CN117043329A - Cargo molecule transduction domain RMAD1, variants thereof, recombinant cargo molecules and cargo molecule transduction methods utilizing the same - Google Patents
Cargo molecule transduction domain RMAD1, variants thereof, recombinant cargo molecules and cargo molecule transduction methods utilizing the same Download PDFInfo
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- CN117043329A CN117043329A CN202280021044.5A CN202280021044A CN117043329A CN 117043329 A CN117043329 A CN 117043329A CN 202280021044 A CN202280021044 A CN 202280021044A CN 117043329 A CN117043329 A CN 117043329A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a cell penetrating peptide derived from human ADARB2 and a cargo molecule transport system using the same, and provides a cargo molecule transduction domain comprising RMAD1 derived from human ADARB2 or a variant thereof, and a method for transporting cargo molecules into cells, wherein the method comprises the step of contacting recombinant cargo molecules fused with the cargo molecule transduction domain with cells. The cargo molecule transduction domain of the present invention is useful for transporting various polymer substances into cells of a human body because it is capable of introducing cargo molecules into cells with high efficiency as compared with conventional cell-penetrating peptides and because it is not necessary to worry about the problem of immune response as a polypeptide sequence derived from human proteins.
Description
Technical Field
The present invention relates to cargo molecule transduction domains RMAD1 derived from human ADARB2, variants thereof, gene constructs, vectors, recombinant cargo molecules encrypting thereof, and cargo molecule transduction methods utilizing the same, providing cargo molecule transduction domains comprising RMAD1 derived from human ADARB2 or variants thereof, and methods of intracellular transport of cargo molecules comprising the step of contacting a recombinant cargo molecule fused to the cargo molecule transduction domains and cargo molecules with cells.
Background
The protein transport technology is called a protein transduction domain (Protein Transduction Domain; PTD) or cell-penetrating peptide (Cell penetrating Peptide; CPP), and is a novel transport system capable of easily transporting a peptide consisting of approximately 5 to 30 amino acids to a living body such as mammalian cells, tissues, blood, etc., after fusing the peptide with a polymer such as a protein or a gene.
Although specific mechanisms have not been clarified, protein transport techniques are often used for transporting therapeutic proteins in vitro and in vivo, and into cells or tissues, and various protein transduction domains are known. In addition to covalent bonds, binding between the protein transduction domain and a biological cargo molecule (e.g., nucleic acid, protein, peptide, small molecule, cytotoxic drug, etc.) can be accomplished by various methods such as ionic bonding, electrostatic bonding, etc.
The protein transduction domain has advantages of low toxicity and less rejection compared to other transporters such as liposomes or polymers. However, there are few protein transduction domains used clinically and the like.
Double-stranded RNA-specific adenosine deaminase B2 (RNA-editing deaminase-2; abbreviated as "RED2" or "ADARB 2") is an enzyme that is encrypted in humans by the ADARB2 gene. The enzyme has insufficient editing activity, prevents other ADAR enzymes from combining with the targeting in the test tube, and reduces the effectiveness of the enzyme. ADARB2 proteins bind not only to dsRNA, but also to ssRNA. ADARB2 is a member of the double-stranded RNA (dsRNA) adenosine deaminase family of RNA editing enzymes. Adenosine deamination of Pre-mRNA results in changes in the amino acid sequence of the gene product, unlike what is predicted by genomic DNA sequence.
However, it has not been reported so far that a part of the sequence of ADARB2 protein is likely to be useful as a cargo molecule transduction domain.
Disclosure of Invention
The object of the present invention is to provide a cargo molecule transduction domain which allows a cargo molecule to permeate into a cell or tissue with high efficiency and which has no or little side effects when used in a human body, a recombinant cargo molecule fused to the cargo molecule transduction domain, and a method for allowing a cargo molecule to permeate into a cell or tissue using the cargo molecule transduction domain.
In order to solve the above problems, as a result of selecting a plurality of candidate peptides derived from human beings, the present inventors have confirmed that a peptide consisting of 15 amino acids derived from CKSKRRRRRRSKRKD of human ADARB2 protein (hereinafter, referred to as "RMAD1" in the present invention) or a peptide in which some amino acids are deleted, substituted and/or added can smoothly permeate a polymer such as a protein, a nucleic acid and the like into a living body such as a cell, a tissue and blood, and that the RMAD1 or a variant peptide thereof is significantly superior to HIV-Tat peptide in terms of cell and tissue permeation ability.
The present inventors have examined the self-permeation efficacy by using the cell-penetrating peptide RMAD1 derived from the human ADARB2 protein, and as a result, they have confirmed that the RMAD1 peptide is very permeable to cells than the HIV-Tat peptide by performing FACS experiments on the synthetic FITC.
In addition, the present inventors have made experiments to evaluate whether or not the RMAD1 peptide is bound to a cargo molecule, so that it is easy to permeate into cells and transport the cargo molecule into cells, and to attach EGFP (enhanced green fluorescent protein; enhanced Green Fluorescence Protein) protein as a cargo molecule for the experiment. The EGFP-RMAD1 fusion protein is proved to have excellent capability of transporting cargo molecules into cells compared with HIV-Tat peptide by using various verification methods such as immunoblotting, FACS and confocal microscopy by preparing and purifying the fusion protein carrier of EGFP and RMAD 1.
Effects of the invention
The cargo molecule transduction domain newly discovered by the present invention is excellent in cell penetration ability, is effectively used as a cargo molecule transduction substance, and is a substance derived from the human body, and thus is safe without risk of eliciting an immune response when administered to the human body.
In addition, the cargo molecule transduction domain of the present invention is remarkably superior to other cargo molecule transduction domains in cell permeation ability, and thus, various substances such as protein drugs, epitopes, etc., which are difficult to permeate cells, can be smoothly permeated, and thus, the cargo molecule transduction domain can be applied to drugs, cosmetics, etc.
Drawings
FIG. 1 is a 2-level structure prediction graph showing the cell-penetrating peptides of RMAD 1. A is the predicted 2-order structure of the peptide using the Pep-fold3 program. B is a graph showing Pepfold of the peptide.
A, B of FIG. 2 is the result of measuring the amount of FITC RMAD1 permeated into cells (fluorescence value) after treating 2.5uM of FITC-conjugated RMAD1 and 2.5uM of FITC-conjugated TAT with two cell lines of 3T3, B16F 10. C is the result of treatment at 2.5uM after conjugation of each of the RMAD1 and its variant with FITC, and then measuring the amount of FITC RMAD1 and its variant (fluorescence value) permeated into the cells. In addition to the untreated control group, a group treated with FITC TAT of 2.5uM was used as a control group.
FIGS. 3a to 3d show vector diagrams of RMAD1 fused with EGFP and purity of purified protein. FIG. 3a shows what restriction enzymes were used by EGFP-RMAD1 for subcloning (sub-cloning) into the pET28a vector. FIG. 3b is a result of confirming the molecular weight of purified EGFP-RMAD1 by Coomassie blue (Coomassie blue). FIG. 3c is an immunoblot result demonstrating that the molecular weight of purified EGFP-RMAD1 can be confirmed as well as a smooth purification. FIG. 3d is the result of comparing the transport rates of EGFP with EGFP, EGFP-TAT using immunoblotting after treatment of purified EGFP-RMAD1, EGFP-TAT to HaCaT cells.
FIGS. 4a to 4d are results of confirming cell permeability using FACS. FIG. 4a is a result of confirming the amount of EGFP transported to cells by FACS after 2.5uM EGFP-RMAD1, EGFP-TAT were treated to HaCaT cells, respectively. Fig. 4b is a graph showing the result of fluorescence values shown in fig. 4a using a histogram. FIG. 4c is the result of confirming the cell permeabilities of EGFP-RMAD1, EGFP-TAT using FACS after 2 hours of treatment at each amount. FIG. 4d is the result of confirming the cell permeabilities of EGFP-RMAD1, EGFP-TAT using FACS after 2.5uM treatment at each time.
FIG. 5a is a graph showing the results of confirming cell permeability by confocal microscopy of images of EGFP transported to cells after 2.5uM of EGFP-RMAD1, EGFP-TAT were treated separately to HaCaT cells. FIG. 5b is a result of confirming the position of the fusion protein by a fluorescence microscope using a lysosome probe (lysotracker) or a mitochondrial fluorescent needle (mitotracker) after the fusion protein was treated by the same method as in FIG. 5 a.
FIGS. 6a to 6e show vector diagrams of RMAD1 fused with SOD1 and purity of purified protein. FIG. 6a shows what restriction enzymes are used by SOD1-RMAD1 for subcloning (sub-cloning) into the pET29a vector. FIG. 6b is a result of confirming the molecular weight of purified SOD1-RMAD1 by Coomassie blue (Coomassie blue). FIG. 6c shows the result of immunoblotting to confirm the molecular weight of purified SOD1-RMAD1 and to confirm the smooth purification. FIG. 6d shows the results of comparing the relative production rates of reactive oxygen species by immunoblotting after treating purified SOD1-RMAD1, SOD1, and SOD1-TAT with HaCaT cells treated with 500ng/ml LPS. FIG. 6e is the result of detecting TNF-. Alpha.after culturing cells on serum-free medium for 1 hour, treating each well with 0.5, 1, 2.5uM SOD1-RMAD1 for 2 hours, then treating each well with 500ng/ml LPS for 16 hours, recovering the medium, centrifuging at 1000g for 10 minutes.
Fig. 7 a-7 e show experiments and experimental results fusing RMAD-1 and peptide antigens to confirm efficacy of an anti-cancer vaccine. Figure 7a shows experimental procedures of injecting TC-1 cells subcutaneously into mice, injecting E7 antigen and MPLA on day 6 and 13, respectively, and sacrificing on day 19. Fig. 7b is a graph showing tumor size changes based on control (Con), E7 antigen-administered (E7), E7-RMAD1 fusion peptide-treated (E7 AD), E7 antigen + MPLA (immune adjuvant and TLR4 agent) -treated (e7 + MPLA), E7-RMAD1 fusion peptide + MPLA-treated (E7 AD + MPLA). FIGS. 7c and 7d are results of analysis of immune cells in rat spleen, confirming E7AD containing RMAD1, and antigen-specific CD8 in E7AD+MPLA treated group + The increase in the number of T cells confirmed the increase in IFN-. Gamma.expression, and thus the CD8 + T cells are activated. FIG. 7E shows the results of comparison and analysis of E7-TAT binding by immunocytes of blood to TAT as the transduction domain of existing cargo molecules, effective in increasing antigen-specific CD8 in the E7-RMAD1 treated group + T cells.
Detailed Description
The main terms used in the description of the present invention, the claims and the like are defined as follows.
"cargo molecule" as a molecule that does not enter the original target cell or does not enter the target cell at the original useful rate, and is not the cargo molecule transduction domain or fragment thereof, refers to the target molecule itself or the target molecule portion of the cargo molecule transduction domain-target molecule complex prior to fusion with the cargo molecule transduction domain. The cargo molecule means a substance selected from the group consisting of proteins including antibodies, peptides including anti-inflammatory or antigenic epitopes, amino acids, nucleic acids, carbohydrates, polymers such as lipids, aptamers, liposomes, exosomes, and mixtures of 1 or more thereof.
"amino acid" and "amino acid residue" refer to natural amino acids, unnatural amino acids, or modified amino acids. When not mentioned, all references to amino acids include references to both specific D and L diastereomers (where such diastereomeric forms of the structure are allowed) either generally or by name. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Unnatural amino acids include deformed amino acid residues that are chemically deformed or reversibly or irreversibly blocked on the N-terminal amino acid or side chain, e.g., N-methylated D and residues in which the L amino acid or side chain functionality is chemically deformed by other functionalities.
"cargo molecule protein" is a term that refers to the case where the cargo molecule is a protein.
As included in the term "cargo molecule," a "target protein" as a molecule that cannot enter the original target cell or cannot enter the target cell at the original useful rate and is not the cargo molecule transduction domain or fragment thereof, refers to the molecule itself or the target molecule portion of the cargo molecule transduction domain-target molecule complex prior to fusion with the cargo molecule transduction domain. As target molecules, polypeptides, proteins, peptides are included. Examples of the target protein included in the target molecule include EGFP (enhanced green fluorescent protein; enhanced Green Fluorescent Protein), superoxide dismutase, epithelial cell growth factor, fibroblast growth factor, catalase, and the like, however, it is only a part of examples of the target protein to those of ordinary skill in the art, and the target protein is not limited thereto.
"recombinant cargo molecule" refers to a complex comprising a cargo molecule transduction domain and one or more cargo molecule portions, and formed by genetic fusion or chemical binding of the cargo molecule transduction domain and the cargo molecule. "fusion protein" refers to a recombinant cargo molecule that is genetically fused or chemically bound to a cargo molecule protein and a cargo molecule transduction domain. In the present specification, the same meaning as recombinant cargo molecule proteins is used.
The term "gene fusion" refers to a linkage formed by linear and covalent bonds formed by gene expression of a DNA sequence encoding a protein. In addition, "target cell" refers to a cell that transports a cargo molecule through a cargo molecule transduction domain, and target cell refers to a cell in vivo or in vitro. That is, the target cell means an in vivo cell, a cell constituting an organ or tissue of a living animal or human, or a microorganism found in a living animal or human. In addition, the target cell is meant to include in vitro cells, i.e., cultured animal cells, human cells, or microorganisms.
The term "cargo molecule transduction domain" as used herein refers to a peptide that forms a covalent bond with a cargo molecule of a polymeric organic compound, such as an oligonucleotide, peptide, protein, oligosaccharide or polysaccharide, so that the cargo molecule can be introduced into a cell or tissue without the need for additional receptors or carriers, energy. In the present specification, the "cargo molecule transduction domain" is used in combination with the "protein transduction domain" or the "cell penetrating domain".
In the present specification, the expression "introducing" a cargo molecule such as a protein or a peptide into a cell or a tissue is used in combination with the expression "penetrating", "transporting" or "permeating".
In the present specification, "conservative substitution" means a deformation of a cargo molecule transduction domain that includes substitution of one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the cargo molecule transduction domain.
In the present specification, a "conservative amino acid substitution" is a substitution in which an amino acid residue is substituted with an amino acid residue having a similar side chain. The class of amino acid residues with similar side chains has been defined in the corresponding technical field and is well known. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
It should be noted that other terms described in the specification, claims and drawings are used in accordance with meanings commonly used by those of ordinary skill in the art to which the present invention pertains unless otherwise specified.
The present invention relates to a cargo molecule transduction domain, wherein as 1) a sequence derived from human ADARM2 consisting of SEQ ID NO:1, a RMAD1 peptide of composition; or 2) a variant RMAD1 peptide consisting of 8 to 50 amino acids, wherein more than one amino acid is deleted, substituted and/or added in the RMAD1 peptide, and the cargo molecule transduction domain is combined with the cargo molecule, and the cargo molecule is transferred into cells or tissues of mammals.
In addition, the present invention relates to a cargo molecule transduction domain, wherein amino acid substitutions are not limited, but preferably conservative amino acid substitutions in the RMAD1 variant peptide.
In addition, the invention relates to a cargo molecule transduction domain, wherein the RMAD1 variant peptide is SEQ ID NO:1 is independently substituted with an arginine residue and/or the amino acid sequence of SEQ ID NO:1 is independently substituted with a lysine residue.
In addition, the present invention relates to a cargo molecule transduction domain in which deletion amino acids are not particularly limited in a peptide sequence in which 1 or more amino acids are deleted in the RMAD1 variant peptide, and preferably 1 to 6 of lysine residues and arginine residues are deleted in the amino acids of the RMAD1 peptide.
In addition, the invention relates to a cargo molecule transduction domain wherein in the RMAD1 variant peptide, more than 1 amino acid is deleted and/or added, and the peptide sequence is deleted and/or added at more than one of the middle, N-terminus and C-terminus of the sequence.
In addition, the invention relates to a cargo molecule transduction domain comprising a sequence derived from human ADARB2 consisting of SEQ ID NO:1, a RMAD1 peptide of composition; or a RMAD1 variant peptide consisting of 8 to 50 amino acids, in which more than one amino acid is deleted, substituted, and/or added to the RMAD1 peptide, the cargo molecule transduction domain binds to the cargo molecule, thereby transducing the cargo molecule into a mammalian cell or tissue. SEQ ID NO:1 is identical to "CKSKRRRRRRSKRKD" in amino acid sequence. In addition, the amino acid variation sequence in the RMAD1 peptide variant refers to the sequence set forth in SEQ ID NO:1, wherein amino acid variations occur individually in each amino acid residue position. In addition, the sequence in which amino acids are deleted in the RMAD1 peptide variant refers to the sequence set forth in SEQ ID NO:1, and at least 1 and at most 7 amino acids in the amino acid sequence of the polypeptide are independently deleted. Amino acid deletions may occur at either end or at the middle of the sequence, and may occur as continuous or discontinuous amino acids.
In addition, the invention relates to a cargo molecule transduction domain, wherein the amino acid variant sequence of the RMAD1 peptide variant is preferably SEQ ID NO:1 is independently substituted with an arginine residue and/or the amino acid sequence of SEQ ID NO:1 is independently substituted with a lysine residue.
In addition, the invention relates to a cargo molecule transduction domain wherein the sequence of at least 1 and at most 7 amino acids deleted in the RMAD1 peptide variant is preferably a sequence of 1 to 7 deletions of consecutive or non-consecutive lysine residues and arginine residues in the amino acids of the RMAD1 peptide.
In addition, the RMAD1 peptide variants of the invention may be amino acid substitutions and/or deletions and/or additions that are repeated. For example, amino acid additions and deletions may also occur on the basis of the RMAD1 amino acid substitution variants. In addition, amino acid additions may occur on the RMAD1 amino acid deletion variants. However, as described above, the cargo molecule transduction function is maintained unchanged even by amino acid substitutions and/or deletions and/or additions of various combinations.
As specific examples of the cargo molecule transduction domain of the present invention, there may be mentioned SEQ ID NO:1 to 11, however, the scope of the present invention is not limited to this definition, as will be apparent to those of ordinary skill in the art to which the present invention pertains.
In addition, the present invention relates to a cargo molecule transduction domain in which i) the RMAD1 peptide or ii) one or more selected from the RMAD1 variant peptides consisting of 8 to 50 amino acids, in which 1 or more amino acids are deleted, substituted and/or added in the RMAD1 peptide, are bound in multimeric form of dimers or more without a linker or via a linker. For example, as the cargo molecule transduction domain of the present invention, there may be mentioned i) the RMAD1 peptide or ii) an RMAD1 variant peptide sequence consisting of 8 to 50 amino acids in which 1 or more amino acids are deleted, substituted and/or added in the RMAD1 peptide, and there may be mentioned the sequence in which the i) or ii) peptide is repeated two or more times, and in addition thereto, there may be mentioned the i) peptide and ii) peptide-linked cargo molecule transduction domain, however, it is clear to those skilled in the art that the cargo molecule transduction domain of the present invention is not limited to the above exemplified peptide.
In addition, in the case of maintaining the activity of the cargo molecule transduction domain, the linker is not particularly limited, and amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, arginine, and the like may be used to join the individual cargo molecule transduction domain monomers, more preferably, a linker in which a plurality of valine, leucine, aspartic acid, glycine, alanine, proline, and the like are joined may be used, and most preferably, glycine, valine, leucine, aspartic acid, and the like may be each joined 1 to 5 in view of convenience of gene manipulation. In addition to the amino acid linkers and peptide linkers described above, chemical linkers may be used as long as the activity of the cargo molecule transduction domain is maintained.
In addition, the present invention relates to a recombinant cargo molecule with enhanced cell membrane permeability, which is a cargo molecule; and one or more of the N-and C-termini of the cargo molecule are fused to the any one cargo molecule transduction domain.
The cargo molecule and cargo molecule transduction domain may be fused in the absence of a linker or in the presence of a linker. In addition, the linker is not particularly limited as long as the cargo molecule transduction activity of the cargo molecule transduction domain and the activity of the cargo molecule are maintained, and preferably, the cargo molecule transduction domain and the cargo molecule may be linked using amino acids such as glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, arginine, etc., more preferably, the linker linking a plurality of valine, leucine, aspartic acid, glycine, alanine, proline, etc., most preferably, the amino acids such as glycine, valine, leucine, aspartic acid, etc., may be linked 1 to 5 each in view of convenience of gene manipulation. In addition to the amino acid linkers and peptide linkers described above, chemical linkers may be used as long as the activity of the cargo molecule transduction domain is maintained.
In addition, the invention relates to a recombinant cargo molecule with enhanced cell membrane permeability, wherein the cargo molecule is a peptide, a protein or a nucleic acid.
In addition, the invention relates to a recombinant cargo molecule with enhanced cell membrane permeability, wherein the cargo molecule is a therapeutic protein, an antigenic protein or an epitope peptide.
In addition, the invention relates to a recombinant cargo molecule with improved cell membrane permeability, wherein the cargo molecule is an antioxidant protein.
In addition, the present invention relates to a medicament for preventing or treating a disease comprising the recombinant cargo molecule.
Furthermore, the present invention relates to a cosmetic product comprising said recombinant cargo molecule. The cosmetic of the present invention may include color cosmetics such as foundations, lipsticks, eye shadows, etc., in addition to basic cosmetics such as lotions, creams, essences, oil-in-water or water-in-oil emulsions, ointments, etc.
In addition, the invention relates to a medical device comprising said recombinant cargo molecule. Medical devices of the present invention may include wound dressings, fillers, composite fillers, and the like.
In addition, the present invention relates to a genetic construct comprising a polynucleotide that encrypts the cargo molecule transduction domain.
As specific examples of polynucleotides encrypting the cargo molecule transduction domains of the present invention, there may be mentioned SEQ ID NO:12 to 22, however, the scope of the present invention is not limited thereto as is clear to one of ordinary skill in the art to which the present invention pertains.
In addition, the invention relates to an expression vector for expressing recombinant cargo molecule proteins with improved cell membrane permeability, which comprises the cargo molecule construct.
In addition, the invention relates to an expression vector for expressing recombinant cargo molecule proteins with improved cell membrane permeability, wherein the vector further comprises a gene for encrypting the cargo molecule proteins so as to express the recombinant cargo molecule proteins fused with the cargo molecule transduction domain.
In addition, the present invention relates to a method of transporting cargo molecules into cells, comprising: a step of preparing a recombinant cargo molecule fused to the cargo molecule transduction domain at one or more of the N-and C-termini of the cargo molecule; and a step of contacting the prepared recombinant cargo molecule with a cell.
In addition, the present invention relates to a method of transporting a cargo molecule into a cell, wherein the cargo molecule is a protein for preventing or treating a disease. The protein for preventing or treating a disease refers to a growth factor such as an epithelial cell growth factor, an antibody drug, a fusion protein including an Fc of an antibody, an antibody-drug complex, a protein drug, an enzyme, and the like, but is not limited to these examples.
In addition, the invention relates to a method of transporting a cargo molecule into a cell, wherein the cargo molecule is an antioxidant protein. Antioxidant proteins such as superoxide dismutase and catalase are shown, but are not limited to these examples.
In addition, the invention relates to a cargo molecule transduction domain, wherein the cargo molecule transduction domain is SEQ ID NO:1 to 11.
In addition, the invention relates to a cargo molecule transduction domain, wherein the genetic construct encrypting the cargo molecule transduction domain is SEQ ID NO:12 to 22.
In addition, the invention relates to a cargo molecule transduction domain, wherein the cargo molecule transduction domain comprises a polypeptide 2-fold.
The cargo molecule transduction domain according to the invention is to be construed as also including variants in which the amino acid residues are conservatively substituted at a specific amino acid residue position of the RMAD1 peptide; or a peptide in which 1 to 5 amino acids are deleted at the N-terminal and/or C-terminal and/or intermediate positions of the RMAD1 peptide or the variant.
The cargo molecule transduction domains of the present invention are predicted to remain active even with conservative amino acid substitutions.
In addition, a cargo molecule transduction domain variant according to the present invention should be interpreted as having substantially the same function and/or effect as a cargo molecule transduction domain according to the present invention, and further comprises a cargo molecule transduction domain variant or fragment thereof having an amino acid sequence homology of 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more.
The present invention is also characterized in that the cargo molecule is selected from the group consisting of nucleic acids, carbohydrates, lipids, and mixtures of 1 or more thereof, such as proteins, peptides, oligonucleotides, polynucleotides, and the like.
In addition, the invention is characterized in that the cargo molecule transduction domain and the chemical bond of the cargo molecule are covalent or non-covalent bonds. The chemical bond may be a covalent bond or a non-covalent bond. The non-covalent bond may include ionic bonds, or bonds through electrostatic attraction, or bonds through hydrophobic interactions, etc. In addition, the substance that can bind to the cargo molecule transduction domain via the ionic bond or electrostatic attraction can be a charged substance such as DNA or RNA.
In addition, the present invention relates to a recombinant cargo molecule that readily penetrates into cells or tissues, wherein the cargo molecule transduction domain is a sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO:11, 1 of the above. It should be clear that the cargo molecule transduction domain of the invention is not limited to SEQ ID NO:1 to 11, representative peptides are shown in table 1 for the convenience of the experiment.
In addition, the present invention relates to a polynucleotide, wherein the polynucleotide sequence for encoding a cargo molecule transduction domain is a sequence selected from the group consisting of SEQ ID NOs: 12 to SEQ ID NO: 22. It should be clear that the polynucleotide encoding the cargo molecule transduction domain of the invention is not limited to SEQ ID NO:12 to 22, representative polynucleotides are shown in table 2 for the convenience of the experiment.
The pharmaceutical composition comprising the recombinant cargo molecule of the present invention, the polynucleotide encoding the same, or a vector comprising the polynucleotide as an active ingredient may be formulated into various dosage forms such as external preparations for skin, oral administration, spray, patch, injection, etc., by a conventional method in combination with a carrier commonly accepted in the pharmaceutical field. For example, oral compositions include tablets and gelatin capsules which may contain, in addition to the active ingredient(s), diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silicon dioxide, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycols), binders (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone), and, if appropriate, disintegrants (e.g. starch, agar, alginic acid or its sodium salts) or boiled mixtures and/or absorbents, colorants, flavourings and sweeteners. Injectable compositions, preferably isotonic aqueous solutions or suspensions, and the compositions mentioned are sterile and/or contain adjuvants (e.g. preservatives, stabilizers, wetting agents or emulsifying agent solution accelerators, salts for regulating the osmotic pressure and/or buffers). In addition, these may contain other therapeutically useful substances.
Pharmaceutical formulations prepared in this way may be administered orally or parenterally, i.e. intravenously, subcutaneously, intraperitoneally or topically, as desired. Dosages may be divided into 1 to several administrations of daily dosages of 0.0001 to 100 mg/kg. The dosage level for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, etc.
Description of the embodiments
The composition of the present invention will be described in more detail below with reference to specific examples. However, it is obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited to the descriptions of the embodiments.
1. Cell culture
HaCaT and RAW264.7 cells were cultured in a cell culture medium supplemented with 10% fetal bovine serum (fetal bovine serum; FBS) and an antibiotic solution (100 units/ml penicillin, 100ug/ml streptomycin). TC-1 cells were cultured by adding cell culture broth of 10% fetal bovine serum (fetal bovine serum; FBS) and antibiotic solution (100 units/ml penicillin, 100ug/ml streptomycin) G418 (0.4 mg/ml) to RPMI medium. Maintaining the temperature at 37deg.C and humidity of 95% and 5% CO 2 When 70 to 80% of the cells are adhered to the dish, treated with trypsin-EDTA and subcultured. HaCaT cells were distributed and cultured in a laboratory taught by Tae-Yoon Kim, university of medical science, caliper, korea, 3T3 and B16F10 cells were sprayed and cultured in a university of head university cell line bank, RAW264.7 cells and TC-1 were cultured in three regionsUniversity pharmaceutical university metallographic paradigms teach laboratory distribution and cultivation.
Construction of RMAD1
The following oligonucleotides were used, annealed at a temperature of 94℃for 5 minutes, and then at a temperature of 25℃for 1 hour.
Sense oligonucleotide:
5’-agctttgcaagtccaagaggaggaggaggcggaggtccaagcggaaagatc-3’(SEQ ID NO：23)
antisense oligonucleotide:
5’-tcgagatctttccgcttggacctccgcctcctcctcctcttggacttgcaa-3’(SEQ ID NO：24)
then, the HindIII and Xho I sites of the pET-28a vector were cut and ligated to the pair of RMAD1 encoding oligonucleotides prepared above. Recombinant RMAD1 was transgenic in E.coli (Escherichia coli) strain DH5 a (RH 618). For the DNA isolated from the transgenic cells, bamHI and HindIII sites were cut and EGFP (Enhanced Green Fluorescence Protein) gene was ligated.
3. Purification of proteins
EGFP-RMAD1, EGFP-TAT proteins were purified using pET28a plasmid vectors containing N-, C-terminal 6X his tags. The BL21-codon plus cells were used for the transgene and colonies were inoculated on the medium for growth. Large scale cells were cultured in LB medium until OD600 reached 0.5, and protein expression was induced using 0.5mM IPTG at a temperature of 4℃for 16 hours. The cell pellet was obtained by centrifugation and subjected to ultrasonic treatment in 50mM Tris buffer (pH 7.5) containing 300mM NaCl, whereby disruption was performed. Then, the mixture was centrifuged at 20000g for 30 minutes to obtain a supernatant. The supernatant was poured onto a Ni-NTA resin column. The washing step was performed at pH7.5 with 50mM Tris buffer containing 300mM NaCl, 5% glycerol and 15mM imidazole. After separating the protein from the column using 10ml of elution buffer (50 mM Tris (pH 7.5), 300mM NaCl, 5% glycerol, 300mM imidazole), endotoxin (endotoxin) was removed using a Mustang column (Pall, MSTG25E 3), and analyzed by LAL (limulus reagent; limulus amebocyte lysate) using an appropriate protein of 0.5EU/mg or less throughout the experiment.
4. Cell introduction of EGFP-RMAD1 fusion proteins Using FACS
To evaluate the cell permeability of RMAD1 in HaCaT skin cells, 1X 10 was attached to a 24-well plate 12 hours ago 5 Amount of HaCaT cells. Then, after washing the cells using serum-free DMEM medium, 2.5uM of protein was treated in serum-free medium for 2 hours. After 2 hours, the cells were removed from the plate by washing 3 times with serum-free medium, and then 150ul of trypsin was treated, and then neutralized by adding 850ul of serum. Then, after centrifugation at 500g for 10 minutes, the medium was removed, and then proteins attached to the cells were removed by 500ul of FACS buffer (1% BSA, 0.1% sodium azide). After repeated washing 2 times, FACS buffer was added to perform FACS analysis.
5. Cell introduction of EGFP-RMAD1 fusion proteins using confocal microscopy
To evaluate the cell permeability of RMAD1 in HaCaT skin cells, 1X 10 was attached after a 9mm cover plate was placed on a 24-well plate 12 hours ago 5 Amount of HaCaT cells. Then, after washing the cells with serum-free DMEM medium, 2.5uM of protein was treated in serum-free medium for 2 hours. After 2 hours, a total of 3 washes with serum-free medium were performed, and hoechst was diluted in PBS at a 1:1000 ratio and stained for 5 minutes. Then, the glass cover plate was separated from the well plate by washing with PBS 5 times, and moisture was removed to perform sealing (mounting). Then, an image was obtained by using a confocal microscope, and fluorescence expression was confirmed. In the case of fluorescence microscopy, 1X 10 was attached to a 96-well plate the previous day 4 The amount of HaCaT cells and then the protein is treated as described above. Then, after 30 minutes before the end of the experiment, washing with serum-free medium, far infrared red mitochondrial fluorescence probe (mitotracker-deep FM (1:2000)) or lysosomal red fluorescence probe (Lysotracer Red DND-99 (1:2000)) was treated, and hoechst was added at a ratio of 1:1000 for 5 minutes before the end of the experimentDiluted in PBS and stained for 5 minutes. Fluorescent signals were then observed using a smart living cell imaging analysis system (Lionheart FX automated microscope) device from the company berteng (BioTek).
6. Immunoblotting
To evaluate the cell permeability of RMAD1 in HaCaT skin cells, 3X 10 was attached to a 12-well plate before 12 hours 5 Amount of HaCaT cells. Then, after washing the cells with serum-free DMEM medium, 1uM of protein was treated in serum-free medium for 2 hours. After 2 hours, in order to remove proteins that may adhere to the cell surface, the cells were washed 3 times with serum-free medium. Then, the protein in the supernatant was quantified by RIPA lysis buffer and centrifugation, and then 30ug of protein was mixed with 5X sample buffer. The prepared protein sample was boiled for 10 minutes, and separated according to molecular weight by using 12% SDS-PAGE gel (gel). After the electrophoresis was completed, proteins were removed by using PVDF membrane, and blocked for 1 hour by using TBS-T buffer containing 5% degreasing oil. Then, in order to measure protein expression, a GFP antibody was used as a 1-time antibody, and then a reaction was performed using an anti-rabbit antibody conjugated with horseradish peroxidase as a 2-time antibody. Then, after washing with TBS-T buffer, the cell permeability of each protein was measured.
7. Antioxidant efficacy improvement evaluation Using SOD1-RMAD1 fusion proteins
To evaluate the antioxidant efficacy of RMAD1 in RAW 264.7 macrophages, 5 x 10 was attached to 24-well plates 12 hours ago 4 Amount of RAW 264.7 cells. Then, after culturing the cells in serum-free medium for 1 hour, 0.5, 1, 2.5uM SOD1-RMAD was treated for 12 hours for each well. Then, 500ng/ml LPS (lipopolysaccharide) was treated for 16 hours for each well. Then, after 150ul of trypsin was treated by washing 3 times with serum-free medium and the cells were dropped, medium mixed with 850ul of serum was added for neutralization. After centrifugation at 500Xg for 10 minutes, the medium was removed and then washed with 500ul of HBSS buffer.Then, after staining with CM-H2DCFDA (Therom, C6827) for 10 minutes, and then washing 2 times with FACS buffer, FACS analysis was performed.
8. Evaluation of antioxidant inhibition of SOD1-RMAD1 TNF- α by SOD1-RMAD1
To evaluate the anti-inflammatory efficacy of RMAD1 in RAW 264.7 macrophages, 5 x 10 was adhered to 24-well plates 12 hours ago 4 Amount of RAW 264.7 cells. Then, after culturing the cells in serum-free medium for 1 hour, 0.5, 1, 2.5uM SOD1-RMAD1 was treated for two hours for each well. Then, 500ng/ml LPS was treated for 16 hours for each well. The medium was then recovered and centrifuged at 1000Xg for 10 minutes to detect TNF- α.
9. Isomouse tumor model and tumor measurement
TC-1 cells were maintained in RPMI supplemented with serum and G418, and after dropping the cells with trypsin, neutralization and washing were performed. Then, 1X 10 was subcutaneously injected into the right side of 6-week-old C57BL/6 mice 5 Cells were subcutaneously injected on days 6 and 13 to the left of mice with 8.8nmol of E7, E7-AD, E7-TAT and 25ug further mixed into the MPLA group for injection. The measurement was performed by using a digital caliper according to (0.52×length×width 2 ) And (5) performing calculation. When the tumor is 1000mm 3 In the above cases, the mice were euthanized.
10. Spleen and blood immune cell analysis
After injection of TC-1 into C57BL/6 mice, antigen and MPLA (Monophosphoryl-Lipid A) were injected separately as described above, and mice were sacrificed on day 19. Then, spleen cells were dissociated using RPMI medium containing 2% fbs and 1% streptomycin. Erythrocyte lysis in spleen and blood by erythrocyte lysis solution (BD, 555899) using CD8, CD3, IFN-gamma, E7 (H-2D) b HPV 16) tetramer (tetramer) antibodies stain individual cells at a temperature of 4 ℃ for 30 minutes. Intracellular staining was performed using a fixative/osmotic solution kit (BD, 555899). In this case, E7 was treated with spleen cells 49-57 (RAHYNIVTF) peptide for 16 hoursRestimulation was performed and then CD8 was detected by FACS + Intracellular IFN-gamma for T cells.
Results 1: RMAD1 peptide secondary structure prediction graph
To predict the peptide structure of RMAD1, the PET-FOLD3 de novo peptide structure prediction (De novo peptide structure prediction) program was used. For the model in which the amino acid sequence of RMAD1 was recorded and predicted by the program, the two-dimensional predicted structure was imaged using the pymol2.4 program (a of fig. 1). In a prediction graph used for analyzing the secondary structure prediction, a bright portion on the lower side represents a spiral, a brightest portion on the uppermost side represents an extension, and a portion between the spiral and the extension represents a coil, thereby deriving a secondary prediction model (B of fig. 1).
Results 2: RMAD1-FITC synthetic peptide cell permeation efficiency
The cell permeation efficiency of RMAD1 and the fluorescent protein FITC bound to its variants was confirmed using synthetic peptides attached with FITC having fluorescent properties, and using cell lines 3T3, B16F10 and HACAT. Since it is difficult to confirm the permeability of the peptide by cells to which FITC is not attached, the cell permeability is confirmed by attaching FITC that exhibits fluorescence. Each cell line was treated with RMAD-FITC, TAT-FITC peptide as FITC-conjugated peptide at the same concentration of 2.5uM for 2 hours. As a result, RMAD-FITC synthetic peptide showed significantly higher cell penetration level (A, B of fig. 2) than TAT-FITC peptide as a control group among 2 cell lines, confirming that HACAT cells maintained excellent penetration efficacy as compared with TAT even by amino acid substitution, removal and addition (C of fig. 2).
Results 3: pattern diagram and purification of EGFP-RMAD1 fusion proteins
To make a fusion protein comprising a cell penetrating peptide from a human source and EGFP, the sequence encoding the RMAD1 polypeptide (tgc aag tcc aag agg agg agg agg cgg agg tcc aag cgg aaa gat; SEQ ID NO: 12) was cloned into the HindIII and XhoI sites of the pET-28a plasmid, the RMAD1 encoding DNA and EGFP cDNA were recombined, the BamHI and HindIII sites were cleaved, and EGFP-RMAD1 was cloned into the pET-28a plasmid (FIG. 3 a). EGFP and EGFP-TAT as control groups were also cloned on pET-28a plasmid as a vector by the method described above, and in the case of a fusion protein comprising his-tag, after binding to Ni-NTA column, washed with a low concentration of 15mM imidazole solution, and eluted with 300mM imidazole solution. For the eluted fusion protein product, a total of 10ug of protein was loaded by SDS-PAGE, and then the molecular weight of the fusion protein purified by coomassie blue (coomassie blue) staining reagent was confirmed (FIG. 3 b). In addition, in order to confirm the expression of EGFP-RMAD1 protein including his-tag by the stained fusion protein, immunoblotting was performed, and it was confirmed by his-antibody that the molecular weight of the fusion protein including his-tag was consistent with the coomassie blue (coomassie blue) staining result (FIG. 3b, FIG. 3 c).
Results 4: introduction of RMAD1 fusion proteins into HaCaT cells
To confirm the cell permeation efficiency of the RMAD1 fusion protein, the efficiency was confirmed by introducing the fusion protein into HaCaT, a cell line of human skin cells. EGFP, EGFP-TAT, EGFP-RMAD1 as recombinant proteins were treated with 1uM at the same concentration for 2 hours on HaCaT cells. As a result, it was confirmed that the EGFP-RMAD1 fusion protein exhibited a significantly high level of cell permeation, and secondly, the EGFP-TAT fusion protein including TAT known as a cell penetrating peptide exhibited a cell permeation rate lower by about 60% than that of EGFP-RMAD 1. In addition, it was confirmed that EGFP protein itself was not permeable to cells (FIG. 3 d).
For cellular efficacy of the RMAD1 fusion protein, intracellular fluorescence was measured using a flow cytometer in order to confirm analysis-based, concentration-based, and time-based efficiencies by other experimental methods. Specifically, in order to analyze the concentration-based introduction efficiency, 4 concentrations of fusion proteins were treated at a 5-fold ratio from 0.1uM to 2.5uM concentration for 2 hours. As a result, it was confirmed that the RMAD1 fusion protein was transported into HaCaT cells in a concentration-dependent manner, and that the cell permeability was significantly higher than that of TAT fusion proteins, as in the immunoblotting results above (fig. 4a, 4b, 4 c). In addition, in order to analyze the introduction efficiency based on the treatment time, the fusion protein at a concentration of 2.5uM was cultured for 30 minutes to 4 hours, thereby performing FACS analysis. As a result, it was confirmed that the RMAD1 fusion protein was transported into HaCaT cells in a time-dependent manner, and that the amount of the protein that was continuously infiltrated for 2 hours was increased. In addition, it was confirmed again that the protein was transported more efficiently than TAT, which is a well-known cell permeable domain (fig. 4 d).
In order to confirm whether the EGFP-RMAD1 fusion protein was actually introduced into cells by confirming the position of the EGFP-RMAD1 fusion protein introduced into cells and to confirm the cell permeation efficacy again, the EGFP-RMAD1 fusion protein was treated with HaCaT cells and then observed by using a fluorescence microscope and a confocal microscope. As a result, it was confirmed that no fluorescent signal was observed in the EGFP protein as a control group, but the fluorescent signal was mainly detected in the cytoplasm as a result of observation by confocal microscopy in EGFP-TAT and EGFP-RMAD1 (fig. 5 a). In addition, a significantly higher fluorescent signal was observed in EGFP-RMAD1 than in EGFP-TAT fusion proteins, consistent with the immunoblotting and FACS results above. The same results were obtained also in fluorescence microscopy, and in most of the introduced EGFP-RMAD1, it was observed that the introduced fusion protein was located in the cytoplasm, as evidenced by the fact that most of the lysosomal tracer (lysotracker) and mitochondrial fluorescent probe (mitotracker) were not located at the same positions as those of the lysosome or mitochondrial positions (FIG. 5 b).
Results 5: pattern diagram and purification of SOD1-RMAD1 fusion proteins
To prepare a fusion protein comprising a cell penetrating peptide from human origin and SOD1, the sequence encoding the RMAD1 polypeptide (tgc aag tcc aag agg agg agg agg cgg agg tcc aag cgg aaa gat; SEQ ID NO: 12) was cloned into the HindIII and Xho I sites of the pET-29a plasmid, the recombinant RMAD1 encoding DNA and SOD1 cDNA, the Nde I and HindIII sites were cleaved, and SOD1-RMAD1 was cloned into the pET-29a plasmid (FIG. 6 a). SOD1 as a control group was also cloned on pET-29a plasmid as a vector by the method described above, and in the case of a fusion protein comprising his-tag, after binding to Ni-NTA column, washed with a low concentration of 15mM imidazole solution, and eluted with 300mM imidazole solution. For the eluted fusion protein product, a total of 10ug of protein was loaded by SDS-PAGE, and then the molecular weight of the fusion protein purified by coomassie blue (coomassie blue) staining reagent was confirmed (FIG. 6 b).
Results 6: introduction of fusion proteins into RAW264.7 cells and evaluation of antioxidant efficacy improvement
Cell penetration performance and whether activity was maintained was evaluated using RAW264.7 cells by fusion with cell penetrating peptide using SOD1 known as antioxidant enzyme. As a result of treating RAW264.7 cells with 0.5uM at the same concentration for 2 hours, it was confirmed that the SOD1-RMAD1 fusion protein showed a significantly high level of cell permeation. It was confirmed that the SOD1-TAT fusion protein including TAT, which is a conventionally known cell penetrating peptide, showed a lower level of cell permeability than that of SOD1-RMAD1, and that SOD1 itself was not permeable to cells (FIG. 6 c).
To confirm whether the fusion protein introduced into the cells was indeed active in the cells, antioxidant and anti-inflammatory effects were evaluated. To evaluate antioxidant efficacy, reactive oxygen species (Reactive Oxygen Species; ROS) removal capability was confirmed. Specifically, after SOD1-RMAD1 fusion protein was treated with RAW264.7 cells at 0.5, 1, 2.5uM concentrations for 2 hours, LPS induced reactive oxygen species was treated for 16 hours. Then, as a result of evaluating the amount of reactive oxygen species by FACS analysis using CM-H2DCFDA, it was confirmed that the SOD1-RMAD1 fusion protein effectively inhibited the production of intracellular reactive oxygen species (FIG. 6 d). In addition, in order to evaluate the anti-inflammatory efficacy, as in the above anti-oxidative efficacy experiment, the fusion protein and LPS were treated, and then by detecting TNF- α as an inflammatory factor in a medium, it was confirmed that the fusion protein effectively improved the anti-inflammatory efficacy compared to SOD1 (fig. 6 e).
Results 7: evaluation of anticancer vaccine efficacy Using peptide anti-inflammatory and RMAD1 fusion peptides
It is predicted that antigen-specific T cells are efficiently produced by rapidly increasing intracellular permeability to prevent peptide antigen decomposition in organisms and recognizing antigens in innate immune cells, thereforRMAD-1 and peptide antigens were fused to confirm anticancer vaccine efficacy. Antigen-specific T cells were evaluated using TC-1 cells overexpressing E7 derived from HPV-16/18 and E7 peptide conjugated to RMAD1, and further using MPLA (Monophosphoryl lipid A), which is known as an immune adjuvant and a TLR4 agent, a decrease in tumor size was confirmed. As a result, it was confirmed that E7AD has a higher anticancer ability than E7, and that the tumor size was significantly reduced as compared with E7+ MPLA in the E7AD + MPLA group to which MPLA was added (fig. 7a and 7 b). In addition, as a result of analyzing immune cells in spleen, it was confirmed that antigen-specific CD8 was found in the E7AD and E7AD+MPLA groups including RMAD1 + An increase in the number of T cells confirmed CD8 by confirming an increase in IFN-gamma expression + T cells were activated (fig. 7c, fig. 7 d). Finally, as a result of analysis using immunocytes of blood in comparison with E7-TAT, which is a conventional cell-penetrating peptide, it was confirmed that antigen-specific CD8 was effectively increased in the group containing RMAD1 + T cells (fig. 7 e).
The following table 1 is a specific example of 11 amino acid sequences in the transduction domains of cargo molecules of the invention, each in the order of the table SEQ ID NO:1 to SEQ ID NO:11.
table 1 below is a list of 11 polynucleotide sequences encoding specific examples of the cargo molecule transduction domains of the invention, each in the order of the table SEQ ID NO:12 to SEQ ID NO:22.
[ Table 1 ]
[ Table 2 ]
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Industrial applicability
The present invention provides a cargo molecule transduction domain which facilitates the transport of cargo molecules into cells by fusing various cargo molecules, including peptides, proteins and nucleic acids, which are hardly permeated into cells, a recombinant cargo molecule using the same, and a method for transporting cargo molecules into cells, and various cargo molecules, such as therapeutic proteins, antigenic proteins, epitope peptides and antioxidant proteins, can be permeated into cells, and thus can be used in therapeutic drugs, cosmetics, therapeutic methods, cell improvement methods, and the like.
<110> Remedi Co., Ltd.
<120> Cargo molecule transport domain RMAD1, variant thereof,
recombinant cargo molecule and cargo molecule transport method
using the same
<130> Remedi-RMAD1
<150> KR 21/062397
<151> 2021-05-14
<150> KR 22/053247
<151> 2022-04-29
<160> 24
<170> KoPatentIn 3.0
<210> 1
<211> 15
<212> PRT
<213> Homo Sapiens
<400> 1
Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys Asp
1 5 10 15
<210> 2
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-1
<400> 2
Cys Arg Ser Lys Arg Arg Arg Arg Arg Arg Ser Arg Arg Arg Asp
1 5 10 15
<210> 3
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-2
<400> 3
Cys Lys Ser Lys Lys Lys Lys Lys Lys Lys Ser Lys Arg Lys Asp
1 5 10 15
<210> 4
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-3
<400> 4
Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser
1 5 10
<210> 5
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-4
<400> 5
Cys Lys Ser Lys Arg Arg Arg Arg Ser Lys Arg
1 5 10
<210> 6
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-6
<400> 6
Ser Lys Arg Arg Arg Arg Ser Lys Arg Lys Asp
1 5 10
<210> 7
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-6
<400> 7
Tyr Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys Asp
1 5 10 15
<210> 8
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-7
<400> 8
Lys Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys Asp
1 5 10 15
Lys Val
<210> 9
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-8
<400> 9
Lys Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys Asp
1 5 10 15
Lys Val Ser
<210> 10
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-9
<400> 10
Leu Lys Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys
1 5 10 15
Asp Lys Val
<210> 11
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> cargo molecule transport domain RMAD1-10
<400> 11
Leu Lys Cys Lys Ser Lys Arg Arg Arg Arg Arg Arg Ser Lys Arg Lys
1 5 10 15
Asp Lys Val Ser
20
<210> 12
<211> 45
<212> DNA
<213> Homo sapiens
<400> 12
tgcaagtcca agaggaggag gaggcggagg tccaagcgga aagat 45
<210> 13
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-1
<400> 13
tgcaggtcca agaggaggag gaggcggagg tccaggcgga gggat 45
<210> 14
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-2
<400> 14
tgcaagtcca agaagaagaa gaagaagaag tccaagcgga aagat 45
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-3
<400> 15
tgcaagtcca agaggaggag gaggcggagg tcc 33
<210> 16
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-4
<400> 16
tgcaagtcca agaggaggcg gaggtccaag cgg 33
<210> 17
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-5
<400> 17
tccaagagga ggcggaggtc caagcggaaa gat 33
<210> 18
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-6
<400> 18
tataagtcca agaggaggag gaggcggagg tccaagcgga aagat 45
<210> 19
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-7
<400> 19
aaatgcaagt ccaagaggag gaggaggcgg aggtccaagc ggaaagataa agta 54
<210> 20
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-8
<400> 20
aaatgcaagt ccaagaggag gaggaggcgg aggtccaagc ggaaagataa agtaagc 57
<210> 21
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-9
<400> 21
ctcaaatgca agtccaagag gaggaggagg cggaggtcca agcggaaaga taaagta 57
<210> 22
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding cargo molecule transport domain RMAD1-10
<400> 22
ctcaaatgca agtccaagag gaggaggagg cggaggtcca agcggaaaga taaagtaagc 60
60
<210> 23
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 23
agctttgcaa gtccaagagg aggaggaggc ggaggtccaa gcggaaagat c 51
<210> 24
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 24
tcgagatctt tccgcttgga cctccgcctc ctcctcctct tggacttgca a 51
Claims (18)
1. A cargo molecule transduction domain, wherein as 1) a sequence derived from human ADARM2 consisting of SEQ ID NO:1, a RMAD1 peptide of composition; or 2) a RMAD1 variant peptide consisting of 8 to 50 amino acids, in which more than one amino acid is deleted, substituted and/or added in the RMAD1 peptide,
The cargo molecule transduction domain binds to the cargo molecule, thereby transducing the cargo molecule into a mammalian cell or tissue.
2. The cargo molecule transduction domain according to claim 1, wherein in the RMAD1 variant peptide, the amino acid substitution is a conservative amino acid substitution.
3. The cargo molecule transduction domain according to claim 1 or 2, wherein the RMAD1 variant peptide is SEQ ID NO:1 is independently substituted with an arginine residue and/or the amino acid sequence of SEQ ID NO:1 is independently substituted with a lysine residue.
4. The cargo molecule transduction domain according to claim 1, wherein the peptide sequence in which more than 1 amino acid is deleted in the RMAD1 variant peptide is a deletion of 1 to 6 of lysine residues and arginine residues in the amino acids of the RMAD1 peptide.
5. The cargo molecule transduction domain according to claim 1, wherein in the RMAD1 variant peptide, a peptide sequence of more than 1 amino acid deletion and/or a peptide sequence of more than 1 amino acid addition is an amino acid deletion and/or addition at one or more of the N-and C-termini.
6. The cargo molecule transduction domain according to any one of claims 1 to 5 wherein,
1) A human ADARB 2-derived polypeptide consisting of SEQ ID NO:1, a RMAD1 peptide of composition; or alternatively
2) A RMAD1 variant peptide consisting of 8 to 50 amino acids in which one or more amino acids are deleted, substituted, and/or added to the RMAD1 peptide; in which one or more of the monomers is bonded to a dimer or more polymer form via a linker.
7. A recombinant cargo molecule having increased permeability of a cell membrane, wherein the cargo molecule; and one or more of the N-and C-termini of the cargo molecule are fused with one cargo molecule transduction domain selected from the group consisting of claims 1 to 6.
8. The recombinant cargo molecule of claim 7, wherein the cargo molecule is a peptide, protein, or nucleic acid.
9. The recombinant cargo molecule of claim 7, wherein the cargo molecule is a therapeutic protein, an antigenic protein, or an epitope peptide.
10. The recombinant cargo molecule of claim 7, wherein the cargo molecule is an antioxidant protein.
11. A medicament comprising a recombinant cargo molecule selected from any one of claims 7 to 10.
12. A cosmetic product comprising a recombinant cargo molecule selected from any one of claims 7 to 10.
13. A genetic construct comprising a polynucleotide that encrypts the cargo molecule transduction domain according to any one of claims 1 to 6.
14. An expression vector for the expression of a recombinant cargo molecule protein having enhanced cell membrane permeability, comprising the genetic construct of claim 13.
15. The expression vector for recombinant cargo molecule protein expression with enhanced cell membrane permeability according to claim 14, wherein the vector further comprises a gene encrypting cargo molecule protein to express recombinant cargo molecule protein fused with cargo molecule transduction domain.
16. A method of transporting cargo molecules into cells, comprising:
a step of preparing a recombinant cargo molecule fused to the cargo molecule transduction domain of any one of claims 1 to 6 at one or more of the N-and C-termini of the cargo molecule; and
contacting the prepared recombinant cargo molecule with a cell.
17. The method of transporting a cargo molecule into a cell of claim 16, wherein the cargo molecule is a therapeutic protein.
18. The method of transporting a cargo molecule into a cell of claim 16, wherein the cargo molecule is an antioxidant protein.
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| PCT/KR2022/006340 WO2022240055A1 (en) | 2021-05-14 | 2022-05-03 | Cargo molecule tranduction domain rmad1, variant thereof, recombinant cargo molecule, and method for tranducing cargo molecule using same |
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