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US20170029798A1 - Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD) - Google Patents

Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD) Download PDF

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US20170029798A1
US20170029798A1 US14/809,279 US201514809279A US2017029798A1 US 20170029798 A1 US20170029798 A1 US 20170029798A1 US 201514809279 A US201514809279 A US 201514809279A US 2017029798 A1 US2017029798 A1 US 2017029798A1
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parkin
protein
amtd
recombinant proteins
cell
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Daewoong Jo
Eunsin Ha
Jieun Lee
Jeongmin Jeon
Kwangjae LEE
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Cellivery Therapeutics Inc
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Cellivery Therapeutics Inc
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Priority to US14/809,279 priority Critical patent/US20170029798A1/en
Assigned to CELLIVERY THERAPEUTICS, INC., JO, DAEWOONG reassignment CELLIVERY THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HA, EUNSIN, JEON, JEONGMIN, JO, DAEWOONG, Lee, Jieun, Lee, Kwangjae
Priority to KR1020187005889A priority patent/KR102132311B1/ko
Priority to CA2993778A priority patent/CA2993778C/fr
Priority to PCT/KR2016/008174 priority patent/WO2017018787A1/fr
Priority to EP16830820.3A priority patent/EP3328996B1/fr
Priority to JP2018503759A priority patent/JP6664464B2/ja
Priority to AU2016299468A priority patent/AU2016299468B2/en
Priority to CN201680044600.5A priority patent/CN108138150B/zh
Publication of US20170029798A1 publication Critical patent/US20170029798A1/en
Priority to US15/879,664 priority patent/US10662419B2/en
Abandoned legal-status Critical Current

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the present invention relates to new protein-based therapeutic agents specially targeted for neurodegenerative disorder based on macromolecule intracellular transduction technology (MITT) enabled with newly advanced hydrophobic CPPs providing cell-permeability of macromolecules in vitro and in vivo.
  • the recombinant protein of this invention has new technical advantages as an intracellular protein therapy for the treatment of Parkinson's disease in that it could resolve blood-barrier permeability, tissue-permeability, and bio-transfer function.
  • Parkinson's disease is one of leading neurodegenerative disease that occurs by instable generation and secretion of dopamine (1).
  • Parkinson's disease there has been damage in dopaminergic neuron in the midbrain; pathological features, such as a formation of lewy body; mobility defect, such as bradykinesia, rest tremor, and rigidity; and non-motor symptoms, such as depression, dementia, and insomnia (2-4).
  • Parkinson's disease is a neurodegenerative disease found mostly in older generations. Statistically, Approximately 1% of people aged more than 55 and 3% in people aged more than 75 have been diagnosed with the disease (5). As the population of aged people increases, patients diagnosed with Parkinson's disease are ever growing in number. Globally, the population of patients with this disease has been projected to increase from 4.1 million individuals in 2005 to 8.7 million individuals by 2030 (6, 7).
  • Parkin gene has been first discovered Japanese stock that has autosomal recessive juvenile Parkinsonism (ARJP) (8). Parkin gene mutation could be discovered from approximately 50% in early-onset hereditary Parkinson's disease and 18% in sporadic patients below the age of 50 (9).
  • Parkin is comprised of 465 amino acid sequences that functions has E3-ligase in ubiquitin-proteasome system.
  • Parkin protein functions to reduce the oxidative stress in the cell by removing damaged, oxidized, and/or irregularly structured protein inside the cell.
  • Parkin mutation When Parkin mutation occurs, it loses its property as an E3-ligase; inclusion body and/or irregular proteins are accumulated inside the cell that lead to reduced secretion of dopamine and apoptosis of dopaminergic neuron (10).
  • Parkinson's disease using the fruit flies that have shown decrease in motor function by the decrease in dopamine secretion due to an inactivation of dopaminergic neuron in which the function of Parkin and PINK1 was revealed (11).
  • Parkin was overexpressed in the fruit fly that did not express PINK1, Parkinson's disease-related symptoms caused by PINK1, such as mitochondrial dysfunction and degradation of dopaminergic neuron, were confirmed to be recovered (11-13).
  • Parkin protein may successfully act as a target protein-based agent to treat Parkinson's related diseases by functioning as a main enzyme in the ubiquitin-proteasome system to destroy inclusion body and suppress apoptosis of dopaminergic neuron by maintaining the function of mitochondria from oxidative stress.
  • Macromolecule such as Parkin protein
  • MMT macromolecule intracellular transduction technology
  • This membrane translocating technology, macromolecule intracellular transduction technology (MITT) using hydrophobic CPP demonstrated its effect in delivering biologically active therapeutic cargo proteins, such as Parkin, into cultured cells and animal tissues.
  • MITT-based hydrophobic CPPs named membrane translocating sequence (MTS) and membrane translocating motif (MTM), derived from the hydrophobic signal peptide of fibroblast growth factor 4 (FGF4) have been reported and used to deliver biologically active peptides and proteins systemically in animals.
  • MTD membrane translocating sequence
  • FGF4 fibroblast growth factor 4
  • CP-Parkin cell-permeable protein-based therapeutics for Parkinson's disease.
  • Parkin has been previously fused to a hydrophobic cell-penetrating peptide (CPP) named macromolecule transduction domain (MTD) to develop MTD-fused Parkin recombinant protein (CP-Parkin).
  • CPP cell-penetrating peptide
  • MTD macromolecule transduction domain
  • CP-Parkin MTD-fused Parkin recombinant protein
  • Cell-/tissue-/BBB-permeable CP-Parkin recombinant protein has proven to have an effect to treat Parkinson's disease by suppressing apoptosis of neuron cells, increasing the secretion of dopamine, and recovering the motor skills.
  • CP-Parkin was not clinically applicable due to its relatively low solubility and yield.
  • iCP-Parkin cell-permeable Parkin recombinant protein
  • aMTD advanced macromolecule transduction domain
  • SD solubilization domain
  • the present invention is devised to develop much enhanced BBB-penetrable Parkin recombinant protein to effectively improve decrease in motor skills from Parkinson's disease by protecting dopaminergic neurons and recovering the formation and secretion of dopamine.
  • this invention iCP-Parkin, has a technical advantage over other previously developed anti-neurodegenerative agents by resolving the setbacks of blood brain barrier penetration and low solubility/yield.
  • An aspect of the present invention pertains to cell-permeable recombinant protein for the treatment of Parkinson's disease based on advanced macromolecule transduction domain (aMTD) sequences capable of mediating the transduction of biologically active macromolecules into live cells.
  • aMTD advanced macromolecule transduction domain
  • An aspect of the present invention relates to Parkin recombinant proteins fused aMTD and SD improved to high solubility and high yield for clinical application possible level.
  • An aspect of the present invention also, cell-permeable Parkin recombinant proteins comprised of aMTD sequences artificially developed with seven critical factors (CFs) and optimized based on in-depth analysis of Parkin and aMTDs association from selected 10 aMTDs and 10 random peptides (rPs or rPeptides).
  • CFs critical factors
  • An aspect of the present invention is related to a list of amino acid sequences of the Parkin recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs)—advanced macromolecule transduction domains (aMTDs) and solubilization domain (SD)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • SD solubilization domain
  • An aspect of the present invention is related to a list of cDNA sequences of the Parkin recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs)—advanced macromolecule transduction domains (aMTDs) and solubilization domain (SD)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • SD solubilization domain
  • An aspect of the present invention is related to a result of analysis with previously developed hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • An aspect of the present invention is related to a result of analysis with newly invented hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • An aspect of the present invention is related to a method for development of the new hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • An aspect of the present invention is related to a method for analysis with previously developed hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • An aspect of the present invention is related to a method for preparation of the Parkin recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs) and solubilization domain (SD)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • SD solubilization domain
  • An aspect of the present invention is related to a method for determination of solubility, yield, cell- and tissue-permeability of the Parkin recombinant proteins fused to newly invented hydrophobic cell-penetrating peptides (CPPs), namely advanced macromolecule transduction domains (aMTDs) and solubilization domain (SD)
  • CPPs hydrophobic cell-penetrating peptides
  • aMTDs advanced macromolecule transduction domains
  • SD solubilization domain
  • cell-/tissue-/BBB-permeable protein-based therapeutics for Parkinson's disease based on an efficient use of aMTD sequences for drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy and peptide therapy.
  • aMTD/SD-fused Parkin recombinant protein could be produced in large quantities.
  • effective BBB-permeability of the recombinant protein overcomes the limitations of previously developed anti-neurodegenerative treatments. Therefore, the present invention, iCP-Parkin, would allow practical applications to efficiently treat Parkinson's related diseases.
  • FIG. 1 shows aMTD321-Mediated Cell-Permeability Compared to Negative Control (rP38) and Previously Developed CPP (MTM12 and MTD85).
  • Cell-permeable potency of a negative control (rPeptide 38) and previously developed hydrophobic CPPs (MTM12 and MTD85) are shown as the references.
  • the cell-permeable potency of aMTD321 was visually compared to that of a SDA only (HSA).
  • the area formed by the graph with the legend of “Vehicle” represents untreated RAW 264.7 cells (vehicle); the line with the legend of “IFTC only” represents FITC-fused cells (FITC only); the line with the legend of “HSA” indicates Histidine fused with SDA with FITC-labeling (HSA); and the line with the legend of “HMSA” shows the negative control (rP38), previously developed CPPs (MTM12 and MTD85), and aMTD-recombinant proteins (HM321SA).
  • FIG. 2 shows aMTD321-Mediated Intracellular Localization Compared to Negative Control (rP38) and Previously Developed CPP (MTM12 and MTD85).
  • rP38 Negative Control
  • MTM12 and MTD85 Previously Developed CPP
  • FIG. 3 shows Schematic Diagram of His-aMTD/SD-Fused Parkin Recombinant Proteins.
  • a schematic Diagram of His-aMTD-SD-Parkin recombinant protein having cell-permeability is illustrated and constructed according to the present invention.
  • Designs (Set 1) of recombinant Parkin fusion proteins contained histidine tag for affinity purification (MGSSHHHHHHSSLVPRGSH (SEQ OD NO: 2), with the legend of “His”), cargo (Parkin, with the legend of “Parkin”), aMTD321 (IVAVALPALAVP (SEQ OD NO: 4), with the legend of “aMTD”), SDA (with the legend of “SDA”) and SDB (with the legend of “SDB”).
  • FIG. 4 shows Agarose Gel Electrophoresis Analysis After Cloning of Parkin Recombinant Proteins. These figures show the agarose gel electrophoresis analysis showing plasmid DNA fragments insert encoding aMTD-SD-Fused Parkin cloned into the pET28a (+) vector according to the present invention.
  • FIG. 5 shows Expression and Purification of Parkin Recombinant Proteins. Expression of Parkin recombinant Protein in E. coli . SDS-PAGE analysis of cell lysates before ( ⁇ ) and after (+) IPTG induction; aliquots of Ni2+ affinity purified proteins (P); and molecular weight standards (M). The size (number of amino acids), yield (mg/L) and solubility of each recombinant protein are indicated. Solubility was scored on a 5-point scale from highly soluble, with little tendency to precipitate (+++++), to largely insoluble proteins (+).
  • FIG. 6 shows Relative Yield of Parkin Recombinant Proteins Compared to Negative Control (HP).
  • the figure shows graphs comparing the yield of aMTD-SD-fused Parkin recombinant proteins with His-Parkin recombinant protein without aMTD (HP).
  • FIG. 7 shows Determination of aMTD-Mediated Cell-Permeability of Parkin Recombinant Proteins. Protein uptake of Parkin recombinant proteins by RAW264.7 cells. Cells were incubated for 1 hour at 37° C. with 10 •M FITC-conjugated Parkin recombinant proteins (FITC-HP, FITC-HM321P, FITC-HM321PSA and FITC-HM321PSB), an equimolar concentration of unconjugated FITC (FITC only) or vehicle (culture medium, DMEM), treated to remove cell-associated but non-internalized protein, and analyzed by flow cytometry.
  • FITC-HP 10 •M FITC-conjugated Parkin recombinant proteins
  • FITC-HM321P 10 •M FITC-conjugated Parkin recombinant proteins
  • FITC-HM321PSA 10 •M FITC-HM321PSA
  • FIG. 8 shows Determination of aMTD-Mediated Intracellular Localization of Parkin Recombinant Proteins.
  • NIH3T3 cells were incubated for 1 hour at 37° C. with 10 •M FITC-conjugated Parkin recombinant proteins (FITC-HP, FITC-HM321P, FITC-HM321PSA and FITC-HM321PSB), an equimolar concentration of FITC only or vehicle.
  • Cell-permeability of Parkin recombinant proteins was visualized by utilizing fluorescence confocal microscopy LSM700 version (top). Nomarski image of the same cells (bottom).
  • FIG. 9 shows Tissue Distribution of Parkin Recombinant Proteins In Vivo.
  • Cryosections (20 uM) of organs were prepared from ICR mice 2 hours after intraperitoneal injection of diluent, FITC only and 600 ug FITC-conjugated Parkin recombinant proteins (FITC-HP, FITC-HM321P, FITC-HM321PSA and FITC-HM321PSB).
  • Tissue distribution of the Parkin recombinant proteins (green staining) was analyzed with fluorescence microscopy.
  • FIG. 10 shows Inhibition of Apoptosis in Dopaminergic CATH.a Cells.
  • CATH.a cells at 70% confluence were incubated with 50 uM 6-hydroxydopamine (6-OHDA, Agonist) for 1 hr, treated for 2.5 hrs with 2.5 uM HP, HM321P, HM321PSA or HM321PSB and assessed for apoptosis by TUNEL staining.
  • the micrographs are representative of three independent experiments, plotted (bottom) as means ⁇ S.D. Experimental differences between groups were assessed by a Student's two-paired t-test (*p, 0.05).
  • FIG. 11 shows Inhibition of Apoptosis in Dopaminergic SH-SY5Y Cells.
  • SH-SY5Y cells at 70% confluence were incubated with 100 uM 6-hydroxydopamine (6-OHDA, Agonist) for 6 hr, treated for 2.5 hrs with 2.5 uM HP, HM321P, HM321PSA or HM321PSB and assessed for apoptosis by TUNEL staining.
  • the micrographs are representative of three independent experiments, plotted (bottom) as means ⁇ S.D. Experimental differences between groups were assessed by a Student's two-paired t-test (*p, 0.05).
  • FIG. 12 shows Schematic Diagram of His-aMTD/SDB-Fused Parkin Recombinant Proteins.
  • a schematic Diagram of His-aMTD/SD-Parkin recombinant protein having cell-permeability is illustrated and constructed according to the present invention.
  • Designs (Set 2) of Parkin recombinant proteins contained histidine tag for affinity purification (with the legend of “His-Tag”), cargo (Parkin, with the legend of “Parkin”), aMTD321 (with the legend of “aMTD”) and SDB (with the legend of “SDB”).
  • FIG. 13 shows Expression and Purification of Parkin Recombinant Proteins. Expression of Parkin recombinant proteins in E. coli . SDS-PAGE analysis of cell lysates before ( ⁇ ) and after (+) IPTG induction; aliquots of Ni2+ affinity purified proteins (P); and molecular weight standards (M). The size (number of amino acids), yield (mg/L) and solubility of each recombinant protein are indicated. Solubility was scored on a 5-point scale from highly soluble, with little tendency to precipitate (+++++), to largely insoluble proteins (+).
  • FIG. 14 shows Relative Yield of SDB-Fused Parkin Recombinant Proteins (HPSB) Compared to Negative Control (HP).
  • HP Relative Yield of SDB-Fused Parkin Recombinant Proteins
  • the figure shows structures of SDB-fused Parkin recombinant proteins and graphs comparing the yield of SDB-fused Parkin recombinant proteins (HPSB) with His-Parkin recombinant protein without aMTD (HP).
  • FIG. 15 shows Determination of aMTD-Mediated Cell-Permeability of HM321PSB. Protein uptake of Parkin recombinant proteins by NIH3T3 cells. Cells were incubated for 1 hour at 37° C. with 10 •M FITC-conjugated Parkin recombinant proteins with or lacking aMTD321 sequence (FITC-HPSB and FITC-HM321PSB), an equimolar concentration of unconjugated FITC (FITC only) or vehicle (culture medium, DMEM), treated to remove cell-associated but non-internalized protein, and analyzed by flow cytometry.
  • FITC-HPSB and FITC-HM321PSB 10 •M FITC-conjugated Parkin recombinant proteins with or lacking aMTD321 sequence
  • FITC-HPSB and FITC-HM321PSB an equimolar concentration of unconjugated FITC
  • vehicle culture medium
  • FIG. 16 shows Determination of aMTD-Mediated Intracellular Localization of HM321PSB.
  • NIH3T3 cells were incubated for 1 hour at 37° C. with 10 •M FITC-conjugated Parkin recombinant proteins (FITC-HPSB and FITC-HM321PSB), an equimolar concentration of FITC only or vehicle.
  • Cell-permeability of Parkin recombinant proteins was visualized by utilizing fluorescence confocal microscopy LSM700 version (top). Nomarski image of the same cells (bottom).
  • FIG. 17 shows In Vivo Cellular Uptake of HM321PSB in PBMC. FACS analysis of PBMC isolated from whole blood of ICR mice 15 min (top) and 30 min (bottom) after intraperitoneal injection of diluent, FITC only and FITC-conjugated Parkin recombinant proteins (600 ug, FITC-HPSB and FITC-HM321PSB).
  • FIG. 18 shows Tissue Distribution of HM321PSB In Vivo.
  • Cryosections (20 uM) of organs were prepared from ICR mice 2 hours after intraperitoneal injection of diluent, FITC only and 600 ug FITC-conjugated Parkin recombinant proteins (600 ug, FITC-HPSB and FITC-HM321PSB).
  • Tissue distribution of the Parkin recombinant proteins (green staining) was analyzed with fluorescence microscopy.
  • FIG. 19 shows Delivery of aMTD-Mediated Parkin Recombinant Protein to the Brain Determined by Western Blot Analysis.
  • FIG. 20 shows Delivery of aMTD-Mediated Parkin Recombinant Protein to the Brain Determined by Immunoblot. Immunoblotting of Parkin recombinant proteins in the cerebellum. Sagittal sections through the cerebellum were immunostained with anti-Parkin antibody 2 hrs after IP injection of 600 ug of diluent alone or His-tagged Parkin recombinant proteins without aMTD or lacking aMTD sequences.
  • FIG. 21 shows Protocol of MPTP-Induced PD Mouse Model. 8-week-old C57BL/6 male and female mice were received intraperitoneal injections of MPTP (15 mg/kg ⁇ 3 times/day, 2 h interval) for three consecutive days. After 3 days, mice were received IP injection of Parkin recombinant proteins (HPSB and HM321PSB, 600 ug/head, a time/day) for five consecutive days, respectively. Urine and brain dopamine levels, gross motor function and brain lesions (TH immunostaining) were analyzed on subsequent days as indicated.
  • HPSB and HM321PSB Parkin recombinant proteins
  • FIG. 22 shows Dopamine of Urine in MPTP-Induced PD mice Treated with Recombinant Proteins.
  • Urine dopamine levels in MPTP-lesioned mice were measured by ELISA 10 hrs after HPSB and HM321PSB protein treatment. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • FIG. 23 shows 3 Dopamine of Brain in MPTP-Induced PD mice Treated with Recombinant Protein. Striatal dopamine levels in MPTP-lesioned mice. Dopamine levels in striatal biopsies were determined by ELISA in lesioned mice without protein treatment or after daily treatments with HM321PSB as shown in FIG. 11 . Dopamine levels in groups of 4 mice are presented as means ⁇ S.D. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • FIG. 24 shows Preservation of Gross Motor Function in MPTP-Lesioned Mice Treated with Parkin Recombinant Proteins.
  • HM321PSB preserves gross motor function of MPTP-lesioned mice. 9 hrs after the last MPTP treatment mice were treated for 3 hrs with 600 ug proteins (IP, HPSB or HM321PSB), and motor ability was assessed by placing the animals in a water bath and video recording subsequent movements. The percentage of time of the mice in each treatment group were engaged in 4 legged motion is presented as means ⁇ S.D. The number of mice in each group was as follows: Diluent, 12; MPTP only, 7; MPTP+HPSB, 14; MPTP+HM321PSB, 12. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • FIG. 25 shows Determination of Footprint Pattern using Gait Test. Parameters measured in footprint analysis with dotted lines representing the direction of progression (DoP) of walking are shown. Footprints of MPTP-lesioned mice were evaluated for stride length (cm) and sway length (cm).
  • FIG. 26 shows Stride Length in Gait Test by Parkin Recombinant Protein. Histograms represent differences in: stride length in groups of 4 mice are presented as means ⁇ S.D. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • FIG. 27 shows Sway Length in Gait Test by Parkin Recombinant Protein. Histograms represent differences in: sway length in groups of 4 mice are presented as means ⁇ S.D. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • FIG. 28 shows Dopaminergic Neuron in Substantia Nigra and Striatum by Parkin Recombinant Protein.
  • HM321PSB reduces MPTP-induced dopaminergic toxicity.
  • MPTM-lesioned mice were treated with Parkin recombinant proteins for 5 days as shown in FIG. 11 (IP, 15 mg/kg) and loss/preservation of dopaminergic neurons was determined by tyrosine hydroxylase (TH) staining.
  • IP Parkin recombinant proteins
  • FIG. 29 shows Recovery Effect of Dopaminergic Neuron in Substantia Nigra by Parkin Recombinant Protein. The percentage of TH-positive cells in each treatment group was calculated. Experimental differences between groups were assessed by a Student's two-paired t-test (*p ⁇ 0.05).
  • the present invention relates to protein-based therapeutics for Parkinson's disease having cell-permeability applicable for the clinical studies that facilitate the transduction of biologically active macromolecules including proteins across the cell membrane.
  • the cell-permeable Parkin recombinant protein of the present invention based on aMTD is artificially developed.
  • the aim is to develop iCP-Parkin by adopting novel hydrophobic CPPs formatted based on the seven critical factors determined based on in-depth analysis to facilitate protein translocation across the membrane.
  • These seven critical factors include the amino acid length (9-13), bending potential based on the proline position and location (6′, 7′, 8′ in the middle and 12′ at the end), rigidity/flexibility (II: 40-60), structural formation (AI: 180-220), amino acid composition (A, V, I, L, and P), and the secondary structure (helix formation recommended).
  • aMTD321 had been discovered to enhance the uptake of a His-tagged coding sequence of solubilization domain A (SDA) in RAW264.7 cells as assessed by flow cytometry. Relative levels of protein uptake was 7 times that of a reference MTM12 protein, which contained 1st generation CPP (membrane translocating motif) and was 2.9 times that of a MTD85 reference protein, which contained 2nd generation CPP (macromolecule transduction domain).
  • Recombinant cargo (parkin) proteins fused to hydrophobic CPP could be expressed in bacteria system, purified with single-step affinity chromatography, but protein dissolved in physiological buffers (e.g. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low yield as a soluble form. Therefore, an additional non-functional protein domain (solubilization domain: SD) has been applied to fuse with the recombinant protein for improving the solubility, yield and eventually cell and tissue permeability.
  • physiological buffers e.g. PBS, DMEM or RPMI1640 etc.
  • the selected domains are SDA and SDB (TABLE 2).
  • the aMTD/SD-fused recombinant proteins have been determined for their stability and stability.
  • solubilization domains (SDs) and aMTDs have greatly influenced in increasing solubility/yield and cell-/tissue-permeability of the protein. Therefore, we have developed highly soluble and highly stable Parkin recombinant protein fused with SD (SDA and SDB) and aMTDs for the clinical application.
  • Protein structures were labeled as follows: (i) a cargo protein only, (ii) a cargo protein fused with aMTD, (iii) a cargo protein fused with aMTD and solubilization domain A (SDA) and (iv) a cargo protein fused with aMTD and solubilization domain B (SDB) ( FIG. 3 ).
  • the aMTD 321 /SD-fused Parkin recombinant proteins have significantly higher cell-, tissue-permeability as compared to the Parkin recombinant proteins lacking aMTD321 sequence (HP and HPSB).
  • HM321PSA and HM321PSB have similar solubility and yield, cellular and systemic delivery activity of aMTD321/SDB-fused Parkin recombinant protein was higher than Parkin recombinant protein lacking aMTD321 sequence. Therefore, aMTD 321 /SD-fused Parkin recombinant protein was determined as the most stable structure of the recombinant proteins.
  • HM 321 PSA and HM 321 PSB showed the highest cell permeability.
  • FIG. 17 we determined in vivo tissue-permeability of Parkin recombinant proteins after 15 min and 30 min of intraperitoneal injection of FITC-labeled proteins.
  • the PBMC analyzed by FACS showed a gain in fluorescence, indicative of the presence of FITC-labeled proteins as compared with control animals that received FITC-labeled HPSB or unconjugated FITC.
  • One of the two Parkin recombinant proteins, HM 321 PSB showed a higher intracellular signal in PBMC.
  • the distribution of FITC-labeled proteins in different organs in cryosections analyzed by fluorescence microscopy FIGS. 9 and 18 ).
  • Parkin recombinant proteins lacking aMTD showed limited tissue permeability in various organs (brain, heart, lung, liver, spleen and kidney).
  • aMTD 321 enhanced the systemic delivery of Parkin recombinant proteins in tissues (heart, lung, liver and kidney).
  • CATH.a and SH-SY5Y cells were treated with 6-hydroxydopamine (6-OHDA). After treatment of 6-OHDA, these cells were pre-treated with Parkin recombinant proteins and TUNEL assays were conducted. A large number of cell death were observed in 6-OHDA only treated group. Similarly to 6-OHDA-treated group, HP lacking aMTD has shown similar percentage of apoptotic cell death with the agonist only group.
  • aMTD 321 /SD-fused Parkin recombinant proteins (HM 321 PSA and HM 321 PSB) have suppressed apoptosis to 19.7 and 14.2% in CATH.a and SH-SY5Y cells, respectively (*p ⁇ 0.05). Similar results have been obtained in both CATH.a cells and SH-SY5Y cells. These results have demonstrated that aMTD 321 /SD-fused Parkin recombinant proteins have neuroprotective effects in cultured neuronal cells ( FIGS. 10 and 11 ).
  • Parkinson's disease—(PD-) animal model that mimics physiological and mental symptoms of Parkinson's disease by using a neural toxin.
  • PD- Parkinson's disease-like symptoms
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine
  • cell-permeable Parkin recombinant proteins have been designed and developed with the aMTD. All Parkin recombinant proteins fused with aMTD and control recombinant proteins lacking aMTD have been confirmed for their quantitative, visual cell-/tissue-permeability and BBB-permeability. We were able to confirm that the cell-permeable aMTD 321 /SD-fused Parkin recombinant proteins had relatively high cell-/tissue-permeability ( FIGS. 7, 8, 9, 15, 16, 17 and 18 ), as well as efficient in the brain tissue delivery by penetrating through BBB ( FIGS. 19 and 20 ).
  • cell-permeable Parkin recombinant protein has anti-apoptotic effect on the neuronal cell death caused by a neurotoxin (6-OHDA) ( FIGS. 10 and 11 ), and it has a recovery effect in the PD-mice model that displayed movement dysfunction induced by neurotoxin (MPTP) ( FIGS. 24, 25, 26 and 27 ).
  • aMTD-based MITT has enabled us to improve the method for developing cell-permeable recombinant proteins.
  • the expression vectors were designed for Parkin proteins fused with aMTD321 and solubilization domain A (SDA) or solubilization domain B (SDB).
  • SDA solubilization domain A
  • SDB solubilization domain B
  • PCR polymerase chain reaction
  • the PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1 ⁇ reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) was digested on the restriction enzyme site between BamHI (5′) and HindIII (3′) involving 35 cycles of denaturation (95° C.) for 30 seconds, annealing (60° C.) for 30 seconds, and extension (72° C.) for 2 min each. For the last extension cycle, the PCR reactions remained for 5 minutes at 72° C. Then, they were cloned into the site of pET-28a (+) vectors (Novagen, Madison, Wis., USA).
  • DNA ligation was performed using T4 DNA ligase at 4° C. overnight. These plasmids were mixed with competent cells of E. coli DH5• strain on the ice for 10 minutes. This mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42° C. for 90 seconds. Then, the mixture added with LB broth media was recovered in 37° C. shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 •g/mL) (Biopure, Johnson, Tenn.) before incubating at 37° C. overnight.
  • kanamycin 50 •g/mL
  • Denatured recombinant proteins were lysed using denature lysis buffer (8 M Urea, 10 mM Tris, 100 mM NaH2PO4) and purified by adding Ni-NTA resin. Resin bound to proteins were washed 3 times with 30 mL of denature washing buffer (8 M Urea, 10 mM Tris, 20 m imidazole, 100 mM NaH2PO4). Proteins were eluted 3 times with 30 mL of denature elution buffer (8 M Urea, 10 mM Tris, 250 mM imidazole).
  • the aMTD 321 -fused Parkin proteins containing SDA or SDB are cloned, expressed, purified, and prepared in a soluble form.
  • Each recombinant protein fused to aMTD and/or SD was determined for their solubility and yield. Solubility was scored on a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (*****) to largely insoluble proteins (*) by measuring their turbidity (A450). Yield (mg/L) in physiological buffer condition of each recombinant protein was also determined. The cell-permeable Parkin recombinant proteins were observed as a single band, where the amount of the final purified protein was 13 mg/L ( FIG. 3 ).
  • Recombinant proteins purified under the denatural condition were analyzed on 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue.
  • Parkin recombinant proteins were conjugated to fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo.).
  • FITC fluorescein isothiocyanate
  • RAW 264.7 cells were treated with 10 •M FITC-labeled recombinant proteins for 1 hour at 37° C., washed three times with cold PBS, treated with proteinase K (5 •g/ml) for 10 min at 37° C. to remove cell-surface bound proteins.
  • Cell-permeability of these recombinant proteins were analyzed by flow cytometry (FACSCalibur; BD, Franklin Lakes, N.J.) using the FlowJo analysis software.
  • NIH3T3 cells were cultured for 24 hours on a coverslip in 24-wells chamber slides, treated with 10 ⁇ M FITC-conjugated recombinant proteins for 1 hour at 37° C., and washed three times with cold PBS.
  • Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 10 minutes at room temperature, washed three times with PBS, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino-2-phenylindole) for nuclear staining.
  • PFA paraformaldehyde
  • DAPI 4,6-diamidino-2-phenylindole
  • ICR mouse 5 weeks old, female
  • IP intraperitoneally
  • FITC FITC only or FITC-conjugated proteins
  • PBMC peripheral blood cells
  • FITC-labeled Parkin recombinant proteins 600 ⁇ g was administered to ICR mice (5 weeks old, female). Two hours later, the mice are sacrificed, and liver, kidney, spleen, lung, heart and brain were isolated and embedded with an OCT compound (Sakura, Alphen anden Rijn, Neetherlands), frozen, and then sectioned to a thickness of 20 ⁇ m.
  • OCT compound Sakura, Alphen anden Rijn, Neetherlands
  • mice 6-week-old ICR female mice were injected intraperitoneally with diluent (PBS) or with 600 •g His-tagged Parkin recombinant proteins. After 2 h, mice was perfused with 0.9% NaCl and fixed with cold 4% paraformaldehyde. After the brains were removed, they were post-fixed with 4% paraformaldehyde and transferred to 30% sucrose. The brains were cut into 30 ⁇ m coronal sections using a freezing microtome.
  • PBS diluent
  • 600 •g His-tagged Parkin recombinant proteins 600 •g His-tagged Parkin recombinant proteins.
  • mice was perfused with 0.9% NaCl and fixed with cold 4% paraformaldehyde. After the brains were removed, they were post-fixed with 4% paraformaldehyde and transferred to 30% sucrose. The brains were cut into 30 ⁇ m coronal sections using a freezing microtome.
  • Brain cryosections (30 •m) are immunostained with anti-Parkin (1:100, Santa Cruz Biotechnology) monoclonal antibodies, followed by biotin-conjugated goat anti-mouse secondary antibody (Vector Laboratories), and developed with Avidin-Biotin Complex kit (Vectastain kit, Vector Laboratories).
  • biotin-conjugated goat anti-mouse secondary antibody Vector Laboratories
  • Avidin-Biotin Complex kit Vectastain kit, Vector Laboratories.
  • mice treated with proteins were perfused with 0.9% NaCl. Brains were isolated, and striatal region was dissected and homogenized in lysis buffer (Intron, Seongnam, Korea). Supernatant from the centrifugation (13,000 rpm for 10 min at 4° C.) is analyzed by western blot that is probed with antibodies against parkin (1:200) and •-actin (1:2,000).
  • the secondary antibody is goat anti-mouse IgG-HRP (all
  • Terminal dUTP nick-end labeling (TUNEL) assays are conducted according to the manufacturers' instructions (Roche).
  • Mouse dopaminergic neuronal (CATH.a) cells (ATCC: American Type Culture Collection) are plated (3 ⁇ 10 4 /well) and pre-treated with 50 •M 6-hydroxydopamine (6-OHDA) for 1 h at 37° C. followed by the treatment with 2.5 •M Parkin recombinant proteins for 2.5 h at 37° C., analyzing the changes in cell survival.
  • SH-SY5Y Human brain tumor (SH-SY5Y) cells (Korea Cell Line Bank) are also cultured, plated (3 ⁇ 10 4 /well) and pre-treated with 100 •M 6-hydroxydopamine (6-OHDA) for 6 h followed by the treatment with 2.5 •M Parkin recombinant proteins for 2.5 h at 37° C., analyzing the alteration.
  • 6-OHDA 6-hydroxydopamine
  • mice 8-week-old C57BL/6 male and female mice housed in plastic cages in a temperature—and humidity—controlled room with a 12-h light/12h-dart cycle.
  • Mice were randomly assigned to one of four experimental groups (Diluent, MPTP only, MPTP+HPSB and MPTP+HM 321 PSB). Three groups of mice except for diluent were received intraperitoneal injections of MPTP (15 mg/kg ⁇ 3 times/day, 2 h interval) for three consecutive days.
  • the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Sigma-Aldrich, St. Louis, Mo.) was dissolved in 0.9% NaCl. Controls are treated with 0.9% NaCl for the same time period.
  • mice in MPTP+HPSB and MPTP+HM 321 PSB groups were received intraperitoneal injection of HPSB, HM 321 PSB recombinant protein (600 ⁇ g/head, a time/day) for five consecutive days, respectively.
  • HPSB HPSB
  • HM 321 PSB recombinant protein 600 ⁇ g/head, a time/day
  • Dopamine synthesized in the urine For measurement of dopamine synthesized in the urine, we collected the urine of mice in all groups on the first day of treatment of Parkin recombinant protein. Dopamine synthesized in the urine is measured by using a commercial ELISA kit according to instructions provided by the manufacturer (GenWay, San Diego, Calif.). In brief, rabbit anti-dopamine antibody is added to urine or tissue extract, and the immune complexes are recovered in wells coated with goat anti rabbit antibody. A second enzyme conjugated anti-dopamine antibody directed against a different epitope produces the reaction products proportional to the amount of antigen as compared against a standard curve.
  • Dopamine synthesized in the brain extracts is measured by using a commercial ELISA kit according to instructions provided by the manufacturer (GenWay, San Diego, Calif.). In brief, rabbit anti-dopamine antibody is added to urine or tissue extract, and the immune complexes are recovered in wells coated with goat anti rabbit antibody. A second enzyme conjugated anti-dopamine antibody directed against a different epitope produces the reaction products proportional to the amount of antigen as compared against a standard curve.
  • mice were allowed to walk along a 50 cm long, 10 cm wide runway with 10 cm high walls into an enclosed box. Stride length and sway length were measured as the average distance of forward movement between each stride and sway.
  • mice On the last day of treatment of Parkin recombinant protein, mice was perfused with 0.9% NaCl and fixed with cold 4% paraformaldehyde. And then, brains were removed, post-fixed with 4% paraformaldehyde, and transferred to 30% sucrose. The brains were cut into 30 ⁇ m coronal sections using a freezing microtome.
  • Dopaminergic neuronal cell marker in brain-tyrosine hydroxylase is immunostained with anti-TH (1:50, Thermo Scientific, Rockford, USA) monoclonal antibody, followed by biotin-conjugated goat anti-rabbit secondary antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, Calif.) and developed with ABC kit (Vectastain kit, Vector Laboratories, Burlingame, Calif.).

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US14/809,279 2015-07-27 2015-07-27 Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD) Abandoned US20170029798A1 (en)

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CN104673823B (zh) * 2015-03-11 2017-11-03 中国农业科学院生物技术研究所 多策略叠加提高甲基对硫磷水解酶在毕赤酵母中分泌表达量的方法
US20170029798A1 (en) * 2015-07-27 2017-02-02 Cellivery Therapeutics, Inc. Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)
WO2017026779A1 (fr) * 2015-08-10 2017-02-16 Cellivery Therapeutics, Inc. Protéine recombinée cre à perméabilité cellulaire améliorée (icp-cre) et son utilisation
EP3337815B1 (fr) * 2015-08-18 2020-12-16 Cellivery Therapeutics, Inc. Protéine recombinante perméable à la cellule (cp)- socs3 et utilisations de ladite protéine

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US20180237485A1 (en) * 2014-08-27 2018-08-23 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (cp-bmp) recombinant protein and use thereof
US10774123B2 (en) * 2014-08-27 2020-09-15 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CP-BMP) recombinant protein and use thereof
US11279743B2 (en) 2014-08-27 2022-03-22 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof
US12378576B2 (en) 2018-04-27 2025-08-05 Spacecraft Seven, Llc Gene therapy for CNS degeneration

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AU2016299468A1 (en) 2018-02-08
CN108138150A (zh) 2018-06-08
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JP2018524994A (ja) 2018-09-06
JP6664464B2 (ja) 2020-03-13
CA2993778A1 (fr) 2017-02-02
CN108138150B (zh) 2022-07-26
CA2993778C (fr) 2020-09-22
EP3328996A4 (fr) 2018-08-01
KR102132311B1 (ko) 2020-07-13
AU2016299468B2 (en) 2019-07-25
US20180171322A1 (en) 2018-06-21
EP3328996A1 (fr) 2018-06-06
US10662419B2 (en) 2020-05-26
KR20180026565A (ko) 2018-03-12

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