WO2018230785A1 - 비바이러스성 유전자편집 crispr 나노복합체 및 이의 제조방법 - Google Patents

비바이러스성 유전자편집 crispr 나노복합체 및 이의 제조방법 Download PDF

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WO2018230785A1
WO2018230785A1 PCT/KR2017/013623 KR2017013623W WO2018230785A1 WO 2018230785 A1 WO2018230785 A1 WO 2018230785A1 KR 2017013623 W KR2017013623 W KR 2017013623W WO 2018230785 A1 WO2018230785 A1 WO 2018230785A1
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crispr
polymer carrier
cells
spcas9
ester
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French (fr)
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정현정
강유경
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한국과학기술원
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Priority to US16/068,161 priority Critical patent/US11319533B2/en
Priority to CA3009389A priority patent/CA3009389C/en
Priority to AU2017390080A priority patent/AU2017390080B2/en
Publication of WO2018230785A1 publication Critical patent/WO2018230785A1/ko

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    • C12N9/14Hydrolases (3)
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    • C12N2310/00Structure or type of the nucleic acid
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    • C12N2310/20Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/80Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the present invention was supported by the task number HI15C1948 with the support of the Ministry of Health and Welfare.
  • the rapid detection and diagnosis of drug-resistant bacteria by the probe ", the host institution is the Korea Advanced Institute of Science and Technology, the period of research is 2015.11.01-2018.10.31.
  • the present invention relates to a non-viral gene editing CRISPR nanocomposite and a method for producing the same. More specifically, a nanocomplex formed by chemically conjugating a carrier material to an enzyme protein of a CRISPR gene editing system and forming a sgRNA, and a method for preparing the same. And a method for transferring the nanocomposite into a cell and using it as a non-viral gene editing system.
  • MRSA Meciillin-resistant Staphylococcus aureus
  • CRE carbapenem-resistant Enterobacteriaceae
  • MRAB multidrug-resistant Acinetobacter baumannii
  • MRPA multidrug-resistant Pseudomonas aeruginosa
  • VRE vancomycin-resistant Enterococci
  • Another problem is that most of the current drugs used to treat bacterial infections are small molecule antibiotics with a broad spectrum.
  • Gene therapy is a powerful tool for designing drugs with high specificity to targets.
  • siRNA When targeting mammalian cells, the use of siRNA to silence certain genes appears to be a promising treatment and is currently used for the treatment of various types of cancers, glaucoma, hemophilia, and family amyloid diseases. Clinical trials are underway; however, direct administration of these gene therapy products will result in immediate enzymatic digestion in body fluids or low transfer efficiency to the target site, thus reducing the effectiveness of cationic polymers and lipid-based substances.
  • Carrier materials such as inorganic nanoparticles, cell permeable peptides and dendrimers have been used to aggregate and deliver biologically active molecules to targets. Attempts to use non-viral gene delivery strategies because of the poor efficiency of transit through You are limited.
  • the target-specific crRNA of sgRNA is 5'-NGG.
  • ZFN zinc finger nuc
  • PAM protospacer-adjacent-motif
  • TALENs transcription activator-like effector nucleases
  • the CRISPR system induces specific cleavage in the target gene.
  • CRISPR has the highest viral editing efficiency with high expression efficiency. It is widely used and has limitations in clinical application due to problems such as vector toxicity, cellular immune response and antibody neutralization reaction.
  • the present inventors have found a novel form that overcomes the low transfer effects and toxicity issues of existing viral gene editing methods and gene editing methods of lipid-based material formulations.
  • the object of the present invention is to provide a polymer carrier substance-conjugated CRISPR (Clustered regularly interspaced short palindromic repeats) enzyme protein.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Another object of the present invention is to provide a method for preparing the polymer carrier material-conjugated CRISPR enzyme protein.
  • Another object of the present invention is to provide a CRISPR nanocomposite comprising a polymer carrier substance-conjugated CRISPR enzyme protein and a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • Another object of the present invention is to provide a method for producing a CRISPR nanocomposite comprising the step of mixing a polymer carrier substance-conjugated CRISPR enzyme protein and sgRNA.
  • Another object of the present invention is to provide a polymer carrier substance-conjugated CRISPR enzyme protein, or a composition for gene editing comprising the CRISPR or a complex.
  • the present invention provides a polymer carrier material-conjugation.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • CRISPR stands for Clustered regularly interspaced short palindromic repeats, the third generation of genes derived from the immune system of bacteria.Bacteria die when infected with a virus, but some survive, and some of their viral DNA remains. Subsequently, when reinfection occurs, small guide RNAs (sgRNAs) are generated based on the stored information, and then combined with an endonuclease called Cas9 to cut external DNA, where the guide RNAs are the desired target genes. Complementary base pairs combine to determine specificity and Cas9 protein cuts target genes
  • the guide RNA acts as a nucleic acid hydrolase.
  • Recent DNA base sequences can be cut according to the sequence.
  • the CRISPR enzyme protein is CRISPR.
  • Enzymes that can cleave or edit target sites on the genome during the gene editing process can cleave or edit target sites on the genome during the gene editing process
  • deaminase or any combination of these proteins, but not limited to.
  • carrier refers to proteins
  • the carrier is a biocompatible polymeric material without cytotoxicity or tissue toxicity, either a synthetic polymer or a natural polymer.
  • the term “cargo” refers to a substance capable of working in vivo, such as physiologically active substances and enzymes of proteins, hormones, and nucleophiles that are bound or conjugated with the carrier and introduced into cells.
  • the term “cargo” in this specification refers to, but is not limited to, gene shear enzyme protein, more specifically CRISPR enzyme protein, and single guide RNA (sgRNA).
  • the polymer carrier material may be branched polyethyleneimine, linear polyethyleneimine, polypropyleneimine, polyamidoamine, polyethylene glycol, polyethylene oxide-polypropylene oxide copolymer, poly- Lactic acid, poly-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, polycaprolactone, polyphosphoester, polyphosphazine, polybetaamino ester, branched
  • Polyaminoester polyaminobutyl-glycolic acid, polyorthoester,
  • Polyhydroxyl ester polyacrylamide, polyvinylpyridone, polyvinyl alcohol, poly (2- (dimethylamino) ethylmethacrylate) (PDMAEMA), dendrimer, hyaluronic acid, alginic acid, chitosan, dextran, cyclodextrin, It may be selected from the group consisting of spermine, poly-Arginine, and poly-Lysine, their copolymers or mixtures, but is not limited to these.
  • the present invention uses a branched polyethyleneimine as the polymer carrier material.
  • the polymer carrier material is conjugated with the CRISPR enzyme protein (conjugation)
  • the polymer carrier material and the CRISPR enzyme are identical to one embodiment of the present invention.
  • Proteins include succinimidyl 4- (N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), sulfo- succinimidyl 4- (N-maleimidomethyl) cyclohexane- 1 -carboxylate),
  • N-a-maleimidoacet-oxysuccinimide ester (AMAS)
  • BMPS N- ⁇ -maleimidopropyl-oxy succinimide ester
  • Ns-malemidocaproyl-oxysuccinimide ester (EMCS), PEG (PEGylated SMCC), succinimidyl 3- (2-pyridyldithio) propionate (SPDP), PEG-SPDP (PEGylated SPDP), DSG (disuccinimidyl glutarate), DCC (Dicyclohexylcarbodiimide) , DSS (disuccinimidyl suberate), BS3 (Bissulfosuccinimidyl suberate), DSP (dithiobis (succinimidyl propionate)), EGS (ethylene glycol bis (succinimidyl succinate)), DMP (dimethyl pimelimidate), BMOE (bismaleimidoethane), BMB (1, 4- bismaleimidobutane),
  • DBCO-PEG-maleimide (Dibenzocyclooctyne-PEG4-maleimide), DBCO-SS-NHS ester (Dibenzocyclooctyne-SSN-hydroxysuccinimidyl ester), DBCO-NHS ester (dibenzocyclooctyne-N-hydroxysuccinimidyl ester), acetylene -PEG-NHS ester acetylene-PEG-NHS ester), and
  • crosslinking agent selected from the group consisting of alkylene-PEG-maleimide, but is not limited thereto.
  • crosslinking agents are chemical crosslinking agents, which induce a binding reaction of the active period of the protein to be bonded to the crosslinking agent of the crosslinking agent so that the polymer carrier material can be conjugated with the CRISPR enzyme protein of the present invention.
  • sulfo-SMCC was used as a chemical crosslinking agent, and sulfo-SMCC was added to branched polyethyleneimine (bPEI).
  • bPEI branched polyethyleneimine
  • the amine group of polyethyleneimine was activated by substitution with a maleimide group, and SpCas9 (SpCas9-bPEI) conjugated with bPEI was prepared by reacting the primary amine group of activated polyethyleneimine with the free sulfhydryl group on the cysteine residue of SpCas9 protein.
  • Polymer carrier material and CRISPR enzyme protein eg
  • the polyethyleneimine is a gene transfer (eg siRNA, plasmid).
  • the carrier material for DNA It is one of the most commonly used carrier materials for DNA and has been selected for the modification of cargo because it can be used in various types of branches or linears.
  • the branched form was selected because the amine group including the primary amine is richer than the linear amines, so that waste packaging and delivery can be efficiently performed.
  • the direct covalent modification of proteins was chosen to minimize the amount of carrier material for administration, thereby addressing the problem of reduced efficacy due to biotoxicity and inadequate release.
  • the present invention provides a method for preparing the polymer carrier substance-conjugated CRISPR enzyme protein, comprising the following steps:
  • the functional group of the polymer carrier material is reacted with a bifunctional chemical crosslinker to activate the functional group of the polymer carrier material.
  • the chemical crosslinking agent refers to a reagent that chemically combines two or more molecules of different kinds, and the bifunctionality refers to a property of inducing a connection between molecules having two same or different functional groups.
  • the activation reaction of the polymer carrier material is DMSO (dimethylsulfoxide),
  • Any solvent selected from the group consisting of chloroform may be used, but is not limited to any solvent available in the art for activation reactions for molecular reactions.
  • the activation reaction of the polymer carrier material is 4 to 60 ° C., specifically 4 to 50 o C,
  • It may be performed under temperature conditions of 4 to 40 ° C, 4 to 30 ° C, or 4 to 25 ° C, but is not limited thereto.
  • the activation reaction of the polymer carrier material is 0.5 to 24 hours, specifically
  • the type of the polymer carrier material in this step is redundant with that described above with respect to the polymer carrier material-conjugated CRISPR enzyme protein and thus the description thereof is omitted.
  • Step 0 ? Molecular Carrier Reactivated by the Functional Group of CRISPR Enzyme Protein Load
  • the conjugate is produced by reacting with the functional groups of the protein.
  • the molar ratio of the polymer carrier material and the CRISPR enzyme protein in step (b) is, for example, 1: 10- 6 to 1: 10 6 , 1: 10 5 to 1: 10 5 , 1: 1 (to 1: 10). 4 , 1: 10- 3 to 1:10 3 , 1: 10- 2 to 1:10 2 , 1: 1 ⁇ - 1 to I:) 1 , but not limited to.
  • At 4 to 60 ° C more specifically, it can be performed under temperature conditions of 4 to 50 o C, 4 to 40 ° C, 4 to 30 ° C, or 4 to 25 0 C, but is not limited to this.
  • this can be 1 to 48 hours, 1 to 24 hours, 1 to 12 hours, or 4 to 12 hours, but is not limited to this.
  • reaction reaction between the polymer carrier material and the CRISPR enzyme protein is pH
  • water soluble solvent 4 to 10
  • water soluble solvent 4 to 10
  • aqueous buffer solution that maintains the above pH range.
  • the CRISPR enzyme protein may be conjugated to the polymer carrier of the present invention.
  • the present invention provides a polymer carrier material-conjugation.
  • CRISPR nanocomposites comprising CRISPR enzyme protein and single guide RNA (sgRNA).
  • the CRISPR nanocomposite is a combination of carrier substance-conjugated CRISPR enzyme protein and sgRNA for a target gene.
  • the complex formed by the physical interaction between the carrier substance-conjugated CRISPR enzyme protein and sgRNA described above is described. it means.
  • the CRISPR nanocomposite refers to a complex composed of carrier material conjugated CRISPR enzyme protein and sgRNA, not a virus or DNA vector, and thus has a feature of being a non-viral nanocomplex.
  • the CRISPR nanocomposite is a complex capable of dispersing in an aqueous solution, and may have a particle size of 1 to 10,000 nm, but is not limited thereto.
  • the CRISPR nanocomposite has a zeta potential of -100 to +100 mV.
  • the sgRNA of the present invention is a ribonucleic acid that guides the CRISPR enzyme protein to a specific site of a target gene on the genome, and CrRNA (CRISPR) for a protospacer, which is a target site proximate to a PAM (protospacer-adjacent motif) sequence.
  • RNA and TracrRNA (trans-activating RNA) sequences.
  • the inventor can select any desired target gene, for example mecA, mecR 1, aph, NDM-1, KPC, oxa, ure, lrg, cap, spl, KPC, GES, IMP.
  • PPP2CA, PPP1CC, PPP2R5C, GFP, RFP, YFP, tdTomato, mCherry, luciferase, etc. may be targeted genes, but it is obvious to those skilled in the art that these are only illustrative.
  • the present invention provides a method for preparing the CRISPR nanocomposite described above.
  • the method for producing the CRISPR nanocomposite comprises the step of mixing the above-described polymer carrier substance-conjugated CRISPR enzyme protein with sgRNAs related to the target gene.
  • the CRISPR the manufacture of nanocomposite carrier material - the molar ratio of the bonded heunhap CRISPR enzyme protein sgRNA is from 1:10 6 to 1:10, 6, in particular from 1: 10-5 to 1 : 10 5 , 1: 10- 4 to 1:10 4 , most specifically 1: 10- 3 to 1:10 3 , but not limited to.
  • the mixing temperature may be 4 to 37 0 C, specifically 4 to 25 ° C, 15 to 37 ° C, 15 to 25 ° C, but is not necessarily limited.
  • the mixed reaction time is 0.1-12 hours, specifically 1-6 hours, 0.1-3 hours, 0.1-1 hours, most specifically 0.1 0.5 hours can be carried out to produce a nanocomposite, but is limited to It is not.
  • the present invention is a polymer carrier as described above.
  • compositions comprising a substance-conjugated CRISPR enzyme protein or a CRISPR nanocomplex described above.
  • the polymer carrier material-conjugated CRISPR enzyme protein contained in the gene editing composition described above corresponds to the invention described in another aspect of the present invention.
  • composition of the present invention is a pharmaceutical composition
  • a pharmaceutically acceptable carrier is included.
  • the pharmaceutically acceptable carrier is usually used at the time of presentation.
  • RO / KR As, lactose, dextrose, sucrose, solbi, manni, starch, acacia rubber, phosphate, alginate, gelatin, silicate, microcrystalline cellulose,
  • compositions of the present invention include, but are not limited to, lubricants, humectants, sweeteners, flavoring agents, Emulsifiers, suspending agents and preservatives may further be included.
  • composition of the present invention may be administered orally or parenterally, such as intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, pulmonary, intrarectal, intradural, It may be administered by eye, skin or transdermal administration.
  • composition of the present invention depends on factors such as formulation method, mode of administration, patient age, body weight, sex, degree of disease symptoms, food, time of administration, route of administration, rate of excretion and response.
  • the doctors who are diverse and usually skilled can easily determine and prescribe the effective dosage for the intended treatment.
  • the dosage is 0.0001-1000 mg / kg, but not limited to this.
  • a “pharmaceutical effective amount” is defined as a significant amount inducing the editing of the desired gene.
  • composition of the present invention may be prepared in unit dose form by preparation using a pharmaceutically acceptable carrier and / or excipient according to an easily accessible method of ordinary knowledge in the technical field to which the present invention belongs. It may also be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, and may further comprise a dispersant or stabilizer.
  • the composition when the composition is administered to an isolated cell, tissue, or individual, the above-mentioned polymer carrier substance-conjugated CRISPR enzyme protein or CRISPR nanocomplex is transferred into a cell and the CRISPR enzyme.
  • Gene editing is induced by protein editing.
  • Dendritic cells MDSC (Myeloid-derived Suppressor Cell), embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSC), vascular endothelial cells, epidermal cells, liver cells, muscle cells, bone cells, fibroblasts Or eukaryotic cells such as chondrocytes, nerve cells, neural stem cells, adpose-derived stem cells (ADSC), mouse embryonic fibroblasts (EF), mullite cells, and parasitic cells, or S. aureus, E coli, P. aeruginosa, K. pneumoniae, A. baumannii, B. subtilis, S. epidermidis, E. faecalis, S. pneumoniae
  • the polymer carrier material-conjugated CRISPR enzyme protein and CRISPR nanocomposite of the present invention were introduced into the cells of the bacterium and exhibited the effect of gene editing. High transfer efficiency.
  • the nanocomposites showed better intracellular transfer efficiency compared with the existing lipid-based carrier materials such as lipofectamine, and thus the editing effect of the target gene was excellent.
  • CRISPR Cas9 protein and a nanoscale complex using the same were formed and confirmed that it can be used as a specific killing target for multidrug-resistant bacteria.
  • the present invention compares with Cas9 in comparison with the previously reported lipid-based non-covalent formulations.
  • RO / KR Fig. La and le show the nucleotide sequences of recombinant Cas9 endonucleases (SpCas9) obtained from cloned Streptococcus pyogenes of the present invention.
  • the sequence is 6x His, FLAG, NLS (nuclear localization sequence) from N to C terminus,
  • FIG. 2 is a diagram showing the SDS-PAGE results of the SpCas9 protein of the present invention and purified.
  • the size of SpCas9 is approximately 190 kDa.
  • FIG. 3 shows the results of fluorescence of GFP fused with SpCas9 protein of the present invention under UV illuminator.
  • sgRNA single guide RNA
  • 5A and 5B show nucleotide sequences of the mecA gene template for constructing sgRNA sequences targeting mecA gene.
  • FIG. 6 shows a primer sequence for amplification of the mecA gene template.
  • FIG. 7 shows the nucleotide sequence of a template for sgRNA synthesis.
  • FIG. 8 shows a primer sequence for synthesis of an sgRNA template.
  • FIG. 11 is a schematic diagram of the conjugation process between SpCas9 and bPEI of the present invention
  • FIG. 12 shows the results of gel delay analysis to confirm successful conjugation of bPEI to SpCas9.
  • Figure 13 shows the results of SDS-PAGE analysis to confirm the successful conjugation of bPEI to SpCas9. '
  • the schematic diagram for the preparation of the complex (natural complex) between the unmodified SpCas9 protein and sgRNA is shown as a control.
  • FIG. 16 shows the results of gel electrophoresis after inducing the cleavage by adding the synthesized target DNA with the Cr-nano complex.
  • FIG. 17 is a diagram showing the results of observation of the intrabacterial delivery of the polymer derivative SpCas9 of the present invention by confocal microscopy.
  • FIG. 18 is a diagram showing the intracellular bacterial transfer efficiency of the polymer derivative SpCas9 of the present invention in comparison with the control group.
  • 19 to 20 and 21a to 21c reconstruct a confocal image section into a 3D image to confirm the influx of polymerized SpCas9 into bacteria.
  • Fig. 22 is a diagram showing the fluorescence signal of SpCas9 and nucleus staining to confirm the influx of polymer-derived SpCas9 into bacteria using a molecular carrier of different molecular weight (molecular weight 25,000).
  • Fig. 23 is a diagram showing the observance and analysis of Cr-nano complex influx in A549 cells using a multifocal fluorescence microscope.
  • Fig. 24 is a diagram showing the observance and analysis of Cr-nano complex influx in HaCat cells with a multifocal fluorescence microscope.
  • Fig. 25 is a diagram showing the presence of the complex in the animal cell from the superposition of the complex and the nuclear staining fluorescence signal by reconstructing the confocal image section into a 3D image to confirm the influx of the Cr-nano complex into the HaCat cells.
  • FIG. 26 shows Cr-nanocomplex influx efficiency in Raw 264.7 cells.
  • Fig. 27 shows the results of observation and analysis of Cr-nano complex influx in Jurkat cells by multifocal fluorescence microscope.
  • FIG. 28 is a diagram illustrating observation of Cr-nano complex influx in neural stem cells by multifocal microscopy.
  • FIG. 30 shows a schematic diagram of an experimental procedure for evaluating dielectric editing efficiency of Cr-nanocomposite of the present invention.
  • Figure 31 shows the growth rate by measuring the OD 600 value after suspension culture of bacteria treated with Cr-nanocomposite of the present invention.
  • 32A and 32B show bacteria in order to further evaluate the dielectric editing pattern.
  • the figure shows the CFU number calculated in the presence or absence of oxacillin and treated with Cr-nanocomplex or control.
  • Fig. 33 shows the relative growth rate when the Cr-nanocomposite of the present invention is treated with bacteria.
  • Fig. 34 is a diagram showing the comparison of dielectric editing efficiency for each concentration of Cr-nanocomposite of the present invention.
  • Fig. 35 shows the results of replica culture experiments of bacteria treated with Cr-nanocomposite of the present invention.
  • the Cr-nanocomplex system of the present invention designed to efficiently deliver genome editing cargo into bacteria, was developed using a polymer-derived Cas9 protein and a sgRNA complex.
  • the SpCas9 is 6x His, FLAG, NLS (nuclear localization from N to C terminus)
  • Lysis buffer 50 mM NaH 2 P0 4 , 300 mM NaCl, 10 mMimidazole, 0.05% ⁇ -mercaptoethanol, ⁇ 8.0
  • Two seconds of pils with a septum coefficient (duty) and 5 seconds of rest were repeated and performed on ice for a total of 30 minutes.
  • the plasmids were obtained by transformation into E. coli competent cells and purification by affinity chromatography.
  • the sequence of cloned SpCas9 is shown in FIG.
  • sgRNA Single guide RNA
  • the protospacer region was adjacent to all of the protospacer-adjacent motif (NGG) sequences, from which the target was cut upstream of the three bases. cleavage will happen.
  • sgRNAs are linked with CrRNA (CRISPR RNA), which targets mecA.
  • TracrRNA trans-activating crRNA
  • Linker GG was also included at the 5 'end.
  • Templates for sgRNAs are HelixAmp Power-Pfu (NanoHelix) and
  • oligonucleotide primer (Bioneer) was used to anneal for 40 seconds at 60 ° C. and extend 30 seconds at 72 0 C, followed by gel extraction (QIAquick, Qiagen). Transcription was performed using phage T7 RNA polymerase (Promega) at 37 0 C for 120 minutes.
  • the DNA template for each sgRNA was first synthesized using the primers of Figure 8 for in vitro transcription.
  • the DNA template for each sgRNA was the T7 promoter region, the template region for CrRNA. And a template region for TracrRNA (FIG. 7).
  • the synthesized DNA template is shown in FIG. 9.
  • FIG. 10 shows that three different types of sgRNAs targeting different regions of mecA: sgRNA (1), sgRNA (2), and sgRNA (3) were successfully synthesized. All sgRNAs are ⁇ 100 bp
  • the functionalities for inducing double strand cleavage of the three sgRNAs prepared above were also investigated.
  • target DNA pure radish by RT-PCR of 1803 bp region in the mecA gene ( Figure 5) from total RNA of cultured MRSA A cellular DNA solution was prepared.
  • the purified native SpCas9 protein and sgRNA (1), sgRNA (2) and sgRNA (3) were mixed and PCR-amplified mecA target DNA was added to endonuclease.
  • sgRNA (3) showed two fragments (648 bp and 1 155 bp) in the gel electrophoresis results in the highest cleavage efficiency, and sgRNA (2) was found in the 1463 bp fragment just below the uncut DNA. The corresponding short fragments were shown, but the other fragments were difficult to observe. In the case of sgRNA (1), none of the cleaved products were shown, so the detection efficiency was evaluated to be too low or sgRNA did not function to induce specific double strand breaks. And further tests for intrabacterial delivery.
  • MRSA strains CCARM 3798, 3803, and 3877 strains were identified by CCARM (Culture).
  • KCTC 3881 an MSSA strain, was obtained from KCTQ Korean Collection for Type Cultures.
  • TSB tryptic soy broth, BD
  • Branched polyethylenimine (bPEI, Mw 2000 and 25000) is a 5 mg sulfo-SMCC (sulfosuccinimidyl) dissolved in ultrapure purified water.
  • the molar concentration ratio of sulfo-SMCC 1: 10).
  • the reaction solution was then dialyzed for 48 hours in deionized water (MWCO 5001000, Spectra / Por).
  • FIG. 11 A schematic diagram of the synthesis process of SpCas9-bPEI is shown in FIG. 11.
  • SpCas9 conjugated with bPEI (SpCas9-bPEI) slightly shifts in the negative (-) direction, as opposed to natural SpCas9, which actually moves in the negative (-) direction.
  • Gel delay analysis of SpCas9-bPEI may be due to changes in mobility due to polymer deformation and clustering of protein molecules.
  • bPEI has a very high density of amine functional groups and has high cationicity.
  • Protein-polymer interactions can induce a pool.
  • FIG. 13 SDS-PAGE analysis to confirm successful conjugation of bPEI to SpCas9 is shown in FIG. 13.
  • SpCas9-bPEI and natural SpCas9 appeared in similar areas.
  • the covalently crosslinked SpCas9 proteins did not appear to be present after conjugation ( Figure 13).
  • the conjugation of SpCas9 and bPEI was successful and it was confirmed that no crosslinking occurred between SpCas9 protein molecules.
  • sgRNA (3) (1.8 ⁇ ) was mixed in desorption water (pH 6.5) and incubated in static conditions at room temperature (25 0 C_ for 15 min.
  • the original SpCas9 (990 nM) was added to sgRNA (3). (1.8 ⁇ ) and under these conditions.
  • Nanoscale complexes with self-assembled Spcas9-bPEI and sgRNA (3) were prepared (FIG. 14).
  • SgRNA ⁇ -MRS A methicillin-resistant Staphylococcus aureus
  • the Cr-nanocomposite showed a particle size (Z average) of 163.3 nm, which is larger than that of the unmodified Cas9 protein and the sgRNA natural complex of 82.6 nm.
  • the results confirm that successful formation of small nanoscale protein-polymer conjugates / RNA complexes by charge interactions between negatively charged sgRNAs and positively charged polymers in SpCas9-bPEI.
  • Each complex will form a larger complex, including several molecules of SpCas9-bPEI and sgRNAs, unlike the unmodified SpCas9, which is predominantly in the form of a single protein bound to a single sgRNA molecule.
  • Anionicity is expected to help improve delivery into bacteria.
  • the amplified template DNA was then treated with spCas9-bPEI or natural SpCas9 complexed with SpCas9-bPEI or sgRNA (3) to achieve a molar ratio of SpCas9: sgRNA: target DNA of 10: 1 and Cas9 nucleic acid decomposed semicoagulant ( 20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, pH 6.5) was treated at 37 ° C. for 1 hour. The final product was agarose gel electrophoresis to confirm the presence and size of the segment of DNA.
  • SpCas9 was able to induce double strand cleavage of the target DNA even after direct covalent modification with bPEI and complex formation with sgRNA.
  • SpCas9-bPEI 200 nM
  • Natural SpCas9 200 nM
  • Concentrated SpCas9 and bPEI Mw 2000
  • natural SpCas9 which is simply mixed with bPEI
  • the Cas9 protein conjugated with polyethyleneimine of the present invention is an unmodified Cas9 protein (SpCas9), or a combination of unreacted polyethyleneimine polymer and Cas9 protein.
  • SpCas9 + bPEI mix
  • the bright green fluorescence of GFPuv was clearly observed adjacent to nuclear staining.
  • SpCas9 did not show any absorption.
  • SpCas9 (SpCas9 + bPEI (mix)) also showed no signs of absorption.
  • the confocal image section was reconstructed into 3D images to show the presence of SpCas9 in bacterial cells from the superposition of SpCas9 and the fluorescence signal of nucleus staining (Fig. 19, FIG. 20).
  • Histograms of fluorescence signals are also scanned in other confocal images.
  • the relative absorption value was 0.4273 for SpCas9-bPEI, natural SpCas9, natural SpCas9 with bPEI, and natural SpCas9 mixed with lipofectamine. It was 0.0041, 0.0001, and 0.0083, respectively.
  • Significant increases in the uptake of Cas9 protein for bPEI conjugation may be due to the high cationic nature of the polymer or the resulting increase in the polarity of the protein.
  • the present inventors have shown that the SpCas9 protein is covalently linked to a cationic polymer.
  • the cationic polymer is expected to be applied to each single molecule of the protein and at the same time ensure that a minimum amount of carrier material is used, unless the modification affects the functional activity.
  • Figure 22 shows that strains with larger bPEI polymers showed no significant absorption of the protein when treated with bacteria. Only strains with small molecular weight bPEI showed high transfer efficiency, thus maximizing transport efficiency. In order to minimize the toxicity and minimize the toxicity, it is important to use the lowest amount of carrier material while using the smallest molecular weight.
  • SpCas9-bPEI25000 / sgRNA (168 nM) or natural SpCas9 / sgRNA (168 nM) complexes were treated in cultured mammalian cells.
  • RNAiMAX Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol (final concentration SpCas9 / s g RNA each-168 nM). Each experimental group was 1- at 37 ° C, 5% C0 2 . After incubation using a cell incubator for 1.6 hours,
  • the conjugated Cas9 protein (SpCas9-bPEI) / sgRNA complex was found to be successfully absorbed into A549 animal cells with significantly higher efficiency compared to the unmodified Cas9 protein (SpCas9) / sgRNA complex.
  • RNAiMAX complex Compared to the RNAiMAX complex, it showed a significantly higher absorption rate. Another control showed no significant intracellular uptake when treated with SpCas9-bPEI25000 / sgRNA, which had a molecular weight of 25,000 and 12.5 times the molecular weight of polyethyleneimine. Similarly when treated with HaCat animal cells, Cas9 conjugated with polyethyleneimine of the present invention, as shown in FIG. 24.
  • RNAiMAX Compared to the combination of protein (SpCas9) / sgRNA complex with another control, Lipofectamine RNAiMAX, it successfully penetrates into animal cells with significantly higher efficiency.
  • the complex was reconstructed from the confocal image section into a 3D image.
  • the SpCas9-bPEI / sgRNA complex showed significantly higher efficiency compared to the unmodified Cas9 protein (SpCas9) / s g RNA complex and showed a slightly higher intracellular absorption rate than the combination with lipofectamine RNAiMAX.
  • the transfer efficiency of SpCas9-bPEI / sgRNA or SpCas9 / sgRNA complex can be confirmed by the GFP signal of SpCas9 recombinant protein, staining the nucleus with DAPI as the counterstain of all cells, and the cytoplasm with rhodamine-phalloidin in the case of FIGS. 28 and 29. It was.
  • the inventors have found that the Cr-nanocomplex can be used to edit bacterial genomes and to provide antibiotic resistance.
  • Cr-nanocomposites formed with sgRNA and SpCas9-bPEI targeting mec A were treated in cultured MRSA (3798 and 3803 strains) in vitro and the growth of the bacteria was examined by subsequent culture in selective medium. Has previously been proven to be resistant to both methicillin and oxacillin.
  • the Cr-nanocomplex of the present invention was tested as an experimental group using SpCas 9- bPEI (990 nM) and sgRNA (3).
  • RNAiMAX Lipofectamine RNAiMAX (15.8 ⁇ L, Thermo Fisher Scientific). Containing only protein (without sgRNA), only SpCas9-bPEI, or only natural SpCas9 990 nM, were also prepared as controls. .
  • the treated bacteria were washed with PBS, treated 100 times in TSB with 6 g / mL oxacillin and for 37 to 90 minutes.
  • the bacterial growth was determined by measuring the OD value ((Nanophotometer, Implen) at 600 nm after 90 min incubation.
  • the bacteria were treated with Cr-nanocomposite or control group, and the CFU number was calculated and shown in FIG. 30 in the presence or absence of oxacillin.
  • relative growth refers to (1) CFU when treated with complexes containing sgRNAs.
  • the Cr-nanocomplex demonstrates that the target DNA can be double-stranded by delivering sgRNA and SpCas9 protein into bacteria with much higher efficiency.
  • FIGS. 32A and 32B various concentrations of Cr-nanocomposites were treated with bacteria to determine the dose-dependent dielectric editing efficiency.
  • Cr-nano of the present invention When compared to natural complexes, the composites treated at low concentrations inhibited relative growth by 18.7 percent, while those at higher concentrations inhibited relative growth by 57.7 percent. Treatments at higher concentrations inhibited growth compared to those treated at medium concentrations. The average was slightly higher, but it was statistically significant only for intermediate treatments.
  • the treatment of low concentrations of Cr-nanocomposites was not sufficient to yield genome editing effects, whereas the treatment of high concentrations of Cr-nanocomposites was bacteriological. It may have interfered with the genome editing process by affecting the function and absorption of stimuli or by stimulating bacterial growth.

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Abstract

본 발명은 비바이러스성 유전자편집 CRISPR 나노복합체,그의 제조방법 등에 관한 것이다. 본 발명에 의한 비바이러스성 유전자편집 CRISPR 나노복합체는 수나노미터~수마이크론 단위의 크기이고, 외부의 물리적인 자극 없이 세포내 전달이 가능하고, 세포 표적유전자에 대해 비바이러스성 경로에 의한 유전자편집에 유용하게 활용할 수 있다. 결과적으로 상기 CRISPR 나노복합체를 동물모델 제작, 미생물공학, 질병 치료를 위한 세포공학 또는 생체 투여 제형에 이용할 경우, 높은 세포내 전달 및 유전자편집 효율을 보이고, 비특이적 편집, 유전자변이, 세포 및 생체독성 유발 등의 문제를 최소화할 수 있다.

Description

명세서
발명의명칭:비바이러스성유전자편집 CRISPR나노복합체및 이의제조방법
기술분야
[1] 본발명은대한민국보건복지부의지원하에서과제번호 HI15C1948에의해 이루어진것으로서,상기과제의연구관리전문기관은한국보건산업진흥원, 연구사업명은 "감염병위기대웅기술개발사업 ",연구과제명은 "나노프로브에 의한다제내성균의신속한검출및진단",주관기관은한국과학기술원, 연구기간은 2015.11.01-2018.10.31이다.
[2] 또한본발명은대한민국보건복지부의지원하에서과제번호 HI14C2270에 의해이루어진것으로서,상기과제의연구관리전문기관은
한국보건산업진흥원,연구사업명은 "세계선도의생명과학자육성사업 'ᅳ, 연구과제명은 "순환종양세포감지에의한암의분자진단법개발",주관기관은 한국과학기술원,연구기간은 2014.12.01-2017.10.31이다.
[3] 또한,본발명은대한민국미래창조과학부의지원하에서과제번호
2015R1C1A1A02036647에의해이루어진것으로서,상기과제의연구관리 전문기관은한국연구재단,연구사업명은 "신진연구자지원사업 ",연구과제명은 "다제내성병원균감염의초민감성진단법개발",주관기관은한국과학기술원, 연수기간은 2015.07.01-2018.06.30이다.
[4] 본특허출원은 2017년 6월 14일에대한민국특허청에제출된대한민국
특허출원제 10-2017-0075053호에대하여우선권을주장하며,상기특허출원의 개시사항은본명세서에참조로서삽입된다.
[5] 본발명은비바이러스성유전자편집 CRISPR나노복합체,그의제조방법에 관한것으로,보다상세하게는 CRISPR유전자편집시스템의효소단백질에 캐리어물질을화학적으로컨쥬게이션하고 sgRNA와형성한나노복합체,그의 제조방법및상기나노복합체를세포내에전달하여비바이러스성유전자편집 시스템으로사용하는방법에관한것이다.
배경기술
[6] 과거수십년동안항생물질의남용은매우증가되어왔으며,그결과다제내성 박테리아의출현및전파가증가되었다.많은경우에이박테리아들은심각한 병원성을획득하고사람들을감염시키며,다른개체,공동체,보건기관,및 병원으로전파될수있다.이들병원체들의대다수는인간의공생
박테리아 (commensal bacteria)에서발생하며 ,면역이억제되거나특정한의학적 조건에처한개체에기회감염을일으킨다.항생제치료가지속될경우항생제 저항성을지닌변미박테리아들의클론들이자연선택되고,이러한항생제 저항성은약제의효소적분해또는약효의억제에수반되는유전자의내재적
대제용지 (규칙제 26조) RO/KR 발현또는수평적전파에의하여획득된다.
전세계적으로가장높은출현율을보이는다제내성박테리아의종류로는
MRSA(methicillin-resistant Staphylococcus aureus), CRE(carbapenem-resistant Enterobacteriaceae) , MRAB (multidrug-resistant Acinetobacter baumannii),
MRPA(multidrug-resistant Pseudomonas aeruginosa), VRE(vancomycin-resistant Enterococci)등이있으며가장최근에는 VRSA(vancomycin-resistant
Staphlyococcus aureus)가줄현하기도하였다.
다제내성박테리아의전파는세균감염증의치료에있어서치료제선택에 제한을가져오며,더강력한약제의사용및개발을필요로한다.그러나,훨씬더 강력한약제를사용하더라도더병원성이강하고더저항성이강한균주만이 생겨날뿐이며,환자치료시더높은독성을야기할수있다.
또다른문제는현재세균감염증치료에사용되는대부분의약제들은광범위한 스펙트럼을가진저분자항생제라는점이다.따라서,특정한병원체를
특이적으로타겟팅할수있는약제의사용은박테리아의성장에있어서선택적 압력 (selective pressure)을최소화하는데홀륭한이점을제공한다.불행히도, 좁은스펙트럼을가진약제또는항체치료제들은시장성의문제와특이적 바이오마커의부재및저항성의획득과같은기술적인문제들로인하여개발에 난항을겪고있다.
유전자치료제는타겟에높은특이성을가진약제를설계함에있어
간편하면서도다양한용도로사용될수있기때문에,종래의저분자약제또는 항체치료제보다혁신적인접근방법으로소개되어왔다.플라스미드 DNA, 안티센스올리고뉴클레오타이드, siRNA,또는바이러스기반백터와같은 형태의유전적약제들은질병의타겟의발현을유도하거나또는억제하기 위하여투여될수있다.바이러스백터들은높은트랜스펙션효율로인하여 유용하지만,또한세포성면역반웅의유도나항체중화와같은문제들로인한 임상적한계를나타내기도한다.
포유류세포를타겟으로하는경우,특정유전자의발현을억제 (silence)시키기 위한 siRNA의사용은치료제로유망한것으로보이며,현재다양한종류의암, 녹내장,혈우병,및가족성아밀로이드성질환등의치료를위한임상시험이 진행중이다.그러나,이러한유전자치료제를직접투여하는경우에는,체액 내에서의즉각적인효소적분해또는표적부위로의낮은전달효율로인해 효능이떨어지게된다.따라서,양이온성고분자,지질 -기반물질,무기나노입자, 세포투과성펩타이 및덴드리머와같은캐리어물질들은생물학적활성이 있는분자를집약시키고,타겟으로전달하는데사용되곤하였다.그러나, 박테리아세포에서는,유전자치료제의낮은효능뿐만아니라유전자가 세포벽을통과하는전달효율이무척떨어지기때문에비바이러스성유전자 전달전략을사용하고자하는시도가심각하게제한된다.
최근새롭게진보된유전자편집기술은모델유기체의유전자조작뿐만
대제용지 (규칙제 26조) RO/KR 아니라유전자치료제의개발에새로운시대를열었다.현재유전자편집기술은 특정유전자를인식하거나변이시키는방식에따라 ZFN(zinc finger nuclease), TALEN(transcription activator-like effector nuclease), CRISPR(clustered regularly interspaced short palindromic repeat)등이있다.이중미생물의획득면역체계로 알려진 CRISPR시스템은계통학적으로 1형, 2형, 3형으로나누어지며,이중 2형인화농성연쇄상구균 pyogens)유래의 Cas9단백질 (SpCas9)은 유전자편집에있어상보적안결합을통해특정유전자를표적으로하는단일 가이드 RNA (sgRNA: single guide RNA)의도움으로 DNA에서이중가닥절단을 일으키는작용을한다.특히, sgRNA의표적특이적 crRNA는 5'-NGG-3'의 서열을지니는 PAM (protospacer-adjacent-motif)염기서열을인식하여높은표적 효율을선보이며,그길이가 20뉴클레오티드정도로초창기유전자가위인 ZFN (zinc finger nuclease)또는 TALEN (transcription activator-like effector nuclease)에 비하여매우짧고,다른통상적인유전자편집법에비해단순화된설계및 제작으로큰이점을제공한다.이렇게 CRISPR시스템은표적유전자내의 특이적절단을유도함으로써동물모델제작에널리활용되고있을뿐만아니라, 표적유전자를억제또는편집하는기능을이용하여치료제로적용하고자하는 연구가지속적으로시도되고있다.하지만현재까지 CRISPR는높은형질발현 효율을지니는바이러스성편집이가장많이이용되고있으며 ,백터의독성, 세포성면역반웅유도및항체중화반웅등의문제로인해임상적적용에있어 한계를가진다.
[13] CRISPR제한효소단백질및 sgRNA의비바이러스성세포내전달은
바이러스성백터를이용하는경우에비해그효율이낮다고보고되고있으며, 이를극복하는것이큰관건이다.특히,일반적으로단백질기반의전달은생체 내즉각적인효소에의한분해또는표적부위에대한낮은전달효율로인해 효능이떨어지게된다.
[14] 따라서 CRISPR를이용한치료에있어안전성과간단한합성단계및높은전달 효율등을장점으로갖는비바이러스성유전자편집의필요성이대두되고 있으며,치료제로의도입을위해,유전자 /약물전달용재료로양이은성고분자, 지질기반물질 (lipofectamine둥 lipid계열소재캐리어 ),무기나노입자,세포 침투성펩티드및덴드리머 (dendrimer)와같은운반체물질이보고되었다.
그러나이들재료는양이은성혹은지용성을지녀생체활성분자를웅집시킬수 있다는한계가있어,비바이러스성 CRISPR시스템의전달을위하여 Cas 단백질과 sgRNA의표적으로의전달및작동효율을증진시키고자하는 노력들이많이보고되었다.상기지질계열소재를이용하여 Cas단백질및 sgRNA를물리적,비공유결합으로봉입시킬경우,낮은봉입효율로인해 실질적인적용이제한되고이로인한높은투여량으로독성문제가야기된다.
[15] 따라서,독성이없으면서도효율적인세포내전달이가능한새로운 CRISPR 전달방법의개발이절실히필요한실정이다.
대제용지 (규칙제 26조) RO/KR 발명의상세한설명
기술적과제
[16] 본발명자들은기존의바이러스성유전자편집방법이나지질기반물질제형의 유전자편집방법의낮은전달효과,독성문제를극복한새로운형태의
비바이러스성유전자편집방법을개발하고자예의연구노력하였다.그결과, CRISPR효소단백질을고분자캐리어물질로접합하고,이를 sgRNA와혼합하여 CRISPR나노복합체를제조하는경우,유전자편집효소의전달효율및유전자 편집효과가우수하고생체독성등의문제가해결될수있음을확인하고,본 발명을완성하였다.
[17] 따라서,본발명의목적은고분자캐리어물질-접합 CRISPR(Clustered regularly interspaced short palindromic repeats)효소단백질을제공하는것이다 .
[18] 본발명의다른목적은상기고분자캐리어물질—접합 CRISPR효소단백질의 제조방법을제공하는것이다.
[19] 본발명의또다른목적은고분자캐리어물질-접합 CRISPR효소단백질및 sgRNA(single guide RNA)를포함하는 CRISPR나노복합체를제공하는것이다.
[2이 본발명의또다른목적은고분자캐리어물질-접합 CRISPR효소단백질및 sgRNA를흔합하는단계를포함하는 CRISPR나노복합체의제조방법을 제공하는것이다.
[21] 본발명의또다른목적은고분자캐리어물질 -접합 CRISPR효소단백질,또는 상기 CRISPR나노복합체를포함하는유전자편집용조성물을제공하는것이다. 과제해결수단
[22] 본발명의일양태에따르면,본발명은고분자캐리어물질-접합
CRISPR(Clustered regularly interspaced short palindromic repeats)효소단백질을 제공한다.
[23] CRISPR는 Clustered regularly interspaced short palindromic repeats의약자로, 박테리아의면역체계에서유래한 3세대유전자가위를말한다.박테리아는 바이러스에감염됐을때대부분죽지만일부는살아남으면서그바이러스 DNA의일부를자신의유전체에저장해놓는다.그후재감염이일어나면저장해 놓은정보를바탕으로작은가이드 RNA(sgRNA: small guide RNA)를만들어낸 후 Cas9이라는엔도뉴클레아제와함께결합하여외부 DNA를자른다.이때 가이드 RNA는원하는표적유전자에상보적염기쌍을이루어결합하여 특이성을결정하고 Cas9단백질은표적유전자를자르는
핵산가수분해효소로서의역할을수행한다.따라서상기가이드 RNA
염기서열에따라어떠한 DNA염기서열도자를수있기때문에최근
CRISPR-Cas9유전자가위를이용한활발한연구가진행되고있다.
[24] 본발명의일구현예에따르면,상기 CRISPR효소단백질은 CRISPR
유전자편집과정에의^유전체상의표적부위를절단또는편집할수있는효소
대제용지 (규칙제 26조) RO/KR 단백질로, Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas9, Csel, Cse2, Cse3, Cse4, Cas5d, Cas5e, Csyl, Csy2, Csy3, Csy4, Cpfl, Csnl, Csn2, Csdl, Csd2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, Csml, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, dCas9 (dead Cas9), Cas9 nickase, C2c2 (Casl 3a), CDA(cytidine deaminase enzyme), APOBEC(apolipoprotein B editing complex) 14, UGI(uracil glycosylase inhibitor),및 AID(activation-induced
deaminase)로이루어진군으로부터선택된단백질,또는이들의재조합단백질 중에서선택된어느하나를사용할수있으나,이에한정되는것은아니다.
[25] 본명세서에서용어 "캐리어 (carrier)", "전달체"또는 "운반체"는단백질,
호르몬,효소,핵산둥과같이생리활성을나타내거나생체내에서활성을 가지고작용할수있는물질을세포막등의생체막을통과시켜세포내로 도입시키기위하여사용되는고분자,단백질,지질등을의미한다.본발명의 목적에따라상기캐리어는세포독성이나조직독성이없는생체적합성고분자 물질로서,합성중합체또는천연중합체이다.
[26] 본명세서에서용어 "카고 (cargo)"는상기캐리어와결합또는접합되어세포 내로도입시키고자하는단백질,호르몬,핵산둥의생리활성물질과효소와 같이생체내에서활성을가지고작용할수있는물질을의미한다.본발명의 목적에따르면본명세서에서용어 "카고 "는유전자가위효소단백질,보다 구체적으로는 CRISPR효소단백질,그리고단일가이드 RNA(sgRNA)를 의미하나,이에한정되는것은아니다.
[27] 또한,본발명의다른구현예에따르면,상기고분자캐리어물질로는가지형 폴리에틸렌이민,선형폴리에틸렌이민,폴리프로필렌이민,폴리아미도아민, 폴리에틸렌글리콜,폴리에틸렌옥사이드-폴리프로필렌옥사이드공중합체, 폴리-락트산,폴리 -글리콜산,폴리 -D,L-락트산 -co-글리콜산,폴리카프로락톤, 폴리포스포에스터 ,폴리포스파진,폴리베타아미노에스터,가지형
폴리아미노에스터,폴리아미노부틸 -글리콜산,폴리오르소에스터 ,
폴리하이드록시프를린에스터,폴리아크릴아마이드,폴리비닐피를리돈, 폴리비닐알코올, poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA), 덴드리머,히알루론산,알긴산,키토산,덱스트란,사이클로덱스트린,스퍼민, 폴리 -Arginine,및폴리 -Lysine,이들의공중합체또는흔합물로이루어진 군으로부터선택될수있으나,이에한정되는것은아니다.
[28] 본발명의일실시예에따르면,본발명은상기고분자캐리어물질로가지형 폴리에틸렌이민을사용하였다.
[29] 상기고분자캐리어물질은 CRISPR효소단백질과접합 (conjugation)되어
박테리아를비롯한원핵세포및진핵세포에대하여 CRISPR효소단백질의표적 세포내전달효율을증진시킨다.
[30] 기존에 CRISPR효소단백질의세포내전달을위해사용되는방법으로는
바이러스성백터를이용한방법과리포펙타민둥비바이러스성캐리어 (carrier,
대제용지 (규칙제 26조) RO/KR 전달체)물질을이용하는방법이있으나,바이러스성백터의경우에는비특이적 반웅이많이일어나고,리포펙타민등지질기반의비바이러스성캐리어물질을 이용하는경우에는진핵세포에서는전달효율이좋으나,박테리아둥
원핵생물에대해서는전달효율이매우불량하여사용이곤란하다는문제가 있었다.
[31] 본발명의상기캐리어물질로접합된 CRISPR효소단백질은수용액상에서
DNA절단또는편집작용기능을가진다.
[32] 본발명의일구현예에따르면,상기고분자캐리어물질과 CRISPR효소
단백질은 SMCC(succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate), sulfo-SMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate),
AMAS(N-a-maleimidoacet-oxysuccinimide ester),
BMPS (N- β-maleimidopropyl-oxy succinimide ester),
G B S (Ν-γ-maleimidobutyryl-oxy succinimide ester),
MB S (m-maleimidobenzoyl-N-hydroxysuccinimide ester) ,
EMCS(N-s-malemidocaproyl-oxysuccinimide ester), SM(PEG) (PEGylated SMCC), SPDP(succinimidyl 3-(2-pyridyldithio)propionate), PEG-SPDP(PEGylated SPDP), DSG(disuccinimidyl glutarate), DCC(Dicyclohexylcarbodiimide), DSS(disuccinimidyl suberate), BS3(Bissulfosuccinimidyl suberate), DSP(dithiobis(succinimidyl propionate)), EGS (ethylene glycol bis(succinimidyl succinate)), DMP(dimethyl pimelimidate), BMOE(bismaleimidoethane), BMB ( 1 ,4-bismaleimidobutane),
DTME(dithiobismaleimidoethane),
EDC(l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride),
NHS(N-Hydroxysuccinimide),
프로파길—숙신이미딜-에스테르 (propargyl-succinimidyl-ester),
DBCO-말레이미드 (Dibenzocyclooctyne-maleimide),
DBCO-PEG-말레이미드 (Dibenzocyclooctyne-PEG4-maleimide), DBCO-S-S-NHS 에스테르 (Dibenzocyclooctyne-S-S-N-hydroxysuccinimidyl ester), DBCO-NHS 에스테르 (dibenzocyclooctyne-N-hydroxysuccinimidyl ester),아세틸렌 -PEG-NHS 에스쩨르 (acetylene-PEG-NHS ester),및
알킬렌 -PEG-말레이미드 (alkyne-PEG-maleimide)로이루어진군으로부터선택된 가교제로접합 (conjugation)될수있으나,이에한정되는것은아니다.
[33] 상기나열된가교제들은화학적가교제들로,가교제의반웅기와접합시키고자 하는단백질의활성기간의결합반웅을유도하여상기고분자캐리어물질과본 발명의 CRISPR효소단백질을접합할수있다.
[34] 상기한화학적가교제들의반웅기와타겟활성기를예를들어설명하면,하기 표 1과같으나,반드시이에한정되는것은아니다.
대제용지 (규칙제 26조) RO/KR [35] [표 1]
Figure imgf000009_0001
[36] 본발명의일실시예에서는화학적가교제로 sulfo-SMCC를사용하였으며, 가지형폴리에틸렌이민 (bPEI)에 sulfo-SMCC를첨가하여가지형
폴리에틸렌이민의아민기를말레이미드기로치환하여활성화하였으며,이어서 활성화된폴리에틸렌이민의 1차아민기과 SpCas9단백질의시스테인잔기상의 자유설프하이드릴기를반웅시킴으로써 bPEI와접합된 SpCas9(SpCas9-bPEI)를 제작하였으며,상기고분자캐리어물질과 CRISPR효소단백질 (예컨대
SpCas9-bPEI)의접합반웅은하기실시예에서성공적으로이루어진것으로 입증되었다.
[37] 본발명에서상기폴리에틸렌이민은유전자전달 (예: siRNA,플라스미드
DNA)에가장널리사용되는캐리어물질중하나이며다양한분자량의가지형 또는선형으로사용할수있기때문에카고 (cargo)의개조를위해선택되었다. 또한,가지형은선형아민류에비해특히일차아민을포함하는아민기가 풍부하여폐키징및전달이효율적으로이루어질수있기때문에선택되었다. 물리적인캡술화대신,단백질의직접공유결합변형을선택하여투여를위한 캐리어물질의양을최소화하여생체독성및불층분한방출로인한효능감소 문제를해결하고자하였다.상기 bPEI는 Cas9엔도뉴클레아제에
공유결합적으로도입되어,정전기상호작용에의한패키징을유도함으로써 sgRNA뿐만아니라,단백질자체의세균내전달을향상시켰다.
[38]
[39] 본발명의다른일양태에따르면,본발명은다음단계를포함하는상기고분자 캐리어물질 -접합 CRISPR효소단백질의제조방법을제공한다:
대제용지 (규직제 26조) RO/KR [40] (a)고분자캐리어물질의기농기를양기능성가교제 (Afunctional crosslinker)로 반웅시켜활성화된고분자캐리어물질을제조하는단계;및
[41] (b) CRISPR효소단백질의기능기를상기활성화된고분자캐리어물질과
반웅시켜접합물질을제조하는단계.
[42] 본발명의상기제조방법을단계별로상세히설명한다.
[43] 단계 (a):고분자캐리어물짐의기능기를양기능성가교제 rbifunctional
crosslinkerᅵ로반음시켜활성화뒤고분자키ᅵ리어 ^짐을체조하는단계
[44] 상기한고분자캐리어물질의기능기를양기능성화학적가교제로반웅시켜 활성화시킨다.상기고분자캐리어물질의기능기를활성화시켜야만다른 분자와연결이가능해진다.
[45] 화학적가교제란,서로다른종류의두개이상의분자를화학적으로결합하는 시약을말하며 ,상기양기능성이란두개의같거나서로다른기능기를가진 분자간의연결을유도하는성질을의미한다.
[46] 상기고분자캐리어물질의활성화반옹은 DMSO(dimethylsulfoxide),
DMF(dimethylformamide),에탄올,메탄올,물,메틸렌클로라이드,및
클로로포름으로이루어진군으로부터선택된용매중에서이루어질수있으나, 이에한정되는것은아니며당업계에서분자간접합반웅을위한활성화반응에 사용가능한모든용매를제한없이사용할수있다.
[47] 상기고분자캐리어물질의활성화반웅은 4~60°C,구체적으로는 4~50oC,
4~40°C, 4~30°C,또는 4~25°C의온도조건하에서수행될수있으나,이에 한정되는것은아니다.
[48] 또한,상기고분자캐리어물질의활성화반웅은 0.5~24시간,구체적으로는
0.5-12시간, 0.5~6시간, 1~24시간, 1~12시간, 1~6시간동안수행될수있으나, 이에한정되는것은아니다.
[49] 상기단계에서고분자캐리어물질의종류는고분자캐리어물질-접합 CRISPR 효소단백질과관련하여상술한것과중복되므로그기재를생략한다.
[50]
[51] 단계 0?ᅵ: CRISPR효소단백짐의기능기를상기활성화되고분자캐리어
물짐과반용시켜접합물짐음제조하는다계
[52] 상기단계 (a)에서활성화된고분자캐리어물질의기능기를 CRISPR효소
단백질의기능기와반웅시켜접합물질을제조한다.
[53] 이때상기 (b)단계의고분자캐리어물질및 CRISPR효소단백질의몰비는 예컨대 1:10-6내지 1: 106, 1: 105내지 1: 105, 1 : 1( 내지 1 : 104, 1:10-3내지 1:103, 1:10-2 내지 1:102, 1:1ο-1내지 I : )1일수있으나,이에한정되는것은아니다.
[54] 또한,상기고분자캐리어물질과 CRISPR효소단백질간의접합반웅은
4~60°C에서,보다구체적으로는 4~50oC, 4~40°C, 4~30°C,또는 4~250C의온도 조건하에서수행될수있으나,이에한정되는것은아니다.
[55] 상기상기고분자캐리어물질과 CRISPR효소단백질간의접합반웅은
대제용지 (규칙제 26조) RO/KR 0.5~48시간,구체적으로는 0.5-36시간, 0.5~24시간, 0.5-12시간,보다
구체적으로는 1~48시간, 1~24시간, 1~12시간,또는 4~12시간동안수행될수 있으나,이에한정되는것은아니다.
[56] 이때상기고분자캐리어물질과 CRISPR효소단백질간의접합반웅은 pH
4~10의수용성용매증에서이루어질수있으며,상기 pH범위를유지하는 수용성완충액이라면제한없이사용될수있다.
[57] 또한상기 CRISPR효소단백질의종류는본발명의고분자캐리어물질 -접합
CRISPR효소단백질과관련하여상술한것과중복되므로그기재를생략한다.
[58] 본발명의또다른일양태에따르면,본발명은고분자캐리어물질 -접합
CRISPR효소단백질및 sgRNA(single guide RNA)를포함하는 CRISPR 나노복합체를제공한다.
[59] 본발명에서상기 CRISPR나노복합체라함은캐리어물질-접합 CRISPR효소 단백질과표적유전자에대한 sgRNA를흔합한제형으로,상술한캐리어 물질-접합 CRISPR효소단백질과 sgRNA의물리적인상호작용으로형성된 복합물을의미한다.
[60] 보다구체적으로상기 CRISPR나노복합체라함은바이러스나 DNA백터가 아닌,캐리어물질접합 CRISPR효소단백질및 sgRNA로구성된복합물을 의미하며따라서비바이러스계나노복합체라는점에특징이있다.
[61] 본발명의일구현예에따르면,상기 CRISPR나노복합체는수용액상에서 분산이가능한복합체로서 ,입도크기가 1내지 10,000 nm일수있으나,이에 한정되는것은아니다.
[62] 본발명의다른일구현예에따르면,상기 CRISPR나노복합체는 -100 ~ +100 mV의제타전위를가진다.
[63] 본발명의상기 sgRNA는 CRISPR효소단백질을유전체상의표적유전자의 특이적부위에안내하는리보핵산이며, PAM (protospacer-adjacent motif)서열과 근접한표적부위인프로토스페이서 (protospacer)에대한 CrRNA(CRISPR RNA) 및 TracrRNA(trans-activating RNA)서열을포함하는것을특징으로한다.
[64] 상기 sgRNA의표적유전자로는발명자가원하는표적유전자를어느것이든 선택할수있으며,예컨대 mecA, mecR 1, aph, NDM- 1, KPC, oxa, ure, lrg, cap, spl, KPC, GES, IMP, VIM, KRAS, Stkl l, TP53, PTEN, BRCA1, BRCA2, Akt, Stat, Stat4, JAK3JAK2, WT1, ERBB2, ERBB3, ERBB4, NFl, NOTCH 1, NOTCH3, ATM, ATR, HIF, HIFl a, HIF3 a, Met, Bcl2, FGFRl, FGFR2, CDKN2 , APC, RB, MEN1, PPAR a, PPAR γ, AR, TSG101, IGF, Igfl, Igf2, Bax, Bcl2, caspase, Kras, Ape, NFl, MTOR, Grml, Grm5, Grm7, Grm8, mGlurRl, mGlurR5, mGlurR8,PLC, AMPK, MAPK, Raf, ERK, TLR4, BRAF, PI3K,F8, F8C, F9, HEMB, KIR3DL1, NKAT3, NKB1, AMB1I, KIR3DS1, IFNG, CXCLI2, IL2RG, SCIDX1, SCIDX, IMD4, CCR5, SCYA5, D17S136E, TCP228, CXCR2, CXCR3, CXCR4, CCR4, CCR6, CCR7, CX3CR1, CD4, CFTR, HBB, HBA2, HBD, HBAl, LCRB, SCNIA,
대제용지 (규칙제 26조) RO/KR CHD8, FMR1, VEGF, EGFR, myc, Bcl-2, survivin, SOX2, Nrgl, Erb4, Cplxl, Tphl, Tph2, GSK3, GSK3a, GSK3 β, El, UBB, PICALM, PSI, SORL1, CR1, Ubal, Uba3, CHIP, UCH, Tau, LRP, CH1P28, Uchll, Uchl3, APP, Cx3crl, ptpn22, TNF-a, NOD2, IL-la, IL-lb,IL-6, IL-10, IL-12, EL-la, IL-lb IL-13, IL-17a, IL-17b, IL-17c, IL-17d, IL-17f, CT1LA4, Cx3cllJ, DM, PINK1, Prp, DJ-1, PINK1, LRRK2, ALAS2, ASB, ANHl, ABCB7,ABC7, ASAT, CDANl, CDAl, DBA, PKLR, PKl, RIPS 19, NT5C3, UMPH1, PSN1, RHAG,RH50A, NRAMP2, SPTB, TBXA2R, P2X1P, 2RX1, HF1, CFH, HUS, MCFD2, Drd2, Drd4, ABAT, BCL7A, BCL7,TALI, TCL5, TAL2, FLT3,NBS1, NBS, ZNEN1A1, TYR, ALDH, ALDH1A1, ADH, FASN, PI, ATT, F5, MDC1C, LAMA2, LAMM, LARGE, MDC1D, FC D, TTID, MYOT, CAPN3, CANP3, DYSF, SGCG, D DA1, SCG3, SGCA,ADL, DAG2, DMDA2, SGCB, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2G, LGMD2H, LGMD2I, SGCD, SGD, CMDIL, TCAP, CMDIN, TRIM32, HT2A, FKRP, POMTl, CAV3, LGMD1CSEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, PPP2R1A,
PPP2CA, PPP1CC, PPP2R5C, GFP, RFP, YFP, tdTomato, mCherry, luciferase등을 표적유전자로할수있으나,이는예시적인것일뿐이에한정되는것이아님은 당업자에게자명하다.
본발명의또다른일양태에따르면,본발명은상술한 CRISPR나노복합체의 제조방법을제공한다.
상기 CRISPR나노복합체의제조방법은상술한고분자캐리어물질 -접합 CRISPR효소단백질과표적유전자에관한 sgRNA를흔합하는단계를포함한다. 본발명의일구현예에따르면,상기 CRISPR나노복합체의제조시캐리어 물질 -접합 CRISPR효소단백질과 sgRNA의흔합몰비는 1:10·6내지 1:106, 구체적으로는 1:10-5내지 1:105, 1:10-4내지 1:104,가장구체적으로는 1:10-3내지 1:103이나,이에한정되는것은아니다.
또한,상기흔합온도는 4~370C,구체적으로는 4~25°C, 15~37°C, 15~25°C일수 있으나반드시이에한정되는것은아니다.
상기흔합반웅시간은 0.1-12시간,구체적으로는으1~6시간, 0.1~3시간, 0.1~1시간,가장구체적으로는 0.1 0.5시간동안수행되어나노복합체를제조할 수있으나,이에한정되는것은아니다.
본발명의또다른일양태에따르면,본발명은상술한고분자캐리어
물질-접합 CRISPR효소단백질,또는상술한 CRISPR나노복합체를포함하는 유전자편집용조성물을제공한다.
상기유전자편집용조성물에포함되는상기고분자캐리어물질-접합 CRISPR 효소단백질과상술한 CRISPR나노복합체는본발명의다른양태에서설명한 발명에해당한다.
본발명의조성물이약제학적조성물인경우,약제학적으로허용되는담체가 포함된다.상기약제학적으로허용되는담체는제제시에통상적으로이용되는
대제용지 (규칙제 26조) RO/KR 것으로서,락토스,덱스트로스,수크로스,솔비를,만니를,전분,아카시아고무, 인산칼슴,알기네이트,젤라틴,규산칼슴,미세결정성셀를로스,
폴리비닐피를리돈,셀를로스,물,시럽,메틸셀를로스,
메틸히드록시벤조에이트,프로필히드록시벤조에이트,활석,스테아르산 마그네슴및미네랄오일등을포함하나,이에한정되는것은아니다.본발명의 조성물은상기성분들이외에윤활제,습윤제,감미제 ,향미제 ,유화제,현탁제, 보존제등을추가로포함할수있다.
[73] 본발명의약제학적조성물은경구또는비경구로투여할수있고,예컨대정맥 내주입,피하주입,근육주입,복강주입,국소투여,비강내투여,폐내투여, 직장내투여,경막내투여,안구투여,피부투여및경피투여등으로투여할수 있다.
[74] 본발명의조성물의적합한투여량은제제화방법,투여방식,환자의연령, 체중,성,질병증상의정도,음식,투여시간,투여경로,배설속도및반웅 감웅성과같은요인들에의해다양하며,보통으로숙련된의사는목적하는 치료에효과적인투여량을용이하게결정및처방할수있다.
[75] 본발명의구체적인구현예에따르면,본발명의약제학적조성물의 1일
투여량은 0.0001-1000 mg/kg이나,이에한정되지않는다.본명세서에서용어
"약제학적유효량 "은원하는유전자의편집을유도하는데층분한양을
의미한다ᅳ
[76] 본발명의조성물은본발명이속하는기술분야에서통상의지식을가진자가 용이하게실시할수있는방법에따라,약제학적으로허용되는담체및 /또는 부형제를이용하여제제화됨으로써단위용량형태로제조되거나,또는다용량 용기내에내입시켜제조될수있다.이때제형은오일또는수성매질중의용액, 현탁액또는유화액형태일수있으며,분산제또는안정화제를추가적으로 포함할수있다.
[77] 본발명의일구현예에따르면,상기조성물은분리된세포,조직,또는개체에 투여되는경우,상술한고분자캐리어물질-접합 CRISPR효소단백질,또는 CRISPR나노복합체를세포내에전달하여 CRISPR효소단백질의유전자편집 작용에의하여유전자편집을유도한다.
[78] 이때상기세포내부로전달이가능한세포로는 HeLa, A549, MDAMB,
SK-BR-3, OVCAR, PC3, PC 12, HEK293, Jurkat, CD4+ T세포, CD8+ Τ세포, RAW264.7,대식세포,단핵구,호증구, ΝΚ세포 (natural killer cell),
수지상세포 (dendritic cell), MDSC(Myeloid-derived Suppressor Cell),배아줄기세포, 중간엽줄기세포,유도만능줄기세포 (iPSC),혈관내피세포,표피세포,간세포, 근육세포,뼈세포,섬유아세포,연골세포,신경세포,신경줄기세포 (neural stem cell), ADSC(adipose-derived stem cell), EF(mouse embryonic fibroblast),곱광이 세포,및기생충세포등의진핵세포이거나,또는 S. aureus, E. coli, P. aeruginosa, K. pneumoniae, A. baumannii, B. subtilis, S. epidermidis, E. faecalis, S. pneumoniae
대제용지 (규칙제 26조) RO/KR 등의원핵세포이다.
[79] 본발명의일실시예에서입증한바와같이,본발명의상기고분자캐리어 물질—접합 CRISPR효소단백질과, CRISPR나노복합체는박테리아의세포 내부에유입되어유전자편집효과를나타내었고,포유류세포에서도높은전달 효율을보였다.
[80] 본발명의고분자캐리어물질 -접합 CRISPR효소단백질과, CRISPR
나노복합체는기존의리포펙타민등의지질기반캐리어물질과비교하여세포 내전달효율이우수하였으며,이에따라표적유전자의편집효과도우수하게 나타났다.
[81] 상술한바와같이,본발명자들은 CRISPR에의한유전체편집을박테리아및 포유류세포를포함하는세포내에높은효율로전달할수있는새로운 전달방법을개발하고자예의연구노력하였으며,직접공유결합변형에의한 고분자유도체화 CRISPR Cas9단백질및이를이용한나노크기의복합체를 형성하였으며 ,이를다제내성박테리아를표적으로하는특이적살상용도로 사용할수있음을확인하였다.
[82] 기존에플라스미드기반 CRISPR게놈편집이탈출돌연변이 (escape mutant)를 일으킨다는보고가있었으나,이들클론의목표부위 (target locus)에서는 유전체적변화가일어나지않았으며,전달백터의돌연변이에의해서유전체 편집기능에장애가일어나박테리아의치사율이낮게나타나는어려움이 있었다.
[83] 본발명은이전에보고된지질기반의비공유적제형과비교하여, Cas9와
고분자의직접적인컨쥬게이션을유도하여 Cas9단백질의각각의단일분자가 캐리어물질에결합되는것을허용하므로이전의비공유적제형의경우에서 발견된로딩효율문제가해결되었으며,하나또는두분자의고분자캐리어 물질 (bPEI)만이각 Cas9분자에컨쥬게이션되었기때문에,최소량의캐리어 물질 (단백질의 2중량 %이하)만을사용하는것이가능하여독성이나부작용을 최소화하고더많은용량을투여할수있다는장점이있으며,기존의플라스미드 기반제형과비교하여더높은전달효율및특이성을가진다는장점이있다. 발명의효과
[84] 본발명에의한비바이러스성유전자편집 CRISPR나노복합체는수
나노미터~수마이크론단위의크기이고,외부의물리적인자극없이세포내 전달이가능하고,세포의표적유전자에대해비바이러스성경로에의한 유전자편집에유용하게활용할수있다.결과적으로상기 CRISPR나노복합체를 동물모델제작,미생물공학,질병치료를위한세포공학또는생체투여제형에 이용할경우,높은세포내전달및유전자편집효율을보이고,비특이적편집, 유전자변이,세포및생체독성유발등의문제를최소화할수있다.
도면의간단한설명
대제용지 (규칙제 26조) RO/KR [85] 도 la내지도 le는클로닝된본발명의 Streptococcus pyogenes로부터얻은 재조합 Cas9엔도뉴클레아제 (SpCas9)의뉴클레오타이드서열을나타낸도이다. 상기서열은 N에서 C말단까지 6x His, FLAG, NLS(nuclear localization sequence),
SpCas9및 GFPuv(green fluorescent protein)을포함하며,소문자로표시된서열은
GFP영역을나타낸다.
[86] 도 2는본발명의발현및정제된 SpCas9단백질의 SDS-PAGE결과를나타낸 도이다. SpCas9의크기는약 190kDa로나타났다.
[87] 도 3은본발명의 SpCas9단백질과융합된 GFP의형광은 UV일루미네이터 하에서관찰한결과를나타낸도이다.
[88] 도 4a내지도 4c는 mecA유전자를표적으로하는단일가이드 RNA(sgRNA) 서열 (1내지 3)을나타낸도이다.
[89] 도 5a및도 5b는 mecA유전자를표적으로하는 sgRNA서열을제작하기위한 mecA유전자주형의뉴클레오타이드서열을나타낸도이다.
[90] 도 6은 mecA유전자주형의증폭을위한프라이머서열을나타낸도이다.
[91] 도 7은 sgRNA합성을위한주형의뉴클레오타이드서열을나타낸도이다.
[92] 도 8은 sgRNA주형의합성을위한프라이머서열을나타낸도이다.
[93] 도 9는합성된 sgRNA주형을전기영동으로확인한결과를나타낸도이다.
[94] 도 10은본발명에서합성한 3종의 sgRNA를전기영동으로확인한결과를
나타낸도이다.
[95] 도 11은본발명의 SpCas9과 bPEI간의컨쥬게아션과정에관한모식도를
나타낸도이다.
[96] 도 12는 SpCas9에대한 bPEI의성공적컨쥬게이션을확인하기위한겔지연 분석결과를나타낸도이다.
[97] 도 13은 SpCas9에대한 bPEI의성공적컨쥬게이션을확인하기위한 SDS-PAGE 분석결과를나타낸도이다. '
[98] 도 14는본발명의 SpCas9-bPEI와 sgRNA간의복합체 (Cr-나노복합체)및
대조군인개질되지않은 SpCas9단백질과 sgRNA간의복합체 (천연복합체) 제조에관한모식도를나타낸도이다.
[99] 도 15는본발명의 Cr-나노복합체및천연복합체의입도크기를비교하여
나타낸도이다.
[100] 도 16은합성된표적 DNA를 Cr-나노복합체와함께첨가하여절단을유도한 후의겔전기영동결과를나타낸다.
[101] 도 17은본발명의고분자유도체화된 SpCas9의세균내전달결과를공초점 현미경으로관찰한결과를나타낸도이다.
[102] 도 18은본발명의고분자유도체화된 SpCas9의세균내전달효율을대조군과 비교하여나타낸도이다.
[103] 도 19내지도 20및도 21a내지도 21c는고분자유도체화된 SpCas9의세균 내로의유입을확인하기위하여공초점이미지단면을 3D이미지로재구성하여
대제용지 (규칙제 26조) RO/KR SpCas9과핵염색의형광신호의중첩으로부터세균세포내의 SpCas9의존재를 나타낸도이다.
도 22는다른분자량 (분자량 25,000)의고분자캐리어를이용하여고분자 유도체화된 SpCas9의세균내로의유입을확인하기위하여 SpCas9과핵염색의 형광신호를증첩하여나타낸도이다.
도 23는 A549세포에서의 Cr-나노복합체유입효율을다초점형광현미경으로 관찰분석하여나타낸도이다.
도 24는 HaCat세포에서의 Cr-나노복합체유입효율을다초점형광현미경으로 관찰분석하여나타낸도이다.
도 25는 Cr-나노복합체의 HaCat세포내로의유입을확인하기위하여공초점 이미지단면을 3D이미지로재구성하여복합체와핵염색의형광신호의 중첩으로부터동물세포내복합체의존재를나타낸도이다.
도 26는 Raw 264.7세포에서의 Cr-나노복합체유입효율을
다초점형광현미경으로관찰분석하여나타낸도이다.
도 27는 Jurkat세포에서의 Cr-나노복합체유입효율을다초점형광현미경으로 관찰분석하여나타낸도이다.
도 28은신경줄기세포 (neural stem cell)에서의 Cr-나노복합체유입효율을 다초점형광현미경으로관찰분석하여나타낸도이다.
도 29는유도만능줄기세포 (iPSC, induce pluripotent stem cell)에서의
Cr-나노복합체유입효율을다초점형광현미경으로관찰분석하여나타낸 도이다.
도 30은본발명의 Cr-나노복합체의유전체편집효율성평가를위한 실험과정의모식도를나타낸도이다.
도 31는본발명의 Cr-나노복합체로처리한세균의현탁배양후 OD600값을 측정하여성장률을나타낸도이다.
도 32a및도 32b는유전체편집패턴을추가적으로평가하기위하여,세균을
Cr-나노복합체또는대조군으로처리하고 oxacillin의존재또는부존재하에 CFU 수를계산하여나타낸도이다.
도 33은본발명의 Cr-나노복합체를박테리아에처리시상대적성장률을 나타낸도이다.
도 34은본발명의 Cr—나노복합체의농도별로유전체편집효율을비교하여 나타낸도이다.
도 35은본발명의 Cr-나노복합체로처리한박테리아의레플리카배양실험 결과를나타낸도이다.
발명의실시를위한형태
이하,실지예를통하여본발명을더욱상세히설명하고자한다.이들실시예는 오로지본발명을보다구체적으로설명하기위한것으로,본발명의요지에
대제용지 (규칙제 26조) RO/KR ᅳ 따라본발명의범위가이들실시예에의해제한되지않는다는것은당업계에서 통상의지식을가진자에있어서자명할것이다.
[119]
[120] 실시예
[121] 실시예 1.키 I리어물짐접합 CRISPR효소단백질의제조
[122] 1-1. SpCas9단백질의발현및정제
[123] 유전체편집카고 (genome editing cargo)를박테리아내에효율적으로전달하기 위해제작된본발명의 Cr-나노복합체 (CRISPR nanocomplex)시스템은고분자 유도체화된 Cas9단백질과 sgRNA와의복합체를이용하여개발되었다.
[124] 먼저 SpCas9단백질을발현시키기위하여 lentiCRISPR (Addgene)로부터얻은 SpCas9유전자를 pET21 a (Novagen)에클로닝하였다. SpCas9에는
ATGACGATAAGATGGCC-3'및
S'-CCCAAGCTTTTTCTTTTTTGCCTGGCCGGCCTTT-S'프라이머를
사용하였고, GFPuv(green fluorescent protein)에는
5'-CCCAAGCTTATGAGTAAAGGAGAAGAAC-3'및
5'-CCCAAGCTTTTATTTGTAGAGCTCATCCA-3' -프라이머로사용하였다.
[125] 상기 SpCas9는 N에서 C말단까지 6x His, FLAG, NLS(nuclear localization
sequence), SpCas9및 GFPuv(green fluorescent protein)을포함하였다.클로닝된 서열은 DNA시퀀싱으로확인하였다.발현백터를 BL21-(DE3) E. coli competent cell로형질전환한후 LB(Luria-Bertani)브로스 (100 μ^πύ ampicillin포함)에 접종하고 30 0C에서밤새배양 (OD600 0.4 )하였고, 0.5 mM IPTG(isopropyl β-D-l-thiogalactopyranoside)를첨가하여 SpCas9발현을유도하였다.세포를 16 시간동안배양한후 5000 rpm에서 10분간원심분리하여수확하고,라이시스 버퍼 (50 mM NaH2P04, 300 mM NaCl, 10 mM이미다졸, 0.05 % β-머캡토에탄올, ρΗ 8.0)로처리하고 41%의중격계수 (duty)로 2초간필스를주고, 5초간정지하는 과정을반복하며총 30분간얼음위에서수행하였다.
[126] 세포파쇄물은 His태깅된 SpCas9가친화성칼럼에결합되도록 Ni-NTA
아가로스비드 (Qiagen)에서배양및세척하였고, 50 mM NaH2P04, 300 mM NaCl, 200 mM imidazole, 0.05 % β-머캡토에탄올 (ρΗ 8.0)을포함하는완충액으로 용출하였다.
[ 127] 용출된액은저장버퍼 (50 mM Tris HC1, H 8.0, 200 mM KC1, 0.1 mM EDTA, 20%글리세를, 1 mM DTT, and 0.5 mM PMSF)에대해 2시간마다완층액을 교환하면서 12시간동안투석하였고, -70 0C에서보관하였다. Streptococcus pyogenes로부터얻은재조합 Cas9엔도뉴클레아제 (SpCas9)는발현
플라스미드를 E.coli competent cell로형질전환시키고친화성크로마토그래피로 정제함으로써수득하였다.클로닝된 SpCas9의시퀀스는도 1에나타내었다.
[128] 정제된 SpCas9는 SDS-PAGE전기영동에의해분석되었다.본발명의정제된
대제용지 (규칙제 26조) RO/KR SpCas9단백질의 SDS-PAGfi결과를보 2에나타내었다. SpCas9의크기는약 190kDa로확인되었다.
[129] 또한, SpCas9의 GFP형광은 UV일루미네이터하에서관찰함으로써
확인되었다 (도 3).
[130]
[131] 1-2. sgRNA (단일가이드 RNA)의설계및합성
[132] sgRNA의설계
[133] MRSA에서 mecA유전자를표적으로하는 sgRNA(Single guide RNA)는
SpCas9에의해세균유전체에서이중-가닥을파괴하도록설계되었다.세가지 다른 sgRNA서열을 mecA유전자내의다양한표적부위에따라
프로토스페이서 (protospacer)로결정하였다 (도 4).프로토스페이서영역은 PAM (protospacer-adjacent motif)서열 (NGG)에모두인접해있었으며,이로부터 3개의 염기의상류 (upstream)부위에서표적절단 (target cleavage)이일어날것이다.
[134] sgRNA는 mecA를타겟으로하는 CrRNA(CRISPR RNA)와
TracrRNA(trans-activating crRNA)를포함한다.링커 (GG)도 5 '말단에포함되었다. sgRNA에대한주형 (template)은 HelixAmp Power-Pfu (NanoHelix)및
올리고뉴클레오티드프라이머 (Bioneer)를사용하여 60 oC에서 40초동안 어닐링하고, 72 0C에서 30초동안연장하는과정을 30사이클반복한후,겔 추출 (QIAquick, Qiagen)에의해제작되었다.인비트로전사는 37 0C에서 120분 동안파지 T7 RNA중합효소 (Promega)를사용하여수행하였다.
증폭물 (amplicon)및프라이머의서열을도 4에나타내었다.전사된 sgRNA를 5
M암모늄아세테이트를사용하여침전시키고에탄올로침전하여정제하였다.
[135] sgRNA를제작하기위해 ,각각의 sgRNA에대한 DNA주형은먼저시험관내 전사를위해도 8의프라이머를사용하여합성되었다.각각의 sgRNA에대한 DNA주형은 T7프로모터영역 , CrRNA를위한주형영역및 TracrRNA를위한 주형영역을포함하였다 (도 7).합성된 DNA주형은도 9에나타내었다.
[136] 시험관내전사는이어서합성된 DNA주형및 T7중합효소를사용하여
수행하여각 sgRNA를생성하였다.도 10은 mecA의다른영역을표적으로하는 세가지다른유형의 sgRNAs: sgRNA (1), sgRNA (2)및 sgRNA (3)가성공적으로 합성되었음을보여준다.합성된 3개의모든 sgRNA는 ~ 100 bp의
뉴클레오타이드크기를갖는것으로나타났다.
[137]
[138] sgRNA의선별
[139] 상기에서제조한세가지 sgRNA의이중가닥분열을유도하기위한기능성 또한조사되었다.표적 DNA로서,배양된 MRSA의총 RNA로부터 mecA 유전자 (도 5)내의 1803 bp영역의 RT-PCR에의해순수한무세포 DNA용액을 제조하였다.정제된천연형 SpCas9단백질과 sgRNA (1), sgRNA (2)및 sgRNA (3)을각각흔합하고 PCR증폭된 mecA표적 DNA를첨가하여엔도뉴클레아제
대제용지 (규칙제 26조) RO/KR 절단을유도하였다.
[140] sgRNA (3)이겔전기영동결과에서두개의단편 (648 bp와 1 155 bp)을모두 나타내어가장높은절단효율을나타내었고, sgRNA (2)는절단되지않은 DNA 바로아래에 1463 bp단편에해당하는명확한단편을보여주었지만다른단편은 관찰하기어려웠다. sgRNA (1)의경우절단된생성물중어느것도보이지 않았으므로검출효율이너무낮거나 sgRNA가특정이중가닥파괴를유도하는 데는기능하지않는다고평가되었다.따라서,상기 sgRNA (3)를본발명의 나노복합체형성및세균내전달에대한추가검사에사용하였다.
[141]
[142] 1-3.세균균주및배양
[143] MRSA균주인 CCARM 3798, 3803,및 3877균주들을 CCARM(Culture
Collection of Antimicrobial Resistant Microbes)으로부터수득하고약제내성을 가진표적세균으로사용하였다. MSSA균주인 KCTC 3881은 KCTQKorean Collection for Type Cultures)에서수득하였고,약제내성이없는균주로
사용하였다.배양을위해,각세균균주들은 TSB(tryptic soy broth, BD
Biosciences)에접종하고 37 °C에서진탕배양기 (shaking incubator)에서 1216시간 동안부유배양하였다.세균의성장및농도는 600 nm파장에서의 OD값으로 측정하였다 (0.4-0.6).
[144]
[145] 짐시예 2.고분자에의해유도체합성되 Cas9의체조
[146] 2-l. SpCas9-bPEI의제조
[147] 유전자편집단백질인 Cas9제한효소에양이온성을지니는캐리어물질인
폴리에틸렌이민을접합하기위하여다음과같은실험을수행하였다.
[148] 가지형폴리에틸렌이민 (Branched polyethylenimine, bPEI, Mw 2000및 25000)은 초순수정제수에용해된 5 mg의 sulfo-SMCC(sulfosuccinimidyl
4-(Nmaleimidomethyl) cyclohexane- 1 -carboxylate, Thermo Scientific)에 16 mg의 bPEI를첨가하여활성화하였고, 25 °C에서 3시간동안반웅시켰다 (bPEI:
sulfo-SMCC의몰농도비 = 1 : 10).다음으로반웅용액을탈이온수 (MWCO 5001000, Spectra/Por)에대하여 48시간동안투석하고,아민기가
말레이미드 (maleimide)로치환된폴리에틸렌이민고분자를동결건조
하였다 (FD8508, IlshinBioBase).
[149] 또한,상술한바와같이유전자편집단백질로 Cas9제한효소를생산하기위해, Streptococcus pyogens유래의 Cas9단백질 (SpCas9)의백터를 E. " 에발현시켜 히스티딘을이용하여정제하였다.
[150] 상기유전자편집단백질 (SpCas9)에폴리에틸렌이민을접합하기위해,상기 정제된 SpCas9단백질 2mg을인산완층용액 (PBS) 1.2ml에녹이고,상기 말레이미드로활성화된폴리에틸렌이민고분자를몰비 1 :100로 4 ° !)11 6.9 에서 4시간동안반웅시키고,최종산물 (SpCas9-bPEI)을 Cas9저장용
대제용지 (규직제 26조) RO/KR 완층용액 (50 mM Tris HC1 at pH 8.0, 200 mM KC1, 0.1 mM EDTA, 20% glycerol, 1 mM DTT, and 0.5 mM PMSF)에대해투석을 24시간수행한후액체질소에서 급속냉각후 80oC에서저장하였다.
[151] 상기 SpCas9-bPEI의합성과정에관한모식도를도 11에나타내었다.
[152]
[153] bPEI의 Cas9에대한컨쥬게이션은 0.5%아가로즈겔및 5% SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)를이용한질지연분석법 (gel retardation)으로확인하였다
[154] SpCas9에대한 bPEI의성공적컨쥬게이션을확인하기위한겔지연분석
결과를도 12에나타내었다.도 12에나타낸바와같이, bPEI와컨쥬게이션된 SpCas9(SpCas9-bPEI)는 (-)방향으로실질적으로이동하는천연 SpCas9와반대 방향인 (-)방향으로약간이동하는것으로나타났다.상기 SpCas9-bPEI의겔 지연분석결과는폴리머와의변형으로인한이동성의변화및단백질분자의 웅집 (clustering)때문일수있다.단백질의분자량을 4000 Da만큼만
증가시킴으로써최대 2개의 bPEI분자가각단백질분자에컨쥬게이션될수 있지만,이경미한변화 (1-2 %)는구조적또는차원적변화로인하여전기 영동하는동안단백질의이동성에실질적으로영향을미칠수있다. GFP융합 SpCas9단백질의이론적인전하는매우음성일것으로예상되었다. bPEI는극히 고밀도의아민작용기를가지고있어서양이온성이높기때문에 bPEI의
SpCas9와의결합은단백질의분자전하에영향을주거나정전기적
단백질 -고분자상호작용에의한웅집을유도할수있다.
[155]
[156] 또한, SpCas9에대한 bPEI의성공적컨쥬게이션을확인하기위한 SDS-PAGE 분석결과를도 13에나타내었다.도 13에나타낸바와같이, SpCas9-bPEI와천연 SpCas9가유사한영역에나타났으며,공유결합으로가교된 SpCas9단백질들이 접합반웅후에존재하지않는것으로나타났다 (도 13).따라서, SpCas9와 bPEI의 컨쥬게이션이성공적으로이루어졌으며 SpCas9단백질분자사이에서는가교가 일어나지않았음을확인하였다.
[157]
[158] 심시예 3. CRISPR유저자편집나노복합체 (Cr-나노복함체ᅵ의계조및특징
[ 159]
[160] 3-1. Cr-나노복합체의제조
[161] 본발명의 Cr-나노복합체를제조하기위하여, SpCas9-bPEI (990 nM)및
sgRNA(3) (1.8 μΜ)을탈이은수 (pH 6.5)에서흔합하여상온 (250C_에서 15분간 정적조건 (static condition)에서배양하였다.대조군으로,원래의 SpCas9 (990 nM)을 sgRNA(3) (1.8 μΜ)과위와같은조건에서흔합하였다.그결과
Spcas9-bPEI와 sgRNA (3)가자기조립된나노크기복합체가제조되었다 (도 14). 상기 sgRNA^- MRS A (methicillin-resistant Staphylococcus aureus)≤항생제내성
대제용지 (규칙제 26조) RO/KR 유전자 mecA를표적으로하는서열을사용하였다.상기나노복합체형성과정 중의 pH는 ~6.4로 bPEI (2 kDa)의 pKa (~8.6)보다낮았고,아민작용가를 양성자화하여음이온성 sgRNA와의정전기적결합을유도하였다.
[162]
[163] 복합체의크기측정및제타전위측정
[164] 동적광산란 (DLS: dynamic light scattering)및제타포텐셜 (zeta potential)측정을 위하여 ,상기복합용액을 PBS또는탈이은수로회석하여,각각 168 nM SpCas9 및 300 nM sgRNA(3)의최종농도로맞추었다. Cr-나노복합체또는자연의 복합체들의유체역학적직경 (hydrodynamic size)및제타전위는 ELSZ-2000ZS (Otsuka)으로측정하였다.
[165]
[166] 동적광산란 (DLS: dynamic light scattering)측정결과, Cr-나노복합체의경우 입도크기 (Z평균)가 163.3 nm로나타나개질되지않은 Cas9단백질과 sgRNA의 천연복합체의크기 82.6 nm에비해큰것으로나타났다 (도 15).상기결과로부터 SpCas9-bPEI내에서음으로하전된 sgRNA와양전하를띤폴리머사이의전하 상호작용에의해작은나노크기의단백질-증합체컨쥬게이트/ RNA복합체가 성공적으로형성되었음을확인할수있었다.각각의복합체는단일 sgRNA 분자에결합된단일단백질의형태로주로존재하는변형되지않은 SpCas9와는 달리,몇분자의 SpCas9-bPEI및 sgRNA들을포함하여더큰복합구조를형성할 것이다.
[167]
[168] Cr-나노복합체와천연복합체의제타전위값은모두단백질표면에결합된 sgRNA의존재로인해음의값을보였으나 Cr-나노복합체 (-12.1 mV)는천연 복합체 (-19.0 mV)에비해상대적으로적은음이온성을나타내었다.또한,복합체 형성전의 SpCas9-bPEI의제타전위는양이은성중합체의도입으로인해양의 값 (+ 4.0 mV)을나타내었으며,이는천연 SpCas9의제타전위 (-17.2 mV)와 비교하여상당히변화된것이었으며,이러한 Cr-나노복합체의더낮은
음이온성은세균내로의전달을향상시키는데도움이될것으로예상되었다.
[169]
[170] 3-2. Cas9엔도뉴클레아제활성측정을위한절단분석 (Cleavage Assay for
Endonuclease Activity)
[171] 본발명의 Cas9제한효소가폴리머유도체화및 Cr-나노복합체형성후이중 가닥 DNA절단을유도하는기능적활성을유지하는지여부를조사하기위하여 , 배양된박테리아로부터유래된 PCR증폭주형 DNA를사용하여인비트로절단 분석을수행하였다ᅳ
[172] 먼저 MRS A및 MSSA균주를배양하고,총 RNA를 Trizol시약 (Invitrogen)
추출한다음, amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT)를 사용하여 60 °C에서 1분간 (denaturation), 25 °C에서 5분간 (어닐링 ), 55 °C에서
대제용지 (규칙제 26조) RO/KR 60분간 (연장),및 85 °C에서 1분간 (비활성화)하여역전사하였다. cDNA는 power pfu중합효소 (Nanohelix)와 mecA유전자에특이적인프라이머 (Bioneer)로 다음조건으로증폭하였다: 95 0C에서 2분간개시; 35사이클 95°C에서 20 초간 (변성), 59 °C에서 40초간 (어닐링), 72 0C에서 3분 38초간 (연장);및 72 °C에서 5분간종결.
[173] 그런다음증폭된주형 DNA를 SpCas9: sgRNA:표적 DNA의몰비가 10: 10: 1이되도록 SpCas9-bPEI또는 sgRNA (3)와복합체로된천연 SpCas9로처리하고 Cas9핵산분해반웅완층액 (20 mM HEPES , 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5)에 37 °C에서 1시간동안처리하였다.최종산물은아가로스겔 전기영동하여 DNA의분절의존재및크기를확인하였다.
[174] 결과는도 16에나타내었다.
[175] 도 16에나타낸바와같이,예상되는크기인 1155 bp와 648 bp에해당하는
하나의큰 DNA단편과하나의작은 DNA단편이나타나, SpCas9는 bPEI와의 직접공유결합적변형및 sgRNA와의복합체형성이후에도표적 DNA의이중 가닥절단을、유도할수있음을확인하였다.
[176]
[177] 3-3.고분자유도체화된 SpCas9의세균내전달및현미경관찰
[178] 본발명의 Cas9단백질의고분자유도체화는천연 Cas9와비교하여세균
내로의흡수 (uptake)를증가시킬것으로기대되었다.본발명자들은세균고분자 유도체화된 SpCas9의세균으로의전달효율을입증하기위해, SpCas9-bPEI를 in vitro배양된 MRSA로처리하고공초점현미경으로관찰하였다. MRSA균주 3798및 3803은상기처리전에배양하였다.
[179]
[180] PBS중의 SpCas9-bPEI (200 nM)또는천연 SpCas9 (200 nM)를 1 x 107의배양된 MRSA에처리하였다.농축 SpCas9와 bPEI (Mw 2000)를흔합하고, 25 °C에서 15 분간배양하고,최종농도가 SpCas9 - 200 nM, bPEI - 3 [ g/mL이되도록 PBS로 (7x)희석하여만든, bPEI와단순히흔합한천연 SpCas9를대조군으로
사용하였다.
[181] 각실험군을 37 °C에서 2시간동안진탕배양기를사용하여부드럽게교반
배양한후,세균을원심분리후 PBS로반복세척하여잔류복합체를제거하였다. 세균을 4%파라포름알데히드용액으로고정시키고 Vectashield (Vector
Laboratories)를사용하여현미경슬라이드에마운팅하고레이저스캐닝공초점 현미경 (LSM780, Carl Zeiss)을사용하여관찰하였다.상대적세균내
전달량 (uptake)을정량화하기위해,상기흔합물로 1.7xKF/mL로 4시간동안 세균을처리하였다.또다른대조군으로,천연 SpCas9를리포펙타민
3000(Thermo Fisher Scientific)과제조사의프로토콜에따라흔합하였다 (최종 농도 SpCas9 400 nM).이로부터저배율영상을공초점현미경으로얻었고녹색 형광신호 (SpCas9)를 ImageJ (NIH)를사용하여적색형광 (H염색)으로
대제용지 (규직제 26조) RO/KR 표준화했다.
[182] 결과는도 17및도 18에나타내었다.
[183] 도 17에나타낸바와같이,본발명의폴리에틸렌이민으로컨쥬게이션된 Cas9 단백질 (SpCas9-bPEI)은개질되지않은 Cas9단백질 (SpCas9)이나,반웅하지않은 폴리에틸렌이민고분자와 Cas9단백질의흔합물 (SpCas9+bPEI (mix))과 비교하여월등하게높은효율로세균내로성공적으로흡수되었음을확인할수 있었다.본발명의 SpCas9-bPEI의경우 GFPuv의밝은녹색형광이핵염색에 인접하여명확하게관찰된반면에,천연 SpCas9는어떠한흡수도나타내지 않았다.대조군으로서 bPEI와단순히 (비공유결합으로)흔합된천연
SpCas9(SpCas9+bPEI (mix))도역시흡수의징후를나타내지않았다.
[184] 또한,고분자유도체화된 SpCas9의세균내로의흡수를더확인하기위하여 공초점이미지단면을 3D이미지로재구성하여 SpCas9과핵염색의형광신호의 중첩으로부터세균세포내의 SpCas9의존재를나타내었다 (도 19,도 20).
[185] 형광신호의히스토그램은또한공초점이미지내에서스캔된다른
영역으로부터얻어졌으며,그결과 SpCas9로부터의신호는핵염색의신호가 존재하는지점에서만나타났다 (도 21).
[186] 도 19에나타낸바와같이 ,상대적흡수값은 SpCas9-bPEI의경우 0.4273으로 나타났으며,천연 SpCas9의경우, bPEI가단순히흔합된천연 SpCas9의경우및 리포펙타민과혼합된천연 SpCas9의경우에각각 0.0041, 0.0001, 0.0083으로 나타났다. bPEI컨쥬게이션에대한 Cas9단백질의흡수가크게증가한것은 폴리머의높은양이온성또는그로인한단백질의극성증가로인한것일수 있다.
[187]
[188] bPEI폴리머는높은분자밀도에서 3차, 2차및 1차아민기를풍부하게갖기 때문에, SpCas9-bPEI와그람양성균의음으로하전된세포벽과의상호작용은 천연 SpCas9과의상호작용에비해실질적으로증가할것으로보인다.또한, SpCas9의표면상에 bPEI가존재하면클러스터의형성또는분자의축합이 가능해진다.전반적으로박테리아세포벽에대한 SpCas9의강화된결합은 아마도펩티도글리칸 (peptidoglycan)을통한침투및연속된세포막을통한 흡수에의한투과에의한것이어서더높은흡수가능성을초래할것이다.또한 bPEI의강한양이온성은박테리아 DNA와정전기적으로상호작용하여
SpCas9의유전체표적에대한분자적인력을허용할것으로기대된다.
[189] 한편,캐리어로서종래의리포펙타민을사용하는것은약물의로딩효율이 낮고세포내로의방출이어렵기때문에한계가있음을보여주었다.
[190] 본발명자들은 SpCas9단백질을양이온성폴리머로공유결합시켜이들
문제를해결할수있을것으로기대하였다.양이온성폴리머는그변형이기능 활성에영향을미치지않는한,단백질의각단일분자에적용되며동시에 최소량의캐리어물질을사용할수있도록해줄것으로기대된다.또다른
대제용지 (규칙제 26조) RO/KR 이점은전달을위한카고물질의방출단계가추가로필요한캐리어물질내로의 캡슐화공정을피할수있다는점에있다.
[191] 더많은증거는분자량 25 000의 bPEI로변형된 SpCas9를사용한치료
실험에서확인할수있었다.도 22는더큰 bPEI폴리머로변형시키면세균에 처리했을때단백질이유의한흡수를보이지않음을나타낸다.작은분자량의 bPEI를사용한변형만이높은전달효율을나타내었기때문에,운반효율을 최대화하고독성을최소화하기위해서는분자량이작으면서도최적량의캐리어 물질을사용하는것이중요함을알수있다.
[192]
[193] 3-4. Cr-나노복합체의동물세포전달및현미경관찰
[194] 본발명의 Cas9단백질의고분자유도체화는천연 Cas9와비교하여
포유류세포 (mammalian cell)내로의흡수 (uptake)를증가시킬것으로기대되었다. 본발명자들은 Cr-나노복합체의포유류세포로의전달효율을입증하기위해, SpCas9-bPEI및 sgRNA를 in vitro배양된동물세포에처리하고공초점
현미경으로관찰하였다. A549, HaCat, Raw264.7동물세포는처리 30시간전에 lxlO4 cells로 8 well chamber에서배양되었고 Jurkat동물세포의경우 48 well cell culture plate에서배양되었다.
[195] PBS증의 SpCas9-bPEI2000/sgRNA (168 nM)또는
SpCas9-bPEI25000/sgRNA( 168 nM)또는천연 SpCas9/sgRNA (168 nM)복합체를 배양된포유류세포들에처리하였다.또다른대조군으로,천연 SpCas9/sgRNA
(168 nM)를리포펙타민 RNAiMAX (Thermo Fisher Scientific)를제조사의 프로토콜에따라흔합하였다 (최종농도 SpCas9/sgRNA각각 - 168 nM).각 실험군을 37 °C, 5% C02에서 1-1.6시간동안세포배양기를사용하여배양한후,
PBS로 3회이상반복세척하여잔류복합체를제거하였다.세포를 4%
파라포름알데히드용액으로 15분간고정시키고 ® Mounting Medium with (Vector
Laboratories)를사용하여현미경슬라이드에마운팅하고레이저스캐닝공초점 현미경 (LSM780, Carl Zeiss)을사용하여관찰하였다.이로부터저배율영상을 공초점현미경으로얻었고녹색형광신호 (SpCas9)와청색형광 (DAPI- nucleous 염색)을확인할수있었다.
[196] 도 23에나타낸바와같이,본발명의폴리에틸렌이민분자량 2000으로
컨쥬게이션된 Cas9단백질 (SpCas9-bPEI)/sgRNA복합체는개질되지않은 Cas9 단백질 (SpCas9)/sgRNA복합체와비교하여월등하게높은효율로 A549 동물세포내로성공적으로흡수되었음을확인할수있었으며,리포펙타민
RNAiMAX와의흔합물에비해서도상당히높은흡수율을보였다.또다른 대조군으로분자량이 25000으로폴리에틸렌이민의분자량이 12.5배차이나는 SpCas9-bPEI25000/sgRNA을처리하였을때는확연한세포내흡수를전혀확인 할수가없었다.또한, HaCat동물세포에처리가되었을때도마찬가지로,도 24에나타낸바와같이,본발명의폴리에틸렌이민으로컨쥬게이션된 Cas9
대제용지 (규직제 26조) RO/KR 단백질 (SpCas9-bPEI)/sgRNA복합체는개질되지않은 Cas9
단백질 (SpCas9)/sgRNA복합체와또다른대조군인리포펙타민 RNAiMAX와의 흔합물에비교하여월등하게높은효율로동물세포내로성공적으로
흡수되었음을확인할수있었다. Cr-나노복합체의세포내로의흡수를더 확인하기위하여공초점이미지단면을 3D이미지로재구성하여복합체
(SpCas9/sgRNA:녹색형광)와핵 (DAPI:파란색형광)염색의형광신호의 중첩으로부터 HaCat동물세포내의복합체의존재를도 25에나타내었다.도 25에서나타낸바와같이,본발명의폴리에틸렌이민으로컨쥬게이션된 Cas9 단백질 (SpCas9-bPEI)/sgRNA복합체의경우녹색형광신호 (SpCas9)와청색 형광 (DAPI- nucleous염색)이가장많이겹쳐짐을확인할수있었다.이는 Raw 264.7동물세포에서도동일한결과를보였으며,도 26에서나타낸바와같이,본 발명의 SpCas9-bPEI/sgRNA복합체의경우 Cas9단백질의 GFPuv밝은녹색 형광이핵염색에인접하여명확하게관찰된반면에,천연 SpCas9/sgRNA 복합체는상대적으로적은형광이관찰되었다.도 27에서나타낸바와같이, 면역세포인 Jurkat동물세포의경우에처리되었을경우에는본발명의
SpCas9-bPEI/sgRNA복합체의경우 Cas9단백질이개질되지않은 Cas9 단백질 (SpCas9)/sgRNA복합체와비교하여상당히높은효율을나타내었으며 , 리포펙타민 RNAiMAX와의흔합물보다다소증가된세포내흡수율을
나타내었다.도 28에서나타낸바와같이,인체유래신경줄기세포에서도본 발명의 SpCas9-bPEI/sgRNA복합체가개질되지않은 SpCas9/sgRNA복합체보다 월등히높은 Cas9형광신호로세포유입효과가훨씬높다는것을관찰할수 있다.인체유래유도만능줄기세포 (induced pluripotent stem cell, iPSC)에서도, 천연 SpCas9/sgRNA복합체의경우세포유입효과가전혀나타나지않았지만,본 발명의 SpCas9-bPEI/sgRNA복합체의경우상당한세포유입효과를보였다 (도 29참조). SpCas9-bPEI/sgRNA또는 SpCas9/sgRNA복합체의전달효율은 SpCas9 재조합단백질의 GFP신호로확인할수있으며,모든세포의 counterstain으로써 핵을 DAPI로,도 28및도 29의경우에는세포질을 rhodamine-phalloidin으로 염색하였다.
[197]
[198]
[199] 3-5. Cr-나노복합체에의한유전체 (genome)편집효율성평가
[200] 본발명자들은 Cr-나노복합체가세균유전체를편집하고항생제내성을
표적화할수있는지여부를조사하였다. mecA를표적으로하는 sgRNA및 SpCas9-bPEI로형성된 Cr-나노복합체를배양된 MRSA(3798및 3803균주)에 시험관내에서처리하고세균의성장을선택배지에서의후속배양으로 조사하였다.배양된 MRSA균주는이전에메티실린과옥사실린모두에 저항성이있다는것이입증되었다.
[201] 실험군으로본발명의 Cr-나노복합체를 SpCas9-bPEI (990 nM)와 sgRNA (3)
대제용지 (규칙제 26조) RO/KR (1.8 μΜ)을흔합하고 15분동¾배양하여제조하였다.대조군으로서상기와 동일한조건에서천연 SpCas9 (990 nM)를 sgRNA (3) (1.8 μΜ)와흔합하였다.또 다른대조군으로기존의지질기반제형은제조사의프로토콜에따라
Lipofectamine RNAiMAX (15.8 μL, Thermo Fisher Scientific)에천연복합체 (50 μί)를첨가하여제조하였다.단백질만 (sgRNA없이 ), SpCas9-bPEI만,또는천연 SpCas9만 990 nM농도로포함하는것들도대조군으로제조하였다.
[202] 모든샘플을 TSB(tryptic soy broth)에서 6배희석 (SpCas9의최종농도: sgRNA = 165: 300 nM)하고, MRSA 5xl06에 37 °C에서 4시간동안부드럽게
진탕배양하며처리하였다.처리된박테리아를 PBS로세척하고, 6 g/mL oxacillin이포함된 TSB에서 100배회석하고 37 에서 90분동안
진탕배양기에서배양하였다.세균성장은 90분간배양후 600 nm에서의 OD 값을측정 ((Nanophotometer, Implen)하여결정하였다.
[203] 나노복합체로처리한세균도 PBS로희석 (105x)하고 6 g/mL oxacillin을
포함하는 MRSA한천평판배지위에스프레딩하여 30 °C에서 21시간동안 배양한후 CFU(colony forming unit)를계수하였다.
[204] Cr-나노복합체로처리한박테리아를현탁배양하거나 oxacillin (6 I mL)을 포함하는한천배지에서배양하는경우,이중가닥 DNA가파괴된클론은성장할 수없고,영향을받지않은클론은세균집락을형성할것이다.상기실험과정에 관한모식도는도 28에나타내었다.
[205]
[206] 본발명의 Cr-나노복합체로처리한세균의현탁배양후 OD6∞값을측정하여 성장를을도 29에나타내었다.도 29에나타낸바와같이,본발명의
Cr-나노복합체처리시 sgRNA가없는 SpCas9-bPEI를대조군으로처리하는것 보다 32 %감소시키켜성장을유의하게억제하는것으로나타났다.
[207]
[208] 또한,유전체편집패턴을추가적으로평가하기위하여,세균을 Cr-나노복합체 또는대조군으로처리하고 oxacillin의존재또는부존재하에 CFU수를계산하여 도 30에나타내었다.
[209] 도 30에나타낸바와같이,본발명의 Cr-나노복합체로처리한결과 Oxacillin의 존재하에서대조군 (SpCas9 only의경우 335 x 106 CFU/mL, SpCas9-bPEI의경우 401 X 106 CFU/mL)과비교하여성장이유의하게감소하였으나 (65 x 106
CFU/mL),천연 SpCas9/sgRNA복합체로처리한경우에는성장감소가더적게 나타났다 (121 10« CFU/mL).또한, sgRNA없이 SpCas9-bPEI만을
처리시 (SpCas9-bPEI Only)에는현저한세균성장의감소를나타내지않아 Cr-나노복합체로처리했을때감소된세균성장이 bPEI의존재에의한독성에서 비롯된것이아니라는것을보여주었다.놀랍게도,천연 SpCas9/sgRNA 복합체의리포펙타민제제는 SpCas9만으로처리한대조군과비교하여서세균 성장에서현저한감소를나타내지않았다 (361 106 CFU/mL).따라서
대제용지 (규칙제 26조) RO/KR SpCas9/sgRNA복합체의전달을위한리포펙타민의사용은포유동물 세포에서는실질적인전달효율을나타내었지만세균세포의경우에는전달 효율이매우불량함을알수있다.또한,생체활성분자 (예를들어, Cas9와같은 단백질)의존재하에세균을배양하는것이이를영양공급원또는자극제로서 작용하여세균의성장에영향을미칠수도있다는사실을고려하였다.
[210] 따라서 "상대적성장"은 (1) sgRNA를포함한복합체로처리했을때의 CFU
수로계산되었고, (2) sgRNA를제외한복합체로처리한경우의 CFU수로 정규화하여도 31에나타내었다.도 31에나타낸바와같이,본발명의
Cr-나노복합체로처리한결과를 SpCas9-bPEI로만처리한결과와비교시 16.3 %의상대적성장감소를보인반면, SpCas9/sgRNA복합체로처리한결과를 SpCas9로만처리한결과와비교시 35.9 %의상대적성장감소를보였다.
리포펙타민의존재하에천연 SpCas9/sgRNA복합체로처리한결과, sgRNA가 없는리포펙타민을함유한 SpCas9와비교하여 71.6%의상대적성장이
나타났다 (도 31).이러한결과는종래의리포펙타민캐리어의사용여부에 관계없이천연 SpCas9복합체를사용하는것과비교하여,본발명의
Cr-나노복합체가훨씬높은효율로 sgRNA와 SpCas9단백질을세균내로 전달하여표적 DNA를이중가닥절단할수있음을입증한다.
[211] 또한,다양한농도의 Cr-나노복합체를세균에처리하여용량-의존적유전체 편집효율을결정하였다 (도 32a및도 32b).도 32a및도 32b에나타낸바와같이, 본발명의 Cr-나노복합체는천연복합체와비교할때,낮은농도에서의처리는 상대적인성장을 18.7 %억제하였고,보다높은농도에서의처리는상대적인 성장을 57.7 %억제하였다.고농도에서의처리는중간농도에서처리한경우에 비해성장을억제하는평균치가약간더높았지만,중간치료의경우에만 통계적으로유의한것으로나타났다.저농도의 Cr-나노복합체를처리하는것은 유의하게유전체편집효능을발휘하기에는층분하지않은반면,고농도의 Cr-나노복합체처리는세균의기능및흡수에영향을미치거나세균증식을 자극하여게놈편집과정을방해했을수있다.
[212] 또다른중요한발견은 oxacillin부존재조건에서박테리아성장을조사한결과 :
Cr-나노복합체의경우 67xl06 CFU/mL,천연복합체의경우 135xl06 CFU/mL, SpCas9-bPEI만의경우 414 x 106 CFU/mL을나타내어 oxacillin이존재할때와 비슷한결과를보였다는것이다 (도 30).
[213] Cr-나노복합체로처리된세균으로부터형성된세균집락의레플리카배양도 수행되었다.여기서 oxacillin을포함하지않는 1차플레이트는 oxacillin을 포함하는 2차플레이트에복제되어 30oC에서 12시간동안배양하고,세균 집락수를계수하였다.그결과비선택성배지에서콜로니를형성한모든클론 또한선택배지에서자랄수있음을보여주었다 (도 33).이러한결과는
Cr-나노복합체에의한유전체편집이박테리아의치사를초래하는반면,상기 Cr-나노복합체처리를견디는박테리아는계속증가함을나타낸다.
대제용지 (규칙제 26조) RO/KR [214]
[215] 통계적분석
[216] 모든통계자료는평균 ±표준편차로계산하여나타내었다.통계적유의성은 Student 's t test를사용하여 p값을얻음으로써결정하였다.
[217]
대제용지 (규직제 26조) RO/KR

Claims

청구범위
[청구항 1] 고분자캐리어물질-접합 CRISPR(Clustered regularly interspaced short palindromic repeats)효소단백질.
[청구항 2] 제 1항에있어서,상기 CRISPR유전자편집효소단백질은 Casl(CRISPR associated protein 1), Cas2, Cas3, Cas4, Cas5, Cas6, Cas9, Csel, Cse2, Cse3, Cse4, Cas5d, Cas5e, Csyl, Csy2, Csy3, Csy4, Cpfl, Csnl, Csn2, Csdl, Csd2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, Csml, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, C2c2(Casl3a), dCas9(dead Cas9), Cas9 nickase, CDA(cytidine deaminase enzyme), APOBEC(apolipoprotein B editing complex) 14, UGI(uracil glycosylase inhibitor),및 AID(activation-induced deaminase)로이루어진 군으로부터선택되는것을특징으로하는,고분자캐리어물질 -접합 CRISPR효소단백질.
[청구항 3] 제 1항에있어서,상기고분자캐리어물질은가지형폴리에틸렌이민, 선형폴리에틸렌이민,폴리프로필렌이민,폴리아미도아민, 폴리에틸렌글리콜,폴리에틸렌옥사이드-폴리프로필렌옥사이드 공중합체,폴리-락트산,폴리 -글리콜산,폴리 -D,L-락트산 -co-글리콜산, 폴리카프로락톤,폴리포스포에스터,폴리포스파진,
폴리베타아미노에스터 ,가지형폴리아미노에스터,
폴리아미노부틸 -글리콜산,폴리오르소에스터 ,
폴리하이드록시프롤린에스터,폴리아크릴아마이드,폴리비닐피를리돈, 폴리비닐알코올, poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA), 덴드리머,히알루론산,알긴산,키토산,덱스트란,사이클로덱스트린, 스퍼민,폴리 -Arginine,폴리 -Lysine,및이들의공중합체또는흔합물로 이루어진군으로부터선택되는것을특징으로하는,고분자캐리어 물질 -접합 CRISPR효소단백질.
[청구항 4] 제 1항에있어서 ,상기고분자캐리어물질과 CRISPR효소단백질은
SMCC(succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate)), sulfo-SMCC(sulfosuccinimidyl
4- (N-maleimidomethyl)cyclohexane- 1 -carboxylate) ,
AMAS(N-a-maleimidoacet-oxysuccinimide ester),
BMPS(N- -maleimidopropyl-oxysuccinimide ester),
GMB S (Ν-γ-maleimidobutyryl-oxysuccinimide ester) ,
MB S (m-maleimidobenzoyl-N-hy droxy succinimide ester),
EMCS(N-8-malemidocaproyl-oxysuccinimide ester), SM(PEG) (PEGylated SMCC), SPDP(succinimidyl 3-(2-pyridyldithio)propionate), PEG-SPDP(PEGylated SPDP), DSG(disuccinimidyl glutarate),
대제용지 (규칙제 26조) RO/KR DCC(Dicyclohexylcafb0diimide), DS S (disuccinimidyl suberate),
B S 3 (B issulf osuccinimidy 1 suberate), DSP(dithiobis(succinimidyl propionate)), EGS (ethylene glycol bis(succinimidyl succinate)), DMP(dimethyl
pimelimidate), BMOE(bismaleimidoethane) , BMB( 1 ,4-bismaleimidobutane), DTME(dithiobismaleimidoethane) ,
EDC(l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS(N-Hydroxysuccinimide),
프로파길 -숙신이미딜-에스테르 (propargyl-succinimidyl-ester),
DBCO-말레이미드 (Dibenzocyclooctyne-maleimide),
DBCO-PEG-말레이미드 (Dibenzocyclooctyne-PEG4-maleimide),
DBCO-S-S-NHS에스테르 (Dibenzocyclooctyne-S-S-N-hydroxysuccinimidyl ester), DBCO-NHS에스테르 (dibenzocyclooctyne-N-hydroxysuccinimidyl ester),아세틸렌 -PEG-NHS에스테르 (acetylene-PEG-NHS ester),및 알킬렌 -PEG-말레이미드 (alkyne-PEG-maleimide)로이루어진군으로부터 선택된가교제로접합 (conjugation)된것을특징으로하는,고분자캐리어 물질 -접합 CRISPR효소단백질.
[청구항 5] 다음단계를포함하는고분자캐리어물질-접합 CRISPR(Clustered
regularly interspaced short palindromic repeats)효소단백질의제조방법:
(a)고분자캐리어물질의기능기를양기능성가교제 (bifunctional crosslinker)로반웅시켜활성화된고분자캐리어물질을제조하는단계;
(b) CRISPR효소단백질의기능기를상기활성화된고분자캐리어물질과 반웅시켜접합물질을제조하는단계.
[청구항 6] 제 5항에있어서,상기 CRISPR효소단백질은 Casl(CRISPR associated protein 1), Cas2, Cas3, Cas4, Cas5, Cas6, Cas9, Csel, Cse2, Cse3, Cse4, Cas5d, Cas5e, Csyl, Csy2, Csy3, Csy4, Cpfl, Csnl, Csn2, Csdl, Csd2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, Csml, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, dCas9(dead Cas9), Cas9, C2c2 (Casl3a), CDA(cytidine deaminase enzyme), APOBEC(apolipoprotein B editing complex) 14, UGI (uracil glycosylase inhibitor),및 AID(activation-induced deaminase)로이루어진군으로부터 선택되는것을특징으로하는,고분자캐리어물질-접합 CRISPR효소 단백질의제조방법.
[청구항 7] 제 5항에있어서,상기 (a)단계의고분자캐리어물질은가지형
폴리에틸렌이민,선형폴리에틸렌이민,폴리프로필렌이민, 폴리아미도아민,폴리에틸렌글리콜,
폴리에틸렌옥사이드 -폴리프로필렌옥사이드공중합체,폴리-락트산, 폴리 -글리콜산,폴리 -D,L-락트산 -co-글리콜산,폴리카프로락톤,
대제용지 (규칙제 26조) RO/KR 폴리포스포에스터,폴리포스파진,폴리베타아미노에스터 ,가지형 폴리아미노에스터,폴리아미노부틸 -글리콜산,폴리오르소에스터, 폴리하이드록시프를린에스터,폴리아크릴아마이드,폴리비닐피를리돈, 폴리비닐알코올, poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA), 덴드리머,히알루론산,알긴산,키토산,덱스트란,사이클로덱스트린, 스퍼민,폴리 -Arginine,폴리 -Lysine,및이들의공중합체또는흔합물로 이루어진군으로부터선택되는것을특징으로하는,고분자캐리어 물질 -접합 CRISPR효소단백질의제조방법 .
[청구항 8] 제 5항에있어서,상기 (a)단계의가교제는 SMCC(succinimidyl
4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate)),
sulfo-SMCC(sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-l-carboxylate),
AMAS(N-a-maleimidoacet-oxysuccinimide ester),
BMPS (N- β-maleimidopropyl-oxysuccinimide ester) ,
GMB S (Ν-γ-maleimidobutyryl-oxysuccinimide ester) ,
MB S (m-maleimidobenzoyl-N-hydroxy succinimide ester),
EMCS(N-£-malemidocaproyl-oxysuccinimide ester), SM(PEG) (PEGylated SMCC), SPDP(succinimidyl 3-(2-pyridyldithio)propionate), PEG-SPDP(PEGylated SPDP), DSG(disuccinimidyl glutarate), DCC(Dicyclohexylcarbodiimide), DS S (disuccinimidyl suberate),
B S 3 (Bissulf osuccinimidyl suberate), DSP(dithiobis(succinimidyl propionate)), EGS (ethylene glycol bis(succinimidyl succinate)), DMP(dimethyl
pimelimidate), BMOE(bismaleimidoethane), BMB(l,4-bismaleimidobutane), DTME(dithiobismaleimidoethane),
EDC(l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), NHS (N-Hydroxysuccinimide) ,
프로파길 -숙신이미딜-에스테르 (propargyl-succinimidyl-ester),
DBCO-말레이미드 (Dibenzocyclooctyne-maleimide),
DBCO-PEG-말레이미드 (Dibenzocyclooctyne-PEG4-maleimide),
DBCO-S-S-NHS에스테르 (Dibenzocydooctyne-S-S-N-hydroxysuccinimidyl ester), DBCO-NHS에스테르 (dibenzocyclooctyne-N-hydroxysuccinimidyl ester),아세틸렌 -PEG-NHS에스테르 (acetylene-PEG-NHS ester),및 알킬렌 -PEG-말레이미드 (alkyne-PEG-maleimide)로이루어진군으로부터 선택되는것을특징으로하는,고분자캐리어물질 -접합 CRISPR효소 단백질의제조방법.
[청구항 9] 제 5항에있어서,상기 (a)단계의고분자캐리어물질의활성화반웅은
DMSO(dimethylsulfoxide), DMF(dimethylformamide),에탄올,메탄올,물, 메틸렌클로라이드,및클로로포름으로이루어진군으로부터선택된용매
대제용지 (규칙제 26조) RO/KR 중에서이루어지는것을특징으로하는제조방법.
[청구항 10] 제 5항에있어서,상기 (a)단계의고분자캐리어물질의활성화반웅은
4~60oC에서, 0.5-24시간동안이루어지는것을특징으로하는제조방법 .
[청구항 11] 제 5항에있어서,상기 (b)단계의고분자캐리어물질및 CRISPR효소 단백질의몰비는 ιο-ι: 1내지 105: 1인것을특징으로하는제조방법 .
[청구항 12] 제 5항에있어서 ,상기 (b)단계의 CRISPR효소단백질의기능기와
활성화된고분자캐리어물질의접합반웅은 4~60oC에서, 1~48시간동안 이루어지는것을특징으로하는제조방법 .
[청구항 13] 제 5항에있어서,상기 (b)단계의 CRISPR효소단백질의기능기와
활성화된고분자캐리어물질의접합반웅은 pH 4~10의수용성용매 증에서이루어지는것을특징으로하는제조방법.
[청구항 14] 제 1항내지제 4항중어느한항의고분자캐리어물질 -접합
CRISPR(Clustered regularly interspaced short palindromic repeats)효소 단백질및 sgRNA(single guide RNA)를포함하는 CRISPR나노복합체 .
[청구항 15] 제 14항에있어서,상기 CRISPR나노복합체는수용액분산상태에서
입도크기가 1내지 10,000 nm인것을특징으로하는 CRISPR나노복합체.
[청구항 16] 제 14항에있어서,상기 CRISPR나노복합체는제타전위가 -100 ~ +100 mV인것을특징으로하는 CRISPR나노복합체.
[청구항 17] 고분자캐리어물질-접합 CRISPR(Clustered regularly interspaced short palindromic repeats)효소단백질및 sgRNA(single guide RNA)를흔합하는 단계를포함하는 CRISPR나노복합체의제조방법 .
[청구항 18] 제 17항에있어서,상기고분자캐리어 -접합 CRISPR효소단백질과
sgRNA의몰비는 1 : 106내지 1 : 106인것을특징으로하는 CRISPR 나노복합체의제조방법.
[청구항 19] 제 1항내지제 4항중어느한항의고분자캐리어물질 -접합 CRISPR 효소단백질,또는제 14항의 CRISPR나노복합체를포함하는유전자 편집용조성물.
[청구항 20] 제 19항에있어서,상기조성물은고분자캐리어물질-접합 CRISPR효소 단백질,또는 CRISPR나노복합체를세포내에전달하여유전자편집을 유도하는것을특징으로하는유전자편집용조성물.
[청구항 21] 제 20항에있어서,상기세포는진핵세포또는원핵세포인것을특징으로 하는유전자편집용조성물.
[청구항 22] 제 21항에있어서,상기세포는 S. aureus, E. coli, P. aeruginosa, K.
pneumoniae, A. baumannii, B. subtilis, S. epidermidis, E. faecalis, S.
pneumoniae, HeLa, A549, MDAMB, SK-BR-3, OVCAR, PC3, PC 12, HEK293, Jurkat, CD4+ T세포, CD8+ T세포, RAW264.7,대식세포, 단핵구,호중구, NK세포 (natural killer cell),수지상세포 (dendritic cell), MDSC(Myeloid-derived Suppressor Cell),배아줄기세포,중간엽즐기세포,
대제용지 (규칙제 26조) RO/KR 유도만능줄기세포 (iPSC),혈관내피세포,표피세포,간세포,근육세포, 뼈세포,섬유아세포,연골세포,신경세포,신경줄기세포 (neural stem cell). ADSC(adipose-derived stem cell), EF(mouse embryonic fibroblast),곰광0 세포,및기생층세포로이루어진군으로부터선택되는것을특징으로 하는유전자편집용조성물. ,
대제용지 (규칙제 26조) RO/KR
PCT/KR2017/013623 2017-06-14 2017-11-27 비바이러스성 유전자편집 crispr 나노복합체 및 이의 제조방법 WO2018230785A1 (ko)

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