KR20220092760A - Composite particles using RNA expression cassettes having a structure capable of cell translation as an active ingredient and uses thereof - Google Patents

Abstract

The present invention relates to: composite particles including an RNA expression cassette having a structure capable of cell translation, a positive ionic polymer, and a negative ionic polymer as active ingredients, forming composite particles by electrostatic interaction between the same, and in which the RNA expression cassette is encapsulated in the formed composite particles; and uses thereof.

Description

Composite particles using an RNA expression cassette having a structure capable of cellular translation as an active ingredient and uses thereof

The present invention is an RNA expression cassette having a structure that can be translated into cells as an active ingredient, comprising a cationic polymer and an anionic polymer, and an electrostatic interaction between them forms a multiparticulate, and the RNA expression cassette is encapsulated in the formed multiparticulate structure It relates to composite particles and uses thereof.

A variety of diseases today are treatments involving the injection of peptide-, protein-, and nucleic acid-based drugs, particularly those involving transfection of nucleic acids into cells or tissues. requires

The full therapeutic potential of peptide-, protein-, and nucleic acid-based drugs is often not fully exploited due to their limited ability to cross the plasma membrane of mammalian cells; As a result, cellular access is poor, resulting in an inadequate therapeutic effect. Today, these difficulties represent significant challenges for biomedical advancement and the commercial success of many biopharmaceuticals.

Nucleic acid-based drugs are nucleic acids such as deoxyribonucleic acid, ribonucleic acid or backbone, polynucleotide derivatives in which sugars or bases are chemically modified or terminally modified, for example, RNA, DNA, RNA- Guided genome editing system, nucleic acids such as siRNA (short interfering RNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA, ribozyme and DNAzyme, that is, anionic There are a wide variety of substances, such as drugs, and different carriers and manufacturing methods are required depending on the type of nucleic acid to be delivered. As an RNA carrier, a formulation with increased stability and production yield and a manufacturing method thereof have been required.

mRNA (messenger RNA) is a material before protein synthesis and contains genetic information. Since mRNA is accessible to various therapeutic agents, it can be used as a vaccine for prevention or treatment, and missing proteins can also be synthesized through mRNA.

For several diseases or disorders, gene therapeutic approaches have been developed as special forms of such treatment. While gene therapy generally involves the injection of one or more nucleic acids or genes into individual cells and tissues to additionally treat diseases such as genetic diseases, such treatments utilize transfection of nucleic acids or genes into cells or tissues, such as Treatment replaces the defective mutant trait with a functional one.

Moving or inserting one or more of these nucleic acids or genes into individual cells still represents a major challenge today, and especially in the field of gene therapy, the excellent therapeutic effect of absolutely nucleic acid-based drugs is definitely not. need.

In order to successfully carry out the transfer of nucleic acids or genes into individual cells, many obstacles must be overcome. The transport of nucleic acids typically occurs through association of nucleic acids with cell membranes, followed by uptake by endosomes. Within the endosome, the introduced nucleic acid is separated from the endosome. When expression occurs in the cytoplasm, these nucleic acids must leave the endosome.

If the nucleic acid does not properly escape the endosomes, the endosomes are fused with lysosomes that cause the degradation of the contents, or the cell membrane and the endosomes are fused to send the contents out of the cell. Escape of endosomes for effective movement of nucleic acids is considered as one of the most important steps to reduce the efficiency of transfection. So far, there are different approaches to dealing with these problems. However, neither approach has been successful in all respects.

Today's transfection agents typically include peptides, various polymers, and lipids, as well as nano- and microparticles. Such transfection agents have typically only been successfully used in in vitro reactions.

When transfecting nucleic acids into cells of living animals in vivo, more conditions must be met. For example, it should be stable in physiological salt solutions against agglomerisation. Furthermore, it should not react with any part of the host's complement system. The complex must protect the nucleic acid from initial extracellular degradation by nucleases that occur anywhere.

For gene therapy applications, the most important thing is that the carrier is not recognized by the adaptive immune system and does not stimulate a non-specific cytokine storm.

The development of methods capable of delivering therapeutic genes to selected cells with appropriate gene expression presents an important challenge towards gene therapy. However, gene delivery and particularly successful transfection of nucleic acids into cells or tissues is not straightforward and generally depends on many factors.

For successful delivery, for example, delivery of a nucleic acid or gene into a cell or tissue must overcome many obstacles. An ideal nucleic acid transport method satisfies three important characteristics: (1) It should protect the target nucleic acid from degradation by nucleases in the intracellular matrix. (2) The target nucleic acid must be able to cross the plasma membrane. (3) There shall be no adverse effect.

This goal can be achieved using a combination of various compounds or vectors (vrctors). In particular, there are several compounds or vectors that overcome some of these obstacles.

In the treatment using anionic drugs including nucleic acids, safe and efficient drug delivery technology has been studied for a long time, and various delivery systems and delivery technologies have been developed. The delivery system is largely divided into a viral delivery agent using adenovirus or retrovirus, etc. and a non-viral delivery agent using cationic lipids and cationic polymers. In the case of a viral carrier, it is known that there are many problems in commercialization because it is exposed to risks such as non-specific immune response and the production process is complicated.

RNA has wide potential as a therapeutic agent. Current clinical efforts are focused on vaccination, protein replacement therapy, and treatment of genetic disorders. Clinical use of mRNA therapeutics has been made possible through advances in the design of RNA manufacturing and intracellular delivery methods.

However, the widespread application of RNA is still limited by the need for improved delivery systems. Therefore, the recent research direction is in the direction of improving the disadvantages by using a non-viral carrier. The non-viral delivery system is inferior in efficiency compared to the viral delivery agent, but has advantages in that there are few side effects in terms of in vivo safety and that the production price is low in terms of economic feasibility.

The most representative non-viral carriers are a lipoplex of a cationic lipid and a nucleic acid using a cationic lipid and a polyplex of a polycationic polymer and a nucleic acid. These cationic lipids or polycationic polymers have been studied in various ways in that they stabilize anionic drugs and increase intracellular delivery by forming a complex through electrostatic interaction with anionic drugs.

However, when the amount required to obtain a sufficient effect is used, it is less than the viral carrier, but causes serious toxicity, resulting in inappropriate use as a pharmaceutical product. Therefore, while reducing toxicity by minimizing the amount of cationic polymers or cationic lipids that can cause toxicity, stable in blood and body fluids, intracellular delivery is possible, and anionic drug delivery technology that can obtain sufficient effects development is needed

Finally, methods and vehicles for intracellular delivery remain major barriers to the widespread application of RNA therapeutics. With some exceptions, intracellular delivery of RNA is generally more difficult than that of small oligonucleotides and requires encapsulation into delivery nanoparticles. This is due to the significantly larger size of mRNA molecules (300–5,000 kDa, ~1–15 kb) compared to other types of RNA (small interfering RNA [siRNA], ~14 kDa; antisense oligonucleotides [ASO]).

The widespread use of natural polysaccharides in pharmaceuticals for drug delivery is their biocompatibility, biodegradability and low toxicity. It has also been used in combination with synthetic materials as a strategy to mitigate the latter toxicity. Their structural diversity makes it possible to select the most suitable polysaccharides in each case according to the needs of the final application, and more importantly, their functional properties can be tuned and optimized by chemical modifications to their functional groups.

Many natural polysaccharides have biological adhesion properties due to the presence of specific functional groups, making them useful starting materials for nanocarrier designs with improved retention times, especially for intestinal and lung applications. The presence of such groups also allows for certain modifications of the polymer structure to enhance its delivery or targeting ability. In addition, natural polysaccharides are neutral (eg, cellulose, pullulan, starch, dextran), negatively (eg, hyaluronic acid, alginate, chondroitin sulfate), or positively charged (eg, chitosan). ) can be

In the present invention, with a focus on biomaterials and delivery strategies, for the clinical use of RNA expression cassette-based therapeutics, a study on a delivery system that can facilitate the clinical use of RNA expression cassette-based therapy was conducted, and protein therapy, gene editing and prophylaxis were conducted. An application of RNA expression cassette-based delivery to inoculation was developed.

In biology, the extracellular matrix (ECM) is a tissue mainly responsible for structural support of animals. The extracellular matrix belongs to the connective tissue of animals. The extracellular matrix is composed of the intercellular matrix and the basement membrane. The intercellular matrix is the matrix that fills the space between several cells. A gel made of polysaccharides and protein fibers are filled between cells, helping to buffer the extracellular matrix. The basement membrane is composed of thin paper, and the epithelial tissue is located on it.

Components of the ECM are produced by the corresponding cells and secreted into the extracellular matrix through exocytosis. The newly created extracellular matrix is secreted and integrated into the existing extracellular matrix. The extracellular matrix is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans. E is composed of plteoglycans such as heparan sulfate, chondroitin sulfate and keratan sulfate, non-proteoglycan polysaccharides such as hyaluronic acid, fibers such as collagen and elastin, fibronectin and laminin. E is mainly composed of a dense collagen network embedded in hyaluronic acid (HA) and glucosaminoglycans gels, creating a strong physical and hydrodynamic barrier in the form of intra-interstitial pressure in E.

These macromolecular components are interconnected to form complex networks that actively communicate with cells through binding to cell surface receptors and/or matrix effectors. E plays a variety of roles, including providing tissues with structural integrity and mechanical properties essential for tissue function, or regulating cellular phenotype and function to maintain tissue homeostasis. E molecular composition and structure vary from tissue to tissue and are significantly altered during normal tissue repair and the progression of various diseases. Indeed, aberrant E remodeling that occurs in pathological conditions drives disease progression by regulating cell-matrix interactions.

Therapeutic strategies for ECM destruction in cancer tissues are rapidly developing clinically, with several clinical trials involving PEG-PH20 (PEGylated rHuPH20 enzyme, recombinant human hyaluronidase enzyme) being conducted. In addition, there is remarkable evidence demonstrating that matrix metalloproteinases (MMPs) play a major role in E-associated tissue destruction. MMPs are a family of zinc-dependent endopeptidases that participate in the degradation of the extracellular matrix and tissue remodeling. There are some 28 species as members of the MMP family, which can be classified into collagenase, gelatinase, stromelysin, membrane-type MMP, matrylysin, enamelysin, and the like. Collagenase, consisting of MMP1, MMP8, MMP13 and MMP18, can degrade the triple-helical fibrillar collagen into distinct 3/4 and 1/4 fragments. In addition, it has been demonstrated that MMP14 can also cleave fibrillar collagen, and MMP2 can also cleave collagen.

A strategy for multivalent presentation of ECM degrading enzymes in polymer nanoparticles has been developed in order to improve the enzyme delivery efficiency and the diffusion of nanoparticles in cancer tissues. However, a possible problem with these nanoformulations is that the properties of the enzyme can be irreversibly changed due to the additional process for enzyme immobilization. In addition, concerns remain about the therapeutic efficacy of chemically modified synthetic nanoparticles having a truncated PH20 form lacking a membrane-bound glycosyl phosphatidylinositol (GPI) domain. GPI anchoring of native PH20 hyaluronidase increases protein mobility at the cell surface, allowing for maximum rate and egg-sperm membrane fusion for successful penetration into the egg vestment.

Despite the research results on nano-drugs for the treatment of cancer, most of the nanoparticles used for drug delivery do not penetrate deeply into E of cancer tissue and are confined to the periphery of blood vessels of E, so their therapeutic efficacy is limited. come. The extent of nanoparticle accumulation in the ECM can be severely limited because of the elevated interstitial fluid pressure and the densely complex extracellular matrix (E).

RNA expression cassettes readily form multiparticulates with cationic and anionic polymers. In order to maximize the biocompatibility and efficacy of the multiparticulates, it is not known which types of polymers with which types of properties can form which multiparticulates under which types of conditions.

In this respect, an object to be solved by the present invention is to provide a novel technology for forming an RNA expression cassette into a multiparticulate.

The present invention relates mainly to RNA expression cassettes, cationic polymers, in particular protamine (PS), cell penetrating peptides (CPP) or derivatives thereof, and RNA expression dependent on interaction with anionic polymers, in particular hyaluronic acid or derivatives of these polysaccharides It is intended to provide a cassette composite particle.

The RNA expression cassette composite particle of the present invention is intended to provide a composite particle including polyelectrolyte composite nanoparticles, nanogel, and polymer micelles.

The object to be solved by the present invention is to secure the in vivo stability of the RNA expression cassette, which is an active ingredient, and the non-toxic and biodegradability of the carrier, in the preparation of composite particles containing the RNA expression cassette, a cationic polymer and an anionic polymer. It is to provide suitable new technology.

The object underlying the present invention is therefore to provide carriers or complexing agents, in particular for the transfection of RNA expression cassettes for gene therapy or other therapeutic applications, which are nucleic acids, preferably RNA It is capable of aggregating with an RNA expression cassette such as an expression cassette, allowing sufficient transfection of the RNA expression cassette into the respective cell line in vitro as well as in vivo. For sufficient release of the RNA expression cassette, uptake by the cell occurs or is provided through an endosomal pathway, such as a carrier or complexing agent. Additionally, it is preferred that the RNA expression cassette delivery and the biodegradable polymer carrier delivery complex prepared by the carrier or complexing reagent exhibit improved resistance to agglomeration. In addition, preferably, in relation to serum containing media, it is possible to give further improved stability to the nucleic acid delivery. Most importantly, in vivo activity is obtained, whereby at least an acute immune response is suppressed. Moreover, a reproducible production and characterization of the carrier must be ensured.

The present invention is to solve various problems including the above problems, and a multiparticulate comprising an RNA expression cassette encoding a more efficient ECM degrading enzyme or a degrading enzyme inhibitor and an extracellular matrix destruction strategy using the same to prevent ECM-related diseases and An object of the present invention is to provide a high anticancer therapeutic effect through an anti-exacerbation effect, in particular, by promoting the movement of an anticancer substance in a tumor tissue. However, these problems are exemplary, and the scope of the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail.

Among the components of the composition according to the present invention, an active ingredient is an RNA expression cassette, a cationic polymer and an anionic polymer form a multiparticulate, and an active ingredient an RNA expression cassette may be encapsulated in the multiparticulate structure. The schematic structure of the polymer composite particle delivery system in which the RNA expression cassette and the cationic polymer composite particles are encapsulated is shown in <Fig. 1>.

The RNA expression cassette is a construct including; a non-amplifying mRNA construct, a self-amplifying mRNA construct, and a trans-amplifying mRNA construct.

Referring to <FIG. 1>, the RNA expression cassette binds to each other through an electrostatic interaction with the cationic polymer to form a cationic polymer complex with the RNA expression cassette. The complex formed by the RNA expression cassette and the cationic polymer may be encapsulated in the multiparticulate structure formed by the anionic polymer.

As shown in <Figure 1>, in the composite particles formed by the anionic polymer in the present invention, in an aqueous environment, the anionic polymer forms the outer wall of the composite particle, and the cationic polymer forms the inner wall of the composite particle, It is a structure in which the RNA expression cassette and the cationic polymer complex are encapsulated inside the formed composite particle.

Among the polymers used in RNA expression cassette-based therapy, the present inventors present the present invention to highlight the applicable properties and characteristic features that are very suitable for RNA expression cassette delivery, the present invention is biodegradable and biocompatible Protamine (PS), chitosan and cells. Cationic polymers such as penetrating peptides (CPP) and anionic polymers such as hyaluronic acid and albumin were selected.

Cationic polymers such as PS, chitosan and CPP can electrostatically interact with RNA expression cassettes to form stably positively charged polyplexes, while neutral and anionic polymers often exhibit more efficient interactions with RNA expression cassettes. It requires the presence of other ingredients in order to be preferred. Anionic polymers are mucoadhesive polymers, but have an opposite charge, greatly affecting their use in nucleic acid delivery. This important difference stems from the chemical structure, which is a positively charged guanidino group in the basic amino acids of protamine, particularly in the arginine monomer, or a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid. to provide.

It can be said that the advantages of the nucleic acid intracellular delivery system proposed in the present invention are clearly achieved by the cationic polymer and complemented by the anionic polymer. First, the efficient physical interaction of cationic polymers with negatively charged nucleic acids protects them from nuclease degradation. Second, they provide a net positive charge, enhancing the interaction of the complex with the anionic surface of the cell. Nevertheless, association with anionic polysaccharides such as albumin and hyaluronic acid in the carrier of the present invention can improve bioavailability by avoiding RNA expression cassette excretion through the glomerular capillary wall. Indeed, anionic material is "unfilterable" due to the repulsive forces established by the negative charge of the glomerular membrane.

Cationic polymers and their derivatives may be used to obtain different types of carriers, and anionic polymers and their derivatives may be used to obtain different types of carriers. Nevertheless, the present invention mainly relies on the interaction of RNA expression cassettes, in particular cationic polymers such as PS, CPP, chitosan, and anionic polymers such as hyaluronic acid, albumin or derivatives of these polysaccharides to provide RNA expression cassette delivery systems. would like to

In this respect, an object to be solved by the present invention is to provide a novel technology suitable for the preparation of multiparticulates containing a cationic polymer, an anionic polymer and an RNA expression cassette.

In the present invention, a nuclear and basic composite particle was prepared with an RNA expression cassette and a cationic polypeptide as active ingredients, and a composite particle was developed that forms an outer layer with anionic polysaccharides.

The present inventors have found that the degree of cationization is important as a property of a cationic polymer capable of forming a basic multiparticulate comprising an RNA expression cassette in order to prepare a multiparticulate, and thus completed the present invention.

The RNA expression cassette and the cationic polymer complex are kept encapsulated in the multiparticulate structure formed by the anionic polymer, so that stability in blood or body fluid is improved.

The cationic polymer is the biodegradable and biocompatible polymeric carrier molecule, for example, sufficient transfection of nucleic acids for gene therapy or other therapeutic applications without loss of activity, particularly in vitro as well as in vivo. for sufficient aggregation of nucleic acids for the purpose of sufficient transfection of the nucleic acids into other cell lines. The polymeric carrier molecule is furthermore not toxic to cells and allows sufficient release of the nucleic acid delivery agent.

In the present invention, additionally, in relation to serum containing media, biodegradable and biocompatible anionic polysaccharides that provide improved stability of nucleic acid delivery and prevent recognition of polymeric carrier delivery complexes by the immune system are used, The reversible addition of particularly hydrophilic polymer chains (eg, PEG-monomers) to the ends of the anionic polysaccharides may also impart improved resistance to aggregation.

Cationic polypeptides such as PS and CPP, although composed of fairly small non-toxic monomer units, form a cationic binding sequence with a defined chain length that provides strong condensation of nucleic acid delivery and complex stability. do. Under reduced conditions of the cytosol (eg, cytosolic GSH), the complexes are rapidly degraded to their monomers, which are further degraded (eg, oligopeptides) or secreted (eg, PEG). . This aids in the deliberation of the nucleic acid transporter within the cell matrix. Because it is degraded into small oligopeptides in the cell matrix, the toxicity known to be observed with high molecular weight oligopeptides, for example, high molecular weight oligoarginine is not observed.

The PEG-coating of the anionic polymer coats the carrier with a hydrophilic coating at its ends, which can prevent salt-mediated aggregation and unwanted interactions with serum contents. Within the cytosol, this coating is easily removed under the reducing conditions of the cell. Again, this effect enhances the induction of nucleic acid delivery in the cell matrix.

The particle size of the composite particles may be in the range of 10 to 1000 nm, and more specifically, in the range of 10 to 200 nm.

In addition, the surface charge of the composite particles may be -20 to 20 mV, more specifically -10 to 10 mV.

Extraction of general conclusions about the polymer of interest. Most of the nanocarriers reported in the examples below correspond to polyelectrolyte composite nanoparticles, nanogels, hydrogels, and polymer micelles.

When the particle size and surface charge of the multiparticulate are maintained at the above levels, it is preferable in terms of stability of the multiparticulate structure, content of components, absorption of the RNA expression cassette in the body, and convenience of sterilization as a pharmaceutical composition.

According to one aspect of the present invention, there is provided a composition for drug delivery comprising an extracellular matrix degrading enzyme and an RNA expression cassette encoding an inhibitor of the degrading enzyme.

According to another aspect of the present invention, there is provided a composition for treating cancer comprising the composition for drug delivery as an active ingredient.

According to another aspect of the present invention, the composition for drug delivery includes an extracellular matrix degrading enzyme and an RNA expression cassette encoding an inhibitor of the degrading enzyme, and a composition for drug delivery in which an anticancer compound is encapsulated as an active ingredient A composition for treating cancer is provided.

It provides a multi-particle comprising an RNA expression cassette encoding a target protein to be expressed of the present invention made as described above, and can provide therapeutic proteins, antigens and antibodies, and extracellular matrix degrading enzyme according to an embodiment Alternatively, the multiparticulate comprising an RNA expression cassette encoding an inhibitor of a degrading enzyme prevents or prevents aggravation of ECM-related diseases, or, in particular, herpes around tumor tissue, thereby interfering with the proximity of immune cells, nucleic acid drugs and anticancer compounds. By decomposing the extracellular matrix, cancer cells can be selectively removed, which can be usefully used in the treatment of cancer.

1 is a conceptual diagram of an RNA expression cassette composite particle as a pharmaceutical active ingredient (a), a basic composite particle (a) composed of the RNA expression cassette and a cationic polymer, and a layer composed of an RNA expression cassette and a cationic polymer as a pharmaceutically active ingredient It is a composite particle (b) comprising (10) and an anionic polymer outer layer (20).
2 is a view showing a method for preparing an RNA expression cassette composite particle having a pharmaceutically active ingredient, and is a positively charged polymer with a focus on the characteristic that the RNA expression cassette itself as a pharmaceutically active ingredient has an anion. A method for preparing particles by coating an RNA expression cassette with a component and additionally with a secondary coating of an anionic polymer is provided.

In the present invention, in nucleotides, nucleosides or polynucleotides (eg, nucleic acids of the invention, eg, mRNA molecules), the term "modified" or suitably "modified" means A, G, U or C-ribonucleotides. Generally, in the present invention, these terms are not intended to refer to ribonucleotide modifications within the naturally occurring 5'-terminal mRNA cap moiety. In polypeptides, the term "modification" refers to a modification compared to a moiety, a canonical set of 20 amino acids.

Variations can be a variety of obvious variations. In some embodiments, where the nucleic acid is an mRNA, the coding region, flanking regions and/or terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. can In some embodiments, a modified polynucleotide introduced into a cell may exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.

Polynucleotides can contain any useful modifications to sugars, nucleobases, or internucleoside linkages (eg, to linking phosphates/to phosphodiester linkages/to phosphodiester backbones). . For example, a major groove face of a polynucleotide, or a major groove face of a nucleobase may include one or more modifications. One or more atoms of the pyridine nucleobase (e.g., on the major groove face) are optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro) or may be substituted. In certain embodiments, modifications (eg, one or more modifications) are present on each sugar and each internucleoside linkage. Modifications according to the present invention are ribonucleic acid (RNA) to deoxyribonucleic acids (DNA) modifications, for example 2'H of 2'OH of ribofuranicil ring, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs) or a hybrid thereof. Further variations are described herein.

As used herein, "genome" encompasses chromosomal DNA present in the nucleus as well as organ DNA present in cellular subcomponents (eg, mitochondria, plastids).

"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. used to refer to a polymer that has been Nucleotides (generally found in the 5'-monophosphate form) are referred to by shorthand names as follows: "A" = adenylate or deoxyadenylate (RNA or DNA, respectively), "C" = cytidylate or deoxycytidylate, "G" = guanylate or deoxyguanylate, "U" = uridylate, "T" = deoxythymidylate, "R" = purine (A or G), "Y" = pyrimidine (C or T), "K" = G or T, "H" = A or C or T, "I" = inosine, "N" = any nucleotide.

The polynucleotide of the present invention does not substantially induce an innate immune response of a cell into which the polynucleotide (eg, mRNA) has been introduced. Characteristics of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and/or 3) termination in protein translation or reduction is included.

"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.

It may be desirable to introduce a modified nucleic acid molecule into a cell so that it is degraded intracellularly. For example, degradation of the modified nucleic acid molecule may be desirable where precise timing of protein production is desired. Accordingly, the present invention provides a modified nucleic acid molecule containing a degradation domain, capable of acting in a direct manner within a cell. In another aspect, the present disclosure relates to binding of a major groove interaction, e.g., binding of a partner to a polynucleotide (e.g., a modified nucleotide, as compared to an unmodified nucleotide, for a major groove interaction partner Polynucleotides comprising nucleosides or nucleotides capable of interfering with binding affinity) are provided. Polynucleotides can be used with other agents (e.g., RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamers, vectors, etc.] may be optionally included. In some embodiments, polynucleotides may comprise one or more messenger RNA (mRNA) having one or more modified nucleosides or nucleotides (ie, modified mRNA molecules).

Nucleic Acid Molecules : A nucleic acid molecule is a molecule that preferably comprises, preferably consists of, a nucleic acid component. The term nucleic acid molecule refers to either DNA or RNA. It is used synonymously with the term "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.

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.

Gene : 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.

Expression is used in its most general sense and 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 expression cassettes, 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.

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. Ordinarily RNA may be obtainable 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.

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.

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. The term “RNA” also includes other encoding RNA molecules, such as viral RNA, retroviral RNA and replica RNA.

Expression Cassette : Expression cassette refers to a structure suitable for expressing a target nucleotide sequence in an organism. Expression refers to the production of a functional product. In the present invention, expression of the nucleotide sequence may refer to translation of RNA into a protein precursor or a mature protein. The expression cassette may be an RNA that can be translated (eg, mRNA). An expression cassette may include a subject nucleotide sequence and a regulatory sequence, which may be derived from different sources, or a subject nucleotide sequence and a translational control sequence, which are derived from the same source but aligned in a manner different from that normally found in nature. .

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.

Sequence of Nucleic Acid Molecules: The sequence of a nucleic acid molecule is generally understood to be a specific and discrete order, ie, a sequence of its nucleotides. 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-identical positions, regardless of their actual positions in alignment.

Stabilized Nucleic Acid Molecules: Stabilized nucleic acid molecules are more susceptible to disintegration or degradation than unmodified nucleic acid molecules, for example by environmental factors or enzymatic digit such as exo- or endonuclease. A more stable, so chemically modified DNA or RNA molecule. In the present invention, a stabilized nucleic acid molecule is stabilized in a cell such as a prokaryotic or eukaryotic cell, a mammalian cell such as a human cell. 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.

G/C chemical modification : The G/C-chemically modified nucleic acid is generally based on a chemically modified wild-type sequence, preferably comprising an increased number of guanosine and/or cytosine nucleotides compared to the wild-type sequence, preferably may be an RNA molecule as defined herein. 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

A “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 RNA by template-independent RNA polymerase after transcription, but is usually not encoded in eukaryotic DNA, whereas the present invention relates to DNA poly(A) sequence encoded by

"Template RNA" refers to RNA capable of being transcribed or replicated by RNA dependent RNA polymerase.

Protein: The protein generally used in the present invention and the protein to be expressed in the present invention are referred to as *?**?*target protein*?**?*, and the two terms are used almost synonymously, and in general, protein is one or more peptides or polypeptides. In general, proteins fold into three-dimensional shapes that may be required for proteins to exert biological functions.

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.

Fragment or part of protein: In the present invention, fragment or part of protein can be generally understood as a peptide corresponding to a continuous part of the amino acid sequence of a protein, preferably from about 6 to about 20 or more amino acids. having a length, e.g., 8, 9, or 10, (or 11), e.g., having a length of e.g. 8, 9, or 10 amino acids , or even 12 amino acids), or fragments processed and presented by MHC class II molecules, preferably about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length, wherein these fragments may be selected from any portion of the amino acid sequence. These fragments can generally be recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, ie the fragments are generally not recognized in their native form. Fragments or portions of proteins as defined herein may also include epitopes or functional sites of these proteins. A fragment or portion of a protein is an antigen, in particular an immunogen, for example an antigenic determinant (also called an 'epitope') or has antigenic characteristics and elicits an adaptive immune response. Accordingly, fragments of proteins or peptides may comprise at least one epitope of these proteins or peptides. Furthermore, it can be understood that a domain of a protein includes fragments of a protein, such as an extracellular domain, an intracellular domain or a transmembrane domain and short or truncated versions of a protein. .

Chimeric Enzyme: 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.

Immunostimulatory RNA: In the present invention, immunostimulatory RNA (isRNA) may generally be an RNA capable of inducing an innate immune response. It generally does not have an open reading frame and thus does not present a peptide-antigen or immunogen, but elicits an innate immune response, for example, by binding to a specific class of toll-like-receptor (TLR) or other suitable receptor. . But, of course, also mRNAs having an open reading frame and encoding peptides/proteins can induce an innate immune response and thus can be immunostimulatory RNAs.

Immune System: The immune system can protect an organism from infection. If the pathogen successfully penetrates the organism's physical barrier and enters the organism, the innate immune system provides an immediate, but non-specific response. If the pathogen evades this innate response, the vertebrate has a second layer of protection, the adaptive immune system. Here the immune system tailors its response during infection to increase its recognition of the pathogen. This enhanced response, in the form of immunological memory, subsequently remains after the pathogen is eliminated, allowing the adaptive immune system to launch a rapid and powerful attack whenever it is encountered. Accordingly, the immune system includes innate and adaptive immune systems. Each of these two parts contains so-called humoral and cellular components.

Adaptive Immune System: The adaptive immune system is essentially responsible for eliminating or preventing pathogen growth. It generally modulates the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember specific pathogens (which generate immunity) and launch a strong attack whenever a pathogen is encountered. The system is highly adaptable due to somatic hypermutation (a process of accelerated somatic mutation), and V(D)J recombination (irreversible genetic recombination of antigen receptor gene segments). This mechanism allows for a small number of genes that, after unique expression on each individual lymphocyte, give rise to a vast number of different antigen receptors. Because gene rearrangement causes irreversible modifications to each cell's DNA, its progeny (progeny), including memory B cells and memory T cells, which are key for long-lived specific immunity. offspring)) will then inherit the gene encoding the same receptor specificity.

Innate Immune System: In general, the innate immune system, also known as the non-specific (or non-specific) immune system, includes cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that cells of the innate immune system recognize and respond to pathogens in a normal way, but unlike the adaptive immune system, they do not confer long-lasting or protective immunity to the host. The innate immune system, for example, toll-like receptors (TLRs) or lipopolysaccharidi, TNF-alpha, CD40 ligand, or cytokines, monokini, lymphokines, interleukins ) or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 , IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL -25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF , ligands of G-CSF, M-CSF, LT-beta, TNF-alpha, growth factor, and hGH, human toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, Ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, ligand of NOD-like receptor, RIG-I like receptor It can be activated by ligands, immunostimulatory nucleic acids, immunostimulatory RNA (isRNA), CpG-DNA, antibiotics, or other auxiliary substances such as antiviral agents.

The pharmaceutical composition according to the present invention may comprise one or more such substances. In general, the response of the innate immune system is to recruit immune cells to the site of infection, through the production of chemical factors, including specialized chemical modulators called cytokines; activation of the complement cascade; the selection and removal of foreign substances present in organs, tissues, blood and lymph by specialized white blood cells; activation of the adaptive immune system; and/or acting as physical and chemical barriers to infectious agents.

Immune response: An immune response is usually a specific response of the adaptive immune system (so-called, specific or adaptive immune response) to a specific antigen or a non-specific response of the innate immune system (so-called, non-specific or innate immune response), or a combination thereof. can be

Adaptive Immune Response: The adaptive immune response is generally understood as an antigen-specific response of the immune system. Antigen specificity allows the development of a response tailored to a specific pathogen or pathogen-infected cell. The ability to initiate these tailored responses is maintained in the body by "memory cells". Pathogens have to infect the body more than once, and these specific memory cells are used to quickly clear them. The first step in the adaptive immune response is the activation of naive antigen-specific T cells or different immune cells capable of inducing an antigen-specific immune response by antigen-presenting cells. It occurs within lymphoid tissues and organs through which pure T cells are constantly passing. The three cell types that can serve as antigen presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in inducing an immune response. Dendritic cells can uptake antigens by phagocytosis and micropinocytosis where they differentiate into mature dendritic cells, e.g. by transporting foreign antigens to local lymphoid tissue by contact with them. can be stimulated by Macrophages are induced by infectious agents or other appropriate stimuli that ingest particle antigens such as bacteria and express MHC molecules. The specific ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important for inducing T cells. MHC molecules are normally responsible for presenting antigens to T cells. Presentation of antigens to MHC molecules results in activation of T cells leading to their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the death of infected cells by CD8+ cytotoxic T cells which together give rise to cell-mediated immunity and the activation of macrophages by Th1 cells, and thus the different types of antibodies, which give rise to a humoral immune response is the activation of B cells by both Th2 and Th1 to produce T cells recognize antigens by their T cell receptors, which do not recognize and bind antigens directly, but instead bind to MHC molecules on the surface of other cells, e.g. pathogen-derived protein antigens, e.g. so-called epitopes ( short peptide fragments of the epitope).

Cellular Immune/Cellular Immune Response: Cellular immunity generally involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to antigens. . In a more general manner, cellular immunity is not based on the activation of cells of the immune system as well as antibodies. In general, a cellular immune response is capable of inducing apoptosis, e.g., within a specific immune cell such as a cell that exhibits an epitope of a foreign antigen on their surface, e.g., a dendritic cell or other cell. It can be characterized by the activation of antigen-specific cytotoxic T-lymphocytes. Such cells may be virally infected, infected with intracellular bacteria, or cancer cells displaying tumor antigens. Also, a feature may be the activation of macrophages and natural killer cells capable of destroying pathogens and the stimulation of cells to release various cytokines that affect the functions of other cells involved in the adaptive and innate immune responses.

Immunogen: In the present invention, an immunogen is generally to be understood as a compound capable of stimulating an immune response. Preferably, the immunogen is a peptide, polypeptide, or protein. An immunogen of the present invention is a product of translation of a given nucleic acid molecule, preferably a nucleic acid molecule as defined herein. In general, the immunogen elicits at least an adaptive immune response.

Antigen: Antigens are generally capable of being recognized by the immune system and induce an antigen-specific immune response by the adaptive immune system, for example through the formation of antibodies and/or antigen-specific T cells as part of the adaptive immune response. substances that can cause In general, the antigen may be or may comprise a peptide or protein capable of being presented by the MHC to a T cell.

Epitope: Epitopes (also called “antigenic determinants”) can be distinguished into T cell epitopes and B cell epitopes. A portion of a T cell epitope or protein may comprise a fragment having a length of about 6 to about 20 or more amino acids, for example a fragment processed and presented by an MHC class I molecule, from about 8 to about 10 amino acids. , e.g., a fragment having a length of 8, 9, or 10, (or even 11, or 12 amino acids), or a fragment processed and presented by an MHC class II molecule, of about 13 or more amino acids, e.g. for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length, wherein these segments can be selected from any portion of an amino acid sequence. These fragments are generally recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, ie the fragments are not generally recognized in their native form. B cell epitopes are generally recognizable by antibodies, i.e., in their native form, having 5 to 15 amino acids, 5 to 12 amino acids, or 6 to 9 amino acids, on the outer surface of a (native) protein or peptide. It is a fragment located in

The epitope of such a protein or peptide may be selected from various variants of such a protein or peptide. Antigenic determinants may be conformational or discontinuous epitopes composed of portions of proteins or peptides, but may be combined with continuous or linear epitopes or three-dimensional structures composed of single polypeptide chains.

Adjuvant/adjuvant component: An adjuvant or adjuvant component in a broad sense is generally a pharmacological and/or immunological agent that can be modified, for example, a drug or other agent such as a vaccine. improve the effectiveness of This should be interpreted in a broad sense and represents a broad spectrum of substances. In general, these substances are capable of increasing the immunogenicity of an antigen. For example, the adjuvant may be recognized by the innate immune system, eg may elicit the innate immune system. An “adjuvant” generally does not elicit an adaptive immune response. To that extent, an “adjuvant” does not qualify as an antigen. Their mode of action is distinct from the effects induced by antigens as a result of the adaptive immune response.

"Transient transformation" or *?**?*transfection*?**?* means that a nucleic acid molecule or protein is introduced into a cell to perform its function without stably inheriting an exogenous gene. In the case of transient transformation, no exogenous nucleic acid sequence is inserted into the genome.

Transformation: Transformation 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, transformation includes any method known to a person skilled in the art for introducing a nucleic acid molecule into a cell, 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. The introduction is non-viral.

Carrier/Polymer Carrier: A 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, less than 9 (eg 5 to 9), or less than 8 (eg 5 to 8), or have a positive charge (cation) of a physiological pH value of less than 7 (eg 5-7), eg 7.3-7.4. Thus, the cationic component may be any positively charged compound or polymer, a cationic peptide or protein that 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 to be an amount sufficient to induce a pharmaceutical effect, such as an immune response, in the case of a hostile situation.

Vaccine : A vaccine is generally understood to be a prophylactic or therapeutic substance that provides at least one antigen, an immunogen. The antigen or immunogen may be derived from any material suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as a bacterial or viral particle (particli), or from a tumor or cancerous tissue. An antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.

Genetic vaccination: Genetic vaccination can generally be understood as vaccination by administration of a nucleic acid molecule encoding an antigen or immunogen or fragment thereof. The nucleic acid molecule may be administered to an individual's body or to an isolated cell of an individual. Upon transformation of specific cells of the body, or upon transformation of isolated cells, antigens or immunogens can be expressed by those cells and subsequently presented to the immune system, eliciting an adaptation, i.e., an antigen-specific immune response. can be Thus, genetic vaccination generally involves a) transformation of a nucleic acid, RNA molecule into a subject, into a patient, or in cells of a patient in vivo/ex vivo or in vitro. administration to isolated cells of a subject, usually a patient; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of the isolated, transformed cells to the subject, the patient, if the nucleic acid was not directly administered to the patient.

I. RNA expression cassette

mRNA is an intermediary between the transcription of DNA encoding protein information and the production of proteins by cytoplasmic ribosomes. To date, there are two main types of RNA vaccines: non-replicating mRNA and virus-derived self-amplifying RNA. Whereas traditional mRNA-based vaccines contain untranslational regions at the 5' and 3' ends, self-amplifying RNA possesses a viral replication mechanism that allows not only antigen but also intracellular RNA amplification and abundant protein expression. have.

Optimal in vitro transcribed mRNA preparation for therapeutic purposes is produced from a linear DNA template using T7, T3 or Sp6 phage RNA polymerase. The RNA prepared in this way should have an open reading frame encoding the target protein, an untranslated section, 5*?**?* Cap, and multiple adenosine tail sequences (poly (A) tail). Thus, the prepared mRNA resembles a fully mature, natural mRNA molecule and exists naturally in the cytoplasm of eukaryotic cells.

A lot of mRNA complexes have been developed recently for in vivo delivery. Naked mRNA (the RNA molecule itself that is not surrounded by a protein-like substance) is quickly degraded by extracellular RNAases (RNases) and does not enter the cell effectively.

Accordingly, various in vitro and in vivo transfection techniques have been developed to promote cellular uptake of mRNA and protect mRNA from degradation. Once the mRNA moves into the cytoplasm, it undergoes post-translational modification to produce a protein that is optimally folded (forming a tertiary structure) and is a fully functional protein. In vitro transcribed mRNA is eventually degraded by normal physiological processes, resulting in a low risk of metabolite toxicity.

There is also an attempt to develop an RNA vaccine platform using the UTR portion of human cell-derived mRNA instead of using the virus-derived UTR to express a desired target gene. A German company, CureVac, developed a platform using various ribosome protein UTRs. For example, 5'-UTR of human ribosomal large protein without 5' terminal oligopyrimidine tract, 3'-UTR of human ribosomal all protein, 64 poly A, poly C with 30 cytidylates, and histone stem-loop sequence were used. An RNA platform has been developed and used.

1. Non-amplifying mRNA

An unamplified mRNA vaccine contains the basic structure of the mRNA and has an open reading frame (ORF) encoding the desired antigen.

The main characteristics of non-amplified mRNA vaccines are (1) the relatively small size of mRNA (~2-3 vs. ~10 kb) compared to self-amplified mRNA vaccines; (2) a system that minimizes the likelihood of causing unwanted immunogenic interactions with the host (associated with viruses) in the absence of additional proteins; (3) It is relatively easy to expand and manufacture, enabling rapid use when diffusion occurs. and (4) easy genetic engineering technology to improve vaccine performance and minimize off-target effects.

The greatest barrier to mRNA vaccine availability is the need for intracellular delivery. Chemical modifications and sequence engineering have improved both the translational and shelf life of synthetic mRNA vaccines.

To achieve an enhanced adaptive immune response, mRNA was electroporated into dendritic cells used for transformation ex vivo. This strategy has been applied in several clinical trials. However, transformation is laborious, difficult to scale, and can lead to adverse immunogenicity. Thus, the field has shifted towards developing more efficient and potentially less toxic mRNA carriers using lipids or polymeric system materials.

Naked RNA vaccine, the simplest structure of RNA vaccine, has a short 5'-UTR region and a 3'-UTR region at both ends, and the genetic information (ORF/GOI) of the protein (target protein) to be expressed between them. .

The advantage of this structure lies in its simplicity. That is, the non-amplified mRNA platform has only UTRs at both ends involved in protein expression regulation, and protein expression does not occur in this part, so basically it cannot interact with the host or affect the immune response.

Therefore, the protein is expressed only in the middle ORF (GOI) region where expression is desired. However, due to the characteristics of cells that try to limit the continuous expression of mRNA, a relatively high concentration of RNA is required to be administered.

The RNA expression cassette of the present invention is mRNA having a structure capable of intracellular translation for increasing the expression of a target protein:

(1) 5'UTR;

(2) RNA encoding the target protein; and

(3) 3'UTR;

1) Structure capable of cell translation

The target protein mRNA for expressing the target protein may include a 5'UTR including a 5'cap or IRES.

The target protein RNA expression cassette, mRNA, mRNA and mRNA molecule 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 is therefore not considered as included in mRNA according to the present invention. This means that the mRNA according to the invention may comprise m7GpppN as 5' cap (CAP), but additionally the mRNA comprises at least one further modification as defined herein.

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

The target protein mRNA of the present invention does not include 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 other than the 5' end of the target protein mRNA sequence, such as a location in the center of the target protein mRNA sequence. is located in the middle of the mRNA 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 target protein mRNA molecule (non-polypeptide-sequence modification 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 the present invention is efficient when the cap is present on the target protein 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 target protein 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 target protein 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).

Examples of additional 5' cap structures include glyceryl, inverted deoxy abasic residues (components), 4',5' methylene nucleotides, 1-(beta-D-erythrofuranosyl)nucleotides, 4 '-thio nucleotide, 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 contained in the mRNA according to the present invention.

Particularly preferred chemically modified 5' cap (CAP) structures are CAP1 (methylation of the ribose of the nucleotide adjacent to m7G), CAP2 (methylation of the ribose of the second nucleotide downstream of the m7G), CAP3 (the ribose of the third 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 (e.g. 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.

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

Similar to IRES in the prior art, the 5' cap on the target protein mRNA is useful for triggering translation initiation of the target protein. 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 target protein 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 target protein mRNA is provided in trans to RNA, it is more efficient for the capped target protein mRNA to induce production of the target protein. 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 by setting the molar ratio of cap to GTP to 5-10 during in vitro transcription.

A 5' cap is a 5' cap analog. It is 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 systems described previously, the target protein mRNA of the present invention preferably comprises a cap analog. Accordingly, translation of the target protein of 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. Thus, 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 Biotechnology)]] It presents an expression system comprising a.

2) chemical modification

"RNA chemical modification" may refer to chemical modifications including sugar modifications or base modifications as well as backbone modifications.

The chemically modified RNA molecules of the present invention may contain nucleotide analogues/modifications, 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.

(1) 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 substituted or 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 Party); 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, mRNA may comprise, for example, nucleotides containing arabinose as a sugar.

(2) 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 made by the substitution of nitrogen (bridged phosphoroamidate), sulfur (bridged phosphorothioate) and carbon (bridged methylene-phosphonate) in the oxygen to which it is linked.

(3) base modifications:

As described herein, chemically modified nucleosides and nucleotides that can be incorporated into a modified RNA can be added to 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 made chemically 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. base-group consisting of 5-methylcytidine-5'triphosphate, 7-deazaguanosine-5'triphosphate, 5-bromocytidine-5'triphosphate, and pseudouridine-5'triphosphate Nucleotides for bases selected from

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 any 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.

mRNA 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:

(4) 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

(1) Variation of open reading frame: Variation of G/C content:

The mRNA 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 mRNA 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 the mRNA 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 the mRNA 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 may 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 the mRNA 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 the mRNA may be increased by a maximum value (ie, 100% of substitutable codons) compared to the G/C content of the wild-type coding region.

(2) Codon optimization:

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 RNA expression cassettes according to the present invention can be adapted.

A further preferred modification of the coding region encoding at least one peptide or protein of the mRNA in the present invention is based on the finding that the translation efficiency is also determined by the different frequencies of appearance of the tRNA in the cell. 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 mRNA is preferably modified compared to the corresponding wild-type coding region so that the codons of the wild-type sequence encoding the tRNA, which are relatively rare in at least one cell, encode the 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. As a result of the modification of the coding region of the mRNA of the present invention by such modification, a codon for using the 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 a tRNA that are relatively rare in the cell can in each case be replaced with a codon encoding a tRNA that is relatively frequent in the cell, 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 inventive mRNA 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.

(3) variants of UTR:

UTR

An untranslated region (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. The UTR may be upstream of the open reading frame and/or downstream of the open reading frame encoding the target protein.

Specifically, in the target protein mRNA of the present invention, "3'UTR" relates to a region located 3' of the coding region for the target protein, and "5'UTR" is 5' of the coding region for the target protein. It relates to the area in which it is located.

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

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

UTR is involved in RNA stability and translation efficiency, which are prerequisites for effective transport using RNA transport. 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 target protein mRNA comprises a 5'UTR and/or a 3'UTR that is not heterologous or native to that from which the target protein 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 target protein 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 the target protein; (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 target protein mRNA of the present invention is activated to increase the translation efficiency and/or stability of the human beta-globin 3'UTR, a fragment thereof, or a human beta-globin 3'UTR variant or fragment thereof, but not a 3'UTR includes

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

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 cleavage, etc. are included. In the present invention, the 3'UTR corresponds to the sequence of a mature mRNA located at the 3' end of the stop codon of the protein coding region, preferably immediately adjacent to the 3' end of the stop codon of the protein coding region, and the poly(A) sequence It extends to the 5' side of the, preferably the nucleotide immediately adjacent to the 5' end of the poly(A) sequence. In the present invention, the 3'UTR of a gene, such as the 3'UTR of alpha or beta globin, is a sequence corresponding to the 3'UTR of a mature mRNA derived from this gene, i.e., obtained by transcription of the gene and maturation of immature mRNA. sequence corresponding to mRNA. 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, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'UTR can be modified post-transcriptionally, for example, by the addition of a 5' cap. In the present invention, the 5'UTR corresponds to the sequence of the mature mRNA located between the 5'cap and the start codon. Preferably, the 5'UTR is from the nucleotide located 3' of the 5' cap, preferably from the nucleotide located immediately 3' of the 5' cap, to 5' of the start codon of the protein coding region. to the nucleotide located, preferably to the nucleotide located immediately 5' of the start codon of the protein coding 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. The 5'UTR of a gene is a sequence corresponding to the 5'UTR of a mature mRNA derived from this gene, ie, 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 start 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, a TOP motif is a nucleic acid sequence corresponding to 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. Preferably, the TOP-motif is at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, even more preferably at least 7 It consists of nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5' end with a cytosine nucleotide. In 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 preferably located at the 5' end of the sequence representing the 5'UTR or at the 5' end of the sequence encoding the 5'UTR. Thus, preferably, if this stretch is derived from the 5' end of the respective sequence, such as from the mRNA according to the invention, from the 5'UTR element of the mRNA according to the invention, or from the 5'UTR of the TOP gene as described herein. A stretch of three or more pyrimidine nucleotides is called a "TOP motif" in the sense of the present invention if it is located in a nucleic acid sequence. In other words, a stretch of 3 or more pyrimidine nucleotides that are not located at the 5' end of the 5'UTR or 5'UTR element and are located anywhere within the 5'UTR or 5'UTR element is preferably not referred to as a "TOP motif" .

TOP genes are generally characterized by the presence of a 5' terminal oligopyrimidine tube. Moreover, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with tissue-specific translational regulation are also known. The 5'UTR of the TOP gene corresponds to the sequence of the 5'UTR of the mature mRNA derived from the TOP gene, and extends from the nucleotide located 3' of the 5'cap (CAP) to the nucleotide located 5' of the start codon. 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). A particularly preferred fragment of the 5'UTR of the TOP gene is the 5'UTR of the TOP gene lacking the 5'TOP motif. The '5'UTR of the TOP gene' preferably refers to the 5'UTR of the naturally occurring TOP gene.

Preferably, the mRNA of the 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 mRNA according to the 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.

The mRNA according to the 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 mRNA 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 the mRNA according to the present invention comprises 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 mRNA according to the 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.

The mRNA according to the present invention is derived from the 3'UTR of a chordoid gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or a chordoid 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 expression cassette. 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 RNA expression cassette with enhanced 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.

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

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:

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 mRNA 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 mRNA to increase protein production.

The mRNA according to the invention may further comprise - with the addition of at least one modification - an internal ribosome entry site (IRES) sequence or an IRES-motif, for example, if the mRNA contains 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 any type of artificial target protein RNA expression cassette.

According to the present invention, the mRNA comprises at least one modification that increases the expression of the peptide or protein encoded by the coding region of the RNA. The mRNA 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 sequences, may also be present in one or more copies in the mRNA according to the invention. can This is especially true for bi- or multicistronic RNAs. The mRNA according to the 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.

poly(A) sequence

The target protein mRNA and m target protein RNA expression cassettes of the present invention include 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, no more than 300, no more than 200, in particular no more than 150 A nucleotides, in particular consists of or comprises 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 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 5-50 in length, preferably 10-30, more preferably 10-20.

A poly(A) cassette consisting essentially of dA nucleotides, but having an even distribution of 4 nucleotides (dA, dC, dG, dT) and interrupted by a random sequence of e.g. 5-50 nucleotides in length, is a DNA It exhibits constant proliferation of plaid DNA in E. coli at the RNA level and is 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 5-50, 10-30, 10-20 in length.

Polyadenylation is generally understood as the addition of a poly(A) sequence to an RNA molecule, eg, a nucleic acid molecule such as an immature RNA expression cassette. 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 includes a hexamer consisting of adenine and uracil/thymine nucleotides, and the hexamer sequence AAUAAA. Other sequences, hexamer sequences, are possible. Adenylic acid polymerization occurs during processing of pre-mRNA (also called immature-mRNA). RNA maturation (from pre-mRNA to mature mRNA) involves the step of adenylate polymerization.

Moreover, for an mRNA 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 serii 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 expression cassette. To be efficiently translated, the poly(A) tail of an exogenously delivered mRNA must consist of at least 20 A residues. Furthermore, mRNA expression has been described to correlate positively with the length of the poly(A) tail (Tavernier et al., J Control Release. 2011 Mar 30;150(3):238-47). Shows improved mRNA stability and translation efficiency compared to shorter poly(A) tails measured with 120 nucleotides (Holtkamp et al., Blood. 2006 Dec 15;108(13):4009-17) . The mRNA of the present invention comprises (optionally in combination with other modifications) a poly(A) tail, wherein the poly(A) tail contains at least 30, preferably more 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 contemplated as at least one modification comprised in the mRNA according to the invention.

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

An mRNA 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.

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

2. Self-amplifying mRNA (SAM)

Self-amplifying mRNA is an RNA called a replicon that encodes an RNA virus genome engineered from benign single-stranded RNA viruses such as alpha viruses and flaviviruses, usually encoding nonstructural proteins that aid in RNA capping and replication. ORFs that express antigens that replace viral structural proteins and ORFs that

SAM vaccines have several attractive features, such as prolonged duration (about 2 months) and magnitude of expression compared to their non-replicating counterparts. However, to maintain self-amplifying activity, these RNA replicons must undergo many synthetic nucleotide modifications and sequence alterations. Other limitations of SAM include (1) inclusion of unrelated proteins that may elicit a potential host response; and (2) large replicon sizes (~10 kb), which may limit cell internalization efficiency.

In general, self-amplifying mRNA provides a system comprising an RNA construct for expressing an alphavirus replicating enzyme and an RNA replicon capable of being trans-replicated by a replicating enzyme, and one RNA in which two types of components are identical It can be divided into a cis-replicon in the cis-replicon and a trans-replicon in two different RNAs.

In a trans-replicon system, there is provided a system comprising an RNA construct for expressing an alphavirus replicating enzyme, an RNA replicon capable of being replicated in trans by a replicating enzyme, wherein the method for expressing an alphavirus replicating enzyme is provided. The RNA construct includes a 5'-cap. The 5'-cap serves the purpose of inducing translation of the replicating enzyme. 5'-caps include native 5'-caps or 5'-cap analogs. The RNA construct for expressing the alphaviral replicator does not include an internal ribosome entry site (IRES) element for directing translation of the replicator.

Thus, a trans-replicon comprises two nucleic acid molecules: a first RNA molecule for expressing (ie, encoding for) a replicator; and a second RNA molecule (replicon). An RNA construct for expressing a replicator is referred to as a "replicate construct" synonymously in the present invention.

An RNA construct for expressing an alphavirus replicator may include (1) a 5' UTR, (2) an open reading frame encoding a replicator, and (3) a 3' UTR. The 5'UTR and/or the 3'UTR is characterized as not unique to the alphavirus inducing replication enzymes. The open reading frame encoding the alphaviral replication enzyme contains the coding region(s) for the nonstructural proteins necessary for RNA replication. The RNA construct for expressing an alphaviral replication enzyme comprises a 3' poly(A) sequence. The RNA construct for expressing the alphavirus replicator cannot be replicated by the replicator.

The RNA replicon comprises: (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. The alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence direct replication of the RNA replicon in the presence of a replication enzyme.

The alphavirus 5' replication recognition sequence and the alphavirus 3' replication recognition sequence are unique to the alphavirus inducing replication enzymes. RNA replicons include heterologous nucleic acids. An RNA replicon comprises an open reading frame encoding a polypeptide of interest. The open reading frame encoding the polypeptide of interest is not unique to the replication enzyme-derived alphavirus.

Expression of the open reading frame encoding the polypeptide of interest is under the control of a subgenomic promoter. The subgenomic promoter is unique to the alphavirus that drives the replication enzyme. Subgenomic promoters are promoters for structural proteins of alphaviruses.

The RNA replicon comprises a 3' poly(A) sequence and a 5'-cap.

It is an RNA construct for expressing an alphavirus replicating enzyme including a 5'-cap for inducing translation of the replicating enzyme. DNA comprising a nucleic acid sequence encoding an RNA construct for expression of an alphaviral replicator, an RNA replicon, or both of the above systems can be prepared.

In the trans-replicon system, the role of the replicator is to amplify the replicon into trans. Therefore, the replicon may be referred to as a trans-replicon. If the replicon encodes a target gene for expression, the expression level and/or expression duration of the target gene can be regulated in trans by changing the level of the replicating enzyme.

The trans-replicon system can be easily prepared. For example, RNA molecules can be transcribed in vitro from a DNA template.

The RNA of the trans-replicon system is in vitro transcribed RNA (IVT-RNA). Accordingly, the system of the present invention comprises an IVT-RNA. Preferably, all RNA molecules of the system of the invention are IVT-RNA. In vitro-transcribed RNA (IVT-RNA) is of particular importance for certain therapies.

A trans-replicon system comprises at least two nucleic acid molecules. 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. It contains exactly two nucleic acid molecules, preferably an RNA molecule, a replicon and a replicator construct.

The system comprises, in addition to the replicatase construct, two or more replicons, each preferably encoding at least one polypeptide of interest. The replicator encoded by the replicatase construct can act on each replicon to induce replication and production of subgenomic transcripts. For example, each replicon may encode a pharmaceutically active peptide or protein. This is advantageous, for example, when it is necessary to vaccinate a subject against several different antigens.

Preferably, the trans-replicon system is not capable of forming viral particles, in particular next-generation viral particles.

Preferably, the replicatase construct is not capable of replicating itself in the target cell or target organism.

Alternatively, although aspects and advantages of RNA are described herein, it is possible for a trans-replicon system to include more than one DNA molecule. Any one or more nucleic acid molecules of the system of the invention may be DNA molecules. It is possible that the replicator construct and/or the replicon is a DNA molecule. In the case of a DNA molecule, it is preferred that the promoter of a DNA dependent RNA polymerase be present, thereby enabling transcription in an infected or vaccinated host cell or host organism.

II. Nucleic acid biodegradable multiparticulates

In relation to the present invention, the nucleic acid of the biodegradable polymer carrier composite particle is any DNA, preferably, but not limited to, for example, genomic DNA, single-stranded DNA molecules, double It may be an appropriate nucleic acid selected from double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, pDNA, and immunostimulating DNA, and any PNA (peptide nucleic acid) may be selected from these, and any RNA, preferably but not limited to, coding RNA, messenger RNA (mRNA), siRNA, shRNA, antisense RNA ), or riboswitches, immunostimulating RNA (isRNA), ribozymes, or aptamers.

In addition, the nucleic acid may be ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), or viral RNA (viral RNA, vRNA). Preferably, the nucleic acid is RNA, more preferably coding RNA.

The nucleic acid may be a linear single-stranded RNA or mRNA. mRNAs typically contain several structural elements, e.g., an upstream ribosome binding site followed by a coding region, an optional 5'-UTR, poly-A-tail (and/or poly-C-tail) RNA containing an optional 3'-UTR that may be followed by mRNA may appear as mono-, di-, or even multicistronic RNA, i.e. one, two or more (same or different) proteins or peptides as defined herein. RNA that carries the coding sequence of In di-, or even multicistronic mRNA, such coding sequences may be separated by at least one internal ribosome entry site (IRES) sequence.

In addition, the nucleic acid of the biodegradable polymer carrier carrier complex may be a single-stranded or double-stranded nucleic acid due to the non-covalent association of two single-stranded nucleic acids (molecules).

or a partially double-stranded or partially single-stranded nucleic acid, at least partially complementary, all of which are partially double-stranded or partially single-stranded; is made by the same two single-stranded nucleic acids, one single-stranded nucleic acid is partially complementary to the other single-stranded nucleic acid, and thus both form a double-stranded nucleic acid molecule at this portion. That is, to form a partially double-stranded or partially single-stranded nucleic acid (molecule).

Preferably, the nucleic acid (molecule) may be a single-stranded nucleic acid molecule. Furthermore, the nucleic acid (molecule) may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.

Transfection of RNA carriers faces extracellular and intracellular barriers. RNA transporters are exposed to serum nucleases and phagocytic uptake, which significantly reduces their biological half-life. In addition, the negative charge of the cell membrane as well as the extracellular matrix (E) prevents the RNA transporter from reaching its target and acting.

Among the many barriers to function, RNA must cross the cell membrane to reach the cytoplasm. Cell membranes are dynamic and powerful barriers to intracellular transport. Various ion pumps and ion channels help to maintain the negative potential. It maintains a negative charge in the cytoplasmic space by regulating the balance of -40 to -80 mV across the cell membrane), most essential metal ions (eg K+, Na+, Ca2+ and Mg2+).

The cell membrane creates a strong barrier to highly negatively charged RNA molecules. Unsaturated lipids, especially cis-double bonded lipids, increase cell membrane fluidity by introducing defects into the membrane structure. The other major component of the bilayer includes sterols (~ 30% of total lipids). Cholesterol, the major sterol in the lipid bilayer, helps to maintain a balance between fluidization and condensation of the lipid bilayer by creating bilayer defects.

Transfection of nucleic acids is performed using viral or non-viral vectors. For successful delivery, these viral or non-viral vectors must overcome the obstacles mentioned above. The most successful gene therapy strategies today rely on the use of viral vectors such as adenoviruses, adeno-associated viruses, retroviruses and herpes viruses. Viral vectors can mediate gene transduction with high efficiency and the potential of long-term gene expression, satisfying two out of three hypotheses. However, the acute immune response, immunogenicity, and insertional mutagenesis revealed in gene therapy clinical trials raise serious concerns about the safety of some commonly used viral vectors.

A solution to this problem may be found in the use of non-viral vectors. Although non-viral vectors are not as efficient as viral vectors, many non-viral vectors offer a more stable alternative to gene therapy.

Non-viral gene delivery methods are being investigated using physical (carrier-free gene delivery) and chemical (gene delivery based on synthetic vector delivery) approaches. Physical approaches usually include needle injection, electroporation, gene gun, ultrasound and hydrodynamic delivery, using physical forces that penetrate cell membranes, and promotes gene introduction into cells. Chemical approaches generally use synthetic or naturally occurring compounds (cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to transport the gene of interest into cells. is used as a carrier.

Although significant advances have been made in basic science and in various non-viral gene delivery systems, the majority of non-viral approaches are still less efficient than viral vectors, particularly in in vivo gene delivery.

In order to achieve steric stability of the RNA of the present invention and improve the BD/PK properties of the carrier, polyethylene glycol conjugated (PEGylated) lipids approved by the FDA for human use in other pharmaceuticals may be used. The entire particle surface may be covered with PEG to minimize any possible immune response and activation of the complement system.

Poly Electrolyte Complex (PEC) is formed spontaneously when two polymers of opposite charges are mixed to form a complex in which macromolecules are bound together mainly by electrostatic interaction. Also, the term “nanoplex” is widely used in the literature to denote a complex formed between a positively charged nanoparticle and a nucleic acid, DNA or RNA. When the positively charged component is based on a cationic polypeptide such as chitosan, a nanoplex can also be defined as a polyplex.

It is well known that PECs are predominantly spherical, contain a stoichiometric mixture of cationic and anionic polymers, and consist of a neutral core surrounded by an excess of polyelectrolyte that allows the formation of a charge stabilizing shell.

Since the self-assembly of PEC is a reversible process, its integrity may be compromised in the presence of other charged species due to electrostatic shielding and polyion exchange between the PEC and the medium. Therefore, the stability of PEC can be severely affected in biological media in the presence of charged proteins and high concentrations of small electrolytes. A strategy to overcome this problem is to improve the stability of PECs by covalently crosslinking the complex polyelectrolyte to prevent separation of the polymer chains from the PEC. PEC-based nanoparticles are attractive because they are easy to fabricate and cost-effective, and the reversibility of electrostatic interactions is attractive because they provide special tunability to the behavior of the carrier while maintaining the integrity of the composite under appropriate conditions.

One of the most developed methods for RNA delivery is a combination formulation with lipid nanoparticles (LNP). LNP formulations generally consist of (1) a polymeric material carrying ionizable or cationic lipids or tertiary or quaternary amines to encapsulate polyanionic RNAs. (2) cationic lipids similar to those of cell membranes (eg, 1,2-dioreoyl-sn-glycero-3-phosphoethanolamine [DOPE]); (3) cholesterol, which stabilizes the lipid bilayer of LNPs; and (4) polyethylene glycol (PEG)-lipids to form a hydration layer on nanoparticles to improve colloidal stability and reduce protein absorption.

The most representative particle manufactured by encapsulation is lipid nanoparticles, and phospholipids are recognized as one of the most promising carriers of RNA due to their biocompatibility, relative ease of large-scale production, and recent approval for use in clinical trials.

Phospholipids are amphiphilic molecules that, when dispersed in water, form spontaneous bilayer structures and trap the dispersed hydrophilic load within the aqueous core of the molding structure. Therefore, chemical modification of the head groups of these molecules with cationic polypeptides facilitates the capture of the negatively charged RNA prepared in the present invention through electrostatic interaction, making it a promising component of a carrier.

The RNA transporter of the present invention is directed to improving the pharmacokinetics of the RNA transporter in terms of plasma stability and circulation time, as well as in terms of cell influx.

The nucleic acid constituting the RNA of the present invention is a strongly negatively charged polymer, and the cationic polymer plays a very important role in the process of preparing the particles. Cationic polypeptides offer several advantages, including the ability to promote complex formation with negatively charged RNA expression cassettes via electrostatic interactions, cellular uptake, and proton sponge-mediated endosomal escape. However, a disadvantage of cationic polymers is that they are highly toxic due to changes in cell membrane integrity and high immunogenicity.

A further promising approach in gene therapy is the use of cationic polymers. Cationic polymers have been found to be useful in transfection of nucleic acids because they form tight complexes and condense negatively charged nucleic acids. Therefore, many cationic polymers are being studied as carriers for gene delivery in vitro and in vivo.

Such cationic polymers include polyethyleneimine (polyethylenimine (PEI)), polyamidoamine dendrimers, polypropylamine dendrimers, polyallylamine, cationic dextran, chitosan, cationic proteins and cationic peptides.

Although most cationic polymers have the function of condensing DNA into small particles and promoting cellular uptake by endocytosis through charge-charge reactions with anionic localization on the cell surface, transfection activity (transfection) There is a marked difference between activity and toxicity.

Cationic polymers exhibit better transfection capacity at higher molecular weights due to stronger complexation of negatively charged nucleic acid carriers. However, the toxicity of the cationic polymer increases as the molecular weight increases. Polyethylenimine (PEI) is perhaps the most active and most studied polymer in the field of gene delivery, but its greatest weakness as a transfection reagent is related to its non-biodegradable properties and toxicity. .

One unusual approach in gene therapy is the use of cationic lipids. However, although many cationic lipids show excellent transfection activity in cell culture, most do not perform well in the presence of serum, and only a few have activity in vivo. When lipoplexes are exposed to overwhelming amounts of negative charge and amphiphilic proteins and polysaccharides present in blood, mucus epithelial lining fluid or tissue matrix, their size, surface charge and lipid Significant changes occur in structure.

Once administered in vivo, lipoplexes react with negatively charged blood components and tend to form large aggregates, which are either adsorbed to the surface of circulating red blood cells or deposited in a thick mucus layer. It can become entrapped or embolize the microvasculature, preventing the lipoplex from reaching its intended target cells at a distal location.

In addition, toxicities associated with gene transfer by lipoplexes include, inter alia, the induction of inflammatory cytokines. In humans, adverse inflammatory reactions of varying degrees, including flu-like symptoms, have been reported among patients receiving lipoplex. Therefore, it remains a question whether Lipoplex can be safely used by everyone.

Moreover, although polyplexes made by polymers with high molecular weight exhibit improved stability under physiological conditions, such polymers can interfere with vector unpacking. For example, 19 and 36 poly L-lysine (PLL) residues are significantly higher than 180 residue poly L-lysine (PLL) residues, resulting in improved short-term gene expression. rapidly dissociates from DNA. At least 6 to 8 cationic amino acids in length are required to pack DNA into structures that are active for receptor-mediated gene transport. However, polyplexes made of short polycations are not stable under physiological conditions and generally aggregate rapidly in physiological saline.

To overcome this negative aspect, linear reducing polycondensation (reducible) produced by oxidative polycondensation of Cys-Lys10-Cys peptide that can be cleaved by the intracellular environment to promote the release of nucleic acids A novel type of synthetic vector based on polycation (RPC) has been developed. They showed that polyplexes made by RPC can be destabilized by a reducing state that allows for efficient release of DNA and mRNA.

The cationic peptide protamine has been shown to protect mRNA from degradation by serum RNases. However, only mRNA complexed with protamine showed limited protein expression and efficacy in cancer vaccine models due to the strong binding force between protamine and mRNA. This problem was addressed by developing an RmRNAtive vaccine platform in which protamine-formulated RNA acts only as an immune activator, not as an expression vector.

CureVac has developed an RNActive vaccine platform based on a protamine-made RNA complex that acts as a TLR 7/8 agonist to induce Th1 T cell responses, whereas nucleotide-modified mRNA functions as antigen producers. This vaccine platform has resulted in favorable immune activation against cancer and infectious diseases in both preclinical animal models and patients.

The interesting prospect that cellular transport of nucleic acids results from the physical assembly or chemical ligation of cell penetrating peptides (CPPs), also expressed as protein-transduction domains (PTDs). was announced CPP corresponds to a short peptide with a sequence of about 10 to 30 amino acids, which can cross the plasma membrane of mammalian cells and thus offer unprecedented possibilities for cell drug transport.

Almost all of these peptides contain a series of cationic amino acids in a combination of sequences that are in the α-helix form at low pH. When the pH is continuously lowered in vivo due to the proton pump, the conformational change of the peptide is usually initiated rapidly.

This helix motif mediates the insertion into the membrane of the endosome leading to the release of the contents into the cytoplasm (cytoplaa). *?**?*The truncated HIV-1 Tat protein basic domain crosses the plasma membrane and accumulates in the cell nucleus*?**?*. Despite these advantages, major obstacles to CPP-mediated drug transport often involve rapid metabolic clearance of peptides when encountering or crossing the enzymatic barriers of epithelia and endothelia. think that

Cellular delivery peptides (CPPs) have been studied for their potential as vectors for intracellular delivery of nucleic acid delivery. Although their internalization mechanism is not fully understood, it is hypothesized that CPPs may promote clustering of negatively charged glycosaminoglycans at the cell surface. This causes macropinocytosis and lateral diffusion or directly disrupts the lipid bilayer. CPPs with arginine-rich amphiphilic RALA sequence repeats have been reported to function as mRNA vectors for dendritic cells and induce T cell immunity.

In conclusion, the metabolic stability of CPPs represents an important biopharmaceutical factor for their cell bioavailability. However, on the one hand, they are stable enough to transport their transporters to the target before they are metabolically cleaved, while on the other hand, they can accumulate and be completely eliminated from the tissue before they can reach toxic levels. CPPs were not previously available.

In addition to the barrier of cell membranes, RNA is subjected to degradation by extracellular ribonucleases, which are abundantly present in the skin and blood. To protect mRNA from degradation by nucleases and to protect its negative charge, amine-containing substances are commonly used as non-viral vectors.

RNA multiparticulates must have the ability to fully protect RNA from enzymatic degradation and have an appropriate size to allow extravasation and retention of the target site while preventing renal clearance. It should also possess appropriate surface properties that allow efficient entry by cells while preventing serum protein interactions and avoiding phagolysosomes. Another desirable property that facilitates clinical application is the easy functionalization of targeting ligands to improve tissue specificity, biocompatibility and reduce toxicity. In addition, the transporter of RNA faces the problem of leaving the endosome in a timely manner to deliver the RNA to the site of protein synthesis located in the cytoplasm.

The backbone of the mRNA constituting the RNA complex particle of the present invention is a nucleic acid and should be protected from nucleases to ensure stability in blood, and the ideal delivery system of the present invention should be able to achieve the above object.

In the present invention, chemical modification of nucleic acids, protection of nucleic acid ends, etc. can be performed, and a method for preparing particles by encapsulating the RNA with a polymer material can be provided.

Cleavage of RPC (reducible polycation) reduced the toxicity of polycations to a level comparable to that of peptides with low molecular weight. A disadvantage of this approach was that it additionally required the endosomolytic drug chloroquine or the cationic lipid DOTAP to improve the transfection efficiency to an appropriate level. Consequently, reducing polycations (RPCs) may contain histidine residues, which are known to have endosomal buffering capacity.

They believe that histidine-rich reducing polycations can be cleaved by an intracellular reducing environment that allows for sufficient cytoplasmic transport of a diverse range of nucleic acids, including plasmid DNA, mRNA, and siRNA molecules, that do not require the endosomal disrupting agent chloroquine. could show that it could.

Unfortunately, it has not been revealed whether it is possible to directly apply many reducing polyions to histidine in vivo. Transfection was performed in the absence of serum to avoid binding of serum proteins to polyplexes that restrict cellular uptake, obscuring the ability of histidine residues to enhance gene transduction. Transfection ability of histidine-rich reducing polycation polyplexes was affected in the presence of 50% reduced serum protein of GFP-positive cells observed in 10% fetal calf serum (FCS).

Other approaches to technology for delivering delivery molecules into cells, such as gene therapy, involve peptide ligands. Peptide ligands may be short sequences separated from larger proteins representing essential amino acids required for receptor recognition, such as EGF peptides used in target cancer cells. Other peptide ligands have been found to include ligands used to target the lectin-like oxidized LDL receptor (LOX-1).

Up-regulation of LOX-1 in epithelial cells is associated with disorders such as hypertension and atherosclerosis. However, peptide ligands cannot be linked to their carrier molecules by complexation or adhesion and require covalent bonding, making them unsuitable for many gene therapy approaches, and generally use crosslinkers with cytotoxic effects in cells. for example.

It is capable of condensing and stabilizing nucleic acids for the purposes of gene therapy and other therapeutic applications and exhibiting effective transfection activity, particularly in terms of the combination of effective release of nucleic acid delivery and low or no toxicity in vivo. No suitable method or carrier has been found to this day.

There is still a strong need in the art to provide carriers for the purpose of gene delivery, and this strong need is that they are stable enough to deliver their delivery to the target before they are metabolically cleaved, on the other hand, that they It must be removed from the tissue before it can accumulate or reach toxic levels.

The therapeutic success of RNA-based agents depends on overcoming many challenges associated with RNA delivery in vivo. The development of safe and effective RNA delivery systems is crucial for the successful application of RNA-based therapies. Nanostructured carriers offer unique advantages such as protection from premature degradation and improved interaction with the biological environment. It also enhances tissue uptake, prolongs RNA retention time, enhances cellular internalization, and enables RNA to migrate into the cytoplasm of target cells.

Systemically delivered LNPs that deliver RNA face multiple barriers to delivery. The mononuclear phagocytosis system (MPS), particularly in the liver and spleen, is a frequent destination for injected nanoparticles because of their intrinsic role in the body's protective function against nano-sized infectious agents. The kidneys filter out naked mRNA or any nanoparticles with a hydrodynamic diameter of less than 5.5 nm.

Most LNPs are about 100 nm in diameter and are large enough not to exit the MPS and reach other organs of interest. The liver, the major organ of MPS, has a porous vasculature and contains phagocytes such as Kupffer cells that carry cationic LNPs. In addition, large cationic LNPs do not leak from the capillaries found in the lungs and cannot be filtered into the bloodstream by the kidneys.

This can lead to buildup of the transmitter in the liver, lungs or other organs. Therefore, clearance and biodegradability of the transporter components are one consideration when developing mRNA transporters.

Ester groups are the most commonly used functional groups to enhance the biodegradability of biomaterials, but the in vivo degradation of other ester bonds may depend on the overall chemistry of the molecule and formulation. It was reported to be rapidly halved in the liver compared to LP-01 and lipid 5 (discussed) compared to DLin-MC3-DMA (t _1/2 > 50 h) with no less protein expression (half-life [t _{1 / 2} ] to 6 h).

In certain tissues, the presence of ester functional groups can accelerate degradation of LNPs, potentially limiting protein expression. For example, OF-Deg-Lin was able to reach liver cells but selectively induced protein expression in the spleen. It was hypothesized that this could be due to the rapid degradation of LNPs by liver enzymes. Rational design of degradable lipids or polymers based on polyester or polycarbonate can better control the degradation of the carrier.

It has proven more difficult to target specific organs or tissues other than the liver with LNPs. Some LNPs accumulate or are absorbed in the liver in an apolipoprotein E-dependent manner. In the case of APE, it has been found that the core structure of the tertiary amino alcohol of the polymer may play an important role in targeting specific organs.

Recently, oligopeptide-modified PBAEs (OM-PBAE) targeting antigen-presenting cells (APCs) were obtained by end-capping the acrylate groups of PBAE with CKKK oligopeptides via Michael addition. These targeting strategies have their limitations and typically require high-throughput screening to find successful targeted solutions for specific tissues. A rationally designed targeting approach, such as incorporating a ligand into a tissue-specific receptor, is an alternative strategy.

We recently reported the in vitro active targeting of sialic acid-terminated glycoproteins on the surface of dendritic cells. A 30-amino acid synthetic peptide with glutamic acid-alanine-leucine-alanine (GALA) is a repeat conjugated to a polyplex carrying EGFP-mRNA. However, any opsonization of LNPs forming a protein corona may be detrimental to active targeting strategies due to masking of the targeting ligand. Therefore, new strategies targeting specific tissues need to be studied to solve clinical challenges.

In the field of delivering RNA-based therapeutics to cells, the final endpoint is endosomal escape. ASO and siRNA delivery to the liver can be addressed by ASGPR-targeted GalNAc-siRNA conjugates, and mRNA and CRISPR delivery by LNPs appear to be promising, but for the delivery of effective and clinically prepared RNA-based therapeutics to the whole body other than the liver. The approach is still at the final endpoint.

Although the exact reason is unknown, ASGPR has properties that are very suitable for the delivery of macromolecular drugs to hepatocytes. ASGPR binds to blood asialoglycoproteins and draws them to the clathrin-coated endosome, where they enter the lysosome for degradation. ASGPR-bound asi-aloglycoproteins are not thought to escape into the cytoplasm.

However, hepatocytes express numerous ASGPRs on the cell surface, which cycle remarkably fast, at rates of every 10 to 15 minutes. A minimum of 5,000 siRNAs are required for a maximal RNAi response. GalNAc binding of ASGPR rarely causes membrane instability, which allows millions of siRNA molecules to enter ASGPR endosomes every 15 minutes, with escape efficiencies lower than 0.01%, resulting in over 5,000 molecules per day via GalNAc delivery. can transmit Unfortunately, no other ligand-receptor system exists that can express the receptor at such a rapid rate or that can rapidly cycle into the endosome.

In fact, most cell surface receptors are expressed in the 10,000-100,000 range (or less), and caveolin and clathrin-mediated cell membrane entrapment are typically rotated every 90 min. Theoretically, a receptor-targeted delivery method could bring more than ~100,000 siRNAs to the cell's endosomes every 2 hours. However, if the endosome escape efficiency is estimated to be less than 0.01%, it becomes difficult to reach the required threshold of at least 5,000. Currently, several research groups are working on solutions to the problem of endosomal escape.

The classic approach is to use chloroquine, a low molecular weight endosomal degradation factor. Chloroquine (chloroquine) passively diffuses through the cell membrane and becomes a proton when the pH falls and is trapped inside the endosome, and the concentration in the endosome sharply increases. Chloroquine inserts a hydrophobic motif into the lipid bilayer and dissolves endosomes at a certain concentration. However, at the concentration showing the effect, these endosome degrading factors non-specifically dissolve endosomes containing siRNA compounds as well as endosomes in other cells, thereby causing toxicity. As a result, these small molecule endosome degrading factors have received attention in the development stage, but present a potential toxicity risk for clinical use.

An alternative endosomal escape approach is to conjugate endosomal degrading peptides or molecules directly to RNA to limit degradation to endosomes containing RNA therapeutics. Arrowhead Pharmaceu-ticals has developed a two-molecule dynamic polyconjugate (DPC) system. siRNA binds to cholesterol to form a large aggregate structure (low-density lipoprotein) in the blood, moves to the liver, and is absorbed by hepatocytes by cell membrane entrapment. The second molecule is a derivative of the pore-forming melittin peptide (derived from bee venom) that dissolves the membrane. Arrowhead synthesized melittin with pH sensitivity and conjugated it to GalNAc.

At low pH of hepatocyte endosomes, melittin is activated and degrades endosomes to release siRNA into the cytoplasm. However, due to melittin toxicity, Arrowhead has decided to discontinue all three melittin-dependent clinical programs with the FDA. This point shows that solving the endosome escape problem is a difficult task.

Ⅲ. Multiparticulate comprising an RNA expression cassette

In the present invention, the RNA expression cassette is an isolated polynucleotide encoding a polypeptide, wherein the isolated polynucleotide comprises: Unmodified mRNA; Modified mRNA; self-amplifying mRNA; And Trans-amplifying mRNA; any one or more selected from the group comprising.

<FIG. 1> is a conceptual diagram of a basic composite particle 10 including an RNA expression cassette according to the present invention and an RNA expression cassette composite particle including the composite particle 20. The basic composite particle 10 is an RNA having a negative charge. The complex (a) is composed of an expression cassette and a cationic polymer through electrostatic interaction, and the complex particle 20 is the layer 21 of the basic complex particle 10 containing an RNA expression cassette and a cationic polymer and the outer layer of the anionic polymer A composite particle (b) comprising a layer (22).

Preferably, the RNA expression cassette multiparticulate of the present invention may be an RNA expression cassette, a multiparticulate by mixing a cationic polymer and an anionic polymer.

The ratio of cationic polymer to RNA expression cassette, ie the net charge of the particle, is important. Therefore, in the present invention, an RNA expression cassette multiparticulate-based delivery platform using cationic polymers and anionic polymers was developed.

In the present invention, a number of novel RNA expression cassette multiparticulates have been developed to facilitate RNA expression cassette delivery, increase protein translation and protect RNA expression cassettes from RNases. The present invention seeks to overcome concerns about rapid extracellular RNA expression cassette degradation and aggregation with serum proteins to present a possibility for systemic administration of RNA expression cassettes.

Another important issue is the biodistribution of RNA expression cassette carriers after systemic delivery. Certain cationic fat nanoparticle complexing agents were delivered via intravenous injection, primarily to the liver, which is not ideal for dendritic cell activation. That is, while positively charged particles mainly target the lungs, negatively charged particles are delivered to secondary lymphoid tissues and bone marrow to activate an immune response.

The composite particle of the present invention may include any one or more selected from the group consisting of an imaging material and a neutral or cationic polypeptide. Alternatively, an imaging material and a neutral molecule, cholesterol, may be attached to the RNA expression cassette. The multiparticulates can simultaneously possess the protein encoding and pharmaceutical function by the contained pharmaceutical active ingredient, the imaging function by the imaging material, and the protection function of the nucleic acid drug by cholesterol.

Since the RNA expression cassette itself is defenseless against an enzyme that degrades nucleic acids composed of nucleotides in the human body, it is difficult to transmit its efficacy completely into the target tissue, and there is a problem in that it is discharged by the renal filtration function. Therefore, the present invention provides a method for overcoming the above problems in order to develop the complex into an excellent drug as follows.

<Figure 2> shows a method for preparing a multiparticulate comprising the RNA expression cassette according to the present invention. In the present invention, paying attention to the characteristic that the RNA expression cassette itself has an anion, the RNA expression cassette is coated with a positively charged molecule to prepare a basic composite particle (10), and additionally, an anionic polysaccharide is second coated to make the composite particle (20) ) to provide a method for preparing

Preferably, the RNA expression cassette multiparticulate of the present invention may be an RNA expression cassette, a multiparticulate by mixing a cationic polymer and an anionic polymer.

More specifically, the cationic polymer forms the basic composite particle 10 condensed by electrostatic interaction with the RNA expression cassette.

The RNA expression cassette consists of n nucleotides and has n-1 negative charges per molecule or 1.8×10 ²⁵ negative charges per mole.

Protamine (PS), a representative material in the cationic polymer, has a molecular weight of 5.8 kDa and is rich in basic amino acids, particularly arginine (70-80%), so that the cation per mole is 1.26 ± 10 ²⁵ . Therefore, when complexed with the complex, PS neutralizes the negative charge of the complex phosphate group. These substances, commonly called Poly-Electrolyte Complexes (PECs), are composed of polymers of biological macromolecules containing groups that can dissociate in water and interact to form charged components.

It is also possible to provide a method in which the charge ratio between the RNA expression cassette and the cationic polymer of the present invention is 1:0.4 or more, that is, RNA is 1 and PS is 0.4 or more.

The binding between the basic multiparticulate particles that aggregate due to the electrostatic interaction of the RNA expression cassette and PS is determined according to the charge of each of the RNA expression cassette and PS, and in the particle of the present invention, the RNA expression cassette and PS (μg /mL) may be 30:15 to 150, preferably 30:15 to 90, more preferably 30:75.

Human serum albumin (HSA), amniotic biopolymer, Any one selected from a salt containing negatively charged nanoparticles or an RNA expression cassette and CaCl ₂ may be used.

In the basic composite particle 10 presented in the present invention, the RNA expression cassette necessarily further contains a cationic polymer to form an ionic complex. That is, the ionic complex formed by the negatively charged RNA encapsulated in the cationic polymer due to the electrostatic interaction with the cationic polymer is located in the core part of the particle. The layer 21 of the basic composite particle 10 formed by the contact of the RNA expression cassette with the cationic polymer has a strong positive charge, so there is a problem in that adhesion to cells is increased and it is difficult to pass through the tissue barrier.

In the present invention, in order to solve this problem, the anionic polymer having good biocompatibility and having a negative charge is used to coat the outer surface of the cationic polymer layer 21 secondarily to form the anionic polymer layer 22 to form the composite particles 20 ) can be prepared.

The anionic polymer layer coating step is a step of adding an anionic polymer and coating the anionic polymer layer 22 on the outer surface of the cationic molecular layer 21 by electrostatic attraction.

The composition and manufacturing method of the basic composite particle 10 and the composite particle 20 will be described in detail as follows.

The RNA expression cassette may have a backbone, sugar, or base chemically modified or terminally modified for the purpose of increasing blood stability or weakening an immune response. Specifically, a part of the phosphodiester bond of RNA is replaced with a phosphorothioate or boranophosphate bond, or a methyl group, methoxyl group at the 2'-OH position of some ribose bases It may include one or more modified nucleotides into which various functional groups such as an ethyl group and fluorine are introduced.

In addition, one or more ends of the RNA expression cassette may be modified with one or more selected from the group consisting of cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms include cholesterol, tocopherols and their respective analogs, derivatives, and metabolites.

In the present invention, the RNA expression cassette may be included in an amount of 0.05 to 10% by weight, more specifically 0.1 to 5% by weight, even more specifically 0.5 to 3% by weight, based on the weight of the total composition. . If the content of the RNA expression cassette is less than 0.05% by weight based on the weight of the total composition, there may be side effects due to the carrier because the amount of the carrier used compared to the drug is too large, and if it exceeds 10% by weight, the size of the nanoparticles becomes too large, the stability of nanoparticles is reduced, and there is a fear that the loss rate during filter sterilization may increase.

In a specific embodiment, the cationic polymer is bound to the RNA expression cassette by electrostatic interaction to form a complex, and the complex is encapsulated within the nanoparticle structure of the anionic polymer. Accordingly, the cationic polymer includes all types of compounds capable of forming a complex by electrostatic interaction with the RNA expression cassette, and may be, for example, cationic lipids or cationic polymers.

cationic polymer

The basic composite particle 10 of the present invention is a complex in which the RNA expression cassette and the cationic polymer are condensed by electrostatic interaction. When the RNA expression cassette having a (-) charge and a cationic polymer having a (+) charge are mixed with each other, they may be condensed. More specifically, molecules having a strong positive charge surround the negatively charged RNA expression cassette and condense or compress to reduce the particle size.

In the present invention, the expression "cationic polymer" denotes a polymer exhibiting cationicity at least in water, and also includes amphoteric polymers exhibiting cationicity as a whole.

In the present invention, a polymer having a cationization degree of 0.2 or more is used. By using the cationic polymer, it is possible to easily form the anionic polymer and the composite particles.

On the other hand, there is no specific upper limit on the degree of cationization of the cationic polymer from the viewpoint of forming multiparticulates with the anionic polymer. As a reference, cationic polymers having a cationization degree of 2 or less can be used.

In this regard, the expression "cationization degree" denotes a value obtainable by calculation according to the following formula.

｛ (the number of cationic functional groups contained in the polymer) - (the number of anionic functional groups contained in the polymer) ｝/ total amount of monomers constituting the polymer

The cationic polymer used in the present invention may be a natural product or a synthetic product as long as it has a cationic functional group.

Examples of the cationic functional group contained in the cationic polymer include an amino group, a guanidino group and an imino group. The cationic polymer of the present invention contains these groups in the side chain or main chain of the polymer.

The cationic polymer includes cationic polypeptides, cationic polysaccharides, cationic lipids, cationic surfactants, cationic molecules and synthetic polymers covalently bound to bile acid or bile acid derivatives, and the cationic polymer is chitosan (chitosan). ), glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethylenimine, dextran, hyaluronic acid ( hyaluronic acid), albumin, high molecular weight polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).

In the present invention, the type of cationic polypeptide is not particularly limited, and the ratio of basic amino acid residues to all amino acid residues is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. , more preferably 99% or more of basic RNA is preferably used.

In particular, a basic cationic polypeptide composed only of basic amino acid residues can be preferably mentioned.

As the basic cationic polypeptide, RNA composed of arginine and lysine can be mentioned particularly suitably.

Specifically, polylysine or polyarginine is preferably used.

In addition, as the cationic polypeptide, a cationic polypeptide composed of a single amino acid residue, or a cationic polypeptide composed of two or more amino acid residues can be used.

The cationic polypeptide of the present invention is a molecule having a weight average molecular weight of 100 to 100,000, and may be, for example, selected from the group consisting of cationic polypeptides, cationic polysaccharides, cationic lipids, cationic surfactants, and synthetic polymers.

The cationic polypeptide may be selected from the group consisting of protamine (PS), cell penetrating peptide (CPP), poly-L-lysine (PLL: poly-L-lysine) and polyamidoamine (PAMAM: polyamidoamine), and , preferably PS.

A candidate for the cationic polypeptide is protamine, a polycationic peptide with various applications. In particular, it is used for drug delivery and has been reported to have the effect of delaying the absorption of an injected insulin suspension. Protamine has a molecular weight of 5.8 kDa and is rich in basic amino acids, especially arginine (70-80%), so it has 1.26 ± 10 ²⁵ cations per mole. Thus, upon forming a complex with DNA, protamine neutralizes the negative charge of the DNA phosphate group. These substances, commonly called Poly-Electrolyte Complexes (PECs), are composed of polymers of biological macromolecules containing groups that can dissociate in water and interact to form charged components. PS is abundantly present in the sperm nucleus of fish, especially salmon, among testis of animals. Like histones, PS is known as a protein involved in the expression of genetic information through association or dissociation with DNA. Usually, the molecular weight of PS extracted from the sperm nucleus of fish is about 4,000 to 10,000, and 70% or more of the constituent amino acids are arginine, but the present invention is not limited thereto.

According to the present invention, the PS of the present invention may be one or two or more proteins selected from the group consisting of Protamine P1, Protamine 1, and Protamine 2).

The PS of the present invention includes both PS itself and a pharmaceutically acceptable salt thereof. Specifically, the salt of PS of the present invention encompasses a salt form with an acidic substance including hydrochloride or sulfate. PS is soluble in water without salt formation, and since the salt form is also soluble in water, it can be used in any form.

In order to obtain a denser 'particle' structure, it is also possible to use a form modified by treating PS with acrylamide (expressed as mPS) and a form modified by treatment with an acrylate group on HA (denoted as mHA), in which case the final gel is It can also be constructed by photocrosslinking through UV irradiation.

Poly-L-lysine (PLL: poly-L-lysine) is linked by a biodegradable peptide bond, so it can form a complex and an ionic complex and can be delivered into the cell. Because there is a limit, it can be used by attaching an endosome-destroying material or an endosome fusion peptide, including CPP and pH-dependent CPP.

Currently, the most commonly used stabilizing material for RNA expression cassettes is polyethylenimine (PEI), which has a positive charge, but has a limitation in its use due to toxicity in the body. In contrast, PS has no cytotoxicity and promotes penetration into negatively charged cell membranes (membrane of endosomes) and endosomal escape of RNA expression cassettes, so that RNA expression cassettes can be safely released into the cytoplasm. There is this.

Cell penetrating peptides (CPPs) are widely used to deliver RNA expression cassettes into cells. CPP increases the stability and bioavailability of RNA expression cassettes in cells. Thus, the use of CPPs to deliver RNA expression cassettes can have significant therapeutic advantages. A common method for binding negatively charged RNA expression cassettes to positively charged peptides is non-covalent electrostatic interaction.

In the cell-penetrating peptide (Cell Penetrating Peptides, CPP), the peptide that activates cell permeability by pH (a) has almost no membrane permeability at a high peptide concentration above the effective concentration (EC) and a physiological pH range (pH 7.0), (b) In the acidic pH range (pH5.5) of endosomes and low peptide concentrations below the effective concentration (EC), macromolecular-sized defects can be efficiently formed in the membrane, and a domain that increases cell membrane permeability and a pH-dependent membrane It is a peptide composed of an active domain.

Cell Penetrating Peptides (CPP) that activates cell permeability by pH type amino acid sequence note Melittin GIGAVLKVLTGLPALISWIKRKRQQ (SEQ ID NO: 1) Membrane activation over a wide pH range LP GWWLALALALALALALALASWIKRKRQQ (SEQ ID NO: 2) Hylin a1 IFGAILPLALGALKNLIK (SEQ ID NO: 3) LPE3-1 GWWLALAEAEAEALALASWIKRKRQQ (SEQ ID NO: 4) at acidic pH
membrane active pHD24 GIGAVLKVLATGLPALISWIKAAQQL (SEQ ID NO: 5)

According to the present invention, Unmodified mRNA; Modified mRNA; self-amplifying mRNA; and Trans-amplifying mRNA; provides an RNA expression cassette/PS basic multiparticulate (10) containing.

According to the present invention, the RNA expression cassette/PS basic composite particle 10 is prepared by mixing the RNA expression cassette and PS in a mass ratio in the range of 1:0.4 to 1:5, has a size of 200 nm or less, and has a positive charge characteristic. There are visible features.

The weight ratio of the RNA expression cassette and PS may be 1:0.2-5, preferably 1:0.4-3.0. In an environment with a w/w ratio of 1:2.5 or less, it is further condensed, and when PS is added at a ratio of 1:5 or more, the particles may have a size of micro or larger.

Although the particles of the present invention remain stable in high-purity water due to their physicochemical stability, the particles can rapidly aggregate when salt is added to the colloidal system. Another disadvantage of these first generation RNA expression cassettes/PS particles, so-called 'particles', may be the low release of the RNA expression cassette from the particle.

To overcome the two problems, biodegradable, non-toxic macromolecules, negatively charged nanoparticles or salts were investigated to prevent aggregation of basic composite particles. In addition, intracellular dissociation between the RNA expression cassette and PS can be enhanced with amphiphilic biopolymers, negatively charged nanoparticles and salts.

The negatively charged nanoparticles are silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), titanium (Ti) ), tantalum (Ta), niobium (Nb), zirconium (Zr), a conductive metal element such as aluminum (Al), or a metal oxide thereof is a nanoparticle selected from the group synthesized by one or more combinations. have.

Human serum albumin (HSA) may be a promising candidate to solve these problems. HSA, with a molecular weight of about 65,000 Da and a slightly negative surface charge under physiological conditions, is a non-toxic macromolecule widely used in particulate formulations and other pharmaceuticals. HSA is a protective colloid. These colloids are trapped only in small amounts in the particle matrix. Most of the macromolecules are left in the surrounding medium to inhibit secondary aggregation of the particles. At a high HSA concentration (1000 μg/ml), the particles can be completely coated with HSA so that the stability of the basic multiparticulate can be extended by this protective colloid.

Examples of cationic polysaccharides include chitosan, glycochitosan, cationic cellulose, cationized guar gum and cationized starch.

Among all polysaccharides used for RNA expression cassette-based therapy with cationic polymers, the present invention specifically selected chitosan to highlight applicable properties and characteristic features that are well suited for RNA expression cassette delivery. It provides a positively charged amino group in the glucosamine monomer of chitosan or a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid.

Cationic polysaccharides such as chitosan can electrostatically interact with RNA expression cassettes to form stably positively charged polyplexes, and the most efficient intracellular delivery of RNA expression cassettes is evident by positively charged nanocarriers. is achieved This can be explained by two factors. First, efficient physical interaction with negatively charged nucleic acids protects them from nuclease degradation. Second, they provide a net positive charge, enhancing the interaction of the complex with the anionic surface of the cell.

Cationic polymer used in the present invention, based on the weight of the total composition, may be included in 1 to 50% by weight, more specifically 2 to 40% by weight, even more specifically 3 to 30% by weight may be included. have. If the content of the cationic lipid is less than 1% by weight based on the weight of the total composition, it is not a sufficient amount to form a complex with the RNA expression cassette, and if it exceeds 50% by weight, the size of the nanoparticles is too large There is a risk that the stability of the particles may be reduced and the loss rate may increase during filter sterilization.

The cationic polymer and the RNA expression cassette bind to each other through electrostatic interaction to form a complex. As a specific example, the ratio of the charge amount of the RNA expression cassette (P) and the cationic polymer (N) (N/P; ratio of the cationic charge of the cationic compound to the anionic charge of the RNA expression cassette) is 0.5 to 100 days may be, more specifically 1 to 50, even more specifically 2 to 20. When the ratio (N/P) is less than 0.5, it is difficult to form a complex including a sufficient amount of the RNA expression cassette, so it is advantageous to be able to form a complex including a sufficient amount of an anionic drug when it is 0.5 or more. On the other hand, when the ratio (N/P) exceeds 100, there is a risk of causing toxicity, so it is preferable to set it to 100 or less.

anionic polymer

The composite particle 20 of the present invention includes an anionic polymer layer 22 coated on the outer surface of the cationic polymer layer 21 .

The cationic polymer layer 21 coating the outer surface of the basic composite particle 10 made of the RNA expression cassette has a strong positive charge, so there is a problem in that it is difficult to pass through the tissue barrier. In the present invention, in order to solve this problem, an anionic polymer having good biocompatibility and a negative charge was used and coated on the outer surface of the cationic polymer layer.

In the present invention, particles may be prepared by mixing an RNA expression cassette, a cationic polymer and an anionic polymer.

In the present invention, a cationic polymer is necessarily further included in order to form an ionic complex with the RNA expression cassette. That is, the negatively charged RNA expression cassette forms an ionic complex enclosed in the cationic polymer due to electrostatic interaction with the cationic polymer, and the formed ionic complex is coated with an anionic polymer to form the anionic polymer layer 22 It is located in the core part of the particle.

The coating step of the anionic polymer layer 22 is a step of adding an anionic polymer and coating the anionic polymer layer on the outer surface of the particle by electrostatic attraction.

The multiparticulate according to the present invention contains an anionic polymer as an essential element. The anionic polymer is not limited as long as there is no harm to the human body, and examples of the anionic polymer are suitably silk protein fibroin, anionic protein including serum albumin, polyglutamic acid and polyaspartic acid anionic poly Amino acids, agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin and carboxymethyl cellulose and anionic polysaccharides such as polyacrylic acid and salts thereof. In particular, hyaluronan, polyglutamic acid and salts thereof may be mentioned suitably.

As the anionic polymer, a commercially available product can be used.

The molecular weight of the anionic polymer used in the present invention is not limited, preferably 100 to 100000 kDa, more preferably 200 to 50000 kDa.

Specifically, as anionic polypeptides, proteins including silk protein fibroin, gliadin, and albumin (when ionized in water of pH 7.4 found in the body) are negatively charged.

Albumin offers several advantages that ensure success in future developments of Abraxane and other types of nanomedicine. 1) Albumin is a component of human blood. It is an endogenous protein (35-50 g/L of human serum), which is not immunogenic and ensures biocompatibility for albumin-based nanoparticles. 2) The half-life of albumin in human blood averages about 19 days. Unlike free drugs, albumin provides a long half-life that helps to improve plasma area under the curve (AUC) values, thus maintaining drug concentration over a relatively long time. 3) Albumin is an excellent cryoprotectant for nanopharmaceuticals in solid form, and the dried nanoparticles can be immediately redispersed in the injection solution. These advantages are advantageous for the development of room-temperature-stable nano-medicines by avoiding drug release and deposit formation, which are common problems in the development of clinically translatable nano-medicines.

There are two types of albumin commonly used to construct nanoparticles: human serum albumin (HSA) and bovine serum albumin (BSA). Both types of albumin are serum albumin proteins (human serum and bovine serum, respectively) and share many properties, including high solubility in water, long half-life in blood, similar molecular weight (65-70 kDa), and similar number. Number of amino acid residues (585 amino acids for HSA, 583 amino acids for BSA). There is no noticeable difference in properties in what constitutes a nanomedicine. Therefore, both types of albumin have been widely used to design multifunctional nanoparticles. Technologies developed with alternative albumin can be easily replaced.

Serum albumin, one of the high molecular substances in the living body, has excellent biocompatibility and is a very stable protein with a half-life of 19 days in the blood. Therefore, serum albumin is chemically bound to various low molecular weight chemical drugs, proteins, antibodies, etc., and can be used to improve the in vivo stability of various drugs and effectively deliver the drugs to target cancer sites. For example, representative hydrophobic anticancer drugs such as doxorubicin (Drug Delivery 6 (1999) 1-7) or methotrexate (MTX; Anticancer Drugs 8 (1997) 835-844) are chemically bound to albumin to target cancer Studies have been reported to improve the drug and improve the stability of the drug in blood. In addition, with respect to the use of albumin as a drug carrier, based on the high targetability of albumin to cancer tissues, fluorescent or radioactive isotope-labeled albumin was injected into small animals transplanted with cancer. It has been reported that positive albumin selectively accumulates in cancer tissues (Cancer Research 46 (1986) 6387-6392), which results in the enhanced permeability and retention (EPR) effect occurring in loose blood vessels around cancer tissues. It can be seen that this is indicated by As such, the superiority of albumin as an anticancer drug carrier has been known, but albumin itself lacks binding force to be used as an siRNA carrier having a strong negative charge. Therefore, while maintaining the unique properties of albumin, modifications are required to increase the binding force.

As salts forming hyaluronan salts and polyglutamate salts, sodium, potassium, calcium, ammonium, magnesium, basic amino acid salts and the like can be mentioned.

More specifically, when hyaluronan or a salt thereof is used as the anionic polysaccharide, hyaluronan or a salt thereof having a molecular weight of preferably 100 to 2000 kDa, more preferably 200 to 1500 kDa may be used.

In addition, when polyglutamic acid or a salt thereof is used as the anionic polymer, polyglutamic acid or a salt thereof having a molecular weight of preferably 500 to 30000 kDa, more preferably 1000 to 20000 kDa, still more preferably 1500 to 15000 kDa is used. can

The degree of anionization of the anionic polymer is not particularly limited, and the lower limit is preferably 0.1 or more, more preferably 0.2 or more, from the viewpoint of promoting the formation of multiparticulates with the cationic polypeptide.

On the other hand, from the viewpoint of forming multiparticulates with the cationic polypeptide, there is no particular upper limit on the degree of anionization of the anionic polysaccharide. As a standard, anionic polysaccharides having an anionization degree of 2 or less can be used.

In this regard, the expression "anionization degree" denotes a value obtainable by calculation according to the following formula.

{(Number of anionic functional groups contained in polymer) - (Number of cationic functional groups contained in polymer) ｝/ Total amount of monomers constituting the polymer

The anionic polysaccharide of the present invention is heparin, hyaluronic acid, chondroitin sulfate, carboxy methyl cellulose, pectin, carbopols, polyacrylates (Polyacrylates) and anionic lipids.

The anionic lipid may be selected from the group consisting of phosphatidylserine, phosphatidic acid, phosphatidylcholine and phosphatidylglycerol.

The anionic polysaccharide has a negative charge and a molecule having biosynthesis can be used without limitation.

Heparin has a structure including a hydroxyl group, a carboxyl group and an amino group. Unfractionated heparin, molecular weight heparin, low molecular weight heparin, heparin fragment, recombinant heparin, heparin analog, heparan sulfate, sulfonated polysaccharide having heparin activity, etc. can be used. have.

The hyaluronic acid (HA) is a large complex oligosaccharide composed of up to 50,000 pairs of disaccharide glucuronic acid-β(1-3) N-acetylglucose-amine β(1-4) as basic units. It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is in the form of a random coil with a diameter of about 50 nm.

Hyaluronic acid (HA) has been used to treat diseases and disorders in the human body, either systemically or locally, because of its ability to target active substances to diseased or diseased sites. It is known that HA can be seen in the damaged carotid artery (for the intact contralateral artery) and colorectal cancer in experimental animals and is maintained in the skin of these animals. The weight average molecular weight of the anionic polysaccharide usable in the present invention may be in the range of 5 to 90 K.

The anionic polysaccharide layer such as hyaluronic acid is coated on the outer surface of the cationic polypeptide layer by electrostatic attraction. The weight ratio of the basic composite particles to the hyaluronic acid may be in the range of 1:0.5 to 2 (basic composite particles: HA).

Hyaluronic acid of the present invention is a complex polysaccharide composed of amino acids and uronic acid, a natural molecular substance without cytotoxicity, has excellent stability in the body, and has various receptors capable of absorbing the hyaluronic acid. Among the hyaluronic acid receptors present in the body, the CD44 receptor is overexpressed in most cancer cells and can be used as a target for therapeutic agents that selectively target cancer.

The anionic polymer layer forms the outermost layer, and thus, the composite particle also exhibits a negative charge on the outside thereof. In the case of normal skin, it is known that the electrostatic property of the particle surface slightly affects the skin permeation, but the cationic property enhances this. However, in the case of skin, the structure of fatty acids in sebaceous lipids and epidermal phospholipids is damaged, and as a result of this, the decrease in acidity of the skin is (-) property as in the present invention. It facilitates the penetration of strong anionic composite particles.

The drug composition of the present invention is composed of an RNA expression cassette (core), a cationic polymer layer (middle layer) and an anionic polymer layer (outermost layer), which are pharmaceutically active ingredients, and the particle size is 50 to 500 nm, preferably 100 to 400 nm, more preferably 150 to 300 nm. The pharmaceutical composition of the present invention has a reduced particle size than the composite hydrogel as a pharmaceutically active ingredient provided as a core.

The present invention can protect the RNA expression cassette from enzymes that degrade the RNA expression cassette in vivo because it further includes an outer layer and a hyaluronic acid layer of the basic composite particle composed of the RNA expression cassette and the cationic polypeptide.

In addition, the present invention reduces the particle size to a nano size by condensing an RNA expression cassette containing a pharmaceutical active ingredient having a micro size or a nano size equivalent thereto, and also hyaluronic acid having a negative charge in the outermost layer It is possible to provide particles that can easily pass through the skin barrier by coating with .

natural polysaccharides

Natural polysaccharides can be obtained from animals (eg chitosan, hyaluronic acid), plants (eg starch, cellulose, cyclodextrin, pectin), algae (eg alginates) or bacteria (eg dextran). It has a wide range of applications, from the agricultural and food industries to the cosmetic, pharmaceutical and biomedical sectors. Polysaccharides are formed by monosaccharides forming neutral or charged, linear or branched and carbohydrate structures with varying degrees of hydrophilicity.

The widespread use of natural polysaccharides in pharmaceuticals for drug delivery is their biocompatibility, biodegradability and low toxicity. It has also been used in combination with synthetic materials as a strategy to mitigate the latter toxicity. Their structural diversity makes it possible to select the most suitable polysaccharides in each case according to the needs of the final application, and more importantly, their functional properties can be tuned and optimized by chemical modifications to their functional groups.

Many natural polysaccharides have biological adhesion properties due to the presence of specific functional groups, making them useful starting materials for nanocarrier designs with improved retention times, especially for intestinal and lung applications. The presence of such groups also allows for certain modifications of the polymer structure to enhance its delivery or targeting ability. In addition, natural polysaccharides are neutral (eg, cellulose, pullulan, starch, dextran), negatively (eg, hyaluronic acid, alginate, chondroitin sulfate), or positively charged (eg, chitosan). ) can be

Types of polysaccharides Animal Polysaccharides Chitin
Chitosan
Glycosamminoglycans Vegetal Polysaccharides Land Vegetal Marine Vegetal Cellulose
Pectins
Galactomannans
Acacia gum
Starch Alginate (Phaeophyceae brown seaweed)
Agar (Rhodophyceae red seaweed)
Carragenans (Rhodophyceae red seaweed)
-
- Microorganis/Fungi Alginate (Pseudomonas aeruginosa and Azotobacter vinelandii)
Dextran (Leuconostoc spp. and Lactobacillus spp.)
Gellan (Pseudomonas elodea)
Pullulan (Aureobasidium pullulans)
Scleroglucan (Sclerotium glucanicum) Xanthomonas campestris
Xanthan (Xanthomonas campestris)

Among all polysaccharides used in RNA expression cassette-based therapy, we specifically selected hyaluronic acid to highlight applicable properties and characteristic features that are well suited for RNA expression cassette delivery. Although hyaluronic acid is a mucoadhesive polymer, it has an opposite charge, greatly affecting its use in nucleic acid delivery. This important difference stems from the chemical structure, which provides a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid.

On the other hand, association of the RNA expression cassette expression cassette with anionic polysaccharides such as hyaluronic acid can improve bioavailability by avoiding RNA expression cassette release through the glomerular capillary wall. Indeed, negatively charged species are "unfilterable" due to the repulsive forces established by the negative charge of the glomerular membrane.

Modified Ionic Molecules

<Modified cation molecule>

According to the present invention, the RNA expression cassette and the cationic polypeptide are coupled by electrostatic interaction, and the bile acid or a derivative thereof covalently bound to the cationic polypeptide has a hydrophilic ionic group on the surface of the particle. can also be positioned.

A bile acid or bile acid derivative comprises one or more cationic polypeptides and complexes covalently bound, wherein the covalently bound bile acid or bile acid derivative has an ionic group.

The particles of the present invention are formed by an electrostatic interaction between a cationic polypeptide and a complex to which a bile acid or a derivative thereof is covalently bound, or an anionic polysaccharide to which a bile acid or a derivative thereof is covalently bound, an electrostatic charge between a cationic polypeptide and an RNA expression cassette Formed by miraculous interactions, it can induce cellular internalization of RNA expression cassettes more safely.

That is, in the basic multiparticulate of the present invention, a cationic polypeptide and an RNA expression cassette are present in the core of the particle, and a bile acid having a hydrophilic ionic group is located on the surface of the particle, and the bile acid is a surface-modified particle.

In order to deliver the RNA expression cassette to a target cell, when the particles on the surface of the bile acid of the present invention are used, the complex is well delivered through the intestinal mucosa, and at the same time, it is very safe in the gastrointestinal tract due to the ionic complex present in the core of the particle. . From this, it can be seen that the particles for delivery of the complex according to the present invention are very suitable for oral administration.

In the present invention, the bile acid or its derivative has a hydrophilic ionic group in addition to a functional group capable of covalently bonding with an ionic molecule. The ionic molecule is deoxycholic acid (DCA), urodeoxycholic acid (taurodeoxycholic acid), taurocholic acid (TCA: taurocholic acid), glycocholic acid (glycocholic acid) and glycochenodeoxycholine acid (glycochenodeoxycholic acid).

The bile acid is absorbed into the liver with a high efficiency of more than 95% through the ileum of the small intestine, which is called 'enterohepatic circulation'. Since it is specifically recognized by ASBT (apical sodium-dependent bile acid transporter) present in the distal intestinal region, it can act as a targeting ligand in oral delivery.

That is, the bile acid, which is absorbed through the ileum, is located on the surface of the particle for delivery of the RNA expression cassette through ASBT, so that the target cell of the particle for delivery of the RNA expression cassette through the intestinal lining, which is the biggest problem of the existing oral administration method through ASBT It acts to increase my absorption. Due to this, the particles for delivery of the RNA expression cassette of the present invention are easily absorbed through the ileum for absorbing bile acid, so they can be used orally, thereby increasing the bioavailability of the drug.

In the present invention, the degree of covalent bonding of the bile acid or its derivative may vary depending on the number of functional groups of the ionic molecule. Preferably, the bile acid or its derivative and the ionic molecule may be covalently bonded in a molar ratio of 1:1 to 1:300.

Preferably, the bile acid or its derivative and chitosan may be covalently bonded in a molar ratio of 1:1 to 1:100.

Preferably, the bile acid or its derivative and heparin may be covalently bonded in a molar ratio of 1:1 to 1:30.

Preferably, the bile acid or its derivative and hyaluronic acid may be covalently bonded in a molar ratio of 1:1 to 1:200.

In the present invention, the RNA expression cassette particles can be absorbed regardless of the particle size, preferably 500 nm or less, more preferably 100 to 300 nm.

In the present invention, the RNA expression cassette and the ionic molecule to which the bile acid or the bile acid derivative is ionically bound may be bound in a mass ratio of 1:0.5 to 1:70, preferably 1:0.5 to 1:5.

In the present invention, the bile acid or the bile acid derivative ion-coupled complex and the cationic polypeptide may be mixed in a mass ratio of 1:0.5 to 1:10, preferably 1:0.5 to 1:5. Because there is a limit to the binding of the cationic polypeptide and delivery is possible only in a range that can have an optimal strong binding ability, the ionic complex according to the electrostatic interaction of the cationic polypeptide and the RNA expression cassette within the above range Formation is well done.

<Modified Anion Molecules>

In addition, the present invention includes an RNA expression cassette/cationic polypeptide particle comprising one or more anionic polysaccharides and an RNA expression cassette covalently bound to a bile acid or bile acid derivative, wherein the covalently bound bile acid or bile acid derivative is ionic It provides a pharmaceutical composition comprising an RNA expression cassette particle having a group as an active ingredient.

Taurocholic acid of the present invention is a kind of bile acid, secreted in the body and known as a substance that helps the absorption of lipids or vitamins during the digestive process, and is characterized by being reabsorbed by more than 90% through the intestinal circulation. Most of the taurocholic acid is absorbed by ASBT (Apical Sodium Dependent Bile Acid Transporter) at the end of the small intestine during digestion. Therefore, if taurocholic acid is combined with hyaluronic acid to prepare particles, the oral absorption rate in the small intestine can be improved, and delivery through enteric circulation is improved.

According to one embodiment of the present invention, the present invention provides a hyaluronic acid-taurocholic acid conjugate.

According to another embodiment of the present invention, in the hyaluronic acid-taurocholic acid conjugate of the present invention, hyaluronic acid and taurocholic acid are added in a molar ratio of 1:1 to 1:100 (feed mole ratio, hyaluronic acid: taurocholine) acid), and has the characteristic of having a negative charge.

If the hyaluronic acid-taurocholic acid conjugate has a positive charge, there is a disadvantage in that it cannot bind to an RNA expression cassette/PS conjugate having a positive charge.

According to the present invention, the hyaluronic acid-taurocholic acid conjugate is prepared by mixing hyaluronic acid and taurocholic acid in a feed mole ratio in the range of 1:1 to 1:100 (feed mole ratio, hyaluronic acid: taurocholic acid). can do. Preferably, the hyaluronic acid-taurocholic acid conjugate can be prepared by mixing hyaluronic acid and taurocholic acid in an addition molar ratio of 1:1∽1:50, and more preferably in an addition molar ratio of 1:10. It can be prepared by mixing.

If it is prepared by mixing the hyaluronic acid and taurocholic acid in an addition molar ratio of less than 1:1, it is not efficient to coat the complex/PS, and the hyaluronic acid and taurocholic acid are added in a molar ratio exceeding 1:100. When prepared by mixing, it is not economically efficient because only a hyaluronic acid-taurocholic acid conjugate having a similar binding ratio is made.

The complex particle for oral administration of the present invention has a form in which the RNA expression cassette/PS is coated with a hyaluronic acid-taurocholic acid conjugate.

In detail, the complex particle for oral administration of the present invention has a form in which the RNA expression cassette / PS is located and the hyaluronic acid-taurocholic acid conjugate is coated on the surface of the RNA expression cassette / PS, and the characteristic of negative charge It has a particle diameter of 250 nm or less, and the RNA expression cassette/PS protein encapsulation rate is 90% or more.

When the diameter of the RNA expression cassette / PS particle exceeds 100 nm, the size of the RNA expression cassette / PS / HA-TCA composite particle prepared by the coating of the following HA-TCA conjugate exceeds 250 nm. In consideration of the above, it is preferable to prepare the RNA expression cassette/PS basic composite particle to have a diameter of 100 nm or less. In addition, when the charge characteristic of the RNA expression cassette PS basic composite particle is negatively charged, binding (coating) with the negatively charged HA-TCA conjugate is difficult, so the RNA expression cassette PS basic composite particle has a positive charge. It is preferable to do

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of the RNA expression cassette PS / HA-TCA multiparticulate.

In addition, the targeting molecule may be covalently linked to the anionic molecule. The composite particles can be used as an active carrier. The targeting molecule may be a cell surface-binding compound, antibody or peptide, aptamer, or a combination thereof.

2. Preparation of multiparticulates containing RNA expression cassettes

In the present invention, the aqueous solution and the anionic polymer of the basic composite particle including the RNA expression cassette and the cationic polymer are separately prepared, and the aqueous solution is mixed with each other to prepare the composite particle for RNA expression cassette delivery, and the first called the method.

In the present invention, first, nuclear and basic composite particles were prepared using an RNA expression cassette as an active ingredient and a cationic polymer.

In the present invention, the cationic polymer is a polypeptide, a peptide. allyl-based amine polymers and/or cationic polysaccharides. By using these cationic polypeptides, the stability of the multiparticulate can be improved.

In the present invention, the cationic polymer forms a basic multiparticulate with the RNA expression cassette. The present invention also relates to a method for producing the above-mentioned composite particles. That is, the production method of the present invention is characterized in that the RNA expression cassette and the cationic polymer having a cationization degree of 0.2 or more are mixed in an aqueous solvent. According to the present invention, the basic composite particles described above can be easily prepared.

The present invention is characterized in that an aqueous RNA expression cassette (A) and an aqueous solution of a cationic polymer (B) are separately prepared, and these aqueous solutions are mixed with each other. Accordingly, it is possible to prevent aggregation of the basic composite particles by separately preparing aqueous solutions of (A) and (B).

In addition, the concentration of the RNA expression cassette in the aqueous solution (A) described above is preferably 1.7 mM or less. In addition, the concentration of the total cationic polymer in the aqueous solution (B) described above is preferably 1.7 mM or less. By setting the concentration of the polymer in the aqueous solutions (A) and (B) in the ranges described above, basic composite particles that are difficult to aggregate can be produced.

The present invention relates to a basic composite particle prepared by the above-described manufacturing method. The basic composite particles of the present invention have excellent stability.

Cationic polypeptides such as protamine, cell-penetrating peptides, and chitosan can electrostatically interact with RNA expression cassettes to form stably positively charged polyplexes, basic complex particles.

Chitin is the second most abundant natural polysaccharide after cellulose and consists of N-acetyl glucosamine. Chitin can be prepared from the crustacean exoskeleton by dissolution of calcium carbonate and protein removal. Chitosan is prepared by full or partial deacetylation of N-acetyl glucosamine residues in chitin. Chitosan consists of a copolymer of glucosamine and N-acetyl glucosamine linked by β (1 → 4) glycosidic bonds and is prepared by partial alkaline deacetylation of chitin. Chitin and chitosan are biocompatible and non-toxic. By “chitosan” is meant a natural cationic biopolymer that generally contains less than 50% N-acetyl glucosamine residues. The solubility, hydrophobicity and electrostatic properties of chitosan depend on the degree of deacetylation and molecular weight. Chitosan is a biodegradable polymer because it is hydrolyzed by lysozyme. The degradation products of chitosan are biocompatible, non-carcinogenic, non-immunogenic, and have biological activities such as antibacterial activity, hypocholesterolemic function, antitumor activity, function of wound healing and stimulation.

According to the present invention, cationic chitosan and anionic RNA expression cassette can produce PEC (basic multiparticulate) by electrostatic interaction, so chitosan can be used as a carrier of the anionic RNA expression cassette of the present invention.

Since chitosan is only soluble in acidic solvents such as diluted hydrochloric acid, acetic acid, propionic acid and ascorbic acid, this reagent is difficult to handle as a biomaterial in wound dressings and bioadhesion therapy. The water solubility of chitosan can be improved through modification over a wide pH range to provide advanced functionality to the polymer. These modifications included modifications to carbohydrate branching and derivatization using various types of disaccharides including lactose, maltose and cellobiose. According to the definition of PEC as a versatile formulation formed by electrostatic interactions between oppositely charged biopolymers, chitosan (and derivatives)-based hydrogels can form PEC. A chitosan-based PEC hydrogel is defined as a network of crosslinked and hydrated chitosan. Chitosan hydrogels are classified into two groups according to this definition: chemical chitosan hydrogels formed by irreversible covalent bonds and physical chitosan hydrogels formed by various reversible bonds. Physical chitosan hydrogels include ionic cross-linked colloids, PEC and ACF-HS with alginate, chitosan and fucoidan. In contrast, in chemical chitosan hydrogels, chitosan and its derivatives are covalently interconnected by low molecular weight crosslinkers to form a three-dimensional (3D) hydration network. The crosslinking density determined by the molar ratio of the crosslinking agent to the repeating units of chitosan and its derivatives affects the properties of the chemically crosslinked chitosan hydrogel. According to the present invention, such a chitosan-based PEG hydrogel can be fully utilized for the RNA expression cassette of the present invention.

The preparation of covalently crosslinked chitosan hydrogels requires the use of a crosslinking agent having two or more reactive groups to crosslink the chitosan chains, such as glutaraldehyde as a dialdehyde. However, it is difficult to completely remove free unreacted dialdehydes from hydrogels, which can lead to toxic effects. Figure 1B shows the preparation of photocrosslinked chitosan hydrogels that form when exposed to visible or ultraviolet light in the presence of a photocrosslinker.

A temperature-sensitive chitosan hydrogel that exhibits a sol-gel transition due to a morphological change at 37 °C can be prepared. Since chitosan does not have inherent heat-sensitive properties, it is necessary to introduce a temperature-sensitive material to make a temperature-sensitive chitosan hydrogel. For example, a temperature-sensitive hydrogel composed of chitosan and β-glycerophosphate or polyethylene glycol (PEG) can produce a sol-gel transition to heat and pH changes. This hydrogel was evaluated as a carrier for cells and functional proteins. According to the present invention, such a chitosan-based PEG hydrogel can be fully utilized for the RNA expression cassette of the present invention.

A photocrosslinkable chitosan derivative (Az-CH-LA) containing both lactobionic acid and photoreactive p-azidobenzoic acid is used as a photocrosslinking agent. The molecular weight of chitosan used is 300 ~ 500 kDa, and the degree of deacetylation is 80%. The lactose moiety was introduced via a condensation reaction with the amino group of chitosan. Chitosan (CH-LA) introduced with 2% lactobionic acid showed excellent water solubility up to 3w% even at neutral pH. In addition, a lamp distance of 2 cm (Spot Cure ML-251C/A with guide fiber unit (SF-101BQ)) and a 250 W lamp (240-380 nm, with a main peak of 340 nm, Usio Electrics Co., Ltd., Tokyo, Japan) ), Az-CH-LA introduced with 2.5% p-azidebenzoic acid by applying ultraviolet (UV) irradiation can prepare an insoluble hydrogel within 30 seconds. 3% or less of Az-CH-LA solution can be injected into the tissue, and an insoluble hydrogel is formed after external irradiation with UV light. In addition, other types of chitosan hydrogels including cross-linked polydopamine/nanocellulose hydrogels, temperature-sensitive chitosan-based injectable hydrogels, PEG poly(L-alanine) thermogels, chitosan-alginate hybrid hydrogels, and hyaluronate/alginate have been developed. It can be made from biomaterials. All these hydrogels, including hydrogels, pH-responsive tannic acid-carboxylated agarose complex hydrogels, catechol functionalized chitosan/pluronic hydrogels, and UV cross-linked biodegradable gelatin, are used in tissue adhesives and wound healing in tissue engineering. It has potential applications for

The chitosan-based PEG hydrogel can be prepared together with the RNA expression cassette of the present invention and self-assembled by electrostatic interaction, so it can be fully utilized as a carrier for the RNA expression cassette of the present invention.

Chitosan accelerates wound healing by promoting rapid dermal regeneration, stimulating macrophages and acting as a chemoattractant for neutrophils, an early event essential for wound healing. Neutrophils improve overall healing by killing microbes, removing dead cells, and stimulating other immune system cells, reducing the chance of infection. Chitosan and its derivatives have been applied as dressings for wound healing. Aqueous chitin/chitosan (WSC) solutions exhibit higher tensile strength and promote faster healing than insoluble chitin and chitosan powders. The high biodegradability and hydrophilicity of the WSC solution compared to the powder can increase the biocompatibility and wound healing stimulating activity. Chitosan also improves the healing process by binding with several functional molecules such as cytokines, GFs and extracellular matrix components. Chitosan improves the healing of pressure ulcer ulcers and wounded meniscus tissue and inhibits scar formation and shrinkage during healing.

According to the present invention, a chitosan-based PEC hydrogel (PCH) containing a GP RNA expression cassette for wound healing can be prepared by self-assembly by regular interaction between components, so PCH is a carrier of the RNA expression cassette of the present invention can be used sufficiently.

Application of photo-crosslinked chitosan hydrogel (PCH) for wound healing in normal mice and rats induces marked wound contraction, accelerating the wound closure and healing process. In addition, PCH exhibits sustained release of angiogenic GFs, acting as a carrier in vivo and promoting angiogenesis. For example, fibroblast growth factor 2 (FGF-2) interacts with chitosan molecules in PCH and is gradually released from PCH during in vivo biodegradation.

FGF-2-incorporated PCH (FGF-2 & PCH) induces significant vascularization and granulation tissue formation and improves wound healing in diabetic db/db mice with impaired healing. Interestingly, FGF-2 & PCH had some effect on the healing of normal db/+ littermates. The cause of this minor effect is not fully understood, but it is likely that the presence of sufficient macrophages has a significant effect on wound granulation tissue formation in db/+ mice. Also, poor wound healing in db/db mice can be explained by a defect in VEGF expression. That is, db/+ mice may have sufficient VEGF for angiogenesis and wound repair.

On the other hand, the most efficient intracellular delivery of RNA expression cassettes can be identified by two factors by positively charged basic complex particles. First, efficient physical interaction with negatively charged RNA expression cassettes protects them from nuclease degradation. Second, they provide a net positive charge, enhancing the interaction of the complex with the anionic surface of the cell.

By mixing the basic composite particles and the anionic polysaccharide in an aqueous solvent of a neutral region at a pH of about 6.5, composite particles can be prepared.

In the present invention, it maintains the characteristics of the basic complex particles composed of cationic polymers such as protamine, cell-permeable peptide, chitosan, and derivatives thereof, and a cationic polymer such as RNA expression cassette, protamine, cell-permeable peptide or chitosan and hyaluronic acid or An RNA expression cassette carrier and multiparticulate that depend on the interaction of anionic polymers such as derivatives of these polysaccharides have been developed.

The multiparticulates of the present invention can be prepared by mixing the basic multiparticulates and the anionic polymer in an aqueous solvent having a pH of around 6.5, comprising an RNA expression cassette, a cationic polymer and an anionic polymer have. Neutral and anionic polymers often require the presence of other components to interact more efficiently with the RNA expression cassette to produce multiparticulates.

The association of the RNA expression cassette with an anionic polymer such as hyaluronic acid in the multiparticulates developed in the present invention can improve bioavailability by avoiding the RNA expression cassette excretion through the glomerular capillary wall. In fact, negatively charged material is "unfilterable" due to the repulsive force generated by the negative charge of the glomerular membrane.

According to the production method of the present invention, it is possible to prepare multiparticulates having a relatively small particle diameter and containing an anionic polymer and an RNA expression cassette.

Preferably, the anionic polymer of the present invention is hyaluronan.

The multiparticulate particles of the present invention can be used for the purpose of penetrating the anionic polymer and the RNA expression cassette into the skin.

The present invention is preferably applied to the production of multiparticulates containing an RNA expression cassette having a useful action on the human body.

In a preferred embodiment of the present invention, an aqueous solution of the RNA expression cassette, an aqueous solution of a cationic polymer, and an aqueous solution of an anionic polymer are separately prepared, and these aqueous solutions may be mixed with each other to prepare a multiparticulate for RNA expression cassette delivery, It was referred to as method 2.

Accordingly, by taking the above embodiment in which the aqueous solution of the RNA expression cassette, the aqueous solution of the cationic polymer, and the aqueous solution of the anionic polymer are separately prepared, aggregation of the multiparticulates can be prevented.

The composite particle preparation is characterized in that an aqueous solution containing an RNA expression cassette, an aqueous solution containing a cationic polymer, and an aqueous solution containing an anionic polymer are separately prepared, and these aqueous solutions are mixed with each other, and the mixture is subjected to an ultrasonicator ) may include dispersing.

It is preferable to use a bath type ultrasonicator as the ultrasonic crusher. Bath type sonicators are known to provide mild sonicating of 20 to 40 Watts/Liter and are known to provide very non-uniform distributions, whereas probe type ultrasonicators provide approximately 20,000 W/L. can be added This means that the probe-type ultrasonic grinder is about 1,000 times superior to the bath-type ultrasonic grinder due to the concentrated and uniform ultrasonic intensity input.

In general, RNA expression cassettes have been known to be impossible or difficult to sonicate due to their instability, but as in the present invention, it is possible to use a sonicator only by encapsulating the RNA expression cassette in polymer nanoparticles as in the present invention. and efficient manufacturing.

In another embodiment, glycosaminoglycans (GAGs) include the heparinoids heparinoids (heparin/heparin sulfate (HS) and other heparin-like molecules), chondroitin Polysaccharide chains such as sulfate, dermatan sulfate, and keratan sulfate, all of which have different densities and different positions and negatively charged Heparinoids are heparin-binding growth factor (GF), cytokines, and cells Interact with a variety of functional proteins, including extracellular matrix components and adhesion molecules.Most biological functions of heparinoids depend on the binding of these functional proteins to polysaccharide chains mediated by specific domains, which bind to different saccharide sequences. For example, the interaction of heparinoids with fibroblast growth factor (FGF)-1 and FGF-2 requires different combinations of sulfate groups and therefore different saccharide sequences. Noid structure interacts with hepatocyte growth factor and vascular endothelial growth factor (VEGF) Electrostatic interactions between oppositely charged polyelectrolytes generate Polyelectrolyte Complexes (PECs).Both synthetic and native PEC proteins Electrostatic interaction of PECs with the RNA expression cassette of the present invention results in the formation of dispersion, soluble complexes, emulsification and/or amorphous precipitates.

According to the present invention, GAG-based PEC (composite particles) can be prepared by self-assembly by self-assembly by electrostatic interaction of anionic RNA expression cassette, cationic polymer, and anionic glycosaminoglycan (GAG). It can be used as a carrier for the RNA expression cassette of the present invention.

The focus is on PEC hydrogels formed by chemical interaction of chitosan and crosslinking agents, such as photo-crosslinked chitosan hydrogels (PCH) formed by the addition of photo-crosslinked chitosan hydrogels, photocrosslinkers. Temperature-sensitive chitosan hydrogel, hydrocolloid] is formed by direct interaction between polymer chains without the addition of cross-linking agents. In addition, the potential medical applications of chitosan-based biomaterials such as PCH and alginate/chitosan/fucoidan complexed hydrocolloid sheets (ACF-HSs) and PEC are well known.

According to the present invention, such ACF-HS and PEC can be prepared as multiparticulates by self-assembly by electrostatic interaction by adding the RNA expression cassette of the present invention, and thus can be fully utilized in the RNA expression cassette of the present invention.

Acidic polymers such as GAG complexed with basic polymers such as protamine and chitosan form PECs through electrostatic interactions. The acidic and basic polymers of PEC bind to various proteins such as cytokines and GFs above and below the protein's isoelectric point. These interactions produce various morphologies such as nanoparticles, colloidal particles, soluble complexes, and amorphous precipitates (Fig. 3). Electrostatic interactions between charged polymers are very interesting because they resemble biological interactions. For example, interactions between nucleic acids and proteins play an important role in transcriptional processes.

According to the present invention, by stabilizing, activating, and gradually releasing the RNA expression cassette from the Glycosaminoglycan (GAG)-based PEC containing the RNA expression cassette, it can be fully utilized for the RNA expression cassette of the present invention.

Chitosan PEC, chitosan/hyaluronate PEC, and chitosan/chondroitin sulfate PEC can be self-assembled through electrostatic interaction by adding an RNA expression cassette during the manufacturing process, so it functions as a carrier for the RNA expression cassette of the present invention do. In addition, insoluble PECs have potential applications such as hydrogels, sheets, microcapsules and scanfolds for tissue regeneration.

CH-LA, which is water-soluble at neutral pH, was prepared by introducing lactobionic acid into an aqueous solution of an acidic polymer. A 2 wt % aqueous solution of CH-LA is highly viscous and gels easily when mixed with acidic polymer solutions such as GAG, especially with heparin (heparin and heparin-like molecules). This product contains non-anticoagulant (NAC)-heparin, 6-O-desulfurized heparin, fucoidan. When NAC-heparin/CH-LA hydrogel containing FGF-2 was subcutaneously injected into the back of rats or mice, fibrous tissue formation and angiogenesis around the injection site were significantly improved. Moreover, the gradual release of FGF-2 from the hydrogel stimulates concomitant circulation and angiogenesis. Conversely, PCH containing paclitaxel, an angiogenesis inhibitor and anticancer drug, effectively inhibits angiogenesis and tumor growth in mice.

The biological properties and applications of GAG-based materials were investigated. GAGs containing heparinoids were deposited on the surface of micro/nanoparticles made of magnets, metals, synthetic polymers and natural biopolymers]. The combination of these micro/nanoparticles with functional molecules such as proteins (eg GF) and DNA could provide particles with potential applications in various biomedical fields. The micro/nano particles bind to GAG to reduce reactivity due to a change in surface properties, thereby improving biocompatibility. Polysaccharides found on the surface of all eukaryotic cells are another interesting class of molecules for foreign coatings.

According to the present invention, by stabilizing, activating, and gradually releasing the RNA expression cassette from the Glycosaminoglycan (GAG)-based PEC containing the RNA expression cassette, it can be fully utilized for the RNA expression cassette of the present invention.

Electrostatic interactions between oppositely charged polyelectrolytes produce polyelectrolyte complexes such as low molecular weight heparin (LH) (Fragmin) and protamine (P). Non-stoichiometric PEC Non-stoichiometric PEC transfer excess charge when these interactions occur unequal. These PECs can interact with and adsorb various functional proteins. Heparinoids such as LH (MW: about 5000 Da) specifically bind to various proteins such as cytokines, GFs, extracellular matrix, and adhesion molecules. In this way, heparinoids may be useful as pharmaceuticals to treat various pathological conditions. However, the use of high-dose heparin carries the risk of excessive bleeding. In contrast, LH offers practical and pharmacological advantages over heparin.

According to the present invention, by stabilizing, activating, and gradually releasing the RNA expression cassette from the Glycosaminoglycan (GAG)-based PEC containing the RNA expression cassette, it can be fully utilized for the RNA expression cassette of the present invention.

The low binding affinity of LH to heparin-binding coagulation factors results in a low and predictable anticoagulant response, eliminating the need to monitor drug concentrations in the laboratory and adjust dosages. Also, because of the long plasma half-life of LH, one or two subcutaneous injections per day are sufficient to maintain therapeutic concentrations. On the other hand, protamine, a mixture of basic proteins produced in fish sperm, forms a stable complex without anticoagulant action to neutralize the blood anticoagulant action of heparin and LH. Protamine (P) is used clinically as an antagonist to the anticoagulant activity of heparin to treat heparin-induced bleeding.

PEC containing low molecular weight heparin/protamine nano/micro particles (LH/P N/MPs) can be prepared by mixing LH and P in a ratio of 6:4. LH/P N/MP has a diameter of 0.1-3 μm and specifically binds to FGF-2, HGF and other GFs secreted from platelets, and can protect and activate various GFs. In addition, LH/P N/MPs are adsorbed to the cell surface and ECM in various tissues. LH/P N/MPs containing GF RNA expression cassettes can significantly induce fibrous tissue formation and vascularization by stabilizing, activating and progressively releasing GFs from GF containing LH/P N/MPs.

Adipose-derived stromal cells (ADSCs) are an attractive potential resource for wound repair and tissue engineering due to their pluripotent properties and their abundance in the human body. ADSC transplantation is used with biomaterials including cell sheets, hydrogels and 3D scaffolds, making the use of ADSCs an alternative to regenerative medicine. ADSCs proliferate efficiently in 3D culture medium consisting of Dulbecco's Modified Eagle's Medium (DMEM) mixed with gels supplemented with 1-6% human serum and LH/P N/MP. Many LH/P N/MPs bind to the surface of adhesive cells, including ADSCs and tumor cells, through interactions with cell surface heparin binding proteins such as integrins. Cell surface interaction with LH/P N/MPs leads to the formation of ADSC-LH/P N/MP-aggregates within 2 h. Cell viability is greatly increased in these in vitro ADSC-LH/P N/MP-aggregates. Injection of LH/P N/MP-aggregates containing ADSC-GF RNA expression cassettes can induce fibrous tissue formation and vascularization in vivo. Therefore, LH/P N/MP containing a GF RNA expression cassette can function as a cell carrier to increase cell viability.

According to the present invention, the RNA expression cassette can be stabilized, activated, and gradually released from LH/P N/MPs containing the FGF-2 RNA expression cassette, so that the RNA expression cassette of the present invention can be fully utilized for adipose-derived stromal cell (ADSC) aggregates. have.

ADSCs are efficiently propagated in 3D culture using a 2% human plasma-DMEM gel containing LH/P N/MP. Inbred rat (IR-) ADSCs were incubated with FGF-2-containing LH/P N/MPs in a plasma gel consisting of inbred rat plasma (IRP) (6%)-DMEM gel, then applied to full-thickness skin wounds on the back . streptozotocin-induced diabetic rats (Figure 5C). Wounds treated with ADSC in IRP-DMEM gel close and heal much faster than wounds not treated with FGF-2-containing LH/P N/MPs. Histological examination of wounds treated with IR-ADSC significantly improved granulation tissue formation, epithelialization and capillary formation. These results suggest that some of the transplanted IR-ADSCs were absorbed into the skin tissue and promoted wound healing, and that IR-ADSCs grown on IRP-DMEM gels were effective in repairing wounds with impaired healing in diabetic mice.

Polyelectrolyte composite hydrogel composed of chitosan and GAG has biological activities such as compatibility and wound healing, antibacterial and antitumor activity, and hypocholesterolemic function. Useful PEC hydrogels can be prepared using positively charged polysaccharides such as chitosan and negatively charged GAGs such as heparinoids and their derivatives. Photocrosslinked chitosan hydrogel (PCH) is formed when exposed to visible or ultraviolet light in the presence of a photocrosslinker and interacts with negatively charged heparinoids and various GFs, cytokines and adhesion molecules. The complex has potential as a functional hemostatic and wound dressing.

The beneficial effects of GAGs, particularly heparinoids, can be utilized by integrating them into the RNA expression cassette delivery system of the present invention. In addition to their anticoagulant action, heparinoids have been implicated in a variety of cytokines and GF biological processes. Because heparinoids are highly water-soluble and disperse in water, an appropriate medium, such as a hydrogel or PEC, may often be required to adsorb or retain the complex. PEC composed of protamine mixed with LH/P N/MP composed of heparinoid and chitosan can be widely used in biomedical applications such as the RNA expression cassette delivery vehicle of the present invention. Therefore, biomaterials containing polysaccharide-based complex PEC hydrogels and N/MPs have potential for RNA expression cassette applications of the present invention.

The present invention also relates to a method for preparing a multiparticulate-containing composition containing the RNA expression cassette multiparticulate prepared by the above-described production method. That is, the method for preparing the multiparticulate-containing composition of the present invention includes the mixing step of the first or second method, and the pH of the composition after mixing may be different from the pH of the aqueous solvent.

The composite particles prepared by the above-described manufacturing method do not disappear even when the pH is changed thereafter. Therefore, according to the present invention, a composition containing multi-particles can be easily prepared.

In addition, the present invention also relates to a multiparticulate comprising a cationic polymer and an RNA expression cassette, wherein the multiparticulate comprises an RNA expression cassette comprising non-amplifying RNA, self-amplifying mRNA and trans-amplifying mRNA. mRNA) may be prepared from one or two or more selected from the group consisting of.

It is characterized in that the particle diameter of the composite particle of the present invention is 500 nm or less.

After the step of removing the water-miscible organic solvent, it may further include a step of freeze-drying by adding a freeze-drying aid.

The manufacturing method may further include a step of sterilizing the polymer nanoparticle aqueous solution with a sterilizing filter before the freeze-drying.

The freeze-drying adjuvant used in the present invention is added to help the freeze-dried composition maintain a cake shape, or to help the anionic polymer composition melt uniformly within a fast time in the process of reconstitution after freeze-drying. Specifically, it may be at least one selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The content of the freeze-drying adjuvant may be 1 to 90 wt%, more specifically 10 to 60 wt%, based on the total dry weight of the freeze-dried composition.

The nanoparticles in the composition in which the RNA expression cassette and the cationic compound complex are encapsulated in the anionic polymer nanoparticle structure, prepared through the production method according to the present invention, are stable in blood, and their size is specifically 10 to 200 It may be nm, and more specifically, it may be 10 to 150 nm.

According to another embodiment, the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a nucleic acid delivery agent as defined herein and a general formula biodegradable polymer carrier delivery complex, optionally with a pharmaceutically acceptable carrier and/or vehicle (vehicle).

As a first important element, the biodegradable pharmaceutical composition comprises a nucleic acid delivery agent and a polymer carrier delivery complex made of biodegradable polymer carrier molecules.

As a second important element, the biodegradable pharmaceutical composition may comprise at least one additional pharmaceutically active ingredient. In this regard, the pharmaceutically active component is a compound that has a therapeutic effect in healing, ameliorate, or prevent a specific symptom, preferably in cancer disease, autoimmune disease, allergy or infectious disease. is a compound with

The biodegradable pharmaceutical composition can be used for humans and also for veterinary purposes, preferably for humans, and can be used as a general pharmaceutical composition or as a vaccine.

As defined above, the biodegradable polymer carrier carrier complex itself is a non-specific innate immune response that allows the biodegradable polymer carrier carrier complex to be used as an immunostimulatory agent (without the addition of other pharmaceutically active components). causes If treated with other pharmaceutically active components, preferably in particular immunogenic components, preferably antigens, the nucleic acids of the present invention may aid in the specific adaptive immune response elicited by the other pharmaceutically active components. The giver acts as an adjuvant.

According to another particularly preferred embodiment, the biodegradable pharmaceutical composition (or biodegradable polymer carrier complex) may be provided or used as a vaccine.

If an RNA expression cassette or mRNA is used as a nucleic acid molecule in a nucleic acid delivery system and a biodegradable polymer carrier delivery complex, the biodegradable vaccine is generally constructed as a biodegradable pharmaceutical composition, preferably the immune response of the immune system of the patient to be treated; For example, it aids or elicits an innate immune response. Furthermore or alternatively, preferably, if the nucleic acid of the nucleic acid carrier and the biodegradable polymer carrier carrier complex encodes any of the above-mentioned antigens or proteins that elicit an adaptive immune response, the biodegradable vaccine is capable of generating an adaptive immune response. can cause

Other additives that may be included in the biodegradable vaccine include emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; It may contain preservatives.

Furthermore, the present invention provides several applications and uses of biodegradable polymer carrier molecules, including nucleic acid delivery and biodegradable carrier delivery complexes made of biodegradable polymer carrier molecules, pharmaceutical compositions comprising them, or kits comprising them ( kits) are provided.

According to one embodiment, the present invention is preferably a gene therapy or a medicament for the treatment of a disease as defined herein, a biodegradable polymer carrier molecule, a nucleic acid delivery agent and a biodegradable polymer made by the biodegradable polymer carrier molecule. It relates to a first medical use of a sex polymer carrier carrier complex, or a kit comprising the same.

The drug may be present in the form of a pharmaceutical composition or in the form of an adjuvant or vaccine, which is a specific form of the pharmaceutical composition. In the context of the present invention, a pharmaceutical composition generally comprises a biodegradable polymer carrier molecule or a nucleic acid delivery agent and a biodegradable polymer carrier delivery complex made of a biodegradable polymer carrier molecule, optionally including other ingredients, for example For example, it preferably comprises a biodegradable nucleic acid as defined herein and optionally a pharmaceutically acceptable carrier and/or vehicle.

Ⅲ. target protein

The target protein to be expressed of the present invention may be any one selected from the group consisting of antigens, antibodies, therapeutic proteins, peptides and fusion proteins. The mRNA comprises at least one open reading frame encoding a therapeutic protein or peptide. Antigens such as pathogenic antigens, tumor antigens, allergens or autoimmune antigens are encoded by at least one open reading frame. Therein, administration of mRNA encoding an antigen is used in a genetic vaccination approach against a disease comprising the antigen. The antibody is encoded by at least one open reading frame of the SM_mRNA according to the invention.

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

fusion protein

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

The former is a heterologous protein polypeptide fused with a linker peptide; And it may be any one selected from the group comprising; and a special-purpose peptide and a polypeptide bound to a protein.

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

Pathogenic antigens:

The mRNA according to the present invention may encode a protein or peptide comprising a pathogenic antigen or a fragment, variant or derivative thereof. Such pathogenic antigens are derived from pathogenic organisms, in particular bacteria, viruses or protozoan (multicellular) pathogenic organisms, which elicit an immune response in a subject, in particular a mammalian subject, more particularly a human. More specifically, preferably the pathogenic antigen is a surface antigen, eg a protein (or a fragment of a protein, eg an external portion of a surface antigen) located on the surface of a virus or bacterial or protozoan organism.

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

The mRNA according to the present invention encodes a rabies virus protein or peptide or antigenic fragment thereof. Preferably, the mRNA according to the present invention is a rabies virus glycoprotein G (RAV-G), nucleoprotein N (RAV-N), phosphoprotein P (RAV-P), matrix protein M (RAV-M) or RNA polymer It encodes an antigenic protein or peptide selected from the group consisting of Rase L (RAV-L), or a fragment, variant or derivative thereof.

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

Tumor antigens:

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

Oncogene (KRAS, p53, etc.) mutant cancer antigen, tumor antigen NY-iO-1, 5T4, MAGE-C1, MAGE-C2, Survivin, Muc-1, PSA, PSMA, PSCA, STEAP and PAP are particularly preferred. The administered mRNA encodes a protein or peptide comprising a therapeutic protein or a fragment, variant or derivative thereof.

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

Thus, therapeutic proteins can be, for example, infectious diseases, neoplasms (eg cancer or tumor diseases), blood and blood-forming organ diseases, endocrine, nutritional and metabolic diseases, neurological diseases, circulatory system diseases, respiratory system diseases. It can be used for a variety of purposes, including the treatment of a variety of disorders (independently hereditary or acquired) such as diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system.

Among them, a particularly preferred therapeutic protein that can be used in the treatment of metabolic or endocrine disorders is understood to be a therapeutic protein since it is intended to treat a subject by replacing its insufficient internal production of a functional protein with a sufficient amount. Accordingly, such therapeutic proteins are generally mammalian, in particular human proteins. Therapeutic proteins can be used for the treatment of blood disorders, circulatory disorders, respiratory disorders, cancer or tumor disorders, infectious disorders or immunodeficiency disorders:

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

therapeutic protein

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

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

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

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

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

A therapeutic antibody is also defined herein as a therapeutic protein. These therapeutic antibodies are inter alia antibodies used for the treatment of cancer or oncological diseases, inter alia antibodies used for the treatment of blood disorders, inter alia antibodies used for immunomodulation, antibodies used for the treatment of diabetes, antibodies used for the treatment of Alzheimer's disease Antibodies, may include antibodies used to treat asthma,

The coding region of the RNA expression cassette according to the invention may occur as mono-, di-, or even multicistronic RNA, ie RNA carrying the coding sequence of one, two or more proteins or peptides. Coding sequences in such di-, or even multicistronic RNAs can induce cleavage of a polypeptide comprising, for example, at least one internal ribosome entry site (IRES) or several proteins or peptides as described herein. can be separated by a signal peptide.

The target protein of the present invention refers to a protein to be expressed in target cells, tissues, and individuals, and can be applied to specific naturally occurring or artificial polypeptides, and there is no limitation to the specific protein. In the present invention, for convenience, ECM degrading enzymes and degrading enzyme inhibitors were exemplarily used.

The effect of ECM on chemotherapeutic resistance of tumor cells was confirmed (BP Toole et al., "Hyaluronan: a constitutive regulator of chemoresistance and malignancy in cancer cells ", Seminars in Cancer Biology 2008, vol. 18, pp. 244 -50). Tumor and stromal cells make ECMs composed of collagen, proteoglycans, and other molecules that make it difficult to move macromolecules into the tumor, and assemble them densely.

The massively organized ECM in all organs of an individual plays a unique role as most, if not all, primary mediators of intracellular events. Thus, almost all known disease mechanisms can be traced back to some of E, in the form of abnormal amounts or activity levels of specific ECM components. These defects significantly affect the downstream signaling axis, resulting in overt cellular dysfunction, organ failure, and death. From skin to bones, blood vessels to brain, eyes to all internal organs, E plays a surprising role as both a cause and a potential means of reversing disease. Chronic human diseases including connective tissue disorders, muscular dystrophy, fibrosis and cancer are all ECM-related diseases. The ECM is mainly responsible for the structural functions of the organism, and through close interaction with cells, it plays an important role in degeneration due to aging and disease, in the regeneration process of damaged tissues (musculoskeletal, liver and lung), and in tumors.

Over the past 25 years, approaches to understanding the biology of solid tumors have changed significantly. Although research has been focused on individual tumor cells for almost a long time, the processes that lead to transformation or transmit malignancies have become increasingly the focus of cancer research due to the total complexity and sheer complexity of tumors. Starting with the concept of seeing a tumor as a complex organ, we use the term tumor microenvironment (TME) to describe the entirety of a tumor component that is not itself malignant. Thus, TME consists of tumor vasculature, connective tissue, infiltrating immune cells, and extracellular matrix (ECM), and all these individual components of TME have become the focus of emerging research communities in the fast-growing field of cancer. These research efforts have already led to the development and successful clinical implementation of TME-targeted drugs, starting with the introduction of anti-angiogenic agents and potentially game-changing immunotherapeutics.

ECM not only influences our efforts to treat cancer, but is also directly involved in tumorigenesis and disease progression. The effect of E-targeted approaches on the efficacy of anti-tumor therapy is often indistinguishable from direct effects on tumor behavior or progression. Therefore, the immediate involvement of E in malignancies and how its transformation affects disease progression should be discussed.

ECM is one of the key factors within solid tumors that limit drug penetration into tumor cells. This aspect of tumor biology has been recognized for decades and has been extensively reviewed in efforts to eliminate E using enzymes or inhibitors of collagen/hyaluronan synthesis. The movement of macromolecular tumor therapies, such as antibodies and nanoparticles, is more sensitive to E than low molecular weight drugs, so efforts to modify E would be most beneficial for the therapeutic activity of these agents.

Several tumors, including breast, pancreas, colorectal, ovarian, and lung, exhibited high-density E with high collagen or hyaluronan content associated with poor prognosis. Cancer-associated fibroblasts produce a large number of ECM components involved in tumor-promoting activities, including angiogenesis, cell proliferation, efflux of metastatic cells, or generation of 'highways' for intravascular invasion of pre-tumor immune cells. Tumor E, composed primarily of collagen and hyaluronic acid (HA), contributes to several aspects of the tumor microenvironment that impede drug delivery, including: 1. Tumors that compress blood vessels, reduce blood perfusion, and restrict adequate vascular access to portions of blood vessels; 2. Limit drug trafficking through the tumor stroma stroma, which prevents the drug from reaching cellular targets. and 3. Maintenance of high interstitial fluid pressure (IFP) generated by plasma leakage from blood, intratumoral mechanical stress, and lack of adequate lymphatic drainage. The proliferation of tumor cells within the confined microenvironment further contributes to mechanical stress within the tumor microenvironment. All of these restrict the movement of drugs from the blood to tumor cells.

In theory, approaches that reduce tumor ECM should increase drug penetration and enable higher drug concentrations within tumor cells. There are subtleties to this approach. Complete removal of all biopolymers from the tumor interstitial space could result in tumor collapse and reduced drug penetration. Therefore, the concept of normalization of E has been proposed as the goal of this treatment. In this review, we highlight the main approaches to E degradation, although these enzymes are less widely used, but because they can be mechanically distinguished from the most advanced form of substrate degradation achieved through the reduction of hyaluronic acid (HA) collagenase. The focus was on ceremonial activity.

1. Hyaluronidase

Hyaluronic acid (HA) is an anionic mucopolysaccharide, a bio-derived material that exists in the vitreous body, joint fluid, cartilage, skin, etc. derived from animals, and has a chemical formula of (C14H21NO11)n, and the molecular weight of hyaluronic acid is limited However, the weight average molecular weight may be in the range of 10,000 to 1,000,000, but may not be limited thereto. For example, the weight average weight is a range between any two values of 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, and 1,000,000. may have, but may not be limited thereto.

Hyaluronic acid (HA), a natural polymer, is a nonsulfated glycosaminoglycan (GAG) composed of N-acetyl-D-glucosamine and D-glucuronic acid, and is a major component of the extracellular structure. It has high biocompatibility. In addition, it is present in the synovial fluid of the vitreous and joint sinus of the eyeball as well as the extracellular matrix of connective tissues such as subcutaneous tissue and cartilage tissue. known to control processes. With these characteristics, hyaluronic acid has therapeutic uses, and is a natural polymer approved by the US Food and Drug Administration (FDA) for use in humans by intravenous injection, oral administration, and skin administration (P. Bulpitt, et al., J. Biomed. Mater. Res. 47, 152 (1999)).

Currently, the scope of its use is increasing as a scaffold for tissue engineering and a hydrogel for drug delivery. In addition, as it was recently revealed that hyaluronic acid is decomposed by active oxygen, it became possible to measure active oxygen through the decomposition of hyaluronic acid.

Hyaluronidase, an enzyme that breaks down hyaluronic acid, has been used for medical purposes for over 60 years. The U.S. Food and Drug Administration (FDA) has approved hyaluronidase as an adjuvant for (1) subcutaneous infusion (hypodermoclysis), (2) accelerating absorption and dispersion of a drug in the subcutaneous tissue, or managing leakage. (3) It is used as an adjuvant to promote absorption of contrast agents in urinary tract angiography (subcutaneous urogiography). It has also been approved and used in Europe to increase hematoma resorption. Hyaluronidase has a variety of uses beyond the approved indications. Current off-label uses include dissolving hyaluronic acid fillers, treating granulomatous foreign body reactions, and treating skin necrosis associated with filler injections. The use of hyaluronidases for these off-label indications is growing significantly.

Human hyaluronidase is present in both organs (testis, spleen, skin, eyes, liver, kidneys, uterus, placenta) and body fluids (tears, blood, semen). There are six known types (hyaluronidase 1-4, PH-20 and HYALP1). Hyaluronidase 1, encoded by the HYAL1 gene, is present in major organs such as the liver, kidney, spleen and heart, as well as in serum and urine. It acts as a major hyaluronidase in plasma and is activated at acidic pH. Hyaluronidase 2 exerts a weaker enzymatic activity than Hyaluronidase 1 and degrades only high molecular weight hyaluronic acid. Hyaluronidase 3 is only found in the testis and bone marrow, and its role is unknown. Testicular PH20 hyaluronidase is found on the surface of human sperm and the acrosome lining and is responsible for breaking down hyaluronic acid in eggs during fertilization.

Hyaluronidases can be classified into three categories according to their mechanism of action. First, mammalian hyaluronidase is an endo-β-N-acetyl hexosaminidase that cleaves β-1,4 glycosidic bonds to form tetrasaccharides. Second, leech/hookworm hyaluronidase is an endo-β-D-glucuronidase that cleaves β-1,3 glycosidic bonds to form pentasaccharides and hexasaccharides. Finally, microbial hyaluronidases are classified as hyaluronate degrading enzymes. Unlike other hyaluronidases, it does not catalyze hydrolysis reactions. Instead, they produce unsaturated disaccharides via a β-elimination reaction at the β-1,4 glycosidic bond.

Hyaluronidases can also be classified into two types according to the pH at which they are most active. Acidic active hyaluronidase is activated at pH 3-4. Neutral active hyaluronidase, including the hyaluronidase enzyme found in snake and bee venom, is activated at pH 5-8.

In the past, medical hyaluronidase was extracted from bovine or sheep testes and used without purification. However, the mammalian hyaluronidase thus obtained is of low purity and contains components that can induce an immune response. After that, purification of mammalian hyaluronidase was performed as a processing step, and microbial hyaluronidase obtained from Streptococcus agalactiae bacteria was also used to reduce side effects.

When hyaluronidase is injected into the body, its activity gradually decreases over time due to dilution, diffusion and inactivation. Inactivation occurs by anti-hyaluronidase activity that proceeds at different rates in subcutaneous tissue and plasma. In experiments using rodents, the subcutaneous tissue half-life of hyaluronidase was less than 30 minutes, and the activity was partially maintained up to 1 hour depending on the experiment. The half-life of intravenous injections of hyaluronidase from plasma into humans was 2 to 3 minutes, and repeated infusions did not result in sustained elevation of serum hyaluronidase levels. The reason for the short half-life of hyaluronidase in human plasma is the presence of numerous hyaluronidase inhibitors in plasma and the metabolism of hyaluronidase in the kidneys and liver.

Hyaluronidase in the body is affected by various drugs. Hyaluronidase antagonists include anti-inflammatory drugs (such as indomethacin, dexamethasone, and salicylates), numerous plant-based compounds (such as flavonoids and antioxidants), antihistamines, mast cell stabilizers, heparin, vitamin C, dicoumaren and Contains radiographic contrast agents.

Levels of hyaluronidase inhibitors may increase depending on the individual's physical condition. In acute phase reactions such as burns, sepsis, and shock, hyaluronidase inhibitor levels are increased to decrease the turnover of hyaluronic acid, preventing circulatory disruption.

Local injections of hyaluronidase can cause side effects such as local pruritus and allergic reactions. The incidence of allergic reactions was reported to be 0.05 to 0.69%, and urticaria and angioedema were also reported to occur at a low frequency (less than 0.1%). Allergic reactions are more likely to occur when hyaluronidase doses are greater than 100,000 IU via intravenous injection, and when the dose is increased to 200,000 IU, the incidence of allergic complications increases by 31.3%.

Most allergic reactions to hyaluronidases are immediate hypersensitivity reactions (type I, immunoglobulin ECM mediated), but delayed hypersensitivity reactions (type IV, T cell mediated) may also occur. Immediate hypersensitivity reaction by hyaluronidase appears as erythematous edema after 1 to 2 hours, and there is no response to antibiotic treatment. In these cases, systemic steroids, antihistamines, and steroid creams may help. Delayed hypersensitivity reactions caused by hyaluronidase can occur even after 24 hours, in which case the skin test does not show a positive reaction within 20 minutes, resulting in a negative diagnosis.

The skin test is performed with 3 IU of hyaluronidase. It is recommended to perform a skin test prior to using hyaluronidase, but this can often be difficult in the general clinic. In general, there is no association between a patient's allergy history and response to hyaluronidase. However, depending on the origin of the hyaluronidase, injections of hyaluronidase should be avoided as cross-reactivity may occur in patients allergic to bovine collagen and bee stings.

2. Substrate metalloproteinase (MMP)

Matrix metalloproteinases (MMPs) consist of a group of zinc endopeptidases with the ability to cleave peptide bonds in the extracellular matrix (E). Peptidase enzymes can be divided into two groups: endopeptidases and exopeptidases. Endopeptidases are divided into six major classes: metalloproteinases, aspartic acid, serine, cysteine, glutamic acid, and threonine peptidases. Most molecular structures of metalloproteases use zinc, but some require cobalt. There are two subgroups of metalloproteinases, metalloexopeptidases or metal carboxypeptidases and metalloendopeptidases, including disintegrin and metalloproteinase proteins and MMPs. MMP can be classified as shown in <Table 1>, collagenase (MMP-1, MMP-8, MMP-13 and MMP-18) gelatinase (MMP-2 and MMP-9), stromelysin ( MMP-3, MMP-10, MMP-11 and MMP-17), matrilysins (MMP-7 and MMP-26), membrane types (MMP-14, MMP-15, MMP-16, MMP-24 and MMP-25) ) and other types (MMP-12, MMP-19, MMP-20, MMP-21, MMP-22, MMP-28, MMP-29), etc.

MMPs have been extensively studied for nearly 40 years and are originally responsible for degrading the ECM. Structurally, MMPs contain similar catalytic inhibition domains. The catalytic domain is specific to a target for degradation of each MMP. The traditional classification of MMPs was based on the first identified degradation targets. Over the years, MMPs have played a significant number of regulatory roles at the cellular level in pathways such as apoptosis, immunity, cell migration and angiogenesis. MMP function is often associated with classical tumor characteristics leading to invasion, immune system evasion and metastasis. Given these properties, MMPs play an important role in carcinogenesis. <Table 1> is an overview of the specific role of each MMP in cancer.

Dysregulation of MMP expression has resulted in numerous studies of protein and RNA levels in many cancer types (Table 3). Often, MMP dysregulation is associated with prognostic differences, a trend that is seen in breast, ovarian, and colon cancers.

Types of MMPs and their roles MMP Role in Cancer Collagenases One Initial invasion, promotes metastasis 8 13 Growth, invasion, and angiogenesis of skin squamous cell carcinoma Matrilysins 7 Contributes to invasive potential, proliferation, anti-apoptotic, immune surveillance 26 Activates MMP-9 in prostate cancer, role in early skin carcinogenesis Metalloelastase 12 Protective inhibition of tumor growth, anti-angiogenic Stromelysins 3 Invasion, metastasis, and epithelial to mesenchymal transition 10 Invasion, migration, and growth; prevents tumor cell apoptosis; produces angiogenic and metastatic factors 11 Produced by peritumoral stromal fibroblasts; regulates early tumor invasion, implantation, and expansion; prevents apoptosis of early cancer cells Gelatinases 2 Proteolytic degradation of extracellular proteins in tumor invasion, collagenolytic pathway driver for lymphatic vessel formation, tumor angiogenesis 9 Proteolytic degradation of extracellular proteins during tumor invasion Enamelysin 20 Synthesized in odontogenic tumors Membrane-Type 14 Cleaves other pro MMPs (mainly MMP2) to activate them, role in invasive blood vessel growth, and promoting metastasis. In vitro has been shown to promote invasion 15 In vitro shown to play role in epithelial to mesenchymal transition, promotes angiogenesis 16 In vitro promotes invasion and metastasis 17 Induce angiogenesis, promote growth and metastasis 24 Progression in brain tumors, aids in migration and metastasis 25 in vitro tumor growth promoter Other 19 In vitro modulates proliferation, adhesion, and metastasis 21 Expression changes associated with cancer prognosis. 23A Expression levels altered in multiple cancers. Urinary levels decreased in renal cell carcinoma. 23B 27 28 Promotes epithelial to mesenchymal transition, promotes invasion and metastasis

Collagenase was first identified in 1962. Collagenase and MMP-13 and MMP-18 are enzymes that degrade four types of collagen (I, II, III, IV), and only protease enzymes can hydrolyze the triple helical domain of collagen under various physiological conditions. can Interstitial collagen fibers are resistant to degradation by most proteases. Only MMP-1, MMP-2 and MMP-3 can initiate degradation of intact triple-helix collagen and collagen types I, II and III into quarter and three-quarter sections. MMP-1 and MMP-13 preferentially degrade collagen types II and III, respectively, whereas collagenase has a cap that is part of matrix specificity. However, MMP-8 degrades type I collagen three times more potently than either MMP-1 or MMP-13. After initial degradation, fibrillar collagen fragments are susceptible to further degradation by various MMPs such as MMP-2, MMP-3 and MMP-9. The moment collagen breaks down into smaller pieces, endogenous enzymes help break down the fibrous material. Because collagenase does not harm cell membranes, it has been widely used for cell dispersion, tissue isolation, and cell culture in the laboratory for many years. For example, Clostridium histolyticum (C. histolyticum) is an FDA-approved drug that breaks down Dupuytren's stiff cord, widely used in human cell isolation, wound healing, and wound healing.

Three types of collagenase enzymes can be isolated and characterized from both microbial cells and animal tissues. Pathogenic microorganisms, primarily C. histolyticum, provide microbial collagenases. These collagenases can split each polypeptide chain of collagen into multiple sites. They function as exotoxins and are known to interfere with connective tissue metabolism due to hydrolysis of collagen in host cells. Recombinant bacterial collagenase can hydrolyze both poorly soluble natural collagen and water-soluble denatured collagen. Unlike microbial collagenases, which have been studied focusing on a single species, tissue collagenases have been isolated and characterized from various tissues of different animals. Because tissue collagenase is a digestive enzyme, it is commonly isolated from the digestive tract of various fish and invertebrates.

Collagenase is a unique enzyme used in the treatment of certain diseases, and excessive collagen aggregation can lead to disorders in the pathogenesis of organelles. Collagen is a major protein of extracellular fibrous tissues such as tendons, skin, cartilage and blood vessels, as well as an organic component of teeth, bones and corneas. Recently, two types of collagenases have been identified. The first type of collagenase is synthesized by some microorganisms. For example, C. histolyticum, a bacterium that causes gas gangrene, produces collagenase that can break down the polypeptide chains of collagen in more than 200 states and hydrolyzes collagen at c-terminal bonds. It can also denature the connective tissue barrier of an organism. Thus, the biological processes of connective tissue depend on collagenase. The second type of collagenase is synthesized by mammals. It can break down the collagen triple helix at specific points and make trophocollagen A and B containing 3/4 and 1/4 trophocollagen molecules, respectively. These broken pieces are converted into gelatin chains. In addition to collagenase, physical factors such as temperature can separate trophocollagen polypeptide chains and produce gelatinous chains, which can be degraded by other proteases. One third of collagen amino acids are composed of glycine, proline and hydroxyproline. Unlike mammalian collagenase, bacterial collagenase, one of the factors of bacterial virulence, attacks and breaks down different parts of the collagen helix. In addition, invasive strains of C. histolyticum and Pseudomonas aeruginosa contain collagenase.

Collagenase is more often used for tissue cell isolation in medical research. These enzymes have been successfully applied to remove and relocate insulin-secreting gland cells (for diabetic patients). Collagenase can also be used to isolate intact liver parenchymal cells, adipocytes and adrenal glands. After isolation, these cells can be used for cell growth. Collagenase has also been applied to G-banding technology to study human chromosomes. Today, collagenase is used as a treatment and may replace invasive treatment for diseases in which excessive collagen deposition disrupts some physiological functions of the body's systems.

There is a difference between bacterial and vertebrate collagenases with respect to the nucleotide sequence of nucleic acids and the function of proteins. The most commonly used microbial collagenase in medical products is C. histolyticum. Collagen makes up one-third of the body's protein. However, changes in production or degradation can cause problems. Collagen may be produced in excess of what is needed or produced in the wrong place, or it may not break down after an appropriate amount of time. In this case, the use of injectable form of collagenase or its ointment may help the breakdown of obstructive collagen.

3. Tissue inhibitors of metalloproteinases (MPs) (TIMPs)

Connective tissue and articular cartilage are maintained in dynamic equilibrium by opposing effects of extracellular matrix synthesis and degradation. Degradation of the substrate occurs mainly by the enzymatic action of metalloproteinases, including matrix metalloproteinases (MMP) and thrombospondin motifs (ADAMTS) and disintegrin-metalloproteinases. Although these enzymes are important in many natural processes (eg, development, morphogenesis, bone remodeling, wound healing, and angiogenesis), abnormal increases in these enzymes are linked to cancer and cardiovascular disease, as well as rheumatoid arthritis and osteoarthritis. It is believed to play a detrimental role in decomposing diseases.

Endogenous inhibitors of metalloproteinases include plasma alpha 2-macroglobulin and tissue inhibitors of metalloproteinases (TIMPs), four of which are known to be encoded in the human genome.

The tissue inhibitors (TIMPs) of metalloproteinases (MPs), the four members of the TIMP-1, 2, 3 and 4 family, were originally described as endogenous inhibitors of secreted metalloproteinases (MPs) and inhibit most tissues decomposition can be inhibited. The proteolytic activity of the extracellular matrix can occur transiently during tissue transformation and is tightly regulated by the standard anti-proteolytic function of TIMPs. In many diseases, MP upregulation and activation is observed as an important disease-inducing mechanism. Increased expression of TIMPs attempts to balance these ECM degrading activities but fails and disease results. While proteolytic activity in cancer supports invasion and metastasis, protease inhibition may be considered to prevent the aggressive features of these malignancies.

IV. Use of multiparticulates comprising RNA expression cassettes

1. mRNA Therapeutic

Eukaryotic cells, including humans, have a membrane surrounding the nucleus containing genetic information, and are largely divided into a nucleus and a cytoplasm. DNA in the nucleus is not directly made into protein, but goes through a process of *?**?*transcription*?**?* that transfers only necessary genetic information from DNA to RNA.

In this case, the RNA involved is mRNA (messenger RNA). The mRNA made in the nucleus comes out to the cytoplasm to synthesize proteins, and this step is called *?**?*translation*?**?*. In other words, mRNA is an intermediate between DNA and protein.

It is the protein that is the final product that regulates human life phenomena. If the protein is modified so that it does not function properly or is not produced, we become ill.

If so, is it effective to target DNA, RNA, or protein to treat diseases? Most of the existing drugs we know of are therapeutics that target proteins. Recently, with the development of biotechnology, treatments that can solve the root cause in terms of prevention beyond the treatment of diseases are being developed. One of them is mRNA-based therapy.

If the protein that causes a specific disease is broken or missing, mRNA encoding the normal protein is injected into the patient's cells so that the translated normal protein functions properly.

Unlike DNA, mRNA does not need to be delivered to the nucleus, and there is no risk of *?**?*Genome Integration*?**?* in which foreign genes are integrated between DNA, so there is little risk. In addition, the delivered mRNA is safe because it temporarily makes a protein and is rapidly degraded. In addition, it is easy to manufacture and the cost is also low.

Despite these many advantages, mRNA is an unstable form that is easily degraded by ribonucleases, an RNA degrading enzyme found in human body fluids (tears, saliva, mucus, sweat, etc.) It was difficult. However, after the study of the structure of mRNA was carried out, the development of mRNA-based therapeutics began as a modified form of mRNA with increased protein translation efficiency and mRNA half-life was prepared.

Basically, all mRNA-based therapeutics start with DNA. mRNAs can be prepared that complementarily bind to the DNA sequence for the deficient protein or antigen that causes infection (by bacteria or viruses) and, in the case of cancer, the protein that causes tumors. Of course, the cost is also less.

The prepared mRNA is directly injected into the patient's body (in vivo transfection), or cells such as immune cells or dendritic cells are extracted from the patient's body and mRNA is injected, and then the transformed cells are put back into the patient's body. (ex vivo transfection).

Cancer-immunotherapies are a typical example. Cancer has tumor-specific antigens, and it varies greatly by cancer type and individual. An mRNA-based cancer vaccine treatment can be used as a personalized anticancer vaccine by expressing specific antigens on dendritic cells or T cells. In addition, as infectious disease vaccines and immunotherapeutic agents, the body can produce antibodies to directly treat diseases. It is also possible to produce a protein that is deficient in my body and directly manufacture a therapeutic agent in my body.

In addition, it is expected to open a new therapeutic market for many diseases that target intracellular space and transmembrane proteins, which could not be achieved with conventional protein-based therapies.

protein therapy

According to the present invention, the use of multiparticulates for mRNA delivery for the expression of therapeutic proteins holds the potential to treat a wide range of diseases. Therapeutic applications include (1) protein replacement to restore the function of a single protein for rare solitary diseases; (2) cellular reprogramming in which mRNA can be used to modulate cell behavior by expressing transcriptional or growth factors; and (3) immunotherapy wherein the mRNA-encoded transcript elicits an immune response specific to a target cell, eg, a therapeutic antibody. In general, mRNAs designed to express therapeutic proteins are engineered to exhibit low immunogenicity, extended stability, and robust translation. Intracellular delivery of mRNA enables the expression of virtually any desired protein inside host cells and tissues, which allows for preservation of post-translational modifications of the protein natively encoded in the host cell. The approach may also address the formulation and delivery difficulties associated with protein-based drugs, particularly those targeting the restoration of intracellular and transmembrane proteins. Other important features of mRNA-based therapeutics include a low risk of insertional mutagenesis, transient production of the encoded protein, and cytoplasmic activity of the mRNA that lowers the cellular barrier.

Repeated administration of mRNA therapeutics is necessary to maintain therapeutic levels of protein. The dosing frequency may vary depending on the half-life of the protein, its activity and the conversion rate of the target cells. The average protein production half-life from the transfected modified mRNA ranges from 50 hours in vitro to 7 to 30 hours in vivo, depending on the route of administration. It has recently been shown that synthetically engineered exogenous circular RNA provides up to threefold. Increased half-life and potent expression of protein production in vitro compared to linearly modified RNA expression cassettes. Most of the mRNA-based protein replacement therapies are currently directed to the liver, lung and heart due to relatively efficient methods for mRNA delivery to these tissues. Access to other organs and cell types may require continued development of new delivery strategies, including novel biomaterials, active targeting, or other methods of administration.

In particular, in certain embodiments of the invention the disease associated with ECM includes cancer, liver fibrosis, cirrhosis, lung fibrosis including lung fibrosis (including ILF), any disease that causes renal fibrosis (eg, ESRD) CKD), peritoneal fibrosis, chronic liver injury, fibrillation, fibrotic disease in other organs, abnormal scarring (keloid) associated with all possible types of accidental and iatrogenic (surgical) skin injury; scleroderma; cardiac fibrosis, fibrosis of the brain; failure of glaucoma filtration surgery; and intestinal adhesions.

In some embodiments, preferred indications include: cirrhosis due to hepatitis C after liver transplantation; cirrhosis due to nonalcoholic steatohepatitis (NASH); idiopathic pulmonary fibrosis (IPF); radiation pneumonia causing lung fibrosis; diabetic nephropathy; includes sclerosis associated with persistent stone peritoneal dialysis (CAPD) and ophthalmoplegia pemphigoid.

Indications for fibrotic liver include alcoholic cirrhosis, hepatitis B cirrhosis, hepatitis C cirrhosis, hepatitis C cirrhosis after orthotopic liver transplant (OLTX), NASH/NAFLD (NASH is non-alcoholic fatty liver disease ( NAFLD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, alpha1 antitrypsin deficiency (alpha1 antitrypsin deficiency, A1AD), Cooper's storage disease (Wilson's disease) ), fructosemia, galactosemia, glycogen hypotonia (especially types III, IV, VI, IX and X), iron overload syndrome (hemochromatosis), lipid abnormalities (e.g. Gaucher disease), peroxisomal disease (eg Zellweger syndrome), tyrosineemia, congenital liver fibrosis, bacterial infection (eg brucellosis), parasites (eg cysticercosis), Budd- Chiari syndrome (hepatic vein occlusive disease).

Pulmonary indications include idiopathic pulmonary fibrosis, silicosis, pneumoconiosis, bronchopulmonary dysplasia after neonatal respiratory distress syndrome, bleomycin/chemotherapy-induced lung injury, brochiolitis obliterans (BOS) after lung transplantation, chronic obstructive pulmonary disease (COPD). , COPD), cystic fibrosis, and asthma.

Cardiac indications include cardiomyopathy, atherosclerosis (such as Burgers' disease), endocardial cardiac fibrosis, atrial fibrillation, and scarring after myocardial infarction (myocardial infarction, MI).

Other thoracic indications include radiation-induced capsule tissue reactions around textured breast implants and oral submucosal fibrosis.

Renal indications include Autosomal Dominant Polycystic Kidney Disease (ADPKD), diabetic nephropathy (diabetic glomerulosclerosis), FSGS (disruption to other histologic variants), IgA nephropathy (Berger's disease), lupus nephritis, Wegener, Scleroderma, Goodpescher's syndrome, tubulointerstitial fibrosis: drug-induced (protective) penicillins, cephalosporins, analgesic nephropathy, membranoproliferative glomerulonephritis (MPGN), Henoho-Schinlein purpura, congenital nephropathy: medullary cysts disease, patellar syndrome and Alport's syndrome.

Bone marrow indications include lymphangioleiomyomatosis (LAM), graft-versus-host disease, polycythemia vera, thrombocythemia vera, and myelofibrosis.

Ocular indications include Retinopathy of Prematurity (RoP), ophthalmoplegia, lacrimal gland fibrosis, retinal adhesion surgery, corneal opacity, herpetic keratitis, pterygium, glaucoma, age-related macular degeneration (AMD/ARMD). , retinal fibrosis associated diabetes mellitus (DM) retinopathy.

Brain indications include fibrosis associated with cerebral infarction.

Gynecological indications include endometriosis in addition to hormonal therapy for the prevention of scarring after STD fibrosis/salpingitis.

Systemic indications include Duftren's contracture, palmar fascia fibromatosis, Peyronie's disease, Lederhorse disease, keloids, multiple fibrosclerosis, systemic nephrotic fibrosis, renal myelofibrosis (anemia).

Injury related fibrotic diseases include burns (including chemical burns) induced skin and soft tissue scars and shrinkage, radiation induced skin and organ scars after cancer treatment radiation treatment, and keloids (skin).

Indications for surgery include peritoneal fibrosis after peritoneal dialysis catheterization, corneal transplantation, cochlear implantation, other implants, intramammary silicone implantation, chronic sinusitis; Adhesive, including intimal hypertrophy of dialysis grafts.

Other indications include chronic pancreatitis. Any disease that may be affected by the present invention is selected from these.

local transfection

Direct injection of the mRNA multiparticulates of the present invention can be used for delivery to cardiac tissue, wherein the modified mRNA is a growth factor (eg, vascular endothelial growth factor-A [VEGF-A], insulin-like growth factor) 1 [IGF1], epidermal growth factor [EGF], hepatocyte growth factor [HGF], transforming growth factor [TGF] b1, TGFb2, stromal cell-derived factor 1 [SDF-1], fibroblast growth factor 1 [FGF-1] , growth hormone [GH] and stem cell factor [SCF]), an intramyocardial injection of a modified RNA encoding human VEGF-A in complex with RNAiMAX.

The therapeutic effect of direct transduction of the mRNA complex of the present invention can also be achieved in other tissues. The surfactant protein B (SP-B) mRNA complex of the present invention may be lyophilized and delivered to the lungs through intratracheal high-pressure spray-protected mice for localized protein synthesis.

2. Vaccination

“Immunification” or “vaccination” refers to a method of treating a subject, generally for therapeutic or prophylactic reasons. Treatment, in particular prophylactic treatment, is preferably or comprises a treatment aimed at inducing or enhancing an immune response of a subject, for example against one or more antigens.

According to the present invention, if it is desired to induce or enhance an immune response by using the RNA as described herein, the immune response can be triggered or enhanced by the RNA.

The present invention preferably provides a prophylactic treatment that is or comprises a vaccination of a subject. The invention wherein the replicon encodes as a polypeptide of interest a pharmaceutically active peptide or protein that is an immunologically active compound or antigen is particularly useful for vaccination.

RNA expression cassettes have been previously described for vaccination against foreign agents, including pathogens and cancer. In contrast to the conventional approaches of the prior art, the RNA expression cassette according to the present invention is a particularly suitable element for efficient vaccination because of its ability to be replicated in trans by the replicator encoded by the replicatase construct of the invention.

The RNA expression cassette multiparticulate-based cancer vaccine and other immunotherapy of the present invention are promising alternative strategies for treating malignant tumors. Cancer vaccines can be made to target tumor-associated antigens that are specifically expressed on cancer cells or antigens of malignant cells that are expressed due to somatic mutations. Most cancer vaccines are aimed at treating cancer rather than prevention, and induce cell-mediated responses such as cytotoxic immune responses that can eliminate or reduce tumors.

The administration route and delivery method of the mRNA multiparticulate vaccine of the present invention can have a significant effect on the effectiveness of the vaccine. A variety of mRNA cancer vaccine formats have been developed using common routes of delivery (intradermal, intramuscular, subcutaneous or intranasal administration) and parts of the vaccination route (intraarticular, intravenous, intravisceral or intratumoral).

Intra-lymphatic administration of the mRNA multiparticulates of the present invention is an unconventional but effective means of vaccine delivery. The mRNA multiparticulates of the present invention directly to secondary lymphoid tissues can effectively deliver antigens to antigen-presenting cells for T cell activation. Several studies have shown that the mRNA multiparticulates of the present invention injected into lymph nodes can be selectively taken up by dendritic cells and induce a potent prophylactic or therapeutic anti-tumor T cell response.

Intratumoral vaccination of the inventive mRNA multiparticulate results in rapid and specific activation of tumor-resident T cells. In an initial study, intratumoral administration of an mRNA multiparticulate of the present invention encoding albumin mRNA or protamine-non-tumor-associated gene (GLB1) would inhibit tumor growth.

The skin is home to a variety of antigen-presenting cells and is an ideal site for vaccination for immunogen delivery. Therefore, delivery of the mRNA multiparticulate vaccine of the present invention via the intradermal route can be widely used in cancer vaccines.

Combining the RNA expression cassette multiparticulate vaccination of the present invention with adjuvant therapies such as traditional chemotherapy, radiation therapy and immune checkpoint inhibitors will increase the effective outcome of vaccination in some preclinical studies.

As evidenced by the very strong expression of the recombinant protein, it is possible for the gene of interest-bearing replicon to reach a high copy number in vaccinated animals. The vaccination according to the invention, in which the replicon encodes a therapeutic protein, proves to be very effective. Thus, the present invention enables effective vaccination using an alphavirus-based trans-replicating RNA system.

In a method comprising vaccination according to the present invention, the medicament of the present invention is administered to a subject, particularly if it is desired to treat a subject having a disease comprising an antigen or at risk of contracting a disease comprising an antigen.

In a method comprising vaccination according to the present invention, the polypeptide of interest encoded by the replicon for use in the present invention is, for example, a bacterial antigen from which an immune response is induced or a viral antigen from which an immune response is induced, or It encodes for a cancer antigen from which an immune response is elicited or an antigen of a unicellular organism against which an immune response is elicited.

The efficacy of vaccination can be assessed by standard known methods, such as measurement of antigen-specific IgG antibodies from an organism. In a method comprising allergen-specific immunotherapy according to the present invention, the polypeptide of interest encoded by the replicon according to the present invention encodes an allergy-associated antigen.

Allergen-specific immunotherapy (also known as desensitization therapy) preferably involves administering an increased dose of an allergen vaccine to an organism having one or more allergies to achieve a condition in which symptoms associated with subsequent exposure to the causative allergen are alleviated. defined as doing The efficacy of allergen-specific immunotherapy can be assessed by standard known methods, such as measurement of allergen-specific IgG and IgE antibodies from the organism.

A medicament of the invention may be administered to a subject for treatment of a subject, including, for example, vaccinating the subject.

The present invention relates to a second medical use of a biodegradable polymeric carrier molecule, or an RNA expression cassette multiparticulate and an RNA expression cassette multiparticulate made by a biodegradable polymeric carrier molecule for the treatment of a disease as defined herein, preferably herein Of the RNA expression cassette for the manufacture of pharmaceuticals for the prevention, treatment and/or improvement of various diseases defined in, or of a biodegradable RNA expression cassette multiparticulate made by a biodegradable polymer carrier molecule, or a pharmaceutical composition comprising them or to the use of a kit comprising them.

The present invention relates to the use of a biodegradable RNA expression cassette multiparticulate, preferably to the use of an RNA expression cassette multiparticulate as defined herein, for immunotherapy, gene therapy, vaccination ( It relates to the use of an RNA expression cassette for vaccination) and biodegradable RNA expression cassette multiparticulates made by a biodegradable polymeric carrier molecule, or to their use as an adjuvant.

The present invention relates to the treatment of diseases as defined herein, preferably to the prevention, treatment and/or amelioration of various diseases as defined herein, preferably comprising a nucleic acid delivery and biodegradable RNA expression cassette multiparticulate. To a biodegradable pharmaceutical composition or vaccine as defined herein or to the use or administration to treat patients in need thereof.

The present invention also treats non-heritable diseases or diseases not summarized in the above categories. Such diseases may include, for example, the treatment of patients in need of certain protein components, such as the therapeutically active proteins mentioned above. This may include, for example, dialysis patients, who are on (regular) kidney or renal dialysis, or a particular therapeutically active protein as defined herein, for example an ECM degrading enzyme or It may include patients in need of enzyme inhibitors and the like.

The present invention provides a kit, in particular at least one biodegradable RNA expression cassette multiparticulate made by at least one RNA expression cassette and a biodegradable polymeric carrier molecule, comprising only components or in combination with other components, herein At least one nucleic acid as defined, at least one pharmaceutical composition and/or at least one kit comprising them, and optionally treatment of an RNA expression cassette, a nucleic acid, a biodegradable RNA expression cassette multiparticulate and/or a biodegradable pharmaceutical composition and a kit, in particular a kit of parts, comprising technical instructions with information on injection. Such a kit, preferably a kit of several components, can be applied, for example, to any of the above-mentioned applications or uses.

Ⅴ. Treatment considerations

Good manufacturing practice production

RNA expression cassettes are produced by in vitro reactions with recombinases, ribonucleotides, triphosphates and DNA templates. Therefore, the production of RNA expression cassettes is fast and simple when compared to traditional subtype protein and live virus vaccine production platforms. The reaction yield and simplicity of RNA expression cassette production enables rapid mRNA production in a small GMP facility. The manufacturing process is independent of sequence and is mainly determined by the length, nucleotide and capping chemistry of the RNA expression cassette and purification of the product. Current experience shows that this process can be standardized to produce almost any coding protein immunogen, making it particularly suitable for rapid response to emerging infectious diseases. All enzymes and reaction components required for GMP production of RNA expression cassettes can be obtained from commercial suppliers as reagents free of synthetic chemicals or bacterially expressed animal components, avoiding safety concerns that adversely affect cell culture-based vaccine manufacturing. have.

Once the RNA expression cassette is synthesized, it is processed through several purification steps to remove reaction components including enzymes, residual nucleotides, residual DNA, and cleaved RNA fragments. Purification on a clinical scale is carried out in a batch or column format with derivatized microbeads. For some RNA expression cassette platforms, removal of dsRNA and other contaminants is critical to the efficacy of the final product. After purification of the mRNA, it is exchanged for a final storage buffer and then filtered sterile for storage in tubes for clinical use. Formulation buffers should be tested to ensure that they are free of contaminating RNase and can contain buffering components such as antioxidants and chelating agents that minimize the effects of reactive free radicals and divalent metal ions inducing RNA expression cassette instability.

Pharmaceutical formulation of RNA expression cassettes is an area that is being actively developed. Although most of the products were stored frozen (-70°C) in early-stage studies, efforts are continuing to develop high-temperature stable formulations more suitable for vaccine distribution. The RmRNAtive platform was reported to be active after lyophilization at 5-25 °C for 3 years and after storage at 40 °C for 6 months. Another report showed that freeze-dried naked mRNA was stable for at least 10 months in refrigerated conditions. The stability of RNA expression cassette products can be improved by packaging them in nanoparticles or combining them with RNase inhibitors.

Regulatory Aspects

There are no specific guidelines from the FDA or the European Medicines Agency (EMA) for RNA expression cassette multiparticulate products. However, a growing number of clinical trials conducted under EMA and FDA oversight indicate that oversight bodies have embraced the idea proposed by various organizations to demonstrate that RNA expression cassette products are safe and acceptable for testing in humans. Because RNA expression cassette multipliers fall within the broad vaccine category of gene immunogens, many of the guiding principles defined for DNA vaccines and gene therapy vectors are directed to RNA expression cassette multipliers through some features that reflect the unique characteristics of RNA expression cassettes. can be applied. As mRNA products become more prominent in the field of vaccines, specific guidelines will be developed for the production and evaluation of new RNA expression cassette multiparticulates.

safety

Because the vaccine is administered to healthy people, the safety requirements for modern prophylactic vaccines are very stringent. Because the manufacturing process of RNA expression cassettes does not require cell culture or toxic chemicals that can be contaminated with adventitious viruses, RNA expression cassette production can be used on other vaccine platforms, including live virus, viral vectors, inactivated virus and subunit protein vaccines. avoid common risks that exist in In addition, the short production time of the RNA expression cassette leaves little chance of contaminating microorganisms. For vaccinated individuals, the theoretical risk of infection or integration of the vector into host cell DNA does not exist for RNA expression cassette multiparticulates.

Potential safety issues that may be evaluated in future preclinical and clinical studies include local and systemic inflammation, biodistribution and persistence of immunogenicity, stimulation of autoreactive antibodies, and the potential toxic effects of all non-natural nucleotides and carrier components. do. Another potential safety issue may arise from the presence of extracellular RNA during RNA expression cassette multiparticulate administration. Extracellular naked RNA has been shown to increase the permeability of tightly bound endothelial cells and may therefore contribute to edema. Another study showed that extracellular RNA promotes blood coagulation and pathological thrombus formation. Therefore, safety needs to be continuously evaluated until other RNA expression cassette multiparticulate methods and delivery systems are utilized in humans.

Since the first report in 1978 detailing proof-of-concept delivery of liposome-encapsulated exogenous mRNA to mouse lymphocytes for functional protein expression, 206 mRNA therapeutics have been published. Various therapeutic applications of mRNA, including protein replacement, gene editing, and vaccination, are currently being investigated by academia and commercial entities. To date, clinical efforts have been mainly focused on vaccine therapy, where mRNA therapeutics have many advantages over conventional strategies. However, mRNA also has strong potential as a vehicle for local and systemic protein replacement therapy. Finally, mRNA has paved the way for nonviral genome-editing therapies by investigating the potential for in vitro and in vivo delivery of genome-editing tools such as ZFNs and CRISPR-Cas nucleases. The widespread application of mRNA is still limited by the need for improved delivery systems. However, we believe that the continued advancement of mRNA nanoformulations using a variety of different materials will ultimately lead to the use of RNA expression cassettes for the treatment of a wide range of diseases.

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 Preparation of RNA Expression Cassettes

The RNA expression cassette of the present invention proposes an RNA having a structure capable of protein synthesis in a cell. The target protein of the RNA expression cassette used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins). In particular, an RNA expression cassette capable of stably expressing a variety of proteins including the target protein, in which the expression cassette is capable of linear nuclear-independent cytoplasmic expression, was prepared as follows.

1-1. Preparation of RNA expression cassettes

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

<Design and manufacture of target protein RNA expression cassette>

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

The target protein DNA of the present invention 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) sequence. The target protein DNA structure encoding the target protein RNA expression cassette may be <T7 promoter - 5'UTR - target protein DNA - 3'UTR muag - A64 - C30 - histone-SL>. where 5'UTR is hAg-Kozak. 3'UTR is any one selected from the group consisting of muag-A64-C30-histone-SL and 2BgUTR-A30LA70. If the target protein is a fusion protein, the linker is (G4S)3-linker and. Sec can be used for the purpose of secreting a target protein to the outside of the cell.

The base sequence constituting the target protein RNA expression cassette backbone DNA or amino acid sequences T7 promoter TAATACGACTCACTATAGGG (SEQ ID NO: 6) hAg-Kozak ATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 7) Sec ATGAGAGTGACCGCCCCCAGAACCCTGATCCTGCTGCTGTCTGGCGCCCTGGCCCTGACAGAGACATGGGCCGGAAGCGGATCC (SEQ ID NO: 8) (M R V T A P R T L I L L S G A L A L T E T W A G S G S; SEQ ID NO: 9) muag - A64 - C30 - Histone S GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCGAGATTA ATAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAATGCA TCCCCCCCCC CCCCCCCCCC CCCCCCCCCC CCAAAGGCTC TTTTCAGAGC CACCA (SEQ ID NO:10) 2 BgUTR Column CCGAGAGCTCGUCGGCCAACAAAAGGTTCUTGCCCAAGTCCAACACAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTACATTGCTGCGTCGAGAGCTCGCTTCTTGCTGTCCAATTTCTATTAAAGGTGTCCTTTGTTCCCTAAGCATTCCAACTATTCAAACTGGGGGATATGAATTGAATTG A30LA70 GAGACCTGGTCCAGAGTCGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 12) (G4S)3-linker GGAGGCGGTGGTAGTGGAGGTGGCGGGTCCGGTGGAGGTGGAAGC (SEQ ID NO: 13) (G G G G S G G G G S G G G G S; SEQ ID NO: 14)

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

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

Kozak consensus sequence ACCAUGG (SEQ ID NO: 15).

<Figure 3> 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.

wherein 3'UTR is the sequence 5'-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3' (SEQ ID NO: 16; muag; underlined nucleotides are mutated compared to wild-type sequence) (alpha-) globin mutated UTR, poly (A) The tail is 64 adenine nucleotides, the poly(C) sequence is 30 cytosine nucleotides, and the histone stem-loop is an RNA sequence consisting of (5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' SEQ ID NO: 17).

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA (SEQ ID NO: 18)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 19)

In the present invention, the target protein DNA fragment was synthesized as <T7 promoter - 5'UTR - target protein DNA - muag - A64 - C30 - histone-SL> (Biosynthesis, USA), and the forward primer 5'- GAATTC was used as a template. Amplification was performed using TAAT ACGACTCACT ATAGGGGAGA CCACAACGGT TTCCCTCTAG AAAGAACC AT GAACACGATT AACA -3' (SEQ ID NO: 20) and reverse primer 5'- GGATCC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 21). 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 transformed into E. coli, and the plasmid was separated from the selected strain, treated with EcoRI/BamHI restriction enzymes, and then electrophoresed (FIG. 4).

The target protein used in the present invention is hyaluronidase, ECM degradation-related protein including MMP and TIMP, therapeutic protein including EPO and G-CSF, rabies virus G protein (RAV-G) and RSV long ( long) a vaccine-related protein including the RSV-F protein of the strain was used, but is not limited thereto.

hyaluronidase

Hyaluronidase (enzyme that degrades HA) includes HYAL1 (NM_153281), HYAL2 (NM_033158), HYAL3 (NM_003549), HYAL4 (NM_012269) and Hyaluronidase PH20 (SPAM1) (NM_001174044), sPH20, and the like.

In the present invention, the enzyme sequence was removed from the sequence corresponding to the carboxy terminal membrane-binding domain to make a soluble enzyme. Upon removal of this carboxy terminal domain, hyaluronidase is secreted into the ECM. Thus, it expresses a secreted hyaluronidase that exhibits enzymatic activity at neutral pH.

<FIG. 5> shows the amino acid sequence (SEQ ID NO: 22) of the PH20 protein and a hydropathic plot according to Kyte-Doolittle's algorithm. Protein PH20 is a membrane protein present in the plasma membrane and hull membrane of the intestine. (a) The amino acid sequence is a hydrophobic sequence (sequence underlined) responsible for the anchorage of the intramembrane protein. In the present invention, the PH20 protein expressed by the virus exhibits a missing hydrophobic tail. Cut points are marked in circles. Due to this deletion, the protein PH20 is released into the extracellular medium. (b) Hydropathic plot of 100 terminal amino acids of the PH20 protein by Kyte-Doolittle. Arrows indicate the beginning of the removed hydrophobicity.

The cDNA corresponding to PH20 is SEQ ID NO:23. SEQ ID NO: 23 is sPH20 in a secreted form as a coding nucleotide sequence for the protein PH20 (isotype, GenBank access number NP_694859.1) from the initiation codon (ATG) to the 1467 position. The nucleotide sequences 1468 - 1527 of the GenBank sequence for PH20 (NM_001174044) encode the hydrophobic tail of the protein, which anchors the protein to the membrane. The sPH20 cDNA sequence was a nucleotide sequence in which the nucleotide sequence corresponding to the hydrophobic tail was deleted, followed by nucleotide 1468 and the translation termination codon TAA was added.

For the preparation of the RNA expression cassette of the present invention, the nucleotide sequences of the selected Hyaluronidase PH20 (SPAM1) (NM_001174044), sPH20 and HYAL1 (NM_153281) are as follows.

Hyaluronidase PH20 (SPAM1) (NM_001174044)

ATGGGAGTGCTAAAATTCAAGCACATCTTTTTCAGAAGCTTTGTTAAATCAAGTGGAGTATCCCAGATAGTTTTCACCTTCCTTCTGATTCCATGTTGCTTGACTCTGAATTTCAGAGCACCTCCTGTTATTCCAAATGTGCCTTTCCTCTGGGCCTGGAATGCCCCAAGTGAATTTTGTCTTGGAAAATTTGATGAGCCACTAGATATGAGCCTCTTCTCTTTCATAGGAAGCCCCCGAATAAACGCCACCGGGCAAGGTGTTACAATATTTTATGTTGATAGACTTGGCTACTATCCTTACATAGATTCAATCACAGGAGTAACTGTGAATGGAGGAATCCCCCAGAAGATTTCCTTACAAGACCATCTGGACAAAGCTAAGAAAGACATTACATTTTATATGCCAGTAGACAATTTGGGAATGGCTGTTATTGACTGGGAAGAATGGAGACCCACTTGGGCAAGAAACTGGAAACCTAAAGATGTTTACAAGAATAGGTCTATTGAATTGGTTCAGCAACAAAATGTACAACTTAGTCTCACAGAGGCCACTGAGAAAGCAAAACAAGAATTTGAAAAGGCAGGGAAGGATTTCCTGGTAGAGACTATAAAATTGGGAAAATTACTTCGGCCAAATCACTTGTGGGGTTATTATCTTTTTCCGGATTGTTACAACCATCACTATAAGAAACCCGGTTACAATGGAAGTTGCTTCAATGTAGAAATAAAAAGAAATGATGATCTCAGCTGGTTGTGGAATGAAAGCACTGCTCTTTACCCATCCATTTATTTGAACACTCAGCAGTCTCCTGTAGCTGCTACACTCTATGTGCGCAATCGAGTTCGGGAAGCCATCAGAGTTTCCAAAATACCTGATGCAAAAAGTCCACTTCCGGTTTTTGCATATACCCGCATAGTTTTTACTGATCAAGTTTTGAAATTCCTTTCTCAAGATGAACTTGTGTATACATTTGGCGAAACTGTTGCTCTGGGTGCTT CTGGAATTGTAATATGGGGAACCCTCAGTATAATGCGAAGTATGAAATCTTGCTTGCTCCTAGACAATTACATGGAGACTATACTGAATCCTTACATAATCAACGTCACACTAGCAGCCAAAATGTGTAGCCAAGTGCTTTGCCAGGAGCAAGGAGTGTGTATAAGGAAAAACTGGAATTCAAGTGACTATCTTCACCTCAACCCAGATAATTTTGCTATTCAACTTGAGAAAGGTGGAAAGTTCACAGTACGTGGAAAACCGACACTTGAAGACCTGGAGCAATTTTCTGAAAAATTTTATTGCAGCTGTTATAGCACCTTGAGTTGTAAGGAGAAAGCTGATGTAAAAGACACTGATGCTGTTGATGTGTGTATTGCTGATGGTGTCTGTATAGATGCTTTTCTAAAACCTCCCATGGAGACAGAAGAACCTCAAATTTTCTACAATGCTTCACCCTCCACACTATCTGCCACAATGTTCATTGTTAGTATTTTGTTTCTTATCATTTCTTCTGTAGCGAGTTTG (서열번호 23)

sPH20

It is a nucleotide sequence from the transcription start (ATG) to position 1467 of the PH20 cDNA that lacks the hydrophobic tail (nucleotides 1468-1527) of the PH20 protein and is a protein secreted by cells in the form of a nucleotide sequence with a stop codon (TAA) added at the end. .

atgggagtgc taaaattcaa gcacatcttt ttcagaagct ttgttaaatc aagtggagta 60

tcccagatag ttttcacctt ccttctgatt ccatgttgct tgactctgaa tttcagagca 120

cctcctgtta ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180

cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga 240

ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg ctactatcct 300

tacatagatt caatcacagg agtaactgtg aatggaggaa tcccccagaa gatttcctta 360

caagaccatc tggacaaagc taagaaagac attacatttt atatgccagt agacaatttg 420

ggaatggctg ttattgactg ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480

aaagatgttt acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540

ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg 600

gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg ttattatctt 660

tttccggatt gttacaacca tcactataag aaacccggtt acaatggaag ttgcttcaat 720

gtagaaataa aaagaaatga tgatctcagc tggttgtgga atgaaagcac tgctctttac 780

ccatccattt atttgaacac tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840

cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900

tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960

cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt aatatgggga 1020

accctcagta taatgcgaag tatgaaatct tgcttgctcc tagacaatta catggagact 1080

atactgaatc cttacataat caacgtcaca ctagcagcca aaatgtgtag ccaagtgctt 1140

tgccaggagc aaggagtgtg tataaggaaa aactggaatt caagtgacta tcttcacctc 1200

aacccagata attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260

ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc 1320

ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt gtgtattgct 1380

gatggtgtct gtatagatgc ttttctaaaa cctcccatgg agacagaaga acctcaaatt 1440

ttctacaatg cttcaccctc cacactatct taa 1473 (SEQ ID NO: 24)

HYAL1 (NM_153281)

ATGGCAGCCCACCTGCTTCCCATCTGCGCCCTCTTCCTGACCTTACTCGATATGGCCCAAGGCTTTAGGGGCCCCTTGCTACCCAACCGGCCCTTCACCACCGTCTGGAATGCAAACACCCAGTGGTGCCTGGAGAGGCACGGTGTGGACGTGGATGTCAGTGTCTTCGATGTGGTAGCCAACCCAGGGCAGACCTTCCGCGGCCCTGACATGACAATTTTCTATAGCTCCCAGCTGGGCACCTACCCCTACTACACGCCCACTGGGGAGCCTGTGTTTGGTGGTCTGCCCCAGAATGCCAGCCTGATTGCCCACCTGGCCCGCACATTCCAGGACATCCTGGCTGCCATACCTGCTCCTGACTTCTCAGGGCTGGCAGTCATCGACTGGGAGGCATGGCGCCCACGCTGGGCCTTCAACTGGGACACCAAGGACATTTACCGGCAGCGCTCACGGGCACTGGTACAGGCACAGCACCCTGATTGGCCAGCTCCTCAGGTGGAGGCAGTAGCCCAGGACCAGTTCCAGGGAGCTGCACGGGCCTGGATGGCAGGCACCCTCCAGCTGGGGCGGGCACTGCGTCCTCGCGGCCTCTGGGGCTTCTATGGCTTCCCTGACTGCTACAACTATGACTTTCTAAGCCCCAACTACACCGGCCAGTGCCCATCAGGCATCCGTGCCCAAAATGACCAGCTAGGGTGGCTGTGGGGCCAGAGCCGTGCCCTCTATCCCAGCATCTACATGCCCGCAGTGCTGGAGGGCACAGGGAAGTCACAGATGTATGTGCAACACCGTGTGGCCGAGGCATTCCGTGTGGCTGTGGCTGCTGGTGACCCCAATCTGCCGGTGCTGCCCTATGTCCAGATCTTCTATGACACGACAAACCACTTTCTGCCCCTGGATGAGCTGGAGCACAGCCTGGGGGAGAGTGCGGCCCAGGGGGCAGCTGGAGTGGTGCTCTGGGTGAGCTGGGAAAATACAAGAACCAAGGAATCATGTC AGGCCATCAAGGAGTATATGGACACTACACTGGGGCCCTTCATCCTGAACGTGACCAGTGGGGCCCTTCTCTGCAGTCAAGCCCTGTGCTCCGGCCATGGCCGCTGTGTCCGCCGCACCAGCCACCCCAAAGCCCTCCTCCTCCTTAACCCTGCCAGTTTCTCCATCCAGCTCACGCCTGGTGGTGGGCCCCTGAGCCTGCGGGGTGCCCTCTCACTTGAAGATCAGGCACAGATGGCTGTGGAGTTCAAATGTCGATGCTACCCTGGCTGGCAGGCACCGTGGTGTGAGCGGAAGAGCATGTGG (서열번호 25)

MMP can be classified as shown in <Table 1>, collagenase (MMP-1, MMP-8, MMP-13 and MMP-18) gelatinase (MMP-2 and MMP-9), stromelysin ( MMP-3, MMP-10, MMP-11 and MMP-17), matrilysins (MMP-7 and MMP-26), membrane types (MMP-14, MMP-15, MMP-16, MMP-24 and MMP-25) ) and other types (MMP-12, MMP-19, MMP-20, MMP-21, MMP-22, MMP-28, MMP-29), etc.

For the preparation of the MMP RNA expression cassette of the present invention, the nucleotide sequences of MMP1 (NM_002421) and MMP9 (NM_004994) selected from various types of MMP are as follows.

MMP1 (NM_002421)

ATGCACAGCTTTCCTCCACTGCTGCTGCTGCTGTTCTGGGGTGTGGTGTCTCACAGCTTCCCAGCGACTCTAGAAACACAAGAGCAAGATGTGGACTTAGTCCAGAAATACCTGGAAAAATACTACAACCTGAAGAATGATGGGAGGCAAGTTGAAAAGCGGAGAAATAGTGGCCCAGTGGTTGAAAAATTGAAGCAAATGCAGGAATTCTTTGGGCTGAAAGTGACTGGGAAACCAGATGCTGAAACCCTGAAGGTGATGAAGCAGCCCAGATGTGGAGTGCCTGATGTGGCTCAGTTTGTCCTCACTGAGGGGAACCCTCGCTGGGAGCAAACACATCTGACCTACAGGATTGAAAATTACACGCCAGATTTGCCAAGAGCAGATGTGGACCATGCCATTGAGAAAGCCTTCCAACTCTGGAGTAATGTCACACCTCTGACATTCACCAAGGTCTCTGAGGGTCAAGCAGACATCATGATATCTTTTGTCAGGGGAGATCATCGGGACAACTCTCCTTTTGATGGACCTGGAGGAAATCTTGCTCATGCTTTTCAACCAGGCCCAGGTATTGGAGGGGATGCTCATTTTGATGAAGATGAAAGGTGGACCAACAATTTCAGAGAGTACAACTTACATCGTGTTGCGGCTCATGAACTCGGCCATTCTCTTGGACTCTCCCATTCTACTGATATCGGGGCTTTGATGTACCCTAGCTACACCTTCAGTGGTGATGTTCAGCTAGCTCAGGATGACATTGATGGCATCCAAGCCATATATGGACGTTCCCAAAATCCTGTCCAGCCCATCGGCCCACAAACCCCAAAAGCGTGTGACAGTAAGCTAACCTTTGATGCTATAACTACGATTCGGGGAGAAGTGATGTTCTTTAAAGACAGATTCTACATGCGCACAAATCCCTTCTACCCGGAAGTTGAGCTCAATTTCATTTCTGTTTTCTGGCCACAACTGCCAAATGGGCTTGAAGCTGCTTACGAAT TTGCCGACAGAGATGAAGTCCGGTTTTTCAAAGGGAATAAGTACTGGGCTGTTCAGGGACAGAATGTGCTACACGGATACCCCAAGGACATCTACAGCTCCTTTGGCTTCCCTAGAACTGTGAAGCATATCGATGCTGCTCTTTCTGAGGAAAACACTGGAAAAACCTACTTCTTTGTTGCTAACAAATACTGGAGGTATGATGAATATAAACGATCTATGGATCCAGGTTATCCCAAAATGATAGCACATGACTTTCCTGGAATTGGCCACAAAGTTGATGCAGTTTTCATGAAAGATGGATTTTTCTATTTCTTTCATGGAACAAGACAATACAAATTTGATCCTAAAACGAAGAGAATTTTGACTCTCCAGAAAGCTAATAGCTGGTTCAACTGCAGGAAAAAT (서열번호 26)

MMP9 (NM_004994)

ATGAGCCTCTGGCAGCCCCTGGTCCTGGTGCTCCTGGTGCTGGGCTGCTGCTTTGCTGCCCCCAGACAGCGCCAGTCCACCCTTGTGCTCTTCCCTGGAGACCTGAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTACACTCGGGTGGCAGAGATGCGTGGAGAGTCGAAATCTCTGGGGCCTGCGCTGCTGCTTCTCCAGAAGCAACTGTCCCTGCCCGAGACCGGTGAGCTGGATAGCGCCACGCTGAAGGCCATGCGAACCCCACGGTGCGGGGTCCCAGACCTGGGCAGATTCCAAACCTTTGAGGGCGACCTCAAGTGGCACCACCACAACATCACCTATTGGATCCAAAACTACTCGGAAGACTTGCCGCGGGCGGTGATTGACGACGCCTTTGCCCGCGCCTTCGCACTGTGGAGCGCGGTGACGCCGCTCACCTTCACTCGCGTGTACAGCCGGGACGCAGACATCGTCATCCAGTTTGGTGTCGCGGAGCACGGAGACGGGTATCCCTTCGACGGGAAGGACGGGCTCCTGGCACACGCCTTTCCTCCTGGCCCCGGCATTCAGGGAGACGCCCATTTCGACGATGACGAGTTGTGGTCCCTGGGCAAGGGCGTCGTGGTTCCAACTCGGTTTGGAAACGCAGATGGCGCGGCCTGCCACTTCCCCTTCATCTTCGAGGGCCGCTCCTACTCTGCCTGCACCACCGACGGTCGCTCCGACGGCTTGCCCTGGTGCAGTACCACGGCCAACTACGACACCGACGACCGGTTTGGCTTCTGCCCCAGCGAGAGACTCTACACCCGGGACGGCAATGCTGATGGGAAACCCTGCCAGTTTCCATTCATCTTCCAAGGCCAATCCTACTCCGCCTGCACCACGGACGGTCGCTCCGACGGCTACCGCTGGTGCGCCACCACCGCCAACTACGACCGGGACAAGCTCTTCGGCTTCTGCCCGACCCGAGCTG ACTCGACGGTGATGGGGGGCAACTCGGCGGGGGAGCTGTGCGTCTTCCCCTTCACTTTCCTGGGTAAGGAGTACTCGACCTGTACCAGCGAGGGCCGCGGAGATGGGCGCCTCTGGTGCGCTACCACCTCGAACTTTGACAGCGACAAGAAGTGGGGCTTCTGCCCGGACCAAGGATACAGTTTGTTCCTCGTGGCGGCGCATGAGTTCGGCCACGCGCTGGGCTTAGATCATTCCTCAGTGCCGGAGGCGCTCATGTACCCTATGTACCGCTTCACTGAGGGGCCCCCCTTGCATAAGGACGACGTGAATGGCATCCGGCACCTCTATGGTCCTCGCCCTGAACCTGAGCCACGGCCTCCAACCACCACCACACCGCAGCCCACGGCTCCCCCGACGGTCTGCCCCACCGGACCCCCCACTGTCCACCCCTCAGAGCGCCCCACAGCTGGCCCCACAGGTCCCCCCTCAGCTGGCCCCACAGGTCCCCCCACTGCTGGCCCTTCTACGGCCACTACTGTGCCTTTGAGTCCGGTGGACGATGCCTGCAACGTGAACATCTTCGACGCCATCGCGGAGATTGGGAACCAGCTGTATTTGTTCAAGGATGGGAAGTACTGGCGATTCTCTGAGGGCAGGGGGAGCCGGCCGCAGGGCCCCTTCCTTATCGCCGACAAGTGGCCCGCGCTGCCCCGCAAGCTGGACTCGGTCTTTGAGGAGCCGCTCTCCAAGAAGCTTTTCTTCTTCTCTGGGCGCCAGGTGTGGGTGTACACAGGCGCGTCGGTGCTGGGCCCGAGGCGTCTGGACAAGCTGGGCCTGGGAGCCGACGTGGCCCAGGTGACCGGGGCCCTCCGGAGTGGCAGGGGGAAGATGCTGCTGTTCAGCGGGCGGCGCCTCTGGAGGTTCGACGTGAAGGCGCAGATGGTGGATCCCCGGAGCGCCAGCGAGGTGGACCGGATGTTCCCCGGGGTGCCTTTGGACACGCACGACGTCTTCCAGTA CCGAGAGAAAGCCTATTTCTGCCAGGACCGCTTCTACCGGCGCGTGAGTTCCCGGAGTGAGTTGAACCAGGTGGACCAAGTGGGCTACGTGACCTATGACATCCTGCAGTGCCCTGAGGAC (SEQ ID NO: 27)

Tissue inhibitors of metalloproteinases (MPs) (TIMPs), four members of the TIMP-1, 2, 3 and 4 family, are endogenous inhibitors of originally secreted metalloproteinases (MPs).

<TIMP>

For the preparation of the TIMP RNA expression cassette of the present invention, the nucleotide sequences of TIMP1 (NM_003254), TIMP2 (NM_003255), TIMP3 (NM_000362) and TIMP4 (NM_003256) are as follows.

TIMP1 (NM_003254)

ATGGCCCCCTTTGAGCCCCTGGCTTCTGGCATCCTGTTGTTGCTGTGGCTGATAGCCCCCAGCAGGGCCTGCACCTGTGTCCCACCCCACCCACAGACGGCCTTCTGCAATTCCGACCTCGTCATCAGGGCCAAGTTCGTGGGGACACCAGAAGTCAACCAGACCACCTTATACCAGCGTTATGAGATCAAGATGACCAAGATGTATAAAGGGTTCCAAGCCTTAGGGGATGCCGCTGACATCCGGTTCGTCTACACCCCCGCCATGGAGAGTGTCTGCGGATACTTCCACAGGTCCCACAACCGCAGCGAGGAGTTTCTCATTGCTGGAAAACTGCAGGATGGACTCTTGCACATCACTACCTGCAGTTTCGTGGCTCCCTGGAACAGCCTGAGCTTAGCTCAGCGCCGGGGCTTCACCAAGACCTACACTGTTGGCTGTGAGGAATGCACAGTGTTTCCCTGTTTATCCATCCCCTGCAAACTGCAGAGTGGCACTCATTGCTTGTGGACGGACCAGCTCCTCCAAGGCTCTGAAAAGGGCTTCCAGTCCCGTCACCTTGCCTGCCTGCCTCGGGAGCCAGGGCTGTGCACCTGGCAGTCCCTGCGGTCCCAGATAGCC (서열번호 28)

TIMP2 (NM_003255)

ATGGGCGCCGCGGCCCGCACCCTGCGGCTGGCGCTCGGCCTCCTGCTGCTGGCGACGCTGCTTCGCCCGGCCGACGCCTGCAGCTGCTCCCCGGTGCACCCGCAACAGGCGTTTTGCAATGCAGATGTAGTGATCAGGGCCAAAGCGGTCAGTGAGAAGGAAGTGGACTCTGGAAACGACATTTATGGCAACCCTATCAAGAGGATCCAGTATGAGATCAAGCAGATAAAGATGTTCAAAGGGCCTGAGAAGGATATAGAGTTTATCTACACGGCCCCCTCCTCGGCAGTGTGTGGGGTCTCGCTGGACGTTGGAGGAAAGAAGGAATATCTCATTGCAGGAAAGGCCGAGGGGGACGGCAAGATGCACATCACCCTCTGTGACTTCATCGTGCCCTGGGACACCCTGAGCACCACCCAGAAGAAGAGCCTGAACCACAGGTACCAGATGGGCTGCGAGTGCAAGATCACGCGCTGCCCCATGATCCCGTGCTACATCTCCTCCCCGGACGAGTGCCTCTGGATGGACTGGGTCACAGAGAAGAACATCAACGGGCACCAGGCCAAGTTCTTCGCCTGCATCAAGAGAAGTGACGGCTCCTGTGCGTGGTACCGCGGCGCGGCGCCCCCCAAGCAGGAGTTTCTCGACATCGAGGACCCA (서열번호 29)

TIMP3 (NM_000362)

ATGACCCCTTGGCTCGGGCTCATCGTGCTCCTGGGCAGCTGGAGCCTGGGGGACTGGGGCGCCGAGGCGTGCACATGCTCGCCCAGCCACCCCCAGGACGCCTTCTGCAACTCCGACATCGTGATCCGGGCCAAGGTGGTGGGGAAGAAGCTGGTAAAGGAGGGGCCCTTCGGCACGCTGGTCTACACCATCAAGCAGATGAAGATGTACCGAGGCTTCACCAAGATGCCCCATGTGCAGTACATCCATACGGAAGCTTCCGAGAGTCTCTGTGGCCTTAAGCTGGAGGTCAACAAGTACCAGTACCTGCTGACAGGTCGCGTCTATGATGGCAAGATGTACACGGGGCTGTGCAACTTCGTGGAGAGGTGGGACCAGCTCACCCTCTCCCAGCGCAAGGGGCTGAACTATCGGTATCACCTGGGTTGTAACTGCAAGATCAAGTCCTGCTACTACCTGCCTTGCTTTGTGACTTCCAAGAACGAGTGTCTCTGGACCGACATGCTCTCCAATTTCGGTTACCCTGGCTACCAGTCCAAACACTACGCCTGCATCCGGCAGAAGGGCGGCTACTGCAGCTGGTACCGAGGATGGGCCCCCCCGGATAAAAGCATCATCAATGCCACAGACCCC (서열번호 30)

TIMP4 (NM_003256)

ATGCCTGGGAGCCCTCGGCCCGCGCCAAGCTGGGTGCTGTTGCTGCGGCTGCTGGCGTTGCTGCGGCCCCCGGGGCTGGGTGAGGCATGCAGCTGCGCCCCGGCGCACCCTCAGCAGCACATCTGCCACTCGGCACTTGTGATTCGGGCCAAAATCTCCAGTGAGAAGGTAGTTCCGGCCAGTGCAGACCCTGCTGACACTGAAAAAATGCTCCGGTATGAAATCAAACAGATAAAGATGTTCAAAGGGTTTGAGAAAGTCAAGGATGTTCAGTATATCTATACGCCTTTTGACTCTTCCCTCTGTGGTGTGAAACTAGAAGCCAACAGCCAGAAGCAGTATCTCTTGACTGGTCAGGTCCTCAGTGATGGAAAAGTCTTCATCCATCTGTGCAACTACATCGAGCCCTGGGAGGACCTGTCCTTGGTGCAGAGGGAAAGTCTGAATCATCACTACCATCTGAACTGTGGCTGCCAAATCACCACCTGCTACACAGTACCCTGTACCATCTCGGCCCCTAACGAGTGCCTCTGGACAGACTGGCTGTTGGAACGAAAGCTCTATGGTTACCAGGCTCAGCATTATGTCTGTATGAAGCATGTTGACGGCACCTGCAGCTGGTACCGGGGCCACCTGCCTCTCAGGAAGGAGTTTGTTGACATCGTTCAGCCC (서열번호 31)

<Therapeutic protein>

EPO

For the preparation of the EPO RNA expression cassette of the present invention, the nucleotide sequence of murine EPO is as follows.

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACCAUGGGCG UGCCCGAGCG GCCGACCCUG 60

CUCCUGCUGC UCAGCCUGCU GCUCAUCCCC CUGGGGCUGC CCGUCCUCUG CGCCCCCCCG 120

CGCCUGAUCU GCGACUCCCG GGUGCUGGAG CGCUACAUCC UCGAGGCCAA GGAGGCGGAG 180

AACGUGACCA UGGGCUGCGC CGAGGGGCCC CGGCUGAGCG AGAACAUCAC GGUCCCCGAC 240

ACCAAGGUGA ACUUCUACGC CUGGAAGCGC AUGGAGGUGG AGGAGCAGGC CAUCGAGGUC 300

UGGCAGGGCC UGUCCCUCCU GAGCGAGGCC AUCCUGCAGG CGCAGGCCCU CCUGGCCAAC 360

UCCAGCCAGC CCCCGGAGAC ACUGCAGCUC CACAUCGACA AGGCCAUCUC CGGGCUGCGG 420

AGCCUGACCU CCCUCCUGCG CGUGCUGGGC GCGCAGAAGG AGCUCAUGAG CCCGCCCGAC 480

ACGACCCCCC CGGCCCCGCU GCGGACCCUG ACCGUGGACA CGUUCUGCAA GCUCUUCCGC 540

GUCUACGCCA ACUUCCUGCG GGGCAAGCUG AAGCUCUACA CCGGGGAGGU GUGCCGCCGG 600

GGCGACCGCU GACCACUAGU GCAUCACAUU UAAAAGCAUC UCAGCCUACC AUGAGAAUAA 660

GAGAAAGAAA AUGAAGAUCA AUAGCUUAUU CAUCUCUUUU UCUUUUUCGU UGGUGUAAAG 720

CCAACACCCU GUCUAAAAAA CAAUAAAUUUC UUUAAUCAUU UUGCCUCUUU UCUCUGUGCU 780

UCAAUUAAUA AAAAAUGGAA AGAACCUAGA UCUAAAAAAA AAAAAAAAAA AAAAAAAAAA 840

AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAUGC AUCCCCCCCC CCCCCCCCCC 900

CCCCCCCCCC CCCAAAGGCU CUUUUCAGAG CCACCAGAAU U 941 (SEQ ID NO: 32)

GM CSF

For the preparation of the GM CSF RNA expression cassette of the present invention, the nucleotide sequence of GM CSF (CSF2) (NM_000758) is as follows.

GM CSF (CSF2) (NM_000758)

ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAG (서열번호 33)

<Vaccination protein>

G protein of rabies virus (RAV-G)

For the preparation of the RAV-G RNA expression cassette of the present invention, the base sequence of the rabies virus G protein (RAV-G) is as follows.

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACCAUGGU GCCCCAGGCC CUGCUCUUCG 60

UCCCGCUGCU GGUGUUCCCC CUCUGCUUCG GCAAGUUCCC CAUCUACACC AUCCCCGACA 120

AGCUGGGGCC GUGGAGCCCC AUCGACAUCC ACCACCUGUC CUGCCCCAAC AACCUCGUGG 180

UCGAGGACGA GGGCUGCACC AACCUGAGCG GGUUCUCCUA CAUGGAGCUG AAGGUGGGCU 240

ACAUCAGCGC CAUCAAGAUG AACGGGUUCA CGUGCACCGG CGUGGUCACC GAGGCGGAGA 300

CCUACACGAA CUUCGUGGGC UACGUGACCA CCACCUUCAA GCGGAAGCAC UUCCGCCCCA 360

CGCCGGACGC CUGCCGGGCC GCCUACAACU GGAAGAUGGC CGGGGACCCC CGCUACGAGG 420

AGUCCCUCCA CAACCCCUAC CCCGACUACC ACUGGCUGCG GACCGUCAAG ACCACCAAGG 480

AGAGCCUGGU GAUCAUCUCC CCGAGCGUGG CGGACCUCGA CCCCUACGAC CGCUCCCUGC 540

ACAGCCGGGU CUUCCCCGGC GGGAACUGCU CCGGCGUGGC CGUGAGCUCC ACGUACUGCA 600

GCACCAACCA CGACUACACC AUCUGGAUGC CCGAGAACCC GCGCCUGGGG AUGUCCUGCG 660

ACAUCUUCAC CAACAGCCGG GGCAAGCGCG CCUCCAAGGG CAGCGAGACG UGCGGGUUCG 720

UCGACGAGCG GGGCCUCUAC AAGUCCCUGA AGGGGGCCUG CAAGCUGAAG CUCUGCGGCG 780

UGCUGGGCCU GCGCCUCAUG GACGGGACCU GGGUGGCGAU GCAGACCAGC AACGAGACCA 840

AGUGGUGCCC CCCCGGCCAG CUGGUCAACC UGCACGACUU CCGGAGCGAC GAGAUCGAGC 900

ACCUCGUGGU GGAGGAGCUG GUCAAGAAGC GCGAGGAGUG CCUGGACGCC CUCGAGUCCA 960

UCAUGACGAC CAAGAGCGUG UCCUUCCGGC GCCUGAGCCA CCUGCGGAAG CUCGUGCCCG 1020

GGUUCGGCAA GGCCUACACC AUCUUCAACA AGACCCUGAU GGAGGCCGAC GCCCACUACA 1080

AGUCCGUCCG CACGUGGAAC GAGAUCAUCC CGAGCAAGGG GUGCCUGCGG GUGGGCGGCC 1140

GCUGCCACCC CCACGUCAAC GGGGUGUUCU UCAACGGCAU CAUCCUCGGG CCCGACGGCA 1200

ACGUGCUGAU CCCCGAGAUG CAGUCCAGCC UGCUCCAGCA GCACAUGGAG CUGCUGGUCU 1260

CCAGCGUGAU CCCGCUCAUG CACCCCCUGG CGGACCCCUC CACCGUGUUC AAGAACGGGG 1320

ACGAGGCCGA GGACUUCGUC GAGGUGCACC UGCCCGACGU GCACGAGCGG AUCAGCGGCG 1380

UCGACCUCGG CCUGCCGAAC UGGGGGAAGU ACGUGCUGCU CUCCGCCGGC GCCCUGACCG 1440

CCCUGAUGCU GAUCAUCUUC CUCAUGACCU GCUGGCGCCG GGUGAACCGG AGCGAGCCCA 1500

CGCAGCACAA CCUGCGCGGG ACCGGCCGGG AGGUCUCCGU GACCCCGCAG AGCGGGAAGA 1560

UCAUCUCCAG CUGGGAGUCC UACAAGAGCG GCGGCGAGAC CGGGCUGUGA GGACUAGUUA 1620

UAAGACUGAC UAGCCCGAUG GGCCUCCCAA CGGGCCCUCC UCCCCUCCUU GCACCGAGAU 1680

UAAUAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1740

AAAAAAAAUG CAUCCCCCCC CCCCCCCCCC CCCCCCCCCC CCCCAAAGGC UCUUUUCAGA 1820

GCCACCAGAA UU 1832 (SEQ ID NO: 34)

RSV-F protein of RSV long strain

For the preparation of the RSV-F RNA expression cassette of the present invention, the base sequence of the RSV-F protein of the respiratory syncytial virus (RSV) long strain is as follows.

RSV-F long (GC)

GGGAGACCAC AACGGUUUCC CUCUAGAAAG AACCAUGGAGC UGCCCAUCCU CAAGGCCAAC 60

GCCAUCACCA CCAUCCUGGC GGCCGUGACG UUCUGCUUCG CCAGCUCCCA GAACAUCACC 120

GAGGAGUUCU ACCAGAGCAC CUGCUCCGCC GUCAGCAAGG GCUACCUGUC CGCCCUCCGG 180

ACCGGGUGGU ACACGAGCGU GAUCACCAUC GAGCUGUCCA ACAUCAAGGA GAACAAGUGC 240

AACGGCACCG ACGCGAAGGU GAAGCUGAUC AACCAGGAGC UCGACAAGUA CAAGAACGCC 300

GUCACCGAGC UGCAGCUGCU CAUGCAGAGC ACGACCGCCG CCAACAACCG CGCGCGGCGC 360

GAGCUGCCGC GGUUCAUGAA CUACACCCUG AACAACACCA AGAAGACGAA CGUGACCCUC 420

UCCAAGAAGC GCAAGCGGCG CUUCCUGGGG UUCCUGCUCG GCGUGGGGAG CGCCAUCGCC 480

UCCGGCAUCG CCGUCAGCAA GGUGCUGCAC CUGGAGGGCG AGGUGAACAA GAUCAAGUCC 540

GCCCUCCUGA GCACCAACAA GGCGGUCGUG UCCCUGAGCA ACGGGGUGUC CGUCCUCACC 600

AGCAAGGUGC UGGACCUGAA GAACUACAUC GACAAGCAGC UCCUGCCCAU CGUGAACAAG 660

CAGUCCUGCC GGAUCAGCAA CAUCGAGACG GUCAUCGAGU UCCAGCAGAA GAACAACCGC 720

CUGCUCGAGA UCACCCGGGA GUUCAGCGUG AACGCCGGCG UGACCACCCC CGUCUCCACG 780

UACAUGCUGA CCAACAGCGA GCUGCUCUCC CUGAUCAACG ACAUGCCCAU CACCAACGAC 840

CAGAAGAAGC UGAUGAGCAA CAACGUGCAG AUCGUGCGCC AGCAGUCCUA CAGCAUCAUG 900

UCCAUCAUCA AGGAGGAGGU CCUCGCCUAC GUGGUGCAGC UGCCGCUGUA CGGGGUCAUC 960

GACACCCCCU GCUGGAAGCU CCACACGAGC CCCCUGUGCA CCACCAACAC CAAGGAGGGC 1020

UCCAACAUCU GCCUGACGCG GACCGACCGC GGGUGGUACU GCGACAACGC CGGCAGCGUG 1080

UCCUUCUUCC CCCAGGCCGA GACCUGCAAG GUCCAGAGCA ACCGGGUGUU CUGCGACACC 1140

AUGAACUCCC UCACGCUGCC GAGCGAGGUG AACCUGUGCA ACGUCGACAU CUUCAACCCC 1200

AAGUACGACU GCAAGAUCAU GACCUCCAAG ACCGACGUGA GCUCCAGCGU GAUCACCUCC 1260

CUCGGCGCGA UCGUCAGCUG CUACGGGAAG ACGAAGUGCA CCGCCAGCAA CAAGAACCGC 1320

GGCAUCAUCA AGACCUUCUC CAACGGGUGC GACUACGUGA GCAACAAGGG CGUGGACACC 1380

GUCUCCGUGG GCAACACCCU GUACUACGUG AACAAGCAGG AGGGGAAGAG CCUGUACGUC 1440

AAGGGCGAGC CCAUCAUCAA CUUCUACGAC CCCCUCGUGU UCCCGUCCGA CGAGUUCGAC 1500

GCCAGCAUCU CCCAGGUGAA CGAGAAGAUC AACCAGAGCC UGGCCUUCAU CCGGAAGUCC 1560

GACGAGCUGC UGCACCACGU CAACGCCGGG AAGAGCACGA CCAACAUCAU GAUCACCACC 1620

AUCAUCAUCG UGAUCAUCGU GAUCCUCCUG UCCCUGAUCG CGGUCGGCCU CCUGCUGUAC 1680

UGCAAGGCCC GCAGCACGCC CGUGACCCUC UCCAAGGACC AGCUGAGCGG GAUCAACAAC 1740

AUCGCCUUCU CCAACUGAGG ACUAGUGCAU CACAUUUAAA AGCAUCUCAG CCUACCAUGA 1800

GAAUAAGAGA AAGAAAAUGA AGAUCAAUAG CUUAUUCAUC UCUUUUUCUU UUUCGUUGGU 1860

GUAAAGCCAA CACCCUGUCU AAAAAACAUA AAUUUCUUUA AUCAUUUUGC CUCUUUUCUC 1920

UGUGCUUCAA UUAAUAAAAA AUGGAAAGAA CCUAGAUCUA AAAAAAAAAA AAAAAAAAAA 1980

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAUGCAUCC CCCCCCCCCC 2040

CCCCCCCCCC CCCCCCCCCA AAGGCUCUUU UCAGAGCCAC CAGAAUU 2087 (SEQ ID NO: 35)

1.2. Preparation of target protein RNA expression cassette

The target protein of the RNA expression cassette used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

In the present invention, an RNA expression cassette including a target protein mRNA containing a capped chemically modified nucleotide was prepared.

In order to synthesize the capped target protein mRNA, first, the prepared target protein pUC19 vector having a T7 promoter-target protein DNA 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. 6).

Using the T7 promoter-target protein DNA template, the target protein DNA PCR product having the T7 promoter prepared above as a template, the highYield T7 ARCA mRNA Synthiis Kit (m5CTP/Ψ-UTP) (Jena BioScience, USA) was used to cap the target protein. A target protein 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 target protein mRNA was purified by 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 target protein vector was performed using the HiScribe ^™ T7 ARCA mRNA Kit (NEB, USA).

Addition and Stability of Cholesterol

7, the prepared capped target protein mRNA (Native RNA), the capped target protein mRNA (modified RNA), and the capped target protein mRNA (modified RNA-choliterol) with choliterol added to the 3' end In vitro stability was confirmed by RT-PCR after processing the RNA prepared in the cell culture medium for various times. The prepared capped target protein mRNA had very superior in vitro safety than the capped target protein mRNA, and the capped target protein mRNA (modified RNA-choliterol) with choliterol added to the 3' end had the highest stability.

For the capped target protein mRNA, a native cap (m7G(5')ppp(5')G; m7GpppG) may be added to the IVT product with the ScriptCap m7G capping system (Epicentre Biotechnologii, USA).

The MDRNA expression cassette prepared above and the prepared Apt EGFR-chol or oligo-chol were designed to hybridize so that a nucleotide sequence capable of complementary binding was present in each.

After mixing 15 pmole of mRNA and 15 pmole of oligo-chol, nuclease free water was added to make a total of 5 ul, and then heated at 95°C for 5 minutes, followed by hybridization at 25°C for 1 hour. The hybridization ratio was 1:1, 1:5, and 1:10 in the molar ratio of RNA expression cassette and oligo-chol, and as a result of retardation assay using 2% agarose gel, it was confirmed as a single band when hybridized at 1:1. .

The stability of the various RNA expression cassette oligo-chol hybrids prepared above was investigated in the serum, and samples were collected over time by processing the various RNA expression cassette oligo-chol hybrids prepared in the serum solution, and the remaining hybrids were analyzed.

7, in the case of the existing mRNA, as a result of the serum stability, it was gradually degraded after 24 hours, and only about 30% of the mRNA remained at 72 hours, but in the case of RNA1-SN38/MMAE/oligo-chol, it was about 40 by 10 days. It was confirmed that the percent remained, and 50 percent of MDrRNA1/Apt EGFR-chol remained until day 5, and about 50 percent of RNA1-SN38/MMAE/Apt EGFR-chol remained until day 5, confirming that the mRNA was more stable than that of mRNA.

Addition of video material

The prepared Rhodamine Labeled Oligo and FITC Labeled Oligo (Biosynthesis, USA) were made to order (Biosynthesis, USA), and the prepared RNA expression cassette and the fluorescent material-labeled oligo were at a constant ratio (W/W, 1:1). ~0.1), it can be used to prepare multiparticulates with Rhodamine B or FITC.

Example 2. RNA expression cassette/PS basic multiparticulate (10)

The target protein of the RNA expression cassette used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

A particle prepared by self-assembly by mixing the selected RNA expression cassette and a cationic polypeptide was referred to as a basic composite particle (10).

The selected RNA expression cassette contains n nucleotides, resulting in n-1 negative charges per molecule or 1.8×10 ²⁵ negative charges per mole. The PS has a molecular weight of 5.8 kDa and is rich in basic amino acids, particularly arginine (70-80%), so that the cation per mole is 1.26 × 10 ²⁵ . PS neutralizes the negative charge of the phosphate group of the RNA expression cassette. A charge ratio between the selected RNA expression cassette and PS was 1:0.4 or more, that is, the RNA expression cassette was 1 and the PS was 0.4 or more to prepare an RNA expression cassette basic composite particle.

RNA expression cassette/PS particle

The size of the basic composite particles prepared in Example 7 was determined by photon correlation spectroscopy (PCS) using a Malvern Zetasizer 3000 HSA (Herrenberg, Germany). The basic composite particle size was expressed as the mean hydrodynamic diameter (dh), and the width of the size distribution was expressed as the polydispersity index (PDI). Software data analysis for particle size distribution calculation was based on the Fitting method of non-negative constrained least-squares (NNLS). Measurement was performed after 1 hour of incubation of the mixture to which the components were added.

According to the research results of the present invention, in the RNA expression cassette-based basic composite particle of the present invention, the weight ratio of the RNA expression cassette and PS (Protamine) (㎍/mL) is 30:15 to 150, preferably 30:15 to 90 , more preferably in distilled water (DW) under the condition of 30:75, possessing the properties of an ideal drug carrier.

When PS was added to the RNA expression cassette solution, an interaction between the cationic PS and the RNA expression cassette occurred, and the mixture of PS and RNA expression cassette also spontaneously formed particles.

As a result of PCS (Photon correlation spectroscopy) measured with a Malvern Zetasizer 3000 HSA (Herrenberg, Germany), an initial complex of RNA expression cassette and RNA expression cassette was formed in the first step, and self-assembly when a strongly negatively charged RNA expression cassette was added. to form an RNA expression cassette/PS particle.

In the present invention, particles were prepared by adding an RNA expression cassette to an aqueous solution composed of HSA and PS, and this was referred to as RNA expression cassette/PS particle-DW.

These results were not limited to selected RNA expression cassettes, but were observed in general with other RNA expression cassettes as well.

Effect of PS on particle size

Referring to FIG. 8 , the RNA expression cassette particle-DW was a very small particle at a low PS concentration, but a large size distribution was observed (RNA expression cassette: PS=30:15). Basic multiparticulate aggregation was observed by increasing the PS concentration from an RNA expression cassette:PS (μg/ml) ratio above about 30:45 mass ratio. Multiparticulates with a small size distribution and most suitable size can be prepared at a PS concentration in which the RNA expression cassette:PS (μg/ml) ratio is higher than 30:60 mass ratio. On the other hand, RNA expression cassette/PS particle- showed a large size distribution (PDI ≤ 0.2) and a dh of about 650 nm at low PS concentration. At PS concentrations where the RNA expression cassette:PS ratio is higher than about 30:60 mass ratio, the RNA expression cassette/PS/HSA particle-sample provides comparable dh and size distribution data to the RNA expression cassette/PS/HSA particle-DW sample. indicated.

As a result of studying the effect of PS concentration on particle size, in order to prepare an RNA expression cassette/PS basic composite particle having a stable small monomodal size distribution, the RNA expression cassette:PS ratio was at least 30:60 by mass ratio of 60 μg/ml. A minimum PS concentration is ideal.

These results were not limited to selected mRNAs, but were generally observed for other RNA expression cassettes as well.

suture and release

Preparation of RNA expression cassette/PS particle-DW mixes positively charged PS and negatively charged RNA expression cassette to form particles according to electrostatic interaction. The formed RNA expression cassette/PS particles were incubated at room temperature for 30 minutes for stabilization. As a solvent for mixing the PS and the RNA expression cassette, a DEPC-treated RNase-free solvent was used to prevent degradation of the RNA expression cassette.

Confirmation of binding of the RNA expression cassette to PS was measured by performing electrophoresis on a 2% agarose gel. The RNA expression cassette was stained with 0.5 μg/ml Etbr (ethidium bromide), and electrophoresis was performed for 20 minutes using 1x TBE buffer under 80 Volt conditions.

In order to confirm the result of encapsulation of the RNA expression cassette by PS, the degree of movement of the band in the electrophoresis image of the RNA expression cassette/PS particle prepared according to the mass ratio of the RNA expression cassette and PS was confirmed. The RNA expression cassette/PS (μg/ml) ratios required to adsorb the RNA expression cassette are 30:15, 30:30, 30:60, 30:75, 30:90 and 30:150 mixing the RNA expression cassette with PS. After centrifugation, the supernatant was irradiated by electrophoresis.

Referring to <FIG. 8>, 82% unbound RNA expression cassette was found at a mass ratio of 30:15 in the RNA expression cassette/PS (㎍/ml) ratio of the basic multiparticulate. At a mass ratio of 30:30, 40% RNA expression cassette remained in solution. In the 30:75, 30:90 and 30:150 mass ratios, the RNA expression cassette is quantitatively present in particle form. It can be judged that, under the influence of positively charged PS, as the amount of PS added increased, it reacted strongly with the RNA expression cassette to form a more stable particle form.

Referring to <Fig. 8>, particle stability and RNA expression cassette release were investigated in PBS at 37°C. After 24 hours, RNA expression cassette release did not occur in the conditions of the RNA expression cassette:PS ratio of 30:90 and 30:150. The 30:75 condition showed a small release of the RNA expression cassette, and 8.0% of the unbound RNA expression cassette was found. The RNA expression cassette of the basic multiparticulate: PS (μg/ml) ratio 30:15 mass ratio ratio 90.0%, and RNA expression cassette of 63.0% at 30:30 mass ratio were found in solution.

These results were not limited to selected RNA expression cassettes, but were observed in general with other RNA expression cassettes as well.

Example 3. RNA expression cassette/PS/HSA multiparticulate (20)

RNA expression cassette/PS/HSA multiparticulate

The target protein of the RNA expression cassette used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

The RNA expression cassette / PS / HSA complex particle of this example used an RNA expression cassette encoding a protein for a therapeutic protein and a protein for a vaccine, but is not limited thereto.

The selected RNA expression cassette, cationic protein protamine (PS) and anionic protein human serum albumin (HSA) solutions were each prepared in distilled water (DW) at a concentration of 2.5 mg/ml.

In another embodiment, the selected RNA expression cassette, PS and HSA isotonic solution was mixed with isotonic RNA expression cassette solution (RNA expression cassette 140 mM), PBS buffer (RNA expression cassette 136.9 mM, KCl 2.68 mM, Na2HPO4 8.1 mM, KH2PO4 1.47 mM) ; pH 7.4) or phenol red-free RPMI 1640 cell medium () at a concentration of 2.5 mg/ml, respectively.

0-2000 μg of HSA was added to 15-150 μg of PS and mixed for 5 seconds. Thereafter, the 30 μg RNA expression cassette prepared above was added to reach a final volume of 1 ml in distilled water, and vortexed for 5 seconds to prepare particles.

0-2000 μg of HSA was added to 15-150 μg of PS and mixed. Then, 30 μg RNA expression cassette solution prepared above was added to a final volume of 1 ml of RNA expression cassette solution, PBS buffer, or Ethyl, and the mixture was vortexed for 5 seconds to prepare particles.

Here, the RNA expression cassette may be any one type selected from the above various types or a mixture of a plurality of types.

RNA expression cassette/PS-TCA/HSA multiparticulate

A stock solution was prepared by dissolving PS-TCA prepared with reference to the present inventor's patent application (Korean Patent Application: 10-2019-0007873) at 2.5 mg/mL in distilled water or the isotonic solution.

0∼2000 μg of anionic protein HSA was added to 15∼150 μg of PS-TCA, and then mixed for 5 seconds. Then, the 30 μg RNA expression cassette prepared above was added to reach a final volume of 1 ml in distilled water, and vortexed for 5 seconds.

Here, the RNA expression cassette may be any one type selected from the above various types or a mixture of a plurality of types.

Then, the reaction was left at room temperature for 1 hour and lyophilized for 2 days to obtain RNA expression cassette/PS-TCA particles prepared.

RNA expression cassette/CS/HSA or RNA expression cassette/CS-TCA/HSA multiparticulate

CS-TCA or CS (Chitosan) prepared with reference to the present inventor's patent application (Korean Patent Application: 10-2019-0007873) was dissolved in distilled water or the isotonic solution at 2.5 mg/mL and used as a stock solution.

0-2000 μg of HSA was added to 15-150 μg of CS or CS-TCA and mixed for 5 seconds. Then, the 30 μg RNA expression cassette prepared above was added to reach a final volume of 1 ml in sterile distilled water, and vortexed for 5 seconds.

Here, the RNA expression cassette may be any one type selected from the above various types or a mixture of a plurality of types.

Then, the RNA expression cassette/CS prepared by leaving the reaction at room temperature for 1 hour and lyophilizing for 2 days or RNA expression cassette/CS-TCA particles were obtained.

Effect of HSA on particle size

The RNA expression cassette/PS/HSA particle-DW forms small particles in the presence of a small amount of HSA, but as the HSA increases, the particles aggregate into a large structure.

Referring to FIG. 9, RNA expression cassette/PS/HSA particle-DW is 30 μg RNA expression cassette, 90 μg PS, 100-500 μg HSA is added to 1 ml of distilled water and then mixed, dh is 250 with PDI ≤ 0.1 It was in the range of ∼280 nm. In RNA expression cassette/PS/HSA particle-DW, when HSA of 1000∼2000 μg was increased, dh increased and PDI became 0.2 or more (red bar graph). These results indicate that the high concentration of HSA negatively affects the manufacturing process in DW, thus forming less stable particles.

Referring to Figure 9, the RNA expression cassette / PS / HSA basic multiparticulates are isotonic RNA expression cassette solution (RNA expression cassette 140 mM), isotonic PBS buffer (mRNAl 136.9 mM, KCl 2.68 mM, Na2HPO4 8. 1 mM, KH2PO4 1.47 mM; pH 7.4) and phenol red free cell medium RPMI 1640 isotonic solution () aggregates to large particles. An increase in HSA added to RNA expression cassette/PS/HSA particles with 30 μg mRNA and 90 μg PS in 1 ml isotonic solution improved stability. The dh of these particles decreases from 1-2 mm to 250-400 nm depending on the isotonic solution used.

Particles with dh = 245 nm and PDI ≤ 0.15 are formed in phenol red-free cell medium RPMI 1640 isotonic (). This was referred to as RNA expression cassette/PS/HSA particle-DW.

The optimized concentration of HSA was 100 μg/ml in RNA expression cassette/PS/HSA particle-DW and 1000 μg/ml in RNA expression cassette/PS/HSA particle-, and the effect of PS was evaluated in the range of 15 to 150 μg.

These results were not limited to selected RNA expression cassettes, but were observed in general with other RNA expression cassettes as well.

Example 4. RNA expression cassette/PS/HA multiparticulate (20)

The target protein of the RNA expression cassette used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

RNA expression cassette/PS/HSA complex particles are extracellular matrix degrading enzymes (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), extracellular matrix degrading enzyme inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP) 4) an RNA expression cassette encoding was used, but is not limited thereto.

Particles formed by self-assembly by mixing the basic composite particles with anionic polysaccharides or particles formed by ultrasonic spraying while mixing mRNA, cationic polypeptides and anionic polysaccharides *?**?*Composite particles (20)* referred to as ?**?*.

RNA expression cassette/PS/HA multiparticulate

For the preparation of the RNA expression cassette multiparticulate or RNA expression cassette/PS/HA multiparticulate, the anionic polysaccharide hyaluronic acid is used for surface modification of the previously prepared RNA expression cassette basic composite particle or RNA expression cassette/PS basic composite particle again. Ronic acid (Hyaluronic acid, HA, 83 kDa) was dissolved in sterile distilled water at 1 mg/mL to prepare a 0.1% stock solution.

The RNA expression cassette: PS (μg/ml) ratio was prepared at a mass ratio of 30:75 and the lyophilized RNA expression cassette/PS basic composite particle was dissolved in sterile distilled water at 1 mg/mL to 0.1% RNA expression cassette/PS basic composite particle solution was prepared.

After mixing the prepared 0.1% [RNA expression cassette/PS] basic composite particle solution and 0.1% HA solution at 1:0.5, 1:1, 1:1.5 or 1:2 feed mole ratio (nmol) Incubated for about 5 minutes and freeze-dried to obtain RNA expression cassette/PS/HA multiparticulate-DW.

Alternatively, the preparation of the RNA expression cassette multiparticulate or RNA expression cassette/PS/HA-DW multiparticulate in another method is the addition molar ratio (feed) of the RNA expression cassette/PS]:HA or [RNA expression cassette/PS]:HA mole ratio, nmol) is adjusted to the particle size for about 5 minutes after mixing the RNA expression cassette, PS and HA according to the compound and HA addition molar ratio 1:0.5, 1:1, 1:1.5 or 1:2 For this purpose, a sonicator or an extruder may be used as appropriate. By freeze-drying, RNA expression cassette/PS/HA multiparticulate-DW was obtained.

RNA expression cassette/PS/HA-TCA multiparticulate

For the preparation of the RNA expression cassette/PS/HA-TCA composite particle, the RNA expression cassette: PS (㎍/ml) ratio prepared earlier was prepared at a mass ratio of 30:75, and the frozen RNA expression cassette/PS basic composite particle-DW was dissolved in sterile distilled water at 1 mg/mL to prepare a 0.1% RNA expression cassette/PS basic multiparticulate solution.

The HA-TCA conjugate prepared with reference to the present inventor's patent application (Korean Patent Application: 10-2019-0007873) is obtained by sterilizing the HA-TCA conjugate prepared under the condition that the addition molar ratio of HA and TCA is 1:10 in sterile distilled water 1mg/ mL to prepare a 0.1% HA-TCA aqueous solution.

Add to the 0.1% RNA expression cassette/PS basic complex particle solution and 0.1% HA-TCA aqueous solution at a molar ratio of 1:0.5, 1:1, 1:1.5 or 1:2 under gentle vortex and incubate at room temperature for 30 minutes , to obtain RNA expression cassette/PS/HA-TCA multiparticulate-DW by lyophilization.

Alternatively, in the preparation of the RNA expression cassette/PS/HA-TCA multiparticulate-DW by another method, HA-TCA was used instead of HA in the method of the RNA expression cassette/PS/HA.

Example 5. RNA expression cassette/PS/HA-aptamer conjugate multiparticulate (20)

Aptamer-HA conjugates were prepared by a coupling reaction between the carboxyl group of HA and the amine group of the aptamer using EDC/NHS chemistry. Briefly, 83 kDa HA (1 g, 20 μmol) was dissolved in 200 mL of degassed deionized water under nitrogen for 5 h. EDC (100.4 mg, 0.52 mmol) and NHS (60.7 mg, 0.52 mmol) were added to the HA solution. After 20 minutes, an aptamer-amine group (99.4 mg, 0.52 mmol) was added slowly. The pH was adjusted to 5.5 with 0.1 M HCl and the solution was reacted at room temperature overnight. Unreacted residues and by-products were removed with a dialysis membrane (MW cut off = 100 kDa) in distilled water (pH 5). The final product was lyophilized and stored at -20 °C.

HA-DAB-aptamer synthesis

The HA-aptamer conjugate was developed as a HA-DAB-aptamer conjugate. Specifically, in the method of synthesizing HA-DAB (1,4-diaminobutane), 1-ethyl-3-[3-(dimethylamino)-propyl ] After activation with carbodiimine (1-ethyl-3- [3- (dimethylamino)-propyl] carbodiimide, EDC) and 1-hydroxy bentriazole monohydrate (HOBt), 1,4 - HA-DAB was synthesized by adding diaminobutane (1,4-diaminobutane, DAB).

At this time, the reaction time was varied to 1 hour, 6 hours, and 24 hours to obtain compounds having different substitution rates. HA-DAB was prepared by filtration purification of the compound obtained as described above using a filtration tube (cut off 100 kDa).

The HA-DAB-aptamer conjugate was synthesized as an HA-aptamer conjugate into which a disulfide bond was introduced. Specifically, the synthesized HA-DAB (20 mg; 4.8 x 10 ^-5 moles) and succinimidyl 3-(2-pyridyldithio) propionate (Succinimidyl 3-(2-pyridyldithio) propionate; SPDP) (5.6 mg; 1.8 x 10 ^-5 mol) was mixed and reacted to introduce a pyridyl group to the HA chain, followed by filtration and purification using a filtration tube (MWCO 100kDa). Purified HA-DAB-SPDP was treated with TCEP (tris(2-carboxyethyl)phosphine) and absorbance was measured at 343 nm to calculate the concentration of the pyridyl group (pyrimidyl group concentration: 34 mM).

The EGFR aptamer used in this Example (Tan Y et al., Acta Pharmacologica Sinica (2013) 34: 1491-1498) is a nucleotide sequence having SEQ ID NO: 37 at the N30 region of SEQ ID NO: 36

5'-GCAATGGTAC GGTACTTCC- (N30)-CAAAAG TGCACGCTAC TTTGCTAA-3' (SEQ ID NO: 36)

5'-TGAATGTTGT TTTTTCTCTT TTCTATAGTA-3' (SEQ ID NO: 37)

The EGFR aptamer was organically synthesized as a single-stranded DNA structure in which a thiol group was introduced at the 3' end (Biosynthesis, USA).

HA containing disulfide bonds by adding an EGFR aptamer having a thiol group at the 3' end to the quantified HA-DAB-SPDP solution in various ratios from 0.2 to 1 times the concentration of the pyrimidyl group. -DAB-aptamer conjugate was synthesized. At this time, the synthesized HA-DAB-aptamer can control the number of conjugated aptamers per 100 kDa HA molecule from 2 to 15.

Identification of HA-DAB-aptamer conjugates

In order to confirm the generation of the HA-DAB-aptamer conjugate synthesized in Example <1-2>, high performance liquid chromatography (HPLC) was used. For the HPLC conditions, a superdex 200 10/300 GL (GE health) column was used, the mobile phase was pH 7.0 50 mM phosphate buffer, the flow rate was 0.5 ml/min, and the absorbance was measured at 210 nm and 260 nm. . The results are described in FIG. 9A, and the compound was separated and purified based on the results. As can be seen from FIG. 9A , it was confirmed that the HA-DAB-aptamer conjugate production rate (reaction rate) was about 64%.

The conjugate production rate (reaction rate) was calculated as follows:

Reaction rate (%) = {(level of aptamer required to form the conjugate)/(level of aptamer added at the beginning of the reaction) }X100

In addition, it was confirmed whether the separated and purified compound was synthesized through agarose gel electrophoresis and whether it was decomposed by a reducing agent (TCEP), and the results are shown in FIGS. 5A to 5D .

10A to 10D are results of analysis of HA-DAB-aptamer conjugates synthesized with two different cross linkers through agarose electrophoresis. Specifically, in FIG. 5a , HA-ss-aptamer means a conjugate including a disulfide bond (-ss-) between HA and aptamer, and HA-aptamer is a conjugate connected by a general covalent bond rather than a disulfide bond. (control) means. As shown in the two columns on the right, when the reducing agent, TCEP, is treated, the aptamer is separated from the conjugate containing the disulfide bond and a band appears at the bottom, whereas in the control group, the aptamer is not separated and remains at the top. .

In addition, FIGS. 10b to 10d show the amount of remaining aptamer through agarose electrophoresis by time by adding FBS to the HA-DAB-aptamer conjugate to evaluate the safety of the HA-DAB-aptamer conjugate (Fig. 5b and c, HA-aptamer conjugate means HA-DAB-aptamer conjugate). As a result, it can be confirmed that the HA-DAB-aptamer conjugate remains undecomposed for a long time compared to the HA-DAB-aptamer unconjugated aptamer (naked aptamer).

Preparation of RNA expression cassette/PS/HA-aptamer conjugate multiparticulates

The RNA expression cassette: PS (μg/ml) ratio was prepared at a mass ratio of 30:75 and the lyophilized RNA expression cassette/PS basic composite particle was dissolved in sterile distilled water at 1 mg/mL to 0.1% RNA expression cassette/PS basic composite particle solution was prepared.

The prepared 0.1% [RNA expression cassette/PS] basic multiparticulate solution and 0.1% HA-aptamer solution were added in a molar ratio of 1:0.5, 1:1, 1:1.5 or 1:2 (feed mole ratio, nmol). After mixing, incubated for about 5 minutes and freeze-dried to obtain RNA expression cassette/PS/HA-aptamer composite particle-DW.

Alternatively, the preparation of the RNA expression cassette multiparticulate or RNA expression cassette/PS/HA-aptamer DW multiparticle of another method is the RNA expression cassette/PS]:HA-aptamer or [RNA expression cassette/PS]:HA -Aptamer addition molar ratio (feed mole ratio, nmol) is based on the compound particle and HA-aptamer addition molar ratio 1:0.5, 1:1, 1:1.5 or 1:2 RNA expression cassette, PS and HA-pressure After mixing the tamer, a sonicator or an extruder may be appropriately used to adjust the size of the particles for about 5 minutes. By freeze-drying, RNA expression cassette/PS/HA-aptamer multiparticulate-DW was obtained.

Example 6. Evaluation of RNA expression cassette/PS/HA multiparticulate (20)

Morphological analysis

The size of the composite particles prepared in Example 4 was determined by photon correlation spectroscopy (PCS) using a Malvern Zetasizer 3000 HSA (Herrenberg, Germany). The particle size was expressed as a mean hydrodynamic diameter (dh), and the width of the size distribution was expressed as a polydispersity index (PDI). Measurement was performed after 1 hour of incubation of the mixture to which the components were added.

The results of examining the size distribution of RNA expression cassettes/PS:HA multiparticulates prepared at various ratios are shown in FIG. 11 and <Table 5>. These results show that the ratio of RNA expression cassette/PS:HA is 1:0.5 and 1:1 when the size of the RNA expression cassette PS/HA complex particle is in the range of 200 nm.

Zetasizer analysis result of RNA expression cassette/PS/HA nanoparticles prepared under various conditions addition molar ratio
(feed mole ratio, nmol) RNA:PS One One One One HA One 1.5 2 2.5 RNA expression cassettePS/HA size(nm) 184.8±7.1 182.3±14.1 493.2±22.6 1259±147 PDI 0.30±0.07 0.34±0.04 0.30±0.02 0.35±0.09 complex efficiency
(% vs RNA) 80.9 71.7 100 100

The RNA expression cassette/PS/HA complex particle or RNA expression cassette/PS/HA complex particle was prepared in a molar ratio of RNA expression cassette/PS];HA addition of 1:1.5, centrifuged, lyophilized, and 5% w/ It was rehydrated with w trehalose solution and the particle size before and after rehydration was investigated.

Looking at <Table 6>, there was little change in particle size before and after rehydration by freeze-drying, and the particle size increased by about 3 to 4% in PBS than in distilled water (DW).

Zetasizer analysis result by rehydration after freeze-drying RNA expression cassette/PS/HA complex particles RNA expression cassette/PS/HA Size(nm) 202.0±30.4 PDI 0.15±0.22 Lyophilization & reconstitution DW PBS Size(nm) 209.7±5.4 224.5±19.7 PDI 0.12±0.06 0.17±0.1 CE(%) 96.4 100

These results were not limited to selected RNA expression cassettes, but were observed in general with other RNA expression cassettes as well.

Multiparticulate Drug Encapsulation and Release Profiles

The preparation of the RNA expression cassette / PS / HA complex particle is a process in which negatively charged HA is coated on the RNA expression cassette / PS basic composite particle formed by electrostatic interaction by mixing positively charged PS and negatively charged RNA expression cassette. . As a solvent for mixing the RNA expression cassette, PS and HA, a DEPC-treated RNase-free solvent was used to prevent degradation of the RNA expression cassette. The RNA expression cassette/PS:HA feed mole ratio (nmol) required to adsorb the RNA expression cassette is 1:0.5, 1:1, 1:1.5 or 1:2 by mixing the RNA expression cassette/PS and HA. After preparation, centrifugation was performed to investigate.

To confirm the RNA expression cassette / PS / HA complex particle, the reaction mixture prepared under various conditions was electrophoresed on 2% agarose gel using 1x TBE buffer at 80 Volt conditions for 20 minutes, and then the RNA expression cassette was 0.5 μg / It was stained with ml of Etbr (ethidium bromide).

Referring to Figure 12, As a result of electrophoresis of the reaction mixture to prepare the RNA expression cassette/PS/HA multiparticle, the RNA expression cassette/PS/HA multiparticle remained in the wells of the 2% agarose gel, and only the unbound RNA expression cassette moved the gel. could see that The stability of the RNA expression cassette/PS/HA particles and the release of the RNA expression cassette were investigated in PBS at 37°C. After 24 hours, 82% unbound RNA expression cassettes were found at a feed mole ratio (nmol) of 1:0.5 [RNA expression cassette]/PS]:HA. At a mass ratio of 1:1, 40% RNA expression cassette remained in solution. At 1:1.5 and 1:2 addition molar ratios, the RNA expression cassette is quantitatively present in the form of particles. The stability of the RNA expression cassette/PS/HA particles and the release of the RNA expression cassette were investigated in PBS at 37°C. After 24 hours, almost no mRNA release occurred at the [RNA expression cassette/PS]:HA addition molar ratio 1:1.5 and 1:2 conditions. [RNA expression cassette/PS]:HA addition molar ratio of 1:0.5 to 90.0% and 1:1 to 63.0% mRNA was found in the solution. The 1:1.5 condition showed a small release of the RNA expression cassette, and 8.0% of the unbound RNA expression cassette was found.

It can be determined that, under the influence of positively charged HA, as the amount of HA added increases, it reacts strongly with the RNA expression cassette to form a more stable particle form.

The drug release profile over time was investigated at the [RNA expression cassette/PS]:HA addition molar ratio of the multiparticulate 1:1.5 mass ratio.

Looking at <Table 7>, the RNA expression cassette/PS/HA composite particle released 100% of the level at which the RNA expression cassette accumulated for 120 hours in PBS solution was sealed, and 50% release was observed between 72 and 96 hours. did

Release profile of RNA expression cassettes over time from RNA expression cassettes/PS/HA multiparticulates Incubation time (hours) 0 4 8 24 48 72 96 120 Band density (%) 20.0 27.9 25.5 36.0 48.0 42.7 41.2 50.2 Release of RNA
(%, normalization) 0 7.9 5.5 17.0 33.8 43.6 65.3 104.9

Example 7. Biological evaluation of RNA expression cassette/PS/HSA multiparticulate 1

<Investigation of G-CSF protein change in G-CSF RNA expression cassette>

293T cells (ATCC CRL-3216™, Thermo Fisher, USA) are grown in DMEM + 2 mM Glutamine + 10% Fetal Bovine Serum (FBS) medium to cover >70% of the culture vessel. 293T cells are transfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of [G-CSF RNA expression cassette:PS]/HA multiparticle.

The secreted huG-CSF concentration in the culture medium is measured in triplicate at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each RNA expression cassette. The secretion of human granulocyte-colony stimulating factor (G-CSF) from transfected 293T cells was quantified using the G-CSF Human Instant ELISA™ Kit (Invitrogen, USA).

<Investigation of EPO protein change in EPO RNA expression cassette>

[EPO of the present invention After transfection of RNA expression cassette:PS]/HA multiparticulates into 293T cells as described in Example 3, respectively, the level of EPO protein expression was analyzed at 0, 24, 48 and 72 hours.

Total protein was isolated at 0, 24, 48 and 72 hours after treatment in the transformant 293T cells by the control group and the unconstitutional group, and the EPO level was determined by ELISA (mouse EPO QuAntikine ELISA kit, R&D Systems), and the The result is shown in <FIG. 13>. The experiment was performed 5 times and the average value was indicated.

<Investigation of RSV-F protein change in RSV-F RNA expression cassette>

After transfection of the [RSV-F RNA expression cassette:PS]/HA multiparticle of the present invention into 293T cells, respectively, the level of target protein expression was analyzed at 0, 24, 48 and 72 hours.

RSV-F synthesis was investigated in cells transformed with [RSV-F RNA expression cassette:PS]/HA multiparticle by ELISA using an anti-RSV-F antibody (ab94968, abcam, USA). 293T cells were treated and the amount of RSV-F strain was measured after 0.24.48 and 72 hours had elapsed. [RSV-F RNA expression cassette: PS] / HA multiparticulates were treated (final concentration of 50 nM mRNA), and after the same time elapsed, ELISA was performed to measure the amount of modification of RSV-F. The experiment was performed 5 times and the average value was indicated.

<Investigation of RAV-G protein change in RAV-G RNA expression cassette>

[RAV-G RNA expression cassette: PS] / HA complex particles of the present invention were incubated with 293T cells for 6 hours, washed, and incubated by adding fresh medium. After incubation, the target protein at 0, 24, 48 and 72 hours The level of expression was analyzed.

RSV-F synthesis was investigated in cells transformed with [RAV-G RNA expression cassette:PS]/HA multiparticulates using the Human Anti-Respiratory syncytial virus IgG ELISA Kit (RSV) (ab108765). 293T cells were treated and the amount of RSV-F strain was measured after 0.24.48 and 72 hours had elapsed. [RAV-G RNA expression cassette: PS] / HA multiparticulates were treated (final concentration 50 nM mRNA) and after the same time elapsed, ELISA was performed to measure the amount of modification of RSV-F. The experiment was performed 5 times and the average value was indicated.

Example 8. Biological evaluation of RNA expression cassette/PS/HA multiparticulate (20) 2

HYAL 1, Hyaluronidase PH20, sPH20

PC3 cell line (ATCC　 CRL-1435™, Thermo Fisher, USA) in F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) + 2mM Glutamine + 10% Fetal Bovine Serum (FBS) in >70% culture medium Incubated until covered. After washing with fresh medium, 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of medium containing PH20 RNA expression cassette/PS/HA complex particles was added, incubated for 6 hours and fresh It was washed with medium and re-cultured in fresh medium. The secreted PH20 concentration in the culture medium is measured in triplicate at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each RNA expression cassette. The secretion of PH20 from the transfected PC3 cell line cells was quantified using the PH20 ELISA™ Kit (Invitrogen, USA) ( FIG. 14 ).

Particle exclusion analysis was performed to visualize the matrix forming around HA producing cell lines in vitro ( FIG. 15 ).

HA-rich human prostate tumor cell line (PC3) and HA-deficient human lung cell line (NCI H460) in F-12K Medium + 2 mM Glutamine + 10% Fetal Bovine Serum (FBS) medium to cover >70% of the culture vessel incubated until After washing the culture medium with fresh medium, 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of a medium containing PH20 RNA expression cassette/PS/HA complex particles was added and at 37° C. Incubated for 6 hours and washed. Then, 0.5 mg/mL of bovine aggrecan (Sigma-Aldrich) (t = 0) was added and incubated together for 1, 2, 8, 16, 24 and 48 hours. Thereafter, the medium was removed and replaced with a suspension with 2% glutaraldehyde fixed 10 ⁹ /mL mouse RBCs in PBS (pH 7.4). Cells were imaged with a phase-contrast microscope coupled with a camera scanner and imaging program (Diagnostic Instruments, Inc.).

In addition, to determine the duration of HA clearance in PC3 cultures after transient exposure of cells to the PH20 RNA expression cassette/PS/HA multiparticulate, followed by a “tracking” period in which the cells were incubated with cell medium in the absence of the enzyme, this A time course study was performed using the assay. As above, the “HALO per cell area” value was determined by measuring the average HALO area (pixels or mm 2 ) divided by the average cell area (pixels or mm 2 ). HALO per cell area before treatment (zero time) was calculated, and the value was set to 100%. For each of the six treated time points, cells were incubated for 1 h in the presence of PH20 RNA expression cassette/PS/HA multiparticulates, followed by a wash period to remove the enzyme and a follow-up period in which the cells were incubated without enzyme. come

(for a total of 2, 4, 8, 16, 24 and 48 hours, respectively). For each time point, HALO per cell area was calculated and the relative HALO fraction was determined compared to the time zero value of 100%. The results are shown in Table 15 below. Numbers listed represent the mean + or - standard error of the mean (SEM) of n = 45 measurements.

MMP: MMP1, MMP 8, MMP 13

Purchase Iscove's Modified Dulbecco's Medium (IMDM), fetal bovine serum (abbreviated as FBS) from Gibco, and non-essential amino acids, sodium bicarbonate, sodium pyrbate, and phosphate buffered saline (abbreviated as PBS) purchased

Human skin fibroblast CCD-986Sk (ATCC　 CRL-1947™) was prepared from 10% FBS, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyrbate under the conditions of 37°C and 5% carbon dioxide. was cultured in IMDM medium (hereinafter abbreviated as cell medium).

Grow human dermal fibroblasts in IMDM medium to cover >70% of the culture vessel. Human skin fibroblasts are transfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of [MMP RNA expression cassette:PS]/HA multiparticulate.

Secreted MMP concentrations in the culture medium are measured in triplicate at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each RNA expression cassette. The secretion of MMP from transfected 293T cells was quantified using the MMP Human Instant ELISA™ Kit (Invitrogen, USA) ( FIG. 16 ).

2ml of cell culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ and then cultured at 37° C., 5% CO 2 environment for 24 hours. Thereafter, the medium containing the MMP1 RNA expression cassette: PS]/HA complex particles (1, 10, and 100 μg/ml, respectively) prepared in Example 1 was replaced and cultured for 3 days.

Then, the culture medium was harvested and centrifuged for 5 minutes at 4 ° C., 7500 rpm environment. Using the ELISA method, the protein expression level of type 1 procollagen (Procollagen Type I C Peptide EIA Kit, Takara, Shiga, Japan) was confirmed (FIG. 5). and Figure 6).

First, in the human fibroblast experiment, it was found that the MMP1 RNA expression cassette:PS]/HA multi-particle treatment group inhibited the production of type 1 procollagen protein in a concentration-dependent manner. This effect was found to be superior when compared to the untreated group with the MMP1 RNA expression cassette:PS]/HA multiparticulate, and compared to the untreated group, the treated group was 109% at doses of 10 and 100 μg/ml, respectively. , and 138%, increased inhibition of type 1 procollagen protein.

Through the above results, the cells transfected with the MMP1 RNA expression cassette:PS]/HA complex particle of the present invention increased the expression of MMP-1, a collagen degrading enzyme, and suppressed the production of type 1 procollagen.

TIMP: TIMP 1, TIMP 2, TIMP 3 and TIMP 4

TIMP controls ECM proteolysis by inhibiting MMP as well as ADAM with the closely related ADAM and ADAMTS. The role of TIMPs in the inhibition of MMP-dependent ECM proteolysis suggests that increased TIMP levels lead to ECM accumulation (or fibrosis), whereas loss of TIMPs leads to enhanced matrix proteolysis.

Human skin fibroblast CCD-986Sk (ATCC　 CRL-1947™) was prepared from 10% FBS, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyrbate under the conditions of 37°C and 5% carbon dioxide. was cultured in IMDM medium (hereinafter abbreviated as cell medium).

2㎖ of IMDM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ and then cultured at 37°C, 5% CO2 environment for 24 hours. Thereafter, the medium containing the TIMP RNA expression cassette: PS]/HA complex particles prepared in Example 1 was replaced and cultured for 6 hours, and then replaced with a fresh medium and cultured for 3 days.

Thereafter, the culture medium was harvested and centrifuged at 4° C., 7500 rpm for 5 minutes, and the TIMP concentration secreted in the culture medium using the ELISA method was 0, 6, 12, 24 after transfection for each concentration of each RNA expression cassette. , and three measurements at 48 hours. The secretion of TIMP from transfected human fibroblasts is quantified using the TIMP Human Instant ELISA™ Kit (Invitrogen, USA).

2ml of DMEM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ , and then cultured at 37°C, 5% CO2 environment for 24 hours. Thereafter, the medium containing the TIMP RNA expression cassette: PS]/HA complex particles (1, 10, and 100 μg/ml, respectively) prepared in Example 1 was replaced and cultured for 3 days.

Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4 ° C., 7500 rpm environment. Using the ELISA method, the protein expression level of type 1 procollagen (Procollagen Type I C Peptide EIA Kit, Takara, Shiga, Japan) was confirmed (FIG. 17). ).

First, in the human fibroblast experiment, it was found that the TIMP RNA expression cassette:PS]/HA multi-particle treatment group increased the production of type 1 procollagen protein in a concentration-dependent manner. This effect was found to be superior when compared to the untreated group with the TIMP RNA expression cassette:PS]/HA multiparticulate, which was 109% at the 10 and 100 μg/ml doses in the treated group compared to the untreated group, respectively. , and 138%, an improved increase in the production of type 1 procollagen protein, indicating that the TIMP RNA expression cassette:PS]/HA multiparticle treatment had a greater effect on increasing the production of type 1 procollagen protein ( FIG. 5 ).

Through the above results, the TIMP RNA expression cassette: PS]/HA complex particle of the present invention acts on the MMP signaling system to promote the production of type 1 procollagen and inhibit the expression of MMP-1, a collagen degrading enzyme. Through the action, it was confirmed that there was an effect of effectively preventing the generation of wrinkles and improving the generated wrinkles.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example 9. Biological evaluation of RNA expression cassette/PS/HA-aptamer multiparticulate 3

RNA expression cassette:PS]/HA-aptamer multiparticulate cancer cell targeting analysis

In order to evaluate the effect of the [RNA expression cassette: PS] / HA complex particle and the [RNA expression cassette: PS] / HA-Apt_EGFR complex particle on cell proliferation according to the expression level of EGFR, the RNA prepared in Example 5 The effect of the expression cassette PS/HA-Apt_EGFR multiparticulate was evaluated.

The cell line used in this Example is an EGFR+ cell line, <lung cancer cell line A549, colon cancer cell line SW48, and skin cancer cell line A431>; And <breast cancer cell line MDA-MB-453, colorectal cancer cell line HT-29 and neuroblastoma cell line SK-N-MC> as EGFR- cell lines.

The [RNA expression cassette: PS] / HA complex particles and [RNA expression cassette: PS] / HA-Apt_EGFR complex particles were added to the cell culture solution, incubated for 4 hours, washed, and cultured for 72 hours for MTT analysis was used.

Referring to Figure 18, the [RNA expression cassette: PS] / HA complex particles without the EGFR ligand have an IC ₅₀ value of about 3.5 nM in EGFR+ lung cancer cell line A549 cells, lung cancer cell line A549, colorectal cancer cell line SW48 and skin cancer cell line A431, In the EGFR- breast cancer cell line, MDA-MB-453 cells, the colorectal cancer cell line HT-29 and the neuroblastoma cell line SK-N-MC, the IC ₅₀ value was about 3.5 nM.

The [RNA expression cassette: PS]/HA-Apt_EGFR multiparticle having the EGFR ligand had an IC ₅₀ value of about 3.5 nM in EGFR+ lung cancer cell line A549 cells, lung cancer cell line A549, colorectal cancer cell line SW48 and skin cancer cell line A431, and EGFR- breast cancer IC ₅₀ values could not be measured in the cell line, MDA-MB-453 cells, colorectal cancer cell line HT-29, and neuroblastoma cell line SK-N-MC.

These cytotoxicity results indicate that apoptosis is induced by selectively delivering [RNA expression cassette: PS]/HA-Apt_EGFR complex particles having an EGFR ligand to the cancer cells depending on the presence or absence of an EGFR target molecule. Mean ± standard deviation (n = 5).

endosomal escape

The endosomal escape analysis of [RNA expression cassette: PS]/HA-Apt_EGFR complex particles with the EGFR ligand was an image taken with LysoTracker Red DNA-99 (ThermoFisher Scientific, USA) with a Confocal laser scanning microscope (CL). was analyzed and performed.

probes는 세포막을 자유롭게 투과하여 살아있는 세포를 표지할 수 있고 중성 pH에서만 부분적으로 protonate되는 약한 염기와 연결된 fluorophore으로 구성되었다. LysoTracker￠

prob는 acidic organelle에 대한 선택성이 높아 살아있는 세포에서 acidic organelles를 표지하고 추적하는 적색 형광 염료로 많이 사용된다.LysoTracker￠

The probes consisted of a fluorophore linked to a weak base that can freely penetrate cell membranes and label living cells and only partially protonate at neutral pH. LysoTracker￠

Prob has high selectivity for acidic organelles, so it is widely used as a red fluorescent dye for labeling and tracking acidic organelles in living cells.

Lung cancer cell line A549 cells were inoculated in a 96-well plate at a concentration of 1 x 10 ⁴ cell/well and then cultured for 24 hours. After administration of the [RNA expression cassette: PS]/HA-Apt_EGFR multiparticulate with EGFR ligand to the cultured A549 cells, the cells were cultured for 0.5 hours, 2 hours, or 4 hours. The EGFR ligand-containing [RNA expression cassette: PS]/HA-Apt_EGFR composite particles were administered to A549 cells cultured with 25 nM Lysotracker for 20 minutes, and the cells were fixed with 4% paraformaldehyde. After that, the CL image was analyzed.

Endosome escape (endosomal escape) is a very important problem in the cell delivery of [RNA expression cassette: PS]/HA-Apt_EGFR multiparticulate with efficient EGFR ligand. In order to prevent the mRNA entering the cell from being degraded by nuclease and for the efficacy of the activator contained in the mRNA, the amount of mRNA that escapes the endosome and is released into the cytoplasm must be sufficient.

19 shows the results of confirming that mRNA delivered into lung cancer cell line A549 cells by [RNA expression cassette: PS]/HA-Apt_EGFR multiparticle with EGFR ligand escapes from endosomes and exists in the cytoplasm. The PS of the present invention has the advantage of improving endosomal escape of mRNA because it has a nucleoprotein characteristic. Through CL image analysis of LysoTracker Red DNA-99(red) (ThermoFisher Scientific, USA), the degree of endosomal escape with respect to mRNA-FITC of mRNA-FITC/PS/HA complex particles was confirmed with time.

[RNA expression cassette: PS]/HA-Apt_EGFR multiparticulate with EGFR ligand was treated with lung cancer cell line A549 cells, and FITC (green) for A549 cells, and CL for LysoTracker (red) after 0.5, 2, and 4 hours The images were analyzed to confirm the cellular uptake of mRNA. When cultured for 0.5 hours after treatment, it was confirmed that the [RNA expression cassette: PS]/HA-Apt_EGFR complex particles with EGFR ligand formed endosomes and absorbed into the cytoplasm, and cultured for 2 hours after treatment It was confirmed that most mRNAs encapsulated in [RNA expression cassette: PS]/HA-Apt_EGFR multiparticle with absorbed EGFR ligand successfully escaped endosomes and released into the cytoplasm. When cultured for 4 hours after treatment, it was confirmed that most of the [RNA expression cassette: PS]/HA-Apt_EGFR multiparticulates with EGFR ligands were cleaved into small molecules.

Therefore, it was confirmed that the [RNA expression cassette: PS]/HA-Apt_EGFR composite particle having the EGFR ligand of the present invention was absorbed into the cell and then successfully escaped the endosome and released into the cytoplasm.

In summary, the above results indicate that the [RNA expression cassette: PS]/HA-Apt_EGFR complex particle with the EGFR ligand of the present invention contacts lung cancer cell line A549 cells to form endosomes, so it was successfully absorbed into cells, and most mRNAs were It is not degraded in the lysosome, suggesting that successful endosomal escape has occurred into the cytoplasm by the nuclear protein function of PS, and the mRNA released into the cytoplasm is judged to be effective in treatment as a pharmaceutical active ingredient.

Example 10. Cell uptake experiment of [RNA expression cassette: PS] / HA-TCA complex particle (20)

In order to evaluate the degree of cellular uptake by the bile acid receptor of the [RNA expression cassette: PS]/HA-TCA (taurocholate) complex particle, an experiment was performed using MDCK and MDCK-ASBT cells (BioIVT, USA). MDCK and MDCK-ASBT cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS.

Whether or not the prepared [RNA expression cassette: PS] / HA-TCA complex particles are cellularly absorbed, [RNA expression cassette: PS] / HA-TCA particles are prepared by the method provided by the Rhodamine Conjugation Kit (abcam, USA). It was prepared using Rhodamine B-conjugated Protamine (Rhod-PS) and investigated through CL image analysis and fluorescence measurement.

Referring to FIG. 20, it can be seen that the complex particles having protamine bound to Rhodamine B are introduced into the cells over time, and the signal by Rhodamine B bound to protamine increases in MDCK-ASBT cells.

The TCA is a kind of bile acid, which is a substance capable of enterohepatic circulation by bile acid receptors, and increases the systemic circulation of the drug. There is this. MDCK-ASBT cells were ASBT receptor overexpressed cells, and MDCK cells were used as a control.

Cellular uptake by bile acid receptors of [RNA expression cassette:PS] basic multiparticle, [RNA expression cassette:PS]/HA multiparticulate and [RNA expression cassette:PS]/HA-TCA multiparticulate in MDCK and MDCK-ASBT cells investigated in It was prepared using Rhodamine B-conjugated Protamine (Rhod-PS) prepared by the method provided by the Rhodamine Conjugation Kit (abcam, USA) and investigated through CL image analysis and fluorescence measurement.

Referring to FIG. 21, as a result of the experiment, there was no significant difference in the presence of [RNA expression cassette: PS]/HA-TCA complex particles in MDCK cells in which ASBT was not expressed, but in MDCK-ASBT cells, the prepared [RNA expression cassette :PS]/HA-TCA composite particles were confirmed to be more pronounced than other sample groups, and it was confirmed that they increased as time passed. In addition, in order to confirm whether the RNA expression cassette PS / HA-TCA particles were absorbed by the ASBT receptor of MDCK-ASBT cells, the [RNA expression cassette: PS] / HA-TCA particles were absorbed after pretreatment with TCA at a high concentration As a result of confirming ([RNA expression cassette: PS]/HA-TCA), it was confirmed that the MDCK-ASBT cell uptake of [RNA expression cassette: PS]/HA-TCA was significantly reduced.

Therefore, it was confirmed that the [RNA expression cassette:PS]/HA-TCA multiparticulate of the present invention has very low cytotoxicity and can enter the enterohepatic circulation through the end of the small intestine.

These results were not limited to selected mRNAs, but were generally observed for other mRNAs as well.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

10: basic composite particles 20: composite particles
21: cationic polymer layer 22: anionic polymer layer

Claims (36)

an RNA expression cassette having a structure capable of translating cells as an active ingredient and comprising chemically modified nucleotides;
any one or more selected from cationic polymers including cationic polypeptides and cationic polysaccharides; and
Any one or more selected from anionic polymers; and
wherein the RNA expression cassette, the cationic polymer and the anionic polymer form a multiparticulate by electrostatic interaction, and the RNA expression cassette is encapsulated in the formed multiparticulate structure, and uses thereof. The method of claim 1, wherein the RNA of the RNA expression cassette is
Multiparticulates comprising an ORF encoding any one selected from the group consisting of therapeutic proteins, peptides, fusion proteins, toxins, antigens and antibodies, and uses thereof. The method of claim 2, wherein the therapeutic protein is
Composite particles, characterized in that at least one selected from the group consisting of extracellular matrix degrading enzymes and degrading enzyme inhibitors, and uses thereof.
Here, MSECM refers to a polypeptide capable of modifying the structure of the ECM. The method of claim 3, wherein the extracellular matrix degrading enzyme is
hyaluronidase (HAase); and
Multiparticulates characterized by any one or more selected from the group comprising matrix metalloproteinases (MMP) and uses thereof. The method of claim 3, wherein the enzyme inhibitor is
Multiparticulates characterized in that any one selected from the group comprising TIMP and uses thereof. The method of claim 1, wherein the RNA expression cassette is
non-amplifying mRNA constructs;
self-amplifying mRNA constructs; and
Multiparticulates characterized in that any one or more selected from constructs comprising a trans-amplifying mRNA (trans-amplifying mRNA) construct and uses thereof. The method of claim 6, wherein the non-amplified mRNA construct comprises at least one selected from the group consisting of natural nucleotides and modified nucleotides,
(a) an open reading frame (ORF) encoding said target polypeptide:
(b) a 5'UTR;
(c) a 3'UTR; and
(d) Multiparticulates characterized by an isolated polynucleotide encoding a polypeptide comprising a 5' cap structure or IRES, and uses thereof. The method of claim 6, wherein the self-amplifying mRNA (self-amplifying mRNA) construct is
mRNA constructs for expressing RNA dependent RNA polymerase (RdRp); and
an isolated polynucleotide comprising an mRNA replicon capable of being replicated by said RdRp and comprising an ORF encoding said polypeptide of interest;
wherein the isolated nucleotide construct comprises a 5'-cap for inducing translation of RdRp, and uses thereof. The method of claim 6, wherein the trans-amplifying mRNA (trans-amplifying mRNA) construct is
mRNA constructs for expressing RdRp; and
an mRNA replicon construct capable of being trans-replicated by said RdRp and comprising an ORF encoding said polypeptide of interest;
wherein the RNA construct for expressing RdRp comprises a 5'-cap for inducing translation of RdRp, and a use thereof. 10. The method of claim 1 to 9, wherein the RNA expression cassette is
A multiparticulate comprising a 5'Cap structure or an internal ribosome entry site (IRES) sequence, and uses thereof. 11. The method of claim 1 to 10, wherein the chemically modified nucleotide is
Multiparticulates comprising any one or more selected from the group comprising sugar modifications, base modifications, backbone modifications and lipid modifications, and uses thereof. 12. The method of claim 1 to 11, wherein the RNA expression cassette is
Includes G/C content, codon optimization (optimizAtion), UTR modification, poly(A) tail with more than 30 adenosine nucleotides, poly(C) sequence, 5' cap (CAP) structure and histone-stem-loop sequence Composite particles comprising at least one selected from nucleotide sequence modifications and uses thereof. 13. The method of claim 12, wherein the 5' cap (CAP) structure is
Multiparticulates characterized by chemically modified ARCA-caps (CAPs) and uses thereof. 15. The method of claim 14, wherein the ARCA-cap (CAP) is
Composite particles characterized by phosphorothioate and uses thereof. 15. The multiparticulate according to claim 1 to 14, wherein the RNA expression cassette is non-covalently or covalently bound to cholesterol and its use. The method of claim 1, wherein the cationic polypeptide is
Protamine, cell penetrating peptide (CPP), chimeric CPP, pH dependent CPP, nucleoline, spermine, spermidine, Poly-L-lysine (PLL), basic polypeptide, poly-arginine, transportan, MPG peptide, HIV (human immunity) Human Immunodeficiency Virus (HIV)-binding peptide, Trans-activating transcriptional activator (Tat), HIV-1 Tat (HIV), Tat-derived peptide (Tat- derived peptide), oligoarginine, penetratin family member, penetratin, Antennapedia-derived peptide, Drosophila Antennapedia peptide (pAntp) , Islet-1 peptide (PIsl), antimicrobial-derived CPP (antimicrobial-derived CPP), Buforin-2 (Buforin-2), Bactenecin 7 peptide fragment 15-24 (bactenecin 7 peptide fragment 15 -24, Bac715-24), syncytinB, SynB, SynB(1), vascular endothelial cadherin derived cell penetrating peptide (pVEC), human calcitonin (hCT)- Derived peptide, sweet arrow peptide (sweet a rrow peptide, SAP), model amphipathic peptide (MAP), KALA, PpTG20, Proline-rich peptide, Lologomere, Arginine-rich peptide , Calcitonin-peptide (Calcitonin-peptide), Fibroblast growth factors (FGF), Lactoferrin (Lactoferrin), histone (histone), VP22 peptide (VP22 peptide), Herpes simplex virus (HSV), Cationic poly containing VP22 (Herpes simplex), protein transduction domain (PTD), lysine-rich peptide, Pep-1, L-oligomer Multiparticulates comprising any one or more selected from peptides and uses thereof. The method of claim 1, wherein the cationic polysaccharide is
chitosan, glycochitosan and artificially derived cationic polysaccharides;
cationic molecules to which bile acids or bile acid derivatives are covalently bound; and
Cationic polysaccharide derivatives or salts, and combinations thereof; Multiparticulates characterized in that at least one selected from the group consisting of, and uses thereof. The method of claim 1, wherein the anionic polymer is
polysaccharides and derivatives thereof having a functional group selected from a carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof;
poly amino acids containing amino acid residues having negatively charged side chains;
PEG derivatives having carboxyl side chains;
synthetic polymers having a functional group selected from a carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof;
carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof; and
Polymers having a functional group selected from an optionally substituted amino group, an ammonium group, or a salt thereof, and combinations thereof; Multiparticulates characterized in that at least one selected from the group consisting of and uses thereof. 19. The method of claim 18, wherein the anionic polymer is
anionic proteins including silk protein fibroin, gliadin and albumin;
anionic polyamino acids including polyglutamic acid and polyaspartic acid;
Agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin and carboxymethyl cellulose and these Anionic polysaccharides including variants of;
polyacrylic acid; and
artificially derived anionic polysaccharides and salts thereof; Multiparticulates characterized in that at least one selected from the group comprising and uses thereof. 20. The method of claim 18 to 19, wherein the anionic polymer is
Serum albumin, hyaluronic acid (hyaluronic acid, HA), or a variant thereof; multiparticulates containing; and uses thereof. 21. The composite particle according to claim 1 to 20, further comprising at least one selected from the group consisting of a polymer containing an anticancer substance, a polymer containing an anticancer substance, and a polymer containing a fluorescent substance. The method of claim 21, wherein the high molecular weight anticancer substance is
Multiparticulates characterized by any one or more selected from the group comprising proteins, antibodies, and nucleic acids, and uses thereof. 23. The method of claim 22, wherein the nucleic acid is
DNA therapeutics including antisense oligonucleotides, DNA aptamers and gene therapy; and
RNA therapeutics including mRNA, micro RNAs, short interfering RNAs, ribozymes, RNA decoys, and circular RNAs; Multiparticulates characterized in that at least one selected from the group consisting of, and uses thereof. extracellular matrix (ECM) degrading enzymes including Hyluronases and MMP; and
MSECM RNA expression cassette encoding any one or more selected from the group comprising; the enzyme inhibitor including TIMP; and
Any one or more selected from the cationic polymer; and
Multiparticulates and uses thereof, characterized in that the RNA expression cassette and the cationic polymer form a multiparticulate by electrostatic interaction, and the RNA expression cassette is encapsulated in the formed multiparticulate structure. extracellular matrix (ECM) degrading enzymes including Hyluronases and MMP; and
MSECM RNA expression cassette encoding any one or more selected from the group comprising; the enzyme inhibitor including TIMP;
any one or more selected from the cationic polymer; and
Any one or more selected from the anionic polymer:
Multiparticulates and uses thereof, characterized in that the RNA expression cassette and the cationic polymer form a multiparticulate by electrostatic interaction, and the RNA expression cassette is encapsulated in the formed multiparticulate structure. drug; and
A composite particle comprising; the composite particle prepared in any one of claims 24 to 25, and a use thereof. 27. The method of claim 26, wherein the drug is
Multiparticulates, characterized in that they are protein drugs, antibodies, small molecules, aptamers, mRNA, RNAi, antisense or cell therapeutics, and uses thereof. 28. The method of claim 24-27, wherein the multiparticulate is administered first,
Multiparticulates and uses thereof, characterized in that different drugs are sequentially administered. 29. The method of any one of claims 1-28,
Multiparticulates comprising an active drug delivery system in which a molecule constituting the outermost layer of the particle and a ligand are bound, and uses thereof. 30. The method of any one of claims 1-29,
Multiparticulates for treating a disease in a subject in need thereof, and uses thereof, comprising administering to the subject a therapeutically effective amount of said multiparticulates, thereby treating the disease. 31. The method of claim 1 to 30, wherein the composite particles are
Composite particles characterized by one selected from the group consisting of injections, creams, and oral mucosal administration, and uses thereof. 32. The method of claim 1 to 31, wherein the composite particles are
Multiparticulates and uses thereof, characterized in that the RNA expression cassette and the cationic polymer form a basic multiparticulate, and the anionic polymer forms an outer layer. 33. The method of claim 1 to 32, wherein the composite particle is
Multiparticulates and uses thereof, characterized in that the RNA expression cassette, a cationic polymer and an anionic polymer are mixed. 34. The method of claim 1 to 33, wherein the composite particle is
Composite particles and uses thereof, characterized in that they are manufactured using any one or more selected from the group including a sonicator and an extruder. 35. Multiparticulates according to claim 1 to 34, characterized in that the multiparticulates are hydrogels and uses thereof Multiparticulates comprising MSECM RNA expression cassette and uses thereof

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