WO2012050402A2 - Protéine parkin recombinante à perméation cellulaire et composition pharmaceutique de traitement des maladies dégénératives du cerveau l'incluant - Google Patents

Protéine parkin recombinante à perméation cellulaire et composition pharmaceutique de traitement des maladies dégénératives du cerveau l'incluant Download PDF

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WO2012050402A2
WO2012050402A2 PCT/KR2011/007682 KR2011007682W WO2012050402A2 WO 2012050402 A2 WO2012050402 A2 WO 2012050402A2 KR 2011007682 W KR2011007682 W KR 2011007682W WO 2012050402 A2 WO2012050402 A2 WO 2012050402A2
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parkin
cell
recombinant protein
seq
mtd
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WO2012050402A3 (fr
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조대웅
김찬기
임정희
최유리
김희현
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주식회사 프로셀제약
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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  • the present invention is for the treatment of degenerative brain disease containing Parkin recombinant protein fused with macromolecule transduction domain (MTD) to the degenerative brain disease therapeutic protein Parkin, and the cell permeable Parkin recombinant protein as an active ingredient. It relates to a pharmaceutical composition and the like.
  • MTD macromolecule transduction domain
  • Parkinson's disease was first reported by British physician James Parkinson in 1817, and is the second most common degenerative brain disease after dementia. It is known to affect about%. As the population ages, the number of patients also increases, but there is insufficient development of effective treatments due to insufficient studies. Characteristic clinical symptoms include movements such as bradykinesia, rest tremor, rigidity, flexed posture, freezing of gait, and postural instability. Symptoms, non-motor symptoms such as demen-tia, depression, anxiety, psychotic symptoms, autonomic neuropathy, and sleep disorders, and are characterized by loss of dopaminergic neurons in the middle brain and Lewy body findings. Pathological findings.
  • Parkinson's disease is reported to be caused by a combination of genetic and environmental factors.
  • Parkinson's genetic factor is responsible for the high prevalence of Parkinson's genetic factors.
  • the Parkin gene was first discovered and named in Japanese families with Autosomal Recessive Juvenile Parkinsonism (ARJP), and (Kitada. T., 1998) early-onset Parkinson's disease with autosomal recessive genetics. 50% of the diseases are known to be caused by Parkin gene mutations (Lucking CB, 2000). Normal Parkin functions as an E3 Ubiquitin Ligase, which is important for the ubiquitin-proteasome system, and removes damaged, oxidized, or abnormally structured proteins in the cell to reduce intracellular stress. It serves to reduce.
  • Parkin Degeneration of Parkin is known to result in the loss of E3 ubiquitin zygotease function, resulting in the accumulation of intracellular inclusion bodies and abnormal proteins, leading to inhibition of dopamine secretion or death of dopamine neurons (Lam YA, 2000). ).
  • Parkin is present in the mitochondrial matrix and is known to be involved in protecting mitochondrial degeneration. Parkin deficient mutant mice have been shown to be highly sensitive to mitochondrial degradation and increased oxidative stress (Greene JC, 2003). Palacino JJ J, 2004). In addition, Parkin gene overexpression has been reported to inhibit the activity of JNK and caspase3 in neurons by 6-OHDA and decrease the activation oxygen (Jiang et al., 2004). In a recent study using fruit flies, Parkin's function was identified, along with another cause of Parkinson's disease, PINK1. Parkinson's dysfunction was a decrease in dopamine secretion due to inactivation, rather than death of dopamine neurons.
  • Parkin may be used as a therapeutic agent for Parkinson's disease.
  • Parkin-lentivirus was administered to Parkinson's animal models in which neurons were damaged by alpha-synuclein mutations.
  • 60% inhibition of dopamine-secreting neurons in black matter was suppressed (Bianco et al., 2004), and 50% inhibition of dopamine neuron death by Parkin-lentivirus in Parkinson's disease animal model using 6-OHDA.
  • allergic disorders were alleviated (Vercammen et al., 2006).
  • Parkin protein plays an important role in destroying inclusion bodies as key enzymes of the ubiquitin-proteasome pathway, maintaining mitochondrial function from oxidative stress, and inhibiting dopamine neuron death. It can be assumed that by inhibiting the dopamine neurons death caused by genetic causes and environmental toxic substances to prevent dopamine neurons degeneration and reactivation. Accordingly, the present inventors have made a thorough study because it is considered that Parkin is of sufficient value as a target material for the development of a therapeutic agent for Parkinson's disease known as degenerative brain disease.
  • macromolecules such as proteins, peptides, antibodies, nucleic acids and the like cannot pass through the double lipid membrane structure or plasma membrane and cannot be transferred into cells.
  • bioactive recombinant proteins, gene therapies, and monoclonal antibodies are unable to pass through the dilipid layer of the plasma membrane of the cell, but also through the blood-brain barrier, an important pathway to the brain.
  • the present inventors have prepared a Parkin recombinant protein that has been imparted with cell permeability by fusion of a macromolecular delivery domain to Parkin, and the recombinant protein is effectively delivered into neurons not only in vitro but also in an in vivo environment, thereby causing a malfunction of a specific protein or It has been confirmed that the present invention can be used as a therapeutic agent for degenerative brain diseases due to deficiency, and thus has been completed.
  • An object of the present invention is to impart cell permeability to the treatment factor for degenerative brain disease, and to introduce it into the cell with high efficiency, thereby inhibiting dopamine neuron death or increasing the release of dopamine. It is to provide a recombinant protein.
  • the present invention provides a cell permeable parkin recombinant protein in which a macromolecular transduction domain (MTD) is fused with a parkin having an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 195. to provide.
  • MTD macromolecular transduction domain
  • the recombinant protein is characterized in that it has an amino acid sequence selected from the group consisting of SEQ ID NO: 394 to SEQ ID NO: 403.
  • the recombinant protein is characterized in that it is prepared using the primers described in the table below.
  • the present invention also provides a polynucleotide encoding the cell permeable parkin recombinant protein.
  • the polynucleotide is characterized in that it has a base sequence selected from the group consisting of SEQ ID NO: 409 to SEQ ID NO: 418.
  • the present invention also provides a recombinant expression vector comprising the polynucleotide.
  • the present invention is a fusion of His-parkin having an amino acid sequence of SEQ ID NO: 389 and macromolecular transduction domain (MTD) having an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 15, 103 and 105 Provides a cell permeable His-parkin recombinant protein.
  • MTD macromolecular transduction domain
  • the recombinant protein is characterized in that it has an amino acid sequence selected from the group consisting of SEQ ID NO: 390 to SEQ ID NO: 393.
  • the recombinant protein is characterized in that it is prepared using the primers described in the table below.
  • the present invention also provides a polynucleotide encoding the cell-permeable His-parkin recombinant protein.
  • the polynucleotide is characterized in that it has a base sequence selected from the group consisting of SEQ ID NO: 405 to SEQ ID NO: 408.
  • the present invention also provides a recombinant expression vector comprising the polynucleotide.
  • the present invention also provides a pharmaceutical composition for treating degenerative brain disease containing the cell permeable parkin recombinant protein as an active ingredient.
  • the degenerative brain disease is characterized in that Parkinson's disease.
  • Parkin recombination protein having a cell permeability of the present invention is a cell-permeable recombinant protein fused with a macromolecular transport domain to Parkin protein, a dopamine neuronal cell death inhibitory factor, in vitro or in vivo using the protein It can be used for the study of Parkinson's disease through the effect of inhibiting apoptosis by transferring into dopamine neurons in vivo) or increasing dopamine secretion, and is expected to be useful as a therapeutic agent for human degenerative brain diseases. .
  • FIG. 1 shows a vector designed for expressing Histidine (His) -tag fusion Parkin recombinant protein.
  • Figure 2 shows a schematic diagram of the combined MTD and His-tag fusion recombinant human full-length Parkin gene.
  • Figure 3 shows the SDS-PAGE results of purified His-tag fusion Parkin recombinant protein.
  • Figure 4 shows the flow cytometry of the His-tag fusion Parkin recombinant protein (HPM01 or HPM13) combined with JO-01 MTD or JO-13 MTD and His-tag fusion Parkin recombinant protein (HP) without MTD. Flow cytometry results are shown.
  • Figure 6 shows the results of comparing the HPM13 and HP mouse blood brain barrier permeability (A. Tissue immunoassay results; B. Analysis using Western blotting).
  • Figure 7 shows the results of TUNEL analysis showing the protective effect of HPM13 and HP on 6-OHDA induced apoptosis of mouse neurons.
  • Figure 8 shows the results of quantifying the dopamine secretion effect of HPM13 and HP in mouse dopamine neurons by ELISA.
  • FIG. 9 shows His-tag fusion Parkin with JO-10, JO-13, JO-29, JO-78 JO-85, JO-103, JO-130, JO-135, JO-151, JO-174 MTD Shown are vectors designed for expression of recombinant proteins (HPM10, HPM13, HPM29, HPM78, HPM85, HPM103, HPM130, HPM135, HPM151, HPM174).
  • Figure 11 shows a vector designed for expression of His-tag non-fusion Parkin recombinant protein.
  • FIG. 12 shows a schematic diagram of a combination of MTD and His-tag non-fusion recombinant human full-length Parkin gene.
  • FIG. 13 shows Parkin recombinant protein HP, HPM13 or unbound PN (His-tag non-fusion Parkin) with JO-10, JO-13, JO-151, JO-174 MTD bound (PNM10, PNM13, PNM151, PNM174) The results of comparing the expression of recombinant proteins are shown.
  • Figure 14 shows the results of SDS-PAGE analysis after purification of PNM10, PNM13, PNM151, PNM174.
  • Figure 15 shows the results of comparing the E3 ligase activity of PNM10, PNM13, PNM151, PNM174 with His6-Parkin protein (control).
  • Figure 16 shows the results of comparing the protective effect of PNM10 against 6-OHDA induced apoptosis of SH-SY5Y neurons.
  • Figure 17 shows the mouse blood brain barrier permeability results of PNM10 (A. Tissue immunoassay results; B. Analysis using Western blotting).
  • Figure 18 shows the neurotoxic effect of PNM10 in the MPTP-induced Parkinson's disease animal model (histoimmunochemical labeling results for A. Tyrosin hydroxylase (TH); B. dopamine quantified by ELISA).
  • the present invention provides a cell permeability through the fusion of Parkin, a dopamine neuronal cell death suppressor, and a macromolecular transduction domain (MTD), thereby introducing a cell-permeable Parkin recombinant protein (CP-Parkin) which introduces Parkin into cells with high efficiency.
  • CP-Parkin cell-permeable Parkin recombinant protein
  • a feature of the present invention is to deliver Parkin into cells with high efficiency by converging specific macromolecular delivery domains (hereinafter abbreviated as "MTD") to Parkin, a macromolecule that is not readily introduced into cells. .
  • MTD specific macromolecular delivery domains
  • the macromolecular delivery domain may be fused only to one end of Parkin, a dopamine neuron cell death inhibitor, or to both ends thereof.
  • a macromolecular delivery domain (MTD) that can be fused to Parkin
  • a dopamine neuronal cell death inhibitor the Parkin recombinant protein having cell permeability by fusion of Parkin to each of the peptide domains that enables the delivery of macromolecules into cells Developed.
  • cell permeable Parkin recombinant protein includes a macromolecular delivery domain and Parkin, a dopamine neuronal cell death inhibitor, and refers to a covalent complex formed by genetic fusion or chemical bonding thereof.
  • genetic fusion is meant a linear covalent linkage formed through the genetic expression of a DNA sequence encoding a protein.
  • a polypeptide having a cell permeability including an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 195 may be used.
  • the macromolecular delivery domain is a cell permeable polypeptide that can mediate the entry of a polypeptide, protein domain, or biologically active molecule comprising a full length protein into a cell through a cell membrane.
  • the macromolecular delivery domain according to the present invention forms helices in signal peptides consisting of three parts of the N-terminal region, the hydrophobic region and the C-terminal secreted protein cleavage site. It is designed to have a hydrophobic region that confers targeting activity.
  • the macromolecular delivery domains that can be fused to Parkin, a neuronal cell death inhibitor, according to the present invention are shown in Table 1 below.
  • Parkin recombinant protein having cell permeability is a macromolecular delivery domain of 12 MTDs (JO-01, JO-10, JO-13, JO-29, JO-78, JO-85, JO- 101, JO-103, JO-130, JO-135, JO-151, and JO-174) are fused to one or both ends of the apoptosis inhibitor Parkin, and easily to one end of this fusion construct.
  • the histidine-tag (His-Tag) affinity domain may be fused.
  • the 12 types of MTD (JO-01, JO-10, JO-13, JO-29, JO-78, JO-85, JO-101, JO-103, JO-130, Full-length forms can be devised as Parkin recombinant proteins using any one of JO-135, JO-151 and JO-174.
  • full length form refers to a form comprising an intact amino acid sequence that does not include the deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1 of the apoptosis inhibitor Parkin. it means. However, not only full-length Parkin, but also Parkin derivatives containing various modifications by deletion, addition, insertion or substitution of one or more amino acid residues in its amino acid sequence within the range not impairing the killing inhibitory effect of Parkin's dopamine secreting cells It can be used in the present invention.
  • control protein has an amino acid sequence of SEQ ID NO: 1, which may be encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 2.
  • His-Parkin in which a histitin label is fused to one end of the cell permeable Parkin recombinant protein is not fused to MTD.
  • the control protein has an amino acid sequence of SEQ ID NO: 389, which may be encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 404.
  • the present invention also provides a recombinant expression vector comprising a polynucleotide encoding the cell-permeable Parkin recombinant protein.
  • a "recombinant expression vector” refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell and comprises an essential regulatory element operably linked to express the gene insert.
  • operably linked refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function.
  • a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the nucleic acid sequence encoding.
  • Operative linkage with recombinant expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.
  • Expression vectors usable in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers. It may be prepared in various ways according to the purpose, including a signal sequence (leader sequence) or a signal sequence for secretion. The promoter of the expression vector may be constitutive or inducible.
  • the expression vector includes a selection marker for selecting a host cell containing the vector, and in the case of an expression vector capable of replication, includes a replication origin.
  • His-Tag is expressed by artificially including six histidine-tags in the N-terminal region of the cell-permeable Parkin recombinant protein for the purpose of facilitating purification of the protein.
  • the nucleotide of the present invention can be cloned into a pET-28a (+) vector having a sequence (Novagen, USA).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the cell permeable parkin recombinant protein as an active ingredient.
  • the composition may further comprise a pharmaceutically acceptable carrier, such as a carrier for oral administration or a carrier for parenteral administration.
  • Carriers for oral administration include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like.
  • the recombinant protein according to the invention can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers.
  • carriers for parenteral administration include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be used by reference to those described in the following references (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • compositions according to the invention can be formulated in a variety of parenteral or oral dosage forms.
  • parenteral formulations are injectable formulations, preferably aqueous isotonic solutions or suspensions.
  • injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • each component may be formulated for injection by dissolving in saline or buffer.
  • oral dosage forms include, for example, tablets and capsules, which include diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and glidants (in addition to the active ingredients).
  • diluents e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine
  • glidants in addition to the active ingredients.
  • the tablets may comprise binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or Disintegrants such as sodium salts, absorbents, colorants, flavors and / or sweeteners may be further included.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally starch, agar, alginic acid or Disintegrants such as sodium salts, absorbents, colorants, flavors and / or sweeteners may be further included.
  • the formulations may be prepared by conventional mixing, granulating or coating methods.
  • compositions of the present invention may further comprise auxiliaries such as preservatives, hydrating agents, emulsifiers, salts for regulating osmotic pressure and / or buffers and other therapeutically useful substances, and may be formulated according to conventional methods.
  • auxiliaries such as preservatives, hydrating agents, emulsifiers, salts for regulating osmotic pressure and / or buffers and other therapeutically useful substances, and may be formulated according to conventional methods.
  • the route of administration of the composition according to the present invention may be administered to humans and animals orally or parenterally, such as intravenous, subcutaneous, intranasal or intraperitoneal.
  • Oral administration also includes sublingual application.
  • Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.
  • the total effective amount of the recombinant protein of the present invention may be administered to a patient in a single dose, and the fractionated treatment protocol in which multiple doses are administered for a long time. It may also be administered by.
  • the composition of the present invention may vary the content of the active ingredient depending on the extent of the disease, but can be repeatedly administered several times a day at an effective dosage of 5 to 20 mg once a single administration based on adults.
  • the effective dose of the recombinant protein may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. .
  • composition according to the present invention is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is shown.
  • an expression vector was produced to produce cell-permeable Parkin recombinant protein fused with His-tag. 4 randomly selected JO-01, JO-13, JO-101, JO-103 (SEQ. ID. NO .: 3, 15, 103, 105) MTD at the full-length Parkin gene C-terminus Amplified the gene.
  • SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 426 (reverse) primer pair was used for the human full-length Parkin-MTD 01 gene
  • SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 427 (reverse) primer pair was used for the human full-length Parkin-MTD 13 gene
  • SEQ. ID. NO .: 424 (forward) and SEQ. ID. NO .: 428 (reverse) primer pair was used for the full length Parkin-MTD 101 gene
  • His-tag allows the MT-D gene to be fused at the C-terminus and ultimately His-Parkin-MTD 01 (HPM 1 ), His-Parkin-MTD 13 (HPM 13 ), His-Parkin-MTD 101 (HPM 101 ), His-Parkin-MTD 103 (HPM 103 ) Parkin recombinant protein was prepared to express, and the results are shown in Figures 1 and 2.
  • CDNA base sequence for the protein expression is SEQ. ID. NO.:404 (His-Parkin), SEQ. ID. NO .: 405 (His-Parkin-MTD 01 ), SEQ. ID. NO .: 406 (His-Parkin-MTD 13 ), SEQ. ID.
  • E. coli BL21 Codon Plus (DE3) (Invitrogen) with a LacI promoter was used to express the cell permeable Parkin recombinant protein, and after transformation, E. coli was 50 ug / mL kanamycine in 500 mL LB. After inoculation into the added culture, when E. coli reached 0.5 at A 600 , it was overexpressed with 0.5 mM IPTG (Isopropyl ⁇ -D-1-thiogalactopyranoside) for 2 hours. All four cell-permeable Parkin recombinant proteins were expressed as inclusion bodies.
  • IPTG Isopropyl ⁇ -D-1-thiogalactopyranoside
  • Parkin recombinant proteins HPM 1 , HPM 13
  • JO-01 and JO-13 MTD were combined in consideration of their expression and physical properties. Purification was performed by selecting Parkin recombinant protein (HP) that did not bind MTD. To purify this, 8 M urea, a potent denaturant, was used to loosen the protein structure. First, the E. coli harvested by centrifugation of the culture was suspended in 20 mL lysis buffer (100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 8.0), which was then equipped with a microtip. E.
  • lysis buffer 100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 8.0
  • coli was crushed by repeating the 30 second-ON and 10 second-OFF conditions for 50 minutes on ice using an ultrasonic crusher. The supernatant was collected by centrifugation at 3,000 rpm at 4 ° C. for 25 minutes and combined with Ni 2+ -NTA agarose resin, followed by centrifugation at 1,000 rpm for 5 minutes at 4 ° C. Removed. To remove the nonspecific adsorbate, it was washed five times with 100 mM NaH 2 PO 4 , 10 mM Tris-HCl, 8 M urea, pH 6.3 buffer.
  • Purified His-tag fusion cell permeable recombinant Parkin protein is denatured by urea to form a released structure and must be refolded.
  • purified recombinant protein was refolded in buffer (0.55 M Guanidine-HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM oxidized glutathione, 0.2 mM reduction).
  • Urea was removed by dialysis at 4 ° C. for 24 hours using glutathione), and cell permeability and in vitro functional tests were performed on stable water-soluble cell permeable Parkin recombinant protein (HPM 1 , HPM 13 , HP).
  • DMEM Dulbecco's Modified Eagle Medium
  • FITC fluorescent material
  • 333 ug was used to bind the protein while shaking for 1 hour at room temperature with little light.
  • Cell permeable recombinant Parkin protein labeled with FITC fluorescence was dialyzed in DMEM medium at 4 ° C. for 1 day to remove unlabeled FITC.
  • the cell permeable Parkin recombinant protein (HPM 1 , HPM 13 ) in which JO-01 MTD and JO-13 MTD are combined is compared to the cell permeable Parkin recombinant protein (HP) without MTD binding. It can be seen that the permeability is high.
  • HPM 1 , HPM13, HP Parkin recombinant protein was treated with NIH3T3 cells (Korea Cell Line Bank, Seoul, Korea) at a concentration of 10 uM and incubated at 37 ° C. for 1 hour. .
  • NIH3T3 cells were cultured in DMEM medium containing 10% FBS, 5% penicillin and streptomycin. After incubation, in order to preserve the fluorescent label of the recombinant protein, 10 uL of mounting media was deposited on the slide, and after 15 minutes, it was observed under a confocal fluorescence microscope (Nikon).
  • MTD-coupled cell permeable Parkin recombinant protein HPM 1 , HPM 13
  • HP Parkin recombinant protein
  • Example 5 Brain-blood barrier permeability test of His-tag fusion cell permeable Parkin protein
  • HPM 13 Parkin recombinant protein with 200 ⁇ g MTD and Parkin recombinant protein with MTD was synthesized in 11-week-old Balb / c mice. 2 hours and 4 hours after subcutaneous injection of HP), the mouse brain was extracted. After fixing the tissue in 4% paraformaldehyde for more than 24 hours, the slide was prepared by frozen sectioning the tissue to 5 ⁇ m thickness. After fixing the sections for 10 minutes using cold acetone, the sections were washed with PBS buffer for 5 minutes and the frozen sections were reacted with 0.2% hydrogen peroxide for 30 minutes.
  • Sections were washed in PBS buffer solution for 5 minutes, then blocked in PBS buffer solution containing 3% normal horse serum for 30 minutes, and then Anti-His mAb (1: 500) and Anti-Parkin mAb (1: 500) The reaction was reacted overnight at 4 ° C. After washing the sections in PBS buffer solution for 5 minutes, reacted with biotin fusion anti-mouse antibody (1: 200, Zymed) for 1 hour, followed by reaction for 30 minutes using ABC kit (Vectastain), Sections were washed in PBS buffer for 5 minutes. After color reaction with DAB (3,3'-Diaminobenzidine) solution, staining was observed under an optical microscope, and the results are shown in FIG. 6A.
  • DAB 3,3'-Diaminobenzidine
  • mice 8 weeks old, females were injected subcutaneously with 200 ⁇ g protein and brain was extracted 2 hours later. After removing residual blood flow with PBS buffer, brain with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, protease inhibitor) was homogenized. After centrifugation at 13,000 rpm for 10 minutes at 4 ° C, the supernatant was taken, followed by 10% SDS-PAGE.
  • RIPA buffer 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, protease inhibitor
  • ParkM antibody positive band was observed to be about twice as thick as HPM 13 compared with HP, indicating that MTD-coupled Parkin recombinant protein has blood-brain barrier permeability.
  • TUNEL assay was performed to confirm the protective effect against neuronal cell death by the neurotoxin 6-hydroxydopamine (6-OHDA).
  • CATHa a mouse dopamine producing neuron, was cultured in DMEM media containing 10% fetal bovine serum, 5% penicillin and streptomycin, and treated with 6-OHDA at a concentration of 50 uM for 30 minutes.
  • JO-13 MTD-coupled Parkin recombinant protein (HPM 13 ) and MTD-coupled Parkin recombinant protein (HP) were each added 2.5 uM and incubated at 37 ° C. for 2 hours and 30 minutes.
  • TUNEL terminal dUTP nick-end labeling
  • the 6-OHDA-only group was observed to kill a large number of cells, whereas HPM 13 , a Parkin recombinant protein with JO-13 MTD, was observed to kill dopamine neurons by 6-OHDA.
  • HPM 13 a Parkin recombinant protein with JO-13 MTD
  • similar results to the group treated with 6-OHDA were observed by HP, a Parkin recombinant protein without MTD binding.
  • mouse dopamine neurons CATHa (Korea Cell Line Bank, Seoul, South Korea) was used. CATHa cells were cultured in DMEM medium containing 10% FBS, 5% penicillin / streptomycin, and dopamine quantification was performed according to the method of GenWay from dopamine ELISA supplier. After 24 h of tyrosine treatment to CATHa cells, HPM 13 recombinant protein fused with JO-13 MTD and recombinant HP protein without MTD were administered to dopamine neuron culture at 2.5 uM, respectively.
  • the dopamine concentration in the medium was quantified by ELISA (GenWay), and the results are shown in FIG. 8.
  • the dopamine content was increased by 366% or more in the culture solution in which HPM 13 was administered compared to HP protein, and after 5 hours, HPM 13 was dopamine compared to Parkin protein without MTD fusion. The content was significantly increased. Therefore, it can be seen that the HPM 13 recombinant protein fused with MTD is transferred into dopamine neurons to increase dopamine secretion ability.
  • the polymerase chain reaction product obtained above was cloned into the pGEM-Tease vector, and then subcloned into the BamHI and HindIII positions of the expression vector pET-28a (+) (Novagen). -At the end, each MTD gene was designed to be fused, which is shown in FIG. 9.
  • the cDNA base sequence of the cloned gene is shown in SEQ. ID. NO .: 2 (Parkin), SEQ. ID. NO .: 409 (Parkin-MTD 10 ), SEQ. ID. NO .: 410 (Parkin-MTD 13 ), SEQ. ID. NO .: 411 (Parkin-MTD 29 ), SEQ. ID.
  • Parkin-MTD 174 Ultimately Parkin-MTD 10 (HPM 10 ), Parkin-MTD 13 (HPM 13 ), Parkin-MTD 29 (HPM 29 ), Parkin-MTD 78 (HPM 78 ), Parkin-MTD 85 (HPM 85 ), Parkin-MTD 103 (HPM 103 ), Parkin-MTD 130 (HPM 130 ), Parkin-MTD 135 (HPM 135 ), Parkin-MTD 151 (HPM 151 ), Parkin-MTD 174 (HPM 174 ) recombinant proteins were prepared and expressed. The results are shown in FIG. As shown in FIG.
  • an expression vector was prepared for the production of His-tag non-fusion cell-permeable Parkin recombinant protein in which JO-10, JO-13, JO-151, and JO-174 MTD were combined in E. coli .
  • Codon Optimization Service was commissioned from GenScript (USA) to produce codon optimized genes cloned at the Nco I and Hind III positions in the pUC57 vector.
  • parkin optimized cDNA (parkin optimized cDNA) is to facilitate the expression of Parkin in the expression system, and to increase the physical properties.
  • pUC57 vector was treated with HindIII restriction enzyme with NcoI, and each of the optimized genes was subcloned into NcoI and HindIII positions of pET-28a (+) (Novagen), and His-tag was removed at the N-terminus, and C -At the end, each MTD gene is designed to be fused, and is shown in FIGS. 11 and 12.
  • it was prepared to express Parkin-MTD 10 (PNM 10 ), Parkin-MTD 13 (PNM 13 ), Parkin-MTD 151 (PNM 151 ), Parkin-MTD 174 (PNM 174 ) recombinant proteins (FIGS. 11 and 12). Reference).
  • the code optimized Parkin DNA was overexpressed in transformed BL21 codon plus (DE3) Escherichia coli, and the results are shown in FIG.
  • the cDNA base sequence of the cloned gene is shown in SEQ. ID. NO .: 419 (PN), SEQ. ID. NO .: 420 (PNM 10 ), SEQ. ID. NO .: 421 (PNM 13 ), SEQ. ID. NO .: 422 (PNM 151 ), SEQ. ID. NO .: 423 (PNM 174 ).
  • E. coli BL21 Codon Plus E. coli 50 ug / mL kanamycine, 500 uM in 500 mL LB
  • overexpression was performed by adding 0.5 mM IPTG for 3 hours.
  • the overexpressed E. coli was harvested by centrifugation, and then repeated 10 sec-on and 20 sec-OFF conditions on ice for 30 minutes, followed by centrifugation to harvest inclusion bodies, 50 mM Tris-HCl (pH 8.0), Washed three times with 100 mM NaCl, 0.1% Triton X-100 buffer. Unpack the suture with 50 mM Tris-HCl (pH 10.0), 8 M urea buffer, and then directly administer to 30 mM Sodium phosphate (pH 8.0), 0.02% Tween-20 buffer at 4 ° C. Stir for 48 hours and refold.
  • the refolded Parkin protein was centrifuged at 9,000 rpm for 30 minutes, followed by Q-sepharose anion exchange resin column chromatography equipped with AKTA purifier for purification. 5 mM column volume was passed through 30 mM Sodium phosphate (pH 8.0) and 30 mM NaCl buffer to remove unbound proteins.
  • PN, PNM 10 , PNM 13 , PNM 151 , and PNM 174 were all identified as a single band of 53 kDa on SDS-PAGE, indicating that they were purified by Q-sepharose chromatography.
  • 1 ug of purified Parkin recombinant protein (PN, PNM 10 , PNM 13 , PNM 151 , PNM 174 ) and 1 ug His 6 -Parkin protein (Boston Biochem) were prepared using 1 uM E1, 50 uM E2, 1 from Boston Biochem.
  • the mixture was mixed with mM His6-Ubiquitin, 10 mM Mg-ATP, and reaction buffer solution (50 mM HEPES, 0.5 M NaCl, 10 mM DTT) for 1 hour at 37 ° C. Nm23 protein purified as a negative control was used. After adding 4x sample buffer to boil to terminate the reaction, 10% SDS-PAGE was performed.
  • Western blotting was performed using an ubiquitin antibody (Enzo life science) at 1: 1,000, and the Parkin recombinant protein to which ubiquitin was conjugated was confirmed, and the results are shown in FIG. 15.
  • TUNEL was performed to confirm the protective effect against neuronal cell death by the neurotoxin 6-OHDA.
  • Human brain cancer dopamine producing neurons SH-SY5Y cells (Korea Cell Line Bank, Seoul, Korea) were cultured in DMEM medium containing 10% fetal bovine serum, 5% penicillin and streptomycin, and 6-OHDA at 100 uM concentration. After 30 minutes of treatment, 2.5 uM of cell-permeable Parkin recombinant protein was added and incubated at 37 ° C. for 2 hours and 30 minutes.
  • TUNEL terminal dUTP nick-end labeling
  • PNM 10 cell permeable Parkin recombinant protein having the highest physical properties and E3 ligase activity was selected and subcutaneous injection to confirm blood brain barrier permeability after PNM 10 protein present in brain Immunohistochemical labeling and Western blotting were performed.
  • the solution was perfused through the heart using a pump to remove blood.
  • Mouse brains were extracted, and half of the brains were placed in 4% Paraformaldehyde solution and fixed at 4 ° C. for at least 24 hours, and immunohistochemical labeling was performed. The other half of the brains were prepared with Western blotting.
  • a fixed brain tissue was put in the OCT compound to make a block, the tissue was frozen in 20 ⁇ m thickness section and stored in PBS buffer solution. After washing three times with PBS buffer 10 minutes each, 3% hydrogen peroxide solution was allowed to stand at room temperature for 30 minutes. After washing three times with PBS for 10 minutes, it was left in 4% normal goat serum and 4% BSA mixture for 1 hour. After removing the mixed solution, the MTD-10 antibody was diluted 1: 1,000 and reacted overnight at 4 ° C. After washing three times with PBS buffer for 10 minutes, the biotinylated anti-Rabbit was diluted 1: 500 and reacted at room temperature for 2 hours.
  • the tissue was weighed and RIPA buffer (Sigma) was added at 0.1 g / mL, followed by crushing the tissue with a homogenizer and standing on ice for 30 minutes. Prepared. After centrifugation at 10,000 rpm for 10 minutes at 4 ° C, the supernatant was quantified using the Bradford quantitative method, and 100 ug of each protein sample was loaded on a 10% SDS-PAGE gel. After transferring the gel to the nitrocellulose membrane, it was blocked for 1 hour at room temperature with 5% skim milk. Parkin antibody (Millipore) and actin (Santacruz) were diluted 1: 1,000 and reacted at room temperature for 90 minutes.
  • RIPA buffer Sigma
  • Anti-mouse-HRP (Santacruz) secondary antibody was diluted 1: 1,000 and reacted at room temperature for 50 minutes.
  • ECL (Amersham) solution was allowed to react with the membrane for 1 minute, followed by LAS-4000 (Fujifilm Life Science) image analyzer. was detected and the result is shown in FIG. 17B.
  • PNM 10 human full-length cell permeable Parkin recombinant protein fused with MTD has blood-brain barrier permeability.
  • Parkinson's disease animal models using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are well known and cause dopamine neuronal death in the brain and striatum of the brain. Therefore, to establish a Parkinson's disease animal model by intraperitoneal injection of MPTP in C57BL / 6 mice, to determine the effect of protecting the death of dopamine neurons by PNM 10 cell permeable Parkin recombinant protein, marker enzyme of dopamine neurons Dopamine in brain tissues was quantitatively analyzed by immunohistochemical labeling and ELISA using antibodies against tyrosine hydroxylase.
  • MPTP was dissolved in 0.9% NaCl solution and injected at 15 mg / kg intraperitoneally three times per day for 2 days. After the last injection, anesthesia was performed with 2.5% avertin on day 7, and then PBS buffer containing 1 mM EDTA and 4 mM metabisulfite was perfused through the heart using a pump to remove blood, and then the brains of the mice were extracted. Half was fixed in 4% Paraformaldehyde solution for more than 24 hours at 4 °C, the other half was used for quantitative dopamine analysis.
  • the immobilized brain tissue was placed in an OCT compound to make a block, and the tissue was frozen in 20 ⁇ m thickness and stored in PBS buffer. After washing three times with PBS buffer for 10 minutes each, 3% hydrogen peroxide solution was left at room temperature for 30 minutes. After washing three times with PBS for 10 minutes, it was left in 4% normal goat serum and 4% BSA mixture for 1 hour. After removing the mixture, the tyrosin hydroxylase antibody (Millipore) was diluted 1: 1,000 and reacted at 4 ° C. overnight. After washing three times with PBS buffer for 10 minutes, the biotinylated anti-Rabbit was diluted 1: 500 and reacted at room temperature for 2 hours.
  • FIG. 18A a large number of dopamine neurons were observed in the melanoma and striatum of mice treated with PNM 10 recombinant protein, compared to the group administered with MPTP alone. It can be seen that there is a neuronal protective function transmitted by intracellular neurotoxic substances.
  • the mouse brain striatum was isolated and quantitatively compared to the dopamine content according to the product manual using ELISA kit (LDN), the results are shown in Figure 18B.
  • the dopamine content was increased by 40% in the PNM 10 cell permeable Parkin recombinant protein treated group compared to the MPTP treated group. Therefore, MTD-coupled cell-permeable Parkin recombinant protein increases dopamine secretion by penetrating the blood brain barrier and protecting dopamine neuron death by MPTP, a neurotoxic substance, through the blood brain barrier in the Parkinson's disease animal model. Able to know.
  • MTD-bound His-tag fusion or non-fusion Parkin recombinant protein is expected to be very likely to develop new drugs as a drug that can treat degenerative brain diseases as well as Parkinson's disease.
  • the cell-permeable Parkin recombination protein according to the present invention is designed to be transferred into cells, and the recombinant protein of the present invention can be used for the purpose of treating Parkinson's disease, and ultimately, can be useful for developing a therapeutic agent for degenerative brain disease in humans. It is expected to be.

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Abstract

La présente invention concerne : une protéine Parkin recombinante présentant des propriétés de perméation cellulaire, où un domaine de transduction macromoléculaire (DTM) est fusionné à une protéine Parkin présentant des propriétés thérapeutiques contre les maladies dégénératives du cerveau ; et une composition pharmaceutique ou similaire, pour le traitement des maladies dégénératives du cerveau contenant la protéine Parkin recombinante présentant des propriétés de perméation cellulaire à titre de principe actif. Pour obtenir la protéine Parkin recombinante présentant des propriétés de perméation cellulaire selon la présente invention, une protéine recombinante à perméation cellulaire est produite dans laquelle un domaine de transduction macromoléculaire est fusionné à une protéine Parkin qui constitue un facteur d'inhibition de la mort neuronale dopaminergique, et, dans la mesure où cette dernière possède la capacité à être transférée dans des cellules, la protéine est en pratique transférée dans des neurones dopaminergiques dans des environnements in vitro ou in vivo de sorte à inhiber la mort cellulaire, ou bien il existe un effet d'augmentation de la sécrétion de dopamine qui permet d'utiliser la protéine dans des études visant à traiter la maladie de Parkinson, et la protéine peut être employée avantageusement en tant qu'agent thérapeutique contre les maladies dégénératives du cerveau humain.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016028036A1 (fr) * 2014-08-17 2016-02-25 Cellivery Therapeutics, Inc. Séquences de domaine de transduction macromoléculaire avancé (amtd) pour l'amélioration de la perméabilité cellulaire, polynucléotides codant pour celles-ci, procédé d'identification des caractéristiques uniques des amtd les comprenant, procédé de développement des séquences d'amtd les comprenant
WO2017018787A1 (fr) 2015-07-27 2017-02-02 Cellivery Therapeutics, Inc. Protéine recombinée de la parkine à perméabilité cellulaire améliorée (icp) et son utilisation
WO2017034330A1 (fr) * 2014-08-27 2017-03-02 Cellivery Therapeutics, Inc. Protéine recombinante (cp-bmp) de protéine morphogénétique osseuse perméable aux cellules et son utilisation
EP3337815A4 (fr) * 2015-08-18 2018-11-07 Cellivery Therapeutics, Inc. Protéine recombinante perméable à la cellule (cp)- socs3 et utilisations de ladite protéine
US10508265B2 (en) 2015-08-10 2019-12-17 Cellivery Therapeutics, Inc. Cell-permeable reprogramming factor (iCP-RF) recombinant protein and use thereof
US10669531B2 (en) 2015-08-10 2020-06-02 Cellivery Therapeutics, Inc. Cell-permeable Cre (iCP-Cre) recombinant protein and use thereof
WO2021195597A3 (fr) * 2020-03-26 2021-11-04 Larimar Therapeutics, Inc. Molécules pour l'administration de protéines spécifiques à des organites
WO2022050778A1 (fr) * 2020-09-04 2022-03-10 Cellivery Therapeutics, Inc. Protéine recombinante de parkine modifiée et perméable aux cellules améliorée pour le traitement de maladies neurodégénératives et son utilisation
WO2024019489A1 (fr) * 2022-07-22 2024-01-25 주식회사 셀리버리 Vecteur aav et plateforme de fusion peptidique de pénétration cellulaire et composition pharmaceutique pour la prévention ou le traitement de maladies cérébrales dégénératives le comprenant
EP4153614A4 (fr) * 2020-05-20 2024-06-26 Spacecraft Seven, LLC Parkine modifiée et utilisations associées

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HIGASHI, Y. ET AL.: 'Parkin attenuates manganese-induced dopaminergic cell death.' JOURNAL OF NEUROCHEMISTRY. vol. 89, no. 6, June 2004, pages 1490 - 1497 *
LIMAN, J. ET AL.: 'Cell-penetrating fragments of the Cdk5 regulatory subunit are protective in models for neurodegeneration.' PHARMACEUTICALS. vol. 3, 2010, pages 1232 - 1240 *
MANFREDSSON, F. P. ET AL.: 'rAAV-mediated nigral human parkin over-expression partially ameliorates motor deficits via enhanced dopamine neurotransmission in a rat model of Parkinson's disease.' EXPERIMENTAL NEUROLOGY. vol. 207, no. 2, October 2007, pages 289 - 301 *
SATO, A. ET AL.: 'Parkin potentiates ATP-induced currents due to activation ofP2X receptors in PC12 cells.' JOURNAL OF CELLULAR PHYSIOLOGY vol. 209, no. 1, October 2006, pages 172 - 182 *

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