KR20140103002A - Peptide for targeting to mitochondria - Google Patents

Abstract

The present invention relates to a peptide for targeting mitochondria, a recombinant vector including the peptide, a method for moving target proteins to mitochondria in order to be distributed using the same, and to a pharmaceutical composition for preventing or treating diseases accompanying mitochondria hypofunction using the same. An amino acid sequence of sequence number 1 in the present invention is targeted to mitochondria so that a peptide including the same can move and distribute a material (compound, protein, gene) exhibiting pharmacological activities in mitochondria. Moreover, the peptide having the amino acid sequence of sequence number 1 serves as a target sequence for moving a particular material to mitochondria. Accordingly, when a composite is produced by binding the peptide with a pharmacologically active material (compound, protein, gene), the pharmacologically active material can be moved to mitochondria in order to directly act therein so that diseases related to mitochondria hypofunction can be prevented or treated using the same.

Description

Peptides for targeting to mitochondria {Peptide for targeting to mitochondria}

The present invention relates to a peptide for targeting mitochondria, a recombinant vector containing the peptide, a method for transferring and distributing a target protein using the peptide to mitochondria, and a pharmaceutical composition for preventing or treating diseases accompanied by mitochondrial dysfunction .

There are numerous cells in our body, and there are small organelles in each cell, including the endoplasmic reticulum, nucleus, mitochondria, peroxysomes, and goliaths. These organelles are surrounded by unique membranes. One cell has about one billion proteins, and these proteins must be transferred to specific organelles, membranes, cytosols, nuclei, etc. in the cell to perform their own functions.

The transport of proteins to specific organelles by cells is called "protein targeting," and for this protein targeting, newly synthesized proteins must be transported sequentially to the endoplasmic reticulum, the Golgi and the final target organelles.

In order for the newly synthesized protein to be transferred to the endoplasmic reticulum, a short amino acid sequence called the signal sequence plays an important role and the signal sequence is made in the early stage of protein synthesis and then transported to the endoplasmic reticulum and then removed. The protein is then transported to the Golgi body, where the sugar, fat, etc. are bound to the protein. In addition, a separate signal sequence must be present in order to be transported to the intracellular organelles such as mitochondria and nucleus, and this sequence may be referred to as a mitochondrial signal sequence or a nuclear signal sequence separately from a signal sequence to be transmitted to an endoplasmic reticulum.

Thus, a protein signal sequence that transfers a target protein functioning in a cell to an intracellular organelle is a very efficient method for delivering a desired protein, and it is very difficult to find a signal sequence capable of targeting to a specific intracellular organelle It is important.

Among the intracellular organelles, mitochondria exist in a net form in the cytoplasm, and they repeat the fusion and division repeatedly and function to produce the energy required for cell survival through cell respiration. Since these mitochondria are the backbone of intracellular energy metabolism, malfunctioning of mitochondria causes various diseases. Diseases caused by dysfunction of the mitochondria include Parkinson's disease, Huntington's disease, and other diseases that frequently occur due to aging. Barth syndrome, Leigh syndrome, Melas syndrome MELAS Syndrome) and rare diseases with low incidence. Recent studies have also shown that mitochondrial abnormalities may be the cause of type 2 diabetes, the metabolic syndrome. These diseases, caused by malfunction of the mitochondria, are caused by an abnormality in the energy supply system of the cell, most of which are accompanied by muscle and brain diseases.

The respiratory function of the mitochondria essential for cell energy production is through the respiratory complex present in the inner membrane of the mitochondria. There are five types of mitochondrial respiratory complexes, each of which receives electrons from specific substrates and ultimately transfers them to oxygen.

Mitochondria are composed of about 1,500 proteins, and mitochondria have their own unique genetic information, unlike other organelles in the cell. In other words, the mitochondria have their own genetic information in the form of mitochondrial DNA (mtDNA) in addition to the genomic DNA that exists in the nucleus and contains all the genetic information of the cell. The size of the mtDNA is about 1600 kbp, and it encodes 13 proteins constituting the mitochondrial respiratory complex. Because mitochondrial respiratory complexes are composed of proteins made from two genetic materials, genomic DNA and mtDNA, their formation processes are controlled by complex regulatory mechanisms compared to other intracellular organelles.

In addition, signal transduction between the mitochondria and the nucleus appears to play a very important role in the formation and function of mitochondria, and research is needed on proteins that can act as mediators.

Therefore, by modulating the function and stability of mitochondria, it is possible to transfer a substance (including chemicals, proteins, and genes) exhibiting pharmacological activity to mitochondria by fusing with peptides, proteins or genes capable of targeting mitochondria If possible, the activity would be doubled.

On the other hand, it is well known that ganglioside, a sialic acid-containing glycolipid, plays an important role not only as a receptor for viral and toxin proteins and microorganisms, but also in cell differentiation, proliferation, growth and signal transduction, have. GM1 is a type of this ganglioside receptor and is an abbreviation of 'ganglioside Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Galβ1-1Ceramide'. Previous studies related to GM1 have disclosed GM1-linked peptides using a random peptide library, but there has been no report on the mitochondrial targeting function of such GM1-linked peptides.

Therefore, the inventors of the present invention have studied a peptide sequence capable of leading to the targeting effect to mitochondria, and have found that a specific sequence known as GM1 (ganglioside Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Galβ1-1Ceramide) The present invention has been completed.

Korean Patent Publication No. 2010-0086164

It is therefore an object of the present invention to provide a peptide having targeting use for mitochondria.

Another object of the present invention is to provide a recombinant vector comprising a gene encoding the amino acid sequence of the peptide.

Yet another object of the present invention is to provide a method for transferring and distributing a target protein to mitochondria using the recombinant vector.

It is still another object of the present invention to provide a pharmaceutical composition capable of preventing or treating a disease associated with mitochondrial dysfunction by using a complex in which the peptide and a pharmacologically active substance are combined.

Yet another object of the present invention is to provide a pharmaceutical composition capable of preventing or treating a disease associated with mitochondrial dysfunction by using a recombinant vector in which a gene encoding the amino acid sequence of the peptide and a gene encoding a target protein are operably linked .

In order to accomplish the above object, the present invention provides a peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present invention, the recombinant vector may be operably linked to a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a gene encoding a target protein.

The present invention also provides a method for transferring and distributing a target protein into mitochondria, which comprises introducing the recombinant vector into a cell.

The present invention also provides a pharmaceutical composition for preventing or treating diseases associated with mitochondrial dysfunction, comprising an effective component of a peptide having a peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 and a pharmacologically active substance- .

In one embodiment of the present invention, the pharmacologically active substance may be a compound, a protein or a gene.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of diseases associated with mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a recombinant vector operably linked to a gene encoding a target protein, Lt; / RTI >

In one embodiment of the present invention, the disease associated with mitochondrial dysfunction is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophy, dystrophic dystrophy, chronic fatigue syndrome, Friedrich's ataxia, epilepsy, peripheral neuropathy, Autonomic neuropathy, nerve-induced intestinal dysfunction, sensory nerve deafness, neurodegenerative bladder dysfunction, migraine, exercise laicosis, renal tubular acidosis, dilated cardiomyopathy, fatty liver disease, hepatic failure, abortion, mitochondrial dysfunction (MELAS), Leber's hereditary optic neuropathy (LHON), MERRF syndrome, MNGIE (mitochondrial neurogastrointestinal encephalopathy syndrome), NARP (neuropathy, ataxia, and retinitis pigmantosa), Barth syndrome Barth Syndrome, Leigh Syndrome, and Kans-Sirens Syndrome.

Since the amino acid sequence of SEQ ID NO: 1 of the present invention has a characteristic of being targeted to mitochondria, the peptide comprising the same can transfer and distribute a pharmacologically active substance (compound, protein, gene) to mitochondria. In addition, since the peptide having the amino acid sequence of SEQ ID NO: 1 functions as a target sequence for transferring a specific substance to mitochondria, when such a peptide is combined with a substance (compound, protein, gene) exhibiting pharmacological activity to form a complex, Since a pharmacologically active substance can migrate directly to mitochondria and act directly, it can be useful in the prevention or treatment of diseases related to mitochondrial dysfunction.

FIG. 1 is a photograph showing that GMs (p3): EGFP is located in the mitochondria. Green is fluorescence emitted by GM3 (p3) protein of the present invention, red is fluorescence emitted by mitochondrial marker MitoTracker, GMs (p3) The part where the protein and MitoTracker fluorescence overlap is yellow.

The terms used in the present invention are defined as follows.

"Mitochondria" are cellular organelles present in most eukaryotic cells. One of the main functions of the mitochondria is oxidative phosphorylation, through which energy derived from the metabolism of fuel substances such as glucose or fatty acids is converted to ATP, which is used to drive a variety of energy-requiring biosynthesis and other metabolic activities . A mitochondrion has its own genome, which is isolated from nuclear DNA and is a circular DNA with about 16,000 base pairs. Each mitochondrion has multiple copies of its genome, and each cell is known to have hundreds of mitochondria.

"Diseases associated with mitochondrial dysfunction" means diseases where the normal function of the mitochondria is due to inherited genetic defects, bacterial infections, disorders, or other causes that result in reduced function or inadequate functioning.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule, and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, a nucleotidyltransferase that occurs in the polymerase chain reaction .

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequences are placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product.

"Transfection, transfection or transfection" refers to the process by which extracellular DNA enters a host cell with and without accompanying entities. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material. And cells into which external DNA is introduced are referred to as " transformants. &Quot;

The term "vector" refers to any nucleic acid that is inserted into a host cell, recombined with the host cell genome, inserted into it, or contains a competent nucleotide sequence that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

"Host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and is understood to refer not only to a particular target cell but also to its progeny or potential progeny. In fact, the offspring are not identical to the parent cell, but are still included within the scope of the term, as used herein, since certain modifications may occur in subsequent generations due to mutations or environmental influences.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a peptide capable of moving and distributing a target substance (compound, protein, gene) into an intracellular mitochondria. More specifically, the present invention provides a peptide having an amino acid sequence of mitochondria targeting It is characterized by providing a peptide for use.

It is well known that ganglioside, a sialic acid-containing glycolipid, plays an important role not only as a receptor for viral and toxin proteins and microorganisms, but also for cell differentiation, proliferation, growth and signal transduction, carcinogenesis and metastasis. GM1 is a type of this ganglioside receptor and is an abbreviation of 'ganglioside Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Galβ1-1Ceramide'. Previous studies related to GM1 have disclosed GM1-linked peptides using a random peptide library, but there has been no report on the mitochondrial targeting function of such GM1-linked peptides.

The present inventors first studied the peptide sequence capable of leading to the targeting effect to the mitochondria, and confirmed for the first time that a specific sequence known as a GM1 binding peptide has a targeting effect to mitochondria.

In the following Example 1 of the present invention, in order to test whether a specific sequence known as a GM1-binding peptide has a targeting effect to a mitochondria, first, a previously studied article (Matubara et al (2007), Langmuir) P3 (MVWRLLAPPFSNRLLP), known as a peptide that binds to GM1, a type of glycosphingolipid, and its modified peptide were named GMs (p3) and GMs (p3) ).

In the following Example 2 of the present invention, polynucleotides (genes) encoding the peptides were amplified by PCR to confirm the intracellular distribution positions of GMs (p3) and GMs (p3) mt peptides, and then EGFP enhanced green fluorescent protein), and these hybrid plasmids were introduced into HEK293T cells, and these plasmids were expressed to identify peptide positions through fluorescence imaging. Green fluorescence by GMs (p3): EGFP and red fluorescence by MitoTracker, a mitochondrial marker, were observed at the same location in the cell, but green fluorescence by GMs (p3) mt: EGFP was completely different from red fluorescence by MitoTracker I showed the pattern. From these results, it was confirmed that the GMs (p3) peptide of the present invention is a peptide having a migration signal to intracellular mitochondria (see FIG. 1).

The present inventors have experimentally proved that GMs (p3) peptide is effective for targeting to intracellular mitochondria through the above experimental results. In the present invention, the amino acid sequence of GMs (p3) peptide is simply represented by SEQ ID NO: Respectively.

The peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be chemically synthesized or obtained using gene recombination technology.

First, the peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be chemically synthesized. When chemically synthesized, it can be prepared by chemical synthesis (Creighton, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983) well known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry (see for example Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press , Boca Raton Florida, 1997; A Practical Approach, Atherton and Sheppard, Eds., IRL Press, Oxford, England, 1989).

Also, the peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be produced by a genetic engineering method. First, a hexane (DNA) sequence encoding the peptide is prepared according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., commercially available from Biosearch or Applied iosystems). The constructed DNA sequence is inserted into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) operably linked to the DNA sequence and controlling the expression of the DNA sequence, and recombinant expression Transform the host cell into a vector. The resulting transformant is cultivated under conditions and conditions suitable for the expression of the DNA sequence to recover a substantially pure peptide encoded by the DNA sequence from the culture. The recovery can be carried out using methods known in the art (for example, chromatography). By "substantially pure peptide" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080,1990.

The present invention also provides a gene encoding the amino acid sequence of SEQ ID NO: 1, or a recombinant vector to which said gene is operably linked.

In the present invention, the gene encoding the amino acid sequence of SEQ ID NO: 1 refers to a polynucleotide encoding a peptide having the amino acid sequence of SEQ ID NO: 1, and the polynucleotide is a polynucleotide, And is a DNA or RNA strand of a certain length or more.

In the present invention, as the recombinant vector, known expression vectors such as a plasmid vector, a cosmid vector, and a bacteriophage vector can be used as a means for expressing the gene encoding the amino acid sequence of SEQ ID NO: 1 of the present invention. Can be readily prepared by those skilled in the art according to any known method using DNA recombinant techniques.

Operably linked "means that the expression control sequence is linked to regulate the transcription and translation of the gene encoding the amino acid sequence of SEQ ID NO: 1, and the expression of the gene (polynucleotide) under the control of the expression control sequence And maintaining the correct decoding frame to produce the peptide of the invention encoded by the gene.

In one embodiment of the present invention, the recombinant vector may be operably linked to a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, or a gene encoding a target protein.

In the present invention, since the gene encoding the amino acid sequence of SEQ ID NO: 1 is effective for targeting mitochondria, a gene encoding the amino acid sequence of SEQ ID NO: 1 of the present invention is operatively linked to a target gene to be targeted to mitochondria, The target protein can be targeted to the mitochondria when it is expressed.

In another embodiment of the present invention, the recombinant vector may be operably linked to a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, a gene encoding a target protein, and a fluorescent protein gene. In this case, the expression of the fluorescent protein can confirm the movement / distribution of the target protein to the mitochondria.

The fluorescent protein may be Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP), but the type of the fluorescent protein is not particularly limited.

The present invention also provides a method for transferring and distributing a target protein to mitochondria, comprising introducing the recombinant vector into a cell.

(A) preparing a hybrid plasmid comprising a gene encoding the amino acid sequence of SEQ ID NO: 1, a gene encoding a target protein, and a gene encoding a fluorescent protein; (b) introducing the hybrid plasmid into a cell and expressing the gene; And (c) confirming the movement and distribution of the target protein expressed in the transformed cells through fluorescence imaging, to determine the migration and spatial distribution of the target protein into the mitochondria in the cell. do.

The step (a) of the present invention is a step of preparing a hybrid plasmid comprising a gene encoding the amino acid sequence of SEQ ID NO: 1, a gene encoding a target protein, and a gene encoding a fluorescent protein.

In the present invention, the gene encoding the amino acid sequence of SEQ ID NO: 1 has a purpose of targeting mitochondria, and the gene encoding the target protein is a gene encoding a specific protein to be transferred and distributed to mitochondria. Has a visualization function that can confirm whether the target protein actually migrates and distributes to the mitochondria.

The step (b) of the present invention is a step of introducing the hybrid plasmid prepared in the step (a) into a cell and expressing the gene.

In the present invention, introduction of a hybrid plasmid into a cell transforms a cell using a hybrid plasmid, and the transforming method can be performed by a method well known to a person having ordinary skill in the art. For example, a method using a chemical substance such as PEG (polyethylene glycol), calcium phosphate, DEAE-dextran; Lipofection methods using cationic lipids; Microinjection method; Electroporation method; Electroporation method, and the like may be used, but the present invention is not limited thereto. It is necessary to optimize the conditions so that the target protein to which the signal protein (late target gene targeting peptide) and the fluorescent protein are linked in the transformed cell is efficiently expressed, It may be appropriately selected depending on the type of fluorescent protein.

In one embodiment of the present invention, the cell may be a eukaryotic cell, and the fluorescent protein may be Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP).

By allowing the fluorescence of a specific wavelength emitted from the protein expressed in the transformed cells to be continuously visualized using the fluorescence microscope in the living state, the expression of the target protein in the cell and the migration to the mitochondria The process can be visualized step by step.

The present invention also provides a pharmaceutical composition for preventing or treating diseases associated with mitochondrial dysfunction, which comprises, as an active ingredient, a complex comprising a peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 and a pharmacologically active substance to provide.

The complex of the present invention may contain a peptide capable of targeting to mitochondria so that a substance capable of acting on mitochondria and exhibiting a direct pharmacological activity can be transferred to the mitochondria. Therefore, it can be usefully used for the prevention or treatment of diseases related to dysfunction (dysfunction) of mitochondria.

The pharmacologically active substance may be a natural product, a compound, a protein or a gene (DNA, RNA or PNA) which can act on mitochondria and exhibit a specific activity.

The present invention also provides a pharmaceutical composition for preventing or treating diseases associated with mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a recombinant vector in which a gene encoding a target protein is operably linked, as an active ingredient .

The diseases associated with the mitochondrial dysfunction that are prevented or treated by the pharmaceutical composition of the present invention include diseases caused by mutation, deletion, or rearrangement of DNA of mitochondria; A disease caused by the nuclear-coded defective protein component of the respiratory chain of mitochondria; Aging-related diseases; A disease caused by administration of a cytotoxic cancer chemotherapeutic agent; Diseases caused by active defects of the mitochondrial complexes I, II, III, IV or V; Congenital mitochondrial disease; Neurodegenerative disease; And neuromuscular degenerative diseases.

Specific examples of such diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, Friedrich's alexithymia, epilepsy, peripheral neuropathy, optic nerve disease, autonomic neuropathy, It has been reported that the incidence of mitochondrial encephalopathy with lactic acidemia and strokelike is higher than that of patients with acute myocardial infarction. (MELAS), Leber's hereditary optic neuropathy (LHON), MERRF syndrome, MNGIE (Mitochondrial neurogastrointestinal encephalopathy syndrome), NARP (neuropathy, ataxia and retinitis pigmantosa), Barth syndrome, Lee's syndrome Leigh Syndrome) and Kans-sirece syndrome.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The present invention also relates to a kit for the preparation of a medicament for preventing or treating diseases associated with mitochondrial dysfunction, comprising (i) a complex comprising a peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 and a pharmacologically active substance, (Ii) a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a recombinant vector operably linked to a gene encoding a target protein as an active ingredient. The composition of the present invention comprising the above complex or recombinant vector as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of diseases related to mitochondrial dysfunction.

The present invention also relates to a method for producing a mitochondrial targeting peptide comprising the steps of: (i) providing a complex comprising a peptide for targeting mitochondria and a pharmacologically active substance comprising the amino acid sequence of SEQ ID NO: 1; or (ii) a promoter, There is provided a method for preventing or treating a disease associated with mitochondrial dysfunction, which comprises administering a recombinant vector operably linked to a gene encoding a gene and a target protein.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

In the therapeutic method of the present invention, the above-described complex or composition comprising the recombinant vector as an active ingredient can be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intramuscularly, transdermally, topically, And can be administered in a conventional manner.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Mitochondrial Targeting  That can lead to an effect Peptides

The present inventors first examined whether a specific sequence known as GM1 (ganglioside Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Galβ1-1Ceramide) binding peptide has a targeting effect on mitochondria, (MVWRLLAPPFSNRLLP), which is a kind of glycosphingolipid, which is known as a GM1 binding peptide, and a modified peptide thereof, were prepared by referring to the above-mentioned method.

GM1 binding peptide and its modified peptide Type of peptide Peptidase sequence Abbreviation GM1 binding peptide MVWRLLAPPFSNRLLP GMs (p3) Modified peptide MV G RLLAPPFSN E LLP GMs (p3) mt

< Example  2>

GM1  Combination Peptides  And its variants Of peptide Intracellular  Check distribution

To confirm the distribution of the GM1-binding peptide and the modified peptide thereof in Table 1, the present inventors amplified each of the polynucleotides (genes) encoding the sequence of Table 1 by PCR and amplified with EGFP (enhanced green fluorescent protein), and then hybrid plasmids were introduced into HEK293T cells. These plasmids were expressed to identify peptide positions through fluorescence imaging.

Specifically, each of the polynucleotides (genes) encoding the GM1-binding peptide and its modified peptide of Table 1 was first prepared (see Table 2) and then amplified by PCR using the specific primer sequence shown in Table 3 below. The conditions of the PCR are shown in Table 4 below. The hybridized plasmid was prepared by ligating the amplified polypeptide to the HindIII / XbaI site of pcDNA3.1 (BD bioscience clontech) and ligating EGFP to the c-terminus. Then, the hybrid plasmid was transfected into HEK293T cells cultured in DMEM medium (10% FBS + Penicillin / streptomycin) at 37 ° C (5% CO 2) using Ca ²⁺ phosphate (Clontech) method. Fluorescence images in HEK293T cells were then measured using a Cal Zeiss confocal microscope as hybrid plasmids were expressed. MitoTracker was used as a mitochondrial marker.

As shown in FIG. 1, the green fluorescence by GMs (p3): EGFP and the red fluorescence by MitoTracker, which is a mitochondrial marker, were observed at the same position in the cell, while the green fluorescence by GMs (p3) Showed a completely different pattern from red fluorescence by MitoTracker (MitoTracker Red FM, Molecular probe). From these results, it was confirmed that the GMs (p3) peptide of the present invention is a peptide having a migration signal to intracellular mitochondria.

The polynucleotide sequence of each GM1-binding peptide and its modified peptide Type of peptide Abbreviation Polynucleotide sequence GM1 binding peptide GMs (p3) 5'-ATG TGG CGA CTG CTG GCA CCT CCT TTC TCA AAC CGA CTG CTG CCT-3 ' Modified peptide GMs (p3) mt 5'-ATG GGC CGA CTG CTG GCA CCT CCT TTC TCA AAC GAG CTG CTG CCT-3 '

The primer sequences used in PCR gene
(Polynucleotide) Primer sequence GMs (p3) sense 5'-CGC CCA AGC TTG CCA CCA TGG TGT GGC GAC TGC TGG CAC CTC CTT T-3 ' antisense 5'-GCT CTA GAA GGC AGC AGT CGG TTT GAG AAA GGA GGT GCC AGC AGT C-3 ' GMs (p3) mt sense 5'-CGC CCA AGC TTG CCA CCA TGG TGG GCC GAC TGC TG-3 ' antisense 5'-GCT CTA GAA GGC AGC AGC TCG TTT GAG AA-3 '

PCR conditions Temperature time Cycle 94 ° C 2 minutes One 94 ° C 20 seconds 30 48 ° C 20 seconds 72 ℃ 40 seconds 72 ℃ 5 minutes 4 4 ℃ ∞ One

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

GM1: ganglioside Galβ1-3GalNAcβ1-4 (Neu5Acα2-3) Galβ1-4Galβ1-1Ceramide

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Peptide for targeting to mitochondria <130> PN1211-516 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mitochondria targeting peptide <400> 1 Met Val Trp Arg Leu Leu Ala Pro Pro Phe Ser Asn Arg Leu Leu Pro   1 5 10 15

Claims (8)

A peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1. A recombinant vector comprising a gene encoding the amino acid sequence of SEQ ID NO: 1. 3. The method of claim 2,
Wherein the recombinant vector is operably linked to a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a gene encoding a target protein. A method for transferring and distributing a target protein to mitochondria, comprising introducing the recombinant vector of claim 2 or 3 into a cell. 1. A pharmaceutical composition for preventing or treating a disease associated with mitochondrial dysfunction comprising, as an active ingredient, a complex comprising a peptide for targeting mitochondria comprising the amino acid sequence of SEQ ID NO: 1 and a pharmacologically active substance. 6. The method of claim 5,
Wherein the pharmacologically active substance is a compound, a protein or a gene. 1. A pharmaceutical composition for preventing or treating a disease associated with mitochondrial dysfunction, comprising a promoter, a gene encoding the amino acid sequence of SEQ ID NO: 1, and a recombinant vector operably linked to a gene encoding a target protein as an active ingredient. 8. The method according to any one of claims 5 to 7,
The diseases associated with mitochondrial dysfunction include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, Friedreich's ataxia, epilepsy, peripheral neuropathy, optic nerve disease, autonomic neuropathy, , Mitral regurgitation, sensory nerve deafness, neurodegenerative bladder dysfunction, migraine, athlete's syndrome, renal tubular acidosis, dilated cardiomyopathy, fatty liver, liver failure, abortion, mitochondrial cerebral apathy MELAS, Leber's hereditary optic neuropathy (LHON), MERRF syndrome, MNGIE, NARP, Barth syndrome, Leigh Syndrome, ) And Kans-sire syndrome. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

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