阅读说明：本技术 细胞穿透肽、包含其的缀合物、及应用 (Cell penetrating peptides, conjugates comprising the same, and uses ) 是由 金商在 于 2013-09-17 设计创作，主要内容包括：本发明公开了细胞穿透肽、包含其的缀合物及其应用。具体而言,本发明公开了具有SEQ ID NO:2至SEQ ID NO:178中一种序列的细胞穿透肽、包含SEQ ID NO:2至SEQ ID NO:178中一种序列的片段的肽的缀合物、或包含与所述肽序列具有至少80％同源性的肽的缀合物、和包含所述缀合物的组合物。(The invention discloses a cell penetrating peptide, a conjugate containing the same and application thereof. Specifically, disclosed are a cell penetrating peptide having one of SEQ ID NO 2 to SEQ ID NO 178, a conjugate of a peptide comprising a fragment of one of SEQ ID NO 2 to SEQ ID NO 178, or a conjugate of a peptide having at least 80% homology with the peptide sequence, and a composition comprising the conjugate.)

1. Use of a peptide for the preparation of a composition comprising a cargo to be delivered, wherein the peptide is a cell penetrating carrier peptide which delivers the cargo into a cell, and

wherein the cell penetrating carrier peptide is a peptide consisting of at least one amino acid sequence of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 5 to 19, SEQ ID NO 23 to 26, SEQ ID NO 28 to 31, SEQ ID NO 34 to 48, SEQ ID NO 50 to 56, SEQ ID NO 58 to 61, SEQ ID NO 63 to 103, SEQ ID NO 110 to 113, SEQ ID NO 115 to 160, SEQ ID NO 162, and SEQ ID NO 164 to 177,

wherein the cargo is a contrast agent.

2. The use of claim 1, wherein the carrier peptide consists of 30 or fewer amino acids.

3. The use of claim 1, wherein the carrier peptide is conjugated to the cargo directly by a covalent bond or indirectly via a linker to the cargo by a covalent bond.

4. The use of claim 1, wherein the carrier peptide and the cargo are conjugated by a non-covalent bond.

5. Use according to claim 1, wherein the contrast agent is selected from the group consisting of radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents and CT contrast agents.

6. Use according to claim 1, wherein the contrast agent is based on iron.

7. Use according to claim 6, wherein the contrast agent is ferrocene carboxylate.

8. The use of claim 1, wherein the composition is a contrast agent.

9. Use according to claim 8, wherein the contrast agent is for contrasting cells.

10. The use of claim 9, wherein the cell is a stem cell.

11. The use according to claim 1, wherein the composition is a pharmaceutical composition for the treatment or prevention of a disease.

12. Use according to claim 1, wherein the composition is a cosmetic composition.

13. Use according to claim 1, wherein the composition is a health food composition.

14. A cell-penetrating peptide, wherein the cell-penetrating peptide consists of an amino acid sequence of any one of SEQ ID NO 2 to SEQ ID NO 77.

15. A polynucleotide encoding the cell penetrating peptide of claim 14.

16. A vector comprising the polynucleotide of claim 15.

17. A transformed cell comprising the vector of claim 16.

Technical Field

The present invention relates to a cell-penetrating peptide derived from human telomerase reverse transcriptase (hTERT), a conjugate of the cell-penetrating peptide and an active ingredient, and a composition comprising the conjugate.

Background

Although low molecular weight substances, nucleic acids, proteins, nanoparticles, etc. have great potential as therapeutic substances at the molecular level, the use of low molecular weight substances, nucleic acids, proteins, particles, etc. is limited in its application due to the inability to penetrate tissues and cell membranes. The development of systems for delivering such substances into cells has been an active area of research over the last two decades. Intracellular transport of substances has become a topic in molecular processing therapies. Low molecular weight substances, nucleic acids or nanoparticles are transported into cells by being tethered by a variety of reagents, electroporation or heat shock. However, it is difficult to find an appropriate method for delivering proteins inside cells without destroying the activity and integrity of the proteins. In the study of the cell-penetrating ability of Human Immunodeficiency Virus (HIV) in the 80's of the 20 th century, it was found that the HIV-TAT protein consisting of a specific 11 amino acid structure plays an important role in the transport into the cell interior. Therefore, in the 90 s of the 20 th century, research to find a suitable method for transporting proteins into cells to transport proteins into cells has been an area of intense research.

Telomeres are known to be repeats of genetic material found at the ends of chromosomes, and prevent chromosome damage or incorporation onto other chromosomes. The length of telomeres is shortened at each cell division, and after a certain number of cell divisions, the telomere length is extremely shortened to such an extent that the cells stop dividing and die. On the other hand, telomere extension is known to prolong the life of cells. As an example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening, thus causing cancer cells to proliferate.

The object of the present invention is to provide a novel peptide.

It is another object of the present invention to provide polynucleotides encoding the novel peptides.

It is another object of the present invention to provide a cell penetrating peptide.

Another object of the present invention is to provide a peptide useful as a carrier for an active ingredient in a cell.

Another object of the present invention is to provide a useful peptide as a carrier for an active ingredient in a cell, particularly a useful peptide for delivering an active ingredient locally to a mitochondria.

Another object of the present invention is to provide useful peptides for delivering an active ingredient to mitochondria to improve, prevent or treat a mitochondrial-related disease or disorder.

It is another object of the present invention to provide conjugates of active ingredients conjugated with cell penetrating peptides.

It is another object of the present invention to provide a composition comprising a conjugate of an active ingredient and a cell-penetrating peptide.

It is another object of the present invention to provide a pharmaceutical composition comprising a conjugate of an active ingredient and a cell-penetrating peptide.

Another object of the present invention is to provide a functional cosmetic composition comprising a conjugate of an active ingredient and a cell-penetrating peptide.

It is another object of the present invention to provide a health food composition comprising a conjugate of an active ingredient and a cell-penetrating peptide.

It is another object of the present invention to provide a contrast agent comprising a conjugate of an active ingredient and a cell-penetrating peptide.

Disclosure of Invention

The conjugate according to one embodiment of the present invention may be a conjugate of a cell penetrating carrier peptide and an active ingredient, wherein the carrier peptide is a peptide comprising at least one amino acid sequence of SEQ ID No. 2 to SEQ ID No. 178, a peptide having at least 80% homology with the above sequence, or a fragment of the above peptide, and wherein the peptide and the fragment having at least 80% homology retain the cell penetrating ability of any amino acid sequence of SEQ ID No. 2 to SEQ ID No. 178.

According to another embodiment of the conjugate of the invention, said fragment may consist of 3 or more amino acids.

According to another embodiment of the conjugate of the invention, the carrier peptide may consist of 30 or fewer amino acids.

According to another embodiment of the conjugate of the invention, the carrier peptide may be a peptide consisting of the amino acid sequence of any one of SEQ ID NO 2 to SEQ ID NO 178 or a peptide having at least 80% homology to the above sequence.

A contrast agent according to an embodiment of the present invention may comprise any of the conjugates described above.

The contrast agent according to one embodiment of the present invention may be used for contrast imaging of cells.

According to another embodiment of the contrast agent of the present invention, the cells may be stem cells.

A composition according to one embodiment of the invention may comprise any of the conjugates described above.

According to another embodiment of the composition of the present invention, the active ingredient may be used for treating or preventing diseases, and the composition may be a pharmaceutical composition.

According to another embodiment of the composition of the present invention, the active ingredient may be an active ingredient for functional cosmetics, and the composition may be a cosmetic composition.

According to another embodiment of the composition of the present invention, the active ingredient may be an active ingredient for a functional health food, and the composition may be a health food composition.

One embodiment of the method according to the invention is a method for delivering an active ingredient into a cell, wherein the method comprises administering a conjugate according to any one of claims 1 to 12 to a subject in need thereof, and wherein the carrier peptide is a cell penetrating peptide for delivering the active ingredient into the cell, and wherein said peptide having at least 80% homology and fragments of the above peptide retain the cell penetrating ability of the peptide consisting of the amino acid sequence of any one of SEQ ID No. 2 to SEQ ID No. 178.

According to another embodiment of the method of the invention, the method can be used for delivering the active ingredient locally to the mitochondria inside the cell.

According to another embodiment of the cell-penetrating peptide of the present invention, the above-mentioned carrier peptide may be a peptide having an amino acid sequence of any one or more of SEQ ID NO 2 to SEQ ID NO 178.

The polynucleotide according to the present invention may encode the above-mentioned cell-penetrating peptide.

The vector according to the present invention may comprise the polynucleotide described above.

The transformed cell according to the present invention may comprise the above-described vector.

Industrial applicability

Active ingredients that are difficult to transport into cells can be easily transported into cells by using the disclosed peptide or conjugate of peptide and active ingredient. This means that the efficacy of the active ingredient can be increased and thus the dosage of the active ingredient can be reduced. Accordingly, side effects due to drug administration can be minimized and the effectiveness of treatment can be increased. In particular, when drugs are delivered locally into the mitochondria, mitochondrial-related diseases or disorders can be ameliorated and the effectiveness of disease prevention and treatment can be increased. In the case of cosmetics, a significant effect can be produced by a small amount of active ingredient. By conjugating the peptide to a contrast agent, the peptide can be used as a contrast agent to monitor the process of cell transplantation or to transplant cells in cell therapy. In particular, the peptides may be used in practice as contrast substances for stem cells injected into the body.

Drawings

FIG. 1 depicts the number of cells taken up by cells in HeLa cells treated after FITC fusion with the peptide Pep1 of SEQ ID No:1 by FACS analysis. Control cells were treated with FITC only.

FIGS. 2 to 29 depict the number of cells taken up by cells in HeLa cells treated after FITC is fused to the peptides of SEQ ID NO:2 to SEQ ID NO:178 by FACS analysis. Control cells were treated with FITC only.

FIGS. 30-51 depict the number of cells taken up by cells in treated Huh7 cells after FITC fusion with the peptides of SEQ ID NO:2 through SEQ ID NO:178 by FACS analysis. Control cells were treated with FITC only.

FIGS. 52 to 69 depict the number of cells taken up by cells in the human T lymphocyte cell line (Jurkat cells) treated after FITC fusion with the peptides of SEQ ID NO:2 to SEQ ID NO:178 by FACS analysis. Control cells were treated with FITC only.

FIG. 70 depicts the results of toxicity and cellular activity of HeLa cells treated after FITC fusion with the peptide Pep1 of SEQ ID No:1 analyzed by FACS (flow cytometry). Control cells were treated with FITC only.

FIGS. 71 to 86 depict the results of FACS (flow cytometry) analysis of the toxicity and cellular activity of HeLa cells treated after FITC fusion with the peptides of SEQ ID NO:2 to SEQ ID NO: 178. Control cells were treated with FITC only.

Detailed Description

Although proteins, nucleic acids, peptides or viruses etc. have great potential as therapeutic substances, the use of proteins, nucleic acids, peptides or viruses etc. is limited by the inability to penetrate tissues and cell membranes. Even though the size of the molecules is small, such molecules cannot penetrate the lipid bilayer due to the structure or properties of the molecules. Therefore, it is attempted to transport proteins, nucleic acids, peptides or viruses into cells by using electroporation, heat shock, or the like; it is difficult to transfer such proteins, nucleic acids, peptides or viruses without disrupting the cell membrane while maintaining the active state of the molecules. Many studies have been conducted to show that Trans-Activating transcription activator (TAT activator) proteins derived from Human Immunodeficiency Virus (HIV) can be used as cell-penetrating peptides which can transport huge active substances into transport cells. Specifically, studies have been made on substances that can transport extremely large molecules such as proteins, nucleic acids, peptides or viruses into cells without causing any toxicity, unlike TAT proteins that produce toxicity inside cells. Accordingly, the present invention has been accomplished by the present inventors' discovery that telomerase-derived peptides have significant efficacy as cell-penetrating peptides without significant toxicity.

Peptides are disclosed in SEQ ID NO:1 through SEQ ID NO:178 shown in Table 1 below through Table 5 below. 179 is the full length sequence of the human telomerase protein. The peptide of SEQ ID NO. 1 is derived from telomerase and consists of a 16 amino acid sequence. The peptides of SEQ ID NO 2 to 77 include the peptide of SEQ ID NO 1. The peptides of SEQ ID NO:78 to SEQ ID NO:178 are fragments of the peptide of SEQ ID NO: 1. The "name" in table 1 below is used to distinguish the peptides. In various specific embodiments of the invention, more than one peptide in SEQ ID NO 1 through SEQ ID NO 178 comprises a "synthetic peptide", i.e., a synthetic peptide of a selected region of telomerase. In the present specification, the term "pep" herein refers to a peptide having the sequence of SEQ ID NO. 1, or any one of the amino acid sequences of SEQ ID NO. 2 to SEQ ID NO. 178, a peptide comprising an amino acid sequence having more than 80% homology with the above sequence, or a fragment of the above peptide.

[ Table 1]

[ Table 2]

41.	pep-RIA-40	[605-627]	EVRQHREARPALLTSRLRFIPKP	23aa
					42.	pep-RIA-41	[605-628]	EVRQHREARPALLTSRLRFIPKPD	24aa
43.	pep-RIA-42	[605-629]	EVRQHREARPALLTSRLRFIPKPDG	25aa
					44.	pep-RIA-43	[605-630]	EVRQHREARPALLTSRLRFIPKPDGL	26aa
45.	pep-RIA-44	[605-631]	EVRQHREARPALLTSRLRFIPKPDGLR	27aa
					46.	pep-RIA-45	[605-632]	EVRQHREARPALLTSRLRFIPKPDGLRP	28aa
47.	pep-RIA-46	[606-632]	VRQHREARPALLTSRLRFIPKPDGLRP	27aa
					48.	pep-RIA-47	[607-632]	RQHREARPALLTSRLRFIPKPDGLRP	26aa
49.	pep-RIA-48	[608-632]	QHREARPALLTSRLRFIPKPDGLRP	25aa
					50.	pep-RIA-49	[609-632]	HREARPALLTSRLRFIPKPDGLRP	24aa
51.	pep-RIA-50	[610-632]	REARPALLTSRLRFIPKPDGLRF	23aa
					52.	pep-RIA-51	[604-627]	AEVRQHREARPALLTSRLRFIPKP	24aa
53.	pep-RIA-52	[604-628]	AEVRQHREARPALLTSRLRFIPKPD	25aa
					54.	pep-RIA-53	[604-629]	AEVRQHREARPALLTSRLRFIPKPDG	26aa
55.	pep-RIA-54	[604-630]	AEVRQHREARPALLTSRLRFIPKPDGL	27aa
					56.	pep-RIA-55	[604-631]	AEVRQHREARPALLTSRLRFIPKPDGLR	28aa
57.	pep-RIA-56	[604-632]	AEVRQHREARPALLTSRLRFIPKPDGLRP	29aa
					58.	pep-RIA-57	[604-633]	AEVRQHREARPALLTSRLRFIPKPDGLRPI	30aa
59.	pep-RIA-58	[605-633]	EVRQHREARPALLTSRLRFIPKPDGLRPI	29aa
					60.	pep-RIA-59	[606-633]	VRQHREARPALLTSRLRFIPKPDGLRPI	28aa
61.	pep-RIA-60	[607-633]	RQHREARPALLTSRLRFIPKPDGLRPI	27aa
					62.	pep-RIA-61	[608-633]	QHREARPALLTSRLRFIPKPDGLRPI	26aa
63.	pep-RIA-62	[609-633]	HREARPALLTSRLRFIPKPDGLRPI	25aa
					64.	pep-RIA-63	[610-633]	REARPALLTSRLRFIPKPDGLRPI	24aa
65.	pep-RIA-64	[611-627]	EARPALLTSRLRFIPKP	17aa
					66.	pep-RIA-65	[611-628]	EARPALLTSRLRFIPKPD	18aa
67.	pep-RIA-66	[611-629]	EARPALLTSRLRFIPKPDG	19aa
					68.	pep-RIA-68	[611-631]	EARPALLTSRLRFIPKPDGLR	21aa
69.	pep-RIA-69	[611-632]	EARPALLTSRLRFIPKPDGLRP	22aa
					70.	pep-RIA-70	[611-633]	EARPALLTSRLRFIPKPDGLRPI	23aa
71.	pep-RIA-71	[611-634]	EARPALLTSRLRFIPKPDGLRPIV	24aa
					72.	pep-RIA-72	[611-635]	EARPALLTSRLRFIPKPDGLRPIVN	25aa
73.	pep-RIA-73	[611-636]	EARPALLTSRLRFIPKPDGLRPIVNM	26aa
					74.	pep-RIA-74	[611-637]	EARPALLTSRLRFIPKPDGLRPIVNMD	27aa
75.	pep-RIA-75	[611-638]	EARPALLTSRLRFIPKPDGLRPIVNMDY	28aa
					76.	pep-RIA-76	[611-639]	EARPALLTSRLRFIPKPDGLRPIVNMDYV	29aa
77.	pep-RIA-77	[611-640]	EARPALLTSRLRFIPKPDGLRPIVNMDYVV	30aa
					78.	pep-RIA-78	[611-625]	EARPALLTSRLRFIP	15aa
79.	pep-RIA-79	[611-624]	EARPALLTSRLRFI	14aa
					80.	pep-RIA-80	[611-623]	EARPALLTSRLRF	13aa

[ Table 3]

81.	pep-RIA-81	[611-622]	EARPALLTSRLR	12aa
					82.	pep-RIA-82	[611-621]	EARPALLTSRL	11aa
83。	pep-RIA-83	[611-620]	EARPALLTSR	10aa
					84.	pep-RIA-84	[611-619]	EARPALLTS	9aa
85.	pep-RIA-85	[611-618]	EARPALLT	8aa
					86.	pep-RIA-86	[611-617]	EARPALL	7aa
87.	pep-RIA-87	[611-616]	EARPAL	6aa
					88.	pep-RIA-88	[611-615]	EARPA	5aa
89.	pep-RIA-89	[611-614]	EARP	4aa
					90.	pep-RIA-90	[611-613]	EAR	3aa
91.	pep-RIA-91	[612-626]	ARPALLTSRLRFIPK	15aa
					92.	pep-RIA-92	[613-626]	RPALLTSRLRFIPK	14aa
93.	pep-RIA-93	[614-626]	PALLTSRLRFIPK	13aa
					94.	pep-RIA-94	[615-626]	ALLTSRLRFIPK	12aa
95.	pep-RIA-95	[616-626]	LLTSRLRFIPK	11aa
					96.	pep-RIA-96	[617-626]	LTSRLRFIPK	10aa
97.	pep-RIA-97	[618-626]	TSRLRFIPK	9aa
					98.	pep-RIA-98	[619-626]	SRLRFIPK	8aa
99.	pep-RIA-99	[620-626]	RLRFIPK	7aa
					100.	pep-RIA-100	[621-626]	LRFIPK	6aa
101.	pep-RIA-101	[622-626]	RFIPK	5aa
					102.	pep-RIA-102	[623-626]	FIPK	4aa
103.	pep-RIA-103	[624-626]	IPK	3aa
					104.	pep-RIA-104	[612-625]	ARPALLTSRLRFIP	14aa
105.	pep-RIA-105	[613-624]	RPALLTSRLRFI	12aa
					106.	pep-RIA-106	[614-623]	PALLTSRLRF	10aa
107.	pep-RIA-107	[615-622]	ALLTSRLR	8aa
					108.	pep-RIA-108	[616-621]	LLTSRL	6aa
109.	pep-RIA-109	[617-620]	LTSR	4aa
					110.	pep-RIA-110	[612-624]	ARPALLTSRLRFI	13aa
111.	pep-RIA-111	[612-623]	ARPALLTSRLRF	12aa
					112.	pep-RIA-112	[612-622]	ARPALLTSRLR	11aa
113.	pep-RIA-113	[612-621]	ARPALLTSRL	10aa
					114.	pep-RIA-114	[612-620]	ARPALLTSR	9aa
115.	pep-RIA-115	[612-619]	ARPALLTS	8aa
					116.	pep-RIA-116	[612-618]	ARPALLT	7aa
117.	pep-RIA-117	[612-617]	ARFALL	6aa
					118.	pep-RIA-118	[612-616]	ARPAL	5aa
119.	pep-RIA-119	[612-615]	ARPA	4aa
					120.	pep-RIA-120	[612-614]	ARP	3aa

[ Table 4]

121	pep-RIA-121	[613-625]	RPALLTSRLRFIP	13aa
					122	pep-RIA-122	[613-623]	RPALLTSRLRF	11aa
123	pep-RIA-123	[613-622]	RPALLTSRLR	10aa
					124	pep-RIA-124	[613-620]	RPALLTSR	8aa
125	pep-RIA-125	[613-619]	RPALLTS	7aa
					126	pep-RIA-126	[613-618]	RPALLT	6aa
127	pep-RIA-127	[613-617]	RPALL	5aa
					128	pep-RIA-128	[613-616]	RPAL	4aa
129	pep-RIA-129	[613-615]	RPA	3aa
					130	pep-RIA-130	[614-625]	PALLTSRLRFIP	12aa
131	pep-RIA-131	[614-624]	PALLTSRLRFI	11aa
					132	pep-RIA-132	[614-622]	PALLTSRLR	9aa
133	pep-RIA-133	[614-621]	PALLTSRL	8aa
					134	pep-RIA-134	[614-620]	PALLTSR	7aa
135	pep-RIA-135	[614-619]	PALLTS	6aa
					136	pep-RIA-136	[614-618]	PALLT	5aa
137	pep-RIA-137	[614-617]	PALL	4aa
					138	pep-RIA-138	[614-616]	PAL	3aa
139	pep-RIA-139	[615-625]	ALLTSRLRFIP	11aa
					140	pep-RIA-140	[615-623]	ALLTSRLRF	9aa
141	pep-RIA-141	[615-621]	ALLTSRL	7aa
					142	pep-RIA-142	[615-620]	ALLTSR	6aa
143	pep-RIA-143	[615-619]	ALLTS	5aa
					144	pep-RIA-144	[615-618]	ALLT	4aa
145	pep-RIA-145	[615-617]	ALL	3aa
					146	pep-RIA-146	[616-625]	LLTSRLRFIP	10aa
147	pep-RIA-147	[616-624]	LLTSRLRFI	9aa
					148	pep-RIA-149	[616-622]	LLTSRLR	7aa
149	pep-RIA-150	[616-620]	LLTSR	5aa
					150	pep-RIA-151	[616-619]	LLTS	4aa
151	pep-RIA-152	[616-618]	LLT	3aa
					152	pep-RIA-153	[617-625]	LTSRLRFIP	9aa
153	pep-RIA-154	[617-624]	LTSRLRFI	8aa
					154	pep-RIA-155	[617-623]	LTSRLRF	7aa
155	pep-RIA-156	[617-622]	LTSRLR	6aa
					156	pep-RIA-157	[617-621]	LTSRL	5aa
157	pep-RIA-158	[617-619]	LTS	3aa
					158	pep-RIA-159	[618-625]	TSRLRFIP	8aa
159	pep-RIA-160	[618-624]	TSRLRFI	7aa
					160	pep-RIA-161	[618-623]	TSRLRF	6aa

[ Table 5]

In one embodiment of the invention, a polynucleotide encodes the following peptides: a peptide comprising at least one amino acid sequence of SEQ ID NO. 2 to SEQ ID NO. 178, a peptide having at least 80% homology with the above sequence or a peptide which is a fragment of the above peptide. The polynucleotides described above enable the peptides to be produced in large quantities. For example, culturing a vector comprising a polynucleotide encoding a peptide allows for the production of large quantities of the peptide.

Peptides disclosed herein may include peptides comprising amino acid sequences having more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% homology. In addition, the peptides disclosed in the present invention may include: a peptide comprising the amino acid sequence of any one of SEQ ID No. 2 to SEQ ID No. 178 or a fragment of said peptide, and a peptide having more than 1 converted (transformed) amino acid, more than 2 converted amino acids, more than 3 converted amino acids, more than 4 converted amino acids, more than 5 converted amino acids, more than 6 converted amino acids or more than 7 converted amino acids.

In one embodiment of the invention, the alteration in the amino acid sequence is a change in the physical and chemical properties of the peptide. For example, amino acid conversions may be performed for improving the thermostability, changing the substrate specificity, and changing the optimal pH of the peptide.

The term "amino acid" herein includes not only the 22 standard amino acids naturally introduced into the peptide, but also the D-isomers and converted amino acids. Thus, in a particular embodiment of the invention, the peptides herein include peptides having D-amino acids. In another aspect, the peptide can include non-standard amino acids, such as those that have been post-translationally modified. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including acetylation, myristoylation, palmitoylation), alkylation, carboxylation, hydroxylation, saccharification, biotinylation, ubiquitination, chemical transformations (e.g., β -removal of deimides, deamidation), and structural transformations (e.g., formation of disulfide bonds). Also included are changes in amino acids that occur as a result of chemical reactions that occur during combination with the cross-linking agent to form the peptide conjugate.

The peptides disclosed herein can be wild-type peptides that have been identified and isolated from a natural source. In another aspect, the peptides disclosed herein may be mutants of artificial mutants comprising one or more amino acids with one or more substitutions, deletions and/or insertional additions when compared to the peptide fragment of any one of the amino acid sequences of SEQ ID NO:2 to SEQ ID NO: 178. Amino acid changes that are changed in the wild-type polypeptide (not only in the artificial mutant) include conservative substitutions of amino acids that do not significantly affect the folding and/or activity of the active protein. Examples of conservative substitutions belong to the group consisting of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that do not generally alter a particular activity are known in the art. The most common changes are alanine/serine, valine/isoleucine, aspartic acid/glutamic acid, threonine/serine, alanine/glycine, alanine/threonine, serine/asparagine, alanine/valine, serine/glycine, tyrosine/phenylalanine, alanine/proline, lysine/arginine, aspartic acid/asparagine, leucine/isoleucine, leucine/valine, alanine/glutamic acid, aspartic acid/glycine, and the reverse. Another example of conservative substitutions is shown in table 6 below.

[ Table 6]

Substantial transformation of the biological properties of peptides is performed by selecting the following significantly different substitutions in terms of efficacy: (a) the efficacy of maintaining the polypeptide backbone structure in the displaced region, such as a sheet-like or three-dimensional helical structure, (b) the efficacy of maintaining the charge or hydrophobicity of the molecule in the target region, or (c) the efficacy of maintaining the integrity of the side chains. Natural residues are divided into the following groups according to general side chain properties:

(1) hydrophobicity: norleucine, methionine, alanine, valine, leucine, isoleucine;

(2) neutral hydrophilicity: cysteine, serine, threonine;

(3) acidity: aspartic acid, glutamic acid;

(4) alkalinity: asparagine, glutamine, histidine, lysine, arginine;

(5) residues that influence chain orientation (chain orientation): glycine, proline; and

(6) aromaticity: tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions may be made by replacing a member of the above-mentioned class with a member of a different class. Any cysteine residue not relevant for maintaining the proper three-dimensional structure of the peptide may typically be substituted with serine, thus increasing the oxidative stability of the molecule and preventing improper cross-linking. Conversely, an improvement in stability may be achieved by adding one or more cysteine bonds to the peptide.

The altered types of amino acid variants of the peptide are those amino acids that have altered the glycosylation pattern of the antibody. The term "altering" as used herein refers to the deletion of at least one sugar residue found in a peptide and/or the addition of at least one glycosylated residue that is not present within a peptide.

Glycosylation in peptides is usually via an N-linkage or an O-linkage. The term "N-linked" as used herein refers to the attachment of a sugar residue to the side chain of an asparagine residue. As tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of sugar residues to the side chain of asparagine. Thus, in the presence of one of the tripeptide sequences in a polypeptide, a possible glycosylation site is created. "O-linked glycosylation" means the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid. The hydroxyamino acids are most often serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the peptide is conveniently performed by altering the amino acid sequence to contain the above-described tripeptide sequence (glycosylation site for N-linkage). This alteration may be made by adding at least one serine or threonine residue to the first antibody sequence or by substitution with those residues (glycosylation sites for O-linkages).

In one embodiment of the present invention, there is provided a cell penetrating peptide comprising a peptide, wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO:2 to SEQ ID NO:178, the peptide has an amino acid sequence that is more than 80% homologous to the above sequence, or the peptide is a fragment of the above peptide.

In one embodiment of the invention, a pharmaceutical composition is provided comprising a peptide as a drug delivery system for the delivery of one or more active ingredients, wherein the peptide comprises an amino acid sequence of any one of SEQ ID No. 2 to SEQ ID No. 178, the peptide has more than 80% homology with the above sequence, or the peptide is a fragment of the above peptide.

A peptide comprising an amino acid sequence of any one of SEQ ID NO 2 to SEQ ID NO 178, a fragment of the above peptide, or a peptide having more than 80% homology with the above sequence is safe and has a significant efficacy as a cell-penetrating peptide. Thus, the peptide may be conjugated to a drug to transport the drug inside the cell.

In one embodiment of the invention, there is provided a conjugate of a peptide comprising an amino acid sequence of any one of SEQ ID NO 2 to SEQ ID NO 178, a fragment of the peptide or a peptide having more than 80% homology with the peptide, and an active ingredient to be delivered. In one embodiment of the present invention, the active ingredient may be at least one selected from the group consisting of: proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast agents, drugs, and compounds. In one embodiment of the invention, the active ingredient may be a peptide. In one embodiment of the invention, the active ingredient may be a cytokine, an antibody fragment, a therapeutic enzyme, a soluble receptor or a ligand.

A cell penetrating peptide as disclosed herein means a peptide that can transport a cargo (cargo) to the interior of a cell from outside the body and/or inside the body. The "cargo" disclosed herein comprises all substances which can be transported inside the cell by conjugation with cell penetrating peptides, for example, all substances intended to increase the cell penetrating efficacy, in particular, active ingredients of drugs, cosmetics or health foods, more particularly substances which cannot be transported inside the cell by general routes, more particularly sugars, nanoparticles, biologicals, viruses, contrast agents or other compounds which may for example have proteins, nucleic acids, peptides, minerals, glucose, but are not limited to those substances. "drug" as disclosed herein is a broad concept including a substance to be delivered for the alleviation, prevention, treatment, or diagnosis of a disease, wound, or specific symptom.

A "carrier peptide" as disclosed herein is a peptide that can transport an active ingredient to a target site by conjugation with the active ingredient.

In one embodiment of the invention, the protein or peptide as cargo comprises one or more of the following: hormones, hormone analogs, enzymes, enzyme inhibitors, signal transfer proteins (or peptides), antibodies, and vaccines, but are not limited to those substances. In one embodiment of the invention, the nucleic acid is the following molecule: can be spontaneous or artificial, single-stranded or double-stranded DNA molecules or RNA molecules. The nucleic acid molecules can be one or more nucleic acids of the same type (e.g., having the same nucleotide sequence) or different types of nucleic acids. The nucleic acid molecule comprises one or more of: DNA, complementary DNA (cDNA), decoy DNA (decoy DNA), RNA, small interfering RNA (siRNA), microRNA (miRNA), small hairpin RNA (shRNA), small sequence RNA (stRNA), small nucleolar RNA (snorNA), small nuclear RNA (snRNA), Pentose Nucleic Acid (PNA), antisense oligo, plasmid, and other modified nucleic acids, but not limited to those. In one embodiment of the invention, the virus comprises a whole virus or a viral core comprising nucleic acid of the virus. In one embodiment of the invention, a chemical substance is a broad indication of comprising a natural or synthetic substance that can act as a drug.

The phenomenon in which specific DNA expression is controlled by double-stranded RNA (dsRNA) during DNA expression is called RNA interference; RNAi. Since this phenomenon was first discovered in C.elegans in 1998, it was found to be common in plants, Drosophila and mammals (Fire et al, Nature 391: 806-164. 811, 1998; Novina & Sharp, Nature 430: 161-164. 2004).

RNA interference is regulated by dsRNA with 19-25bps, which enters the cell and then binds to an RNA-induced silencing complex (RISC). Binding of dsRNA to the antisense strand of complementary messenger RNA (mRNA) sequences triggers degradation of the target messenger RNA by endonucleases found in the RISC complex (Rana, T.M., Nat. Rev. mol. cell biol., 8:23-36, 2007; Tomari, Y. and Zamore, P.D., Genes Dev., 19:517-529, 2005). In other words, small interfering RNA by inhibiting the production of specific proteins and thus interfering with DNA expression and included in RNA interference. Small interfering RNAs consisting of 19 to 23 nucleotides form base pairs according to the complementary sequence of messenger RNA to the particular mRNA that forms the double stranded RNA. Subsequently, double-stranded RNA is specifically cleaved off at the same time as messenger RNA is removed from the cells. Small interfering RNAs have received attention as agents for gene therapy because they have shown significant effects on inhibiting the expression of specific DNA in recent animal studies. Small interfering RNAs with higher activation and precise selection of DNA have been studied over the last 20 years and are expected to replace antisense oligonucleotides that are currently being used as therapeutics. Thus, many pharmaceutical companies are now developing small interfering RNA based therapeutics. Compared to existing antisense oligonucleotides, small interfering RNA are known to inhibit gene expression in 10-fold fewer quantities and only inhibit the target gene with significant selectivity of the gene. Small interfering RNA technology, particularly for therapeutic purposes, has significant benefits, as it can be easily designed compared to other drugs and has properties such as high target selectivity and inhibition of specific gene expression. Also, since inhibition of gene expression by RNA interference utilizes a naturally occurring mechanism in vivo, toxicity is low. However, small interfering RNA has the disadvantage of not being able to easily transport the small interfering RNA into cells, because small interfering RNA is anionic and cannot penetrate cell membrane and is easily decomposed in a short period of time due to low stability in vivo. This disadvantage of small interfering RNAs can be addressed by conjugation to the carrier peptides disclosed herein.

In one embodiment of the invention, the efficacy of the active ingredient (cargo) is cancer cells, immune cells or fibroblasts. Specifically, the cancer cell includes any one selected from the group consisting of: hepatoma, breast cancer and leukemia cells; the above immune cell comprises any one immune cell selected from the group consisting of: t lymphocytes, B cells and monocytes.

In one embodiment of the invention, the active ingredient will be localized in the cytoplasm and the carrier peptide will locally transport the active ingredient to the cytoplasm.

In one embodiment of the invention, the active ingredient will be localized in the mitochondria and the carrier peptide will locally deliver the active ingredient to the mitochondria.

In one embodiment of the present invention, the drug delivered inside the cell by the cell penetrating peptide may comprise one or more drug delivery vehicles, such as a lipophile, micelle, nanoparticle, magnetic particle, or quantum dot.

The term "contrast material" is disclosed herein as a broad indication that encompasses all materials used to contrast structures or fluids within the body in medical images. Suitable contrast media include, but are not limited to, radiopaque contrast media, paramagnetic contrast media, superparamagnetic contrast media, Computed Tomography (CT), and other contrast media. For example, radiopaque contrast agents (for X-ray imaging) will include inorganic and organic iodine compounds (e.g., diatrizoate), radiopaque metals and salts of the radiopaque metals (e.g., silver, gold, platinum, etc.), and other radiopaque compounds (e.g., calcium salts, barium salts such as barium sulfate, tantalum, and tantalum oxide). Suitable paramagnetic contrast agents (for MR imaging) include gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) and Gd-DTPA derivatives, other gadolinium, manganese, iron, dysprosium, copper, europium, erbium, chromium, nickel and cobalt complexes, such as 1,4,7, 10-tetraazacyclododecane-N, N ', N ", N '" -tetraacetic acid (1,4,7, 10-tetraazacyclododecane-N, N ', N ", N '" -tetraazacyclododecane acid; DOTA), ethylenediaminetetraacetic acid (ethylene diamine tetraacetic acid; EDTA), 1,4,7, 10-tetraazacyclododecane-N, -N ', N "-triacetic acid (1,4,7, 10-tetraazacyclododecane-N, -N ', N" -triacetic acid; DO3, 3A), 1,4,7-triazacyclononane-N, -N ', N "-triacetic acid, n ', N "-triacetic acid (1,4, 7-triazacyclonane-N, N', N" -TRIACETIC ACID; NOTA), 1,4,8,10-tetraazacyclotetradecane-N, N ', N ", N'" -tetraacetic acid (1,4,8,10-tetraazacyclotetradecane-N, N ', N ", N'" -tetraacetic acid; TETA), hydroxybenzylethylenediamine diacetic acid (HBED). Suitable superparamagnetic contrast agents (for MR imaging) include magnetite, superparamagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) and monocrystalline iron oxide. Other suitable contrast media are iodinated, non-iodinated, ionic and non-ionic CT contrast media, contrast media like rotational markers or diagnostically effective agents.

Other examples of contrast agents include beta-galactosidase, green fluorescent protein, cyan fluorescent protein, luciferase (but are not limited to those agents), and marker genes encoding proteins that can be readily detected when expressed within a cell. Various labels may be used, such as radioisotopes, flurs, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens).

In one embodiment of the present invention, the contrast agent is ferrocene carboxylic acid of chemical formula 2 below. The structure of ferrocene is shown in chemical formula 1.

[ chemical formula 1]

[ chemical formula 2]

In one example of the present invention, the conjugate of the cell-penetrating peptide and the contrast medium is ferrocenecarboxy-pep 1 represented in chemical formula 3 below.

[ chemical formula 3]

In one embodiment of the invention, the peptide or composition may be fused to one or more detectable labels. The label may be a compound that is detectable in a chemical, physical or enzymatic reaction or a compound that directly or indirectly generates a signal in a reaction. Labeling and detection can then be performed according to methods well known in the art (e.g., Sambrook, J. and Russel, D.W. (2001); and Lottspeich, F. and Zorbas H. (1998) Bioanalytik, Spektrum Akademischer Verlag, Heidelberg/Berlin, Germany). Labels include fluorescent labels, enzyme labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals and colloidal gold, but are not limited to those labels. All forms of the indicia are well known in the art of this work and are commercially available from various suppliers.

In one embodiment of the invention, the cargo may be directly bound to the peptide. In another embodiment of the invention, the cargo may be bound to the peptide by various types of bonds, such as covalent or non-covalent bonds. For example, in one embodiment of the invention, the cargo may be bound to the N-terminus or C-terminus of the peptide. For example, the cargo may be bonded to the peptide by a disulfide bond or a covalent bond. The covalent bond is a bond that can bond the cargo to the alpha-amine of the N-terminal glutamic acid or the amine of the C-terminal lysine residue. Also, the peptide and cargo may be bound by non-covalent bonds, which may allow the peptide or cargo to be encapsulated within each other.

In another embodiment of the invention, the peptide may be bound to the cargo by a linker. For example, a peptide may be bound to a cargo by binding the cargo to a linker after introducing the linker, such as a hydrazinium nicotinamide (6-hydrazinopyridine-3-carboxylic acid) linker (Hynic (6-hydrazinopyridine-3-carboxylic acid) linker), to the amine of the alpha-amine of the N-terminal glutamic acid or the C-terminal lysine residue.

In another embodiment of the present invention, when the cargo is DNA or RNA, an SH group (thiol group) is introduced into the peptide and a maleimide group is introduced into the DNA or RNA, and then, the SH group of the peptide and the maleimide group of the DNA or RNA are bound, thereby generating binding between the cargo and the peptide.

In another embodiment of the invention, when the cargo is a peptide or protein, the DNA of the expression cargo is bound to the DNA of the expression vector peptide, and by expressing the DNA binding, the cargo can be bound to the peptide in the form of a fusion protein. Specific examples of binding by fusion proteins are as follows: when primers are made for producing the fusion protein, nucleotides encoding the carrier peptide are attached in front of nucleotides expressing the cargo, and the obtained nucleotides are embedded in a vector such as Polyethylene Terephthalate (pET) vector using restriction enzymes, and the nucleotides are expressed by transformation into cells such as BL-21(DE 3). At this time, the fusion protein will be efficiently expressed by treating the fusion protein with an expression-inducing agent like isopropyl-1-thio- β -D-galactopyranoside (IPTG). Subsequently, the expressed fusion protein was purified by His-tag purification, and dialyzed with PBS, and concentrated by centrifugation under such conditions of 2000rpm to 4000rpm, 5 to 20 minutes, by adding to the kit.

In one embodiment of the invention, the carrier peptide is conjugated to a staining substance, a Fluorescent substance, in particular Fluorescein Isothiocyanate (FITC) or Green Fluorescent Protein (GFP). In one embodiment of the invention, FITC is conjugated to the amine group of lysine (NH3+) at the N-terminus or C-terminus of the carrier peptide. In the case of peptides in which lysine is not present at the end of the peptide, the peptide may be conjugated to FITC through a linker that includes lysine.

The carrier peptide disclosed herein, which is a peptide comprising an amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:178, or a peptide having an amino acid sequence more than 80% homologous to the above peptide, or a fragment of the above peptide, can bind to the cargo at a mole fraction of 1:1, but the carrier peptide can also bind to the cargo at a mole fraction different from 1: 1. For example, the mole fraction of CPP to cargo may be greater than 2:1, specifically, greater than 2:1, greater than 3:1, greater than 4:1, greater than 5:1, greater than 6:1, greater than 7:1, greater than 8:1, greater than 9:1, or greater than 10: 1. This means that a large number of carrier peptide molecules can be bound to the cargo molecule. A large number of carrier peptide molecules can be combined in series or in parallel. By "serially bound" is meant that the carrier peptide and the cargo molecule will be bound at the terminal amino acid. By "parallel binding" is meant that the carrier peptide and the cargo molecule will bind at a site other than the terminal amino acid. In another aspect, the molar fraction of carrier peptide to cargo can be greater than 1: 2. This means that the carrier peptide molecule can be bound to a large number of cargo molecules. For example, the molar fraction of carrier peptide to cargo can be 1:2, specifically, greater than 1:2, greater than 1:3, greater than 1:4, greater than 1:5, greater than 1:6, greater than 1:7, greater than 1:8, greater than 1:9, or greater than 1: 10.

The moving pathway of the peptide bound to fluorescein isothiocyanate can be easily found. Thus, the carrier peptide in one embodiment of the invention will be used for cellular imaging or detection of drug delivery pathways inside cells.

In one embodiment of the invention, there is provided the use of a peptide as a drug delivery vehicle for delivery of one or more active ingredients, wherein the peptide comprises an amino acid sequence of any one of SEQ ID No. 2 to SEQ ID No. 178, or the peptide is a fragment of the aforementioned peptide, or the peptide has an amino acid sequence which is more than 80% homologous to the aforementioned peptide. Use may refer to therapeutic use or non-therapeutic use.

In one embodiment of the present invention, there is provided a method of delivering a drug to the interior of a cell of a subject, the method comprising the steps of administering a composition comprising the drug and a peptide; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, or the peptide is a fragment of the peptide, or the peptide has an amino acid sequence with more than 80% homology with the peptide.

In one embodiment of the present invention, a method of detecting a drug delivery route is provided, the method comprising the steps of applying a peptide and a contrast agent to a subject; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, or the peptide is a fragment of the peptide, or the peptide has an amino acid sequence with more than 80% homology with the peptide.

In one embodiment of the present invention, a method of detecting a drug delivery route is provided, the method comprising the steps of applying to a subject a conjugate of a peptide and a contrast agent; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, or the peptide is a fragment of the peptide, or the peptide has an amino acid sequence with more than 80% homology with the peptide.

In one embodiment of the invention, a kit for delivering a drug into a cell of a subject is provided, the kit comprising a composition comprising a conjugate of a peptide of the invention and a drug for delivery, wherein the peptide comprises an amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO:178 or is a fragment of the aforementioned peptide, or has an amino acid sequence with more than 80% homology to the aforementioned peptide, and instructions, wherein the instructions comprise at least one of: dosage administered, route of administration, frequency of administration, and composition.

In one embodiment of the present invention, a cosmetic or food composition comprising an active ingredient and a peptide is provided; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, has an amino acid sequence which is more than 80% homologous to the above sequence, or is a fragment of the above peptide. In another embodiment of the present invention, there is provided a cosmetic or food composition comprising a conjugate of a peptide and an active ingredient; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, has an amino acid sequence which is more than 80% homologous to the above sequence, or is a fragment of the above peptide.

In one embodiment of the present invention, there is provided a pharmaceutical, cosmetic or food composition having a significant ability to transport an active ingredient into the interior of a cell, the pharmaceutical, cosmetic or food composition comprising a conjugate of a peptide and an active ingredient; wherein the peptide comprises any one of the amino acid sequences of SEQ ID NO 2 to SEQ ID NO 178, has an amino acid sequence which is more than 80% homologous to the above sequence, or is a fragment of the above peptide.

Mitochondria, the central organelle in the energy metabolism of eukaryotic cells, are the first well-known intracellular organelles associated with human diseases (Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B: A case of non-thyroid hormone with a defect in the international of mitochological respiratory control: a associated clinical, biochemical and morphological studies), J toxin 41: 1776-.

Since mitochondria play an important role in controlling energy metabolism and apoptosis of cells, they serve as main targets of various therapeutic drugs. Also, this organelle is involved in controlling calcium concentration inside the cell, the mitochondrial respiratory chain acts as an electron transport system important in energy generation, and the mitochondrial respiratory chain causes the production of reactive oxygen species. Therefore, abnormal mitochondrial action has a close relationship with adult diseases such as diabetes insipidus, cardiomyopathy, infertility, blindness, kidney/liver diseases, and stroke (modia-Napolitano KS, Singh KK: March mitochondri as targets for detection and treatment of cancer.) Expert Rev Mol Med 11:1-19, 2002). Also, it is being proposed to include mitochondrial genetic mutations in outbreaks of aging, degenerative neuronal diseases and cancer, among others.

The mitochondrial targeting delivery system provided according to an embodiment of the present invention may comprise any one of the conjugates described above, wherein the carrier peptide moves locally into the mitochondria and performs the role of local intracellular mitochondrial delivery of the active ingredient, wherein the peptide having an amino acid sequence at least 80% homologous to the above sequence and the fragment are peptides that retain the mitochondrial targeting delivery system, and the above mitochondrial targeting peptide may be a peptide having an amino acid sequence of any one of SEQ ID No. 2 to SEQ ID No. 178.

There may be provided a mitochondrial activity modulating composition, wherein the composition comprises a conjugate of the peptide of the present invention and a carrier peptide for delivery, wherein the carrier peptide moves locally into intracellular mitochondria and performs a role of delivering the active ingredient locally to intracellular mitochondria, wherein a peptide having an amino acid sequence at least 80% homologous to the above sequence and a fragment of the peptide are peptides that maintain a mitochondrial targeting delivery system, and the above mitochondrial targeting peptide may be a composition having an amino acid sequence of any one of SEQ ID NO:2 to SEQ ID NO: 178.

A mitochondrial activity modulating composition of one embodiment of the invention for use as a pharmaceutical composition for treating a disease or disorder associated with mitochondria, preventing, inhibiting disease progression, or alleviating symptoms; wherein the active ingredient is used for treating a disease or disorder associated with mitochondria, preventing, inhibiting the progression of a disease, or alleviating a symptom.

The "Mitochondrial-related diseases" disclosed herein include Huntington's disease, amyotrophic lateral sclerosis, Mitochondrial Encephalomyopathy with lactacidemia and Stroke-like syndromes (Mitochordrial Enphalyopathia with Lactic acidity and Stroke-like isomers; MELAS); contracture epilepsy with red color \35124, tattered myofibrosis (Myoclonus, epilesy, and myopathy with strained red fibers; MERRF); neuromuscular relaxation, disorders, retinitis pigmentosa/hereditary Leigh syndrome (Neurogenic muscular pain, ataxia, retinitis pigmentosa/matricial inherited leigh syndrome; NARP/MILS); leber's optic neuropathy (Lebers hereditary optical neuropathy; LHON); Kearns-Sayre Syndrome (Kearns-Sayre Syndrome; KSS); pearson Marrow-Pancreas Syndrome (PMPS); chronic Progressive External Opthalmopoligia (CPEO); (ii) the rapi syndrome; alper's syndrome; a plurality of mitochondrial DNA deletion syndromes; mitochondrial DNA depletion syndrome; defect of the complex I; complex II (succinate dehydrogenase; SDH)) deficiency; defect of complex III; cytochrome c oxidase (COX, complex iv) deficiency; composite V defect; adenine Nucleotide Transporter (ANT); pyruvate Dehydrogenase (PDH) deficiency; ethylmalonic acid acidic urine with lactic acidemia; 3-methylpentenedioic acid acidic urine having lactic acidemia; refractory epilepsy manifested as attenuation during infection; asperger syndrome, manifested as attenuation during infection; autism manifested as attenuation during infection; attention Deficit Hyperactivity Disorder (ADHD); cerebral palsy manifested as attenuation during infection; misregistration manifested as attenuation during infection; maternally inherited thrombocytopenia; leukemia; MNGIE (mitochondrial myopathy, peripheral and autonomic neuropathy, gastrointestinal dysfunction and epilepsy); MARIAHS syndrome (mitochondrial disorders, recurrent infections, aphasia, hypouricemia/myelin reduction (hypomyelination), seizures, and dicarboxylic aciduria); ND6 dystonia; periodic vomiting symptoms manifested as decay during infection; 3-hydroxyisobutyric acid acidic urine having lactic acidemia; diabetes insipidus with lactic acidemia; uridine Reactive Neural Syndrome (URNS); familial Bilateral Striatal Necrosis (FBSN); hearing loss associated with aminoglycosides; relaxed cardiomyopathy; splenic lymphoma; symptoms of tungsten; multiple mitochondrial DNA deletion symptoms; and renal tubular acidemia/diabetes insipidus/disorder symptoms, but are not limited to those diseases.

In another embodiment of the present invention, there is provided a nucleic acid molecule encoding the above polypeptide. For example, the nucleic acid molecule has the base sequence GAA GCG CGC CCG GCG CTG CTG ACC AGC CGC CTG CGC TTT ATT CCG AAA. The nucleic acid may be introduced into the host cell according to methods well known to those skilled in the art. For example, a well-known method may be a transformation method by: calcium phosphate methods, lipoplasts, electroporation, contacting viruses and cells, or microinjection directly into cells, etc. The host cell is a higher eukaryotic cell (e.g., a mammalian cell), or a lower eukaryotic cell (such as a yeast cell), or a prokaryotic cell (such as a bacterial cell). Prokaryotic host cells suitable for transformation may be of a species belonging to: for example, Escherichia coli, Bacillus subtilis, Salmonella, Pseudomonas, Streptomyces, and Microbacterial species.

The vector comprising the above-described nucleic acid molecule is typically a recombinant expression vector and comprises an origin of replication enabling host cell transformation and a selectable marker (e.g., dihydrofolate reductase for eukaryotic cell culture, or neomycin tolerance, tetracycline or ampicillin tolerance in E.coli, the yeast TRP1 gene), and a promoter for controlling transcription of the protein-coating sequence. For example, expression vectors that can be used are: bacterial plasmids such as SV40, derivatives of pcDNA; and well-known bacterial plasmids such as colE1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al, Gene 67:31-40 (1988)); plasmids such as RP4, a derivative of pMB9 and pMB 9; phage DNA identical to numerous derivatives of phage I, such as NM 989; phage DNA such as M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., phage DNA or vectors induced from the combination of a modified plasmid using expression suppression sequences with phage DNA. Mammalian expression vectors contain an origin of replication, an appropriate promoter, and an enhancer. Likewise, the vector may comprise a forced ribosome binding site, a polyadenylation site, splice donor and acceptor moieties, transcription termination sequences and 5' flanking (cloning) non-transcribed sequences. Mammalian expression vectors can comprise inducible promoters, e.g., vectors comprising a dihydrofolate reductase promoter, any expression vector comprising a DHFR expression cassette or a DHFR/methotrexate co-amplification vector, such as pED (Randal J, kaufman, 1991, Randal J. Kaufman, Current Protocols in Molycular Biology, 16, 12 (1991)). Alternatively, the following vectors may be used: glutamine synthetase/methionine sulfonimide co-amplification vectors, for example, pEE14(Celltech corporation), human herpesvirus type IV (Epstein-Barr-Virus; EBV); or vectors which direct episomal expression under the control of nuclear antigen (EBNA), for example, pREP p4 (Invitrogen), pCEP4 (Invitrogen), pMEP4 (Invitrogen), pREP p8 (Invitrogen), pREP p9 (Invitrogen), and pEBVHis (Invitrogen). The mammalian expression vectors can be selected from Rc/CMV (Invitrogen), pRc/RSV (Invitrogen), and the like. Vaccinia virus mammalian expression vectors useful in the invention are pSC11, pMJ601, pTKgptF1S, and the like.

The yeast expression vector system to be used in the present invention is a non-fusion pYES2 vector (Invitrogen corporation), fusion pYESHISA, B, C (Invitrogen corporation), pRS vector, or the like.

The above vectors can be introduced into various cells such as mammalian cells (particularly human-derived cells) or bacterial, yeast, fungal, insect, nematode and plant cells. Examples of suitable cells are: VERO cells; HELA cells, e.g., ATCC No. ccl 2; CHO cell lines, e.g., ATCC No. ccl61; COS cells, such as COS-7 cells and ATCC No. CRL 1650 cells; w138, BHK, HepG2, 3T3, e.g., ATCC No. crl 6361; a549, PC12, K562 cells; 293 cells; sf9 cells, e.g., ATCC No. crl 1711; and Cv1 cells, such as ATCC No. ccl70, and the like.

Other suitable cells to be used in the present invention are prokaryotic host cell strains, for example, strains belonging to the genera Escherichia coli (e.g., DH 5-alpha strain), Bacillus subtilis, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.

In one embodiment of the invention, the composition may contain from 0.1 μ g/mg to 1mg/mg, specifically from 1 μ g/mg to 0.5mg/mg, more specifically from 10 μ g/mg to 0.1mg/mg of the following peptides: a peptide comprising an amino acid sequence of any one of SEQ ID NO 2 to SEQ ID NO 178, a peptide comprising an amino acid sequence having more than 80% homology with the above sequence, or a fragment of the above peptide. When the peptide is contained within the above range, all safety and stability of the composition can be satisfied and are appropriate in terms of cost effectiveness.

In one embodiment of the invention, the composition is applicable to all animals, including humans, dogs, chickens, pigs, cows, sheep, guinea pigs, and monkeys.

In one embodiment of the invention, the pharmaceutical composition may be administered in a bone marrow, epidural or subcutaneous means by: oral, rectal, transdermal, intravenous, intramuscular, intraperitoneal.

Oral administration may be in the form of, but is not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Non-oral administration may be in the form of, but is not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, aqueous emulsions, suppositories, patches or sprays.

In one embodiment of the present invention, the pharmaceutical composition may contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, aromatics or sweeteners, if necessary. In one embodiment of the invention, the pharmaceutical composition may be manufactured by industrial methods well known in the art.

In one embodiment of the invention, the active ingredients of the medical composition may vary according to the following aspects: age, sex, weight, pathology and condition of the patient, route of administration or judgment of the prescriber. The dosage based on this factor is determined within the level of the skilled person and the daily dosage may be, for example, but not limited to, 0.1 to 1 g/kg/day, specifically 1 to 10 mg/kg/day, more specifically 10 to 1 mg/kg/day, more specifically 50 to 100 μ g/kg/day. In one embodiment of the present invention, the pharmaceutical composition may be administered one to three times per day, but is not limited thereto.

In one embodiment of the present invention, the cosmetic composition may be provided in all forms suitable for topical application. For example, the form may be provided as a solution, an aqueous emulsion obtained by dispersion of an oil phase in water, an aqueous emulsion obtained by dispersion of water in an oil phase, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol. This form can be manufactured by industrial methods well known in the art.

In one embodiment of the present invention, the cosmetic composition may include other ingredients that may desirably increase the primary effect, at levels that do not impair the primary effect. In one embodiment of the present invention, the cosmetic composition may additionally comprise an emollient cream, emollient, surfactant, UV absorber, preservative, fungicide, antioxidant, pH adjuster, organic or inorganic pigment, fragrance, coolant or antiperspirant. The formulation ratio of the above-mentioned ingredients may be determined by those skilled in the art within a level not impairing the objects and effects of the present invention, and may be 0.01 to 5% by weight, specifically 0.01 to 3% by weight, based on the total weight of the cosmetic composition.

In one embodiment of the invention, the food composition is not limited to the form, but may be in the form of granules, powder, liquid and solid, for example. Each form may be composed of ingredients (in addition to the active ingredient) commonly used in the industry, appropriately selected by those skilled in the art, and may have an increased effect with other ingredients.

The determination of the dosage of the above active ingredients is within the level of the skilled person, and the daily dosage may be, for example, from 1. mu.g/kg/day to 10 mg/kg/day, more specifically from 10. mu.g/kg/day to 1 mg/kg/day, more specifically from 50. mu.g/kg/day to 100. mu.g/kg/day, but is not limited to this amount and may vary depending on age, health status, morbidity and other various factors.

The terminology used herein is intended to be used to describe embodiments and not to limit the invention. The preceding term of a number of countless is not intended to limit the quantity but rather to mean that there may be more than one of the things that the term is used. The terms "comprising," "having," "consisting of," and "containing" are to be construed in an open-ended fashion (i.e., "including, but not limited to").

The recitation of ranges of quantities is used instead of specifying separate quantities within the range, and thus each quantity can be read as a separate quantity incorporated herein, unless specifically stated otherwise. The endpoints of all ranges are inclusive of the range and independently combinable.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., using a language such as "similar") is intended to more clearly describe the invention and not to limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the invention. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The preferred embodiments of the present invention are the best modes known to the inventors for carrying out the invention. It will be clear to one skilled in the art that the statements are read in advance of the variations in the preferred embodiments. The inventors expect skilled artisans to employ such variations as appropriate, and the invention will be practiced otherwise than as specifically described herein. Accordingly, this invention includes equivalents of the subject matter recited in the claims appended hereto and variations of the equivalents as permitted by applicable law. Moreover, all possible variations in any combination of the above-described components are encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, 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.

Example 1: synthesis of peptides

The peptides having SEQ ID NO 2 to SEQ ID NO 178 were synthesized according to the existing method of solid phase peptide synthesis. In detail, the peptide was synthesized by coupling each amino acid from the C-terminus using ASP48S (Peptron Corp., Tada. City, Korea) using Fmoc Solid Phase Peptide Synthesis (SPPS). Those peptides were used as follows, those peptides were attached to the resin at the first amino acid of the C-terminus:

NH 2-lysine (Boc) -2-chloro-trityl resin

NH 2-alanine-2-chloro-trityl resin

NH 2-arginine (Pbf) -2-chloro-trityl resin

All amino acid materials of the synthetic peptide were protected at the N-terminus by Fmoc, and the amino acid residues were protected by Trt, Boc, t-Bu (t-butyl ester), Pbf (2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl), which are soluble in acid. Such as:

Fmoc-alanine-OH, Fmoc-arginine (Pbf) -OH, Fmoc-glutamic acid (OtBu) -OH, Fmoc-proline-OH, Fmoc-leucine-OH, Fmoc-isoleucine-OH, Fmoc-phenylalanine-OH, Fmoc-serine (tBu) -OH, Fmoc-threonine (tBu) -OH, Fmoc-lysine (Boc) -OH, Fmoc-glutamine (Trt) -OH, Fmoc-tryptophan (Boc) -OH, Fmoc-methionine-OH, Fmoc-asparagine (Trt) -OH, Fmoc-tyrosine (tBu) -OH, Fmoc-aminocaproic acid-OH, Trt-thioglycolic acid.

HBTU [2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium hexafluorophosphate ]/HOBt [ N-hydroxybenzotriazole ]/NMM [ 4-methylmorpholine ] was used as coupling reagent. Fmoc was removed using Dimethylformamide (DMF) containing 20% piperidine. To remove the protection from the residue or to separate the synthesized peptide from the resin, a cleavage mixture [ trifluoroacetic acid (TFA)/triisopropylsilane (trisisopropylsilane; TIS)/dithioglycol (EDT)/H2O ═ 92.5/2.5/2.5 ] was used.

Peptides were synthesized by adding each amino acid in the following sequential process using a solid phase molecular framework: amino acid protection, coupling reaction, washing and deprotection. After cleavage of the synthesized peptide from the resin, the synthesized peptide was purified by High Performance Liquid Chromatography (HPLC) and the synthesis was confirmed by Mass Spectrometry (MS) and then lyophilized.

Specific peptide synthesis procedures are described below with examples of pep1 (EARPALLTSRLRFIPK).

1) Coupling of

Amino acid (8 eq) protected with NH 2-lysine (Boc) -2-chloro-trityl resin was melted in coupling reagent HBTU (8 eq)/HOBt (8 eq)/NMM (16 eq) and after addition of DMF, the reaction mixture was incubated at room temperature for 2 hours, followed by sequential washes with DMF, MeOH and DMF.

2) Fmoc deprotection

After addition of 20% piperidine in DMF, the reaction mixture was incubated at room temperature for 2 times for 5 minutes, followed by washing with DMF, MeOH and DMF in that order.

3) The basic framework of the peptide is formed by repeating reaction 1 and reaction 2.

4) Cutting: the cleavage mixture is added to the fully synthesized peptide and the peptide is separated from the resin.

5) Pre-cooled ether was added to the mixture, and the reaction mixture was then centrifuged to precipitate the peptide.

6) After purification by Prep-HPLC, the molecular weight was checked by LC/MS and lyophilized to obtain the peptide in powder form.

Example 2:pep(CPP)-FITCpreparation of conjugates

(1) Preparation of FITC-CPP conjugate

Conjugates of peptides having SEQ ID NO:2 to SEQ ID NO:178, e.g., a conjugate of pep1 and FITC, conjugated with FITC, in other words, FITC-linker-pep 1, were made as follows.

Basic framework NH 2-linker-e (otbu) - ｃA-r (pbf) -P- ｃA-L-t (tbu) -s (tbu) -r (pbf) -L-r (pbf) -F-I-P-k (boc) -2-chloro-trityl resin of the peptide obtained according to the manufacturing method described in example 1 was reacted with FITC. Specifically, fluorescein-5-isothiocyanate (FITC) (8 equivalents) was melted in DMF with N, N-Diisopropylethylamine (N, N-Diisopropylethylamine; DIPEA) (16 equivalents). DMF solution was added and reacted at room temperature for 2 hours, followed by washing with DMF, MeOH and DMF sequentially. Thus, FITC-linker-E (OtBu) -A-R (Pbf) -P-A-L-T (tBu) -S (tBu) -R (Pbf) L-R (Pbf) -F-I-P-K (Boc) -2-chloro-trityl resin was obtained. The linker herein is 6-aminocaproic acid (Ahx). TFA/TIS/H2O ═ 95/2.5/2.5 was added to the peptide composed on the resin, and the conjugate was separated from the resin. Pre-cooled ether was added to the obtained mixture and the peptide conjugate was precipitated using centrifugation. After purification by Prep-HPLC, purity was determined by analytical HPLC and molecular weight was determined by LC/MS. The peptide synthesized as described above was verified as FITC-pep1 by determining the molecular weight by LC/MS. The conjugate was then lyophilized. Conjugates of peptides of SEQ ID NO 2 through SEQ ID NO 178 fused to FITC were also prepared in the same manner as described for pep 1.

(2) Preparation of CPP-FITC conjugate

The basic framework of the peptide (NH2-e (otbu) - ｃA-r (pbf) -P- ｃA-L-t (tbu) -s (tbu) -r (pbf) -L-r (pbf) -F-I-P-k (dde) -2-chloro-trityl resin) was produced according to the manufacturing method described in example 21 (1). For the selective introduction of FITC to the C-terminus of the peptide, the N-terminus of the peptide was protected from Boc. Subsequently, di-tert-butyl dicarbonate (30 equivalents) and DIPEA (30 equivalents) were melted in DMF. The DMF solution was added to the peptide and incubated at room temperature for 2 hours, and the peptide was washed sequentially with DMF, MeOH and DMF. Thus, Boc-E (OtBu) -A-R (Pbf) -P-A-L-T (tBu) -S (tBu) -R (Pbf) L-R (Pbf) -F-I-P-K (Dde) -2-chloro-trityl resin was obtained. DMF with 2% hydrazine was used to remove Dde, which is a protecting group for the C-terminal residue lysine, to add FITC to the C-terminus of lysine. Subsequently, FITC (8 equivalents) and DIPEA (16 equivalents) were melted in DMF, which was added to the peptide reaction mixture, and the mixture was incubated at room temperature for 2 hours, followed by sequential washes with DMF, MeOH, DMF. Thus, Boc-E (OtBu) -A-R (Pbf) -P-A-L-T (tBu) -S (tBu) -R (Pbf) L-R (Pbf) -F-I-P-K (FITC) -2-chloro-trityl resin was obtained. TFA/TIS/H2O ═ 95/2.5/2.5 was added to separate the peptide from the resin. Pre-cooled ether was added to the mixture and the peptide was precipitated using centrifugation. After purification by Prep-HPLC, purity was determined by analytical HPLC and molecular weight was determined by LC/MS. The material obtained was verified as pep1-FITC by LC/MS confirmation of molecular weight. The conjugate was then lyophilized. peptide-FITC conjugates of SEQ ID NO 2 through SEQ ID NO 178 were also prepared in the same manner as described above.

Example 3: penetrability of ferrocenecarboxy-CPP conjugates

Amino acid protected with NH 2-lysine (Boc) -2-chloro-trityl resin (8 eq) and coupling reagent HBTU (8 eq)/HoBt (8 eq)/NMM (16 eq) were melted in DMF for coupling. Add DMF solution and react for 2 hours at room temperature, followed by DMF, MeOH, DMF washes in that order. Subsequently, DMF with 20% piperidine was added for Fmoc deprotection and reacted at room temperature for 5 min 2 times, followed by DMF, MeOH, DMF washes in that order. By repeating the above reaction, basic frameworks (NH2-E (OtBu) -A-R (Pbf) -P-A-L-T (tBu) -S (tBu) -R (Pbf) L-R (Pbf) -F-I-P-K (Dde) -2-chloro-trityl resin) constituting the peptides were prepared. DMF with 2% hydrazine was added to remove Dde, which is a protecting group for the C-terminal lysine residue. Subsequently, ferrocene carboxylic acid (Sigma Aldrich cat. # 46264, 16 equivalents) and the coupling agent HBTU (16 equivalents)/HoBt (16 equivalents)/NMM (32 equivalents) were melted in DMF. The DMF solution was added and the reaction was carried out at room temperature for 2 hours, followed by washing with DMF, MeOH and DMF in that order. TFA/TIS/H2O ═ 95/2.5/2.5 was added to the peptide resin synthesized above to separate the peptide from the resin. Cooled ether was added to the resulting mixture and centrifugation was used to precipitate the aggregated peptide. The precipitate was purified by HPLC and determined by MS. Subsequently, the peptide was freeze-dried.

Example 4: cell penetration experiments with pep-FITC conjugates

(1) Cell penetration experiments in HeLa cell line

Cell culture

HeLa cell line human cervical adenocarcinoma cells, purchased from ATCC. Cells were cultured in Minimal Essential Medium (MEM) containing 10% fetal bovine serum (Invitrogen, USA), Erie's salt, optional amino acids, sodium pyruvate, 100. mu.g/mL penicillin and 10 units/mL streptomycin in a 5% CO2 incubator at 37 ℃.

Flow cytometry and confocal microscopy analysis of cell penetration

Flow cytometry and confocal microscopy analyses were performed to compare the extent of cellular uptake of cells treated with peptides of SEQ ID NO:2 to SEQ ID NO:178, pep (cpp), and control.

The cell lines were split in 6-well culture plates and cultured in medium containing 10% fetal bovine serum (Invitrogen, USA), 100. mu.g/ml penicillin, 100 units/ml streptomycin at 37 ℃ for 12 hours in a 5% CO2 incubator. After washing the cell lines with PBS, starvation was induced in the minimum essential medium for one hour. 20uM of each carrier peptide was treated and incubated at 37 ℃ for up to one hour. After repeating the step of washing the cells with PBS three times, the trypsin-EDTA was treated at 37 ℃ for 10 minutes to isolate the carrier peptide outside the cells. Cells were collected with chilled PBS and centrifugation was performed to repeat the step of washing cells up to three times. Subsequently, the cells were suspended in 0.5ml PBS containing 4% paraformaldehyde and the fluorescence of the cells was analyzed using FACS Calibur (BD corporation, usa). The cellular uptake pattern of the control and the various peptides bound to FITC were compared and analyzed by Mean Fluorescence Intensity (Mean Fluorescence Intensity; MFI).

The results are shown in fig. 2 to 29. FIG. 1 shows the results for pep1 of SEQ ID NO 1 to which reference was made. The analysis results shown in fig. 2 to 29 are given in detail in table 7 below.

[ Table 7]

(2) Cell penetration in the Huh7 cell line

Cell culture

The Huh7 (human hepatocellular carcinoma) Cell line was purchased from the American Type Cell Culture Collection (ATCC) and used as suspension cells. The cells were cultured in MEM medium with 10% fetal bovine serum (Invitrogen, USA), Erl's salt, optional amino acids, sodium pyruvate, 100. mu.g/ml penicillin and 10 units/ml streptomycin in a 5% CO2 incubator at 37 ℃.

Screening assay for cell penetration by flow cytometry

To confirm the cell penetration of the peptides, the Huh7 cell line was treated with SEQ ID NO:2 to SEQ ID NO:178 and analyzed by flow cytometry. The assay was also confirmed by the manner described in example (1) for HeLa cells. The results of the analysis are shown in fig. 30 to 51.

(3) Cell penetration experiments in human T lymphocyte cell lines

Cell culture

The Jurkat cell line (human T cell leukemia cell line) was purchased from ATCC and used as suspension cells. And the cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, USA), Erl's salt, optional amino acids, sodium pyruvate, 100. mu.g/ml penicillin and 10 units/ml streptomycin in a 5% CO2 incubator at 37 ℃. Human-derived lymphocytes were isolated from blood (50ml) of a healthy person, and then layers of Peripheral Blood Mononuclear Cells (PBMCs) and lymphocytes were collected using a Biocoll isolation solution (Biochrom AG, berlin, germany).

Screening analysis of cell penetration by flow cytometry

To confirm the cell penetration of the peptides, human T cell lymphocyte cell lines were treated with SEQ ID NO:2 through SEQ ID NO:178 and analyzed by flow cytometry. The assay was also confirmed by the manner described in example (1) for HeLa cells. The results of the analysis are shown in fig. 52 to 69.

(4) Analysis of cell Activity and cytotoxicity

HeLa cells were cultured by the same manner as in example 4(1) above, divided into 96-well culture plates, and cultured in a medium containing 10% fetal bovine serum (Invitrogen, USA), 100. mu.g/ml penicillin and 10 units/ml streptomycin in a 5% CO2 incubator at 37 ℃ for 12 hours. After washing the cells with PBS, starvation was induced in the minimal essential medium. 20 μ M of each carrier peptide was treated at 37 ℃ and incubated for 1 hour. After cell culture, cell viability and cytotoxicity were analyzed by MTT assay. The results are shown in fig. 70 to 86.

<110> Zhenbaiskel Co., Ltd

Jin Shangzai

<120> cell-penetrating peptide, conjugate comprising the same, and use

<130> OF13P178/CN

<150> KR10-2012-0104173

<151> 2012-09-19

<150> KR10-2012-0104144

<151> 2012-09-19

<150> KR10-2012-0109207

<151> 2012-09-28

<150> KR10-2012-0109216

<151> 2012-09-28

<150> KR10-2013-0017046

<151> 2013-02-18

<160> 186

<170> PatentIn version 3.2

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<210> 51

<211> 23

<212> PRT

<213> Homo sapiens

<400> 51

Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10 15

Lys Pro Asp Gly Leu Arg Pro

20

<210> 52

<211> 24

<212> PRT

<213> Homo sapiens

<400> 52

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro

20

<210> 53

<211> 25

<212> PRT

<213> Homo sapiens

<400> 53

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp

20 25

<210> 54

<211> 26

<212> PRT

<213> Homo sapiens

<400> 54

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly

20 25

<210> 55

<211> 27

<212> PRT

<213> Homo sapiens

<400> 55

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu

20 25

<210> 56

<211> 28

<212> PRT

<213> Homo sapiens

<400> 56

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg

20 25

<210> 57

<211> 29

<212> PRT

<213> Homo sapiens

<400> 57

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro

20 25

<210> 58

<211> 30

<212> PRT

<213> Homo sapiens

<400> 58

Ala Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5 10 15

Arg Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25 30

<210> 59

<211> 29

<212> PRT

<213> Homo sapiens

<400> 59

Glu Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg

1 5 10 15

Leu Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25

<210> 60

<211> 28

<212> PRT

<213> Homo sapiens

<400> 60

Val Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu

1 5 10 15

Arg Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25

<210> 61

<211> 27

<212> PRT

<213> Homo sapiens

<400> 61

Arg Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg

1 5 10 15

Phe Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25

<210> 62

<211> 26

<212> PRT

<213> Homo sapiens

<400> 62

Gln His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5 10 15

Ile Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25

<210> 63

<211> 25

<212> PRT

<213> Homo sapiens

<400> 63

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5 10 15

Pro Lys Pro Asp Gly Leu Arg Pro Ile

20 25

<210> 64

<211> 24

<212> PRT

<213> Homo sapiens

<400> 64

Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10 15

Lys Pro Asp Gly Leu Arg Pro Ile

20

<210> 65

<211> 17

<212> PRT

<213> Homo sapiens

<400> 65

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro

<210> 66

<211> 18

<212> PRT

<213> Homo sapiens

<400> 66

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp

<210> 67

<211> 19

<212> PRT

<213> Homo sapiens

<400> 67

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly

<210> 68

<211> 21

<212> PRT

<213> Homo sapiens

<400> 68

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg

20

<210> 69

<211> 22

<212> PRT

<213> Homo sapiens

<400> 69

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro

20

<210> 70

<211> 23

<212> PRT

<213> Homo sapiens

<400> 70

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile

20

<210> 71

<211> 24

<212> PRT

<213> Homo sapiens

<400> 71

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val

20

<210> 72

<211> 25

<212> PRT

<213> Homo sapiens

<400> 72

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn

20 25

<210> 73

<211> 26

<212> PRT

<213> Homo sapiens

<400> 73

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn Met

20 25

<210> 74

<211> 27

<212> PRT

<213> Homo sapiens

<400> 74

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp

20 25

<210> 75

<211> 28

<212> PRT

<213> Homo sapiens

<400> 75

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr

20 25

<210> 76

<211> 29

<212> PRT

<213> Homo sapiens

<400> 76

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val

20 25

<210> 77

<211> 30

<212> PRT

<213> Homo sapiens

<400> 77

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

20 25 30

<210> 78

<211> 15

<212> PRT

<213> Homo sapiens

<400> 78

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10 15

<210> 79

<211> 14

<212> PRT

<213> Homo sapiens

<400> 79

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5 10

<210> 80

<211> 13

<212> PRT

<213> Homo sapiens

<400> 80

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5 10

<210> 81

<211> 12

<212> PRT

<213> Homo sapiens

<400> 81

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg

1 5 10

<210> 82

<211> 11

<212> PRT

<213> Homo sapiens

<400> 82

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu

1 5 10

<210> 83

<211> 10

<212> PRT

<213> Homo sapiens

<400> 83

Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg

1 5 10

<210> 84

<211> 9

<212> PRT

<213> Homo sapiens

<400> 84

Glu Ala Arg Pro Ala Leu Leu Thr Ser

1 5

<210> 85

<211> 8

<212> PRT

<213> Homo sapiens

<400> 85

Glu Ala Arg Pro Ala Leu Leu Thr

1 5

<210> 86

<211> 7

<212> PRT

<213> Homo sapiens

<400> 86

Glu Ala Arg Pro Ala Leu Leu

1 5

<210> 87

<211> 6

<212> PRT

<213> Homo sapiens

<400> 87

Glu Ala Arg Pro Ala Leu

1 5

<210> 88

<211> 5

<212> PRT

<213> Homo sapiens

<400> 88

Glu Ala Arg Pro Ala

1 5

<210> 89

<211> 4

<212> PRT

<213> Homo sapiens

<400> 89

Glu Ala Arg Pro

1

<210> 90

<211> 3

<212> PRT

<213> Homo sapiens

<400> 90

Glu Ala Arg

1

<210> 91

<211> 15

<212> PRT

<213> Homo sapiens

<400> 91

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10 15

<210> 92

<211> 14

<212> PRT

<213> Homo sapiens

<400> 92

Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10

<210> 93

<211> 13

<212> PRT

<213> Homo sapiens

<400> 93

Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10

<210> 94

<211> 12

<212> PRT

<213> Homo sapiens

<400> 94

Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10

<210> 95

<211> 11

<212> PRT

<213> Homo sapiens

<400> 95

Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10

<210> 96

<211> 10

<212> PRT

<213> Homo sapiens

<400> 96

Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5 10

<210> 97

<211> 9

<212> PRT

<213> Homo sapiens

<400> 97

Thr Ser Arg Leu Arg Phe Ile Pro Lys

1 5

<210> 98

<211> 8

<212> PRT

<213> Homo sapiens

<400> 98

Ser Arg Leu Arg Phe Ile Pro Lys

1 5

<210> 99

<211> 7

<212> PRT

<213> Homo sapiens

<400> 99

Arg Leu Arg Phe Ile Pro Lys

1 5

<210> 100

<211> 6

<212> PRT

<213> Homo sapiens

<400> 100

Leu Arg Phe Ile Pro Lys

1 5

<210> 101

<211> 5

<212> PRT

<213> Homo sapiens

<400> 101

Arg Phe Ile Pro Lys

1 5

<210> 102

<211> 4

<212> PRT

<213> Homo sapiens

<400> 102

Phe Ile Pro Lys

1

<210> 103

<211> 3

<212> PRT

<213> Homo sapiens

<400> 103

Ile Pro Lys

1

<210> 104

<211> 14

<212> PRT

<213> Homo sapiens

<400> 104

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10

<210> 105

<211> 12

<212> PRT

<213> Homo sapiens

<400> 105

Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5 10

<210> 106

<211> 10

<212> PRT

<213> Homo sapiens

<400> 106

Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5 10

<210> 107

<211> 8

<212> PRT

<213> Homo sapiens

<400> 107

Ala Leu Leu Thr Ser Arg Leu Arg

1 5

<210> 108

<211> 6

<212> PRT

<213> Homo sapiens

<400> 108

Leu Leu Thr Ser Arg Leu

1 5

<210> 109

<211> 4

<212> PRT

<213> Homo sapiens

<400> 109

Leu Thr Ser Arg

1

<210> 110

<211> 13

<212> PRT

<213> Homo sapiens

<400> 110

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5 10

<210> 111

<211> 12

<212> PRT

<213> Homo sapiens

<400> 111

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5 10

<210> 112

<211> 11

<212> PRT

<213> Homo sapiens

<400> 112

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg

1 5 10

<210> 113

<211> 10

<212> PRT

<213> Homo sapiens

<400> 113

Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu

1 5 10

<210> 114

<211> 9

<212> PRT

<213> Homo sapiens

<400> 114

Ala Arg Pro Ala Leu Leu Thr Ser Arg

1 5

<210> 115

<211> 8

<212> PRT

<213> Homo sapiens

<400> 115

Ala Arg Pro Ala Leu Leu Thr Ser

1 5

<210> 116

<211> 7

<212> PRT

<213> Homo sapiens

<400> 116

Ala Arg Pro Ala Leu Leu Thr

1 5

<210> 117

<211> 6

<212> PRT

<213> Homo sapiens

<400> 117

Ala Arg Pro Ala Leu Leu

1 5

<210> 118

<211> 5

<212> PRT

<213> Homo sapiens

<400> 118

Ala Arg Pro Ala Leu

1 5

<210> 119

<211> 4

<212> PRT

<213> Homo sapiens

<400> 119

Ala Arg Pro Ala

1

<210> 120

<211> 3

<212> PRT

<213> Homo sapiens

<400> 120

Ala Arg Pro

1

<210> 121

<211> 13

<212> PRT

<213> Homo sapiens

<400> 121

Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10

<210> 122

<211> 11

<212> PRT

<213> Homo sapiens

<400> 122

Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5 10

<210> 123

<211> 10

<212> PRT

<213> Homo sapiens

<400> 123

Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg

1 5 10

<210> 124

<211> 8

<212> PRT

<213> Homo sapiens

<400> 124

Arg Pro Ala Leu Leu Thr Ser Arg

1 5

<210> 125

<211> 7

<212> PRT

<213> Homo sapiens

<400> 125

Arg Pro Ala Leu Leu Thr Ser

1 5

<210> 126

<211> 6

<212> PRT

<213> Homo sapiens

<400> 126

Arg Pro Ala Leu Leu Thr

1 5

<210> 127

<211> 5

<212> PRT

<213> Homo sapiens

<400> 127

Arg Pro Ala Leu Leu

1 5

<210> 128

<211> 4

<212> PRT

<213> Homo sapiens

<400> 128

Arg Pro Ala Leu

1

<210> 129

<211> 3

<212> PRT

<213> Homo sapiens

<400> 129

Arg Pro Ala

1

<210> 130

<211> 12

<212> PRT

<213> Homo sapiens

<400> 130

Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10

<210> 131

<211> 11

<212> PRT

<213> Homo sapiens

<400> 131

Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5 10

<210> 132

<211> 9

<212> PRT

<213> Homo sapiens

<400> 132

Pro Ala Leu Leu Thr Ser Arg Leu Arg

1 5

<210> 133

<211> 8

<212> PRT

<213> Homo sapiens

<400> 133

Pro Ala Leu Leu Thr Ser Arg Leu

1 5

<210> 134

<211> 7

<212> PRT

<213> Homo sapiens

<400> 134

Pro Ala Leu Leu Thr Ser Arg

1 5

<210> 135

<211> 6

<212> PRT

<213> Homo sapiens

<400> 135

Pro Ala Leu Leu Thr Ser

1 5

<210> 136

<211> 5

<212> PRT

<213> Homo sapiens

<400> 136

Pro Ala Leu Leu Thr

1 5

<210> 137

<211> 4

<212> PRT

<213> Homo sapiens

<400> 137

Pro Ala Leu Leu

1

<210> 138

<211> 3

<212> PRT

<213> Homo sapiens

<400> 138

Pro Ala Leu

1

<210> 139

<211> 11

<212> PRT

<213> Homo sapiens

<400> 139

Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10

<210> 140

<211> 9

<212> PRT

<213> Homo sapiens

<400> 140

Ala Leu Leu Thr Ser Arg Leu Arg Phe

1 5

<210> 141

<211> 7

<212> PRT

<213> Homo sapiens

<400> 141

Ala Leu Leu Thr Ser Arg Leu

1 5

<210> 142

<211> 6

<212> PRT

<213> Homo sapiens

<400> 142

Ala Leu Leu Thr Ser Arg

1 5

<210> 143

<211> 5

<212> PRT

<213> Homo sapiens

<400> 143

Ala Leu Leu Thr Ser

1 5

<210> 144

<211> 4

<212> PRT

<213> Homo sapiens

<400> 144

Ala Leu Leu Thr

1

<210> 145

<211> 3

<212> PRT

<213> Homo sapiens

<400> 145

Ala Leu Leu

1

<210> 146

<211> 10

<212> PRT

<213> Homo sapiens

<400> 146

Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5 10

<210> 147

<211> 9

<212> PRT

<213> Homo sapiens

<400> 147

Leu Leu Thr Ser Arg Leu Arg Phe Ile

1 5

<210> 148

<211> 7

<212> PRT

<213> Homo sapiens

<400> 148

Leu Leu Thr Ser Arg Leu Arg

1 5

<210> 149

<211> 5

<212> PRT

<213> Homo sapiens

<400> 149

Leu Leu Thr Ser Arg

1 5

<210> 150

<211> 4

<212> PRT

<213> Homo sapiens

<400> 150

Leu Leu Thr Ser

1

<210> 151

<211> 3

<212> PRT

<213> Homo sapiens

<400> 151

Leu Leu Thr

1

<210> 152

<211> 9

<212> PRT

<213> Homo sapiens

<400> 152

Leu Thr Ser Arg Leu Arg Phe Ile Pro

1 5

<210> 153

<211> 8

<212> PRT

<213> Homo sapiens

<400> 153

Leu Thr Ser Arg Leu Arg Phe Ile

1 5

<210> 154

<211> 7

<212> PRT

<213> Homo sapiens

<400> 154

Leu Thr Ser Arg Leu Arg Phe

1 5

<210> 155

<211> 6

<212> PRT

<213> Homo sapiens

<400> 155

Leu Thr Ser Arg Leu Arg

1 5

<210> 156

<211> 5

<212> PRT

<213> Homo sapiens

<400> 156

Leu Thr Ser Arg Leu

1 5

<210> 157

<211> 3

<212> PRT

<213> Homo sapiens

<400> 157

Leu Thr Ser

1

<210> 158

<211> 8

<212> PRT

<213> Homo sapiens

<400> 158

Thr Ser Arg Leu Arg Phe Ile Pro

1 5

<210> 159

<211> 7

<212> PRT

<213> Homo sapiens

<400> 159

Thr Ser Arg Leu Arg Phe Ile

1 5

<210> 160

<211> 6

<212> PRT

<213> Homo sapiens

<400> 160

Thr Ser Arg Leu Arg Phe

1 5

<210> 161

<211> 5

<212> PRT

<213> Homo sapiens

<400> 161

Thr Ser Arg Leu Arg

1 5

<210> 162

<211> 4

<212> PRT

<213> Homo sapiens

<400> 162

Thr Ser Arg Leu

1

<210> 163

<211> 3

<212> PRT

<213> Homo sapiens

<400> 163

Thr Ser Arg

1

<210> 164

<211> 7

<212> PRT

<213> Homo sapiens

<400> 164

Ser Arg Leu Arg Phe Ile Pro

1 5

<210> 165

<211> 6

<212> PRT

<213> Homo sapiens

<400> 165

Ser Arg Leu Arg Phe Ile

1 5

<210> 166

<211> 5

<212> PRT

<213> Homo sapiens

<400> 166

Ser Arg Leu Arg Phe

1 5

<210> 167

<211> 4

<212> PRT

<213> Homo sapiens

<400> 167

Ser Arg Leu Arg

1

<210> 168

<211> 3

<212> PRT

<213> Homo sapiens

<400> 168

Ser Arg Leu

1

<210> 169

<211> 6

<212> PRT

<213> Homo sapiens

<400> 169

Arg Leu Arg Phe Ile Pro

1 5

<210> 170

<211> 5

<212> PRT

<213> Homo sapiens

<400> 170

Arg Leu Arg Phe Ile

1 5

<210> 171

<211> 4

<212> PRT

<213> Homo sapiens

<400> 171

Arg Leu Arg Phe

1

<210> 172

<211> 3

<212> PRT

<213> Homo sapiens

<400> 172

Arg Leu Arg

1

<210> 173

<211> 5

<212> PRT

<213> Homo sapiens

<400> 173

Leu Arg Phe Ile Pro

1 5

<210> 174

<211> 4

<212> PRT

<213> Homo sapiens

<400> 174

Leu Arg Phe Ile

1

<210> 175

<211> 3

<212> PRT

<213> Homo sapiens

<400> 175

Leu Arg Phe

1

<210> 176

<211> 4

<212> PRT

<213> Homo sapiens

<400> 176

Arg Phe Ile Pro

1

<210> 177

<211> 3

<212> PRT

<213> Homo sapiens

<400> 177

Arg Phe Ile

1

<210> 178

<211> 3

<212> PRT

<213> Homo sapiens

<400> 178

Phe Ile Pro

1

<210> 179

<211> 1132

<212> PRT

<213> Homo sapiens

<400> 179

Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser

1 5 10 15

His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly

20 25 30

Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg

35 40 45

Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro

50 55 60

Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu

65 70 75 80

Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val

85 90 95

Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro

100 105 110

Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr

115 120 125

Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val

130 135 140

Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val

145 150 155 160

Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr

165 170 175

Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly

180 185 190

Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg

195 200 205

Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg

210 215 220

Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg

225 230 235 240

Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp

245 250 255

Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val

260 265 270

Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala

275 280 285

Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His

290 295 300

Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro

305 310 315 320

Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly

325 330 335

Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro

340 345 350

Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser

355 360 365

Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln

370 375 380

Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His

385 390 395 400

Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg

405 410 415

Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln

420 425 430

Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu

435 440 445

Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe

450 455 460

Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser

465 470 475 480

Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser

485 490 495

Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met

500 505 510

Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys

515 520 525

Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe

530 535 540

Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe

545 550 555 560

Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr

565 570 575

Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His

580 585 590

Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln

595 600 605

His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile

610 615 620

Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val

625 630 635 640

Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser

645 650 655

Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg

660 665 670

Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg

675 680 685

Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro

690 695 700

Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile

705 710 715 720

Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln

725 730 735

Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His

740 745 750

Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp

755 760 765

Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser

770 775 780

Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu

785 790 795 800

Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His

805 810 815

Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro

820 825 830

Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp

835 840 845

Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu

850 855 860

Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala

865 870 875 880

Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys

885 890 895

Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu

900 905 910

Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe

915 920 925

Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser

930 935 940

Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe

945 950 955 960

Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly

965 970 975

Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn

980 985 990

Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln

995 1000 1005

Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln

1010 1015 1020

Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala

1025 1030 1035 1040

Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu

1045 1050 1055

Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp

1060 1065 1070

Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr

1075 1080 1085

Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser

1090 1095 1100

Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn

1105 1110 1115 1120

Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp

1125 1130

<210> 180

<211> 48

<212> DNA

<213> Homo sapiens

<400> 180

gaagcgcgcc cggcgctgct gaccagccgc ctgcgcttta ttccgaaa 48

<210> 181

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA sense

<220>

<221> variation

<222> (20)..(21)

<223> n is deoxythymidine (dT)

<400> 181

cuuacgcuga guacuucgan n 21

<210> 182

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> siRNA antisense

<220>

<221> variation

<222> (20)..(21)

<223> n is deoxythymidine (dT)

<400> 182

ucgaaguacu cagcguaagn n 21

<210> 183

<211> 239

<212> PRT

<213> Artificial Sequence

<220>

<223> Green Fluorescent Protein

<400> 183

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 184

<211> 720

<212> DNA

<213> Artificial Sequence

<220>

<223> Green Fluorescent Protein

<400> 184

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

720

<210> 185

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> siCont sense

<220>

<221> variation

<222> (20)..(21)

<223> n is deoxythymidine (dT)

<400> 185

gcaccuauaa caacgguagn n 21

<210> 186

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> siCont antisense

<220>

<221> variation

<222> (20)..(21)

<223> n is deoxythymidine (dT)

<400> 186

cuaccguugu uauaggugcn n 21