KR20180037602A - PARK2 knock-out porcine Models for Parkinson' s disease and the Use thereof - Google Patents

Abstract

The present invention relates to a transgenic pig for a Parkinson′s disease model, a method for producing the same, and a use thereof. More specifically, the present invention relates to a use of a transgenic pig having a PARK2 gene knocked-out as an animal model of Parkinson′s disease.

Description

PARK2 Gene Knockout Parkinson's Disease Model Pigs and Their Uses {PARK2 knock-out porcine Models for Parkinson's disease and the Use thereof}

The present invention relates to a transgenic pig for a Parkinson's disease model, a method for producing the same, and a use thereof, and more particularly to the use of a transgenic pig having a Park2 gene knocked out as an animal model of Parkinson's disease .

Dementia is a disease with general impairment of systemic functions such as memory impairment and loss of judgment. About 50% of the dementias are of dementia of Parkinson type, about 30% of them are dementia of vascular type, alcoholic dementia and Parkinson's disease, about 20% Is known to develop amphotericism such as Parkinsonian dementia and vascular dementia.

Parkinson's disease (PD) is the second most common aging-related disease among senile dementia in the degenerative neurological disease. About 2% of elderly people aged 65 or older suffer from this disease and there are about 1 million patients in the US alone. This disease is accompanied by emotional disturbances such as hand tremor, stiffness, slow motility, instability of the posture, and emotional disturbance and fear. The progression of the disease is progressive and pathologically severe degeneration (about 70-80%) of dopaminergic neurons in the brain of the brain is observed. Within these cells, an abnormal deposition of a protein called the Lewy body is observed.

The population of the elderly in Korea is rapidly increasing, and the number of patients with Parkinson 's disease is also increasing. The cause of Parkinson's disease is not yet clear. However, Parkinson's disease has been reported to occur due to the combined action of genetic factors and environmental factors. In recent years, research on genetic factors of Parkinson's disease has been developing very rapidly.

In this context, the use of animal models to discover new therapies for neurodegenerative diseases is an essential element in discovering new therapeutic targets and performing drug testing at preclinical stages.

The primary concern of neuroscientists is the choice of the most appropriate animal model useful for achieving the research objectives. Researchers are based on mechanisms that are not essential to their patients, but they often face choices between animal models based on known pathogenetic mechanisms, although models with underlying pathologic features of the disease and not all clinical features are reproduced. Therefore, researchers are challenging the development of animal models that are compatible with pathogenesis, symptoms, treatment, and physiological basis. Studies of these animal models will play an important role in accurately identifying the characteristics of dementia, the temporal and spatial changes of abnormal brain cells, and the mechanism of brain dysfunction, and verifying the effectiveness of various new therapeutic targets and new therapies.

Until now, most of the disease models for drug therapy and mechanism studies of degenerative brain diseases have been using rodents, but the pathological patterns and symptoms of animal disease models are much different from those observed in humans. Based on the results from rodent disease models There have been many problems in clinical trials.

That is, a model animal that expresses both symptoms of Parkinson's disease (PD) and Parkinson's (PD) in human enough to be used as an effective disease model has not yet been established.

Therefore, a clear distinction between dimenzon, reproduction, lifespan, and behavior with humans has raised the need for animal models of disease using more human species, and in the case of primates, the cost and difficulty of rarity and breeding management Therefore, there is a growing demand for biomedical research as a new model animal for pigs that can study incurable diseases at a relatively low cost and at a relatively low cost.

Pigs have been recognized for similarity with human anatomy physiologically and have already been used for the pathological mechanism and treatment of various diseases. Especially, when pigs are valued as economic animals for a long time, It can avoid ethical problems and has a stable breeding system, so it is easy to maintain and manage when developing animal models.

Therefore, the present inventors used a pig recognized to be similar to a human gene, but applied a transgenic somatic cell cloning technique using a nuclease that recognizes and cleaves a specific sequence of the human Park2 gene, thereby knocking out the Park2 gene It is possible to produce an animal model of an effective Parkinson's disease, and the present invention has been completed.

1. Lees AJ, Hardy J, Revesz T. Parkinson's disease. Lancet 2009; 373: 2055-2066. 2. Dawson TM, Ko HS, Dawson VL. Genetic animal models of Parkinson's disease. Neuron 2010; 66: 646-661. 3. Dawson TM, Dawson VL. Neuroprotective and neuroresthetic strategies for Parkinson's disease. Nat Neurosci 2002; 5 Suppl: 1058-1061. 4. Bohlhalter S, Kaegi G. Parkinsonism: heterogeneity of a common neurological syndrome. Swiss Med Wkly 2011; 141: w13293. 5. Lang AE, Lozano AM. Parkinson's disease. First of two parts. N Engl J Med 1998; 339: 1044-1053. 6. Lang AE, Lozano AM. Parkinson's disease. Second of two parts. N Engl J Med 1998; 339: 1130-1143. 7. Yue Z, Lachenmayer ML. Genetic LRRK2 models of Parkinson's disease: Dissecting the pathogenic pathway and exploring clinical applications. Mov Disord 2011; 26: 1386-1397. 8. Blesa J, Phani S, Jackson-Lewis V, Przedborski S. Classic and new animal models of Parkinson's disease. J Biomed Biotechnol 2012; 2012: 845618. 9. Hawkes CH, Del Tredici K, Braak H. A timeline for Parkinson's disease. Parkinsonism Relat Disord 2010; 16: 79-84. 10. Brandt I, Gerard M, Sergeant K, et al. Prolyl oligopeptidase stimulates the aggregation of alpha-synuclein. Peptides 2008; 29: 1472-1478. 11. Fearnley JM, Lees AJ. Aging and Parkinson ' s disease: substantia nigra regional selectivity. Brain 1991; 114: 2283-2301. 12. Oliveras-Salva M, Van der Perren, Casadei N, et al. rAAV2 / 7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration. Mol Neurodegener 2013; 8: 44. 13. Gombash SE, Manfredsson FP, Kemp CJ, et al. Morphological and Behavioral Impact of AAV2 / 5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System. PLoS One 2013; 8: e81426. 14. Eslambolia A, Romero-Ramos M, Burger C, et al. Long-term consequences of human alpha-synuclein overexpression in the primate ventral midbrain. Brain 2007; 130: 799-815. 15. Glud AN, Hedegaard C, Nielsen MS, et al. Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the Gottingen minipig CNS. Acta Neurobiol Exp 2011; 71: 508-518. 16. Swindle MM, Machine A, Herron AJ, Clubb FJ, Jr., Frazier KS. Swine as models in biomedical research and toxicology testing. Vet Pathol 2012; 49: 344-356. 17. Koo OJ, Park HJ, Kwon DK, Kang JT, Jang G, Lee BC. Effect of recipient breed on delivery rate of cloned miniature pig. Zygote 2009; 17: 203-207. 18. Moon J, Lee C, Kim SJ, et al. Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases. Mol Ther Nucleic Acids 2014; 3: e166. 19. Ramaker C, Marinus J, Stiggelbout AM, Van Hilten BJ. Systematic evaluation of rating scales for impairment and disability in Parkinson's disease. Mov Disord 2002; 17: 867-876. 20. Moon JH, Kim JH, Im HJ, et al. Proposed Motor Scoring System in a Porcine Model of Parkinson's Disease Induced by Chronic Subcutaneous Injection of MPTP. Exp Neurobiol 2014; 23: 258-265. 21. Freichel C, Neumann M, Ballard T, et al. Age-dependent cognitive decline and amygdala pathology in alpha-synuclein transgenic mice. Neurobiol Aging 2007; 28: 1421-1435.

It is an object of the present invention to provide a pig for a Parkinson's disease model and a method for producing the same.

Another object of the present invention is to provide a recombinant vector, a cell transformed with the recombinant vector, and a nuclear transfer embryo necessary for the method for producing pigs for the Parkinson's disease model.

It is another object of the present invention to provide a gene knockout or knockdown method using an artificial nuclease system necessary for the method for producing pigs for the Parkinson's disease model.

Another object of the present invention is to provide various uses of pigs for the Parkinson's disease model.

It is another object of the present invention to provide a method for screening for a preventive and therapeutic agent for Parkinson's disease using the pig for the Parkinson's disease model.

In order to solve the above problems,

Provides an artificial manipulation of the Park2 gene and its use for the production of pigs for the Park2 deficiency disease model. More specifically, the present invention relates to a method for producing a pig having a Park2 deficient phenotype by using an artificial manipulation or modification of the Park2 gene and its use. The Park2 deficient phenotype may appear as Parkinson's disease.

Therefore, the present invention can provide a pig model and its use useful for studying the progression and pathological mechanism of Parkinson's disease.

The present invention provides a composition for manipulating or modifying the Park2 gene and a method for using the composition for a specific purpose.

In one embodiment, a Park2 deficient disease pig model is provided wherein the Park2 gene knocked out by deformation in the nucleic acid sequence of the Park2 gene, and Park2 protein expression is inactivated.

Parkin (Park2) is a protein composed of 465 amino acids and is a kind of ubiquitin-protein ligase 3 The amino terminus is similar to ubiquitin, and the carboxy terminus has two ring finger domains. An example is shown in SEQ ID NO: 1 herein.

Modifications in the nucleic acid sequence may include deletion, substitution, insertion or inversion of one or more nucleic acids.

In one embodiment, the modification of the nucleic acid may have occurred in the exon 6 region of the Park2 gene.

In one embodiment, modifications in the nucleic acid sequence can be artificially caused by the nuclease agent.

For example, the nuclease agent may use one or more of the following nuclease agents:

(a) zinc finger nuclease (ZFN);

(b) Transcription activator-like effector nuclease (TALEN);

(c) meganuclease; or

(d) RNA-Guided Endonuclease (RGEN)

In one embodiment, the modification in the nucleic acid sequence can be artificially engineered by a transcription activator-like effector nuclease (TALEN) or an RNA-guided endonuclease (RGEN). The subject can be a target nucleic acid, gene, chromosome or protein.

The TALEN also includes a form in which a TAL effector DNA binding domain and a DNA cleavage domain are linked through a linker. "TALEN targeting PARK2 gene" means TALEN which results in cleavage of the PARK2 gene by binding to a specific sequence of the PARK2 gene. The TALEN targeted to the PARK2 gene preferably specifically cleaves the exon 6 of the PARK2 gene, more preferably TALEN which binds to SEQ ID NO: 2 or 3 site located in exon 6 of the PARK2 gene.

The RNA-guided endonuclease may be selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces viridachromogenes, Streptomyces spp. Streptosporangium roseum, Streptosporangium roseum, Alicyclobaclus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, and the like. , Exiguobacterium si Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphtalenibolans (Bacillus spp.), Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Cynokocorsus sp. For example, in the order of the genus Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natra nna The present invention relates to a method for the treatment and / or prophylaxis of a disease or condition selected from the group consisting of Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, For example, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, (Eg, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthr sp. ospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipo africanus, Thermosipho africanus) or Acaryochloris marina (Acaryochloris marina). Additional examples of the Cas9 family are described in WO 2014/131833, the entire contents of which are incorporated herein by reference. It may be a Cas9 protein derived from Streptococcus pyogenes.

For example, the Park2 gene can be genetically engineered by TALEN (transcription activator-like effector nuclease)

Among the nucleic acid sequences constituting the Park2 gene,

Deletion or insertion of one or more nucleotides;

Substitution with one or more nucleotides that is different from the wild type gene;

Insert one or more nucleotides from outside

Lt; RTI ID = 0.0 > of < / RTI >

On the other hand, in the present invention, the modification of the Parkin (Park2) nucleic acid can occur at multiple sites in two or more chromosome whole genome sequences.

In a specific embodiment, at least one or more sites, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides may be altered in the Park2 gene nucleic acid sequence. Whereby the Park2 gene can be knocked out or knocked down.

Modifications of the nucleic acid can occur in the promoter region of the gene.

Modifications of the nucleic acid can occur in the exon region of the gene. Preferably in the exon 6 region.

Modifications of the nucleic acid can occur in the intron region of the gene.

The modification of the nucleic acid may occur in the enhancer region of the gene.

Also, in another embodiment, it provides a sequence recognizable by TALEN in the nucleic acid sequence of the Park2 gene.

In another embodiment, the present invention provides a guide nucleic acid capable of forming a complementary bond, respectively, to a target sequence in the nucleic acid sequence of the Park2 gene.

The sequences may be at least one of the nucleotide sequences of the Park2 gene.

The sequence recognizable by TALEN may be, but is not limited to, nucleotides of 18-25 bp, 18-24 bp, 18-23 bp, 19-23 bp, 19-23 bp, or 20-23 bp.

For example, a sequence recognizable by the following TALEN can be provided:

5-TGAGTGCCAGTCTCCAAACT-3 (SEQ ID NO: 2)

5- AGCAGTAAGTACTGGTCAAA- 3 (SEQ ID NO: 3)

Further, in an embodiment, the present invention may provide an artificially engineered cell or embryo comprising said artificially engineered Park2 gene and at least one of the products expressed therefrom.

The cell or embryo may contain an inactivated Park2 gene that has undergone a modification in the nucleic acid sequence and may develop into an individual having a phenotype thereof.

Further, in an embodiment, the present invention can provide a composition for genetically manipulating the Park2 gene and its use.

In one embodiment, a guide nucleic acid capable of forming a complementary bond to at least one site of the nucleic acid sequence constituting the Park2 gene; And an RNA-guide endonuclease or a nucleic acid encoding the same.

In another embodiment, there is provided a Park2 gene manipulation composition comprising a transcription activator-like effector nuclease (TALEN) capable of recognizing and cleaving at least one region of a nucleic acid sequence constituting the Park2 gene or a nucleic acid encoding the same.

In certain embodiments, the invention provides a composition for Park2 gene knockout comprising a TAL effector nuclease pair comprising a first and a second TAL effector nuclease monomer or a nucleic acid encoding it.

The TAL effector DNA binding domain of the first TAL effector nuclease monomer binds to the target sequence 5-TGAGTGCCAGTCTCCAAACT-3 (SEQ ID NO: 2), and the TAL effector DNA binding domain of the second TAL effector nuclease monomer binds to the target sequence 5-AGCAGTAAGTACTGGTCAAA-3 (SEQ ID NO: 3).

The DNA cleavage domain in each TAL effector nuclease monomer can be located at the C-terminus of the TAL effector DNA binding domain. The DNA cleavage domain of the TAL effector nuclease monomer may be the DNA-cleavage domain of FokI-endonuclease.

In one embodiment,

There is provided a method for producing a cell or embryo in which the nucleic acid sequence of the Park2 gene has been altered, comprising contacting the Park2 gene-expressing composition with a cell or an embryo comprising the target genomic locus on the Park2 gene.

The TALEN, RNA-guide endonuclease, guide RNA, and the like may be present in one or more vectors each in the form of a nucleic acid sequence, or may be present by forming a complex by binding of a guide nucleic acid and an RNA-guide endonuclease .

The contacting step may be carried out in vivo or ex vivo. Preferably, in vitro.

The step of contacting may be carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods.

The viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.

In an embodiment,

The present invention provides a cell line transformed with a recombinant vector for producing a disease pig model knocking out the Park2 gene, and a pig for a disease model in which the Park2 gene is knocked out. At this time, the transformation can be performed by somatic cell nuclear transfer (SCNT).

The present invention includes all the transformation vectors, cells, and nuclear transfer embryos necessary for modeling Park2 deficiency diseases which can be obtained in the process of the method for producing pigs for the Park2 deficiency disease model.

For example, it is possible to provide a transformed cell into which a recombinant vector for constructing a Park2 deficiency disease pig model containing a sequence encoding an artificial nuclease having an activity of recognizing and cleaving a Park2 gene-specific sequence is introduced, The nucleus of the transformed porcine-derived cells can also be provided in a porcine nuclear transfer embryo formed by implantation into a enucleated oocyte.

In an embodiment, the invention can provide a method and such an entity for obtaining an individual produced by said method.

The subject may be one or more animals selected from the group consisting of non-human primates, cows, horses, pigs, sheep, chickens, birds, rabbits, goats, dogs, cats and fish.

The present invention also provides a variety of uses for pigs for Park2 deficiency diseases such as Parkinson's disease models.

As an example, there is provided a method of screening for a Park2 deficiency disease, for example a Parkinson's disease preventive and therapeutic agent, comprising:

Administering a prophylactic and therapeutic agent candidate to the pig for the Park2 deficiency disease model; And after administering the candidate substance, analyzing the tissue of the pig compared to a control group to which the candidate substance is not administered.

Thus, the present invention provides animal models of Park2 deficiency disease using pigs, which are physiologically similar to humans, and their various uses.

The pig for the Parkinson's disease model of the present invention is an animal model that is genetically similar to human, useful for genetic analysis and useful for studying precise cause and mechanism of disease, and also suitable for toxicity and safety evaluation. , The search for therapeutic substances, the development of diagnostic methods, and the like, so that it is very useful as an animal model of Parkinson's disease.

FIG. 1 shows the result of indirectly measuring the mutation efficiency of a blastocyst prepared by in vitro culture.
FIG. 2 shows the result of analysis of genomic DNA and blastocysts produced in in vitro condition using mismatch-sensitive nuclease assay to verify efficiency of donor cells.
Figure 3 is a photograph of the cleft, liver, brain, heart, lung, kidney and spleen of PDM5.
4 is a photograph of the PDM 10 and the PDM 11 replicating the PDM 3 again.
FIG. 5 shows gene expression analysis results of pigs (PDM1 to PDM11) for the Parkinson's disease model of the present invention.
FIG. 6 is a photograph of the PET and CT images of a 1-year-old Parkin KO mini-pig 18F-FDG.
Fig. 7 is a photograph of PET-CT of a 1-year-old Parkin KO mini-pig with FP-CIT.
Fig. 8 shows the image of a 1-year-old Parkin KO mini-pig.
FIG. 9 is a photograph of MRI of a 1-year-old Parkin KO mini pig.
FIG. 10 is a photograph showing the ingestion of feces showing a difference from a normal pig and a state in which the tongue is exposed to the outside.

Representative terms used in the present invention are as follows.

"Engineered nuclease" is a term collectively referred to as an artificial nuclease and / or a system containing it, which is capable of binding to an arbitrary gene and capable of damaging or destroying a specific nucleic acid site, for example, a specific DNA site . May also be referred to as "target-specific (endo) nuclease" or "engineered nuclease. &Quot; The gene scissors are sequence-specific endonuclease. "Sequence-specific endonuclease" or "sequence-specific nuclease" refers to a polynucleotide that recognizes and binds a polynucleotide, eg, a target gene, in a particular nucleotide sequence, unless specifically stated otherwise, ≪ / RTI > refers to a protein that catalyzes single- or double-strand breaks in E. coli. Since the nucleic acid can be cleaved by specific recognition of a specific sequence, it is very useful for genome editing technology. For example, ZFN (zinc finger protein), TALEN (transcription activator-like effector protein), and RGEN (RNA-guided endonuclease) including CRISPR / Cas system.

"Cell "," host cell ", "transformed host cell" and the like refer to a progeny or potential progeny of such a cell as well as a specific target cell. Such a offspring, in fact, would not be identical to the parent cell, but is still included within the scope and range of terms used in the present invention, since certain modifications may occur later in time by mutation or environmental effects.

The term "modified" or "engineered ", as applied to nucleic acids, is used interchangeably herein to refer to the artificial alteration of a nucleic acid sequence constituting a gene. Such alterations may include deletion, substitution, insertion or inversion of one or more nucleic acids, and the like. A modified or engineered nucleic acid sequence may appear as an alteration, for example, an increase, enhancement, decrease or loss of expression and / or functional activity of these nucleic acids or an expression product thereof.

The terms "damage" and "destruction ", as applied to nucleic acids, are used interchangeably herein to refer to any genetic modification that reduces or eliminates the expression and / or functional activity of a nucleic acid or an expression product thereof. For example, destruction of a gene is included within the scope of any genetic modification that reduces or eliminates the expression of the gene and / or the functional activity of the corresponding gene product (e.g., mRNA and / or protein). Genetic modification includes complete or partial inactivation, repression, deletion, disruption, containment or downregulation of a nucleic acid (e.g., a gene).

The term "cleavage " applied to a nucleic acid means to disassociate the covalently bonded backbone of the nucleotide molecule of the gene.

"Disease model animal" refers to an animal with a disease that closely resembles a human disease. Diseases in the study of human disease The significance of model animals is due to their physiological or genetic similarities between humans and animals. Biomedical disease models in disease studies Animals provide research materials for various causes of diseases, pathogenesis and diagnosis, and research on disease-related animals to identify disease-related genes and understand the interactions between genes And the basic efficacy and toxicity test of the developed new drug candidates will provide basic data for judging the possibility of practical use.

An "animal" or "laboratory animal" means any mammal other than a human. The animal includes animals of all ages, including embryos, fetuses, neonates and adults. Animals for use in the present invention can be used, for example, from commercial sources. Such animals include, but are not limited to, laboratory animals or other animals, rabbits, rodents (such as mice, rats, hamsters, gerbils and guinea pigs), cows, sheep, pigs, goats, horses, (Eg, chickens, turkeys, ducks, geese), primates (eg, chimpanzees, monkeys, rhesus monkeys). The most preferred animal is a pig.

&Quot; Knock-out " refers to a modification of a gene sequence that results in a degradation of a target gene, preferably by which target gene expression is not detectable or meaningless. In the present invention, the knockout of the Parkin (Park2) gene means that the function of the Parkin (Park2) gene is substantially lowered so that expression can not be detected or exists only at a meaningless level. The knockout transformant may be a transgenic animal having a heterozygous knockout of the Parkin (Park2) gene or a homozygous knockout of the Parkin (Park2) gene. In knock-outs, for example, in the case of exposing the animal to a substance that promotes target gene degeneration, introducing an enzyme that promotes recombination at the target gene site, or other methods of directing target gene modification after birth, Conditional knock-outs in which deformation can occur are also included.

A " target gene " refers to a nucleic acid encoding an intracellular polypeptide unless specifically stated otherwise. The term "target sequence ", as used herein, refers to a portion of a target gene, for example, one or more exon sequences of a target gene, an intron sequence or a control sequence of a target gene or an exon and intron sequence of a target gene, And control sequences, exons and control sequences, or combinations of exons, introns, and control sequences.

&Quot; Sequence homology " refers to sequences that align sequences with respect to donor and target gene polynucleotide sequences and, if necessary, introduce a gap to achieve maximum percent sequence identity, and then, compared to nucleotide residues in the target gene sequence Is defined as the percent identity of the nucleotide residues in the donor sequence. Alignment to determine percent nucleotide sequence homology can be accomplished in a variety of ways that are within the skill of the art, for example, using commercially available computer software BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) have. The skilled artisan will be able to determine appropriate parameters for the ordering of the sequence, such as all the algorithms necessary to achieve maximal alignment over the entire length of the sequences to be compared.

'Nuclear transplantation' refers to a genetic manipulation technique that allows an enucleated oocyte to artificially bind other cells or nuclei to the same trait. "Nuclear oocyte" refers to an oocyte into which nuclear donor cells are introduced or fused.

'Cloning' is a genetic manipulation technique for creating a new individual having the same gene set as an individual. In particular, in the present invention, a somatic cell, an embryo cell, a fetal cell and / or an adult cell of a pig is substantially And has the same nuclear DNA sequence. The present invention utilizes a technique of replicating pigs using nuclear transfer technology. In particular, somatic cell nuclear transfer technology is a technology that can produce offspring without passing through the meiosis and hemispheric retention cells that are common in the reproductive process. As a technology, transplanted somatic cells are transplanted into the nucleus- And the embryo is transplanted in vivo to generate a new individual.

'Nuclear donor cells' refers to nuclei of cells or cells that deliver nuclei to recipient oocytes, which are nuclear receptors. The term " oocyte " preferably refers to a mature oocyte that has reached the middle stage of the second meiosis. In the present invention, porcine somatic cells or stem cells can be used as the nuclear donor cells.

The term "somatic cell" refers to a cell other than a germ cell that constitutes a multicellular organism, a cell that is specialized for a specific purpose and does not become any other cell, and a cell having several different functions Cells that have the ability to differentiate.

"Stem cell" refers to a cell that can develop into any tissue. There are two basic features: self-renewal, which creates itself by repeated division, and the ability to differentiate into cells with specific functions depending on the environment.

'Cultivation' is the cultivation of living organisms or parts of organisms (organ, tissue, cells, etc.) under moderately artificially controlled environmental conditions. In this case, external conditions such as temperature, humidity, light, gas phase composition The most important direct effect on the organism cultivated is the culture medium, which is a direct environment for the organism and a supply point for various nutrients necessary for survival and growth.

"In vitro culture" means a series of laboratory processes in which cells are cultured in an incubator in a laboratory in a manner similar to the environment in the body, in a manner different from that in which the cells grow.

The term "medium" or "medium composition" refers to a mixture for growth and proliferation of cells, etc., which is essential for growth and proliferation of cells such as sugar, amino acids, various nutrients, serum, growth factors and minerals.

"Living offspring" refers to an animal that can survive outside the uterus. Preferably, it refers to an animal that can survive for 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months, or 1 year. The animal does not require an intrauterine environment for survival.

&Quot; Treatment " refers to an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction &Quot; Treatment " refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. Palliating the disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression compared to when the disease is not treated, It means to lose.

By "about" is meant a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprises "and" comprising ", when not explicitly required in the context, include a stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to an animal model of Parkin (Park2) gene deficiency disease.

More specifically, the present invention relates to a method for artificially transforming a nucleic acid sequence of Parkin (Park2) gene and its use.

[Parkin-deficient pig model]

As a model animal, pigs have a longer survival period than conventional rodent models and are known to have a similar biological mechanism to humans, and are thus attracting attention as a next-generation disease model animal species that can overcome the limitations of the prior art.

Therefore, the present inventors developed and used a more reliable animal model of Parkin (Park2) deficiency disease through production and behavior of a disease model that exceeds the rodent model using the large animal pig. For example, it may be an animal model of Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, most often occurring between the ages of 50 and 60 years, with a frequency of occurrence between men and women similar or slightly higher in men (Tanner and Aston, 2000). The four major signs of Parkinson's disease are tremor, rigidity, bradykinesia, and poor postural reflexes (Benatru et al., 2008). The cause of Parkinson's disease is not known precisely yet, but it is known to be caused by a lack of dopamine, a secreted substance in the substantia nigra to control the function of the basal ganglia (Di Monte, 2000 ). The main symptoms of Parkinson's disease are physical problems such as muscle stiffness, gait disturbances, balance disorders, and cognitive dysfunction (Emre, 2003), and psychological problems such as depression and self-efficacy decline after onset (Hantz et al., 1994), ultimately reducing the quality of life and increasing the burden of dependents (Ray et al., 2006; Olanow et al., 2009). There are no clear remedies or remedies available to cure this disease.

Parkinson's disease is characterized by the degenerative changes and death of substantia nigra dopamine neurons and the Lewy body in which cytoplasmic inclusion of α-synuclein occurs, resulting in various Parkinsonian symptoms . In recent years, pathological findings have been observed not only in specific areas such as black and basal ganglia but also in general brain areas including the brain stem. In addition to exercise symptoms, autonomic symptoms and mental psychological symptoms are also important, Making a model is very difficult

An animal model of Parkinson's disease is a neurotoxic model induced by the administration of environmental toxins or synthetic toxins and a genetic animal model produced by genetic modification of α-synuclein, Parkin, Pink1, DJ-1, LRRK2, .

Animal models using neurotoxic drugs have been used to administer 6-OHDA (6-hydroxydopamine), a dopaminergic neurotoxin, to black blood cells, or as a neurotoxic compound to make Parkinson's rat and monkey models, MPTP (1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine, a method using a pesticide rotenone or a herbicide paraquat, and the like.

The neurotoxicity model has limitations in the finding of progressive neuronal degeneration, leucovorosis, and non-motor symptoms. Therefore, we are studying to make an animal model similar to the mechanism of disease induction in patients with Parkinson's disease through a genetic model.

Genetic model animals for the study of existing degenerative brain diseases are mostly rodent models employing transformation techniques, and other animal species have many limitations. Thus, the existing Parkinson animal model is limited to mice, and the need for PD disease model production is rapidly increasing for various reasons such as genetic similarity with humans and fertility, and thus, the Parkinson pig model has not been developed to date.

Therefore, the present inventors developed a more reliable animal model of Parkinson's disease by utilizing the large animal pig, confirming the induction of Parkinson's disease through the production and behavior of a disease model that exceeds the rodent model, and developed the animal model of Parkinson's disease.

In one aspect, the present invention relates to a transgenic animal model knocked out the Parkin (Park2) gene, preferably a pig model of Parkinson's disease.

In one embodiment, the invention relates to a genetically modified animal, in particular a recombinant pig knockout of the Parkin (Park2) gene, preferably a mini pig.

Pigs are anatomically similar in size to organs and are suitable as heterogeneous organs, as well as physiologically and genetically similar to humans. In addition, it has high breeding capacity, which makes it suitable for the development of biological materials for production and treatment as well as an animal model that is good for toxicity and safety evaluation.

The pigs to be used in the present invention can be any known kind which can be appropriately selected and used by a person skilled in the art.

Pigs (Scientific name: SusscrofPDomestica) belong to the subspecies, descent descent, emerald-side subspecies, right titles (including cattle, chlorine) and wild boar, and the number of chromosomes is 2n = 38. Originally, pigs have attracted much attention as experimental animals as well as ovarian animals raised with cattle, with a similar interest in physiological anatomical findings and a high interest in animal welfare in recent years. The pigs are attracting much attention as physiological anatomy. Pigs are widely used throughout the study because they are similar to humans in many physiological and anatomical points.

Particularly, in the present invention, a small pig (typical example: Pittman, Moore) having a maximum body weight of 60-70 kg when maturing is used, or a small money (adult: 30-40 kg) A yukatan mini pig, a micro pig, or the like may be used. In recent years, there has been production of a near-mini-pig (NIH system) not only having a small body shape but also having a characteristic. In one embodiment of the present invention, a yucatan mini pig was used

[PARK2 genetic modification or modification]

The Parkinson's disease pig model of the present invention is characterized in that the Parkin (Park2) gene is transformed to knockout.

Parkin (Park2) is a protein composed of 465 amino acids and is a kind of ubiquitin-protein ligase 3 The amino terminus is similar to ubiquitin, and the carboxy terminus has two ring finger domains.

Each nucleic acid encoding Parkin (Park2) can be used without limitation as long as it has a nucleotide sequence encoding PARK2 known in the art.

The nucleic acid encoding Parkin (Park2) can also be produced by a recombinant method known in the art. For example, PCR amplification for amplifying a nucleic acid from a genome, chemical synthesis, or a technique for producing a cDNA sequence.

The nucleic acid may also have a nucleotide sequence encoding each functional equivalent of Parkin (Park2).

The functional equivalent means a sequence homology of at least 70%, preferably 80%, more preferably 90% or more with the amino acid sequence represented by SEQ ID NO: 1, 2 or 3 as a result of addition, substitution or deletion of amino acids Refers to a polypeptide having physiological activity substantially equivalent to the SNCA of the present invention of the present invention.

At this time, deletion or substitution of the amino acid is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. In a preferred embodiment of the present invention, the amino acid sequence encoding Parkin (Park2) is represented by SEQ ID NO: 1.

Any of the known methods may be used to knock out the Parkin (Park2) gene, for example, using known gene scissors. For example, ZFN (zinc finger protein), TALEN (transcription activator-like effector protein), and RGEN (RNA-guided endonuclease) including CRISPR / Cas system.

In another embodiment, manipulation of the Parkin (Park2) gene may be applied to the cell or embryo to inactivate it.

In one embodiment, the cell or embryo is an in vitro cell or embryo that specifically binds to a chromosomal target site of a cell or an embryo to cause double stranded DNA breakage to interfere with the gene to selectively inhibit the germ cell formation process, The agent may be selected from the group consisting of known gene scissors (targeting endonuclease) and recombinant enzyme fusion proteins.

In another embodiment, the manipulation of the Parkin (Park2)

For example, a targeted change in the polynucleotide of interest, including a targeted change in the Parkin (Park2) gene or a targeted change in other desired polynucleotides.

Such targeted modifications include the addition of one or more nucleotides, deletion of one or more nucleotides, substitution of one or more nucleotides, knockout of the polynucleotide of interest or a portion thereof, dissolution of the polynucleotide of interest or a portion thereof, Substitution with a nucleic acid sequence, or combinations thereof. In a specific embodiment, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are altered to form a targeted genomic modification

In an illustrative example, a target sequence is a nucleotide sequence that is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%.

The Parkin (Park2) gene manipulation of the present invention can be performed in consideration of the process of regulating gene expression.

In embodiments, selection may be made of suitable manipulation means for each step in transcription regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translational regulation or protein modification regulation.

In embodiments, RNAi (RNA interference or RNA silencing) can be used to prevent small RNAs (sRNAs) from interfering with mRNA, degrading stability and, in some cases, destroying protein synthesis information, Expression can be controlled. In an illustrative example, the expression product comprises a target region corresponding to the nucleotide sequence of the Parkin (Park2) gene and contains an RNA molecule (e.g., siRNA, shRNA, miRNA, dsRNA) that weakens or destroys the expression of the Parkin Etc.).

In embodiments, wild-type or variant enzymes that can catalyze the hydrolysis (cleavage) of DNA or RNA molecules, preferably bonds between nucleic acids in DNA molecules, can be used.

In certain embodiments, a meganuclease; Zinc finger nuclease; TALE-nuclease; RNA-Guided Endonuclease (RGEN), for example, one or more nucleases selected from the group consisting of CRISPR / Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) The expression of the genetic information can be controlled.

In one aspect, compositions and methods for targeting and genetically manipulating some or all of the non-coding or coding regions of the Parkin (Park2) gene can be provided.

The composition and method

In embodiments, for inactivation of the desired Parkin (Park2), some of the Parkin (Park2) gene nucleic acid sequences involved may be engineered or modified. As a result of the operation, it includes knock down, knock out, and knock in forms.

In embodiments, a promoter region, or a transcription sequence, such as an intron or exon sequence, may be targeted. A cipher sequence, for example a cipher region, an initial cipher region, can be targeted for alteration of expression and knockout.

In an embodiment, the nucleic acid modification is carried out in the presence of one or more nucleotides, such as 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, Deletion, and / or insertion of 1 bp nucleotides.

In an embodiment, it can be targeted to include deletions or mutations in one or more of the Parkin (Park2) genes.

In an embodiment, the gene knockdown can target the transcription site of the unwanted Parkin (Park2) gene.

In certain embodiments, exon regions can be targeted. For example, the exon 6 region can be targeted.

In an embodiment, it can be used to alter the expression or activity of the Parkin (Park2) gene by targeting a non-coding sequence of a promoter, enhancer, intron, 3'UTR, and / or polyadenylation signal.

The genetic modification may be a regulation, eg, inactivation, of a gene by deletion, substitution, and / or insertion of one or more nucleotides, all or a portion of the gene (eg, one or more nucleotides). The gene inactivation means that the gene is inhibited from expression or downregulation of the gene or modified to encode a protein whose original function is lost. In addition, gene regulation can be accomplished by structural alteration of the protein resulting from deletion of the Exon site by simultaneously targeting both intron regions surrounding one or more exons of the target gene, expression of dominant negative forms, competitive inhibitors secreted in soluble form Expression of the gene may result in a change in function of the gene

The gene modification may be to inactivate the gene so that the protein encoded from the gene is not expressed in the form of a protein having the original function

In one example, the transgenesis may be by inactivating the targeted gene by catalyzing a single stranded or double stranded cleavage of a specific site in a gene targeted by a genetic correction system comprising a targeting endonuclease .

Nucleic acid strand breaks catalyzed by a targeting endonuclease can be repaired via mechanisms such as homologous recombination or nonhomologous end joining (NHEJ). In this case, when the NHEJ mechanism occurs, a change is made in the DNA sequence at the cleavage site, whereby the gene can be inactivated.

In this case, when the NHEJ mechanism occurs, a change is made in the DNA sequence at the cleavage site, whereby the gene can be inactivated. Repair via NHEJ results in short gene fragment substitutions, insertions or deletions and can be used to induce corresponding gene knockouts.

The variant of the Parkin (Park2) gene may be derived by one or more of the following:

(1) deletion of all or part of the target gene (hereinafter referred to as a "target gene"), such as 1 to 30 nucleotides, 1 to 27 nucleotides, 1 to 25 nucleotides, 1 to 23 nucleotides, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide,

2) a nucleotide of 1 bp or more, such as 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5 , One to three, or one nucleotide to the original (wild type) and to a different nucleotide, and

3) one or more nucleotides, such as from 1 to 30, from 1 to 7, from 1 to 25, from 1 to 23, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5, 3, or 1 nucleotide (each independently selected from A, T, C, and G) at any position of the target gene.

A portion of the target gene that is modified (the 'target site') may be at least 1 bp, 3 bp, 5 bp, 7 bp, 10 bp, 12 bp, 15 bp, 17 bp, 20 bp, 3bp to 30bp, 5bp to 30bp, 10bp to 30bp, 12bp to 30bp, 15bp to 30bp, 17bp to 30bp, 20bp to 30bp, 1bp to 27bp, 3bp to 27bp, 5bp to 27bp, 7bp to 27bp, 27bp, 15bp to 27bp, 17bp to 27bp, 20bp to 27bp, 1bp to 25bp, 3bp to 25bp, 5bp to 25bp, 7bp to 25bp, 10bp to 25bp, 12bp to 25bp, 15bp to 25bp, 17bp to 25bp, 20 bp to 23 bp, 3 bp to 23 bp, 5 bp to 23 bp, 7 bp to 23 bp, 10 bp to 23 bp, 12 bp to 23 bp, 15 bp to 23 bp, 17 bp to 23 bp, 20 bp to 23 bp, 1 bp to 20 bp, 3 bp to 20 bp, 20bp, 7bp to 20bp, 10bp to 20bp, 12bp to 20bp, 15bp to 20bp, 17bp to 20bp, 21bp 25 bp, 18 bp to 22 bp, or 21 bp to 23 bp.

Therefore, in an exemplary embodiment of the present invention,

Park2 (Park2) gene function is reduced or eliminated by genetically modifying or manipulating a target sequence region, that is, a site where nucleic acid modification can occur, in at least one region of the nucleic acid sequence constituting the Parkin (Park2) gene.

The target sequence may simultaneously target two or more sites in the nucleic acid sequence constituting the Parkin (Park2) gene.

In one embodiment, one or more of the nucleic acid sequences constituting the Parkin (Park2) gene may be targeted using one or more TAL effector nuclease monomers. Thereby knocking out or knocking down the Parkin (Park2) gene.

In one embodiment, at least one site of the nucleic acid sequence constituting the Parkin (Park2) gene can be targeted using one or more guide RNAs. Thereby knocking out or knocking down the Parkin (Park2) gene.

In one embodiment, the level and / or deactivation of the Parkin (Park2) gene is altered by alterations in the nucleic acid sequence that make up the Parkin (Park2) gene, such as in a non-coding region, coding region, and / Can be attributed.

Such Parkin (Park2) gene nucleic acid sequence modifications include, but are not limited to, addition, deletion or substitution of nucleotides into the genome.

Insertion of one or more nucleotides into Parkin (Park2) gene nucleic acid sequences,

Deletion of one or more nucleotides from the Parkin (Park2) gene nucleic acid sequence,

Substitution of one or more nucleotides in Parkin (Park2)

Knockout of a Parkin (Park2) gene nucleic acid sequence or a portion thereof,

Substitution of the endogenous nucleic acid sequence of a Parkin (Park2) gene or a portion thereof with a heterologous nucleic acid sequence, or

And Parkin (Park2) gene nucleic acid sequence changes including the combination.

Thus, in a specific embodiment, it can be reduced or eliminated by destroying a gene or the like that encodes a protein or polypeptide having the activity of the Parkin (Park2) gene. In a specific embodiment, at least one, at least one, two, three, four, five, seven, eight, nine, ten or more nucleotides are changed in the Parkin (Park2) gene nucleic acid sequence. Using various methods, it is possible to cause additional targeted deformations.

In certain embodiments, the level and / or deactivation of the Parkin (Park2) protein may be due to genetic modification of the Parkin (Park2) gene (i.e., regulatory region, coding region, and / or genetic modification in the intron, etc.) .

Such genetic modifications include, but are not limited to, addition, deletion or substitution of nucleotides into the genome.

Such genetic modification may, for example,

Insertion of one or more nucleotides into the Parkin (Park2) gene,

Deletion of one or more nucleotides from the Parkin (Park2) gene,

Substitution of one or more nucleotides in the Parkin (Park2) gene,

Knockout of the Parkin (Park2) gene or its part,

Parkin (Park2) gene changes, including the mutation of a Parkin (Park2) gene or a portion thereof, replacement of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence, or a combination thereof.

Thus, in a specific embodiment, the activity of the Parkin (Park2) polypeptide can be degraded or eliminated by disrupting the gene encoding the Parkin (Park2) polypeptide. In a specific embodiment, at least 1, 2, 3, 4, 5, 7, 8, 9, 10 or more nucleotides are altered in the Parkin (Park2) gene. Using a variety of methods, additional targeted genetic modifications can be made.

[NUCLEASE SYSTEM]

In embodiments of the invention, the following targeted nuclease system or components of such a system may be used to modify the genome of the Parkin (Park2) gene. It is collectively referred to as nuclease preparation. In a preferred example, a transcription activator-like effector nuclease (TALEN) or an RNA-guided endonuclease (RGEN) system can be used.

Zinc finger nuclease system

Zinc-Finger Nuclease (ZFN) is an artificial restriction enzyme created by fusion of zinc finger DNA-binding domains. The zinc finger domain can be engineered to target the desired DNA sequences, which allows the zinc-finger nuclease to target specific sequences within the complex genome. Taking the advantage of an endogenous DNA repair machine, the reagents can be used to transform the genome of higher organisms. ZFN can be used to inactivate genes.

The zinc finger DNA-binding domain has about 30 amino acids and folds into a stable structure. Each finger binds primarily to the triplet in the DNA matrix. Amino acid residues at critical locations contribute to most of the sequence-specific interactions with DNA sites. These amino acids can be changed while preserving the remaining amino acids to preserve the essential structure.

Binding to longer DNA sequences is accomplished by binding several domains simultaneously. Other functional groups such as non-specific FokI cleavage domain (N), transcriptional activator domain (A), transcriptional repressor domain (R), and methylase (M) are fused to ZFP to produce ZFN, A factor (ZFA), a zinc finger transfer inhibitory factor (ZFR), and zinc finger methylase (ZFM).

Materials and methods for using zinc finger and zinc finger nuclease to produce genetically engineered animals are described, for example, in U.S. Patent No. 8,106,255, U.S. Patent Application No. 2012/0192298, U.S. Patent No. 2011/0023159, 0.0 > 2011/0281306. ≪ / RTI >

TALEN System

"TALEN (Transcription activator-like effector nuclease)" is a protein consisting of DNA-binding domain and DNA cleavage domain (Nuclease domain, endonuclease domain).

The DNA binding domain located at the N-terminal of TALEN can utilize transcription activator-like effectors (TALEs) proteins found in plant pathogenic bacteria, Xanthomonas spp.

TALE protein is a structure consisting of 33 to 35 amino acid repeats, and one module recognizes one base pair. The base pairs recognized by the module are determined by the type of amino acids at the 12th and 13th positions of the module, and these amino acids are called RVD (Repeat variable digitsidues). The RVD unit used in TALE is 20 units, each of which binds to one DNA base in the major groove of the DNA chain.

RVD (Single Spell Code) RVD (Three Spell Codes) Nucleotide (s):

NH Asn-His G

HD His-Asp C

NG Asn-Gly T

NI Asn-Ilea

NN Asn-Asn R (G, A)

NK Asn-Lys G

NS Asn-Ser N (A, C, G, T)

"RVD sequence" is understood to be a contiguous sequence of RVD in the TAL effector binding domain, in which case the sequence is specified in the NC-direction, i.e. from the N-terminus to the C- have. It will be appreciated by those skilled in the art that the RVDs of a single "RVD sequence" are not directly contiguous in that direction, and even if they are not directly covalently bonded to one another, ) Are directly coupled to each other, so that the RVDs are separated by the amino acids of the basic structure of the repeating units.

The DNA cleavage domain located at the C-terminus of TALEN uses the nuclease region of the FokI restriction enzyme. In the case of FokI restriction enzymes, two FokI restriction enzymes must work together for double-strand breaks. Therefore, TALEN requires both left TALEN and right TALEN to work, which greatly increases TALEN's Specificity.

"TAL effector nuclease monomer" means a TAL effector nuclease consisting of a single polypeptide chain. A "TAL effector nuclease pair" or "TALEN pair" is understood to be TALEN consisting of two TAL effector nuclease monomers. Monomers represent the left or right arm of TALEN that binds to the opposing strand of one DNA and together causes DNA fragmentation at one point. Within one TALEN pair, a TALEN monomer is inserted in pairs, i. E. One monomer causes strand breaks within the double stranded DNA by linking one target sequence to its target strand while the other TALEN pair The monomers can be used to link a target sequence to a complementary object strand that is complementary to its target strand, resulting in the nuclease domains being aligned with respect to each other, and the shared DNA between target sequences referred to as " Lt; RTI ID = 0.0 > single-stranded < / RTI >

TALEN cloning can induce gene deletion at any site, including promoters, ORFs, and enhancers in genomic DNA, because it can synthesize artificial proteins that are needed for the target and induce DNA cleavage at that site. Since TALEN has the feature that one DNA base attaches one-to-one to RVD unit of TALE, RVD unit can be arranged according to the target sequence so that the experimenter can easily design the desired TALEN. In addition, there is no special condition other than to start with the thymine in determining the target sequence, which is advantageous in that it can be used for easy and various targets.

A "target sequence" is understood to be a single nucleotide sequence, generally a single DNA sequence, that is joined by a TAL effector binding domain.

In one embodiment of the present invention, the Parkin (Park2) gene was knocked out using this TALEN method.

For example, a specific sequence of the Park2 gene can be specifically recognized and cleaved using a pair of TAL effector nucleases comprising the first and second TAL effector nuclease monomers.

Each TAL effector nuclease monomer comprises a TAL effector DNA binding domain having an endonuclease domain with type II endonuclease activity and a plurality of repeat units, each of which may comprise a variable amino acid pair RVD have.

In one embodiment of the present invention, the TAL effector DNA binding domain of the first TAL effector nuclease monomer is capable of binding to the target sequence 5-TGAGTGCCAGTCTCCAAACT-3 (SEQ ID NO: 2), wherein the TAL of the second TAL effector nuclease monomer The effector DNA binding domain can bind to the target sequence 5-AGCAGTAAGTACTGGTCAAA-3 (SEQ ID NO: 3).

TALEN bound to a specific site binds Fok1 nuclease activity and can induce DNA cleavage near the target site. Although the repair mechanism is activated by the cleavage at the target DNA site, mutation of the DNA occurs due to the restoration mechanism that can not return the DNA sequence to the original state such as NHEJ (nonhomologous end joining). Specifically, the mutation may be a mutation by substitution, deletion, insertion, or a combination thereof.

Each nucleic acid encoding the TALEN gene having the activity of recognizing and cleaving the Park2 gene-specific sequence may be any of those having a nucleotide sequence encoding the TALEN gene having an activity of recognizing and cleaving the Park2 gene-specific sequence known in the art Can be used.

In addition, the nucleic acid encoding the TALEN gene can be produced by a recombinant method known in the art. For example, PCR amplification for amplifying a nucleic acid from a genome, chemical synthesis, or a technique for producing a cDNA sequence.

In addition, the nucleic acid may have a base sequence encoding each functional equivalent of the TALEN gene. The functional equivalents are those having a sequence homology of at least 70%, preferably 80%, more preferably 90% or more, with the amino acid sequence as a result of amino acid addition, substitution or deletion, and recognize a specific sequence of the park2 gene Refers to a polypeptide having activity of cleavage.

In certain embodiments, the nucleic acid encoding the TALEN gene of the invention can be operatively linked to an expression control sequence and inserted into the vector.

&Quot; Operably linked " means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. An expression control sequence also refers to a DNA sequence that controls expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. All of them can be expressed as 'a DNA construct containing a nucleic acid encoding the TALEN gene'.

RNA-guided endonuclease (RGEN) system

According to another aspect of the invention, the Park2 gene knockout can also be achieved via RGEN.

A representative example of RGEN (RNA-guided engineered nuclease) is the CRISPR system including CRISPR-related nuclease Cas9 and sequence-specific guide RNA (gRNA).

The components of the CRISPR system, including the CRISPR-related nuclease Cas9 and the sequence-specific guide RNA (gRNA), can be introduced into cells in one or more vectors, e.g., coded on a plasmid.

The CRISPR / Cas system includes transcripts and other elements involved in the expression or activation of Cas genes. The CRISPR / Cas system may be a Type I, Type II or Type III system. The methods and compositions disclosed herein use the CRISPR / Cas system by using the CRISPR complex (including the guide RNA (gRNA) complexed with the Cas protein) for site-specific cleavage of the nucleic acid.

Cas protein

Cas proteins generally comprise at least one RNA recognition or binding domain. These domains can interact with the guide RNA (gRNA, described in more detail below).

The nuclease domain has catalytic activity for nucleic acid cleavage. Cleavage involves the destruction of the covalent bond of a nucleic acid molecule. Cleavage can produce a smooth or staggered end and can be single-stranded or double-stranded.

Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12) (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 (CasA), Casse, CasH, Csy1, Csy2, Csy3, Cse1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1 and Cu1966, A modified version.

Cas proteins can be derived from a Type II CRISPR / Cas system. For example, the Cas protein may be a Cas9 protein or may be derived from a Cas9 protein.

The Cas9 protein may be, for example, selected from the group consisting of Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobac Hlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, and Lactobacillus spp. , Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp. Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus desulforudis, Clostridial botulinum, Clostridium botulinum, Clostridium botulinum, Clostridium difficile, Finegoldia magna, But are not limited to, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium, vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, The compounds of the present invention can be used for the treatment of Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp ), Arthrospira maxima, Arthrospira platensis, Arthrospira sp. For example, Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, It can be derived from the Acaryochloris marina. Additional examples of the Cas9 family are described in WO 2014/131833, the entire contents of which are incorporated herein by reference. The Cas9 protein or derivative thereof from Streptococcus pyogenes is a preferred enzyme.

The Cpf1 protein may be selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. For example, Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp (GW2011_GWA_33_10), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus methanoplasma termitum and Eubacterium eligens. . (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens But is not limited thereto.

The Cas protein can be a wild-type protein (i.e., one that occurs in nature), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. The Cas protein may also be an active variant or fragment of the wild type or modified Cas protein. The active variant or fragment is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or more sequence identity, wherein the active variant retains the ability to cleave at the desired cleavage site, thus retaining nick-inducing or double-strand break inducing activity. Methods for measuring nick induction or double-strand break induction activity are known.

The Cas protein may be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity and / or enzyme activity. Cas proteins may also be modified to alter other activities or properties of the protein, such as stability. For example, one or more nucleotide domains of the Cas protein may be modified, deleted or inactivated, or the Cas protein may be cleaved to remove domains that are not essential for protein function or to optimize the activity of the Cas protein.

One or two nucleases domains may be deleted or mutated, resulting in either no longer functional or a decrease in nuclease activity.

When one nucleotide domain is deleted or mutated, the resulting Cas protein (e. G., Cas9) may be referred to as a nickase and single strand cleavage in a CRISPR RNA recognition sequence in double- Can be generated.

When two nuclease domains are deleted or mutated, the resulting Cas protein (e. G. Cas9) will degrade the ability to cleave two strands of double stranded DNA. An example of a mutation that converts Cas9 to nick carase is D10A or H939A or H840A in the RuvC domain of sp. Cas9.

The Cas protein may also be a fusion protein. For example, the Cas protein can be fused to a cleavage domain, a reproductive deformation domain, a transcription activation domain, or a transcription repression factor domain. The fused domain or heterologous polypeptide may be located at the N-terminus, the C-terminus, or within the Cas protein.

In addition, the Cas protein can be fused to a heterologous polypeptide. Heterologous peptides include, for example, a nuclear localization signal (NLS) such as the SV40 NLS for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, an ER retention signal, and the like. Such a signal may be located anywhere in the N-terminus, the C-terminus or the Cas protein.

Cas proteins can also be linked to the cell permeability domain. For example, the transmembrane domain may be derived from a HIV-1 TAT protein, a TLM cell permeable motif from human hepatitis B virus, MPG, Pep-1, VP22, a cell permeable peptide from simple herpes virus, or a polyarginine peptide sequence .

The Cas protein may also comprise a heterologous polypeptide, e. G. A fluorescent protein, a purified tag or an epitope tag, to facilitate tracking or purification.

The Cas protein may be provided in any form. For example, the cas protein may be provided in the form of a protein such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid encoding a Cas protein, such as RNA (e. G., Messenger RNA (mRNA)) or DNA. Optionally, the nucleic acid encoding the Cas protein can be codon-optimized for efficient translation into a protein from a particular cell or organism.

The nucleic acid encoding the Cas protein can be stably integrated into the genome of the cell and can be operably linked to a promoter that is active in the cell. Alternatively, the nucleic acid encoding the Cas protein can be operably linked to the promoter of the expression construct.

The guide RNA (gRNA)

A "guide RNA" or "gRNA" includes RNA molecules that bind Cas proteins and target Cas proteins at specific locations within the target DNA. The guide RNA may comprise two segments, a "DNA target segment" and a "protein binding segment. &Quot; A "segment" includes a segment, section or region of a molecule, such as a continuous stretch of nucleotides of RNA. Some gRNAs include two separate RNA molecules: the "activator-RNA" crRNA and the "targeter-RNA" tracrRNA. Another gRNA is a single RNA molecule (a single RNA polynucleotide), also referred to as a "single molecule gRNA", "single guide RNA" or "sgRNA".

The crRNA and the corresponding tracrRNA hybridize to form gRNA. The crRNA provides a single stranded DNA targeting segment that hybridizes to a CRISPR RNA recognition sequence. If used for transformation in a cell, the complete sequence of a given crRNA or tracrRNA molecule can be designed such that the RNA molecule is specific to the species in which it will be used.

The DNA targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to the sequence of the target DNA.

The DNA targeting segment of the gRNA interacts with the target DNA in a sequence-specific manner through hybridization (i.e., base pairing). As such, the nucleotide sequence of the DNA targeting segment can be different and determines the location in the target DNA with which the gRNA and the target DNA will interact. The DNA targeting segment of the subject gRNA may be modified to hybridize to the desired sequence in the target DNA.

The DNA targeting segment may have a length of from about 12 nucleotides to about 100 nucleotides. For example, the DNA targeting segment may comprise about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, about 12 nt to about 30 nt , From about 12 nt to about 25 nt, from about 12 nt to about 20 nt, or from about 12 nt to about 19 nt.

The tracrRNA can be in any form (e. g., full-length tracrRNA or active tracrRNA) and in various lengths.

The complementarity ratio between the DNA targeting sequence in the target DNA and the CRISPR RNA recognition sequence is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , At least 97%, at least 98%, at least 99%, or 100%).

The guide RNA may have additional desirable characteristics, for example, modified or controlled stability; Intracellular targeting; Tracking with fluorescent labels; A binding site for a protein or protein complex, and the like.

Examples of such modifications include, for example, 5 'caps (e.g., 7-methyl guanylate cap (m7G)); 3 'polyadenylated tail (i.e., 3' poly (A) tail); Riboswitch sequences (e.g., considering controlled stability and / or controlled access by protein and / or protein complexes); Stability control sequence; dsRNA < / RTI > duplex structure (i. e., a hairpin), and the like.

The guide RNA can be provided in any form. For example, a gRNA may be provided in the form of a complex of two molecules (isolated crRNA and tracrRNA) or an RNA form as one molecule (sgRNA) and optionally a Cas protein. gRNA can also be provided in the form of DNA encoding RNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or a separate RNA molecule (e. g., isolated crRNA and tracrRNA).

The DNA encoding the gRNA can be stably integrated into the genome of the cell and can be operably linked to a promoter that is active in the cell. Alternatively, the DNA encoding the gRNA may be operably linked to the promoter of the expression construct. For example, a human U6 promoter can be used.

Cleavage of nucleic acid

The Cas protein can cleave nucleic acids within or outside the nucleic acid sequence present in the target DNA to which the DNA targeting segment of the gRNA will bind.

"Cleavage site" includes nucleic acid sites where the Cas protein causes single strand breaks or double strand breaks. For example, CRISPR complex formation may be performed at or near the nucleic acid sequence present in the target DNA to which the DNA targeting segment of the gRNA will bind (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs).

The cleavage site may be on only one or two strands of the nucleic acid. The cleavage site may be at the same position (producing a smooth end) of the two strands of the nucleic acid, or at different sites of each strand (creating a staggered terminus).

Site-specific cleavage of the target DNA by Cas9 can occur in the target DNA at positions determined by (i) base pairing complementarity between the gRNA and the target DNA and (ii) short motifs referred to as PAM.

PAM (Protospacer adjacent motif) can be flanked by CRISPR RNA recognition sequences. Optionally, CRISPR RNA recognition sequences can be flanked by PAM. For example, the cleavage site of Cas9 can be from about 1 to about 10, or from about 2 to about 5 base pairs (e.g., three base pairs) upstream or downstream of the PAM sequence.

[Genetically Modified Composition]

In one embodiment of the invention, there is provided a composition for genetic manipulation or modification of the Parkin (Park2) gene and methods of using the same.

In one embodiment, a composition for genetic manipulation or modification of the Parkin (Park2) gene may comprise a component of the TALEN (transcription activator-like effector nuclease) system.

For example, a specific sequence of the Park2 gene can be specifically recognized and cleaved using a pair of TAL effector nucleases comprising the first and second TAL effector nuclease monomers.

Each TAL effector nuclease monomer comprises a TAL effector DNA binding domain having an endonuclease domain with type II endonuclease activity and a plurality of repeat units, each of which may comprise a variable amino acid pair RVD have.

In one embodiment of the present invention, the TAL effector DNA binding domain of the first TAL effector nuclease monomer is capable of binding to the target sequence 5-TGAGTGCCAGTCTCCAAACT-3 (SEQ ID NO: 2), wherein the TAL of the second TAL effector nuclease monomer The effector DNA binding domain can bind to the target sequence 5-AGCAGTAAGTACTGGTCAAA-3 (SEQ ID NO: 3).

The TALEN monomers forming the TALEN pair according to the present invention may also be contained in two nucleic acids in a separated state. The nucleic acids constituting the first and second TALEN monomers may each be mRNA, particularly preferably stabilized mRNA. In some cases, other elements besides the first and second TALEN monomer nucleic acids may be included, such as one or more promoters, and polyadenylation signals, and the like. Two TALEN arms (right arm and left arm) can be sent into one cell simultaneously or separately.

Carrying the TALEN pair through mRNA has a number of crucial advantages for clinical applications. The use of DNA-based gene transfer mediators can be avoided and this situation makes the manufacturing process and the actual application critical and simple. The mRNA-mediated expression of TALEN is only relatively short, because the mRNA is rapidly degraded in the target cell. By doing so, the risk of off-target effect is further reduced. Furthermore, target cells can only be cultured in vitro for very short periods of time. Corresponding engineering techniques can easily be matched to GMP requirements. Unlike viral mediators or plasmid mediators, adverse effects (off-target effects, activation of TALEN-specific immune responses) by long-term TALEN expression due to side effects (such as insertional mutagenesis) Not expected.

In another embodiment, a composition for genetic manipulation or modification of the Parkin (Park2) gene may comprise a component of the RNA-Guided Endonuclease (RGEN) system.

For example, there is provided a genomic engineering composition comprising a guide RNA or a nucleic acid molecule encoding it and an RNA-Guided Endonuclease (RGEN) or a nucleic acid molecule encoding the same.

The composition may be provided in the form of an expression cassette.

Recombinant vector for Park2 gene knockout

In one embodiment, the invention relates to a recombinant vector for constructing an animal model of Parkinson's disease knocking out Parkin (Park2), most preferably a Parkinson's disease pig model.

The term "vector" or "expression vector" of the present invention refers to a plasmid, a viral vector or other medium known in the art capable of introducing a nucleic acid encoding the structural gene and capable of expressing the nucleic acid in a host cell it means. Preferably a plasmid vector.

As a means for knocking out the Park2 gene of the present invention by introducing the vector of the present invention into a cell, a known expression vector such as a plasmid vector, a cosmid vector, and a bacteriophage vector may be used. And can be easily produced by those skilled in the art according to known methods.

In one embodiment, the vector comprises a gene scissor having the activity of recognizing and cleaving a Park2 gene-specific sequence, wherein the gene scissors include Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN; RNA-guided engineered nuclease (RGEN) or the like can be used, but most preferably, TALEN (transcription activator-like effector nuclease) or RGEN (RNA-guided engineered nuclease) can be used.

In addition, the recombinant expression vector may use a promoter of the present invention that expresses a gene.

The term " promoter " is a regulatory sequence that is a nucleic acid sequence region in which transcription initiation and rate are regulated. These promoters may contain regulatory proteins such as RNA polymerase and other transcription factors and genetic elements capable of binding molecules to initiate specific transcription of the nucleic acid sequence. The terms " effectively positioned, " " effectively combined, " " under control, " and under " transcriptional control " Orientation. ≪ / RTI >

Meanwhile, the Park2 gene knockout vector may include a reporter gene, and the reporter gene may include a reporter system.

Examples of reporter systems are surrogate reporters, surrogate reporters for flow cytometry, surrogate reporters for magnetic seperation, and surrogate reporters for antibiotic selection. The basic structure of the surrogate reporter has a common constitution starting from the CMV promoter up to the mRFP gene, the target nucleotide sequence, and the stop codon. The eGFP gene, H-2kk gene, and hygromycin resistance gene are inserted after the termination codon for each surrogate reporter.

In addition, the recombinant expression vector may include a selection marker, and the selection marker includes an antibiotic resistance gene such as a kanamycin resistance gene and a neomycin resistance gene, and a fluorescent protein such as a green fluorescent protein and a red fluorescent protein. It is not limited.

In addition, the vector of the present invention may further include a tag sequence for protein separation purification or confirmation. Examples of the tag sequence include GFP, Glutathione S-transferase (GST) -tag, HA, His-tag, Myc-tag and T7-tag. However, the tag sequence of the present invention is not limited by these examples. In the preferred embodiment of the present invention, expression and expression level were confirmed using GFP.

The recombinant vector for Park2 gene knockout can induce Parkinsonism by causing knockout mutation of Park2 gene in pig cells.

Vector introduction into nuclear donor cells

One embodiment of the present invention relates to a method for introducing a composition for genetic manipulation or modification of the Parkin (Park2) gene into the cells of pigs.

For example, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection Transgenic animals can be transfected by DEAEDextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acid into cells. Can be introduced into cells for production.

In one embodiment of the invention, various nucleic acids are introduced into the cell for the genetic manipulation or modification, for example to obtain knockout, gene inactivation, or expression of a particular gene, or for other purposes .

Nucleic acid means both RNA and DNA, including, for example, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA as well as naturally occurring and chemically modified nucleic acids, such as synthetic bases or alternating backbones . The nucleic acid molecule may be double-stranded or single-stranded.

The nucleic acid may be included in a vector.

A vector can refer to an expression vector or vector system and is a set of components necessary to cause DNA insertion into a genomic or other targeted DNA sequence, such as an episome, a plasmid, or even a virus / phage DNA segment.

Viral vectors used for gene transfer in animals can be used. Non-viral vectors may also be used.

Many different kinds of vectors are known. For example, plasmids and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids are typically derived from a recombinant plasmid containing a replication origin, a suitable promoter, and an optional enhancer, and any necessary ribosome binding sites, a polyadenylation site, a splice donor and acceptor site, a transcription termination sequence, and a 5 ' Sequence. Examples of vectors are: plasmids (which may be carriers of other types of vectors), adenoviruses, adeno-associated viruses (AAV), lentiviruses (e.g., modified HIV-1, SIV or FIV), retroviruses (E.g., ASV, ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).

In one embodiment, the target nucleic acid sequence can be operably linked to a regulatory region, such as a promoter

In one embodiment, additional regulatory regions may be introduced together. But are not limited to, polyacetylation sequences, detoxification control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements or introns.

In one embodiment, nucleic acid constructs encoding signal peptides or selectable markers may be used.

In one embodiment, the exogenous nucleic acid encodes a polypeptide. and may include a tag sequence encoding "tag ".

In addition, the nucleic acid can be delivered in a non-viral vector delivery system.

For example, TAL effector nuclease monomers in the form of mRNA can be packaged and delivered to lipid nanoparticles. The most widely known cationic materials for delivering nucleic acids are naturally-occurring polymers, synthetic polymers, and lipids

In one embodiment, the present invention can provide pig cells with an inactivated or removed Parkin (Park2) gene.

For example, a recombinant vector comprising a composition for Parkin (Park2) knockout is introduced into a nuclear donor cell, and a cell into which the recombinant vector is introduced.

For example, the present invention relates to a method for introducing an RNP complex comprising a composition for Parkin (Park2) knockout into a nuclear donor cell, and a cell into which the RNP complex is introduced.

In one embodiment, the cell may be an embryonic cell, somatic cell, or stem cell of a porcine.

In certain embodiments, for example, but not limited to, cumulus cells, epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, cartilage cells, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal derived cells, embryonic cells and embryonic cells. Also, adult stem cells derived from various origins can be used, such as adult stem cells such as fat, uterus, bone marrow, muscle, placenta, cord blood or skin (epithelium). Non-human host embryos can generally be embryos comprising a 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage, a 32-cell stage, a 64-cell stage, .

More preferably, it may be a fetal-derived cell, an adult fibroblast, or a cumulus cell. Most preferably, fibroblasts isolated from fetal and adult pigs are used. The characteristics of these cells are that they can obtain a large number of cells at the initial separation, have relatively easy cell culture, and are easy to cultivate and manipulate in vitro.

The embryonic cells, somatic cells or stem cells provided as nuclear donor cells can be obtained from a method for preparing surgical specimens or biopsy specimens using conventional methods known in the art.

Meanwhile, the nuclear donor cell transformed with the knockout vector of Parkin (Park2) gene for the production of a Parkin (Park2) gene deficiency disease pig model according to the present invention can be proliferated and cultured according to a method known in the art have.

Suitable media are those which have been developed for the cultivation of animal cells and in particular mammalian cells, or for any use which may be produced in the laboratory together with suitable components necessary for animal cell growth, such as assimilable carbon, nitrogen and / or micronutrients Possible media can be used.

The medium may be any basic medium suitable for animal cell growth, including but not limited to MEM (Minimal Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium) and K-SFM (Keratinocyte Serum Free Medium). In addition, there is no limit to the use of any medium used in the industry. Preferably, the cells are cultured in a culture medium such as? -MEM medium (GIBCO), K-SFM medium, DMEM medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO), DMEM / F12 medium, PCM medium, M199 / (GIBCO), and MSC expansion medium (Chemicon).

To this base medium may be added an assimilative source of carbon, nitrogen and micronutrients, as a non-limiting example, a serum source, a growth factor, an amino acid, an antibiotic, a vitamin, a reducing agent, and / or a sugar source.

It will be apparent to those skilled in the art that suitable media may be selected or combined and cultured appropriately in a known manner. In addition, it is apparent that culturing can be carried out while adjusting conditions such as a suitable culture environment, time, temperature and the like on the basis of ordinary knowledge in this field.

[Transgenic Animal]

In one embodiment, the present invention provides a method for producing a transgenic pig for a Parkinsonian model in which a nucleus of a nuclear donor cell into which a recombinant vector has been introduced is transplanted into an enucleated oocyte to produce a knockout animal, wherein the Park2 gene is knocked out, The present invention relates to pigs for disease models.

In certain embodiments, the embryo can be conceived under suitable conditions to produce a pig in which Parkin (Park2) protein expression is inactivated.

For example, the somatic cell nuclear transfer method (SCNT) described above can be used using somatic cells transformed into pigs.

In certain embodiments, the present invention produces cloned animals of the Parkinson's disease model of the invention by somatic cell nuclear transfer (SCNT) using a transformed cell line knocked out of the Parkin (Park2) gene.

In one embodiment, the method for producing a pig for a Parkinson's disease model of the present invention comprises:

(a) a step of preparing a nuclear donor cell comprising culturing somatic cells or stem cells isolated from a tissue of a pig;

(b) introducing Park2-targeted nuclease (gene scissors) into said nuclear donor cell;

(c) removing the nucleus from the oocyte of the pig to produce a enucleated oocyte;

(d) injecting and fusing nuclear donor cells of step (b) into the enucleated oocytes of step (c);

(e) activating the oocyte fused in step (d); and

(f) transplanting the activated oocyte into the tubal duct of the surrogate mother.

The conventional technology for each step can be understood with reference to a conventional method for producing a cloned animal using a somatic cell nuclear transfer technique known in the art.

As a specific example, a method for preparing a porcine-derived somatic cell transformed with a composition knocking out the Parkin (Park2) gene, a nucleus of an embryo cell or a stem cell into a enucleated oocyte, Nuclear transfer embryos can be provided.

In another embodiment, there is provided a method for producing a transformed pig for a Parkinson's disease model knocking out a Parkin (Park2) gene comprising transplanting the nuclear transfer embryo into a tubal duct of a surrogate mother to produce a live animal, and Park2 (Parkin) deficiency disease characterized by knockout of the gene, for example, a pig for Parkinson's disease model.

In certain embodiments, the invention encompasses a method of producing a GM animal,

The method includes exposing an embryo or cell to an mRNA encoding a targeting nuclease (e.g., a meganuclease, zinc finger, TALEN, RGEN, recombinant enzyme fusion molecules)

Cloning a cell in a surrogate mother, or transplanting embryos in a surrogate mother, wherein the targeting nuclease specifically binds to an embryonic or intracellular target chromosomal region to cause a change in a cell chromosome, Genetically engineered animals can be conceived.

In one embodiment of the present invention, the gene expression analysis of the Park2 knockout transformed pig in the present invention confirmed that nucleotide loss, insertion or point mutation occurred.

The above-described Park2 gene knockout-confirmed transgenic pig cells are used to produce pigs for Parkinson's disease model in which Park2 gene is knocked out through replication.

There is no limitation on the method of replication, but, for example, somatic cell nuclear transfer (SCNT) can be used using somatic cells of transformed pigs.

Uses of Parkinson pig model

In another aspect, the present invention provides a method for utilizing UPDRS (Unified Parkinson's Disease Rating Scale) for determining the clinical condition of a pig for the Parkinson's disease model.

UPDRS is a method of expressing the symptoms of Parkinson's disease as time points and treatment-related deterioration and improvement scores by expressing the motor symptoms and daily life performance of Parkinson's patients as scores. The UPDRS is divided into four parts: Part 1 is an indicator of mental and mood, Part 2 is the daily life ability, Part 3 is the motor symptoms of Parkinson's disease, and Part 4 is the side effect of the treatment. Because the main symptoms of Parkinson's disease are motor symptoms, only Part 3 may be assessed and recorded. Part 1-4 may also be used as needed.

The UPDRS (Unified Parkinson's Disease Rating Scale) method can be suitably modified and applied to the pig for the Parkinson's disease model of the present invention to confirm its usefulness as an animal model of Parkinson's disease. The mobility was evaluated in one embodiment of the present invention.

As described above, the pig for the Parkinson's disease model of the present invention exhibits a pathological mechanism very similar to that of the human Parkinsonian patient, and is very useful as an animal model for the study of human parkinson therapy.

The transgenic pig for the Park2 gene knockouted Parkinson model according to the present invention is capable of producing live plants through crosses and is capable of transferring the external gene in the future.

Therefore, a pig characterized by knockout of the Park2 gene of the present invention is advantageous as an animal model of Parkinson's disease, because it has an advantage of easy reproducibility.

That is, it is possible to confirm the induction of Parkinsonism using the Parkinson's disease model mini pig of the present invention, to compare the onset of the Parkinson's disease model developed by a method other than the administration of neurotoxic substances, It will be possible to use various methods such as searching for substances and developing diagnostic methods.

Therefore, the present invention, in a different aspect, includes various uses of pigs for the Parkinson's disease model produced by the above method.

For example, an animal model according to the present invention can be used in a research required to treat or prevent Parkinson's disease, for example, as a method of screening a test agent.

In one embodiment, the screening method of the present invention comprises

1) administering Parkinson ' s disease model pigs with Parkinson ' s prophylactic and therapeutic agents;

2) After administration of the candidate substance, the tissue of the pig may be analyzed in comparison with a control group to which the candidate substance is not administered.

Preferably, the candidate substance is any one selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and plasma. The compound may be a novel compound or a well-known compound. These candidate substances may form salts.

The method of administering the candidate substance may be suitably selected in accordance with the symptoms of the subject animal, the nature of the candidate substance, etc., for example, by oral administration, intravenous injection, subcutaneous administration, intradermal administration or intraperitoneal administration. The dose of the candidate substance can be appropriately selected in accordance with the method of administration, the nature of the candidate substance, and the like.

As described above, the present invention is very useful for the development and practical use of Parkinson's disease treatment technology by providing an animal model of Parkinson's disease using a pig, which is the most physiologically similar animal to a human, in Parkinson's disease considered to be an incurable disease.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

Primary cell culture (primary cell culture)

Adult female Yucatan kidney (16 months old) was transferred to the laboratory to construct experimental cells. The whole kidney was washed 3 times in PBS and the experiment was performed according to the following procedure. The washed kidneys were chopped into small pieces with trypsin and the trypsin-treated tissues were incubated for 30 min at 37 [deg.] C.

Well dissociated tissues were centrifuged at 1500 rpm for 2 minutes. The supernatant was discarded and the pellets were resuspended in PBS and then centrifuged at 1500 rpm for 2 minutes. These procedures were repeated twice and finally the supernatant was discarded and the pellet was resuspended in 15% FBS (Gibco), 1% penicillin / streptomycin (P / S) (Gibco), 1% nonessential amino acid (NEAA) (Gibco, Carlsbad, Calif., USA) supplemented with 100 mmol / l? -Mercaptoethanol (ME).

The cells resuspended in the medium were allowed to stand at room temperature (about 25 ° C) for 5 minutes, and the suspension was transferred to a cell culture dish and incubated for about 10 days while changing the culture medium every 2-3 days. These primary cells were cultured and expanded and frozen at -196 ° C for use. The cell cultures were maintained in DMEM with 15% FBS, 1% P / S, 1% NEAA and 100 mmol / l beta-ME.

2. Establishment of Parkin KO (knock out) cell line

The mismatch-senstive nuclease assay was used to verify the conditional test to optimize the introduction of gene scissors into pig primary cells.

Using established conditions, we produced knockout donor cells from various pig primary cell lines using TALEN and reporter system, which were verified against pig PARK2 among PD related genes.

An example of the amino acid sequence of the PARK2 gene of the present invention is as follows.

(SEQ ID NO: 1)

MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELRNDWTVQNCDLDQQSIVHIV

QRPWRKGQEMNATGGDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGR

SIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWDDVLIPNRMSGECQSPHCPGTSAEFFF

KCGAHPTSDKETSVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDRQFVHD

PQLGYSLPCVGTGDTVVLRGALGGFRRGVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVL

CPRPGCGAGLLPEPDQRKVTCEGGNGLGCGYGQRRTK

Specific sequences (SEQ ID NOS: 2 and 3) of the PARK2 gene recognized by TALEN nuclease are as follows.

(SEQ ID NO: 2)

5-TGAGTGCCAGTCTCCAAACT-3

(SEQ ID NO: 3)

5-AGCAGTAAGTACTGGTCAAA-3

3. Production of transformed Parkin KO replication mini pig

Using TALEN or CRISPR / Cas9, the cells knocked out the parkin gene (cells expressing GFP) were selected, and the cells were used as donor cells for somatic cell nuclear transfer.

Hereinafter, this will be described in more detail.

3-1. Preparation of recipient oocytes for somatic cell nuclear transfer

The pig ovary was transferred from the slaughterhouse into physiological saline solution at 28-31 ° C and transferred to the laboratory. Using a 10-ml syringe equipped with an 18-gauge needle from follicles having a diameter of 3-6 mm, the cumulus-oocyte complex , COC) were recovered. In vitro fertilization was induced by in vitro maturation culture for 40 hours in a 5% CO 2 incubator at 39 ℃.

The cells were immersed in 0.1% (v / v) hyaluronidase in TALP (Tyrode's albumin lactate pyruvate) medium and then repeatedly pipetted using a micro-glass pipette to obtain cumulus cells . Each oocyte was then fixed with a holding micropipette and incubated in a TALP medium supplemented with 5 ug / mL of bisbenzimide (Hoechst 33342) and 5 ug / mL of cytochalasin B using a micro manipulator (Nikon-Narishige , Tokyo, Japan).

The first polar and middle-II chromosomes stained with bezuanimide were removed using an aspiration pipette, and enucleated oocytes were placed on PZM (Porcine zygote media) -5 and used for somatic cell nuclear transfer.

3-2. Preparation of donor cells for somatic cell nuclear transfer

The process of producing transgenic cloned embryos using pig2 gene knockout cells was performed as follows.

Park2 gene knockout cells were thawed for somatic cell nuclear transfer, cultured until 100% confluency, trypsinized for about 3 minutes, separated from a single layer, recovered as a single cell, and used as a nuclear donor cell Respectively. Reproduced embryos were prepared using the fact that the cells expressing the reporter gene, GFP, are the cells that are knocked out by park2 gene.

3-3. Production of PD cloned embryos using somatic cell nuclear transfer technique

Donor cells expressing a single GFP were injected into the perivitelline space of the prepared enucleated oocytes and the couplets were precipitated in a fusion medium containing 0.26 M mannitol, 0.1 mM MgSO4, 0.5 mM HEPES , And fused using needle-shaped electrodes.

The single cell-oocyte complex was placed between two opposing electrodes attached to a micro manipulator (Nikon-Narishige, Tokyo, Japan), and the contact surface between the oocyte cytoplasm and the nucleus donor cells was placed in parallel with the electrode, The cells were electrostimulated with an electro-cell fusion SNCA apparatus (NEPA GENE Co., Chiba, Japan). After 30 minutes of electrical stimulation, the fusion between the nuclear donor cell and the oocyte cytoplasm was observed under a stereomicroscope.

Fused embryos were selected and placed in a chamber containing active medium supplemented with 0.26 M mannitol, 0.5 mM HEPES, 0.1 mM CaCl ₂ , and 0.1 mM MgSO ₄ , and the electrodes were connected to each other using a BTX electro-cell Manipulator 2001 machine for 1.5 lt; RTI ID = 0.0 > kV / cm < / RTI > Five to six embryos were cultured in 20 μL of PZM-5 microdrops applied with mineral oil.

3-4. Transplantation into a surrogate mother of an embryo made by somatic cell nuclear transfer

A surrogate mother with synchronized estrus is supplied from the farm. The surrogate mothers were anesthetized by administering 1 mg / kg of ketamine and 0.5 mg / kg of xylazine to the pigs. After taking the combination, the patient fell for a while and then the unconscious pig was moved to the operating table, and isopulllan was used to maintain general anesthesia.

After the surgical site was cleaned with soap, the hair was cleanly removed using a razor, and the surgical site was disinfected in the order of povidone, alcohol, povidone, alcohol, and povidone, followed by complete sterilization Surgery was performed.

First, the middle part of the lower abdomen was cut open, the uterus was pulled, and the ovaries and tubal status were visualized. Transplanted embryos were transplanted into the fallopian tubes. Using a Tomcat catheter, the catheter was directly inserted into the fallopian tube with a sharp needle, and the catheter loaded with the transgenic cloned embryos was inserted and the oocyte was transplanted into the surrogate mother. The transplanted embryos of this transgenic embryo thus returned the uterus and sutured the lacerations.

After the suture was completed, the surrogate was disinfected with betadine, then moved to the waiting room after the operation, and the patient was post-operatively cared for. In addition, a wide range of antibiotics were administered to prevent infection after surgery. When the anesthesia was completely broken and the stamina was restored, it was transferred to the pregnant woman who was originally inhabited and went into individual management.

4. Reproduction of Parkin KO replica mini pig production

We cloned the parkin gene knockout entity through replication using the cells of individuals whose parkin gene knockout was confirmed. It has the advantage of producing 100% knockout objects.

First, somatic cell nuclear transfer was performed using PDM5 cells identified by homo knockout, and PDM9 was produced.

Like PDM5, PDM9 also had cleft palate, which determined sacrifice.

Next, somatic cell nuclear transfer was carried out using PDM3 cells confirmed with hetero KO, and it was confirmed that PDM10 and PDM11 were produced to grow well.

5. Parkin KO replication mini-pig gene expression analysis

Sequencing was carried out for all the borns.

6. Behavioral and imaging analysis of cloned mini pigs

6-1. Imaging analysis

Park2 gene knockout replication The following tests were performed to obtain brain image data of mini pigs.

The brain 18F-FDG PET images were taken using HRRT-PET (Siemens Medical Systems and CTI, Inc) for the study of brain PET images. F-18 FDG (500 μCi / 100 g of body weight) was intravenously injected before general anesthesia, and 30 minutes later, 18F-FDG PET imaging was performed using isoflurane inhalation anesthesia via bronchial intubation. Functional evaluation in the area of the brain associated with the PD, and the shape of the brain were compared and analyzed to determine the extent of the disease.

Using the normal mini pig as a control, the same brain imaging data were obtained.

6-2. Behavior analysis

The scoring profile for replicating pigs was based on UPDRS (United Parkinson's disease rating scale).

The UPDRS is the most widely accepted clinical scale for the evaluation of PD patients. The replicate pigs' motor scores were evaluated three times. (From the sixth month to the first year of April)

Example 1: Mutation detection of transformed donor cells and blastocysts

1-1. Indirect analysis of mutation efficiency of blastocysts

After 48 h of production of transformed donor cells using pig cells, somatic cell nuclear transfer was performed using GFP positive cells in the cells. The mutation efficiency was verified by T7E1 and the mutation efficiency was indirectly measured and analyzed by transferring the blastocyst prepared by in vitro culture. (Fig. 1)

FIG. 1 shows that reporter gene is expressed by the expression of park2 TALEN in the blastocyst. Therefore, the mutation of the park2 gene occurred, and it was possible to somehow grasp the mutation generation efficiency of the donor cell of the present invention.

1-2. Verification of efficiency of produced donor cells

In order to verify the efficiency of the produced donor cells, genomic DNA and blastocysts produced in vitro conditions were transferred and analyzed using a mismatch-sensitive nuclease assay. The results are shown in FIG.

As shown in FIG. 2, a total of 44 blastocysts were analyzed, and it was confirmed that mutation was observed at an efficiency of 20% by confirming 8 positive results.

This implies that the possibility of the mutation of the park2 gene occurs in about 20% of the expressed donor cells by the expression of inserted park2 TALEN.

Example 2: Production and characteristics of Parkin KO replication mini pigs

A total of 8 litters were produced, named PDMs 1 to 8, respectively.

In the postnatal sequencing analysis, 3 knockouts of parkin gene knockout (Hetero Knockout; PDM3, homo knockout, PDM5 and PDM6) were detected in 8 dogs. The remaining 5 animals were found to be of wild type.

The confirmed knockout efficiency of the parkin gene was 37.5%.

To date, PD knockout animals have produced a total of 11 live organisms through somatic cell nuclear transfer (Table 1).

8 from PDM1 to PDM8 were born by transfection of TALEN into cells and KO-induced cells were used as donor cells to carry out somatic cell nuclear transfer, and 3 mice from PDM9 to PDM11 were transfected with somatic cells They are the living relatives born again.

Among the animals from PDM1 to PDM8, 3 animals with parkin KO were PDM3 (hetero KO), PDM5 (homo KO) and PDM6 (homo KO). Based on this, the efficiency of KO was estimated to be 37.5%. The remaining 5 were identified as wild type.

Since the KO confirmed individuals were present, somatic cell nuclear transfer, which proceeds further, was carried out using somatic cells derived from KO-confirmed individuals.

First, PDM9 was born in the production of livestock using PDM5, a homo-KO-confirmed individual. As a result of the nucleotide sequence confirmation, it was confirmed that homo KO was exactly same as PDM5, but it had cleft lip and palate as same as PDM5.

Figure 3 is a photograph of the cleft, liver, brain, heart, lung, kidney and spleen of PDM5, which was born with cleft palate.

On the autopsy, no other abnormalities were found other than cleft palate.

Next, replication was performed on heterozygous PDM3, and two PDM10 and PDM11 cells were born using PDM3 cells. In the case of PDM10, the tongue was found to be malformed, but in PDM11 it was healthy (Fig. 4) and both were born healthy with over 900g.

The knockout efficiency of the parkin gene in the Parkinson's disease pig model according to the present invention was 37.5%, which was high in three out of eight mice, and the knockout of the parkin gene (PDM3, 5) , It is possible to produce pigs with Parkinson's disease with 100% efficiency of knockout of parkin gene.

Example 3: Analysis of Parkin KO replication mini-pig gene expression

The sequencing results for all the born subjects are shown in FIG. The wild type sequence of the target site before inducing KO is the top sequence shown in FIG.

In case of PDM1, PDM2, PDM4, PMD7 and PDM8, we confirmed that the wild type and sequence match perfectly.

On the other hand, in the sequencing results of TALEN transfected cells, PDM3 showed heterozygous KO (7bp deletion in one allele and point mutation in A in the opposite allele where G is mutated) , Homo KO for 4bp deletion / 7bp insertion in PDM5, and homo KO replication mini pig for 78bp deletion / 14bp deletion in PDM6.

We confirmed that the sequencing result with PDM5 was exactly the same as that of PDM9 produced by replicating the PDM5. We also confirmed that the sequencing result of PDM10 and PDM11 We confirmed that sequencing results with PDM3 were exactly the same.

In addition, in PDM3, the point mutation was changed to A in the opposite allele where KO did not occur, and it was confirmed that the same point mutation existed in the rewritten PDM10 and PDM11.

Sequence analysis of the Park2 gene knockout in 6 out of 11 pig models produced by the method of the present invention revealed that the Park2 gene knockout animal model can be effectively produced Respectively.

Example 4: Behavioral and imaging analysis of Parkin KO replication mini pig PDM11

4-1. Symptoms of behavior analysis

The PARK2 KO gene, which was born in March 2015, showed a continuous decrease in behavioral and motor status as a result of three consecutive behavioral observations from September 2013 until July 2016. (Evaluation individual score: 7, 6, 6)

As a result, it was confirmed that the Park2 knockout pig model produced by the present invention exhibited similar behavioral symptoms as Parkinson's disease.

4-2. Image analysis symptoms

Parkin KO Mini-pig One-year-old PDM-11 showed decreased bilateral occipital cortex metabolism in FDG PET images compared to normal mini-pigs.

In addition, FP-CIT PET images showed a significant decrease in both putamen intake and increased Cortical atrophy and ventricle size.

Therefore, it was confirmed that the Park2 knockout pig model produced by the present invention exhibited image analysis symptoms similar to that of Parkinson's disease.

Example 5: Imaging evaluation and analysis of PET and CT images of the brain of Parkin KO mini-pig (PDM-11) and normal mini-pig at the first year of life

5-1 Experimental Method

Imaging evaluation and analysis of PET and CT in the brain of Parkin KO mini-pig (PDM-11) and normal mini-pig at the first year of life

We performed brain PET and brain CT of Parkin KO mini - pig and normal mini - pig using PET and CT imaging equipment of Seoul National University Hospital Cancer Hospital Department of Nuclear Medicine to obtain brain imaging data of Parkin KO mini pig and normal mini pig. The brain PET images were taken 1 hour after intravenous infusion of the fluoro-CIT (18F) FP-CIT radioisotope intravenous injection, a dopamine transporter, and 2) glucose analogue FDG (Fluorodeoxyglucose (18F ), [18F] FDG, and 18F-FDG) radioisotopes were injected intravenously.

<Types of Parkin KO replica mini pigs used for PET and CT imaging>

FP-CIT (PDM-11): Yucatan, male / 28 weeks old

FP-CIT (normal pig): Yucatan, ♂ / 30 weeks old

FDG (PDM-11): Yucatan, male / 28 weeks old

FDG (normal pig): Yucatan, ♂ / 30 weeks old

Imaging evaluation and analysis of brain MRI and CT scan of Parkin KO mini-pig (PDM-11) and normal mini-pig at the first year of life

Brain CT and brain MRI of Parkin KO mini pigs and normal mini pigs were performed using CT and 1.5T MRI imaging equipment at Ilsan Helix Animal Hospital to obtain brain imaging data of Parkin KO mini pigs and normal mini pigs.

<Types of Parkin KO mini pigs used for MRI and CT imaging>

PDM-11: Yucatan, ♂ / 28 weeks old

Normal pig: Yucatan, ♂ / 30 weeks old

5-2 Experimental results

PET, CT, and MRI of the Parkin KO mini pig (PDM-11) and the normal mini-pig at the first year of life were evaluated.

As a result, PET and CT images of 18F-FDG in Parkin KO mini pigs showed a decrease in bilateral occipital cortex metabolism in FDG PET images compared to normal mini pigs (Fig. 6).

PET and CT images of the FP-CIT of Parkin KO mini pigs showed a significant reduction in both-side putamen uptake in the FP-CIT PET image compared to the normal mini-pig (FIG. 7).

In addition, imaging findings of Parkin KO mini pigs (PDM-11) and normal mini-pigs at 1 year of age, , There was no significant abnormal finding in Parkin KO mini pig (Fig. 8).

And, imaging findings on the MRI of parkin KO mini pigs showed increased Cortical atrophy and ventricle size in Parkin KO mini pigs compared to normal mini pigs.

In the case of Parkin KO 1-year-old PDM-11, a decrease in bilateral occipital cortex metabolism was observed in FDG PET image compared with the normal mini pig, and the FP-CIT PET image showed a significant decrease in both putamen intake Cortical atrophy and ventricle size were observed to increase (Fig. 9).

Example 6: PARK2 KO PD model Mini pig behavior evaluation

PARK2 KO PD model In order to evaluate the behavioral characteristics of the mini pig, the following experiment was conducted.

One PARK2 KO mini-pig (male) delivered on March 6, 2015 was stockpiled as a shielding facility on November 5, 2015. The weight at the time of stocking was about 35 kg. Behavioral observations were carried out three times in total since January 2016 after implantation and brain imaging, and the results of the observation items were obtained through scoring.

The behavior of PDM-11 individuals was compared with that of normal mini-pigs with the same sex and same age and PDM-11 individuals. Are shown in the following table.

In the case of PDM-11, there was no significant difference in other items from the normal subjects. However, there was a large difference between the experiment of pulling out the foot from the cylinder and the sticking of the sticker to the skin. This is a big difference from normal individuals who try to pull their feet out and shake their bodies to remove stickers.

In addition, in addition to the behavioral observation item, a state in which the difference from the normal subject was observed was consumed in the feces of other mini pigs, and the tongue was exposed to the outside even during normal times (FIG. 10).

As described above, it was confirmed that the Park2 knockout pig model manufactured by the present invention can be useful as a Parkinson's disease model.

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Claims (15)

Park2 gene knocked out by a modification of the nucleotide sequence of the Park2 gene,
Park2 disease pig model with inactivated Park2 protein expression. The method according to claim 1,
Wherein the strain in the nucleic acid sequence comprises deletion, substitution, insertion or inversion of one or more nucleic acids. The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the exon 6 region of the Park2 gene. The method according to claim 1,
Wherein the strain in the nucleic acid sequence is artificially caused by a nuclease agent. The method according to claim 1,
Wherein the nuclease agent is a transcription activator-like effector nuclease (TALEN) or an RNA-guided endonuclease (RGEN). The method according to claim 1,
The Park2 gene is genetically engineered by TALEN (transcription activator-like effector nuclease)
Among the nucleic acid sequences constituting the Park2 gene,
Deletion or insertion of one or more nucleotides;
Substitution with one or more nucleotides that is different from the wild type gene;
Insert one or more nucleotides from outside
Lt; RTI ID = 0.0 &gt;
Wherein the Parkinson disease pig model comprises: [Claim 2] The Parkinson's disease model according to claim 1, wherein the Park2 gene comprises the amino acid sequence of SEQ ID NO: 1. A Park2 gene knockout composition comprising a TAL effector nuclease pair comprising a first and a second TAL effector nuclease monomer or a nucleic acid encoding the same,
The TAL effector DNA binding domain of the first TAL effector nuclease monomer binds to the target sequence 5-TGAGTGCCAGTCTCCAAACT-3 (SEQ ID NO: 2)
Wherein the TAL effector DNA binding domain of the second TAL effector nuclease monomer is capable of binding to the target sequence 5-AGCAGTAAGTACTGGTCAAA-3 (SEQ ID NO: 3). 9. The method of claim 8,
Wherein the DNA cleavage domain in each TAL effector nuclease monomer is located at the C-terminus of the TAL effector DNA binding domain. 10. The method of claim 9,
Wherein the DNA cleavage domain of the TAL effector nuclease monomer is the DNA-cleavage domain of FokI-endonuclease. In porcine cells or embryos containing the target genomic locus on the Park2 gene,
Comprising contacting a composition comprising TALEN (transcription activator-like effector nuclease) that recognizes SEQ ID NO: 2 and SEQ ID NO: 3, with a Park2 nucleic acid sequence altered. 12. The method of claim 11,
Wherein the step of contacting is carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods. 14. The method of claim 13,
Wherein the viral vector is at least one selected from the group consisting of retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV). 12. The method of claim 11,
Characterized in that the step of contacting is introduced into pig cells in vitro 12. The method of claim 11,
Wherein the method is performed by somatic cell nuclear transfer (SCNT).

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