAAGGACTGC TTACGCTGGC GAAAGGTAAA CCAATCGGTA AGGAAGGTTA CTACTGGCTG 1380

AAAATCCACG GTGCAAACTG TGCGGGTGTC GATAAGGTTC CGTTCCCTGA GCGCATCAAG 1420

TTCATTGAGG AAAACCACGA GAACATCATG GCTTGCGCTA AGTCTCCACT GGAGAACACT 1480

TGGTGGGCTG AGCAAGATTC TCCGTTCTGC TTCCTTGCGT TCTGCTTTGA GTACGCTGGG 1540

GTACAGCACC ACGGCCTGAG CTATAACTGC TCCCTTCCGC TGGCGTTTGA CGGGTCTTGC 1620

TCTGGCATCC AGCACTTCTC CGCGATGCTC CGAGATGAGG TAGGTGGTCG CGCGGTTAAC 1680

TTGCTTCCTA GTGAAACCGT TCAGGACATC TACGGGATTG TTGCTAAGAA AGTCAACGAG 1740

ATTCTACAAG CAGACGCAAT CAATGGGACC GATAACGAAG TAGTTACCGT GACCGATGAG 1820

AACACTGGTG AAATCTCTGA GAAAGTCAAG CTGGGCACTA AGGCACTGGC TGGTCAATGG 1880

CTGGCTTACG GTGTTACTCG CAGTGTGACT AAGCGTTCAG TCATGACGCT GGCTTACGGG 1940

TCCAAAGAGT TCGGCTTCCG TCAACAAGTG CTGGAAGATA CCATTCAGCC AGCTATTGAT 2000

TCCGGCAAGG GTCTGATGTT CACTCAGCCG AATCAGGCTG CTGGATACAT GGCTAAGCTG 2060

ATTTGGGAAT CTGTGAGCGT GACGGTGGTA GCTGCGGTTG AAGCAATGAA CTGGCTTAAG 2120

TCTGCTGCTA AGCTGCTGGC TGCTGAGGTC AAAGATAAGA AGACTGGAGA GATTCTTCGC 2180

AACGGTTGCG CTGTGCATTG GGTAACTCCT GATGGTTTCC CTGTGTGGCA GGAATACAAG 2240

AAGCCTATTC AGACGCGCTT GAACCTGATG TTCCTCGGTC AGTTCCGCTT ACAGCCTACC 2300

ATTAACACCA ACAAAGATAG CGAGATTGAT GCACACAAAC AGGAGTCTGG TATCGCTCCT 2360

AACTTTGTAC ACAGCCAAGA CGGTAGCCAC CTTCGTAAGA CTGTAGTGTG GGCACACGAG 2420

AAGTACGGAA TCGAATCTTT TGCACTGATT CACGACTCCT TCGGTACCAT TCCGGCTGAC 2480

GCTGCGAACC TGTTCAAAGC AGTGCGCGAA ACTATGGTTG ACACATATGA GTCTTGTGAT 2540

GTACTGGCTG ATTTCTACGA CCAGTTCGCT GACCAGTTGC ACGAGTCTCA ATTGGACAAA 2600

ATGCCAGCAC TTCCGGCTAA AGGTAACTTG AACCTCCGTG ACATCTTAGA GTCGGACTTC 2660 GCGTTCGCGT AATAA 2675 (SEQ ID NO:33)

wherein the 3'UTR is a mutated (alpha-)globin comprising the sequence 5'-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3' (SEQ ID NO: 34; muag; the underlined nucleotides are mutated compared to the wild-type sequence) UTR, poly(A) tail consists of 64 adenine nucleotides, poly(C) sequence consists of 30 cytosine nucleotides and histone stem-loop is (5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' SEQ ID NO: 35) according to the RNA sequence.

muag - A64 - C30 - histone SL: GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG AGATTA ATAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AATTAATGCA TCCCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA (SEQ ID NO: 37)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 38)

In the present invention, the SM_DdRp DNA fragment was synthesized with <T7 promoter - 5'UTR - T7Pol - muag - A64 - C30 - histone-SL> (Biosynthesis, USA), and the forward primer 5'- GAATTC TAAT ACGACTCACT ATAG GGGAGA was used as a template. It was amplified using CCACAACGGT TTCCCTCTAG AAAGAACC AT GAACACGATT AACA -3' (SEQ ID NO: 39) and reverse primer 5'- GGATCC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 40). Here the target sequence of T7pol is underlined. Both ends of the primers used include EcoRI/BamHI recognition sequences.

The amplified PCR product was digested using EcoRI/BamHI enzymes, and then cloning reaction was performed using the pUC19 vector linearized with EcoRI/BamHI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with EcoRI/BamHI restriction enzymes, and then electrophoresed (FIG. 7).

The recombinant pUC19 prepared above can synthesize RNA by DdRp synthesized from a DdRp RNA expression cassette in prokaryotes and can synthesize DdRp by a protein synthesis tool, so it can be used as a DdRp expression cassette. However, in the present invention, in eukaryotic cells, there is no origin of replication in eukaryotic cells, and thus serves as an expression vector for a short time.

<Preparation of DdRp RNA expression cassette>

In order to start self-transcription of T7 polymerase in the RNA/DNA system of the present invention, the first T7 polymerase supply is required. In the present invention, T7pol mRNA containing nucleotides (CM) with capped chemical modifications to solve the problem of low nuclear membrane permeation A DdRp RNA expression cassette comprising a was prepared.

In order to synthesize capped CM_T7pol mRNA, first, the prepared SM_DdRp pUC19 vector having a T7 promoter-T7 polymerase sequence was amplified using a forward primer (SEQ ID NO: 39) and a 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. 8).

CM/SM_T7pol capped using the HighYield T7 ARCA mRNA Synthiis Kit (m5CTP/Ψ-UTP) (Jena BioScience, USA) using the SM_DdRp PCR product with the T7 promoter prepared above as a T7 promoter-T7 polymerase DNA template as a template A DdRp RNA expression cassette containing mRNA was synthesized in vitro. Specifically, a reaction solution containing anti-reverse cap analog [(ARCA) (3′-O-Me-7mG(5′)ppp(5′)G cap analog)] and chemically modified nucleotides (6 mM In vitro transcription was performed in 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 CM/SM_T7pol mRNA was purified by LiCl extraction and ethanol precipitation.

As a control, the synthesis of capped T7pol mRNA in which a 5'cap structure was added to the native RNA transcript in the SM_DdRp vector was performed using the HiScribe ^™ T7 ARCA mRNA Kit (NEB, USA).

As shown in <Fig. 9>, the prepared capped SM_T7pol mRNA (Native RNA), the capped CM_T7pol mRNA (modified RNA), and the capped CM_T7pol mRNA (modified RNA-choliterol) with choliterol added to the 3' end were added to the cell culture medium. In vitro stability was confirmed by RT-PCR after processing the prepared RNA for various times. The prepared capped CM_T7pol mRNA had very superior in vitro safety than the capped T7pol mRNA, and the capped CM_T7pol mRNA (modified RNA-choliterol) with cholesterol added to the 3' end had the highest stability.

Capped CM_T7pol mRNA can also be added with a native cap (m7G(5')ppp(5')G; m7GpppG) to the IVT product with the ScriptCap m7G capping system (Epicentre Biotechnologii, USA).

1-2. Preparation of auto_DdRp and auto_chimeric DdRp expression cassettes

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. For the expression cassette used in the self-transcribed RNA/DNA system of the present invention, a promoter corresponding to DdRp commonly selected for prokaryotes or progenitors, particularly mammals, plants, and the like may be used. For example, when DdRp is T7 Polymerase, it can be prepared using the T7 promoter.

The auto_DdRp DNA expression cassette and the auto_(DdRp+DNAge) expression cassette of the present invention can be prepared using a plasmid or a viral vector. The auto_DdRp DNA expression cassette and the auto_(DdRp+DNAge) expression cassette are stored in a plasmid or viral vector with an auto_DdRp DNA fragment (T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone- SL> was inserted.As shown below, the synthesized DdRp and chimeric DdRp DNA fragments were PCR amplified as a template to obtain a large amount of DdRp and chimeric DdRp DNA fragments that are easy to clone. recombined.

The expression vectors used in this example were mammalian expression plasmid vector pCI-neo vector (Promega, USA), pShuttle in AdEasy Adenoviral Vector System (Agilent Technologii, USA), ViraSafe™ Lentiviral Exprision System (Cell Biolabs, USA). pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector, shuttle vector pSMPUW, binary vector for plant expression (Ti plasmid), etc., but not limited thereto. In this example, only the plasmid vector was described by limiting it, and the viral vector can be sufficiently prepared with reference to Examples 1-4 below.

<auto_DdRp expression cassette>

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. For the expression cassette used in the self-transcribed RNA/DNA system of the present invention, a promoter corresponding to DdRp commonly selected for prokaryotes or progenitors, particularly mammals, plants, and the like may be used. For example, when DdRp is T7 Polymerase, it can be prepared using the T7 promoter.

In this example, an auto_SM_DdRp expression cassette including a DNA fragment providing DdRp was designed using a plasmid. The pCI-neo vector (Promega, USA) capable of constructing a stable cell line with a mammalian expression vector was digested with BglII/HpaI restriction enzyme treatment and <T7 promoter followed by 5'UTR, DdRp-encoding DNA fragment, muag, A64 The auto_SM_DdRp expression cassette was prepared by inserting an SM_DdRp DNA fragment consisting of a poly(A) sequence, a poly(C) sequence (C30), and a histone stem-loop sequence (histone-SL) sequence>.

Nuclear localization signals (NLS) may be located in the DdRp. DdRp without NLS mostly remains in the cytoplasm, whereas protein containing NLS is localized in the nucleus. DdRp without NLS can be cytoplasmic or hybrid DdRp with NLS can be enzymatically activated in the nucleus to selectively start transcription at the T7 promoter placed on chromosomal or plasmid DNA and stop at a specific 3*?**?*UTR. have.

The base sequence of the unit elements constituting the SM_DdRp DNA fragment is as follows.

The SM_DdRp DNA fragment synthesized in Example 1-1 was forward primer 5'- AGATCT TTAA TACGACTCAC TATAGGGGAG ACCACAACGG TTTCCCTCTA GAAAGAACC A TGAACACGAT TAACA -3' (SEQ ID NO: 43) and reverse primer 5'- GTTAAC TGGT GCTCTAAAA (SEQ ID NO: 44) was used to amplify. Here the target sequence of T7pol is underlined. Both ends of the primers used include BglII/HpaI recognition sequences.

The PCR product containing the amplified SM_DdRp DNA fragment was digested using BglII/HpaI enzymes, and then subjected to cloning reaction using a pCI-neo vector linearized with BglII/HpaI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and then electrophoresed (FIG. 10).

In the above <T7 promoter followed by 5'UTR, DNA fragment encoding DdRp, muag, A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) sequence> The recombinant pCI-neo vector constructed from the SM_DdRp DNA fragment was referred to as an auto_DdRp expression cassette.

<auto_chimeric DdRp expression cassette>

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. For the expression cassette used in the self-transcribed RNA/DNA system of the present invention, a promoter corresponding to DdRp commonly selected for prokaryotes or progenitors, particularly mammals, plants, and the like may be used. For example, when DdRp is T7 Polymerase, it can be prepared using the T7 promoter.

The auto_chimeric DdRp expression cassette of the present invention may use a plasmid or a viral vector. In this Example, an auto_chimeric DdRp expression cassette including a DNA fragment providing DdRp was designed using a plasmid.

A pCI-neo vector (Promega, USA) capable of constructing a stable cell line as a mammalian expression vector was digested with BglII/HpaI restriction enzyme treatment and <T7 promoter followed by 5'UTR, a DNA fragment encoding chimeric DdRp, muag; The auto_chimeric DdRp expression cassette was prepared by inserting an SM_DdRp DNA fragment consisting of A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) sequence>.

The DNA fragment encoding the chimeric DdRp of the present invention is the ORF of NP868R-T7RNAP (SEQ ID NO: 21). One plasmid encoding a fusion between NP868R, the mRNA capping enzyme of African swine fever virus, and the wild-type phage DdRp of bacteriophage T7 was synthesized. This capping enzyme was fused to the amino terminus of T7 RNA polymerase by a (Gly3Ser)4 linker.

The nucleotide sequence for the ORF of NP868R-T7RNAP is shown in SEQ ID NO: 21 below.

NP868R-T7RNAP:

ATGGAATTCG CCAGCCTGGA CAACCTGGTG GCCAGATAACC 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 TCAACACCCT 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

TTCCAGCCCT GCGTGGTGCC TCCTAAGCCC TGGACAGGCA TCACAGGCGG CGGATACTGG 3540

GCCAACGGCA GACGCCCTCT GGCTCTGGTG 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 AGAGGGCTAC TACTGGCTGA AGATCCACGG CGCCAACTGC 4080

GCTGGCGTGG ACAAGGTGCC CTTCCCAGAG CGGATCAAGT TCATCGAGGA AAACCACGAG 4140

AACATCATGG CCTGCGCCAA GAGCCCTCTA GAAAACACTT GGTGGGCCGA GCAGGACAGC 4200

CCCTTCTGCT TCCTGGCCTT 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

ACAGTGGTGG 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: 45)

Nuclear localization signals (NLS) may be located in the chimeric DdRp. The chimeric DdRp without NLS mostly remains in the cytoplasm, whereas the protein containing NLS is confined to the nucleus. Chimeric DdRp without NLS can be activated enzymatically in the nucleus, either in the cytoplasm or chimeric DdRp with NLS to selectively start transcription at the T7 promoter placed in chromosomal or plasmid DNA and stop at specific T7 terminator sequences.

The base sequence of the unit elements constituting the SM_chimeric DdRp DNA fragment is as follows.

In the present invention, a SM_DdRp DNA fragment consisting of <T7 promoter - 5'UTR - NP868R-T7RNAP - muag - A64 - C30 - histone-SL> was synthesized (Biosynthiis, USA), and the forward primer 5'-AGATCT TAAT ACGACTCACT was used as a template. Amplification was performed using ATAG GAGACC ACAACGGTTTC CCTCTAGAAA GAACC ATGGAATTC GCCAGCCTGG A -3' (SEQ ID NO: 43) and reverse primer 5'-GTTAAC TGGT GGCTCTAAAA GAGCCTTTGG -3' (SEQ ID NO: 44). Here the target sequence of T7pol is underlined. Both ends of the primers used include BglII/HpaI recognition sequences.

The PCR product containing the SM_DdRp DNA fragment in which DdRp is NP868R-T7RNAP was digested using BglII/HpaI enzymes, and then a cloning reaction was performed using a pCI-neo vector linearized with BglII/HpaI enzymes and T4 DNA ligase. The reaction solution was transfected into E. coli, and the plasmid was isolated from the selected strain, treated with BglII/HpaI restriction enzymes, and then electrophoresed (FIG. 11).

In the above <T7 promoter followed by 5'UTR, DNA fragment encoding NP868R-T7RNAP, 3'UTR, A64 poly(A) sequence, poly(C) sequence (C30) and histone stem-loop sequence (histone-SL) The recombinant pCI-neo vector constructed with the SM_DdRp DNA fragment consisting of sequence> was referred to as auto_SM_chimeric DdRp expression case.

1-3. Genome editing tool DNA (DNAge) expression cassette

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. For the expression cassette used in the self-transcribed RNA/DNA system of the present invention, a promoter corresponding to DdRp commonly selected for prokaryotes or progenitors, particularly mammals, plants, and the like may be used. For example, when DdRp is T7 Polymerase, it can be prepared using the T7 promoter.

In the present invention, the DNAge expression cassette encoding the genome editing tool comprises a DNAen fragment encoding a targeting endonuclease that can be transcribed by DdRp or NP868R-T7RNAP synthesized in the DdRp RNA expression cassette or the auto_DdRp expression cassette and a guide nucleic acid. It may consist of an encoding DNAsg fragment. As shown in <Figs. 3 and 5>, the DNAen fragment and DNAsg fragment of the present invention may be produced in independent expression cassettes, or may be produced in one expression cassette.

The DNAge fragment produced in one expression cassette is in the form of <DNAen + DNAsg> (T7 promoter - 5'UTR - <NLS + targeting endonuclease + NLS> - muag - A64 - C30 - histone SL) and It can be composed of a DNA fragment encoding (T7 promoter - sgRNA and T7 transcription termination sequence).

The targeting endonuclease may include RNA-guided endonuclease, DNA-guided endonuclease, and a fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase. The guide nucleic acid may be of an sgRNA type, a tRNA-sgRNA type, a phosphorylated single-stranded DNA type, and a pegRNA type.

The DNAge fragment produced in one expression cassette is first in the form of <DNAen+DNAsg>, and the DNAen is (T7 promoter - 5'UTR - <NLS + targeting endonuclease + NLS> - muag - A64 - C30 - histone SL). The constructed DNA fragment was prepared by dividing it into (T7 promoter - 5'UTR), (muag - A64 - C30 - histone SL) and <NLS + targeting endonuclease + NLS>. BglII recognition sequence-(T7 promoter - 5'UTR)-BamHI recognition sequence, SalI recognition sequence-(muag-A64-C30-histone SL)-EcoRI recognition sequence was prepared by organic synthesis (Biosynthesis, USA), and BamHI Recognition sequence-<NLS+targeting endonuclease+NLS>-SalI recognition sequence was prepared by the PCR method described below using a commercial plasmid as a template.

DNAen obtained by recombination of three DNA fragments with restriction enzymes and ligases was amplified by PCR. At this time, BglII/EcoRI at both ends using a forward primer (5'- AGATCT TAAT ACGACTCACT ATAG GGAGACCAC AACGGUUUCC CUCU -3', SEQ ID NO: 43), and a reverse primer (5'- GAATTC GGT GGCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 51) PCR products with recognition sequences were obtained in large quantities.

In addition, DNAsg is a DNA fragment encoding (T7 promoter + guide nucleic acid + T7 transcription termination sequence), which was organically synthesized and mass amplified by PCR. At this time, EcoRI / HpaI at both ends of the PCR product using a forward primer (5'- GAATTC TAAT ACGACTCACT ATAG-3', SEQ ID NO: 74) and a reverse primer (5'- GTTAAC GGT GGCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 44) A recognition sequence was assigned.

After treating the amplified PCR products with EcoRI restriction enzyme, T4 DNA ligase was used to bind the two DNA fragments.

The product obtained by re-amplifying the bound DNA fragment was treated with BglII and HpaI to prepare an auto_(DdRp+DNAge) DNA fragment having a BglII recognition sequence at the 5' end and an HpaI recognition sequence at the 3' end, followed by BglII/HpaI restriction enzymes. The DNA fragment prepared by ligation reaction to the linearized pCI-neo vector as shown in <Table 2> was cloned.

The composition of the DNAge DNA fragment in the recombinant expression vector, pCI-neo vector is <T7 promoter - 5'UTR - DNAen - muag - A64 - C30 - histone-SL- T7 promoter - DNAsg - T7 transcription termination sequence>.

Composition of Ligation Reaction ingredient volume DNA fragment (0.5-50 μmol/L) 2 μl CI-neo (~100 ng/μl) 2 μl 10× T4 DNA Ligase Buffer (NEB) 1.5 μl 10x BSA 1.5 μl T4 DNA Ligase (HC, NEB) 1 μl ddHO 7 μl Total volume 15 μl

E. coli was transformed with 5 μl of the reaction mixture, and clones were selected on the ampicillin LB agar plate (FIG. 12).

In addition to the pCI-neo vector (Promega, USA) that can construct a stable cell line using the prepared DNAge DNA fragment as a mammalian expression vector, AdEasy Adenoviral Vector System (Agilent Technology, USA) and ViraSafe™ Lentiviral Exprision System (Cell Biolabs) , USA) to prepare a DNAge expression cassette.

As shown in <Fig. 13>, the target genes of the DNAge in the present invention include VEGF, PD-L1 and HEK3 genes, and DNAsg for their target sequences was prepared. It is not limited to these.

(a) DNA (DNAen) fragment encoding the targeting endonuclease

To prepare the DNAen fragment of the present invention (T7 promoter + 5'UTR - <NLS + targeting endonuclease + NLS> - muag - A64 - C30 - histone SL), a DNAen fragment including histone SL was prepared by organic synthesis (Biosynthesis) , United States of America). For NLS, the nucleotide sequence of <Table 3> was used.

NLS sequence Kinds base sequence SEQ ID NO: NLS PKKKRKV
(N-terminal) CCCAAGAAGA AGAGGAAGGT G (SEQ ID NO: 35) 21 CCAAAGAAGA AGAGGAAGGT T (SEQ ID NO: 36) 21 SGGSPKKKRKV
(C-terminal) TCGGGGGGGA GCCCAAAGAA GAAGCGGAAG GTG (SEQ ID NO: 37) 33

This was amplified by PCR as a template. At this time, BglII/HpaI recognition sequences were given to both ends using PCR primers. The amplified DNA fragment was treated with BglII/HpaI to prepare a BglII cleavage site at the 5' end and an HpaI cleavage site at the 3' end. After pCI-neo vector (Promega, USA) was treated with BglII/HpaI restriction enzymes, the prepared DNA fragment was cloned by ligation reaction as shown in <Table 2> using T4 DNA ligase.

Looking specifically at the endonuclease of <NLS + targeting endonuclease + NLS> in the DNAen fragment of the present invention,

① RNA-guided endonuclease: In Korean Patent Publication No. 10-2019-0120287, Cas9 nuclease variant eSpCas9 (1.0) (K810A / K1003A / R1060A), eSpCas9 (1.1) (K848A / K1003A / R1060A) and Cas9 nuclease It has been reported that the clease mutant SpCas9-HF1 (N497A/R661A/Q695A/Q926A) can significantly lower the off-target ratio during genome editing, that is, it has high specificity.

The SpCas9-HF1 (N497A/R661A/Q695A/Q926A) DNA used in the present invention can significantly lower the off-target ratio during genome editing, that is, a Cas9 nuclease variant with high specificity, purchased from Addgene (USA). It was prepared by amplifying the corresponding nucleotide sequence in one pJSC111 (Addgene #101209).

② DNA-guided endonuclease: In the present invention, the HuAgo nucleotide sequence of Argonaute used in the present invention was confirmed in Korean Patent Application Laid-Open No. 10-2019-0102070. The gene sequence of NgAgo was modified, and this modification improved the expression and/or activity of the algonut polypeptide in mammalian cells. It was referred to as 'HuAgo' and acts as a DNA-guided gene-editing nuclease in mammalian cells.

HuAgo DNA was prepared by amplifying the <NLS-NgAgo-NLS> sequence from NLS-NgAgo-GK (Addgene #78253) purchased from Addgene.

③ Fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase: Recently, a prime editing system that enables target insertion, deletion and point mutations without double-strand breaks or donor DNA repair templates has been developed in mammals. It has been shown to function efficiently in cells (Anzalone, AV et al. Nature 576, 149--157 (2019)). In this report, a third generation of prime editor (PE3) inserted a mutated M-MLV-RT (Moloney murine leukemia virus reverse transcriptase) into the C terminus of a catalytically impaired Cas9 (H840A) (Cas9 nickase, nCas9). designed by fusion. Cas9 enzyme is modified to cut only one strand of DNA, and one reverse transcriptase plays a role in creating new DNA by copying one RNA template. When the processed pegRNA (prime editing guide RNA) moves the editor to the target site, Cas9 cuts one strand of DNA. It is programmed with a prime editing guide RNA (pegRNA) consisting of a single chimeric guide RNA (sgRNA) targeting a specific site, a primer binding site (PBS) and a reverse transcription (RT) template encoding the desired editing.

Looking at the mechanism of action of PE3, in order to transfer the 'sequence to be edited' in the pegRNA to the target DNA, the reverse transcriptase reads the RNA and attaches the corresponding DNA letter to the end of the cut DNA. An endonuclease in the cell cuts the original DNA sequence and corrects the genome with new nucleotides. Now, one edited strand and one unedited strand remain at the target site. To resolve this discrepancy and permanently mount the edited DNA, another guide RNA guides the prime editor to cut the unedited DNA strand. This cleavage prompts the cell to repair the cleaved strand, and the cell uses the edited strand as a template to complete the editing.

The fusion protein DNA of M-MLV reverse transcriptase and Cas9 H840A nickase is described in Anzalone, A. V. et al. The nucleotide sequence obtained by referring to Nature 576, 149--157 (2019) was used.

NLS - SpCas9H840A -linker-M_MLV_RT- NLS

atgaagaggacagccgatggcagcgagttcgagagccctaagaagaagaggaaggtg agcggcgggag caagcggactgcggatgggtctgagttcgagccaaaagaagaagaggaaggtga

The fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase is Cas9 H840A nickase Cas9 enzyme has two nuclease domains capable of cleaving a DNA sequence, a RuvC domain cleaving a non-target strand and a target Contains HNH domains that cleave strands. Introduction of the H840A substitution in Cas9 in which the histidine at amino acid position 840 is replaced with alanine inactivates the HNH domain. With only the RuvC functional domain, a catalytically damaged Cas9 can introduce a single strand nick. M-MLV reverse transcriptase is an enzyme that synthesizes DNA from a single-stranded RNA template.

M-MLV reverse transcription by amplifying the <NLS+Cas9(H840A)-M-MLVrt(D200N, T306K, W313F, T330P, L603W)+NLS> sequence from the pCMV-PE2 plasmid (Addgene #132775, Addgene, USA) by PCR A BamHI recognition sequence-DNA fragment-SalI recognition sequence including a fusion protein of the enzyme and Cas9 H840A nickase was obtained.

(b) a DNA (DNAsg) expression cassette encoding a guide nucleic acid

The DNAsg fragment of the present invention was composed of a T7 promoter, guide nucleic acid and T7 transcription termination sequence, and was prepared by organic synthesis (Biosynthesis, USA), which was amplified by PCR as a template. At this time, BglII/HpaII recognition sequences were given to both ends using PCR primers. The amplified DNA fragment was treated with BglII/HpaI restriction enzymes to prepare a BglII cleavage site at the 5' end and an HpaI cleavage site at the 3' end. After pCI-neo vector (Promega, USA) was treated with BglII/HpaI restriction enzymes, the prepared DNA fragment was cloned by ligation reaction as shown in <Table 5> using T4 DNA ligase.

The type of guide nucleic acid encoded in the DNAsg fragment will be described in detail below.

① The guide nucleic acid DNAsg fragment used in Cas9 genome editing of the present invention may include a DNA fragment encoding an sgRNA type. The sgRNA used in this example is a 23 bp target nucleotide sequence (5'N ₂₀ NGG-3') within the exon of genomic DNA used for genome editing. Target specificity can be evaluated and selected on the site (http://www.rgenome.net/cas offinder/offinder/). The target sites of the genome-wide prediction website of CRISPR/Cas9 target sites in plants can be retrieved at http://www.genome.arizona.edu/crispr/CRISPRsearch.html.

In the present invention, all RNAs used to cut the target nucleic acid sequence using the CRISPR complex are designed to recognize 18bp of the base PAM sequence of the region to be captured. In this example, 'NGG' (one of N = A, T, C, G) was used as the PAM sequence, and this NGG sequence is a PAM sequence specifically recognized by streptococcus pyogenes, A, T, There is any one base of C or G.

In order to obtain a guide nucleic acid by transcription in the cytoplasm of a cell introduced by DdRp provided by the genome editing RNA/DNA system prepared in the present invention, as shown in <Fig. 13>, sgRNA in which the binding portion is designed as above It was prepared by binding the T7 promoter capable of initiating transcription by binding with T7 RNA polymerase to the encoding template DNA.

The T7 promoter used at this time is 'GGATTCTAATACGACTCACTATAGG' (SEQ ID NO: 1), and the rest of the sgRNA scaffold except for the 18bp sequence that binds to the target nucleic acid in the sgRNA template sequence has the following sequence: 5'-GTTTTAGAGCTAGAAATAGGTGAAGTTTTAAAAGGCTAGTCGTGTTATCAACTTGAATCAGTGTTATCAACTTGAAAAAGTG SEQ ID NO: 38)

Between the T7 promoter sequence of SEQ ID NO: 1 and the sgRNA scaffold of SEQ ID NO: 38, an 18bp target sequence corresponding to 'NNNNNNNNNNNNNNNNNNNN' (N = one of A, T, C, G, SEQ ID NO: 39) is located. Such a target sequence varies depending on the position of the nucleotide sequence to be cut.

As a result, the sequence of the synthesized template DNA is the same as SEQ ID NO: 40 in which the T7 promoter, the target sequence, and the sgRNA template sequence are sequentially combined.

5'-GGATTCTAAT ACGACTCACT ATAGGNNNNN NNNNNNNNNN NNNGTTTTAG AGCTAGAAAT AGCAAGTTAA AATAAGGCTA GTCCGTTATC AACTTGAAAA AGTGGCACCG AGTCGGTGCT TTTTTT-3' (SEQ ID NO: 40)

Transcription termination sequence: 5'-TTTTTT-3' (SEQ ID NO: 41)

In another embodiment, the RNA Poly III type human U6 and H1 promoters may be used in place of the T7 promoter.

U6 promoter (249 nucleotides): 5'-GAGGGCCTAT TTCCCATGAT TCCTTCATAT TTGCATATAC GATACAAGGC TGTTAGAGAG ATAATTGGAA TTAATTTGAC TGTAAACACA AAGATATTAG TACAAAATAC GTGACGTAGA AAGTAATAAT TTCTTGGGTA GTTTGCAGTT TTAAAATTAT GTTTTAAAAT GGACTATCAT ATGCTTACCG TAACTTGAAA GTATTTCGAT TTCTTGGCTT TATATATCTT GTGGAAAGGA CGAAACACC-3 '(SEQ ID NO: 42)

Through the above process, a DNAsg fragment including a DNA fragment encoding the T7 promoter, sgRNA and T7 transcription termination sequence was synthesized by organic synthesis (Biosynthesis, USA).

② DNAsg used for Cas9 genome editing of the present invention may include a DNA fragment encoding a tRNA-sgRNA type, and the nucleotide sequences of tRNA and sgRNA are as follows.

In the present invention, each and all RNA used to cut several target nucleic acid sequences to be captured using the CRISPR complex can be designed to recognize 18bp of the base PAM sequence in the region to be captured, and these are multiple gRNAs as shown in FIG. 14 . Expression cassettes (a) and polycistronic tRNA-gRNA DNA fragments (b) can be prepared.

The nucleotide sequence of the DNA fragment constituting the polycistronic tRNA-gRNA DNA fragment of the present invention is as follows.

Base sequence of the DNA fragment encoding the tRNA used in the present invention: 5'-AACAAAGCAC CAGTGGTCTA GTGGTAGAAT AGTACCCTGC CACGGTACAG ACCCGGGTTC GATTCCCGGC TGGTGCA-3' (SEQ ID NO: 43).

Base sequence of DNA fragment encoding sgRNA: 5'- GAAATTAATA CGACTCACTA TA GGN ₁₈ GTTTT AGAGCTAGAA ATAGCAAGTT AAAATAAGGC TAGTCCGTTA TCAACTTGAA AAAGTGGCAC CGAGTCGGTG CTTTT- 3' (SEQ ID NO: 44).

The initial 22 bp (indicated in italics) is the promoter of T7 RNA polymerase used for in vitro transcription. This is followed by the GGN ₁₈ sequence (underlined), representing 20 bp consistent with the target (spacer) sequence. The remaining 80 bp (type in bold) encodes a chimeric fusion of crRNA and tracrRNA.

As the target sequence, sgRNA was designed for the target sequence 5'-GGTGAGTGAG TGTGTGCGTG TGG - 3' (SEQ ID NO: 45) in the human VEGFA gene.

Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signaling protein produced by cells that stimulates blood vessel formation. VEGF is a subfamily of growth factors, the platelet-derived growth factor family of cystine knot growth factors. They are important signaling proteins involved in angiogenesis (new formation of the embryonic circulation) and angiogenesis (growth of blood vessels from the existing vasculature).

Multiple isoforms of VEGF-A exist from alternative splicing of mRNA from exon 8 of the VEGFA gene. In humans, there are VEGF121, VEGF121b, VEGF145, VEGF165, VEGF165b, VEGF189 and VEGF206.

The normal function of VEGF is to create new blood vessels during embryonic development, new blood vessels after injury, muscles after exercise, and new blood vessels to bypass clogged blood vessels (class circulation). Solid cancers cannot grow beyond their limited size without an adequate blood supply. Cancers capable of expressing VEGF can grow and metastasize. Overexpression of VEGF can lead to vascular disease in the retina of the eye and other parts of the body.

For DNAsg of Cas9 genome editing of this example, DNA fragments were prepared in this order by the T7 promoter, the tRNA-sgRNA encoding DNA, and the T7 transcription termination sequence.

③ For genome editing using DNA-guided endonuclease, HuAgo, phosphorylated single-stranded DNA type was used in the DNAge expression cassette instead of DNAsg, and it was organically synthesized (Bioneer, Korea).

A DNA-guided endonuclease, HuAgo, can bind to phosphorylated ssDNA and guide it to a target site and perform DNA double-strand repair at gDNA sites. In addition, phosphorylated ssDNA guide nucleic acid was a sequence of 24 specific targets of the target genome, and phosphorylated ssDNA was synthesized at the 5*?**?* ends.

PD-L1 is a protein located on the surface of cancer cells or in hematopoietic cells. Also called CD274, B7-H1. When PD-L1 and PD-L2, which are proteins on the surface of cancer cells, bind to PD-1, which is a protein on the surface of T cells, T cells cannot attack cancer cells. Immunotherapy inhibits the evasion function of cancer cells by binding to the PD-1 receptor on T cells. This is how MSD's Keytruda and Opdivo work.

To test whether HuAgo can be used to modify endogenous human genes such as CD274 or PDL-1 for potential applications in cancer therapy, the guide DNA oligo GD5 (5'-pGCGAATTACT) complementary to the region of exon 3 of the CD274 gene GTGAAAGTCA ATGG-3') (SEQ ID NO: 46) was used.

5' phosphorylation of the guide DNA oligo was performed using T4 polynucleotide kinase (New England BioLab) to prepare guide nucleic acids.

④ Prime editing guide nucleic acid DNAsg identifies the target sequence to be edited and instructs to nick the Cas9 H840A nickase portion of the fusion protein with a sequence that can encode new genetic information replacing the target sequence to the unedited DNA strand contains a DNA fragment encoding a prime editing guide RNA (pegRNA) composed of a single guide RNA (sgRNA) that .

In the prime editing approach, reverse transcriptase and catalytically impaired Cas9 nickase are fused to a protein PE complex and a target site, and a multifunctional prime editing guide that additionally serves as a template for reverse transcription (RT) Two major components of the multifunctional prime editing guide (pegRNA) are involved. The pegRNA is similar to a standard single-guide RNA (sgRNA), but has an additional 3' end extension sequence. The 3' end extension sequence consists of an RT template encoding the desired edit and a primer binding site (PBS) that anneals to the target genomic site to prime the RT reaction. These additional components significantly increase the complexity of pegRNA design compared to standard sgRNA.

In this example, pegFinder (http://pegfinder.sidichenlab.org), a simplified web tool for rapidly designing candidate pegRNAs, can be used.

The pegRNA consisted of an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a reverse transcriptase (RT) template sequence. During genome editing, the primer binding site allows the 3' end of the nick DNA strand to hybridize to the pegRNA, whereas the RT template serves as a template for the synthesis of the edited genetic information.

In this example, the HEK3 gene having the target nucleotide sequence to be edited encodes a member of the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors are mediated throughout the developmental system, particularly in the nervous system. The protein encoded by this gene functions as a receptor for ephrins A2, A3 and A5 and plays a role in short-range contact-mediated axon guidance during development of the mammalian nervous system.

The pegRNA of this example is composed of sgRNA target sequence-sgRNA scaffold-3'end extension (RT-PBS). The components of the nucleotide sequence of the guide nucleic acid and pegRNA of this embodiment are in <Table 4>, and the nucleotide sequence is as follows.

Components of the pegRNA used name base sequence sgRNA (20 nt) GGCCCAGACT GAGCACGTGA (SEQ ID NO: 48) RT template (13 nt): TCTGCCATCA AAG (SEQ ID NO: 49) PBS (primer binding sequence, 13 nt): CGTGCTCAGT CTG (SEQ ID NO: 50) 3' end extension (26 nt) TCTGCCATCA AAG CGTGCTC AGTCTG (SEQ ID NO: 51)

Base sequence of pegRNA: 5'-GGCCCAGACT GAGCACGTGA GTTTTAGAGC TAGAAATAGC AAGTTAAAAT AAGGCTAGTC CGTTATCAAC TTGAAAAAGT GGCACCGAGT CGGTGC TCTG CCATCAAAG C GTGCTCAGTC TG -3' (SEQ ID NO: 47)

Target sequence of PE3 2° nicking sgRNA: TGGGCCCCAA GGATTGACCC

PE3 2° nicking sgRNA has a target sequence of 20 bp between the T7 promoter sequence of SEQ ID NO: 1 and the sgRNA scaffold of SEQ ID NO: 38. Such a target sequence varies depending on the position of the nucleotide sequence to be cut.

As a result, the sequence of the synthesized template DNA is the same as SEQ ID NO: 40 in which the T7 promoter-target sequence-sgRNA scaffold sequence is sequentially combined.

PE3 2° nicking sgRNA: 5'-TAATACGACT CACTATAGGT GGGCCCCAAG GATTGACCCG TTTTAGAGCT AGAAATAGCA AGTTAAAATA AGGCTAGTCC GTTATCAACT TGAAAAAGTG GCACCGAGTC GGTGCTTTTT TT-3' (SEQ ID NO: 40)

Transcription termination sequence: 5'-TTTTTT-3' (SEQ ID NO: 41)

The pegRNA and sgRNA cassettes.

T7 promoter-sgRNA target sequence-sgRNA scaffold-3'end extension-T7 terminator-T7 promoter-sgRNA target sequence-sgRNA scaffold-T7 terminator

TAATACGACT CACTATAGGN NNNNNNNNNN NNNNNNNNGT TTTAGAGCTA GAAATAGCAA GTTAAAATAA GGCTAGTCCG TTATCAACTT GAAAAAGTGG CACCGAGTCG GTGCNNNNNN NNNNNNNNNN NNNNNNNNNN NNNTTTTTTA ATACGACTCA CTATAGGNNN NNNNNNNNNN NNNNNNGTTT TAGAGCTAGA AATAGCAAGT TAAAATAAGG CTAGTCCGTT ATCAACTTGA AAAAGTGGCA CCGAGTCGGT GCTTTTTT

Prime editing guide nucleic acid, DNAsg fragment was prepared. <T7 promoter, DNA encoding pegRNA, T7 transcription termination sequence> and <T7 promoter, DNA encoding PE3 2° nicking sgRNA, T7 transcription termination sequence> in order A DNA fragment encoding the constructed <pegRNA+sgRNA> type was synthesized (Biosynthesis, USA).

DNA fragment encoding the guide nucleic acid of Prime editing for HEK3 gene:

TAATACGACT CACTATAGGG CCCAGACTGA GCACGTGAGT TTTAGAGCTA GAAATAGCAA GTTAAAATAA GGCTAGTCCG TTATCAACTT GAAAAAGTGG CACCGAGTCG GTGC TCTGCC ATCAAAG CGT GCTCAGTCTG TTTTTTAATA CGACTCACTA TAGGTGGGCC CCAAGGATTG ACCCGTTTTA GAGCTAGAAA TAGCAAGTTA AAATAAGGCT AGTCCGTTAT CAACTTGAAA AAGTGGCACC GAGTCGGTGC TTTTTT

1-4. Preparation of auto_DdRp or DNAge virus expression cassettes

The auto_DdRp DNA expression cassette or DNAge expression cassette of the present invention can be prepared using a plasmid or a viral vector. In this example, auto_DdRp and DNAge virus expression cassettes were prepared using viral vectors. Auto DdRp DNA fragment is [T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone-SL], DNAge is <DNAge+DNAsg> fragment and <T7 promoter - 5 'UTR - DNAen - muag - A64 - C30 - histone-SL - T7 promoter - DNAsg - T7 transcription termination sequence>.

Auto_DdRp and DNAge virus expression by inserting the prepared auto_DdRp and DNAge DNA fragment into the AdEasy Adenoviral Vector System (Agilent Technologii, USA) and ViraSafe™ Lentiviral Exprision System (Cell Biolabs, USA) that can construct a stable cell line as a mammalian expression vector Cassettes can be prepared.

The DNAen of the present invention may include NLS-SpCas9-HF1(N497A/R661A/Q695A/Q926A)-NLS, NLS-NgAgo-NLS and NLS-SpCas9H840A-linker-M_MLV_RT-NLS.

In this example, NLS-SpCas9H840A-linker-M_MLV_RT-NLS was used.

In the present invention, the target gene of the DNAge includes, but is not limited to, VEGF, PD-L1 and HEK3 genes. The type of guide nucleic acid is also determined according to the selected target endonuclease. sgRNA type if the selected target endonuclease is NLS-SpCas9-HF1(N497A/R661A/Q695A/Q926A)-NLS, phosphorylated ssDNA type if NLS-NgAgo-NLS, and <pegRNA type if NLS-SpCas9H840A-linker-M_MLV_RT-NLS +sgRNA> type.

<Adenovirus system>

In general, prokaryotes and prokaryotes use different promoters for expression cassettes. The expression cassette used in the self-transcribed RNA/DNA system of the present invention may use a promoter corresponding to DdRp commonly selected for prokaryotes and prokaryotes, particularly mammals, plants, and the like. For example, when DdRp is T7 Polymerase, it can be prepared using the T7 promoter.

In this example, the auto_DdRp DNA or DNAge fragment was recombined in the pShuttle of the adenovirus system, and this was called "auto_DdRp or DNAge adeno expression cassette". The transfection system using capped SM DdRp RNA@LS and auto_DdRp DNA or DNAge adeno expression cassette was called auto_DdRp DNA or DNAge adeno expression system. In addition, the auto_DdRp DNA fragment was recombined in the adenovirus system and this was called "auto_DdRp adeno expression cassette". The transfection system using the capped SM DdRp RNA@LS and the auto_DdRp adeno expression cassette was called the auto_DdRp adeno expression system.

In this example, the pShuttle vector in the AdEasy Adenoviral Vector System (Agilent Technologii, USA) was used.

To prepare the auto_DdRp DNA or DNAge adeno expression cassette, PCR with the auto_DdRp or DNAge expression cassette as a template to convert the auto_DdRp DNA fragment to T7 promoter - 5'UTR - DdRp DNA (or chimeric DdRp DNA) - muag - A64 - C30 - histone-SL ) or DNAge fragments were prepared in the form of <DNAen+DNAsg> in large quantities.

First, the <T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone-SL> region was amplified using the auto_DdRp expression cassette prepared in Example 1-2 as a template. At this time, both ends of the PCR product using a forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG GGAGACCAC AACGGUUUCC CUCU -3', SEQ ID NO: 43) and a reverse primer (5'- GTCGAC GGT GGCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 51) XhoI/SalI recognition sequence was given.

And the DNAge target gene includes VEGF, PD-L1 and HEK3 genes, and in this example, a DNAge fragment targeting HEK3 was prepared by PCR.

The DNAge fragment of the present invention is in the form of <DNAen+DNAsg> with reference to Example 1-3 (T7 promoter - 5'UTR - <NLS-SpCas9H840A-linker-M_MLV_RT-NLS> - muag - A64 - C30 - histone SL) One DNA fragment consisting of a DNA fragment encoding (T7 promoter - pegRNA-sgRNA - T7 transcription termination sequence) and a DNA fragment encoding This was amplified by PCR as a template. At this time, using a forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG-3', SEQ ID NO: 74) and a reverse primer (5'- GTCGAC AAAAAA GCACCGACTCGGTGCCACTTTTTCAAGTTG-3', SEQ ID NO: 44) at both ends using XhoI / SalI A PCR product having a recognition sequence was obtained.

The amplified PCR products were treated with XhoI/SalI restriction enzymes and cut to prepare an auto_DdRp or DNAge DNA fragment having an XhoI recognition sequence at the 5' end and a SalI recognition sequence at the 3' end. The digested auto_DdRp DNA or DNAge fragment was inserted into the pShuttle vector linearized with XhoI/SalI restriction enzymes using T4 DNA ligase. 2-3 μl of the pShuttle vector recombinant solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated at 37° C. overnight. Pick 6 to 12 colonies and inoculate them in 3 ml of LB growth medium containing 50 μg/ml of ampicillin, respectively. Incubate overnight on a shaker at 37°C. Recombinant pShuttle vectors were isolated from auto_DdRp DNA and DNAge fragments using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen, USA). The recombinant plasmid was digested with XhoI/SalI restriction enzymes and gel electrophoresis was performed to confirm clones containing auto_DdRp DNA and a DNAge expression cassette (FIG. 15).

The prepared auto_DdRp DNA or DNAge fragment is placed in the pShuttle vector, and the composition of auto DdRp DNA and DNAge fragment is <T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64 - C30 - histone- SL> and <T7 promoter - 5'UTR - DNAen - muag - A64 - C30 - histone-SL- T7 promoter - DNAsg - T7 transcription termination sequence>.

Production of the recombinant auto_DdRp or DNAge adenovirus of the present invention was performed according to the instructions of the AdEasy Adenoviral Vector System (Agilent Technologii, USA). First, the recombinant auto_DdRp or DNAi expression cassettes were linearized with Pme I. Linearized auto_DdRp or DNAi expression cassettes were co-transformed into BJ5183 bacteria with pAdEasy-1, a supercoil viral DNA plasmid. Transformants are selected for kanamycin resistance. Recombination was confirmed by the method using a restriction enzyme (FIG. 16). Restriction of recombinant Ad plasmid DNA with Pac I resulted in large fragments of ~30 kb and small fragments of 3.0 kb (if recombination occurred between the left arm) or 4.5 kb (if recombination occurred at the original replication site).

When recombination was confirmed, it was mass-produced using the XL10-Gold strain without recombination ability and digested with Pac I. Purified recombinant Ad plasmid DNA was digested with Pac I and inverted terminal repeat (ITR) was exposed. To the tube containing the DNA, 25 μl of ViraPack Transfection Kit's Solution I and 250 μl of Solution II were added and incubated at room temperature for 10 minutes. . Plates containing AD-293 cells in MBS-containing medium were prepared in an incubator. The DNA suspension was gently mixed by pipetting up and down to resuspend the DNA precipitate, then the DNA suspension was added dropwise to the plate to treat AD-293 (or HEK293) cells.

After the tissue culture plates were incubated in an incubator at 37° C. for 3 hours, the medium was removed from the plates and replaced with 4 ml of growth medium supplemented with 25 μM chloroquine (see medium and reagent preparation). After the plate is incubated for an additional 6 h in a 37 °C incubator, the growth medium containing 25 μM chloroquine is removed and replaced with 4 ml growth medium (chloroquine).

Culture plates were incubated at 37° C. for 7-10 days and growth medium was replenished as needed (based on media color). When the cells appeared to adhere well to the plate, the medium was replaced with 4 ml of fresh medium. Alternatively, when cells isolated from the growth medium were observed, the same volume of fresh medium was added to the existing medium and cultured. A primary virus stock was obtained from this cell culture solution.

The growth medium was carefully removed from the adenovirus producing AD-293 plates and the cells were washed once with PBS. 0.5 ml of PBS was added to each plate of cells to be harvested. Cells were collected by holding the plate at an angle and scraping the cells into a pool of PBS with a cell lifter. The cell suspension was transferred to a 1.7 ml screw cap microcentrifuge tube. The cell suspension was frozen/thawed 4 times by alternating the tubes between the dry ice-methanol bath and the 37°C water bath and vortexing briefly after each thawing. Cell debris was collected by microcentrifugation at 12,000 × g for 10 min at room temperature. The supernatant (primary virus stock) was transferred to a fresh screw cap microcentrifuge tube. This primary virus stock can be stored at -80°C for over 1 year.

The titer of the prepared primary virus stock was determined by the method of assay 1 (Korea Food and Drug Administration) of adenovirus-derived vector gene therapy, and was maintained at 10 ⁷ -0 ⁸ pfu/ml.

<Retivirus system>

In this example, the auto_DdRp DNA or DNAge fragment obtained by PCR using the auto_DdRp or DNAge expression cassette prepared in the <Plasmid expression cassette> above was recombined in the shuttle vector of the retivirus system in this example, and this was called "auto_DdRp or DNAge lenti expression cassette". A transfection system using capped SM DdRp RNA@LS and an auto_DdRp or DNAge lenti expression cassette was referred to as an auto_DdRp or DNAge lenti expression system. In addition, the auto_DdRp DNA fragment was recombined in the retivirus system and this was called "auto_DdRp lenti expression cassette". The transfection system using the capped SM DdRp RNA@LS and the auto_DdRp lenti expression cassette was called the auto_DdRp lenti expression system.

In this example, the pSMPUW-IRES-Blasticidin Lentiviral Exprision Vector, the shuttle vector pSMPUW, from the ViraSafe ^™ Lentiviral Exprision System (Cell Biolabs, USA) was used.

In this example, the pShuttle vector in the AdEasy Adenoviral Vector System (Agilent Technologii, USA) was used.

In order to prepare an auto_DdRp DNA or DNAge lenti expression cassette, the auto_DdRp DNA fragment and the target gene of the DNAge include VEGF, PD-L1 and HEK3 genes, and in this embodiment, the DNAge fragment targeting HEK3 is PCR method was prepared with

The DNAge fragment of the present invention is in the form of <DNAen+DNAsg>, first, using the auto_DdRp expression cassette prepared in Example 1-2 as a template <T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - muag - A64-C30-histone-SL> sites were amplified. At this time, both ends of the PCR product using a forward primer (5'- GGATCC TAAT ACGACTCACT ATAG GGAGACCAC AACGGUUUCC CUCU -3', SEQ ID NO: 43), and a reverse primer (5'- GTCGAC GGT GGCTCTAAAA GAGCCTTTGG-3', SEQ ID NO: 51) BamHI / BglI recognition sequence was given.

With reference to Example 1-3 (T7 promoter - 5'UTR - <NLS-SpCas9H840A-linker-M_MLV_RT-NLS> - muag - A64 - C30 - histone SL) and (T7 promoter - pegRNA-sgRNA) - One DNA fragment consisting of a DNA fragment encoding T7 transcription termination sequence) was synthesized organically (Biosnthesis, USA). This was amplified by PCR as a template. At this time, BamHI / BglI at both ends using a forward primer (5'- GGATCC TAAT ACGACTCACT ATAG-3', SEQ ID NO: 74) and a reverse primer (5'- GTCGAC AAAAAA GCACCGACTCGGTGCCACTTTTTCAAGTTG-3', SEQ ID NO: 44) A PCR product having a recognition sequence was obtained.

The amplified PCR products were treated with BamHI/BglII restriction enzymes and cut to prepare an auto_DdRp or DNAge DNA fragment having a BamHI recognition sequence at the 5' end and a BglII recognition sequence at the 3' end. The digested auto_DdRp DNA and DNAge fragments were inserted into the pSMPUW vector linearized with BamHI/SalI restriction enzymes using T4 DNA ligase. 2-3 μl of the pSMPUW vector recombinant solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated overnight at 37°C. Pick 6 to 12 colonies and inoculate them into 3 ml of LB growth medium containing 50 μg/ml of ampicillin, respectively. Incubate overnight on a shaker at 37°C. Recombinant pShuttle vectors were isolated from auto_DdRp DNA and DNAge fragments using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen, USA). The recombinant plasmid was digested with BglII/HpaI restriction enzymes and gel electrophoresis was performed to confirm clones containing auto_DdRp DNA and a DNAge expression cassette (FIG. 17).

Recombinant auto_DdRp and DNAge Lenti expression cassette virus production of the present invention was performed according to the instructions of pSMPUW Universal Lentiviral Exprision Vector (Cell Biolabs, USA) and Lipofectamine LTX Reagent (ThermoFisher, USA) as follows.

One day before transfection, plate enough 293T cells or 293LTV cells (Cat. # LTV-100) and incubate to cover 70-80% of the culture vessel on the day of transfection. The molar ratio of pSMPUW, envelope and packaging plasmids prepared in the present invention as a transfer plasmid to Lipofectamine ^™ Plus (Invitrogen), that is, pSMPUW: pCMV-VSV-G: pRSV-REV: pCgpV vector Cells are transfected by mixing 3:1:1:1.

Harvest the lentiviral supernatant 36-72 hours after transfection. The supernatant can be harvested 2-3 times every 12 hours. Store at 4°C during the collection period. Collect the collected supernatant and centrifuge at 1500 rpm for 5 min to remove cell debris and filter at 0.22 µm. The supernatant can be used directly or purified/concentrated if necessary. For long-term storage, aliquot the supernatant and store at -80°C.

The harvested and stored lentivirus solution was highly concentrated in virus using the ViraBind ^™ Lentivirus Concentration and Purification Kit (Cat. No. VPK-090, Cell Biolabs, USA) before transfection into host cells. To ensure consistent viral transduction into host cells for the auto_(DdRp+DNAge) lentivirus of the present invention, lentivirus using p24 ELISA, QuickTiter ^™ Lentivirus Titer Kit (Cat. No. VPK-107, Cell Biolabs, USA) The titer was measured and the titer of the purified and concentrated virus stock was maintained at 10 ⁶ -0 ⁷ pfu/ml.

It is difficult for target cells to be infected with the auto_DdRp and DNAge lentivirus of the present invention, and transduction efficiency may be low without auxiliary reagents. ViraDuctin ^™ Lentivirus Transduction Kit (catalog number LTV-200, Cell Biolabs, USA) can be used to promote virus and cell interaction to increase transduction efficiency.

<Binary vector for plant transfection>

In this example, the auto_(DdRp+DNAge) DNA fragment was recombined in a binary vector (Ti plasmid) for plant transfection, and this was called “auto_(DdRp+DNAge) Ti expression cassette”. The transfection system using the capped DdRp RNA@LS and auto_(DdRp+DNAge) Ti expression cassette was called the auto_(DdRp+DNAge) Ti expression system.

Point mutations in the acetolactate synthase (ALS) gene, which encodes a key enzyme in the biosynthetic pathway of branched-chain amino acids, may confer resistance to ALS inhibitors such as chlorsulfuron . /doi.org/10.1101/2020.06.18.159111 ).

In this example, an auto_(DdRp+DNAge) Ti expression system was prepared by targeting the potato StALS gene in order to introduce simultaneous conversion and transfer mutations in specific genes with the CRISPR-mediated plant prime editing (PPE) RNA/DNA system of the present invention did

To prepare the auto_DdRp expression cassette and the auto_(DdRp+DNAge) Ti expression cassette including the DNAge fragment targeting the potato StALS gene, the following procedure was performed.

First, the DNAge fragment in this example is <T7 promoter - 5'UTR - NLS-SpCas9H840A-linker-M_MLV_RT-NLS - Nos-terminator-T7 promoter-T1-sgR-rtT-PBS-T7 terminator-T7 promoter- T2 - sgR-T7 terminator > structure was organically synthesized (Biosynthesis, USA).

Nos- Termination sequence:

GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGA ATCCTGTTGC CGGTCTTGCG 60

ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATG TAATAATTAA CATGTAATGC 120

ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCC CGCAATTATA CATTTAATAC 180

GCGATAGAAA ACAAAATATA GCGCGCAAAC TAGGATAAAT TATCGCGCGC GGTGTCATCT 240

ATGTTACTAG ATC 253

<FIG. 18> is a prime editing strategy for correct modification of the potato ALS gene, and the configuration of PE3 pegRNA is as follows.

DNA fragments encoding pegRNA and sgRNA;

T7 promoter - T1-sgR-rtT-PBS-T7 terminator-T7 promoter- T2- sgR-T7 terminator

TAATACGACT CACTATAG GNNNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TTTTTTT TAAT ACGACTCACT ATAG GNNGTNNNNTTAGATAGCTAGAAGATTAGCTTAGACTCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGA

DNA fragment encoding PE3 potato ALS pegRNA:

TAAT ACGACTCACT ATAG GCTATTACGGGTCAAGTGCCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAATCATCCTTCTGGACACTTGACCCGTA TTTACTTT TAAT ACGACTCACT ATAG GTAAGTCACGTAACCAGCTAGAGTAGTCGATTAGAGCTAAGAGTAGTTAGTTAGAGCTAAGCAGTT

<T7 promoter - 5'UTR - NLS-SpCas9H840A-linker-M_MLV_RT-NLS - Nos-terminator-T7 promoter-T1-sgR-rtT-PBS-T7 terminator-T7 promoter- For the preparation of the T2 -sgR-T7 terminator>structure, the DNA fragment synthesized earlier was amplified by PCR as a template. At this time, EcoRI / XhoI recognition sequences at both ends using a forward primer (5'- GAATTC TAAT ACGACTCACT ATAG-3', SEQ ID NO: 72), a reverse primer (5'- CTCGAG AAAAAA GCACCGACTCGGTGCCACTTTTTCAAGTTG-3', SEQ ID NO: 73) PCR products were obtained.

With reference to Example 1-2, an auto_DdRp DNA fragment (T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R-T7RNAP DNA) - Nos termination sequence) having an XhoI / SalI site at the end was prepared, and PCR method was amplified to It is also possible to insert NP868R-T7RNAP DNA in place of the DdRp DNA sequence. At this time, a forward primer (5'- CTCGAG TAAT ACGACTCACT ATAG-3', SEQ ID NO: 72) and a reverse primer (5'- GTCGAC GAT CAGTAACAT AGATG-3', SEQ ID NO: 73) were used at both ends of the XhoI / SalI recognition sequence A PCR product with this was obtained.

The prepared DNAge fragment and auto_DdRp DNA fragment were digested with XhoII restriction enzyme and ligated with ligase to auto_(DdRp+DNAge) fragment (T7 promoter - 5'UTR - DdRp DNA (or chimeric NP868R) with EcoRI/SalI sites at both ends. -T7RNAP DNA) - Nos terminator - T7 promoter - 5'UTR - NLS-SpCas9H840A-linker-M_MLV_RT-NLS - Nos-terminator-T7 promoter-T1-sgR-rtT-PBS-T7 terminator-T7 promoter- T2- sgR-T7 terminator) was prepared.

This was cloned into the EcoRI/SalI site in pCAMBIA1305.1 (AbcAm, USA) and designated as the auto_(DdRp+DNAge) Ti expression cassette. In addition, a binary vector (Ti plAsmid) containing an auto_DdRp DNA fragment was referred to as an auto_DdRp Ti expression cassette.

2-3 μl of the recombinant vector cloning reaction solution was transduced into E. coli, plated on a selection plate, and incubated overnight at 37°C.

Pick 6 to 12 colonies and inoculate them into 3 ml of LB selection medium at 50 μg/ml each. Incubate overnight on a shaker at 37°C. Plasmid DNA was isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen). Recombinant binary vector (Ti plAsmid) was digested with EcoRI/SalI and gel electrophoresis was performed to screen auto_(DdRp+DNAge) Ti expression cassette clones (FIG. 19).

The auto_(DdRp+DNAge) Ti expression cassette thus obtained was transferred to E. coli DH5α and amplified. The auto_(DdRp+DNAge) Ti expression cassette was then introduced into AGrobActerium tumefAciens LBA4404 using a freeze-thaw method to prepare an auto_(DdRp+DNAge) Ti expression system.

The auto_(DdRp+DNAge) Ti expression cassette of the present invention can be transformed into an autologous lineage of tomato plants through the A. tumefAciens LBA4404 mediated method.

1-5. Preparation of vector for nuclear-dependent mRNA expression based on CMV promoter

Using the pCI-neo Mammalian Exprision Vector (Promega, USA), pCI_DNAge capable of expressing DNAge was prepared in the nucleus by CMV immediAte-early enhAncer/promoter reGion and β-Globin/IGG chimeric intron.

For genome editing, target endonucleases including SpCas9-HF1, NLS-NgAgo-NLS, M-MLV reverse transcriptase and Cas9 H840A nickase, and guide nucleic acids corresponding thereto were used.

With reference to Example 1-3 (T7 promoter - 5'UTR - <NLS-SpCas9H840A-linker-M_MLV_RT-NLS> - muag - A64 - C30 - histone SL) and (T7 promoter - pegRNA-sgRNA) - One DNA fragment consisting of a DNA fragment encoding T7 transcription termination sequence) was amplified by PCR as a template. At this time, using a forward primer (5'- CTGGAC TAAT ACGACTCACT ATAG-3', SEQ ID NO: 74) and a reverse primer (5'- TCTAGA AAAAAA GCACCGACTCGGTGCCACTTTTTCAAGTTG-3', SEQ ID NO: 44) at both ends using XhoI/XbaI A PCR product having a recognition sequence was obtained.

The amplified PCR products were treated with XhoI/XbaI restriction enzymes and cut to prepare a DNAge DNA fragment having an XhoI recognition sequence at the 5' end and an XbaI recognition sequence at the 3' end. The cut DNAge fragment was inserted into the pCI-neo vector linearized with XhoI/XbaI restriction enzymes using T4 DNA ligase. 2-3 μl of the pSMPUW vector recombinant solution was transduced into E. coli, plated on an ampicillin selection plate, and incubated at 37° C. overnight. Pick 6 to 12 colonies and inoculate them in 3 ml of LB growth medium containing 50 μg/ml of ampicillin, respectively. Incubate overnight on a shaker at 37°C. Recombinant pCI-neo vectors with DNAge fragments were isolated using standard procedures or a commercial Qiaprep Miniprep kit (Qiagen, USA). The recombinant plasmid was digested with XhoI/XbaI restriction enzymes and gel electrophoresis was performed to confirm clones containing auto_DdRp DNA and a DNAge expression cassette (FIG. 20).

Example 2. Preparation of liposome delivery system

A liposome delivery system was prepared by using the linear DdRp RNA expression cassette prepared in the present invention, various types of circular plasmid expression cassettes, and combinations thereof with Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA).

1 μg of the mixture of the prepared DdRp RNA expression cassette, plasmid expression cassette, and a combination thereof (same molar ratio) and 100 μl of Opti-MEM were mixed in a 1.5 ml tube, and 5 μl of Lipofectamin2000 and 100 μl of serum-free medium were mixed with another 1.5 ml After mixing in a tube, it was left at room temperature for 5 minutes. Thereafter, 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 replaced with a new medium after 4 hours.

Auto(-)_DNAge@LS, a complex of the DdRp RNA/DNAge system and liposome, was referred to as “DdRp RNA/DNAge@LS”, a complex of the DdRp RNA expression cassette and DNAge expression cassette and liposome. T7 polymerase repeatedly synthesized from the stabilized capped CM_DdRp mRNA can be maintained at high concentrations in the cytoplasm for a long time.

In addition, the DdRp RNA/auto_DdRp+DNAge system or the DdRp RNA/auto_(DdRp+DNAge) system and auto_DNAge@LS, which is a complex of the system, is a DdRp RNA expression cassette, auto_DdRp expression cassette, and “DdRp RNA, a complex of a DNAge expression cassette and liposome” /auto_DdRp+DNAge@LS or DdRp RNA expression cassette, DdRp RNA/auto_(DdRp+DNAge)@LS, a complex of auto_(DdRp+DNAge) expression cassette and liposome. By binding to the T7 promoter of the auto_DdRp expression cassette by T7 polymerase or auto_(DdRp+DNAge) expression cassette by T7 polymerase synthesized from initially stabilized capped CM DdRp mRNA, T7 polymerase can be maintained at a high concentration in the cytoplasm for a long time as a result of autotranscription.

As a control, DOPC liposome nazoparticles loaded with pCI-neo_DNAge were prepared, and were called pCI-neo_DNAge@LS, a complex of pCI-neo_DNAge and liposomes.

Example 3. Transfection of expression-repressed RNA/DNA system

The self-transcribed RNA/DNA system of the present invention is composed of an RNA component and a DNA component, and the carrier thereof can be variously used, such as a linear nucleic acid fragment, a circular plasmid, and a virus, or a combination thereof. Various methods can be selected when the carrier of the prepared self-transcribed RNA/DNA system is non-viral. In this example, the liposome type was selected, but the present invention is not limited thereto.

Transfection of the target cells of the expression-suppressing RNA/DNA system of the present invention was performed as follows in order to smoothly obtain stabilized transformed cells. Of course, transformation can be performed on the same.

The first transfection is a step in which an expression cassette containing a DNAge encoding the mRNA of interest is transfected and transformed cells are selected using a selective medium, and the second transfection is the cell stabilized in the selective medium by the first transfection. In this step, a DdRp RNA expression cassette is introduced into the target to express the mRNA of interest and the protein they encode by the DdRp synthesized in the transformed cell.

<1st transfection>

- Plasmid carrier

In the case of the plasmid delivery system, according to the instructions of pCI-neo Mammalian Exprision Vector (Promega, USA) and Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA), target cells were prepared and 1x10 ⁵ cells were seeded into 96-wells. The next day, the liposome gene delivery system prepared in Example 2 was prepared in a total volume of 100 μl with a cell culture medium, and then treated in each well. 37 ℃, 5% CO ₂ After culturing for 24 hours in an incubator, the cells were washed with 1X PBS, treated with trypsin to remove the cells, and then transferred to a 100 mm culture dish and incubated.

For transfection of the plasmid delivery system, transfection can be carried out by making the RNA/DNA system together as a liposome delivery system, or by dividing them, each liposome can be produced and each transfected.

For the selection of stable transformed cells by the liposome delivery system, after transfection, the transfected cells were selected with antibiotic G-418. After transfection, cells were inoculated at a low cell density, and G-418 antibiotics were added at a concentration of 400 to 600 μg/ml for selection, and at a G-418 concentration of 200 to 400 μg/ml to maintain stable transformants. processed.

- virus carrier

In the case of the viral carrier, 1x10 ⁵ target cells were prepared according to the instructions of AdEasy Adenoviral Vector System (Agilent Technologii, USA), pSMPUW Universal Lentiviral Exprision Vector (Cell Biolabs, USA) and Lipofectamine 2000 Transfection Reagent (ThermoFisher, USA). Dog cells were seeded into 96-wells. According to the multiplicity of infection (MOI) to be treated in the cells the next day, the viral gene carrier was made into a cell culture medium in a total volume of 100 μl, and then treated in each well. After treatment with the viral gene carrier, after culturing for 24 hours at 37° C., 5% CO 2 in an incubator, the cells were washed with 1X PBS, and the cells were removed by treatment with trypsin and then transferred to a 100 mm culture dish and cultured.

In the case of lentivirus, the medium containing the antibiotic blasticidin at a concentration of 5 μg/ml was changed once every 2 to 3 days.

<Secondary transfection>

The second transfection is a step of transfecting the first transfected cells with the DdRp RNA@LS prepared in Example 2 to provide the first T7 Polymerase acting on the T7 promoter. DdRp RNA@LS transfection was performed on the cells immediately after inoculation of the selected stabilized cells and adenovirus gene carrier after treatment with the plasmid and the lentiviral carrier of the first transfection.

Example 4. T7 polymerase expression persistence experiment

Controls of the present invention 5 pmol pCI-neo_RSV-F@LS, 5 pmol DdRp RNA/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_DdRp/RSV-F DNAi@LS, 5 pmol DdRp RNA/auto_(DdRp/RSV) -F DNAi)@LS, 3x10 ⁴ MOI auto_(DdRp/RSV-F DNAi) adeno expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) adeno expression cassette] or 10 ⁴ MOI auto_( DdRp/RSV-F DNAi) lenti expression system [system consisting of DdRp RNA@LS and auto_(DdRp/RSV-F DNAi) lenti expression cassette] was transfected into 293T cells by the method of Example 3, respectively, 0, 24 , T7 polymerase mRNA and protein in 293T cells were analyzed at 48 and 72 hours. In this example, auto_DdRp of DdRp was used as a self-transcribed RNA/DNA system with T7RNAP or NP868R-T7RNAP.

- T7 polymerase mRNA analysis

For T7 polymerase mRNA analysis of the transgenic 293T cells, total RNA was isolated from 293T cells at 0, 24, 48 and 72 hours after the second transfection and analyzed by quantitative PCR (qPCR) with SYBR Green.

Specifically, total RNA was extracted from the transfected 293T cells using the RNeAsy mini kit (Qiagen). A total of 150 ng of total RNA was reverse transcribed into cDNA (SuperScript II Reverse Transcriptase, Invitrogen), and the cDNA was used for analysis using KAPA SYBR FAST qPCR Kits (Sigma aldrich, USA).

Before preparing the qPCR reaction, KAPA PROBE FAST Bio-Rad iCycler ™ qPCR Master Mix (2X), template DNA, primers and probes were thoroughly mixed as presented in Table 3.

qPCR constitutive reagents Final Concentration 20 μl rxn PCR grade water up to 20 μl 7.2 μl qPCR Master Mix (2X) 1X 10 μl Forward Primer (10 μM) 200 nM 0.4 μl Reverse Primer (10 μM) 200 nM 0.4 μl Template DNA (10 ng/1 μl) 1 ng/μl 2.0 μl

Forward primer 5'-TCACGACTCC TTCGGTACCA T-3' (SEQ ID NO: 90) and reverse primer 5'-CATAGTTTCG CGCACTGCTT T-3' (SEQ ID NO: 91) were used.

The qPCR protocol is a Bio-Rad iCycler (Bio -Rad, USA) and analyzed the data according to the manufacturer's instructions of the equipment used. In this example, the highest expression value, the value obtained at 72 hours of DdRp RNA/auto_DdRp/RSV-F DNAi@LS, was 100%, and the result of expressing the value as a percentage in proportion to it is shown in <Fig. 17>. The experiment was performed 5 times and the average value was indicated.

As in <Fig. 21>, as a result of analyzing the auto_DdRp DdRp transformed cells using T7RNAP, there was no expression of T7 polymerase mRNA in the control pCI-neo_RSV-F@LS, and DdRp RNA/RSV-F DNAi@LS transformed cells It was confirmed that the transfected 5'Cap structure and the stabilized DdRp RNA were slowly degraded in the cell, DdRp RNA/auto_DdRp/RSV-F DNAi@LS; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; In cells transformed with the auto(+) DdRp expression system including the auto_(DdRp/RSV-F DNAi) adeno expression system or the auto_(DdRp/RSV-F DNAi) lenti expression system, the first It was confirmed that the T7 polymerase binds to the T7 promoter of the auto_DdRp expression cassette, and the self-transcription loop that synthesizes DdRp in the downstream region acts to stably synthesize DdRp.

As a result of comparing transformant cells with auto_DdRp DdRp of T7RNAP or NP868R-T7RNAP, NP868R-T7RNAP transfected cells had on average 5 to 6 times higher T7 polymerase mRNA expression than T7RNAP. became (data not shown).

- T7 polymerase assay

The transfected 293T cells were collected at 0, 24, 48 and 72 hours, lysed with RIPA buffer containing cocktail protease inhibitor, and then protein samples were prepared by centrifugation. After coating these samples on an ELISA plate at 4°C for 12 hours and blocking in 0.2% Tween-20 and 5% dry milk to prevent non-specific staining, Anti-T7 RNA Polymerase antibody (CABT-B8990, Creative diagnostic, UK), secondary staining with HRP-conjugated secondary antibody, and analysis with Promega™ Microplate Reader (Promega, USA). The experiment was performed 5 times and the average value was indicated.

As in <Fig. 22>, as a result of analyzing the DdRp of auto_DdRp transformed cells using T7RNAP, in conclusion, the auto(-) DdRp expression system and DdRp RNA/auto_DdRp/RSV-F, which are DdRp RNA/RSV-F DNAi@LS DNAi@LS; DdRp RNA/auto_(DdRp/RSV-F DNAi)@LS; T7 polymerase synthesized in cells transformed with an auto_(DdRp/RSV-F DNAi) adeno expression system or an auto(+) DdRp expression system including an auto_(DdRp/RSV-F DNAi) lenti expression system is at least in the cytoplasm. It is maintained at a high concentration for more than 72 hours.

As a result of comparing the auto_DdRp DdRp transformant cells with T7RNAP or NP868R-T7RNAP, NP868R-T7RNAP transformed cells had on average 10 to 15 times higher T7 polymerase expression than T7RNAP, which was thought to be the effect of NP868R. (data not shown).

Example 4. Cell introduction and T7E1 analysis of genome editing system

A genome editing system prepared by the method of Example 3 was prepared, and this was referred to as a genome editing expression system. The prepared genome editing expression system was mixed in an RNase-free Eppendof tube and stored on ice until used for transfection.

HEK293T (ATCC CRL-3216) cells were inoculated at about 20,000 cells/cm ² by the method of Example 3 for primary transfection of the prepared genome editing expression system. HEK293T (ATCC CRL-3216) cells were cultured in DMEM high glucose medium supplemented with 10% fetal calf serum, 2 mM GlutaMax, 1X non-essential amino acids and 100 U/ml penicillin and 100 ug/ml streptomycin. One day prior to transfection, cells were seeded in 12-well plates. The prepared DdRp RNA liposome was transfected when the cells were about 60% confluent.

Cells were harvested 3 days after transfection and genomic DNA (gDNA) was purified with QIAamp DNA Blood Mini Kit (Qiagen, USA). Then, using 200 ng input gDNA, the genomic region surrounding the guide nucleic acid target site was amplified by PCR with the corresponding primers.

The degree of genome editing was confirmed by T7E1 analysis of the PCR product.

The T7E1 assay (mismatch detection assay) was performed by heating the harvested cells at 65° C. for 15 min and in 500 μL extraction buffer (10 mM Tris, pH 8; 2 mM EDTA; 0.2% Triton X-100; 200 μg/mL protein After extraction with K), it was carried out on the PCR product obtained by PCR on the isolated genomic DNA.

The target region was amplified by PCR with EconoTaq PLUS GREEN 2X Master Mix (Lucigen, USA) or AccuPrime Taq DNA Polymerase, High Fidelity (TermoFisher, USA) according to the manufacturer's protocol.

The PCR product was denatured at 95° C. for 10 minutes, annealed at -2° C. per second down to 85° C., and then annealed again at -1° C. per second and down to 25° C. again.

The formed heterogeneous complex PCR product (5 μL) was reacted with 5U T7E1 enzyme (New England Bio Labs, USA) at 37° C. for 20 minutes. Products from the mismatch assay were electrophoresed on Novex 10 % TBE gels (Invitrogen, USA).

① RNA-guided endonuclease

The genome editing liposome was used for genome editing targeting human VEGF gene in HEK293 cells, including the DdRp RNA expression cassette, an expression cassette encoding Cas9 mutant SpCas9-HF1, and a tRNA-sgRNA (sgRNA targeting VEGF) expression cassette The performance of genome editing liposomes was evaluated. As a control group, an expression cassette encoding a Cas9 mutant SpCas9-HF1 without a DdRp RNA expression cassette and a tRNA-sgRNA (sgRNA targeting VEGF) expression cassette were used.

Cells were harvested 3 days after transfection and genomic DNA (gDNA) was purified with QIAamp DNA Blood Mini Kit (Qiagen, USA). Then, the genomic region surrounding the pegRNA target site was amplified by PCR with the following primers using 200 ng input gDNA.

Forward Primer, 5'TCCAGATGGCACATTGTCAG-3' (SEQ ID NO: 55)

Reverse Primer, 5'AGGGAGCAGGAAAGTGAGGT-3' (SEQ ID NO: 56)

The results of T7E1 analysis in <FIG. 23 a> are the results of T7E1 in genome editing by RNA-guided endonuclease, untreated group (first lane) and control group (second lane) treated with liposomes without DdRp RNA expression cassette It was confirmed that genome editing did not occur because there was no reaction product by the enzyme. In the experimental group (third lane), there is genome editing by a transcription-targeting genome editing system comprising an expression cassette encoding the treated Cas9 mutant SpCas9-HF1 and a tRNA-sgRNA (sgRNA targeting VEGF) expression cassette. When the PCR product containing the hybridization was performed, a heterogeneous complex double DNA was formed, and as a result, it was confirmed that cleavage occurred by the T7E1 enzyme, and the degree was confirmed to be very high.

② DNA-guided endonuclease

The genome editing was performed on the human CD274 gene in HEK293 cells, and the performance of the genome editing liposome containing the DdRp RNA expression cassette, the HuAgo-encoding RNA expression cassette and the guide nucleic acid GD54 was evaluated. As a control group, an RNA expression cassette encoding HuAgo and a guide nucleic acid GD5 without a DdRp RNA expression cassette were used.

Cells were harvested 3 days after transfection and genomic DNA (gDNA) was purified with QIAamp DNA Blood Mini Kit (Qiagen, USA). Then, the genomic region surrounding the pegRNA target site was amplified by PCR with the following primers using 200 ng input gDNA.

Forward Primer, 5'CCTGGCTGCACTAATTGTCTAT-3' (SEQ ID NO: 57);

Reverse Primer, 5'CTGTGTTGTTTGTTCTGGATTTC-3' (SEQ ID NO: 58)

was generated by PCR.

The T7E1 analysis result of <FIG. 23b> confirmed that genome editing by DNA-guided endonuclease did not occur in the control group (first lane) but occurred at a very high level in the experimental group (second lane).

③ Fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase

The genome editing was performed by targeting the human HEK3 gene from the human HEK3 gene, and the auto_DdRp DNA expression cassette and a DNAen expression cassette encoding a fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase, pegRNA-sgRNA (HEK3 Stable cell lines were selected by transfection with a genome editing system comprising a DNAsg expression cassette encoding targeting peg RNA and sgRNA). Selected stable cell lines were transfected with a DdRp RNA expression cassette to induce and evaluate genome editing.

As a control group, a fusion protein mRNA of M-MLV reverse transcriptase and Cas9 H840A nickase without a DdRp RNA expression cassette and a pegRNA-sgRNA (peg RNA and sgRNA targeting HEK3) RNA expression cassette were used.

Cells were harvested 3 days after transfection and genomic DNA (gDNA) was purified with QIAamp DNA Blood Mini Kit (Qiagen, USA). Then, the genomic region surrounding the pegRNA target site was amplified by PCR with the following primers using 200 ng of gDNA.

Forward Primer, 5'-AGGGAAACGCCCATGCAATTAGTCT-3' (SEQ ID NO: 59)

Reverse Primer, 5'-CTAGCCCCTGTCTAGGAAAAGCTGTC-3' (SEQ ID NO: 60)

The T7E1 analysis result of <Fig. 23c> showed that genome editing by the fusion protein of M-MLV reverse transcriptase and Cas9 H840A nickase did not occur in the control group (first lane), but occurred at a very high level in the experimental group (second lane). was confirmed.

Claims (32)

A system having at least one endogenous genome editing and transcribeable component in a cell, the system comprising:
RNA (DdRp RNA) expression cassette comprising DNA dependent RNA Polymerase (DdRp) mRNA with a structure that enables intracellular translation; and
A genome editing RNA/DNA system comprising a; DNA (DNAge) expression cassette encoding a genome editing tool that can be transcribed by the DdRp. The structure of claim 1 , wherein the structure enabling intracellular translation is
5'Cap structure or IRES (internal ribosome entry site) sequence, characterized in that it comprises a genome editing RNA / DNA system. The genome editing RNA/DNA according to claim 1 or 2, wherein the cell is any one selected from the group consisting of prokaryotic cells, plant cells, eukaryotic cells, mammalian cells and human cells. system. The method according to claim 1 to 3, wherein the DdRp RNA expression cassette is
(1) 5'UTR;
(2) RNA encoding the DdRp; and
(3) 3'UTR; genome editing RNA/DNA system characterized in that it comprises an RNA. 5. The method of claim 1 to 4, wherein the DdRp RNA expression cassette is
A genome editing RNA/DNA system comprising chemically modified nucleotides (CM). According to claim 5, wherein the chemically modified nucleotide (CM) is
An expression RNA/DNA system comprising any one or more selected from the group comprising sugar modifications, base modifications, backbone modifications, and lipid modifications. According to claim 1 to 6, wherein the RNA / DNA system for self-transcription of the DdRp mRNA,
In addition, a promoter to which DdRp binds and an auto_DdRp expression cassette comprising a DNA encoding DdRp; and a genome editing RNA/DNA system, characterized in that it is configured to be operably linked. The method of claim 6, wherein the auto_DdRp expression cassette is
(1) a promoter to which DdRp binds;
(2) 5'UTRs;
(3) a DNA fragment encoding the DdRp; and
(4) 3'UTR; genome editing RNA/DNA system comprising DNA. The method of claim 1, wherein the DNAge expression cassette is
a DNAen expression cassette comprising DNA encoding a targeting endonuclease; and
A genome editing RNA/DNA system comprising a; RNA related to a specific targeting nucleotide sequence of a selected genome, and a DNAsg expression cassette comprising DNA encoding sgRNA. 10. The method of claim 9, wherein the DNAen expression cassette is
(1) a promoter to which DdRp binds;
(2) 5'UTR;
(3) DNA encoding the targeting endonuclease; and
(4) 3'UTR; genome editing RNA/DNA system comprising DNA. 11. The method of claim 9 to 10, wherein the targeting endonuclease is
meganuclease;
zinc-finger nucleases (ZFNs);
TAL-effector nucleases (TALENs);
RNA-guided endonuclease;
DNA-guided endonuclease; and
Moloney Murine Leukemia Virus (M-MLV) a fusion protein of reverse transcriptase and Cas9 H840A nickase; a genome editing RNA/DNA system characterized by at least one selected from the group comprising. 12. The method of claim 11, wherein the RNA-guided endonuclease is
single component class 2 CRISPR systems such as type II Cas9 or type V Cas12a (Cpf1); and
A genome editing RNA/DNA system characterized by any one selected from the group comprising; a multi-component class 1 CRISPR system such as a multiprotein type Cas3. 12. The method of claim 11, wherein the DNA-guided endonuclease is
A genome editing system comprising an Argonaute. 10. The method of claim 9, wherein the DNAsg expression cassette is
(1) any one selected from the group consisting of a promoter to which DdRp binds and a promoter to which RNA Polymerase III binds;
(2) a DNA fragment encoding a guide nucleic acid; and
(4) 3'UTR; A genome editing RNA/DNA system comprising a DNA comprising: 15. The method of claim 14, wherein the RNA Polymerase III (pol III) is bound promoter (RNA polymerase III promoters)
A genome editing RNA/DNA system characterized by any one selected from the group consisting of U6 promoter, tRNA promoter and H1 promoter. 15. The method of claim 14, wherein the DNA fragment encoding the guide nucleic acid is
A genome editing RNA/DNA system comprising one or more guide nucleic acids. 17. The method of claim 1 to 16, wherein the DNAen expression cassette and the DNAsg expression cassette are
A genome editing RNA/DNA system, characterized in that it resides on more than one plasmid. 18. The method of claim 1 to 17, wherein the DNAge expression cassette
If there is no guide nucleic acid related to the specific targeting nucleotide sequence of the selected genome, the guide nucleic acid related to the specific targeting nucleotide sequence of the selected genome artificially synthesized from the outside may be injected into the cell, or
Or a genome editing RNA/DNA system characterized in that the DNAge related to the specific targeting nucleotide sequence of the selected genome artificially synthesized from the outside is injected into the cell. The system of claim 1 , wherein the system edits at least one endogenous genomic sequence in a cell, the system comprising:
A genome editing RNA/DNA system, characterized in that it is regulated by anti-CRISPR proteins and compounds. 20. The method of claim 19, wherein the anti-CRISPR protein is
A genome editing RNA/DNA system characterized by any one or more selected from the group comprising AcrIF1, AcrIIA4, AcrIE1 and AcrIIC1. 21. The method of claim 1 to 20, wherein the DNAen expression cassette and the DNAsg expression cassette and the auto_DdRp expression cassette are
A genome editing RNA/DNA system and a composition thereof, characterized in that it is on the same one or more plasmids. 22. The method according to claim 1 to 21, wherein the DdRp RNA expression cassette, the auto_DdRp expression cassette, the DNAen expression cassette and the DNAsg expression cassette are
The former comprises a poly(A) tail or a poly(T) tail. 23. The method of claim 1-22, wherein the DdRp RNA expression cassette, the auto_DdRp expression cassette, the DNAen expression cassette and the DNAsg expression cassette are
G/C content, codon optimization, UTR modification, poly(A) tail with more than 30 adenosine nucleotides, poly(C) sequence, 5' cap (CAP) structure except m7GpppN and histone-stem-loop A genome editing RNA/DNA system comprising at least one selected from nucleotide sequence modification (SM) consisting of a sequence. 24. The method according to claim 1 to 23, wherein the DdRp RNA expression cassette, the auto_DdRp expression cassette, the DNAen expression cassette and the DNAsg expression cassette are
A genome editing RNA/DNA system characterized by any one selected from the group comprising various types or combinations thereof, including linear nucleic acid fragments, circular plasmids, lentiviruses, adeno-associated viruses and ribonucleoprotein (RNP) complexes . 25. The method of any one of claims 1-24,
A genome editing RNA/DNA system, characterized in that by delivering the RNA/DNA system, the genome editing tool continuously expresses for a long time in the cytoplasm. (a) preparing the DdRp RNA expression cassette;
(b) preparing the DNAge expression cassette or auto_(DdRp/DNAge) expression cassette; and
(c) transfecting the RNA/DNA system comprising the prepared DdRp RNA expression cassette and DNAge expression cassette or auto_(DdRp/DNAge) expression cassette;
Here, the genome editing RNA/DNA system, characterized in that the auto_DdRp expression cassette is additionally prepared and co-transfected. 27. The genome editing RNA/DNA system of any one of claims 1-26, wherein the self-transcribeable genome editing RNA/DNA system is
an expression system comprising the DdRp RNA expression cassette and a DNAge expression cassette;
an expression system comprising the DdRp RNA expression cassette, an auto_DdRp expression cassette, and a DNAge expression cassette; and
A genome editing RNA/DNA system kit comprising any one selected from the group comprising; an expression system comprising the DdRp RNA expression cassette and an auto_(DdRp/DNAge) expression cassette 28. The cell, tissue or subject according to any one of claims 1 to 27 transduced with a genome editing RNA/DNA system capable of self-transcription. 29. The method of any one of claims 1-28, wherein DdRp is
any one selected from the group comprising T7 RNA polymerase, T3 RNA polymerase and SP6 polymerase; or
A self-transcribed RNA/DNA system comprising a chimeric enzyme comprising at least one catalytic domain of DdRp. 30. The method of claim 29, wherein the chimeric enzyme is
at least one catalytic domain of RNA triphosphatase,
at least one catalytic domain of guanylyltransferase,
at least one N ⁷ -guanine methyltransferase catalytic domain and
A self-transcripting RNA/DNA system comprising a chimeric enzyme comprising at least one catalytic domain of a DdRp. 31. The method of claim 29-30, wherein the chimeric enzyme is
At least two catalytic domains selected from the group consisting of the catalytic domain of RNA triphosphatase, the catalytic domain of guanylyltransferase, the catalytic domain of N ⁷ -guanine methyltransferase and the catalytic domain of DdRp are linked by a linking peptide. Self-transcription RNA / DNA system, characterized in that. 32. The method according to claim 1 to 31, wherein the DNAi expression cassette comprises a DNA indicative of an enzyme or catalytic domain capable of adding a 5'Cap structure to mRNA synthesized using a portion of the DNA of the DNAi expression cassette as a transcript. Self-transcription RNA / DNA system, characterized in that.

Images