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WO2020130546A1 - Domaine de pénétration de membrane cellulaire dérivé de la protéine clk2 humaine - Google Patents

Domaine de pénétration de membrane cellulaire dérivé de la protéine clk2 humaine Download PDF

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Publication number
WO2020130546A1
WO2020130546A1 PCT/KR2019/017828 KR2019017828W WO2020130546A1 WO 2020130546 A1 WO2020130546 A1 WO 2020130546A1 KR 2019017828 W KR2019017828 W KR 2019017828W WO 2020130546 A1 WO2020130546 A1 WO 2020130546A1
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Prior art keywords
protein
cargo
cell membrane
peptide
recombinant
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PCT/KR2019/017828
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English (en)
Korean (ko)
Inventor
김성준
김균도
황인수
구근본
김천생
김범태
안대균
김해수
권영찬
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Korea Research Institute of Chemical Technology KRICT
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Korea Research Institute of Chemical Technology KRICT
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Application filed by Korea Research Institute of Chemical Technology KRICT filed Critical Korea Research Institute of Chemical Technology KRICT
Publication of WO2020130546A1 publication Critical patent/WO2020130546A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers

Definitions

  • the present invention relates to a cell membrane permeation domain derived from human CLK2 protein and an intracellular delivery system comprising the same.
  • Biopharmaceuticals include therapeutic proteins, antibodies, vaccines for prevention or treatment, gene therapy, and cell therapy.
  • recombinant protein drugs are drugs that are produced in large quantities using gene recombination technology for therapeutic proteins that are difficult to produce through a living body. Growth hormone, erythropoietin, interferon, colony stimulating factor (CSF), and blood coagulation factor (Recombinant human insulin) for the treatment of diabetes in the early 1980s were first approved by the U.S. FDA.
  • a representative method is a protein transduction domain (PTD) or cell permeation peptide.
  • PTD protein transduction domain
  • CPP Cell Penetrating Peptides
  • MPG GLFLGFLGAAGSTMGAWSQPKKKRKV
  • bovine prion protein-derived BPrPr MVKSKIGSWILVLFVAMWSDVGLCKKRP
  • human Hph-1 protein-derived Hph-1 YARVRRRGPRR
  • human NLBP protein-derived NP2 KIKKVKKKGRK
  • TAT-JBD20 cell-penetrating peptides are developed for the treatment of cerebral vascular disease (Cerebral ischemia) and degenerative brain disease (Alzheimer's disease) using TAT-JBD20, the treatment of amyotrophic lateral sclerosis using TAT-BH4, MPG-8/siRNA and TAT -Preclinical experiments such as cancer treatments using DRBD/siRNA are in progress.
  • TAT- ⁇ PKC inhibitor a treatment for hearing loss and inflammation using TAT-JBD20
  • a treatment for brain tumor using p28 are in clinical trials.
  • the existing cell permeation peptides are mainly invented from non-human proteins such as viruses and Drosophila or have low permeation efficiency
  • cell permeation peptides having high permeation efficiency among human derived peptides human CLK2 (CDC Like Kinase 2)
  • the protein-derived cell permeation peptide was invented, and similarity to the existing cell permeation peptides was confirmed for peptides present in human-derived proteins, thereby completing the present invention.
  • 6.dNP2 is a blood-brain barrier-permeable peptide enabling ctCTLA-4 protein delivery to ameliorate experimental autoimmune encephalomyelitis. Nat Commun. 8244 (2015), pp. 1-13
  • An object of the present invention is to provide a novel cell membrane permeation domain that exhibits high cell permeation efficiency and can minimize side effects.
  • Another object of the present invention is to provide an intracellular delivery system including a cell membrane permeation domain to which a cargo of an intracellular transport target is bound to the end of a peptide.
  • Another object of the present invention is to provide a method for transporting a cargo into a cell, the method comprising contacting a cell membrane permeation domain to which a cell of the intracellular transport target is bound to the cell at the end of the peptide.
  • the present invention is to provide a cell membrane permeation domain comprising a polypeptide of any one of SEQ ID NOs: 1 to 11 derived from human CLK2 (CDC Like Kinase 2) protein.
  • cell penetrating peptide refers to a peptide having the ability to carry a cargo (Cargo) of a transport target into a cell in vitro or in-compensation, the term “cell membrane permeation domain ”.
  • the term'peptide' or'peptide' means a polymer in the form of a chain formed by combining 4 to 1000 amino acid residues by a peptide or a peptide bond, and mixed with'polypeptide' or'polypeptide' It is possible.
  • the present invention in order to minimize the side effects that occur when the cargo material is delivered through the cell permeation peptide into the human body, unlike the cell permeation peptide derived from a virus or other species, it is a peptide existing in a human-derived protein.
  • a novel human-derived cell membrane permeation domain or cell permeation peptide capable of minimizing side effects when treated to the human body and having a very high delivery efficiency into cells.
  • the present invention provides a recombinant cargo with improved cell membrane permeability, including a cargo fused to the cell membrane permeation domain and the N-terminal or C-terminal of the cell membrane permeation domain.
  • the cargo may be a recombinant cargo that is a protein, nucleic acid, lipid, or compound.
  • the cargo may be an adjuvant or antigen.
  • the cargo is a hormone, an immunoglobulin, an antibody, a structural protein, a signaling peptide, a storage peptide, a membrane peptide, a transmembrane peptide, an inner peptide, an outer peptide, a secretory peptide, a viral peptide, a native peptide , Glycated protein, fragmented protein, disulfide peptide, recombinant protein, chemically modified protein, and any one selected from the prion group.
  • the term'recombination cargo means a complex formed by recombination of cell membrane permeation domains and one or more cargoes by genetic fusion or chemical bonding.
  • the term'contact' means that the cargo is in contact with eukaryotic or prokaryotic cells, and the cargo is transferred into the eukaryotic or prokaryotic cells.
  • the cargo may be any one of a recombinant cargo selected from the group consisting of nucleic acids, coding nucleic acid sequences, mRNA, antisense RNA molecules, carbohydrates, lipids, and glycolipids.
  • the cargo may be a recombinant cargo that is a contrast agent, drug, or chemical.
  • the term “contrast material” refers to all materials used for imaging biological structures or fluids in medical imaging.
  • the contrast material may include, but is not limited to, radiopaque contrast material, paramagnetic contrast material, superparamagnetic contrast material, CT contrast material, or other contrast material.
  • it may include radiopaque metals and salts thereof (e.g., silver, platinum, platinum, etc.) and other radiopaque chemicals (e.g., calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide). have.
  • Paramagnetic contrast materials include gadolinium proliferatives, MR imaging, and other gadolinium, manganese, iron, dysprosium, copper europium, erbium, chromium, Nickel and cobalt complex hydroxybenzylethylene diamine diacetic acid (HBED).
  • the superparamagnetic contrast material may include magnetite, superparamagnetic iron oxide, ultrafine, superparamagnetic iron oxide, and monocrystalline iron oxide.
  • Other suitable contrast materials may include iodized and non-iodinated, ionic and nonionic CR compositions, and contrast materials such as spin-labels, or other diagnostic actives. When expressed in a cell, it may include a marker gene encoding an easily detectable protein.
  • Various labels such as radionuclides, fluorescent substances, enzyme cofactors, and enzyme inhibitors can be used.
  • the cargo according to the present invention is a protein or a peptide
  • the transport peptide and the transport peptide form a fusion protein form a fusion protein form You can combine objects.
  • a specific example of binding by a fusion protein is as follows: When preparing a primer for producing a fusion protein, a nucleotide encoding a transport peptide is attached to a nucleotide expressing a moving target, and then the obtained nucleotide is vectored as a restriction enzyme. (Example: PET vector), and BL-21 (DE3) is introduced into cells and expressed.
  • the carrier peptide is a dye or fluorescent substance, specifically fluorescein ithiothiosia Nate (FITC, fluorescein isothiocyanate), luciferase (Luc), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Tag GFP, Superfolder GFP, PA GFP , AcGFP, PS-GFP2, Yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), SYFP, TagYFP, PhiYFP, Azurite, mKalamal, blue fluorescent protein (Cyan fluorescent protein, CFP), enhanced Cyan fluorescent protein (ECFP), TagCFP, PS-CFP2, Red fluorescent protein (RFP), Tag RFP, Tag RFP657, mRFP1, PaTagRFP, Turbo RFP, RFP693, tdRFP
  • FITC fluorescein ithiothiosia Nate
  • FITC fluorescein isothiocyanate
  • Luc green fluorescent protein
  • GFP enhanced green fluorescent protein
  • BLA beta-lactamase
  • GUS beta-glucuronidase
  • alkaline phosphatase chloramphenicol acetyltransferase or peroxidase
  • the present invention provides a gene construct comprising a polynucleotide encoding the cell membrane permeation domain.
  • the present invention provides an expression vector for expressing a recombinant cargo protein having improved cell membrane permeability, including a gene construct.
  • the present invention also includes the step of administering to a subject a pharmaceutical composition comprising a conjugate with a bioactive peptide bound to the cell membrane permeation domain and a pharmaceutically acceptable carrier, cervical cancer, prostate cancer, ovarian cancer, and uterus. It is to provide a method of preventing or treating diseases such as endometrial cancer, uterine cancer, bladder cancer, esophageal cancer, head and neck cancer Wilms' tumor, soft tissue sarcoma, stomach cancer, pancreatic cancer, breast cancer, and bronchial cancer.
  • diseases such as endometrial cancer, uterine cancer, bladder cancer, esophageal cancer, head and neck cancer Wilms' tumor, soft tissue sarcoma, stomach cancer, pancreatic cancer, breast cancer, and bronchial cancer.
  • the pharmaceutical composition according to the present invention can be used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup aerosol, external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. have.
  • Carriers, excipients and diluents that may be included in the composition include lactose, textrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in the extract. Is prepared. Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • non-aqueous solvent suspension propyl glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • injectable ester such as ethyl oleate
  • Witepsol Macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin may be used.
  • the preferred dosage of the composition of the present invention depends on the patient's condition and body weight, the degree of disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is administered at 0.0001 to 500 mg/kg per day, preferably 0.001 to 250 mg/kg per day. Administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.
  • the cell permeation peptide of the present invention exhibits significantly improved permeation efficiency or cell permeability compared to conventional peptides, thereby allowing the carried cargo or biologically active molecule to be introduced into the cell, effectively maintaining activity, and significantly reducing cost. .
  • FIG. 1 is a schematic diagram of a gene composite for making an expression gene composite of CLK2P-EGFP recombinant fusion protein.
  • FIG. 2 shows that a gene composite expressing a protein in which a human permeable peptide derived from human CLK2 protein (CLK2P) and Enhanced Green Fluorescent Protein (EGFP) is fused is observed through agarose gel electrophoresis.
  • CLK2P human permeable peptide derived from human CLK2 protein
  • EGFP Enhanced Green Fluorescent Protein
  • FIG. 3 shows that the result was confirmed by processing a restriction enzyme on the bound gene composite by inserting the CLK2P-EGFP gene composite into the protein expression vector pET28a+.
  • FIG. 4 shows that CLK2P-EGFP-pET28a+ vector was transformed into Rosetta (DE3) receptor cells, followed by inducing protein production, followed by sonication, and observed through polyacrylamide gel electrophoresis.
  • FIG. 5 shows that CLK2P-EGFP recombinant fusion protein produced using Rosetta (DE3) receptive cells was purified and observed through polyacrylamide gel electrophoresis.
  • FIG. 6 shows the results of comparing the permeation efficiency in cells with a flow cytometer after treating the recombinant protein fused with the existing cell permeation peptide with EGFP and the CLK2P-EGFP recombinant protein in a human T immune cell line (Jurkat).
  • Figure 9 shows the distribution pattern of CLK2P-EGFP recombinant protein permeated into cells using a confocal microscope after treating the CLK2P-EGFP recombinant protein on the cervical cancer cell line (HeLa).
  • Figure 10 shows the results of confirming the efficiency of introduction into cells by flow cytometry (FACS) after processing the intracellular delivery efficiency of the CLK mutant (EG) fusion protein of the present invention to a human T immune cell line (Jurkat).
  • FACS flow cytometry
  • the present invention provides a cell membrane permeation domain or cell permeation peptide comprising the polypeptide of any one of SEQ ID NOs: 1 to 11 derived from human CLK2 (CDC like Kinase 2) protein.
  • CLK2P peptide (RRKHRRRRRRSR, SEQ ID NO: 1) consisting of the 115th to 126th amino acid sequences in human CLK2 (CDC like kinase 2) protein, which is expected to have cell permeability among human-derived peptides.
  • the gene sequence information expressing CLK2P in the base sequence of human-derived CLK2 protein was cggaggaagcacagacggcggaggaggcgcagccgg (SEQ ID NO: 12) and was converted to cgcagaaaacaccgccgtcgcagaagacgctccaga (SEQ ID NO: 13) through codon optimization during gene synthesis.
  • the base sequence expressing the EGFP fluorescent protein linked after the base sequence expressing the CLK2P peptide is represented by SEQ ID NO: 14, and the base sequence of the linker portion between the CLK2P peptide and the EGFP protein is ggaggtgggggctcg (SEQ ID NO: 15). to be.
  • the cell permeation peptides or cell membrane permeation domains represented by SEQ ID NOs: 2 to 11 of the present invention are mutant cell permeation peptides of the CLK2P cell permeation peptide, and the histidine, the fourth amino acid of CLK2P, and serine, the 11th amino acid, are different.
  • the cell membrane permeation domain or cell permeation peptide derived from human CLK2 protein of the present invention is shown in Table 1 below.
  • Example 1 Construction of a plasmid vector expressing a protein in which a cell-penetrating peptide (CLK2P) derived from human CLK2 protein and EGFP fluorescent protein are fused
  • CLK2P cell-penetrating peptide
  • the present invention provides a gene construct comprising a polynucleotide encoding a cell membrane permeation domain (CLK2P cell permeation peptide, CLK2P), and the gene construct containing the recombinant cargo protein expression with improved cell membrane permeability
  • a dragon expression vector is provided.
  • the vector is characterized in that it further comprises a gene encoding a cargo protein, so that a recombinant cargo protein in which the cargo of the cell membrane permeation domain and the protein is fused can be expressed.
  • the present invention comprises the steps of preparing a recombinant cargo fused to the N- terminal or C- terminal of the cell membrane permeation domain; And contacting the prepared recombinant cargo with cells.
  • the CLK2P base sequence was converted to a base sequence expressing the same peptide through codon optimization during artificial gene synthesis, and was synthesized by attaching a gene expressing the EGFP fluorescent protein to the base sequence of the CLK2P peptide to verify cell permeability (FIG. 2). .
  • linker sequencing was added between the cell-penetrating peptide and the EGFP protein to increase flexibility.
  • the artificial synthetic gene was introduced into the pET28a+ plasmid vector treated with NheI restriction enzyme and XhoI restriction enzyme through T4 ligase after treatment with NheI restriction enzyme and XhoI restriction enzyme.
  • the pET-28a plasmid vector used herein allows protein purification through Ni-NTA Resin by expressing 6 histidines (6 x His) at the N-terminus in front of the CLK2P-EGFP fusion protein.
  • the pET28a+ plasmid vector into which the CLK2P-EGFP fusion protein artificial gene has been introduced is transformed into Top10 recipient cells and cultured in LB agar plates containing Kanamycin to incubate grown colonies in LB medium for about 16 hours.
  • a recombinant cargo in which cargo was fused to the cell membrane permeation domain (CLK2P) was prepared.
  • the method of delivering cargo to cells of the present invention comprises the steps of: 1) preparing the recombinant cargo; And 2) contacting the recombinant cargo prepared in the step with cells. Preparation 2) The contact between the cargo and the cell can be performed in vitro or in vivo.
  • the contact is made by culturing the cell by treating the medium containing the recombinant cargo to cells in vitro.
  • the recombinant cargo is intramuscular, intra-peritoneal, intravenous, oral, intranasal, subsutaneous ), subcutaneous (intradermal), mucosal (muscosal), or inhalation (inhale) is injected into the body in vivo (injection), the cell is in contact with the recombinant cargo in vivo.
  • the plasmid vector prepared in Example 1 above was transformed into Rosetta (DE3) recipient cells and cultured for about 16 hours in LB medium containing Kanamycin. Here, 100 times more LB medium was added, incubated until the OD value reached 0.4-0.6, and then IPTG (Isopropyl ⁇ -D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, followed by 5 hours at 37°C. Or incubate for 16 hours at 20 °C.
  • the transformed Rosetta (DE3) recipient cells were cultured under the same conditions, and then the culture solution was centrifuged to obtain a cell pellet and lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole ). After supernatant, the supernatant was recovered by centrifugation, filtered through a 45 ⁇ m filter, and the supernatant was passed through a Ni-NTA agarose column to bind the recombinant protein.
  • lysis buffer 50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole
  • the plasmid vector expressing the protein in which the EGFP fluorescent protein is fused with the cell permeation peptides TAT, Hph-1 and NP2, respectively express the CLK2P-EGFP fusion protein It was prepared in the same manner as the plasmid vector. Expression and purification of the conventional CPP-EGFP fusion protein was also performed in the same manner as CLK2P-EGFP fusion protein.
  • Jurkat cells were dispensed into a 24 well plate at 1.5 X 10 6 /well, and then suspended in 2 ml of RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin to bring the CPP-EGFP fusion protein to a final concentration of 5 ⁇ M. After treatment, the cells were cultured in a 5% CO 2 incubator maintained at 37° C. for 2 hours. Thereafter, the medium containing the cells was transferred to a 5 ml tube, followed by centrifugation to remove the supernatant and washing with PBS solution was repeated 3 times.
  • EGFP, TAT-EGFP, and CLK2P-EGFP fusion proteins were treated with Jurkat cells at a final concentration of 5 ⁇ M, respectively, and cultured for 0.5, 1, 2, 4, and 6 hours, respectively. It was confirmed (Fig. 8). Through this, it was confirmed that the CLK2P-EGFP fusion protein was introduced into cells in a time-dependent manner, and it was confirmed that it showed a significantly higher delivery efficiency at all times compared to the TAT-EGFP fusion protein.
  • HeLa cells are dispensed into a 24 well cell culture slide with 2 X 10 4 /well, and 10% Fetal Bovine Serum and 1% penicillin/streptomycin are included.
  • the cells were suspended in 500 ⁇ l of DMEM medium and cultured for 18 hours in a 5% CO 2 incubator maintained at 37°C. Thereafter, EGFP and CLK2P-EGFP fusion proteins were treated with cells at a final concentration of 1 ⁇ M, and then cultured for 1 hour. After removing the culture solution, washed 3 times with PBS solution, and fixed by treating the 4% PFA solution at room temperature for 30 minutes.
  • CLK2P mutants each of which is substituted with histidine, the fourth amino acid of CLK2P, and serine, the eleventh amino acid, with different amino acids, and the CLK2P mutant with the N-terminal and C-terminal amino acids removed one by one, are designed.
  • a plasmid vector expressing the CLK2P mutant-EGFP fusion protein was produced and then purified by expressing the protein.
  • Jurkat cells were dispensed into a 24 well plate at 1.5 X 10 6 /well, and then suspended in 2 ml of RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin to bring the CPP-EGFP fusion protein to a final concentration of 5 ⁇ M. After treatment, the cells were cultured in a 5% CO 2 incubator maintained at 37° C. for 2 hours. Thereafter, the medium containing the cells was transferred to a 5 ml tube, and centrifugation was performed to remove the supernatant and washing was repeated 3 times with PBS solution.
  • the Jurkat cells were suspended in PBS of 650 ⁇ l, and the fluorescence of CLK2P mutant-EGFP fusion proteins introduced into the cells was measured through a flow cytometer to confirm the efficiency of cell introduction.
  • CLK2P mutants showed a change in the introduction efficiency through mutation, but it was confirmed that the cell permeation ability was maintained (FIG. 10).

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Abstract

La présente invention concerne un domaine de pénétration de membrane cellulaire dérivé de la protéine CLK2 humaine et, plus particulièrement, un domaine de pénétration de membrane cellulaire dérivé de la protéine CLK2 humaine (domaine de pénétration de membrane cellulaire CLK2P) ou un peptide de pénétration cellulaire, et un système d'administration intracellulaire l'utilisant. Le domaine de pénétration de membrane cellulaire dérivé de la protéine CLK2 humaine selon la présente invention peut être administré de manière intracellulaire et des substances de charge, telles que des composés, des biomolécules, et divers matériaux polymères, peuvent être administrées de manière intracellulaire à l'aide de celui-ci. Le domaine de pénétration de membrane cellulaire CLK2P selon la présente invention présente une efficacité de pénétration cellulaire plus élevée que celle des peptides de pénétration cellulaire classiques et est dérivé de la protéine humaine, et peut ainsi empêcher l'apparition d'effets secondaires et de réponses immunitaires qui sont provoquées par des peptides dérivés de protéines exogènes, et par conséquent, peut être efficacement utilisé en tant que procédé pour administrer efficacement, dans des cellules, des composés, des biomolécules, et divers matériaux polymères, qui doivent être appliqués à un corps humain.
PCT/KR2019/017828 2018-12-19 2019-12-16 Domaine de pénétration de membrane cellulaire dérivé de la protéine clk2 humaine Ceased WO2020130546A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180165437 2018-12-19
KR10-2018-0165437 2018-12-19
KR10-2019-0165873 2019-12-12
KR1020190165873A KR102282692B1 (ko) 2018-12-19 2019-12-12 인간 clk2 단백질 유래 세포막 투과 도메인

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6797513B2 (en) * 1996-12-19 2004-09-28 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Nucleic acid encoding CLK2 protein kinases
US7943732B2 (en) * 2006-06-05 2011-05-17 Intrexon Corporation AKT ligands and polynucleotides encoding AKT ligands
US9217146B2 (en) * 2011-06-15 2015-12-22 Dicerna Pharmaceuticals, Inc. Phase changing formulations of nucleic acid payloads
WO2016146262A1 (fr) * 2015-03-16 2016-09-22 Amal Therapeutics Sa Nouveau complexe comprenant un peptide de pénétration cellulaire, un cargo et un agoniste des peptides tlr pour le traitement du cancer colorectal

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6797513B2 (en) * 1996-12-19 2004-09-28 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Nucleic acid encoding CLK2 protein kinases
US7943732B2 (en) * 2006-06-05 2011-05-17 Intrexon Corporation AKT ligands and polynucleotides encoding AKT ligands
US9217146B2 (en) * 2011-06-15 2015-12-22 Dicerna Pharmaceuticals, Inc. Phase changing formulations of nucleic acid payloads
WO2016146262A1 (fr) * 2015-03-16 2016-09-22 Amal Therapeutics Sa Nouveau complexe comprenant un peptide de pénétration cellulaire, un cargo et un agoniste des peptides tlr pour le traitement du cancer colorectal

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI . GenBank 26 July 2016 (2016-07-26), ANONYMOUS: "CDC-like kinase 2, isoform CRA_b, partial [Mus musculus]", XP055720643, retrieved from Protein Database accession no. EDL15234.1 *

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