WO2024005457A1 - Activateur immunitaire à base d'acide nucléique formant une structure secondaire - Google Patents

Activateur immunitaire à base d'acide nucléique formant une structure secondaire Download PDF

Info

Publication number
WO2024005457A1
WO2024005457A1 PCT/KR2023/008736 KR2023008736W WO2024005457A1 WO 2024005457 A1 WO2024005457 A1 WO 2024005457A1 KR 2023008736 W KR2023008736 W KR 2023008736W WO 2024005457 A1 WO2024005457 A1 WO 2024005457A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
sequence
control element
acid molecule
expression control
Prior art date
Application number
PCT/KR2023/008736
Other languages
English (en)
Korean (ko)
Inventor
남재환
방유진
박효정
Original Assignee
가톨릭대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 가톨릭대학교 산학협력단 filed Critical 가톨릭대학교 산학협력단
Publication of WO2024005457A1 publication Critical patent/WO2024005457A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

  • the present disclosure relates to an adjuvant, and more particularly, to a nucleic acid-based adjuvant into which an expression control element is inserted and a vaccine composition containing the same.
  • the immune system refers to the biological structure and process that detects pathogens and tumor cells within living organisms, removes them, and protects living organisms from diseases.
  • the immune system can be largely divided into the innate immune system (innate immune system) and the adaptive immune system (acquired immune system).
  • the innate immune system is a cell or mechanism that protects the host from infection in a non-specific way, and is an immune system that responds immediately without remembering specific pathogens. All types of animals and plants have an innate immune system, while plants, fungi, insects, etc. only have an innate immune system.
  • the adaptive immune system is specific for antigens or pathogens and requires recognition of non-self antigens through the antigen presentation process. Accordingly, a specific immune response against a specific antigen or antigen-infected cells is possible in the adaptive immune system.
  • memory cells which constitute the adaptive immune system, can recall an immune response that has already been carried out, so when the same pathogen invades the body multiple times, they can be quickly eliminated.
  • the immune system can be divided into humoral immunity and cellular immunity.
  • humoral immunity B lymphocytes derived from the bone marrow recognize an antigen, differentiate, and secrete antibodies made of a glycoprotein called immunoglobulin (Ig), and pathogens are infected by the secreted antibodies.
  • Ig immunoglobulin
  • pathogens are infected by the secreted antibodies.
  • An immune response occurs that eliminates this.
  • cell mediated immunity is an immune response in which T lymphocytes derived from the thymus recognize an antigen and secrete lymphokine or directly kill infected cells.
  • Vaccines that inoculate all or part of a pathogen as an antigen to induce an immune response against a pathogen are used to prevent or treat various diseases. At this time, various immune responses that can be caused by the vaccine antigen can be induced.
  • Sub-unit vaccines with clear structures and components have been developed recently instead of the attenuated live bacteria vaccines or inactivated dead bacteria vaccines that were developed initially. Sub-unit vaccines have lower immunogenicity compared to existing vaccines, so they do not induce an immune response. To increase immunity, an adjuvant is used.
  • the adjuvants used in human vaccines to prevent diseases are 1) alum, a metal salt such as aluminum hydroxide/aluminum phosphate/aluminum hydroxide phosphate sulfate, and 2) MF59, an adjuvant in the form of an oil-in-water emulsion based on squalene. etc. are being used.
  • conventional adjuvants mainly have excellent humoral immune response activity, but their cellular immunity inducing activity is very low. Therefore, conventional adjuvants can only be used when protection against infection is possible with antibodies alone, and have limitations in that they are not suitable for use in vaccines that require a cellular immune response.
  • the cell walls of microorganisms which are representative pathogens, contain types of pathogenicity-related molecules consisting of glycoproteins such as lipopolysaccharides (LPS), ⁇ -1,3-glucan, and peptidoglycans. It has pathogen-associated molecular patterns (PAMPs). These PAMPs are recognized by specific proteins that make up the host's immune system, such as pattern recognition receptors (PRRs) or pattern recognition proteins (PRPs).
  • PRRs pattern recognition receptors
  • PRPs pattern recognition proteins
  • TLRs Toll-like receptors
  • LPS an endotoxin
  • TLR9 agonists can enhance various immune responses, and oligonucleotides containing a CpG motif as TLR9 agonists are being developed as immune enhancers.
  • LPS and CpG motifs used as TLR agonists are highly toxic and have side effects such as causing inflammatory reactions in vivo, which poses a safety issue.
  • the purpose of the present disclosure is to provide a nucleic acid-based adjuvant designed to induce humoral immunity and cellular immunity in a balanced manner and a vaccine composition containing the same.
  • Another object of the present disclosure is to provide an adjuvant and a vaccine composition containing the same that do not cause toxicity or side effects to the living body when injected into the living body.
  • the present disclosure is an immune adjuvant comprising a nucleic acid molecule having an expression regulatory element with Internal Ribosomal Entry Site (IRES) activity derived from Solenopsis invicta virus (SINV). provides.
  • IRS Internal Ribosomal Entry Site
  • the nucleic acid molecule may further include at least one restriction enzyme recognition sequence or restriction enzyme cleavage sequence operably linked to the expression control element.
  • the nucleic acid molecule may further comprise a coding region operably linked to the expression control element.
  • the expression control element may include a first translation control element and a second translation control element located downstream of the first translation control element.
  • the first translation control element may include SEQ ID NO: 1 or a transcript sequence thereof.
  • the second translation control element may include SEQ ID NO: 2 or a transcript sequence thereof.
  • the expression control element may further include a transcription control element located upstream of the first translation control element.
  • the nucleic acid molecule may further include at least one nucleotide sequence selected from the group consisting of a first polyadenylation signal sequence or a first polyadenosine sequence located downstream of the expression control element.
  • the nucleic acid molecule may further include a second polyadenylation signal sequence or a second polyadenosine sequence located downstream of the first polyadenylation signal sequence or the first polyadenosine sequence.
  • the nucleic acid molecule is located downstream of the second polyadenylation signal sequence or the second polyadenosine sequence and may further include a nucleotide sequence of Formula 1 below or a transcript sequence thereof.
  • G represents guanosine and X is one of adenosine, cytinine, and thymidine; m, p, r and t are each independently an integer from 1 to 10; n, q and s are each independently integers from 0 to 10.
  • the expression control element may include a nucleotide sequence having IRES activity derived from Solenopsis invictavirus 1 (SINV-1).
  • the present disclosure provides a vaccine composition comprising an immunogen and an adjuvant comprising a pharmaceutically effective amount of the above-described nucleic acid molecule.
  • the vaccine composition may further include at least one of lipid nanoparticles, nucleic acid stabilizers, and other immune enhancers.
  • the present disclosure provides a method of enhancing immune activity in a subject comprising administering to the subject a pharmaceutically effective amount of the above-described nucleic acid molecule.
  • a nucleic acid molecule comprising an expression control sequence derived from Solenopsis invictavirus (SINV) and, optionally, at least one restriction enzyme recognition sequence or restriction enzyme cleavage sequence and/or a coding region efficiently induces an immune response.
  • Nucleic acid molecules can be used as an adjuvant to enhance the immune response caused by immunogenic substances.
  • Nucleic acid molecules can induce an immune response in a different pathway than the immune response caused by immunogenic substances.
  • nucleic acid molecules When nucleic acid molecules are used as adjuvants, the entire immune response caused by immunogenic substances can occur in a balanced manner in vivo. Additionally, nucleic acid molecules have no toxicity or side effects in vivo, ensuring safety.
  • nucleic acid molecules can be used as an immune enhancer and used as a vaccine composition to prevent various diseases.
  • FIG. 1 is a schematic diagram schematically showing the structure of a nucleic acid molecule according to an exemplary embodiment of the present disclosure.
  • Figure 2 is a schematic diagram schematically showing the structure of a nucleic acid molecule according to another exemplary embodiment of the present disclosure.
  • Figures 3 and 4 are graphs showing the results of measuring immunoglobulins produced in the serum of mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to an exemplary embodiment of the present disclosure.
  • Figures 5 and 6 are graphs showing the results of measuring neutralizing antibody titers in mice immunized by injection of nucleic acid molecules, respectively, according to an exemplary embodiment of the present disclosure.
  • Figures 7 and 8 are graphs showing the results of measuring cytokines secreted from mice immunized by injecting nucleic acid molecules using ELISPOT, respectively, according to an exemplary embodiment of the present disclosure.
  • Figures 9 to 11 are graphs showing the results of FACS measurement of cytokines produced in mice immunized by injection of nucleic acid molecules, respectively, according to exemplary embodiments of the present disclosure.
  • Figures 12 and 13 are graphs showing the results of measuring cytokines produced in mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to an exemplary embodiment of the present disclosure.
  • Figures 14 and 15 are graphs showing the results of measuring immunoglobulins produced in the serum of mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 16 and 17 are graphs showing the results of measuring neutralizing antibody titers in mice immunized by injecting nucleic acid molecules, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 18 and 19 are graphs showing the results of measuring immunoglobulins produced in the serum of mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 20 and 21 are graphs showing the results of measuring neutralizing antibody titers in mice immunized by injecting nucleic acid molecules, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 22 and 23 are graphs showing the results of measuring immunoglobulins produced in the serum of mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 24 and 25 are graphs showing the results of measuring neutralizing antibody titers in mice immunized by injecting nucleic acid molecules, respectively, according to another exemplary embodiment of the present disclosure.
  • Figure 26 is a graph showing the results of measuring cytokines secreted from mice immunized by injecting nucleic acid molecules using ELISPOT, according to another exemplary embodiment of the present disclosure.
  • Figures 27 to 29 are graphs showing the results of FACS measurement of cytokines produced in mice immunized by injecting nucleic acid molecules, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 30 to 32 are graphs showing the results of measuring cytokines produced in mice immunized by injecting nucleic acid molecules using ELISA, respectively, according to another exemplary embodiment of the present disclosure.
  • Figures 33 to 35 are graphs showing the results of measuring dendritic cells and the degree of activation of dendritic cells in human cell lines cultured by stimulating them with nucleic acid molecules using FACS, according to another exemplary embodiment of the present disclosure, respectively. am.
  • Figures 36 to 40 are graphs showing the results of measuring the secretion of cytokines in human cell lines cultured by stimulating them with nucleic acid molecules, respectively, according to another exemplary embodiment of the present disclosure.
  • amino acid is used herein in the broadest sense and is intended to include naturally-occurring L-amino acids or residues. Commonly used one-letter abbreviations and/or three-letter abbreviations for naturally-occurring amino acids may be used herein.
  • Amino acids include D-amino acids as well as chemically-modified amino acids, such as amino acid analogs, naturally-occurring amino acids that are not normally incorporated into proteins, such as norleucine, and chemically modified amino acids that have properties known in the art to be characteristic of amino acids. -Includes synthesized compounds.
  • analogs or mimetics of phenylalanine or proline are included within the definition of amino acids that allow for conformational restriction of peptide compounds identical to native Phe or Pro. Such analogs and mimetics are referred to herein as “functional equivalents” of amino acids.
  • amino acid analogs include 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric acid (Abu) for Met, Leu and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminobutyric acid (Aib) for Gly; cyclohexylalanine (Cha) for Val, Leu and Ile; Homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dap) for Lys
  • peptide includes all proteins, protein fragments, and peptides isolated from naturally occurring ones, or synthesized chemically or by recombinant techniques.
  • the peptide of the present disclosure consists of at least 5 amino acids, for example, at least 10 amino acids.
  • variants of a compound such as peptide variants with one or more amino acid substitutions
  • peptide variants refer to the original amino acid in which one or more amino acids are substituted, deleted, added, and/or inserted into the amino acid sequence of the peptide. This means that it exerts almost the same biological function as the peptide composed of.
  • a peptide variant must have at least 70%, for example, at least 90%, or at least 95% identity with the original peptide.
  • substitutions may include amino acid substitution agents known as “conservative”.
  • variant polypeptide may also contain nonconservative changes.
  • sequence of a variant polypeptide differs from the original sequence by substitution, deletion, addition, or insertion of five or fewer amino acids.
  • variants may also be altered by deletion or addition of amino acids that have minimal effect on the immunogenicity or secondary structure of the peptide.
  • “Conservative” substitution means that there is no significant change in the properties such as secondary structure and hydropathic nature of the polypeptide even when one amino acid is replaced with another amino acid.
  • Amino acid mutations are based on the relative similarity of amino acid side chain substitutions, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature. It can be obtained by doing this.
  • amino acids can be classified according to their common side chain properties as 1) hydrophobic (norleucine, methionine, alanine, valine, leucine, isoleucine), 2) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine), and 3) acidic ( Aspartic acid, glutamic acid), 4) basic (histidine, lysine, arginine), 5) residues that affect chain direction (glycine, proline), 6) aromatic (tryptophan, tyrosine, phenylalanine).
  • a conservative substitution would involve exchanging a member of one of each of these classes for another member of the same class.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.
  • hydrophobic index of the amino acid may be considered.
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobic index.
  • a substitution is made between amino acids showing a difference in the hydrophobicity index within ⁇ 2, within ⁇ 1, or within ⁇ 0.5.
  • Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).
  • the most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • the peptides (including fusion proteins) and polynucleotides referred to herein are isolated.
  • An “isolated” peptide or polynucleotide is one that has been removed from its original environment. For example, a protein that exists in its natural state is separated by removing all or part of the substances that exist together in that state. Such polypeptides should be at least 90% pure, for example, at least 95% pure or at least 99% pure.
  • Polynucleotides are isolated by cloning into a vector.
  • polynucleotide or “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length and generically include DNA (e.g., cDNA) and RNA molecules.
  • Nucleotide a structural unit of a nucleic acid molecule, refers to deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or those that can be incorporated into a polymer by DNA or RNA polymerase or by synthetic reactions. It can be any substrate.
  • Polynucleotides may include modified nucleotides, analogues with modified sugars or bases, such as methylated nucleotides and analogs thereof.
  • the nucleotides may include 5-modified cytidine and/or 5-modified uridine.
  • 5-Modified cytidines include 5-halocytidine (e.g., 5-iodocytidine or 5-bromocytidine), 5-alkynylcytidine, and or 5-heterocyclyluridine.
  • 5-Modified uridine may include 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine, and/or 5-heterocyclyluridine. You can.
  • 5-modified uridine is a nucleoside containing 2-deoxyribose.
  • nucleotides may not cause mutations in proteins. These nucleic acids include functionally equivalent codons, or codons that encode identical amino acids (e.g., due to codon degeneracy, there are six codons for arginine or serine), or codons that encode biologically equivalent amino acids. Contains nucleic acid molecules that Additionally, variations in nucleotides may lead to changes in the protein itself. Even in the case of a mutation that changes the amino acid of a protein, it can be obtained that shows almost the same activity as the protein of the present disclosure.
  • nucleic acid molecule or polynucleotide of the present disclosure has the characteristics, for example, the effect as an immune enhancer
  • the peptide and nucleic acid molecule of the present disclosure are not limited to the amino acid sequence or base sequence described in the sequence listing. It's clear.
  • a biological functional equivalent that may be included in a coding region operably linked to an expression control sequence and/or a recombinant protein/peptide expressed therefrom exhibits equivalent biological activity to the coding region and/or recombinant protein described above. It may be a polynucleotide having a nucleotide sequence mutation and/or a protein/peptide having an amino acid sequence mutation.
  • the nucleic acid molecule encoding the peptide and/or protein according to the present disclosure is interpreted to also include sequences showing substantial identity with the sequences listed in the sequence listing.
  • the above substantial identity is at least 61% when aligning the sequence of the present disclosure and any other sequence to correspond as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art.
  • Homology means sequences that exhibit, for example, at least 70% homology, at least 80% homology, or at least 90% homology. Alignment methods for sequence comparison are known in the art.
  • vector refers to a structure that can be delivered to a host cell, for example, to enable the expression of one or more genes or sequences of interest. Additionally, a specific vector can direct the expression of a gene in the form of an ORF to which the vector is operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”).
  • expression control/regulation sequence or “expression control/regulation element” refers to transcription of a nucleic acid and/or translation from a nucleic acid in the form of a transcript. It may refer to a nucleic acid sequence that regulates. Meanwhile, the term “transcription control/regulation sequence” or “transcription control/regulation element” refers to a nucleic acid sequence that regulates transcription of nucleic acids. Transcriptional control sequences include promoters such as constitutive promoters or inducible promoters, or enhancers.
  • translation control/regulation sequence or “translation control/regulation element” will be used for the nucleic acid sequence that regulates the translation of a nucleic acid in the form of a transcript into a protein or peptide. You can. These expression control sequences/elements, transcription control sequences/elements and/or translation control sequences/elements are operatively linked to the sequence to be expressed, eg, the nucleic acid sequence to be transcribed or translated.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, etc.) and another nucleic acid sequence. , whereby the regulatory sequence regulates transcription and/or translation of the other nucleic acid sequences.
  • a nucleic acid expression control sequence e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, etc.
  • nucleic acid molecule comprising an immunogenic target sequence operably linked to an expression control sequence can be utilized as an adjuvant by efficiently enhancing the immune response caused by an immunogen.
  • 1 is a schematic diagram schematically showing the composition of a nucleic acid molecule that can be used as an immune enhancer, according to an exemplary embodiment of the present disclosure.
  • the nucleic acid molecule contains an expression control element (ECE).
  • EEE expression control element
  • the nucleic acid molecule is operably linked to an expression control element (ECE) and may include at least one restriction enzyme recognition sequence or restriction enzyme cleavage sequence.
  • a nucleic acid molecule may contain multiple cloning sites (MCS) containing multiple restriction enzyme recognition sequences or restriction enzyme cleavage sequences.
  • MCS multiple cloning sites
  • the nucleic acid molecule may comprise a coding region (CR) operably linked to an expression control element (ECE).
  • the coding region (CR) may include genes in the form of an open reading frame (ORF) that encode immunogenic and/or oncogenic peptides or proteins.
  • ORF open reading frame
  • the expression control element contains a nucleotide sequence with Internal Ribosomal Entry Site (IRES) activity derived from Solenopsis invicta virus (SINV).
  • the expression control element may include a nucleotide sequence having internal ribosome binding site activity derived from Solenopsis invictavirus 1 (SINV-1).
  • an expression control element (ECE) is operably linked to a multiple cloning site (MCS) and/or a coding region (CR) and is a translation control element with IRES activity from SINV, e.g., SINV-1. (translation control element, TLCE).
  • the expression control element is operably linked to a multi-cloning site (MCS) and/or a coding region (CR) and is located upstream of a translation control element (TLCE).
  • MCS multi-cloning site
  • CR coding region
  • TCCE translation control element
  • the nucleic acid molecule may further comprise at least one nucleotide sequence (PA) of a polyadenylation signal sequence or a polyadenosine sequence located downstream of an expression control element (ECE), for example, downstream of a translation control element (TLCE). there is.
  • PA nucleotide sequence
  • the translation control element may comprise a nucleotide sequence with IRES activity from SINV operably linked to the multiple cloning site (MCS) and/or coding region (CR).
  • a translation control element is a first translation control element (U-TLCE) located upstream of a multiple cloning site (MCS) and/or a coding region (CR) and/or a multiple cloning site (MCS) and /or may comprise a second translation control element (D-TLCE) located downstream of the coding region (CR).
  • the first translation control element may be all or part of the 5'Untranslated Region (5'-UTR) with IRES activity derived from SINV
  • the second translation control element may be all or part of the 3'-UTR from SINV-1.
  • the translation regulatory element may include a nucleotide sequence having IRES activity of the Intergenic Region (IGR) of SINV or a transcript sequence thereof.
  • the first translation control element may be a nucleotide sequence upstream of the nucleotide sequence having IGR IRES activity of SINV or a transcript sequence thereof.
  • the second translation control element may be a nucleotide sequence downstream of the nucleotide sequence having IRES activity of SINV or a transcript sequence thereof.
  • the first translation control element may include the nucleotide sequence of SEQ ID NO: 1 or its transcript sequence
  • the second translation control element may include the nucleotide sequence of SEQ ID NO: 2. It may include a sequence or a transcript thereof, but the present disclosure is not limited thereto.
  • the first translation control element is a region where the translational initiation complex binds during the translation of peptides and/or proteins expressed from the coding region (CR), and the IRES is a region where the secondary and It is a cis-acting base sequence that induces translation of the target gene by forming a complex tertiary structure.
  • IRES has a unique secondary or tertiary structure and is classified into Group 1 to Group 4 based on the need for special translation factors for translation and the location of the translation start codon. can be distinguished.
  • IRES derived from SINV-1 belongs to Group 1.
  • Group 1 IRES binds directly to ribosomes and does not require translation initiation factors.
  • the first translation regulatory element (U-TLCE) is all or part of a nucleotide sequence with IRES activity derived from SINV, for example, upstream of the nucleotide sequence with IGR IRES activity of the first translation regulatory element (U-TLCE).
  • the nucleic acid molecule optionally contains a second translation control element (Multiple Cloning Site (MCS)) and/or a second translation control element ( D-TLCE) may include the downstream nucleotide sequence or its transcript sequence among the nucleotide sequences having IRES activity of SINV.
  • MCS Multiple Cloning Site
  • D-TLCE second translation control element
  • the nucleic acid molecule may be all or part of a 3'-UTR from SINV.
  • a second translation control element may be included.
  • the first translation control element (U-TLCE) and the second translation control element (D-TLCE) determine the translation efficiency of the ORF or its transcript located in the multiple cloning site (MCS) and/or coding region (CR). It improves and plays an important role in maintaining the mRNA, a transcript, stably without being destroyed within the cell.
  • the first translation control element (U-TLCE) may be located upstream of the multiple cloning site (MCS) and/or coding region (CR)
  • the second translation control element (D-TLCE) may be located upstream of the multiple cloning site (MCS) and/or coding region (CR).
  • MCS multiple cloning site
  • D-TLCE coding region
  • the nucleic acid molecule may contain one or more cloning sites, such as multiple cloning sites (MCS).
  • One or more cloning sites may include one or more restriction endonuclease recognition sites and/or sequences cleaved by restriction enzymes.
  • Restriction enzymes include natural restriction enzymes found in bacteria and archaea, as well as artificially manufactured restriction enzymes (e.g., restriction enzymes based on the DNA binding site of zinc finger nuclease or TAL effector or PNA-based PNAznymes, etc.) may include.
  • Naturally occurring restriction enzymes are 1) Type I restriction enzymes (cuts at a site separate from the recognition site and requires ATP, S-adenoxyl-L-methionine, and magnesium ions), 2) Type II restriction enzymes. (cuts a specific site within the recognition site or slightly away from the recognition site, and mostly requires magnesium ions), 3) Type III restriction enzyme (cuts a site slightly away from the recognition site, requires ATP, but requires magnesium ions) (no need for hydrolysis), 4) Type IV restriction enzyme (targets modified sites such as methylation, hydroxymethylation, or glucosyl-hydroxymethylation), 5) Type V restriction enzyme (CRISPRsdml cas9-gRNA complex), etc. It can be divided into:
  • the multiple cloning site may include, but is not limited to, a site recognized by the restriction enzymes below and/or a restriction enzyme cleavage site.
  • the multiple cloning site may include, but is not limited to, at least one restriction enzyme
  • the coding region may be composed of a nucleotide sequence in the form of an ORF encoding an immunogen to be described later or a transcription sequence thereof, but is not limited thereto.
  • Codon usage can usually affect protein/peptide expression in various species, but it is known that codon usage bias in humans does not usually have a significant effect on protein/peptide expression, so it is necessary to develop nucleic acid vaccines or gene therapeutics for humans. It is not a consideration when
  • the initial codon can have a Kozak sequence, and the nucleotide sequence near the stop codon also needs to be optimized.
  • the third part of the codon sequence of the gene to be expressed in the coding region (CR) or the mRNA, which is its transcript can be changed to GC to increase the GC% of the target gene without changing the amino acid, thereby increasing the stability of the mRNA.
  • the nucleic acid molecule can be further inserted with nucleotide sequences that can increase the expression efficiency of the coding region (CR) inserted in the form of an ORF.
  • a nucleic acid molecule may have a translation control element (TLCE), such as a transcription control element (TCCE) adjacent to a first translation control element (U-TLCE) that promotes transcription of the nucleic acid molecule.
  • TCCE transcriptional control element
  • TCE transcriptional control element
  • TCE transcriptional control element
  • TCE transcriptional control element
  • TCE transcriptional control element
  • TECS1 first translational control element
  • the transcriptional regulatory element may be a promoter that promotes transcription of a cytokine encoded in the form of an ORF in the coding region (CR).
  • Transcriptional regulatory elements can operate in animal cells, more specifically mammalian cells, to regulate transcription of genes or fragments thereof encoded in the coding region (CR).
  • a transcriptional regulatory element includes a promoter derived from a mammalian virus, a promoter derived from the genome of a mammalian cell, or a promoter derived from a bacteriophage.
  • transcriptional regulatory elements include the cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, T7 bacteriophage promoter, T3 bacteriophage promoter, SM6 promoter, and RSV.
  • Promoter EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF
  • promoters of genes include, but are not limited to, promoters of genes, cancer cell-specific promoters (e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter) and tissue-specific promoters (e.g., albumin promoter).
  • cancer cell-specific promoters e.g., TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, and AFP promoter
  • tissue-specific promoters e.g., albumin promoter
  • any transcription control element capable of transcribing mRNA from a linearized DNA template, such as the T7 bacteriophage promoter, T3 bacteriophage promoter, SP6 bacteriophage promoter, etc., is adjacent to the first translation control element (U-TLCE), Specifically, it may be located upstream of the first translation control element (U-TLCE).
  • the nucleic acid molecule can induce the expression of an ORF consisting of a gene encoded in the coding region (CR) or its transcription sequence, in addition to the above-mentioned translation regulatory element (TLCE), coding region (CR), and transcriptional regulatory element (TCCE). Nucleotide sequences may be inserted. In one exemplary embodiment, the nucleic acid molecule is inserted between a transcriptional regulatory element (TCCE) or a first translational regulatory element (U-TLCE) and the multiple cloning site (MCS) and/or initiation codon of the coding region (CR). It may contain a Kozak sequence.
  • TCCE transcriptional regulatory element
  • U-TLCE first translational regulatory element
  • MCS multiple cloning site
  • MCS multiple cloning site
  • initiation codon of the coding region (CR) It may contain a Kozak sequence.
  • a polyadenylation signal sequence and/or a polyadenosine sequence (PA) that can further improve the translation efficiency of the ORF consisting of may be further inserted.
  • the polyadenosine sequence can be from approximately 25 to approximately 400, e.g., 30 to 400, 50 to 250, or 60. It may be a nucleic acid sequence consisting of 250 adenosines.
  • the polyadenylation signal sequence (PA) may be located downstream of the multiple cloning site (MCS) and/or coding region (CR).
  • MCS multiple cloning site
  • CR coding region
  • the polyadenylation signal sequence (PA) is derived from SV40, human growth factor (hGH), bovine growth hormone (BGH), or rabbit beta-globine (rbGlob). Any one may be used, but the present disclosure is not limited thereto.
  • the polyadenylation signal sequence or polyadenosine sequence contains a plurality of adenosines, e.g., 25 to approximately 400, e.g., 30 to 400, 50 to 250, or 60 to 250. It may consist of a sequence in which a signal sequence such as 5'-GATCATCAGT-3' is inserted between two nucleotides consisting of adenosine or its transcript sequence.
  • the nucleic acid molecule may be a DNA nucleic acid molecule or an RNA nucleic acid molecule.
  • nucleic acid molecules according to the present disclosure may have the form of RNA.
  • a nucleic acid molecule is in the form of RNA, it is advantageous compared to a nucleic acid molecule in the form of DNA.
  • nucleic acid molecules in the form of DNA do not need to enter the nucleus of the host cell for transcription into mRNA.
  • Nucleic acid molecules in the form of RNA have no possibility of inserting into the host chromosome within the nucleus, antibiotic resistance genes, which are selection markers used for selective production in host cells, are unnecessary for the production of RNA nucleic acid molecules, and RNA has a half-life compared to DNA. Because it is short, it does not induce long-term genetic transformation.
  • RNA-type nucleic acid molecules can induce a desired in vivo immune response even when used in relatively small amounts compared to DNA-type nucleic acid molecules. Additionally, when producing nucleic acid molecules in the form of RNA, all manufacturing processes can be artificially controlled, so they can be safely produced in small-scale GMP (good manufacturing practice) production facilities without the risk of biological contamination. When producing RNA nucleic acid molecules, there is no need to directly deal with the infectious agent. Instead, only the nucleic acid sequence of the neutralizing antibody-inducing part (neutralizing epitope) of the infectious agent to be expressed is artificially synthesized and mass-produced using in vitro transcription (IVT). can produce RNA nucleic acid molecules. Recently, reagents related to IVT reactions, especially DNA-dependent RNA polymerase, have been improved, making it possible to rapidly produce large amounts of RNA within 1 to 2 weeks using a small amount of DNA template.
  • IVT in vitro transcription
  • Nucleic acid molecules in the form of RNA can induce a stronger immune response than naked DNA nucleic acid molecules, and the RNA nucleic acid molecule itself produces a complex antigen within the cell, which is the major histocompatibility complex (MHC) of the antigen-presenting cell. ; Major histocompatibility complex) class II and can function as an ideal immune enhancer.
  • MHC major histocompatibility complex
  • multiple antigens to induce an immune response can be produced simultaneously, mixed, and then immunized, and there are no special restrictions on the gene length of the antigen to be expressed, which can increase the applicability and simplicity of producing RNA nucleic acid molecules.
  • an appropriate transcriptional regulatory element (TCCE) to enable IVT may be placed upstream of the first translational regulatory element (U-TLCE). Since nucleic acid molecules in the form of RNA can be synthesized through the IVT process, there is no need to directly deal with live viruses or pathogenic microorganisms used in the production of general live or killed vaccines, and yeast, which must be used to produce recombinant proteins/peptides, Cultivation of host cells such as E. coli or insect cells is not necessary.
  • TCCE transcription control element
  • TLCE translation control element
  • the nucleic acid molecule can be injected into the body on its own, or alternatively, the nucleic acid molecule can be inserted into a vector and injected into the body in the form of an expression construct or vector, which is a gene carrier.
  • Vectors that can be used as gene carriers can be produced in various forms, including viral vectors, DNA or RNA expression vectors, plasmids, cosmids, or phage vectors. , DNA or RNA expression vectors linked to cationic condensing agents (CCA), e.g., packaged DNA or RNA expression vectors packaged in liposomes or niosomes containing plasmids, specific cells such as producer cells. Includes eukaryotic cells.
  • CCA cationic condensing agents
  • nucleic acid molecules of the present disclosure are formulated to enter and be expressed in mammalian cells. Such compositions may be particularly useful for use in the treatment and/or prevention of disease. There are many methods for expressing nucleic acid molecules in host cells, and any suitable method can be used. For example, nucleic acid molecules according to the present disclosure can be applied to any expression construct or gene delivery system.
  • vector refers to a circular double-stranded DNA loop within which additional DNA segments can be ligated.
  • a phage vector refers to a viral vector in which additional DNA segments can be ligated into the viral genome.
  • a particular vector is capable of autonomous replication within the host cell into which it is introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • expression vectors useful in recombinant DNA technology often exist in the form of plasmids.
  • a nucleic acid molecule can be inserted into a host cell using a viral gene transfer system.
  • Viral vectors into which nucleic acid molecules can be inserted include adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia or other pox viruses (e.q., avian pox virus). ), lentivirus, and herpes simplex virus-derived vectors.
  • viral vectors include vectors derived from lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV); These include, but are not limited to, retroviral vectors derived from murine retroviruses, gibbon ape leukemia virus, adeno-associated viruses (AAVs), and adenoviruses.
  • retroviral vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), and ecotropic retroviruses are widely used.
  • the vector system according to the present disclosure can be constructed through various methods known in the art.
  • the above-mentioned nucleic acid molecule can be inserted into an appropriate vector and then transformed into an RNA-type nucleic acid molecule through in vitro transcription (IVT).
  • IVTT in vitro transcription
  • Retroviral vectors contain target sites (such as genes for selectable markers that facilitate identification or selection of transduced cells and/or genes encoding ligands that serve as receptors for specific target cells). targeting moiety) can be additionally inserted. Targeting can also be achieved by known methods using antibodies.
  • vectors available and known in the art can be used for the purposes of this disclosure. The choice of an appropriate vector will largely depend on the size of the nucleic acid molecule inserted into the vector and the particular host cell being transformed with the vector. Each vector contains a variety of components depending on its function (amplification or expression of heterologous polynucleotides, or both) and compatibility with the particular host cell in which the vector resides. Vector components typically include, but are not limited to, an origin of replication (especially if the vector is inserted into a prokaryotic cell), a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and/or a transcription termination sequence. does not
  • the expression vector according to the present disclosure may include expression control sequences that can affect the expression of a gene in the form of an ORF encoded within the coding region (CR) or its transcription sequence, such as an initiation codon, a stop codon, an enhancer, etc. , signal sequences for membrane targeting or secretion, etc.
  • Enhancer sequences are nucleic acid base sequences that are located at various sites in a promoter and increase transcriptional activity compared to the transcriptional activity caused by the promoter in the absence of the enhancer sequence.
  • the host is a bacterium of the Escherichia genus
  • the PhoA signal sequence, OmpA signal sequence, etc. can be used, and if the host is a bacterium of the genus Bacillus , the ⁇ -amylase signal sequence, subtilisin signal sequence, etc. can be used. there is.
  • the host is yeast, the signal sequence can be used as the MF- ⁇ signal sequence, SUC2 signal sequence, etc.
  • the host is an animal cell, the insulin signal sequence, a-interferon signal sequence, or antibody molecule signal sequence can be used.
  • the vectors of the present disclosure can typically be constructed as vectors for cloning or vectors for expression.
  • the vector of the present disclosure can be constructed using prokaryotic cells or eukaryotic cells as hosts.
  • vectors that can be used in the present disclosure include plasmids (e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phages (e.g., ⁇ gt4 ⁇ B, ⁇ -Charon) that are often used in the art. , ⁇ z1, ⁇ GEM.TM.-11, and M13, etc.) or viruses (e.g., SV40, etc.).
  • plasmids e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.
  • phages e.g., ⁇ gt4 ⁇ B, ⁇ -Charon
  • Constitutive or inducible promoters may be used in the present disclosure depending on the needs of the particular situation as can be ascertained by one skilled in the art.
  • a large number of promoters recognized by a variety of possible host cells are well known.
  • the selected promoter is selected by removing the promoter from the source nucleic acid molecule through restriction enzyme digestion and inserting the isolated promoter sequence into a selection vector, thereby having a coding region (CR) consisting of an ORF of a gene or transcript encoding an immunogenic peptide or protein.
  • CR coding region
  • Both native promoter sequences and multiple heterologous promoters can be used to direct amplification and/or expression of the genes or transcripts that make up the coding region (CR).
  • Heterologous promoters generally allow greater transcription and higher yields of the expressed gene of interest compared to native promoters.
  • the vector of the present disclosure is an expression vector and a prokaryotic cell is used as a host
  • a strong promoter capable of advancing transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter
  • rac5 promoter amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • ECS expression control sequence
  • the promoter and operator regions of the E. coli tryptophan biosynthetic pathway and the left-handed promoter of phage ⁇ (pL ⁇ promoter) can be used as regulatory regions.
  • a promoter derived from the genome of a mammalian cell e.g., metallothionein promoter
  • a promoter derived from a mammalian virus e.g., adenovirus
  • Viral late promoters e.g., vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV
  • bacteriophage-derived promoters e.g., T7 promoter, T3 promoter, SM6 promoter
  • a sequence it generally has a polyadenylation signal sequence and/or a polyadenosine sequence (PA).
  • PA polyadenosine sequence
  • the recombinant vector of the present disclosure when it is an expression vector capable of replication, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated. Additionally, the recombinant vector may include a selection marker.
  • the selection marker is used to select cells transformed with a vector, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins may be used.
  • the vector of the present disclosure is a selection marker and includes antibiotic resistance genes commonly used in the art, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and There is a gene for resistance to tetracycline.
  • selection markers include auxotrophic markers such as ura4, leu1, and his3, but the above examples do not limit the types of selection markers that can be used in the present disclosure.
  • RNA polymerase-based techniques are known to amplify sequences subcloned into expression vectors. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), Q ⁇ -replicase amplification, and other RNA polymerase-based techniques.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q ⁇ -replicase amplification Q ⁇ -replicase amplification
  • the vector of the present disclosure may be fused with other sequences to facilitate purification of the recombinant protein or peptide expressed therefrom.
  • Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6 x His (hexahistidine; Quiagen, USA). Because of the additional sequences required for purification, proteins expressed in the host are quickly and easily purified via affinity chromatography. If necessary, sequences encoding Fc fragments may be fused to facilitate extracellular secretion of these recombinant proteins.
  • the fusion protein expressed by a vector containing the fusion sequence is purified by affinity chromatography.
  • affinity chromatography For example, when glutathione-S-transferase is fused, glutathione, the substrate of this enzyme, can be used, and when 6 x His is used, a Ni-NTA His-linked resin column (Novagen, USA) can be used to Recombinant proteins can be obtained quickly and easily.
  • Host cells capable of stably and continuously cloning and expressing the above-described vectors are known in the art and any host cell can be used, such as E. coli JM109, E. coli BL21(DE3), E. coli RR1, Bacillus genus strains such as E. coli LE392 , E. coli B, E. coli There are intestinal bacteria and strains such as .
  • yeast Saccharomyce cerevisiae
  • insect cells e.g., SF9 cells
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, etc.
  • COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines can be used.
  • the vector of the present disclosure can be used to genetically modify cells inside or outside the cell ( in vivo, ex vivo ) or in vitro ( in vitro ).
  • Methods for genetically modifying cells include infection or transduction of cells with viral vectors, calcium phosphate precipitation, and bacterial protoplasts containing DNA into recipient cells.
  • Method of fusion method of processing liposomes or microspheres containing DNA in receptor cells, endocytosis (DEAE dextran, receptor-mediated endocytosis), electroporation, microinjection Several methods are known, including micro-injection.
  • the host cell when it is a prokaryotic cell, it may be performed by the CaCl 2 method, the Hananhan method, and/or the electroporation method.
  • the vector when the host cell is a eukaryotic cell, the vector is introduced into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and/or gene bombardment. It can be injected.
  • a vector injected into a host cell can be expressed within the host cell, in which case a large amount of recombinant protein or peptide is obtained. For example, if the expression vector contains the lac promoter, gene expression can be induced by treating the host cell with IPTG.
  • nucleic acid molecule of the present disclosure in addition to the above-described configuration, may include other nucleotide sequences through which the gene of interest or its transcription sequence can be expressed stably and efficiently.
  • Figure 2 is a schematic diagram schematically showing the structure of a nucleic acid molecule according to another exemplary embodiment of the present disclosure.
  • the nucleic acid molecule illustrated in Figure 2 has a first polyadenylation signal sequence or a first polyadenylation signal sequence downstream of an expression control element (ECE), specifically a second translation control element (D-TLCE).
  • ECE expression control element
  • D-TLCE second translation control element
  • a polyadenylation signal sequence (PA1) and a second polyadenylation signal sequence or a second polyadenosine sequence located downstream of the first polyadenylation signal sequence or the first polyadenosine sequence (PA1) via a linker Includes PA.
  • the nucleic acid molecule may further comprise a second polyadenylation signal sequence or a G-Quadruplex sequence located downstream of the second polyadenosine sequence (PA2).
  • the first and second polyadenylation signal sequences or the first and second polyadenosine sequences may each have the same structure as the polyadenylation signal sequence or polyadenosine sequence (PA) described in FIG. 1.
  • the linker located between the first and second polyadenylation signal sequences or the first and second polyadenosine sequences (PA1, PA2) may consist of approximately 3 to 20 arbitrary nucleotides.
  • the G-Quadruplex sequence may consist of the base sequence of Formula 1 below or its transcription sequence, but is not limited thereto.
  • G represents guanine and X is any of adenine, cytosine, guanine, and thymine; m, p, r and t are each independently an integer from 1 to 10; n, q and s are each independently integers from 0 to 10.
  • Equation 1 m, p, r and t are each independently an integer from 2 to 5, for example an integer from 3 to 5, and n, q and s are each independently an integer from 1 to 7.
  • the G-Quadruplex sequence having the nucleotide sequence of Formula 1 may include, but is not limited to, the telomeric repeat sequence of yeast of the genus Candida .
  • the present disclosure relates to a composition for enhancing immune activity, such as a vaccine composition comprising a pharmaceutically effective amount of the above-described nucleic acid molecule or a gene carrier containing the above-described nucleic acid molecule as an adjuvant.
  • the present disclosure includes a pharmaceutically effective amount of the above-described nucleic acid molecule or a gene carrier comprising the above-described nucleic acid molecule as an immune adjuvant, and a pharmaceutically acceptable carrier, optionally pharmaceutically. It relates to a vaccine composition comprising an active substance.
  • pharmaceutically effective amount means an amount sufficient to achieve the efficacy or activity of the nucleic acid molecule according to the present disclosure.
  • the above-described nucleic acid molecule can be administered directly to the subject or in the form of a gene delivery system.
  • the present disclosure relates to a method of enhancing immune activity in a subject in vivo, comprising administering to the subject a pharmaceutically effective amount of the above-described nucleic acid molecule.
  • the vaccine composition may include a nucleic acid stabilizer.
  • the nucleic acid molecules described above can be stabilized in vaccine compositions using cationic polymers, cationic peptides, or cationic glandular polypeptides.
  • Cationic (poly)peptides that can be used as stabilizers can be polycationic polymers such as polylysine or polyarginine, cationic lipids or lipofectants. More specifically, the stabilizers include histones, nucleolins, protamines, oligofectamines, spermine or spermidine and cationic polysaccharides, especially chitosan, TDM, MDP, muramyl dipeptide, pluronic and/or these.
  • Histones and protamines are cationic proteins that naturally compact DNA.
  • Histones that can form complexes with nucleic acid molecules used as adjuvants according to the present disclosure include histones H1, H2a, H3 and H4, and protamine may be protamine P1 or P2, especially the cationic partial sequence of protamine. , but is not limited to this.
  • the vaccine composition may further comprise other compounds.
  • Other compounds capable of forming complexes with the nucleic acid molecules according to the present disclosure may be other adjuvants used additionally. Additional adjuvants enhance the immunological activity of the pharmaceutically active substance and/or nucleic acid molecule used as an adjuvant.
  • adjuvants included in the vaccine composition include protamine, nucleolin, spermine, spermidine and cationic polysaccharides, and stabilizing cationic peptides or polypeptides, especially chitosan, TDM, MDP and muramyl dipeptide.
  • PRRs pattern recognition receptors
  • TLRs Toll-like receptors
  • RIG-I-like receptors RLRs
  • NLRs NOD-like receptors
  • the vaccine composition can be prepared as a sustained-release formulation.
  • sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing nucleic acid molecules or gene carriers, which matrices are in the form of molded articles, for example films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide, L-glutamic acid, and gamma-ethyl- copolymers of L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers and poly-D-(-)-3-hydroxybutyric acid.
  • hydrogels e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)
  • polylactide e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)
  • L-glutamic acid e.glutamic acid
  • gamma-ethyl- copolymers of L-glutamate e.glutamic acid
  • non-degradable ethylene-vinyl acetate e.
  • the vaccine composition may include lipid nanoparticles (LNPs) that can protect nucleic acid molecules used as active ingredients and improve bioinjection activity.
  • LNPs lipid nanoparticles
  • Lipid nanoparticles contain multiple lipid molecules physically associated with each other, including microspheres (including unilamellar and multilamellar vesicles such as liposomes), dispersed phases in emulsions, micelles, or internal phases in suspension.
  • Lipid nanoparticles can be used to encapsulate nucleic acid molecules or peptides (proteins) expressed from nucleic acid molecules of the present disclosure for delivery.
  • Agents containing cationic lipids are useful for delivering polyanions such as nucleic acid molecules.
  • Other lipids that may be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and lipids that increase the length of time the nanoparticles can remain in vivo. It is a stealth lipid.
  • neutral lipids i.e., uncharged or zwitterionic lipids
  • anionic lipids i.e., helper lipids that enhance transfection
  • lipids that increase the length of time the nanoparticles can remain in vivo. It is a stealth lipid.
  • suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids are disclosed in WO 2016/010840 A1, incorporated herein by reference.
  • lipids for encapsulation can be cationic lipids, biodegradable lipids.
  • such a lipid may be (9Z,12Z)-3((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy )methyl)propyloctadeca9,12-dienoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl ) Bis(decanoate), 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9 'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), 3-(((3-(di)(d
  • Suitable neutral lipids may include neutral, uncharged or zwitterionic lipids.
  • Neutral phospholipids include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), and dimyristoylphosphatidylcholine.
  • DMPC phosphatidylcholine
  • PLPC phosphatidylcholine
  • DAPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • PE phosphatidylethanolamine
  • EPC nonphosphatidylcholine
  • DLPC dimyristoylphosphatidylcholine
  • PMPC 1-palmitoyl-2-myristoylphosphatidylcholine
  • PSPC 1,2-diarachidoyl-sn-glycero-3-phosphocholine
  • SPPC 1,2 die It may include, but is not limited to, eicosenoyl-sn-glycero-3-phosphocholine (DEPC
  • Helper lipids can enhance transfection and/or enhance membrane fusogenicity.
  • Helper lipids include steroids, sterols, and alkyl resorcinols.
  • helper lipids may include cholesterol, 5-heptadecyl resorcinol, and cholesterol hemisuccinate.
  • Stealth lipids can aid the formulation process by reducing particle aggregation and controlling particle size, or modulate the pharmacokinetic properties of LNPs.
  • stealth lipids include PEG (polyethylene glycol or polyethylene oxide), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acid, and poly N-( It may include a polymer having a hydrophilic head such as 2-hydroxypropyl) methacrylamide.
  • the stealth lipids are PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), and PEG-dimethylglycerol.
  • Nucleic acid molecules of the present disclosure that can be used as adjuvants can induce a non-antigen specific immune response.
  • T-lymphocytes differentiate into T-helper 1 (Th 1 ) cells and T-helper 2 (Th 2 ) cells, and the immune system responds to intracellular (Th1) and extracellular (Th2) pathogens (e.g. For example, antigens) can be destroyed.
  • Th1 cells support cellular immune responses by activating macrophages and cytotoxic T-cells.
  • Th 2 cells promote humoral immune responses by augmentation of B-cells for conversion into plasma cells and formation of antibodies (e.g. against antigens).
  • the Th 1 / Th 2 ratio is very important in the immune response, and the nucleic acid molecule of the present disclosure enhances and induces the Th 1 immune response, that is, a cell-mediated immune response. Therefore, when the nucleic acid molecule according to the present disclosure is administered to a living body together with a pharmaceutically active substance, for example, an immune enhancing ingredient, the nucleic acid molecule of the present disclosure enhances a specific immune response induced by the pharmaceutically active substance. It can act as an immune enhancer.
  • a pharmaceutically active substance for example, an immune enhancing ingredient
  • the vaccine composition may include pharmaceutically active substances in addition to the nucleic acid molecules described above as adjuvants.
  • the pharmaceutically active substance is an immune enhancing ingredient, such as an immunogen.
  • a pharmaceutically active substance may be a compound that has therapeutic and/or preventive effects against cancer, infectious diseases, autoimmune diseases, or allergies.
  • pharmaceutically active substances include peptides, proteins, nucleic acids, therapeutically active low molecular weight organic or inorganic compounds, sugars, antigens or antibodies, therapeutic agents known in the art, antigen cells, fragments of antigen cells. , cell fragments, and pathogens (such as viruses or bacteria) that have been modified (e.g. attenuated or inactivated) by chemical or light irradiation.
  • an antigen which is one of the pharmaceutically active substances, may be peptides, polypeptides, proteins, cells, cell extracts, polysaccharides, complex polysaccharides, lipids, glycolipids, and carbohydrates.
  • surface antigens of tumor cells and surface antigens especially those of viral pathogens, bacterial pathogens, fungal pathogens or protozoan pathogens, may be in secreted form.
  • the antigen may exist, for example, inside a nucleic acid molecule according to the present disclosure, or may also exist as a hapten bound to a suitable carrier.
  • Other antigenic components, inactivated or attenuated pathogens may also be used.
  • tumor antigens are, among others, tumor-specific surface antigens (TSSA), such as 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5 -beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/ abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA,
  • the class of tumor antigens is suitable for the purpose of the present disclosure, for example, tumor antigens known to be involved in neovascularization, affecting extracellular matrix structure, etc.
  • the tumor antigen may be provided in the vaccine composition as a protein or peptide antigen, or as a tumor antigen or an epitope thereof, or as mRNA or DNA encoding the tumor antigen.
  • a pathogenic antigen can be derived from a pathogenic organism that elicits an immune response by an individual, such as a mammalian individual, especially a human.
  • pathogenic antigens may be derived from bacterial, viral, or protozoological (multicellular) pathogenic organisms.
  • the pathogenic antigen is a surface antigen, e.g., a protein (or fragment of a protein, e.g., an external portion of the surface lower body) located on the surface of an organism, such as a virus, bacterium, or protozoa. It can be.
  • Pathogenic antigens are protein or peptide antigens derived from pathogens associated with infectious diseases.
  • pathogens associated with infectious diseases include: Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, and Ancylostoma braziliense.
  • Ancylostoma duodenale Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus ), Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus , Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei ), Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae (
  • Epstein-Barr Virus EBV
  • Escherichia coli O157:H7, O111 and O104:H4 Fasciola hepatica and Fasciola gigantica
  • FFI prion Filarioidea superfamily
  • Flaviviruses Francisella tularensis, Fusobacterium genus ( Fusobacterium genus), Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenza, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus), Hepatitis A virus A Virus), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunode
  • VZV Varicella zoster virus
  • VZV Varicella zoster virus
  • Variola major or Variola minor vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholera (Vibrio cholera), West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica , Yersinia pestis, Yersinia pseudotuberculosis and Zika virus.
  • influenza viruses respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papilloma virus (HPV), human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, and dengue viruses.
  • RSV respiratory syncytial virus
  • HSV herpes simplex virus
  • HPV human papilloma virus
  • HIV human immunodeficiency virus
  • Plasmodium Staphylococcus aureus
  • dengue viruses Chlamydia trachomatis
  • CMV cytomegalovirus
  • HBV hepatitis B virus
  • Mycobacterium tuberculosis rabies virus
  • yellow fever virus Middle East respiratory syndrome coronavirus (MERS-CoV)
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • Zika virus coronaviruses.
  • the vaccine composition according to the present disclosure may include a pharmaceutically acceptable carrier in addition to nucleic acid molecules and pharmaceutically active substances used as adjuvants.
  • the pharmaceutically acceptable carrier may include, for example, pyrogen-free water; isotonic saline or buffered (aqueous) solutions such as phosphates, citrates, etc.; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of the cacao fruit; For example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; It may contain polyols such as arginic acid.
  • an aqueous buffer containing sodium salt, calcium salt, and optionally potassium salt may be used.
  • Sodium, calcium, and potassium salts may exist in the form of halogens such as chlorine, iodine, or bromine, or in the form of hydroxides, carbonates, bicarbonates, or sulfates.
  • the pharmaceutically acceptable carrier may include a solid carrier such as a solid filler, liquid filler, or diluent, and an encapsulating compound suitable for administration to a living body may also be used.
  • pharmaceutically acceptable solid carriers include sugars such as lactose, glucose, and sucrose; Starches, for example corn starch or potato starch; Cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; Powdered tragacanth; malt; gelatin; tallow;
  • solid lubricants such as stearic acid and magnesium stearate; Calcium sulfate, etc.
  • a pharmaceutically acceptable carrier may be selected depending on the manner in which the vaccine composition according to the present disclosure is administered.
  • Vaccine compositions according to the present disclosure can be administered, for example, systemically.
  • Routes of administration include, for example, oral, subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracardial, intraarterial, and sublingual ( Includes sublingual topical and/or intranasal routes.
  • the appropriate amount of vaccine composition that should be used can be determined by routine experiments using animal models. Such models include, without limitation, rabbit, sheep, mouse, rat, dog, and non-human primate models.
  • Unit dosage forms for injection include sterile aqueous solutions, physiological saline, or mixtures thereof. The pH of such solution should be adjusted to approximately 7.4.
  • Carriers suitable for injectable use include hydrogels, controlled release devices, delayed release devices, polylactic acid, and collagen matrices.
  • Pharmaceutically acceptable carriers suitable for topical use include those suitable for use in lotions, creams, gels and the like. If the compound is to be administered perorally, tablets, capsules and the like are in unit dosage form. Pharmaceutically acceptable carriers for the preparation of unit dosage forms that can be used for oral administration are well known in the art.
  • the vaccine composition according to the present disclosure may additionally include one or more auxiliary substances.
  • auxiliary substances are compounds that allow maturation of dendritic cells (DC), such as lipopolysaccharide, TNF-alpha or CD40 ligand, GM-CFS and/or cytokines.
  • DC dendritic cells
  • auxiliary substances include compounds that allow maturation of dendritic cells (DC), such as lipopolysaccharide, TNF-alpha or CD40 ligand, GM-CFS and/or cytokines.
  • various interleukins, interferons, monokines, lymphokines, etc. that promote immune responses, such as growth factors such as GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, and hGH.
  • Cytokines such as interleukins or keokines.
  • the vaccine composition according to the present disclosure may additionally contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, lubricants, processing aids, colorants, sweeteners, fragrances, flavoring agents, Diluents, and other known additives that provide an attractive appearance to the drug (i.e., the nucleic acid molecule that is the active ingredient of the present disclosure, gene carrier, or vaccine composition) or assist in the manufacture of pharmaceutical products (i.e., medicaments).
  • emulsifiers such as Tween; Wetting agents, for example sodium lauryl sulfate; coloring agent; Taste-imparting agents, agents forming tablets; stabilizer; antioxidant; It is a preservative.
  • nucleic acid molecules used as an adjuvant according to the present disclosure is not particularly limited.
  • nucleic acid molecules according to the present disclosure may be used in a vaccine composition at a concentration of about 1 to about 1000 ⁇ g/ml, for example, but not limited to, about 10 to about 1000 ⁇ g/ml. .
  • T-lymphocytes typically differentiate into two subpopulations: T-helper 1 (Th 1 ) cells and T-helper 2 (Th 2 ) cells, which are capable of protecting against intracellular (Th 1) pathogens and extracellular (Th 1 ) pathogens.
  • Th 1 T-helper 1
  • Th 2 T-helper 2
  • Th 1 T-helper 1
  • Th 2 T-helper 2
  • Th 1 T-helper 1
  • Th 2 T-helper 2
  • Th 2 has an immune system that can destroy pathogens (e.g. antigens).
  • the two Th cell populations differ in the patterns of effector proteins (cytokines) produced by them.
  • Th 1 cells support cellular immune responses by activation of macrophages and cytotoxic T-cells, primarily associated with humoral immunity.
  • Th 2 cells mainly initiate humoral immune responses by augmenting B-cells for conversion to plasma cells in relation to cellular immunity and by the formation of antibodies (e.g., antibodies against antigens). promotes Therefore, the Th 1 / Th 2 ratio is quite important in the immune response. Nucleic acid molecules according to the present disclosure enhance both Th 1 and Th 2 immune responses.
  • the vaccine composition according to the present disclosure can be used to prevent tumors and infectious diseases by inducing a tumor-specific or pathogen-specific immune response.
  • the vaccine composition may be used for the prevention of allergic disorders or diseases, but is not limited to autoimmune diseases.
  • Vaccine compositions according to the present disclosure may be administered in any convenient dosage form, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc.
  • These vaccine compositions may contain ingredients customary for pharmaceutical preparations, such as diluents, carriers, pH adjusters, sweeteners, bulking agents and further active agents.
  • the vaccine composition of the present disclosure is manufactured in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the invention pertains. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersant or stabilizer.
  • the formulations herein may also contain more than one active compound, for example, compounds with complementary activities that do not adversely affect each other, if required for the particular indication to be treated.
  • the vaccine composition may comprise agents that enhance its function, such as, for example, cytotoxic agents, cytokines, chemotherapeutic agents, or growth-inhibiting or growth-enhancing agents. These molecules are suitably present in combination in amounts effective for the intended purpose.
  • a gene carrier comprising a nucleic acid molecule of the present disclosure may be included in a vaccine composition.
  • a gene delivery system is designed to transport and express nucleotides encoding the desired cytokine.
  • the transcript of the gene of interest can be presented in a suitable expression construct.
  • the transcript of the gene of interest encoding the cytokine can be operably linked to a transcriptional regulatory element (TCCE).
  • TCCE transcriptional regulatory element
  • the expression construct may be an expression vector into which the above-described nucleic acid molecule is inserted.
  • the vector may include a nucleic acid molecule, and the nucleic acid molecule may combine with another nucleotide sequence to encode a fusion protein or fusion peptide.
  • the method of introducing the above-described gene delivery system into cells can be carried out through various methods known in the art.
  • the gene delivery vehicle when the gene delivery vehicle is produced based on a viral vector, it is carried out according to a viral infection method known in the art.
  • the gene delivery system in the present disclosure is a naked recombinant DNA molecule or plasmid, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, DEAE-dextran treatment method), and gene bombardment method Genes can be transfected into cells by this method.
  • Example 1 Construction of nucleic acid molecules of RNA platform
  • RNA nucleic acid molecule with IRES activity derived from SINV-1 was produced.
  • the template DNA was designed as follows.
  • the template DNA was cloned into the pGH vector, linearized with restriction enzymes, and a nucleic acid molecule (SINV-1-MCS) in the form of an RNA platform was produced through in vitro transcription (IVT).
  • Comparative Example 1 Construction of nucleic acid molecules of RNA platform
  • a nucleic acid molecule (CrPV-MCS) in the form of an RNA platform was produced by repeating the procedure of Example 1, except that the design of the template DNA was modified as follows:
  • Comparative Example 2 Construction of nucleic acid molecules of RNA platform
  • a nucleic acid molecule (EMCV-MCS) in the form of an RNA platform was produced by repeating the procedure of Example 1, except that the design of the template DNA was modified as follows:
  • Wild type Balb/c mice (5 per group) were injected intramuscularly with the immunogenic Influenza subunit vaccine (SKY cell flu, 0.6 ⁇ g) and each nucleic acid molecule produced in Example 1 and Comparative Examples 1-2 (20 ⁇ g each). , was immunized. Nucleic acid molecules were purified with cellulose without removing endotoxin. The second immunization was performed 2 weeks after the first immunization. Immunoglobulin production was measured 2 weeks after the first immunization and 1 week after the second immunization, blood was collected from the mouse 1 week after the second immunization, the mouse was euthanized using carbon dioxide, and an autopsy was performed via abdominal vein. Blood and organs were extracted and analyzed. The control group and experimental group were divided as shown in Table 1 below.
  • H1N1 neutralizing antibodies against A/California/04/2009
  • H1N1 a virus homologous to H1N1
  • Mouse serum was mixed with RDE (receptor destroy enzyme) in a ratio of 1:3 and reacted overnight at 37°C. After inactivation at 56°C for 30 minutes, it was diluted 1/10 by adding PBS. 25 ⁇ l of PBS was added to a 96-well v-bottom plate. 25 ⁇ l of mouse serum diluted 1/10 was added to the first column, and sequentially diluted two-fold to the next column.
  • Influenza subunit vaccine Influenza virus (A/California/04/2009) homologous to the vaccine strain was diluted to 8 HAU (hemagglutinating unit)/50 ⁇ l, and then 25 ⁇ l each was added to each well and reacted for 30 minutes.
  • red blood cells were agglutinated.
  • the reason red blood cells agglutinate is because of the HA antigen of the virus, and the reason red blood cells do not agglutinate is because there are HA antibodies in the serum.
  • MDCK cells were spread in a 96-well plate at 3x10 4 cells/well. 12 ⁇ l of mouse serum was prepared and inactivated at 56°C for 30 minutes. The inactivated serum was diluted 10 times with medium and 80 ⁇ l each was added to the first row of an empty 96-well plate. 40 ⁇ l of medium was added to other wells, and the first line of serum was diluted two-fold, 40 ⁇ l each. 40 ⁇ l of 100 TCID50 (Tissue Culture Infective Does 50%) virus was added to each well and reacted for 1 hour in an incubator at 37°C.
  • TCID50 Tissue Culture Infective Does 50%
  • 50 ⁇ l of the reacted virus and serum were placed on MDCK cells in a 96-well plate from which the medium had been removed, and the cells were infected in a 37°C incubator for 2 hours. After 2 hours, 50 ⁇ l of MEM complete medium containing 2% FBS was added to each well and placed in an incubator at 37°C. Cells were observed over time, and if cell lesions occurred, all supernatant was removed, 4% formaldehyde solution was added, and the cells were fixed for more than 3 hours. Afterwards, 2% crystal violet solution was added to each well and stained for over 30 minutes, washed with water, and neutralizing antibody titer was measured.
  • mice 1 week after secondary immunization were ground using a 40 ⁇ m strainer to produce as many single cells as possible.
  • 50 ⁇ l of ground spleen cells were placed in a pre-coating ELISpot plate at 5x10 5 cells/well.
  • For non-stimulation add 50 ⁇ l of RPMI-1640 complete medium containing 10% FBS and 1% antibiotics, and for protein stimulation, add 50 ⁇ l of the vaccine used for immunization at a concentration of 500 ng/well to the complete medium, respectively.
  • IFN-gamma, IL-4, and specific antibody (1 st antibody) were diluted 1:1000 in FBS 0.5% PBS, added at 100 ⁇ l/well, and incubated at room temperature for 2 hours. After washing with PBS 5 times, Stratavidin antibody was diluted 1:1000 with FBS 0.5% in PBS and added at 100 ⁇ l/well. After incubation at room temperature for 1 hour, the cells were washed 5 times with PBS. BCIP/NBT (substrate solution) was added to each well after filtering to develop color, and the reaction was stopped by washing with running tap water.
  • BCIP/NBT substrate solution
  • the spleen of the mouse removed from the mouse 1 week after the secondary immunization was ground using a 40 ⁇ m strainer to produce as many single cells as possible.
  • After placing 1x10 6 cells/well in a 96-well round bottom plate add 50 ⁇ l of the vaccine used for immunization to the complete medium at a concentration of 500 ng/well, stimulate it, and place in an incubator at 37°C for 16 hours.
  • Cultured After staining with T cell-specific antibodies and antibodies specific for IFN-gamma, IL-2, and TNF-alpha, the number of T cells producing IFN-gamma, IL-2, and TNF-alpha was measured using flow cytometry. Measured. The measurement results are shown in Figures 9 to 11. The number of T cells secreting specific cytokines was improved in mice immunized with the nucleic acid molecule prepared in Example 1.
  • the spleen of the mouse removed from the mouse 1 week after the secondary immunization was ground using a 40 ⁇ m strainer to produce as many single cells as possible.
  • the vaccine used for immunization at a concentration of 500 ng/well was added to the complete medium for stimulation by adding 50 ⁇ l each, and then cultured in an incubator at 37°C for 72 hours. Afterwards, the cell culture supernatant was obtained and the experiment was conducted according to the protocol provided by the manufacturer.
  • the analysis results are shown in Figures 12 and 13. It was confirmed that the secretion of IFN-gamma, TNF-alpha, IL-2, and IL-6 cytokines was significantly improved in the spleen of mice immunized with the nucleic acid molecule prepared in Example 1.
  • the procedure was carried out except that a template DNA was designed in which a linker (SEQ ID NO: 4) and 50 adenosines were additionally inserted between the 50 adenosines constituting the nucleic acid molecule of Example 1 and the Not1 recognition sequence at the 3' end.
  • the procedure of Example 1 was repeated to produce a nucleic acid molecule (SINV-1L-MCS) in the form of an RNA platform.
  • a linker (SEQ ID NO: 4), 50 adenosines, and a G-quadruplex (SEQ ID NO: 5) are additionally inserted between the 50 adenosines constituting the nucleic acid molecule of Example 1 and the Not1 recognition sequence at the 3' end.
  • a nucleic acid molecule (SINV-1LG-MCS) in the form of an RNA platform was produced by repeating the procedure of Example 1, except that one template DNA was designed.
  • Comparative Example 3 Construction of nucleic acid molecules of RNA platform
  • nucleic acid molecules constituting the nucleic acid molecule of Comparative Example 2 except that a template DNA was designed in which a linker (SEQ ID NO: 5) and 50 adenosines were additionally inserted between 50 adenosines and the Not1 recognition sequence at the 3' end. And, the procedure of Comparative Example 2 was repeated to produce a nucleic acid molecule (EMCVL-MCS) in the form of an RNA platform.
  • EMCVL-MCS nucleic acid molecule
  • Comparative Example 4 Construction of nucleic acid molecules of RNA platform
  • nucleic acid molecules constituting the nucleic acid molecule of Comparative Example 2 a linker (SEQ ID NO: 5), 50 adenosines, and a G-quadruplex (SEQ ID NO: 5) were formed between 50 adenosines and the NotI recognition sequence at the 3' end.
  • a nucleic acid molecule (EMCVLG-MCS) in the form of an RNA platform was produced by repeating the procedure of Comparative Example 2, except that an additionally inserted template DNA was designed.
  • Wild type Balb/c mice (5 per group) were administered the immunogenic Influenza subunit vaccine (SKY cell flu, 1.0 ⁇ g) and each nucleic acid molecule (20 ⁇ g each) prepared in Examples 2-3 and Comparative Examples 3-4 into the muscle.
  • immunization was performed. Nucleic acid molecules were purified with cellulose after removing endotoxin.
  • the second immunization was performed 2 weeks after the first immunization. Immunoglobulin production was measured 2 weeks after the first immunization and 2 weeks after the second immunization, blood was collected from the mouse 2 weeks after the second immunization, the mouse was euthanized using carbon dioxide, and an autopsy was performed via abdominal vein. Blood and organs were extracted and analyzed. The control group and the experimental group were divided as shown in Table 2 below.
  • an ORF-type coding region (sequence) encoding type A Hemagglutinin (SEQ ID NO: 10) of influenza virus A nucleic acid molecule (SINV-1L-FluA) in the form of an RNA platform was produced by repeating the procedure of Example 2, except that a template DNA was additionally inserted (identification number: 11).
  • an ORF-type coding region (SEQ ID NO: 13) encoding type B Hemagglutinin (SEQ ID NO: 12) of influenza virus is formed between the Cla I recognition sequence and the Pac I recognition sequence constituting the MCS.
  • a nucleic acid molecule (SINV-1L-FluB) in the form of an RNA platform was produced by repeating the procedure of Example 2, except that an additionally inserted template DNA was designed.
  • Comparative Example 5 Construction of nucleic acid molecules of RNA platform
  • an ORF-type coding region (sequence) encoding type A Hemagglutinin (SEQ ID NO: 10) of influenza virus A nucleic acid molecule (CrPV-FluA) in the form of an RNA platform was produced by repeating the procedure of Example 2, except that a template DNA was additionally inserted (identification number: 11).
  • Comparative Example 6 Construction of nucleic acid molecules of RNA platform
  • an ORF-type coding region (SEQ ID NO: 13) encoding type B Hemagglutinin (SEQ ID NO: 12) of influenza virus is formed.
  • a nucleic acid molecule (CrPV-FluB) in the form of an RNA platform was produced by repeating the procedure of Example 2, except that an additionally inserted template DNA was designed.
  • Wild type Balb/c mice (5 per group) were administered the immunogenic Influenza subunit vaccine (SKY cell flu, 1.0 ⁇ g) and each nucleic acid molecule produced in Examples 4-5 and Comparative Examples 5-6 (20 ⁇ g each) into the muscle. By injection, immunization was performed. Nucleic acid molecules were purified with cellulose after removing endotoxin. The immunization schedule was conducted according to the same procedure as in Experimental Example 7. The control group and the experimental group were divided as shown in Table 3 below.
  • mice immunized with the nucleic acid molecules prepared in Examples 4 and 5 after the first and second immunizations the production of both IgG1, an immunoglobulin related to the Th 2 immune response, and IgG2a, an immunoglobulin related to the Th 1 immune response, increased. did.
  • Wild type Balb/c mice (5 per group) were immunized by intramuscular injection with the immunogenic Influenza split vaccine (Covaxflu quadrivalent PF strain, 1.0 ⁇ g) and the nucleic acid molecule prepared in Example 3 (20 ⁇ g). Nucleic acid molecules were purified with cellulose after removing endotoxin.
  • the immunization schedule was conducted according to the same procedure as in Experimental Example 7. The control group and experimental group were divided as shown in Table 4 below.
  • cytokines secreted from the mouse spleen 2 weeks after the secondary immunization were analyzed using FACS according to the same procedure as Experimental Example 5. The analysis results are shown in Figures 27 to 29.
  • CD4 effector/memory T cells, memory B cells, as well as specific cytokines such as IFN-gamma, TNF-alpha, IL-2, and IL-4 were produced. The number of secreting T cells increased significantly.
  • cytokines secreted from the mouse spleen 2 weeks after the secondary immunization were analyzed using ELISA according to the same procedure as Experimental Example 6.
  • the analysis results are shown in Figures 30 to 32. It was confirmed that the secretion of cytokines was significantly increased in the spleen of mice immunized with the nucleic acid molecule prepared in Example 3.
  • Human peripheral blood mononuclear cells were placed in a 96-well plate at a number of 5x10 5 cells/well, and 200 ⁇ l of the nucleic acid molecules prepared in Example 3 at a concentration of 20 ⁇ g/ml were added to the complete medium. After stimulation, the cells were cultured in an incubator at 37°C for 48 hours. As a positive control group, 200 ⁇ l of LPS was added at a concentration of 1 ⁇ g/ml. After 48 hours, the cell culture supernatant was obtained and the experiment was performed according to the protocol provided by the manufacturer. The results of measuring cytokine secretion from cultured cell lines are shown in Figures 36 to 40.
  • cytokines related to Th 1 and Th 2 immune responses such as IFN-gamma, IL-2, IL-6, IL-10, and TNF-alpha, were measured in cells stimulated with the nucleic acid molecules produced in Example 3. Secretion was greatly increased and maintained.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un activateur immunitaire comprenant une molécule d'acide nucléique, l'activateur comprenant un élément de commande d'expression avec une activité de site d'entrée ribosomique interne (IRES) dérivée du virus de Solenopsis invicta (SINV). En utilisant l'activateur immunitaire à base d'acide nucléique, les deux réponses immunitaires Th1 et Th2 peuvent être induites. Un immunoadjuvant à base d'acide nucléique peut être utilisé dans des médicaments tels que des vaccins.
PCT/KR2023/008736 2022-06-29 2023-06-23 Activateur immunitaire à base d'acide nucléique formant une structure secondaire WO2024005457A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20220079941 2022-06-29
KR10-2022-0079941 2022-06-29

Publications (1)

Publication Number Publication Date
WO2024005457A1 true WO2024005457A1 (fr) 2024-01-04

Family

ID=89380844

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/008736 WO2024005457A1 (fr) 2022-06-29 2023-06-23 Activateur immunitaire à base d'acide nucléique formant une structure secondaire

Country Status (1)

Country Link
WO (1) WO2024005457A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102075581B1 (ko) * 2018-04-27 2020-02-10 가톨릭대학교 산학협력단 바이러스성 발현 조절 서열이 삽입된 핵산 분자를 포함하는 면역보강제 및 이를 포함하는 약학 조성물
US10736957B2 (en) * 2017-12-19 2020-08-11 President And Fellows Of Harvard College Enhanced immunogenicity of mRNA with co-encoded adjuvant sequences
WO2020237227A1 (fr) * 2019-05-22 2020-11-26 Massachusetts Institute Of Technology Compositions et procédés d'arn circulaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10736957B2 (en) * 2017-12-19 2020-08-11 President And Fellows Of Harvard College Enhanced immunogenicity of mRNA with co-encoded adjuvant sequences
KR102075581B1 (ko) * 2018-04-27 2020-02-10 가톨릭대학교 산학협력단 바이러스성 발현 조절 서열이 삽입된 핵산 분자를 포함하는 면역보강제 및 이를 포함하는 약학 조성물
WO2020237227A1 (fr) * 2019-05-22 2020-11-26 Massachusetts Institute Of Technology Compositions et procédés d'arn circulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HYEONGJUN PARK; HYE-JUNG KIM; HAE LI KO; HYE WON KWAK; DONG-HOON WON; DA-BIN HWANG; YOO-SUB SHIN; JUN-WON YUN; JAE-HWAN NAM: "Evaluation of the safety profile of ssRNA adjuvant derived from the IGR IRES region of CrPV. ", THE JOURNAL OF IMMUNOLOGY., vol. 202, no. 1_Supplement, 1 May 2019 (2019-05-01), pages 196.25, XP009551313 *
KWAK HYE WON; PARK HYO-JUNG; KO HAE LI; PARK HYELIM; CHA MIN HO; LEE SANG-MYEONG; KANG KYUNG WON; KIM RHOON-HO; RYU SEUNG ROK; KIM: "Cricket paralysis virus internal ribosome entry site-derived RNA promotes conventional vaccine efficacy by enhancing a balanced Th1/Th2 response", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 37, no. 36, 29 July 2019 (2019-07-29), AMSTERDAM, NL , pages 5191 - 5202, XP085759216, ISSN: 0264-410X, DOI: 10.1016/j.vaccine.2019.07.070 *

Similar Documents

Publication Publication Date Title
KR102075581B1 (ko) 바이러스성 발현 조절 서열이 삽입된 핵산 분자를 포함하는 면역보강제 및 이를 포함하는 약학 조성물
JP7212468B2 (ja) 標的免疫調節抗体および融合タンパク質に基づく組成物および方法
JP6515182B2 (ja) Ｗｔ１抗原ペプチドおよび免疫調節剤の併用
CN1212155C (zh) 用于治疗肿瘤的cd40结合分子和ctl肽
JP2019059774A (ja) 新生児及び乳幼児におけるワクチン接種
US20070212337A1 (en) Polypeptide-nucleic acid conjugate for immunoprophylaxis or immunotherapy for neoplastic or infectious disorders
US20030118588A1 (en) Induction of anti-tumor CTL immunity through in vivo triggering of 4-1BB and/or CD40
JP2017529841A (ja) キメラ抗原受容体
JP2021515577A (ja) ５’キャップトリヌクレオチドまたはより高級のオリゴヌクレオチド化合物、ならびにｒｎａの安定化、タンパク質の発現および治療法におけるそれらの使用
US20210100886A1 (en) Wt1 cancer antigen peptides and peptide conjugates comprising the peptides
AU2021402007B2 (en) Covalently modified antigens for improved immune response and/or stability
US10588952B2 (en) Conjugate vaccine using trimming function of ERAP1
US20210315992A1 (en) Adjuvant effect of the tlr1/2 agonist diprovocim synergizes with checkpoint-inhibiting antibodies to eliminate disease
KR102007926B1 (ko) 발현 조절 서열이 삽입된 핵산 분자 및 이를 포함하는 발현 벡터
US20200016255A1 (en) Wt1 helper peptides and combinations of wt1 helper peptide and conjugate of cancer antigen peptides
WO2024005457A1 (fr) Activateur immunitaire à base d'acide nucléique formant une structure secondaire
WO2019131722A1 (fr) Conjugué de peptide dérivé de wt1 et composition le comprenant
WO2023008814A1 (fr) Immunoadjuvant à base d'acide nucléique et composition de vaccin le comprenant
WO2023008881A1 (fr) Système d'expression et composition pharmaceutique à base d'acide nucléique le comprenant
JP7162675B2 (ja) 注射用組成物
RU2811940C2 (ru) 5'-кэп-тринуклеотидные соединения или 5'-кэп-соединения с большим количеством нуклеотидов и их применение для стабилизации рнк, экспрессии белков и в терапии
RU2819805C2 (ru) Композиции для т-клеточной терапии с химерным антигенным рецептором и их применения
US20050255106A1 (en) Induction of anti-tumor ctl immunity through in vivo triggering of 4-1bb and/or cd40
TW202200188A (zh) 用以處置癌症之醫藥組合物
KR20240034061A (ko) 변형에 의해 발현 효율을 증가시킨 핵산 분자, 이를 포함하는 발현 벡터 및 hpv 유래 질환 치료 또는 예방용 약학 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23831817

Country of ref document: EP

Kind code of ref document: A1