KR102007457B1 - Nanocomplex comprising Cas9 protein, guide RNA and amphipathic peptide, and uses of the same - Google Patents

Abstract

The present invention relates to a nanocomplex comprising Cas9 (CRISPR associated protein 9) protein, guide RNA and amphipathic peptide and uses thereof, including Cas9 protein, guide RNA and amphipathic peptide, wherein the Cas9 protein and guide RNA are Formation of hybrids, the amphiphilic peptide has a micelle structure, by binding the micelle structure to the outer surface of the hybrid, it is possible to stably and efficiently transfer gene shears in vivo, by applying to brain cells brain It can prevent or treat neurological diseases and various other diseases, can solve the problems of the virus delivery method used to deliver conventional gene shears into cells, and can be stably delivered into cells to correct genes in cells, Nanocomplexes comprising Cas9 protein, guide RNA and amphipathic peptide It relates to the application.

Description

Nanocomplex comprising Cas9 protein, guide RNA and amphipathic peptide, and uses of the same}

The present invention relates to nanocomposites comprising Cas9 protein, guide RNAs and amphiphilic peptides and uses thereof, and more particularly, to the use of the nanocomposites for the treatment of brain diseases.

According to the World Health Organization (WHO), patients with neurological diseases such as Alzheimer's, Parkinson's, and Lou Gehrig's disease are increasing over time, and rare neurological diseases are increasing in some countries.

Gene therapy is the fundamental treatment of neurological diseases. In Korea, there are still many guidelines for gene therapy research because there are no guidelines for gene therapy. However, more than 170 gene therapies are being carried out worldwide. It is going forward and the scope is getting wider. In particular, CRISPR / Cas9, which is recently attracting attention as a life science technology, is used as a key tool for genetic correction.

CRISPR / Cas9 is a technology derived from the adaptive immunity of microorganisms. Specifically, an immune system that remembers fragments of bacteriophage as DNA for bacteriophage infection and cuts it off with Cas9 (CRISPR associated protein 9: RNA-guided DNA endonuclease enzyme), a nuclease that acts as a genetic scissors when reinfected. Started from. This has evolved into a genetic correction technique that can cut and fix a desired site as long as the specific sequence can be recognized by a guide RNA (gRNA) in the genome.

This is in the spotlight as a way to treat the root cause of the disease in diseases induced by gene abnormalities that were separated into incurable diseases. However, there are problems to be solved, such as efficient intracellular delivery of gene editing systems and off targeting to cut genes other than target genes. In particular, the gene editing system using the Cas9 plasmid, which is an early method, required verification of safety of antibiotic resistance and various immune responses in the body delivery. Recently, a system for making and delivering protein scissors (Cas9) and guide RNA made in vitro has been applied as an alternative, but this also raises the issue of efficient intracellular delivery and stability of proteins and RNA.

Therefore, many attempts have been made to treat diseases by enabling fundamental genetic correction through gene therapy, but how to introduce a gene delivery vector capable of such genetic correction in vivo stably and efficiently Much research is needed.

Korean Patent Publication No. 2013-132795

An object of the present invention is to provide a nanocomposite capable of stably and efficiently delivering genetic scissors in vivo.

The present invention aims to provide a nanocomposite capable of preventing or treating brain neurological diseases and various other diseases by applying to brain cells.

An object of the present invention is to provide a nanocomposite that can solve the problems of the virus delivery method used to deliver the conventional genetic scissors into cells.

An object of the present invention is to provide a nanocomposite that can be stably delivered into cells to correct intracellular genes.

1. Cas9 protein where the guide RNA is located on the surface; And an amphiphilic peptide of micellar structure bound to the surface.

2. The method according to the above 1, wherein the guide RNA and Cas9 protein 1: 1 to form a hybrid in a molar ratio of 0.5, nanocomposite.

3. In the above 2, wherein the hybrid and the amphiphilic peptide is included in the nanocomposite in a molar ratio of 1: 2 to 4, nanocomposites.

4. The nanocomposite of 3 above, wherein the amphiphilic peptide is SEQ ID NO: 2 (R7L10) peptide.

5. The nanocomposite of 1 above, wherein the guide RNA has a sequence complementary to at least one selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

6. The pharmaceutical composition for the treatment of brain neurological diseases comprising the nanocomposite of any one of the above 1 to 5.

7. In the above 6, wherein the brain neurological disease is Alzheimer's, therapeutic pharmaceutical composition.

8. Kit for cutting a target DNA comprising the nanocomposite of any one of the above 1 to 5.

According to one embodiment of the present invention, the nanocomposite can stably and efficiently deliver genetic scissors in vivo.

According to an embodiment of the present invention, the nanocomposite may be applied to brain cells to prevent or treat brain neurological diseases and various other diseases.

According to one embodiment of the invention, the nanocomposite can solve the problem of the virus delivery method used to deliver the conventional genetic scissors into the cell.

According to one embodiment of the invention, the nanocomposites can be stably delivered into cells to correct intracellular genes.

Figure 1a schematically shows a method for intracellular gene correction using gene shears (CRISPR / Cas9) nanocomposites.
Figure 1b is the result of confirming the genetic scissors nanocomposite by AFM image.
Figure 2a is a result of confirming the operation of the gene scissors (CRISPR / Cas9) nanocomposites targeting the TH (tyrosine hydroxylase) in the cells through a surveyor assay.
Figure 2b is the result of confirming the degree of change of the target DNA and non-target DNA through the next generation sequencing (NGS) after hitting the intracellular TH gene with the nanocomposite according to an embodiment of the present invention.
Figure 2c is a result of confirming through the surveyor by hitting the two different target DNA with different gene shears (CRISPR / Cas9) nanocomposite, the results show that the gene shears (CRISPR / Cas9) nanocomposites can operate for a variety of genes.
Figure 2d is a result of hitting human dedifferentiated stem cells (hiPS), human embryonic stem cells (hESC), HEK-293T, human neural stem cells (hNSC) through the gene scissors (CRISPR / Cas9) nanocomposite and confirmed through a surveyor . The results show that the gene shear (CRISPR / Cas9) nanocomposites are operable in various cells.
Figure 3a schematically shows a method for intracellular gene correction using gene shears (CRISPR / Cas9) nanocomposites.
Figure 3b is the result of confirming the reduction of the TH gene through staining and fluorescence microscopy by injecting the gene scissors (CRISPR / Cas9) nanocomposites targeting the TH gene into the mouse brain.
Figure 4a is a result of confirming the reduction of the BACE1 gene by staining and fluorescence microscopy by injecting the gene scissors (CRISPR / Cas9) nanocomposites targeting the BACE1 gene associated with Alzheimer's disease into the mouse brain.
FIG. 4B shows ThS staining and fluorescence microscopy for reduction of amyloid plaque, a major pathological feature of Alzheimer's disease, by injecting the gene scissors (CRISPR / Cas9) nanocomposites targeting the BACE1 gene associated with Alzheimer's disease into the mouse brain. This is the result confirmed.
Figure 4c is a result showing that the cognitive function of the mice injected with the gene scissors (CRISPR / Cas9) nanocomposite in the Morris water maze behavioral test used as a memory test of Alzheimer's disease mice restored.

The present invention relates to a nanocomplex comprising Cas9 (CRISPR associated protein 9) protein, guide RNA and amphipathic peptide and uses thereof, including Cas9 protein, guide RNA and amphipathic peptide, wherein the Cas9 protein and guide RNA are Formation of hybrids, the amphiphilic peptide has a micelle structure, by binding the micelle structure to the outer surface of the hybrid, it is possible to stably and efficiently transfer gene shears in vivo, by applying to brain cells brain It can prevent or treat neurological diseases and various other diseases, can solve the problems of the virus delivery method used to deliver conventional gene shears into cells, and can be stably delivered into cells to correct genes in cells, Nanocomplexes comprising Cas9 protein, guide RNA and amphipathic peptide It relates to the application.

Hereinafter, the present invention will be described in detail.

The term “base sequence” is a deoxyribonucleotide or ribonucleotide present in single- or double-stranded form and includes analogues of natural or non-natural nucleotides unless specifically stated otherwise.

Unless otherwise defined with respect to a particular disease or condition, the term “treating” or “treating” means (i) disease, disease and / or disease in an animal or human who is susceptible to disease but has not yet been diagnosed with the disease, Preventing the occurrence of a disease or illness; (ii) inhibit a disease, disorder or condition, ie inhibit its progression; And / or (iii) alleviate a disease, disorder or condition, ie cause degeneration of the disease, disorder and / or disease.

The term "complementary" when used in reference to a nucleic acid, means a nucleic acid with a unidirectional polarity that contains a polynucleotide sequence whose bases bind with the bases of a polynucleotide of another nucleic acid of opposite polarity. . For example, a nucleic acid having the sequence GCAT in the 5 'to 3' direction is complementary to a nucleic acid having the sequence CGTA in the 3 'to 5' direction. In this specification, the term complementary is used to include substantially complementary nucleic acids. Substantially complementary means that not all nucleotides are base paired with nucleotides of another nucleic acid, but nevertheless both nucleic acids have sequences capable of forming stable hybrids under certain conditions.

The term "cleavage" refers to the breakage of the covalent backbone of a nucleotide molecule, wherein cleavage of the Cas9 protein may be, but is not limited to, site-specific DNA double helix cleavage.

Unless specifically defined in the present invention, the term "cell" is a concept that encompasses not only prokaryotic cells, eukaryotic cells, but also individuals including DNA such as viruses. Prokaryotic cells may include Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota, and Thaumarchaeota, and eukaryotic cells include mammalian cells, fish cells, and plants. There may be various cells such as cells, insect cells, and the like.

The present invention includes a Cas9 protein, guide RNA and amphiphilic peptide, wherein the Cas9 protein and guide RNA forms a hybrid, the amphiphilic peptide has a micelle structure, the micelle structure on the outer surface of the hybrid It provides a bonded nanocomposite.

Hereinafter, the structure contained in a nanocomposite is divided and demonstrated, respectively.

"Cas9 protein" means an essential protein element in the CRISPR / Cas system and is active endonuclea when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). To form agents or nickases. Cas9 gene and protein information can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

Cas9 protein can be linked to a protein transduction domain. The protein transfer domain may be, but is not limited to, a poly-arginine domain, a nuclear localization sequence (NLS), or a TAT protein derived from HIV.

"Cas9 protein" may be a variant thereof, in which case a mutant form of Cas9 in which the catalytic aspartate residue is changed to any other amino acid. For example, the other amino acid may be alanine, but is not limited thereto.

In addition, the Cas9 protein was identified as Streptococcus sp. (Streptococcus sp.), Such as, but not limited to, isolated from organisms such as Streptococcus pyogens or recombinant proteins. "Recombinant" refers to, for example, a cell, nucleic acid, protein or vector, etc., when used to introduce a heterologous nucleic acid or protein or to alter a native nucleic acid or protein, or to a cell derived from a modified cell. Cells, nucleic acids, proteins, or vectors modified by the present invention. Thus, for example, a recombinant Cas protein can be made by reconstructing a sequence encoding the Cas protein using a human codon table.

Cas protein-encoding nucleic acid may be in the form of a vector such as a plasmid comprising Cas-encoding sequences under a promoter such as CMV or CAG. When the Cas protein is Cas9, the Cas9 coding sequence may be from Streptococcus sp. And may preferably be from Streptococcus pyogenes. For example, the Cas9 encoding nucleic acid may comprise the nucleotide sequence of SEQ ID NO: 1. Moreover, the Cas9 encoding nucleic acid may comprise a nucleotide sequence having at least 50% homology with the sequence of SEQ ID NO: 1, preferably at least 60, 70, 80, 90, 95, 97, Nucleotide sequences having 98, or 99% homology, but are not limited thereto.

"Guide RNA" refers to RNA specific for target DNA, which can form a complex with Cas9 protein and brings Cas9 protein to target DNA.

In the present invention, the guide RNA may be composed of two RNAs, namely CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single generated by fusion of an essential portion of crRNA and tracrRNA. It may be a single-chain RNA (sgRNA).

Any guide RNA can be used in the present invention as long as the guide RNA includes essential portions of crRNA and tracrRNA and sequences complementary to the target gene. According to one embodiment of the invention, the guide RNA is TH gene (NCBI ACCESSION: NM_199292.2 (SEQ ID NO: 3), NM_009377.1 (SEQ ID NO: 4)) and BACE1 gene (NCBI ACCESSION: NM_011792.6 (SEQ ID NO: 5) It may have a sequence complementary to at least one selected from the group consisting of)). When using a guide RNA having a sequence complementary to at least any one of SEQ ID NO: 3 to 5 in the present invention, it is possible to correct the target DNA causing Alzheimer's, thereby preventing or treating Alzheimer's. According to one embodiment of the invention, the guide RNA may be complementary binding to at least one sequence selected from the group consisting of SEQ ID NO: 6 to 8. In this case, SEQ ID NO: 6 is contained in SEQ ID NO: 3, SEQ ID NO: 7 is included in SEQ ID NO: 4 and SEQ ID NO: 8 so that the guide RNA can accurately target the target gene of TH, BACE1, through which Cas9 Accurate correction by protein can be induced.

The target DNA may be endogenous DNA or artificial DNA, for example, may be endogenous DNA for treatment, or may be artificial DNA for screening, DNA production, and the like, but is not limited thereto.

Guide RNA can be modified using methods known in the art to achieve the purpose of improving affinity with the target DNA or binding to the Cas9 protein. For example, the guide RNA may further comprise one or more additional nucleotides (eg, guanine) at the 5 ′ end of the crRNA of the single-chain guide RNA or duplex RNA. In addition, the guide RNAs may be backbone modified nucleotides such as peptide nucleic acids (PNA), phosphorothioate DNA, phosphorodithioate DNA, phosphoramidate DNA, amide-linked DNA, MMI-linked DNA, 2′- O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-0-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-0-alkyl DNA, 2′-O-allyl DNA, 2′-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines (substituents Fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethylyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, pyri Di-containing), 7-deazapurine with C-7 substituents (substituents are fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl- , Alkenyl , Thiazolyl-, imidazoyl-, pyridyl-), inosine, diaminopurine, and the like, but is not limited thereto. In addition, the guide RNA may include, but is not limited to, natural and / or non-natural nucleotides.

In the present invention, the Cas9 protein and the guide RNA form a hybrid. Hybridization may be performed in a form in which the guide RNA may move the Cas9 protein to the target DNA. For example, the guide RNA may be located on the surface of the Cas9 protein. This is not restrictive. According to one embodiment of the present invention, hybridization may be performed by mixing Cas9 protein and guide RNA in a molar ratio of 1: 0.5 to 3, but is not limited thereto. According to one embodiment of the present invention, hybridization may be performed by mixing Cas9 protein and guide RNA in a molar ratio of 1: 0.5 to 1.5 (eg, 1), but is not limited thereto.

When the Cas9 protein and the guide RNA form a hybrid within the above range, more accurate gene targeting becomes possible, and genetic correction can be performed with excellent accuracy.

If it is out of the range, when the content of Cas9 protein is low, gene cleavage may be incomplete, and when the content of guide RNA is low, there may be a problem that the probability of hitting the target DNA may be lowered.

In the present invention, the guide RNA included in the hybrid may be hybridized with one kind of target DNA, or may be hybridized with two or more kinds of target DNAs, and the term "hybridized with two or more kinds of target DNAs" means This means that the guide RNA is not one but two or more types, and that there is a base sequence capable of hitting two types of target DNA in one type of guide RNA.

"Amphiphilic peptide" means a peptide comprising a polar amino acid and a non-polar amino acid, wherein the polar amino acid and the non-polar amino acid are partially dense with each other to represent a polar portion and a non-polar portion, and the polar amino acids include cysteine, glutamine, threonine, Tyrosine, serine and asparagine, and the like, and the nonpolar amino acids include, but are not limited to, phenylalanine, tryptophan, methionine, proline, valine, isoleucine, leucine, glycine and alanine.

In the present invention, the amphiphilic peptide may be used as a medium for efficiently delivering a hybrid of Cas9 protein and guide RNA into a cell. The polar and nonpolar amino acids in amino acid sequence may be in a compact form, and the polarity in amino acid sequence. Although amino acids and non-polar amino acids are not dense with each other, when the peptide including the amino acids forms a three-dimensional structure, the polar and non-polar amino acids may be dense with each other by the three-dimensional structure, but is not particularly limited thereto. Does not. For example, an amphiphilic peptide may be in the form of a combination of a plurality of identical or non-identical polar amino acids and a plurality of identical or non-identical non-polar amino acids.

According to an embodiment of the present invention, the amphiphilic peptide may be a sequence in which a plurality of combinations of the aforementioned polar amino acids are continuously connected and a sequence in which a plurality of combinations of the aforementioned nonpolar amino acids are continuously connected. For example, an amphiphilic peptide is a combination of two or more amino acids of cysteine, glutamine, threonine, tyrosine, serine, and asparagine, which are linked one by one, or another type of amino acid that is continuously linked after one type of amino acid is continuously linked. Can be joined to form a polar moiety and can be joined by crossing one or more amino acids of any one or more of phenylalanine, tryptophan, methionine, proline, valine, isoleucine, leucine, glycine and alanine, or in sequence of one amino acid sequence and then continuous Other types of amino acids bonded to each other may be combined to form a non-polar site, and the polar and non-polar sites thus formed may be combined.

In addition, according to an embodiment of the present invention, the amphiphilic peptide is any one of the above-described polar amino acids may form a polar region, any one of the above-mentioned non-polar amino acids may form a non-polar region, the polarity thus formed The site and the nonpolar site may be bound.

According to one specific embodiment of the present invention, the amphiphilic peptide may be a sequence in which a plurality of arginine (R) sequences are continuously linked and a sequence in which a plurality of leucine (L) sequences are combined. For example, the amphiphilic peptide may be a sequence in which 5 to 10 arginine (R) is continuously coupled, and 7 to 13 leucine (L) are continuously coupled, and more specifically R7L10 (SEQ ID NO: 2). : RRRRRRRLLLLLLLLLL), but is not limited thereto.

In the present invention, the amphiphilic peptide may form a micelle structure by itself, and may be disposed on the surface of the above-mentioned hybrid body in the state in which the micelle structure is formed. The hybrids described above are negatively charged by the guide RNAs, and since the amphiphilic peptides formed with the micelle structure are positively charged, the amphiphilic peptides formed with the guide RNAs and the micelle structure may be bound by ionic bonds. It is not limited.

According to one embodiment of the invention, the above-described hybrid and amphiphilic peptides may be included in the nanocomposite in a molar ratio of 1: 2 to 4. When included in the nanocomposite within the molar ratio range, the probability of hitting the nanocomposite of the present invention against the intracellular genes increases, and at the same time, the cell death rate decreases, which is suitable for intracellular gene correction.

When the amphiphilic peptide is included in the nanocomposite below the molar ratio, the probability of hitting the intracellular gene may be drastically lowered. When the amphiphilic peptide is included in the nanocomposite above the molar ratio, the cell death rate is excessive. May be high, and may not be suitable for intracellular genetic correction.

The nanocomposites of the present invention can specifically cleave the target DNA due to the specificity of the guide RNA to the target DNA and the endonuclease or kinase activity of the Cas9 protein, and the amphiphilic peptide is composed of the guide RNA and the Cas9 protein. Positioning on the surface of the hybrid can greatly increase the stability and delivery efficiency of the hybrid in delivery into cells. This allows for further refinement of the target DNA in the cell, further increasing the effect of the target treatment on diseases caused by the mutant DNA.

Nanocomposites of the present invention may further comprise zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the like, where “comprises”. Is meant to include, but is not limited to, all but included instead of the Cas9 protein, or included with the Cas9 protein.

In another aspect, the present invention provides a pharmaceutical composition for treating brain neurological diseases comprising the nanocomposite described above.

"Cerebral neurological disease" is defined as a disorder of the brain nervous system, the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including the brain nerve), and the autonomic nervous system (some of which are located in both the central and peripheral nervous system) Includes disorders associated with

Brain neurological diseases in the present invention may be any disease that can be prevented or treated with the above-described nanocomposites, that is, diseases caused by DNA damage.

According to one embodiment of the present invention, Alzheimer's dementia due to damage of TH, BACE1, ApoE, Preslinin-1 or -2 genes, Carpenter syndrome due to damage of ZC4H2 gene, etc., UCH-L1, Parkin, DK-1 , PINK1, LRRK2, HTrA2, ATP13A2, such as Parkinson's disease due to gene damage, and other diseases associated with the development of brain neurological diseases SUMO, NEDD8, ISG15, FAT10, BDNF may be a disease caused by damage. In addition, diseases such as Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease, advanced nuclear palsy, spinal cerebellar degeneration, cerebellar atrophy, multiple sclerosis, Lewy body dementia, and frontal lobe dementia may be included in brain neurological diseases caused by DNA damage. This is not restrictive. According to an embodiment of the present invention, the brain neurological disease may be Alzheimer's dementia, but is not limited thereto.

Guide RNA included in the pharmaceutical composition of the present invention may have a base sequence that can hit one or more genes associated with each disease, such a base sequence may have a sequence complementary to each target gene. Complementary herein includes substantially complementary, and substantially complementary need not be a sequence that is completely complementary to the target gene, but sufficient complementarity within a range that can be hybridized with the target gene to act as a unique guide RNA it means. For example, the base sequence of the guide RNA may have 90% or more complementarity with the target DNA, preferably 95% or more, more preferably 99% or more complementarity, but is not limited thereto.

The pharmaceutical compositions of the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

In another aspect, the present invention provides a kit for cleaving target DNA in eukaryotic cells, organisms or biological samples comprising the nanocomposites described above, or for inducing targeted mutations. The mutation can be a naturally occurring mutation or an artificial mutation.

Kits of the present invention may include some additional elements needed to deliver nanocomposites to eukaryotic cells, organisms or biological samples. For example, the kit may include, but is not limited to, an injection buffer, such as a DEPC-treated injection buffer, and materials needed to analyze mutations in the target DNA.

Eukaryotic cells or organisms may be yeast, fungi, protozoa, plants, higher plants and insects, or cells of amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1, for example For example, cultured cells (in vitro), transplanted cells and primary cell cultures (invitro and ex vivo), and in vivo cells, which are commonly used in the art, It may also be a mammalian cell including a human, but is not limited thereto.

The kit of the invention can be used for cleavage of target DNA of eukaryotic cells or other biological samples other than the organism. A "biological sample" includes, but is not limited to, a sample for analysis such as tissue, cells, whole blood, Semm, plasma, saliva, sputum, cerebrospinal fluid or urine.

Kits of the invention can be used for genotyping analysis, wherein the term “genotyping” refers to “limited fragment length polymorphism (RLFP) assay”. That is, the kit of the present invention can be used for 1) detection of indels induced by genetic scissors in cells or organisms, 2) genotyping of naturally occurring mutations or mutations in cells or organisms, or 3) viruses or bacteria. It can be used for genotyping of DNA of infected pathogenic microorganisms.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Development and Analysis of Genetic Scissors (CRISPR / Cas9) Nanocomposites

The present invention is a technique for using gene therapy in the treatment of diseases. As an example, as shown in FIG. 1, an amphiphilic polymer is conjugated to gene scissors to prepare gene scissors (CRISPR / Cas9) nanocomposite. Genetic scissors (CRISPR / Cas9) nanocomposites were used to correct and treat genes. More specifically, in the present embodiment, a complex was prepared by conjugating an amphiphilic polymer and CRISPR / Cas9, which is a gene shear, and analyzed the manufactured gene shear (CRISPR / Cas9) nanocomposite.

1-1 Genetic Scissors (CRISPR / Cas9) Nanocomposite Analysis

CRISPR / Cas9, a gene shear, and R7L10, an amphiphilic polymer, were conjugated to prepare a "genetic shear (CRISPR / Cas9) nanocomposite", and then analyzed for the formation of complexes through AFM images.

As a result, as shown in FIG. 2, it was confirmed that the gene shear (CRISPR / Cas9) nanocomposite was formed as a larger size as about 125 nm compared to the sgRNA or Cas9 protein + sgRNA control.

Example 2. Confirmation of Genetic Correction Using Genetic Scissors (CRISPR / Cas9) Nanocomposites

In this example, genetic scissors (CRISPR / Cas9) nanocomposite-based in vitro calibration was possible.

Specifically, an RNA that complementarily binds to a sequence of at least one of SEQ ID NOS: 6 to 8 and a guide RNA complementarily binding to a TH gene (SEQ ID NO: 3 or 4) and a BACE1 gene (SEQ ID NO: 5) associated with a neuron ) Was confirmed that the nanocomposite nanocomposite that is attached to the TH gene and the BACE1 gene can be precisely hit.

In addition, the genetic scissors (CRISPR / Cas9) nanocomposites were used to determine whether the gene could be hit by various cell lines.

As a result, as shown in Figure 2a, various ratio-specific gene shears (CRISPR / Cas9) nanocomposites were prepared to confirm the cellular gene hit rate through a surveyor assay.

As a result, the ratio of Cas9-sgRNA: R7L10 = 1: 5 was the highest in the gene hit ratio, but since the cell death rate was high, the most suitable and stable ratio was confirmed to be the 1: 3 ratio.

In addition, the cell gene hit ratio is expressed by the next generation sequencing technology (NGS). Next-generation sequencing is a method of quickly deciphering genome information by breaking down the genome into a myriad of fragments and reading each piece and combining them. Therefore, through this analysis, by enabling full-seasoning, the gene hit rate through the gene shear (CRISPR / Cas9) nanocomposite was accurately confirmed (FIG. 2B).

As shown in FIG. 2B, when both on-target and off-target rates were confirmed, the on-target rate was high as about 24%, and the off-target rate was little or no similar to that of the control group.

As shown in Figure 2c, by hitting the genes TH and BACE1 associated with neurons, it was confirmed that the gene is hit through the gene shear (CRISPR / Cas9) nanocomposites regardless of the gene type.

In addition, in FIG. 2d, various cell lines, human dedifferentiated stem cells, embryonic stem cells, 293T cells, and neural stem cells, were hit through gene scissors (CRISPR / Cas9) nanocomposites and confirmed by a surveyor assay.

As shown in FIG. 2D, gene scissor (CRISPR / Cas9) nanocomposites showed that gene targeting was possible not only for various genes but also for various cell lines.

Example 3 Genetic Scissors (CRISPR / Cas9) Nanocomposite Alzheimer's Disease Mouse Model Intracellular Correction and Therapeutic Effect Confirmation

In this example, the intracellular gene targeting of the present invention was confirmed using an Alzheimer's disease animal model. For this purpose, it was confirmed that Alzheimer's disease is alleviated by forming a complex and injecting into the brain cells as shown in the schematic diagram shown in Figure 3a. As a control group, B6 mice, B6 mice injected with Saline, and 5X FAD Alzheimer's mice were prepared.

As a result, as shown in Figure 3b, the TH gene was produced through the gene scissors (CRISPR / Cas9) nanocomposite and injected into the mouse brain, the reduction of the TH gene was confirmed by immunostaining and fluorescence electron microscopy.

In addition, as shown in Figure 4a, the result of injecting a gene scissors (CRISPR / Cas9) nanocomposite prepared using a guide RNA containing a nucleotide sequence complementary to the BACE1 gene that produces ab plaque in Alzheimer's disease into the mouse brain , bace1 was reduced.

In addition, as shown in 4b, when ab plaque, the main symptom of Alzheimer's disease, was confirmed by ThT staining, ab plaque was also remarkably shown as a result of injecting the BACE1 gene scissors (CRISPR / Cas9) nanocomposite mentioned in FIG. 4A into the mouse brain. The decrease was confirmed by fluorescence microscopy.

In FIG. 4C, the main symptom of Alzheimer's cognitive decline was confirmed through a water maze behavioral experiment.

As a result, it was found that the group of mice injected with the gene scissors (CRISPR / Cas9) nanocomposite into the brain compared to the control group improved the cognitive ability compared to the group of 5X FAD Alzheimer's mice (control) not injected with the nanocomposite. .

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Nanocomplex comprising Cas9 protein, guide RNA and amphipathic          peptide, and uses of the same <130> 17P04013 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 4107 <212> DNA <213> Artificial Sequence <220> <223> Cas9-coding sequence <400> 1 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg 60 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc 120 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag 180 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc 300 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc 360 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag 420 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac 480 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc 660 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc 900 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc 960 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc 1020 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc 1080 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg 1140 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc 1200 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac 1260 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc 1320 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc 1380 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag 1440 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag 1500 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg 1620 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc 1680 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc 1740 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc 1920 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc 1980 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg 2040 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac 2100 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg 2160 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc 2220 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg 2280 atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc 2340 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc 2400 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc 2460 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac 2520 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc 2580 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag 2640 aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg 2700 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag 2760 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac 2820 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc 2880 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac 2940 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag 3060 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc 3120 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc 3180 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc 3240 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc 3360 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc 3420 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg 3480 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac 3540 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg 3660 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc 3720 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag 3780 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg 3840 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag 3900 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag 4020 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc 4080 gacctgagcc agctgggcgg cgactaa 4107 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> R7L10 <400> 2 Arg Arg Arg Arg Arg Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu   1 5 10 15 Leu     <210> 3 <211> 8077 <212> DNA <213> Artificial Sequence <220> Homo sapiens tyrosine hydroxylase (TH) <400> 3 gtgggggatg taaggagggg aaggtggggg acccagaggg ggctttgacg tcagctcagc 60 ttataagagg ctgctgggcc agggctgtgg agacggagcc cggacctcca cactgagcca 120 tgcccacccc cgacgccacc acgccacagg ccaagggctt ccgcagggcc gtgtctgagc 180 tggacgccaa gcaggcagag gccatcatgg taagagggca ggtaggtgcc cggcggccgc 240 agtggaccgg agcccagggc tggtgccagc tgcctctgct actccccagc ctggctggca 300 gccccaggct cagggtccat gcaaacccct gggacgcggc gtggatgtgg aggcctgggc 360 acagcggcat cccctgtgcc tggtgtttga gtccctgttg ggggagggtg aggtgatgcc 420 tgtccctgtg tgtgcccctc ttaggccgac ctctctcggg ggtcgtgtgg gtctctgtgt 480 cttgtttcat cttgaatctt aacgatcgga atgtggaaac aaatccatcc aaaaaatcca 540 agatggccag aggtccccgg ctgctgcacc cagcccccac cctactccca cctgcccctg 600 cctccctctg ccccagctgc cctagtcagc accccaacca gcctgcctgc ttggggaggc 660 agccccaagg cccttcccag gctctagcag cagctcatgg tggggggtcc tgggcaaata 720 gggggcaaaa ttcaaagggt atctgggctc tggggtgatt cccattggcc tgttcctccc 780 ttatttccct cattcattca ttcattcatt cattcattca ccatggagtc tgtgttccct 840 gtgacctgca ctcggaagcc ctgtgtacag gggactgtgt gggccaggct ggataatcgg 900 gagcttttca gcccacagga ggggtcttcg gtgcctcctt gggcactcag aaccttgggc 960 tccctggcac atttaaaatg ggtttttatt tatggacctt gattgaaatg tggtgtgagt 1020 tgtagcagtg tcatttccag gtaccttctc agggacacag ggcgccctcc cccgtcctcc 1080 cccgccctcc cctaccctcc cccaccaggc tccccatcag gcatcccctc cccagggcgc 1140 cccggggccc agcctcacag gctctccgtg gcctggaact gcagccccag ctgcatccta 1200 cacccccacc ccaagggtaa gtaagagggg actctgggag gggcttctgc tgctcccctt 1260 catgttccac aaccctggaa gctcaggatg aagctgattc ttctcttaca aggggcccag 1320 agccttcttg ggagttcagc tccaagggat gagccccagg tgtctgccaa gtccccctct 1380 gtccaggcct gggacggctc tgggatcgag gggtcagagg cgctgagccc agggagagac 1440 acctgcgccc agagctatga caaagggtgg agggatgaca aggcagccag gagcgggcgc 1500 ctgcggggtg gcacagaggg gcagggcccg aggacaggtg tcctgatggg agtgtgagaa 1560 agggtcccct gtgcggcagc caggagggta ggggggttgt tcactggggc cctgtggggg 1620 cagctccttc ctgagctgcc gttccctccc cggcagccga tgccactgtc catcaagaca 1680 tcgccctctt cccatcacta atccagttag cgcctggcct ggggatgagt gacacagcgt 1740 ctctgtctgt ctgctcgcca cagagtgggg agcaggcgag caccttccca gcccccactc 1800 ctcccccacc accactgctt ctgactgggc tgcccccatc gggaagggcg tgcaatgccc 1860 gcaggcacct cggctagcat ctgccccagc aggcacacag taggcgctca aaaacgtgct 1920 ctcatcccct gcctctgtgt gccatcagcg ctgcccgact gtgggaccag ctgtgggtgg 1980 aggtccccgg gtctcagcag gtggaggagg catgggtgcc ccttgtcccc acagtccccg 2040 cggttcattg ggcgcaggca gagcctcatc gaggacgccc gcaaggagcg ggaggcggcg 2100 gtggcagcag cggccgctgc agtcccctcg gagcccgggg accccctgga ggctgtggcc 2160 tttgaggaga aggaggggaa ggccgtgcta aacctgctct tctccccgag ggccaccaag 2220 ccctcggcgc tgtcccgagc tgtgaaggtg tttgaggtga gctggtggcc ttcgtgtccc 2280 tggggcaagt tcacctgtgg gtggggctgt gtgggctgag ttcctgaccc ctctatagca 2340 gaggtgcagc tgcccaggcc cccgaggccg gcacaggatg cagcagggga gtctcaggcc 2400 tcagctcagc ccccatggca tctagccaca cccccgtgtt tttgagggat cctgagccca 2460 cccctagggc tgaggctacc aagccccact gtgcctcttg ccttgcccat cccctggatc 2520 cccctcaccc accatttccc acgtgggggg ctcccagcag ggcagcacaa gaggcagggg 2580 cagggcagtg tgccctctcc cacccaccca gcacagtggc tcaggtgacc actgattgca 2640 ttagtcactc cggccccact gtgccccggg aggcaggtga cccagctccc ggaagaagct 2700 cccaaatgac attaaagcca gactccccgc cccccagctc ccagagccag ttttgtggcc 2760 cgagggccac tgcgacccac cgcccttgtt gctaggcaac aggaggtggg ggtggagcgg 2820 acccttctgg ccagtgtcct ggacgctcag gggccagtga gactcagggc ccatcctaca 2880 aacctggatg aggccaccag ggttgggggc accttctgac cagtggctga ggagccggac 2940 tgtgtggcat ggccttggga cacacacacc gagccgccca gaaccaggtt aagcctcaag 3000 cggtgacaac tcctggttag gcacgtaaca caaaatccaa cttgccagtg gcaaaccctg 3060 gcctggtggc cgacagctga cctgagcctg gaagaacggg atctgtgtgc tgctagcaca 3120 aaagtcaagg gcagggcctg gccagccagc cagatgtgcc tcctccccgc ccaccccacc 3180 ctctctctcc atctctgtct ctttctcctt ctctctctct tcctgctttt gctccctaag 3240 acgtttgaag ccaaaatcca ccatctagag acccggcccg cccagaggcc gcgagctggg 3300 ggcccccacc tggagtactt cgtgcgcctc gaggtgcgcc gaggggacct ggccgccctg 3360 ctcagtggtg tgcgccaggt gtcagaggac gtgcgcagcc ccgcggggcc caagggtgag 3420 gcggttttct gtccttgagg gccaccaaat gaccttgaga ggctggggtg caggggctcc 3480 tgcaggggga ccctacagtg accacgtggt ggtggcctgg ttccctctct gcgggctcca 3540 ctccgcaccc cgttttgcta cacatccgtg tccgggcctg gggccactcc aggatccccc 3600 cgcagctctc acagccccgg ctgcctctgc cccccggaag tcttgtaggg gaggctgctt 3660 caaggtgggt gacacagccc cacggctccg agctcaccaa gatctcttcc tccatcaccc 3720 ataaagtccc ctggttccca agaaaagtgt cagagctgga caagtgtcat cacctggtca 3780 ccaagttcga ccctgacctg gacttggacc acccggtgag tggtgcgccc ctcactcagg 3840 cctcctgccc ctgatcacat cccctaccct tagcccaacc ctggacagga gtctgtcggc 3900 tccaggagcc tccgtggcct gtgcccccac cccagcacag cctcctgacc cgtgcatccc 3960 ctctgccctc agggcttctc ggaccaggtg taccgccagc gcaggaagct gattgctgag 4020 atcgccttcc agtacaggca gtgaggggcc cctgcgctcg ggacccagac tccgtcctgc 4080 aggctgacgc tggacctggg gggtgggagg gaaggacaaa ggggaggacc catcttgtca 4140 ccagcatcag tgcctcctgc caggcagctc tgctccaggg ctttccatgt ccccaaatcc 4200 cagtggggaa actgaggccc aggggggcta gagcaacctg ccgaggccac atagccggct 4260 cacggcacag tcagctgggg tgcaccctcc tgtccatcct ccaacccaaa ggcctcgctg 4320 cactaggcgg gtgtggacct gtgcccagtg aagctccctc cctccctcct gcccttctca 4380 ctccccgagg ggacctgctg accactggcc ccctccccag cggcgacccg attccccgtg 4440 tggagtacac cgccgaggag attgccacct ggtgagacct ccgtgcagct aggggctggg 4500 gaggagcccg ggggatgcct cctggaatcc tggcgtgtga gggccgcctc cagggacctt 4560 ggcacaacag gagagactaa ggccgggaag aagagggact tgcagggctc agaatgttgg 4620 gttgggagga agaggctacc catcctgtcg ggccatcccc agtgtgctga gggaccgccc 4680 ctcatggccc cctatcccct gggattccct aaagccacca gcaaaagccc ctcccggggg 4740 cctgggtctt caggggtccc caagaggcct gcgttggtag gggctcaggc aggcagaggc 4800 acccacagtt caggaggggg gtttcgggca ctggggtggg gcattagagg gccctgagcc 4860 tggctgcccg caggaaggag gtctacacca cgctgaaggg cctctacgcc acgcacgcct 4920 gcggggagca cctggaggcc tttgctttgc tggagcgctt cagcggctac cgggaagaca 4980 atatccccca gctggaggac gtctcccgct tcctgaaggg tgtgcccaga cgggaggggc 5040 gcagagccgg ggggccgggg atggtcagcc aagcgcccca ccccagcgcg gctccagccc 5100 gtcccggctc ggcagtgacc cgcgtggccc cttgcagagc gcacgggctt ccagctgcgg 5160 cctgtggccg gcctgctgtc cgcccgggac ttcctggcca gcctggcctt ccgcgtgttc 5220 cagtgcaccc agtatatccg ccacgcgtcc tcgcccatgc actcccctga gccgtgagtg 5280 cgcgccctgg ccgccagccc gagggtgggg ggtgcgacgg gcggcccctc agcccccttc 5340 tccctcctac gcgcagggac tgctgccacg agctgctggg gcacgtgccc atgctggccg 5400 accgcacctt cgcgcagttc tcgcaggtac gccgcggcct cggagggagc cggggtcacc 5460 caggggctgg cttggcgccg ggggcgggcg gggatcgatg tgcgggtggg tgaagtgtgc 5520 tgcctgctcc cgggccccgc caaggaggct cggcgccccg agggtcgcgc ggcatagggc 5580 ggggctggag cggagcctcc cacggcctgt gctgccacct gccggctacc tgggaacggc 5640 gcccacgggc ttaggaatgt ggtcaaggag ggctgcctgg aggaggaggc ccggtggagg 5700 tgcggatcct gggcggccag ggaaggtctc tgccgccagg gaagtgtccc agagacccct 5760 ggaggggctg ctgacacccc cggtgccccc acctcgagca tgacccaggg ctgcctctcc 5820 ccatccttca tcctccctgc tccacaggac attggcctgg cgtccctggg ggcctcggat 5880 gaggaaattg agaagctgtc cacggtgggt tgacccctcc ctgcagggcc tggggtgtgg 5940 gtttgggggt ctgaatccag gcctcaccct cttgccgtcc aggctgaggc ctctccttcc 6000 acccacgaat tgtgaccctc accctggcct gcctgcatcc tggcctggcc tccctggggg 6060 tggtatcctg gtcacgggtg accaggggct gcccggtggg cggcagctgt ctctgggctg 6120 atgctgcccg gcttccccgc agctgtactg gttcacggtg gagttcgggc tgtgtaagca 6180 gaacggggag gtgaaggcct atggtgccgg gctgctgtcc tcctacgggg agctcctggt 6240 gagagtctct ccttgctgca gcccccagca gaggggcagg gctgggggac ggtgcaggga 6300 ggggacaggc tcccagtggg aggaaactga ggcctggacc tccaggactc aggctctgtt 6360 tgggagaagg cttgtctctg cccagtcctc accccacatt atcccaggcc tccgaaggcc 6420 cggcggggga gatgggggtg actctaccca aggaacccac ccagcgtcag gccacggtgc 6480 cccagttccc tcggggacct gggtgcagtg gagtcagtga tgccattggc ctcctgccag 6540 cactgcctgt ctgaggagcc tgagattcgg gccttcgacc ctgaggctgc ggccgtgcag 6600 ccctaccaag accagacgta ccagtcagtc tacttcgtgt ctgagagctt cagtgacgcc 6660 aaggacaagc tcaggtgggc taggctgcta gggcaagccc cccatggtgc ccccaaactg 6720 ggccagccag gccttccttc tggccttgag cagggctgga cctgtgagcc caggtcacag 6780 atgagaaaac cgacccctgg ttgcagcagc ccccacacag cagggacacc atccgtgaga 6840 aggaccccag cgtctgggga ggggcagacc tacaggactg ggggctgctg ggtggccggg 6900 tcaaggccag tcttggaggt gctgacagag cctgagcttt gtgaggacgt cctgtggaac 6960 ctgtcccggc cccctgccct gggatgggga gaagtcaggg ggatagacag agtcaaggtg 7020 ggggacaggg cgggagtggg gtccccaggg ctgggggcct ttggtgcagt gaccagagtg 7080 tcaggagagg ggagcaaagc cctctagcct catcctcata aaaggtctca tcattttccc 7140 tccagcctct tatgcactgg ggaaactgag gccaggggct atgtgtccag cggacagggg 7200 tgctgaattc cacccacagg cttagggata tggtcaagga aagcttcctg gaggaggccc 7260 agtggaggtt cagggaggga tggggtgccc ggcagtctct agtggaaaag gcgcctagcc 7320 tatctccccc atgaaccccc tcacccagcc ctggaagagg cctcagtgtc ccgcctgtga 7380 ccagttggct cagaaaagcc ctgggagctc tgagccactg tgaaggtgga aacgcggccc 7440 ctggcctccc ctctcctgga ggctgcagac tctgcccgcc agttgacgag ggctctgccg 7500 ctctcctccc caggagctat gcctcacgca tccagcgccc cttctccgtg aagttcgacc 7560 cgtacacgct ggccatcgac gtgctggaca gcccccaggc cgtgcggcgc tccctggagg 7620 gtgtccagga tgagctggac acccttgccc atgcgctgag tgccattggc taggtgcacg 7680 gcgtccctga gggcccttcc caacctcccc tggtcctgca ctgtcccgga gctcaggccc 7740 tggtgagggg ctgggtcccg ggtgcccccc atgccctccc tgctgccagg ctcccactgc 7800 ccctgcacct gcttctcagc gcaacagctg tgtgtgcccg tggtgaggtt gtgctgcctg 7860 tggtgaggtc ctgtcctggc tcccagggtc ctgggggctg ctgcactgcc ctccgccctt 7920 ccctgacact gtctgctgcc ccaatcaccg tcacaataaa agaaactgtg gtctctacac 7980 ctgcctggcc ccacatctgt gccacagaga cagaccctgg gatcctcaga ctcccacacc 8040 cccaccccag cctcactcag aggtttcgcc ctggcct 8077 <210> 4 <211> 7391 <212> DNA <213> Artificial Sequence <220> <223> Mus musculus tyrosine hydroxylase (Th) <400> 4 aggaggtggg ggacccagag gggctttgac gtcagcctgg cctttaagag gccgcctgcc 60 tggcaagggc tgtggagaca gaactcggga ccaccagctt gcactatgcc cacccccagc 120 gcctcctcgc cacagcccaa gggcttcaga agagccgtct cagagcagga taccaagcag 180 gccgaggctg tcacggtgag gatgacatgc tgtcacccag gggtatctgg agcaacttct 240 gcacctataa atgggtggat accccaggct gggagtcttt ccagtgtctg gtacctaggg 300 ttggagtggg tcttagttct atcccgtctc agggttagtt tgggtcctag ctctggttct 360 gtgcttgggt aaatgcttct gttgggtcct gctggactcc gagagggaca gccaggacct 420 gtttcactgt gccaggcttc tgtgggtcct attttgtgaa gtaagggaaa atcccccctg 480 ggtggtcttg ggagagagcc ccatgatcca agtcccccca gtagccctcc ctactcatct 540 gctcccttgg gttctctagc cttgcctgtc atagagtgtg gtgttcctgt aatacacact 600 tctgagcaca cctggtcagc aaatggctgg tctgcctgat gctgtcctgt gcaaatgggg 660 cccaggttct accatacctc ttcagccccc caaactctgg gaaaatctct gtgtctcctt 720 tttcccttct caccttccac cagtctgctt cggtgttccc taagagtagg acttaggaag 780 ccacagggac caaggggaga tgctaccaag acatcaaaca gtcccttttg tcgggggcag 840 ggggagaatc agctcataag aagggctttc agattatcct tggggcccca tctaactatt 900 gccctgggtt gacagagacc ttaatgaaac cagtgtgacc tttagaaatt taatttgtag 960 gcacttctcc ggggacaact gtggggtcaa aagtgcccct tcatacttct tagctgcatg 1020 actctcttct cagctccctg ggaaccaccc tgtctcatgt tctccccagc gagcctgggg 1080 ctctggctgt attcacagtg tcctgctgga ggtccggagg gggctccaga gggcatgttt 1140 ctccccatga ctaggacgtt cctagaaccc aggacccaga accttttggg gagttcaact 1200 cccagattag agttcaggct tctcctctct gaagtccagg tagttctgag tgttggggga 1260 ttaggaatac tggttcacag ctgtggcaga cacagagcca gtggagtcca tacagaggta 1320 ctcagacaag ggaggagggg gagacatttt gagggggcta tttccaggta ggaggacagc 1380 ctagacagga cacctggcta agaaccactt attttttcaa tgatgatgtg tccacagggc 1440 tcagcctctc agagagttgg gagggaaagg atgcttgttt aagctgtgta cagtgctctc 1500 tacctgaatg gaccacccaa gctattcact ccccatgagg tctgaggctt tgtgctccta 1560 cagtctctgg gcgctgctca catacatcag gaaaaacaac ctgagtcaca gccctctatc 1620 actaattcag ttaggtctaa gcctgggagg taggtgacag ggcacctcta tcttgggaag 1680 atggcagcta ggagcccctg ctttccagtt ctgccatggc tctccagccc tagctaccca 1740 ccatgccctg tcagaacact gattagcatg ttagtcctcc actcctacat ggcacccagt 1800 ggtttgacca gagccctgga ggcgggatca gcagaggcag cagcatggag atacaggcat 1860 gtctcttgac tctcagtccc caaggttcat tggacggcgg cagagtctca tcgaggatgc 1920 ccgcaaggag cgggaggcag cagcagctgc agcagcggct gcggtagcct ccgcggaacc 1980 tgggaaccca ttggaggctg tggtattcga ggagagggat ggaaatgctg ttctcaacct 2040 gctcttctcc ttgaggggta caaaaccctc ctcactgtct cgggctttga aagtgtttga 2100 ggtgggttgg cttggccttc cggggtagca tgcataagat atgtaagaat gtctgtgtcc 2160 tccccccacc gccagggctg aaatatggct gctcaggcca tcagaggctg ggtctcaggg 2220 actacctttc agctcctcca gttaatctca gctccctgac acccactgct atatatacac 2280 agcctcttac ccagctgccc aggggtcaat gaagcagctg attacactgt tgctttggct 2340 aatgacctgc cccccttccc ctgccccaga aaaactctca aatgacatta aagccagaac 2400 cctgtgagct cccagaattg acagcctggc ctaagtgttt tgttgctggg caacagaagg 2460 tgaagatgaa gttgacccct gtccaccagt cctgagtttc ctatttaccc tgtcatggcc 2520 tgagacttgt gtccttgata tggtggagtg tgatccaggc ctttgggcgt ggccagtaag 2580 tcaaattgag accattaggg gatagcgcct tctgctatgc tttctagaca aacagaactg 2640 tccagaaagg ggaccaaagg cacagctgtg atgattcctg gatggataca caagacaaaa 2700 tccaatctat tggattttga caccttcaaa attcataggc aagggtaaaa acaaaaacca 2760 aacgaacaaa caaaaaccaa acgaacaagc aaacaactga cagtgcctac cagggtcctc 2820 cgtctccctc attcccacta ttatgtctct ccatctctcc ctacctgtca tcatctctct 2880 ctcttcatct tctccaccct ctctccattg ctccttcccc atctgtctct ctccttctct 2940 ctcttcctgc ttttgctcct gaagacattt gaagccaaaa tccaccactt agagacccgg 3000 cctgcccaga ggccactggc aggaagcccc cacctggagt actttgtgcg cttcgaggtg 3060 cccagtggcg acctggctgc cctcctcagt tctgtgcgtc gggtgtctga cgatgtgcgc 3120 agtgccagag aggacaaggg tgaggatgga tttctgtccc tggcctgggg gtgggaggca 3180 gagtatcctg ctgggcgggg gggacgaccc tgaagtgtcc acagagttgg accaccacgc 3240 tggctctacc ttctactttt ttttcatgaa tcctagtaaa ctctgccctc taggaggtcc 3300 ctgggggagg ctggacccag catggccaaa ccttattatg gacccatgct tacctgggcc 3360 cctttgcctc tcttgcttgt gaagttccct ggttcccaag gaaagtgtca gagttggata 3420 agtgtcacca cctggtcacc aagtttgacc ctgacctgga cctggaccat ccggtgagct 3480 tgtgtcccag agctgagtcc tgtggggtgg gcacaggtag ggactgaccc tggtctttgg 3540 agcagggaat gcccaacttc ctcctgatgt gtgttccttg gccatcaggg cttctctgac 3600 caggcgtatc gccagcgccg gaagctgatt gcagagattg ccttccaata caagcagtaa 3660 ggagcctgcc tgtggggcag agtccccaga ctgtagagag aggaggagag gagggaggag 3720 gtgactggtt agctgtcaca cagctttaca tgtcagcttg tctgtctctg ctctgtgctt 3780 ggcattattc ttagtcacag ctaaggagac tggggcagag aggctaagca gctggtttgg 3840 gtcagtgtac ccaaaccagt ccactccata ctccaaaggc ctctgaacag ctgatacagc 3900 ttcaaacgaa gcctctcacc ccatctttcc tactctccga gatggcggga ggggggttgc 3960 cctattgacc actgatcccc tccacagggg tgagccaatt ccccacgtgg aatacacaaa 4020 ggaggaaatt gctacctggt aagaccctgc tgatggttgg gttgggcagg gagggctggg 4080 cttgagaaga gccagggaga cggttcccag aatccctgtc tgtgagggcc atctctggaa 4140 actttggtac aatagaagaa gagactaaag ccagttagaa gaggggcttc cagtggccca 4200 aaataatcag gtcattcaga gatgtcgcta tcggcccttt cccagcctct gggttccctc 4260 aaaccacagg aatgccctat gtattgatgg gggcccacag ttcaggagga tggtggaagc 4320 aggcaaagcg tccatggggt ggtcatgggg agaagaaccc cagcagctag taattcccta 4380 agcccctgtg gcctacagga aggaggtata cgccacgctg aagggcctct atgctaccca 4440 tgcctgccgg gaacacctgg aggctttcca gcttctggaa cggtactgtg gctaccgaga 4500 ggacagcatt ccacagctgg aggatgtgtc tcacttcttg aagggtgaga tgcagaggaa 4560 gtagggcagg gaggggcatc ccctgtgcta tttctgatgc gaggtccctt acagaacgga 4620 ctggcttcca gctgcgaccc gtggccggtc tactgtctgc ccgtgatttt ctggccagtc 4680 tggccttccg tgtgtttcag tgcacacagt acatccgtca tgcctcctca cctatgcact 4740 cacccgagcc gtgagtatgc tgcctagggc agaggaccct gaacttgttg cccagtaata 4800 tccgtgtgca atgatggcag aggtgtggag gtgctaaagt ttccttgtct ccggggaagg 4860 acagagagcc ttcccattcc atcttccttt tctcgtggcc tcacacagaga actgctgcca 4920 cgagctgctg ggacacgtac ccatgttggc tgaccgcaca tttgcccagt tctcccaggt 4980 atgtttaggg cctcaacgca gaaaccagag gttatttggt tgagccagag attcaaaagt 5040 tttacttgtt ggggtactcc tccagctaga cagtgtgtct tcatggattc atgggacttg 5100 taggtagcca aacaccgaag cagagactgt ttctgtaaca gggagggact aactagtaac 5160 acaagtccca ggggaaaggg atttttgtgg ggtttgatgc cacctgctgg taattctggg 5220 aactgcaggc cctgagctct tggctgactt ggcgtgtggt gaagtgtatg ttgcagggtc 5280 ccctggagaa gtagcatcca gtgcccttgc ctcaagcaca atgtcagagc agcgtctttt 5340 catactttag cctctttctc tacccacagg acattggact tgcatctctg ggggcttcag 5400 atgaagaaat tgaaaaactc tccacggttc gttttcagag ccctgggggg gggggggggg 5460 ggaagttcag acctttcttt ctgtccccaa gaacagcctt acctgttgtg ggtgctacac 5520 acagaggggc tatgcgcggc accaggcatt ttaggtgatc cttatgatta tctggcaggt 5580 gtactggttc actgtggagt ttgggctgtg taaacagaat ggggagctga aggcttacgg 5640 tgcagggctg ctgtcttcct atggagagct cctggtgaga ttaccctgct cccagccccc 5700 agcaaaggag atcagggctg gaggatcagg gtgtggaggg ccctgctctc atcagaaggg 5760 tccttggctc aggtctgaga ttcaggctgt tctagagagt gtatgcctct ggccagaccc 5820 cactgtacag tagcccagaa ccctgcggga ggggggagcc ccacacccat ggcacccatt 5880 tgccttgagc gctgcttggg tgtgatacct gtggcctctt tctagcactc cctgtcagag 5940 gagcccgagg tccgggcctt tgacccagac acagcagccg tgcagcccta ccaagatcaa 6000 acctaccagc cggtgtactt cgtgtcagag agcttcagtg atgccaagga caagctcagg 6060 tgggtggagc catcaggagt cagccctgcc cagcttcacc agtgcaagcc aggccttgtc 6120 ttcacttgag agcccattta gtacactcag caacaccccc ttgtaacaag agttcatgct 6180 tgagattacc ctgctcctgg cccctcagca aaggagacca gggctggggg gtgagggtat 6240 ggagtgccct gctcccatca gaaagtccga gaagagagag accagcagca tagggtcaag 6300 gcaaagtcaa ggtcaggtcc aggccagctg tctgtgcctc agcagagcct gagtctctca 6360 ttctcagcag cacgggctgg agtgggatgg ggacagataa gagagaaaaa gaggcagagt 6420 ggcaggtaag agctgccacg agggcaaaga ttatagaggg agtggcagcc caaaggcagg 6480 agacacagtt atacaggggt ctgcagagga gaaaggagac tggtaggttg gtgcttatca 6540 cttctcccag cctgctgtgc tcatctggca tgtccactcc agtttaggga ctctctaggg 6600 cacacagtca cggggagctt cccgaaggaa gaagcccagc tgagaaacgg ggtgctttgc 6660 agaggcctac ccacaagaat cgctgaggct ttagtctcct gggtgtctag aatgggcttt 6720 aggggtctgg gaaactcaga aaagccattt ccccattgcc cttggagatt gcagatccta 6780 tctggagcta cctaactcta gcctgctttc tcatgcccat atccaatctc ctgtgattga 6840 aggattcata ctggctcccc ccccccccac acacaggaac tatgcctctc gtatccagcg 6900 cccattctct gtgaagtttg acccgtacac cctggccatt gatgtactgg acagtcctca 6960 caccatccgg cgctccttag agggggtcca ggatgagctg cacaccctga cccaagcact 7020 gagtgccatt agctaaatgc atagggtacc acccacaggt gccaggggcc tttcccaaag 7080 ttcccagccc cttctccaac ctttcctggc ccagaggctt tcccatgtgt gtggctggac 7140 cctttgatgg gctgctcttg gtccccctcc tccactgctt ctcaaccaca tcttactact 7200 gcatgcgctc caggatggtc ctgcattcct cctgcccttc atgctgtatt ctaccctgat 7260 tattatctca ataaaggaag gaaaggtctc caggagtgtt cctggcctct gtgtcaccac 7320 aatgatagct cctagcactt gtatatgccc tggcttcatt tggaggcttc tctctgatct 7380 ctttcgtcca a 7391 <210> 5 <211> 24140 <212> DNA <213> Artificial Sequence <220> <223> Mus musculus beta-site APP cleaving enzyme 1 (Bace1) <400> 5 cagagtgaac tccatctcgg cagagggcat cccagacccc tttcagctcc ggaagctggg 60 ctgtctgcca tgggaagact acacttccca gagatcccag ggaaaagcaa aaaccctttg 120 gctttgacag ccaccgccac aagcctttcc gcctccccag cctgcctagg tgctgggagc 180 tgggagctgg attatggtgg cctgagcagc cgacgcagcc gtaggagccc ggagtccctg 240 tcggtcccca agctgcaaag cccgcctgga agaccccgaa agctacgggc tcggatagcc 300 atgcccgccc ctcccagccc cacaaggggc ccgatccccc cgctggcggc cggcgtccag 360 atgtagctgg gtcccctgga tcgccatcgt cgtctcctct cgtgcgctac ggatttctcc 420 tgcccactct ccgccgcctg gaccgggaac tgagcgaggg gcctgcagac tctgcagtcc 480 tgatgccgcc gaggccgctc tcctgagaga agccaccacc acccagactt aggggcaggc 540 aagagggaca gtcaccaacc ggaccacaag gcccgggctc actatggccc cagcgctgca 600 ctggctcctg ctatgggtgg gctcgggaat gctgcctgcc cagggaaccc atctcggcat 660 ccggctgccc cttcgcagcg gcctggcagg gccacccctg ggcctgaggc tgccccggga 720 gaccgacgag gaatcggagg agcctggccg gagaggcagc tttgtggaga tggtggacaa 780 cctgagggga aagtccggcc agggctacta tgtggagatg accgtaggca gccccccaca 840 gacggtaagg tggtcagatc tagcaaccct ccttcatgca tggtagaggg gtgatagggt 900 agagatgaag cagcctgccc tgataaaatg gtttgtttag tgggtgcaga atgaccgccc 960 gctcctattc ctccaggatc cggttgggag tagcctttcc atttcccgcc ccctctcact 1020 ggcatccaag ccccaggact gactactcac ctctcactcc cactgcccca tccccccacg 1080 cccaagaaag cacttcagtg gtggctgacc ctggggaagg gccaggcacg gggacactgt 1140 aacaggtgtc agtggccgga gtctctcata ttccatctcc acactctccc ctactctagt 1200 gttgcggggg gggggggggt atctaagatc cccaccttca tcttcttgag aacccaaaat 1260 cctggcacca gttggatcaa tcagcccctt actctaagtc tgggctgcca taagccaaca 1320 aagtgagagt cttagtaaca tccatgtagc ctccaagtcc caatagactc tggcttcccc 1380 tagtcccttc ctcctttatc ccaaccccca ctctctacac tctagctcct cccccaccca 1440 tgcctcagaa catggccaga agagagctgt actgagtctg agaaagataa gcctagggga 1500 gccagaaacc ccttgcaagg gttctgagac ggatgggcac acagccatgg gaagggactt 1560 ctccctgtct tttcccgtct tttccaacag tttccaaggt acctggggtg gctggtggag 1620 gttgtgtcct ctacctgtgc tgactgggag tgtgggcttg agtcagctat ggcccatgtg 1680 gggttgctgg ctgacctctc tgagtgcatc tgccacctcc gtgggcgagt ctgggtactg 1740 gttggtggca gtgtgattct gcgtgtgctt ctgtgtgtca ccttgagtct gatcctgtgt 1800 atctctgtga gctaccctca gtggctccgg accgtgtcca caaagcttgt ccacggaggg 1860 tgtggaaaga tgggagtgtg tgcgctgggg tcagggagga gacagtcaca cttcctttca 1920 ctcaggcagt tggatatggg agaaccacag acccctgctt tcttgttact tgctgggtct 1980 ataccactca agcttgcttt gggagctctg aagtcctggt ctcccccact cacccctcct 2040 ctttgctgta caccataact tcccagaagt cgtatggaat ccgggggtcg gagccaagtc 2100 agagctagag ttcagtctga gccaagagaa ggtaagatgt cagggaaggg gacatgtgga 2160 gacgtgggac tctagtgacg tgctgtgcaa ctgcacagat ctgtgataaa gccacaagga 2220 ctttagggtc agacaggcta gaatctgggg gtctgcctag caccctagcc ttcccttttc 2280 ccagcagtgt gaccttaagc aacttatata actcttgttg gctattgcct tctcttcaaa 2340 acatgggaaa tggggcctcc tttgcaagac aattgtgagg attaaatgaa accacgtagg 2400 ggaccatgcc tagaagaaat ccctgggtta attgacctcc aataaatatt acctccttct 2460 ccttaactgg cctggccact tctgcctctt ctgtgctatg caatgaagca taaaatctgg 2520 accgtgttga gtatatttag tacatacttc cctggtcccc tgagatgcaa tgtaggtgct 2580 ccagcccagc ccttggcaga gagtagctgt tgagggctag tcagtgccct cccctcccag 2640 ctagaagtca gagcaggggg ctggaggtca cacactagcc ctgggaaccc tgagtagagg 2700 cactctgtgg ttgcacgtgc ctctgatccc gaccctgttc cagccagcct cggctctgtg 2760 gctattttgt tccttccctc agcagcctgt gagaaggatg agccttcttc ttacccaacg 2820 gggctatcag aagcagtgaa atgggatgga gggtggcaga gttctatgaa cacaaacctc 2880 tggaatttgg agggttactg cccatgcttg gtggtctaat ctacaaaaca atggaagaca 2940 cacctctgca tgaaaggctt taaaaaggaa gtaaaatgta agtactcctt tgacaaaagt 3000 gatgctctgt agattcttat ctttagaacc cgctaaacat gtaaattctc atgatcccac 3060 cccagagtct gggagtctgc aggtcttaag gctgagaagc accgatctat gaggagaaat 3120 aatgatgcca aaggtagagt tggctcaagg gacttgatgg agtatgtgtt cattgagtga 3180 ggtcctggag ggtggaaagg aatggggcca tcctagccag gacctggcgc ttctcccaac 3240 acaccgtaat tgcttctcac ttgctcccca ctctggccca gagactatga gaaacgagag 3300 acgaatgagt tgtgtgtttg agcttatgtg tgtttgttca ggagacaagt gaaagccagg 3360 ccagttgata ctgcagggtt atgctgagac cagatcaatt taggtaaata aaacggagcc 3420 caactgtcaa aaagccttaa atgtcaggct atatggtttg gacttcggct ctgagggggg 3480 aaattgagat catttgtact ggagaatgat atgaagcaag caagcagacg tctgaatctt 3540 agggagagtg gaatatagag ctacgggaga cgcgaagctc actttctgtg gagaaggcgt 3600 gaggttgcgg taagagggag agagaaagga ggccaggatg caaggaatgt cacaaaacag 3660 gcgagcaggc tggggacatc gtgtggaggg tggagaagaa ggaaggcatc agaggtaact 3720 tggtgattct gacatggcgt aagttgaaat ggaccattgg tggaccagga tacagggcag 3780 gagggcatgg ttggtataca cacttgtttt attttaattc agtgtgcata tctttgtttg 3840 ttctggtttg tttgttttgc tgcatccctc actcctaccc cttgctatgt agcccctggc 3900 tagacacaat tcatgagcct tctgccaaag cctttagcat ggtgggacta gaagtgtacg 3960 ccatcacacc cagcttcttc ttttgttctg ttgcttggct taagacaggt cctcactgtg 4020 tagccctggc tgatctgaat ttaccatgta gacatgctag cctcaaactc caaaagatcc 4080 acctgcctct gcctccctag tgctagaatt aaggctgtgt gccaccaagc ccagtttact 4140 tggttctttg tttatttgtt ctttctttct gcgatcatgt atatgtatat gagtatgtat 4200 atgcatgtat acaaacacac atatagacgc tctacattat tcatagcttt cagaaggaaa 4260 aacaggctct gaaaggtaaa cccttggtca cgtgctgtgt cagtaagctg aggccctccc 4320 tactttaccc tttgttctcc tacaatggta ttgtcttgaa tagcctgttg agagcatccc 4380 tctgctgtga tgtctcccct acagttgtgt agtgtgctat gactattttt gtttccttca 4440 tcaaacttca agctcttggg actagggatg tagtctagtg gtagcacact agcctagcaa 4500 gcacatggct ctaggctcat tcctcagcac cacacaactt taagttcatt ttttattaat 4560 taattaatta attagtgtgt gcatatacca caaactgcat gtggaggtta gaagacagtc 4620 tggaggattc tccctttcta ccgtgtgggt tccagggtct gaactcagct tggcaggtgt 4680 agctgcaagt accttccccc actgagccac ctcaccaacc caaaagctca tattctaata 4740 tgtatgataa tttctagctc agacatctgc tgagtgaatg aatccgtgag ctacaggatg 4800 ttgaaataga gctgttaact gggcctggtg actcaggtct gtaacctcag ctcctgaggc 4860 tgaggcagca ggatcagaag gtcaagactt agctgggcta caaagtgagt tcgggggaaa 4920 ctcccgaaat tacacaaagc ctggggaatc tgacgagacc ctgtctcaag taagaaagaa 4980 aagtcagggt gggctaggga tatagtttaa ttatactgca ccagctagca cccctggtcc 5040 ccaaatgtac agtgcgcatg cactccaggt tccttggtgc tgagatcaca ggcatgaaac 5100 cttaatttcc gcactttcct atcctgcctc attttcactt caaatgaaaa gctgcacctc 5160 attgcattag gataagcatc actgacccaa cactgaagag tagtttgtta ggaaggaaat 5220 ttggtaacag tcatttcaat atgataaata tttattgagc aactgttgtg ttcacaggag 5280 aaacagtccc agcctttggg gtgcttacaa tttggtgtga aatacggatg tataaacatc 5340 actgatgccc aagaaggatg agtccaggaa atggcacaaa gctacttctg atagggcaca 5400 ggctggctgc tccactctat ggggagaggt gcatgagctg tagtgagaag ctggaagttg 5460 taaggatgct gcgggccttg ttctggaagc agctggcaga cctcataagc ctatgaaaga 5520 ggggtggtgg tgtctaattt tggtttcagg aaaacaactt tagtgtggag gagggcttgg 5580 atagggtagt gtgtgagaca agcatctgtt agacaagggc ctgatgaggg aggaggcagt 5640 gaggggagag aggagagaag gtctcaggat gctgacagtc acagctcagg tcaaccagcc 5700 ctgctctgtc cctatgagct aaggggtgag agaaagggcg agggaaggag ttggtccctt 5760 ctccctaacc tctcagtttg tttgtatagg tgaacagcaa aggggtggag gcacatgcct 5820 ttgatcctag tacttgggaa gcagaagtag gtggatctgt gattttgaga ccagcctggt 5880 ctacagagta agttccagga tagccatagc tacacagaaa aatcctgtct tgggagggca 5940 gggggaggaa agcttttaaa acgtagtatg atttacctag tgccttacac atttatatct 6000 ctctctctct cctctctctc tctctctctc tctctctctc tctctctctc tctctctctc 6060 tctctctctc tctctctttg gccaggggat ccggtaagac aggggtatcc tagaacttgt 6120 gacctaggcc atcctagaac tcactatact gccaggctga cctcgaattt gcagtaatcc 6180 tcttacctca gcttccaggg attgattaca ggtgggccac cgtgcctggc ttaaacagtt 6240 acttttctgg tttaatgata tggcctaggt attagtatgc tcattttggt ggtaagaaaa 6300 ccagggctga aagagattaa gggggtttca tgaagttact cagctaggat taacacagca 6360 gaaattcaag cccttatctt tagttccaaa gtgctgggtc aatttccatt ttatcatctt 6420 gaacatgtta attgacttgg aaatgaacca tccttatggg tgaacctggc cactactggc 6480 cacgggcatc tggaaactgg tgggagatgg gactcaactg atagacttgg tgatcacctc 6540 gcagagtcag caaatgatct tgttcctcga gagagaatgg aaagaaacga ggatgatggt 6600 ggaggaagaa tattacgtat agacaggagg ccatcagaaa tgggggcctt ccgggtctga 6660 gctacagatg gcaccagccc ttcctattgt ttcaaacact tctctgcctt cccagctaaa 6720 gaaagacagg ttcatttggg agaacgtttc attcgtttga gagcgatgtc ctctgactga 6780 acatttctag acaattctgg taaaaagcaa agcagaaaat tctaattgaa aatccaagaa 6840 gagatctccc agggaagccc cacgagagtc tctctctgct gtagtccatc tcaatggtgt 6900 gactgagcaa aagccaggct gttttcctgg tcccagagag gggttctcct tctgggaggg 6960 ccccggaggg tggaaggcat tcactgatgt tgatccacca tatccaccgg gtcttaggag 7020 agtgagatgg gtgttctgtg gaaggatgct gaatcattgg tctccagagc attctgaccc 7080 agcgagcaga ggctcccagg ctgttctccc agactatggg ttttctgtca tccagtagtt 7140 ggcctctctg ctgctcactg taaccccacc gtggccagga tggctgtttt ctgctggggt 7200 ctcccctcag aatcagtgac cctagcatta attccttttc agggcctgtg aagattttgc 7260 cagggaacag agaaggtgaa cgctagcttt ccccagtggg tgagcatgac tcaaggacca 7320 gagggaaatt gagctgatgc cagagaagga ttcactgtct gacagtgaca agcatgtagc 7380 taatagctgc tctggggaag ggctttctgg gtggcttcta cagagaatga aggaacaaaa 7440 gaggtcctag gaaccagcac aagggatggg tctgttgctc ttgtagagta cgcagttagc 7500 acgcatgggg ccttggttcc atccctagca tcaggagtaa agtttaaaag ccatcctgtc 7560 aattagacat gtggtttttt tttgtttgtt tgtttgtttt ttgactctgc taccctactg 7620 atagatggca aaagaactgc cctggaggct ggaggaaatg gaataccctg gccaaagaca 7680 gaagggtgca ctggataagt ttggtttggg gtgaccctgc cctttaagat cccttaaaaa 7740 tctagccaga gttggtgcgt gatgcttgtt tgcaggcccc agctactgcc tggaaaacaa 7800 agaaagtaga ggatatagct gagaaagaag tctgcagacc tagggtcagc tgcctttact 7860 cacagttgca gggcagcctg aggcaggagg gacaaacagg gtaaactcga ggtaaactgc 7920 ttcaaggaga gccaggcaaa gctcttaacc acagagaaat cacttagtca tgactggcta 7980 aacaagaaag accatttaaa gagcaactac catgttgtag gccttgaggc agacactagc 8040 caacacccta gcccccacca caaactcaga gagctcttag gagaagcctc agggtattgt 8100 attttaaggt aagctgtttg gccacaaaca gtagatatcc cattccagga gcctctggtc 8160 agaggatagg caggcgtgac tcaatgattg aggtttgagg agtggtaacc agagaaggca 8220 ggtgatactg aggacctgca ggtagaggag cttcatcttg gcacactctt gagagacagg 8280 atatttgacc aggacttgaa agttgaacat gtgtcaggtg tacagcagaa catgccagct 8340 aaggaatgga gctgtgtgtc ccagcccagg caaggccatc atgctcctca gcgatggatg 8400 tcacaggaac cacacggagg aaggggagct cctggggagt gaccaggaaa tagccagtag 8460 ctctcaatgt caagggtcct aggatttcat tcagataggt agcctaatgc tgggcaagaa 8520 acctcccaat tactgtgact taaaaacgca agtcattttc caggatcctg tgggctgact 8580 tgaggtcctc ctgtttgaac ttattcttgt ggctacagac agctggcaag tcagctagag 8640 gccaagttgt cccaatttgc taggtttgtg gctgcaacac ttgggctcac tgctggactg 8700 caatccctgt atgtaagtgc cacctcacca gcccctcctt gcctttactg atttctcact 8760 ggccaagccc agagtcactg tggagggaat ctcacacaaa ggcctgtgaa tcaccagtat 8820 acccatttac cacatacttc ctctccagat ggaaggccaa gaagatcaaa ggccgttact 8880 acttagtggt cggttagagc cggtggaggc tggtattggc tttgtggctt ggttgtaaga 8940 tggccacgtt gctcatactc tctgggtctg tttctttctc ttcagattgg aagaaagcct 9000 cccccatctc agctttacac caaaggcttg ttcttgctta gcccccagct ctaaggtaca 9060 tacggaagaa agcagatgtc ttctttggga ggcttggatg agaacctctg ccctagaggc 9120 ttgcaaaagc tgtagagcct gagataggct gcaaggaagt ttgtggggca ggggaggtgc 9180 gttttattct tgtgctgaac tggggacgga atacctgagc ccttgagcat agacgattct 9240 gagctatccc tgtctgtggg ggttaagtcc tgtaggattg agggaatcca aacccacctt 9300 aaatttgcca gtctcgaact taactgcatc tgctaagatc tattgctgag cacagtagag 9360 cgaacctaag caggtgtgcc tctcccagcg gggcttgtta ggccttccct agaagctgcc 9420 ttctctcaga ggtcccaagg agcacatttg tttgttaagt gattggcact gacctagtcc 9480 tgaccccaag ttaaatctcc tgggcatggg actcctaggt ctccctaggc caggggacaa 9540 ggagaaagct acaacacagg tggtctatct catgtcaaga caatggaaac tgggttaagg 9600 aaggaaggta gatcataaaa ctaaccagct attgggagcc tcagagggac tttctccccc 9660 actgtgtggt ttttgttgtt gttgttgttg ttgttgtttt tgtttttgtt ttggtttttt 9720 gagacaagag tttctctgta tagtcctggc tgtcctggaa ctcactctgt agaccaggct 9780 ggccttgaac tcagaaattc acctgcccca agaaacgcct agtgtttttt ttttttttta 9840 agggaaaaaa aaaaaaagct tgaaaaacca gtttttcaag agacacctat tgtcaggcac 9900 taggatacca aacagggcta cactgagttc ttagacactg ggaaggtaag agaggcagat 9960 ggtaagggag cctatagtca gttacagtta agggaatgga gcctggtctt cctcccaagg 10020 gaggaagttt atgctcaggc ttaaagatga agaagtaaga cacagaacca agaagaggag 10080 gggccaggca gagccagtgg ccagtgacac tctatgggat gggcccagga ctggcaggag 10140 cagaccatgt gtggtgggga ttggagattt gcctgaggct gagcatgcaa gcagggtagg 10200 ttggaaatgc cctgaaggaa gtatgcaagc cagaagtttc cctagtcatc ttatcgacat 10260 ctcctgcctt agggggacca gtcggttcac atttgacctt gggaaggtta ctcaggccaa 10320 cagtccagaa gacagaatgg tgaggtccaa ggcagacatg tcaagatagt tgagattgga 10380 ggctgcacag tggtttaaga gcgcattgct gctcttgcag agacggttca gttctaagca 10440 cccacatggt ggctcccaac catctgtaac tccagttcca ggggatttga caacgtattc 10500 tgaccttttc atcctcctgt acacatatag gagacatgca tgtacacata taacattgta 10560 tatgataaat tatgtcatgt gtgagatgtg tgtgtgtgtg tgtgtgtgtg acagttaagg 10620 ttgagaagtc ctgtaatatc ccagggtaaa atgatgccaa ctgggccaaa gggtggtaaa 10680 ggggacggga agaaacaggc acacaaacac tctttaaagg gtggtgcctg gacaggactg 10740 agggagcact agggacagat atgaagaaga ccattaaaga gtctctgagg ttgttgcctt 10800 acagaacaga ctggataatg ccaccactta ccaatacaga gaataccgaa gaggaccagg 10860 tagaggttgg ctttgggcat ccctacgtgg aggtgcctac aggaaagtag tctggggaag 10920 aaggcaaagc caagctgggg gtgtgagcct gtgaggaccc aagaggtgac atgggagtga 10980 aagaaagatc tctcatgtat ctgtccctgt gaaagcctca gcaactgtag taaaggctgc 11040 ttgatagtgg cgggatgcag gcaaagccag ttttagaatg gcttgagaaa gagtaggagg 11100 tgaagaaatg aagacagctt ggctgagagc ttagcttgtg aagagagggt agctggaggc 11160 gggtgtgaga ttcaggaagg tttgtgttgt ttttaatgaa gagactcggc atttgtaaag 11220 gccagtggga aggatttgga ttctttggtg ctgggatgga ggagcgggag cccatgtagc 11280 acggtgattg aattagcctg aggaaaaggg gatcatgtct agaatgggtg cctggcaaag 11340 gagcgaggag ggaattgtct tcccagccca gatgaaaact ggggcctgag ccagggggaa 11400 ggctcgagtg gctggggcag cctcaggccc aggaaggcta gagtaacaag actagagtgg 11460 gctcagggcc caggagatag gaggcttctg caactaatcc ccccccacac caccactaac 11520 tgccttgttt ggcagtaaga gtactgggtg gctcagctcc tgcctcaggc ctgcaccaaa 11580 ctgaaaggga gaggctgcca agagctgccc aaggggctca gttagaaatg ttgaaagaca 11640 gctccagaaa tcatttctca gcagcaggac cacatgcata gcaggagcag ccagacattg 11700 ctgcggaggg agtctcaacg ttttcctgtt ttgttgtgga gagggagagg caagagcgat 11760 aggagtctga agccctggaa aagggcaggc tggggaagga gaggggccca ggagtgagga 11820 aagttgttct ttcttggcct tagccctgtt tgtctgagag aaaccttcct caggtggaag 11880 agtcagactg ggaccagaga gaaaacaagg ctcaggacct tgtggtagag aaggaggaac 11940 tctgaagaga ggagagacag gaaaaggccc tggacaacgg ggtccctgga tagaggtttc 12000 agtctagtgt aaagaagtgg ccaatctgct caagctagcc tttacacaga tcccaagtag 12060 tgtccctccc atgaacaggt tacagggact gtgtcaggag gggacccatt tctggtgctc 12120 aaagctaagc cctaaaggta cagaaagggg tcccggtaag cggggtgagg ccttttaacc 12180 attaacccag ctggttgaaa tctgccatgc ggagcctcca ggctctatcg attattttaa 12240 aagcacagcc actgttaact ggtggctgcc cagaaaccag ggagaggcac agaagtagag 12300 gtggggagaa gcaggcaggg aggaaaatgg gagcccagcc aggagtggga gccggagagg 12360 aggtctgagg taggattcac ggcttggcca tcaatgtagg ggaggctttg ggggtgatca 12420 gaaaacctaa gctggcattg gaatcagcca gcacagccca tgggcactgc tgagccctga 12480 agccaggaaa gggcctggca gatgggacaa aacaagctca tgaccatggt gaccttcgca 12540 aggtgactag gtcagagctt tgctgacttc actgagaaag gaagggaagg ccactgggaa 12600 agctgaggtg cagggagaga gagggcagag agagtccatg acaggctgct gggttttcct 12660 gaggacaggg ccctttccct gatcctccta tacagcaaaa acatcacggt ctggtgtggc 12720 tgagggcagg gtcaggccat cagagtctga ctgcacctgg cctgctatgt atgcactctg 12780 cttcatagtt agttttacaa agctaatttc atttgtgagg tggctaaatg gggaggtgag 12840 gaaccctcag actaaagctg gcttctggag aagatcaggt tgaaggatgc ttgtgtgata 12900 aagaaacagg gttgctataa cttggatcta aactgtaccc caacggacca tgtgttttca 12960 gtgtggtgct agtgggagga agtcaggtaa ctgggggtat actctaggag gagatattag 13020 gactctggct ccttcctctt cctctgcttc ttggctgcgg tggcaggtcc cctttgtcac 13080 acgacgtact atgttgacac ataccaaagc aatgtggcct agtgaccaca gactgaaaca 13140 tctgaaaccc taagctatgt agttgaggtg gggaggaagg ggcaggcaca cggtgggtgg 13200 catgtgcctc agcatcctgg aggttgggga agaataaagg atcaaagtca ctctcagttt 13260 caaagtgagc tcaagagcca ttttgggtga caggagaccc ttcttttaaa aaaaaaaaaa 13320 aaaggcaagg aagagaagga agaaaagaag gaagaggagg aggaaaccag agacacaaaa 13380 tacattttta cactttccta agttgcttta ggcaggtgtc accatggtgg aagctaatac 13440 aagtcacagt ggaaagtgac tgcaggtaag gaaaggtgaa atgagggaca gtctgcacat 13500 gcataaactt catgactcat cacaggatgc atacattggc aggatttgcg gacagcggtt 13560 ttggctctgt gatatcatct ggtcattttt gggagtccag ggtcagtggt ttcaaccagt 13620 ttgacctaga aggcatctcc aggtttgaaa aacaatttta aaacaacaac caaaatcctg 13680 aacaaccctt accaaggtgg gtaaccaaac agaagaggct gctatttttt tcctttatta 13740 gttaagcgac ttctaaaatc agtgatgagc acaggtaatt gaaagcaaat gaagatacag 13800 agtttatttg ggtttttcat gagtgtaatg ttctctttgg tttttacaat ttaaagagga 13860 tggtccaatc ctctttgaaa aggaaaagaa tatgattcct ttcattaaga ggagttgatc 13920 acaagcagta gagcggggga cacattagta gaggtgctag gagagagctg tgcacacaca 13980 gccaggacag agacacagct gtctgtccac tctcagccat ggcactcctg gatgcccatt 14040 ctttctgacg atggcacaca taagcttaag taactcagga gaaaccacat tatgggtgtg 14100 gggcatggag tcccacacac acctgggtac caatgtgccc tacccactga tcccacacat 14160 ctttccttgc cagtgccaga tggagtggat cagccgcagc agaggcctct tgatggcaga 14220 gagaaatagt gacgatcagg tgacaggaaa cactagcatg tcacagacag acgcaagtgc 14280 agtctctgag gctcaggagg accctcaaag tctgcctgtg gccttggtgc cagtgctgtc 14340 ggcctggctg ctcttattcc gtggacaggt ccaccaggac cacaggccgg tgggtccaga 14400 gccgtctgtc tgcaatgtct tttctctccc ctttctctga cagctcaaca tcctggtgga 14460 cacgggcagt agtaactttg cagtgggggc tgccccacac cctttcctgc atcgctacta 14520 ccagaggcag ctgtaagtcc tggggagggg tgggagaggg ttcaagaaca agagcctttc 14580 ttctactagg gagggggaaa gcaaaaggag gcagctgggg ccaggaagaa agacagtgtg 14640 ggaaagggtg ggttaagggg cagacatgaa gtgcttctgc cccaaggctc ccagagcaga 14700 acatgaacca tctcattttc tgtctatatg gacaggcagt agggccaacc cactgtcata 14760 gctagagttt agaaggctga acaggctagt ggttagtgcc tttcaccatg ttctcaccgg 14820 ggactggggg ctgctctggg tagttacacg tgatttaagc aagatgaagg aactaggcat 14880 gttggtatat accagtaatc ccagcatttg gaggactgtg gagactacac agcaactctc 14940 aaagagccaa aaaaacggga gctgggagat gactcagtga ctaagaactt actatgaaag 15000 caagaagact gagttcaagt tctagtacct acctaaagcc tgccatggct tgcatgcttg 15060 taaccccaat attgacaggc taagataggc agatctagct ttctggacag catagctgaa 15120 acttcaggtt tagtgagaga ccctgtctca aaaaataaaa tggagagcga ttatcctctg 15180 gcttctgcat gcacatgtgt gcacatactc catgtacaac aaaaaccata ctcttctcca 15240 gttttaaaaa gccagaaaat aagaaggaaa gagtctttca tgacccccac tgaattgtct 15300 ccaggtacca ctacaactgc caccagctgg gttccgctca gtggtgggag atggactaga 15360 atgggttttt atgtaagaga gacatgagga gtgttgtgaa agactggtta taaaagcaga 15420 ttgaggatgt ggtgaggaga atctagtgtt ggcagtatgt ctgtatgaga caggaggaag 15480 gtgtggcctc cacatagtac aggcttacac cctaaccttg gacttgtagg aaagtacaaa 15540 accactgtcc tgtttccttt tgccctaaga acacagttgg ggaaggagta agctgggtaa 15600 gatcagtgcg aggacatcca ttataagttt tcatgcagtg ccatcagccc tgggtagtta 15660 catgatgaag agaaacagca ggggcattgc acagcagtga gtgcaactac tcttgtggat 15720 tccaacccct gcctgctgtg ccaggacact tgctcggatt ttctgtagag gttattccat 15780 ggcttccaca gtcactctgc ctagttctct ccccaccact cagttggtac cactgggccc 15840 caaggcagca agagagtcaa tggctccttt tctctgtgag tgtgatggat ctggggtggg 15900 ggaggagatg tccaatagct gtctttgcag cagccataga cacaatgtac aaagcccatc 15960 ttgttcttta gcctgagctt caggtccaac aatatacact atggggacag gagagcaatc 16020 cccagctaca gctatctatc tgcagggatg cttgacattc tgctctgggt aacagggcta 16080 gggttgttca gctgagcaga ttctagaatg ccatatagag tgcatttggg aatatatctg 16140 atgacacaca tctattgtta cagctttgct tcctttttat aggtccagca catatcgaga 16200 cctccgaaag ggtgtgtatg tgccctacac ccagggcaag tgggaggggg aactgggcac 16260 cgacctggtg agcatccctc atggccccaa cgtcactgtg cgtgccaaca ttgctgccat 16320 cactgaatcg gacaagttct tcatcaatgg ttccaactgg gagggcatcc tagggctggc 16380 ctatgctgag attgccaggg tgagagaatc aagcctgttg cctgcatgtt gctcactgca 16440 tgctacacaa ggaaggagcc aagagagaga agggggaagg atggattgaa agggtaaaca 16500 aggggctgga gagatggctc agtgagaata gtgcttaccc agcagtcatg agtttaatcc 16560 tcacaactaa acttttcaaa actgctgggt gtgatactat gtacttataa tcccagctct 16620 ggaaagccaa ggtcaagtaa atcctggggc ttcctggcca ttaagcctag cttactagac 16680 cagttcccag ccaatgagag accctgcctt agaaaccgag gtagaatgcc gctagagaga 16740 tggctcagca cttgctgctc ttccaagggg cccagggtta attcctagca tccatgtgcc 16800 agctcacaac tccacttcca gggatccaat gccctctttt ggtctctaga agcaacaagc 16860 acatgaatgg tgcacaggca tagaggcagg caaaacaccc atattcatag aaacacattt 16920 tttttttttt ttttgagaca gggtttctct gtatagccct ggctgtcctg taactctcta 16980 cgtagaccag gctggggata aactcataga gattcccctg cccctgcctc ccaggtgctg 17040 ggataaaagg catgtgccac ttctgcccag cataaaaaca aaatccattt taaaacaaaa 17100 caaactaggg ctagtgagat ggcttggtgt gttaggaatc cttgccaaag cctaagagcc 17160 atgtggtgga aggaacgaac tcatttctgc acaggcctct ctctcacctt cacacccttg 17220 ctgcacaagc cagtgcacac acagggaata ttcttaaagg tacacagatt ctcaagaata 17280 atatccaaga ttgtcctttg gtctccaaat gtgtgtgtgc acagacacat gcacacacac 17340 acacacacac acacacacac acacacacac acacacgata taggaagagg aacaggtgct 17400 acaggaacag gaggaggctg tccccaaatc tgaaacaagt gaatcacgtg gggctggagg 17460 aaggtgctat ggcctcgaaa cacctgcctc tgcactgcag tatctcttat ccttgctcag 17520 tacttccttg gtctttcctg aggaacatcc ccaagctttg actttgccct tgagtaccga 17580 caggaagcgg acctgtctgg tagcccctgt gtgaggattt attcctgttt ctctctgcca 17640 cagcccgacg actctttgga gcccttcttt gactccctgg tgaagcagac ccacattccc 17700 aacatctttt ccctgcagct ctgtggcgct ggcttccccc tcaaccagac cgaggcactg 17760 gcctcggtgg gagggagcat ggtgagtagg ccctcagagg gccatgtgcc ttctccctca 17820 tggccggaat caagaaaccc ctttaaagtc ccgattcttg cttccctcct ttccttttct 17880 tcattgctgt tcttgaaagg aattgaatct ctcatggaag ggatcagaaa caattaggta 17940 catttcaaag aaaatgaaaa ttagtcctgt agattactgt ctgagtctga gaacagtatg 18000 taaggactct gttaagagcg cattaatggg ctggagagat ggctcagcag ataagagcac 18060 tgactgcttt tccagagatc ctgagttcaa ttcacaataa ccacatggtg gctcacaacc 18120 atctgtaatg ggatctgatg ccctcttctg gtgtgtctga agacagcgac agtgtacttg 18180 tataaaaaca aagaaacaaa caaacagagc atgttaaggc tggtatctgg agccatgact 18240 gacccagaac tcactatgta gtccaggctg gcctcagata tgcagcactc cctccttcca 18300 ttttccaagt atatgccacc agactcagtt caagcccatt tattaattag gacagtaaca 18360 agtattctcc aacaagcaaa gacaagaaaa tgtatgaaag cagcatacaa tcagttcatt 18420 ccacgtttat tgtactagct taggcttaaa taactttcgg tcaagggcta tagattttga 18 480 tacaaataat cccgatgtgt ttcttctcct gaacacattt aggaaactaa atattaccat 18540 gcatggggca tgatgggaga ttagagagaa tcttggcacc ccaagaaatg gctgatctta 18600 ttatttagtc ttgggtaact tgctctcctt ctgtgctagg acataggtgt ctgtctcttt 18660 ttcttttaga tcattggtgg tatcgaccac tcgctataca cgggcagtct ctggtacaca 18720 cccatccggc gggagtggta ttatgaagtg atcattgtac gtgtggaaat caatggtcaa 18780 gatctcaaga tggactgcaa ggaggtaatg ggatggaggg gattctcgtg agcataaggg 18840 gagtctcaca gtgtgtgaga gccatgaggg tggaagcaca ggatcacgag gcaatcccta 18900 aggtgaaggg cggggcctgg cagagcagtc acagagcctg gaccacatga cttaagtata 18960 gtaatgaaaa catgaaagag ttcctttctg gactctagtc actgcctctt cagccttggg 19020 caaggctttt gtgaggcaga ggttggacct gactagagtc ctgctgttac taagtgccct 19080 caggctcctc actacagtgc cagactcctc atctacaagg cagaggtaga aatacacaag 19140 cgttctcaca gactgtaaag gtcaaagcca atgtatgaag tgttagtcgt tatgcttggc 19200 atacccaaat gccaacactt agggtctctt tccctagatt ttgttttaca tttattggga 19260 tgaggatggg gcatgtgcca cagcttgtgt atggaggtta gaggacaatt ttcagtaact 19320 agtaggtctc tctggggttg aactcaggtc atcagacttg gtagcaggtg cccttaccta 19380 tctcaacagc cccctttttt ggcttttttg aggatcttat gtagcccacg ctgacctcag 19440 acttgctagg tagctaaaga tgaccttgaa ctcctggtcc tcctttctct gactctcaag 19500 tgtctctcta caggcatata ccaccatgca gcatcagagc tcaagctcat ggctttgtgc 19560 gtgctgagcc acccttctct ccattataat gtatggtgaa tgagaataac tacataaaag 19620 tactagaagt tacaaatacc acacaaggca tttctgcttt agatttagag aacacctacc 19680 cgcacaaggt gacgaagtta gtacttacca agtgcctggg atctgccagg acctacttag 19740 tgctttcagc cttttgacct atgtaatcct tatggtaacc tttgaggtag gcactgttat 19800 tatcttcaat ttagaacaag agaacaggta cacagaggtt aagcaacgtg cccaaggacg 19860 cacatataca aaatgggaaa gctaagatgc caaccccagt aggatgctta gctgtcagat 19920 tcacccctcc cacctctccc cccctctctc ctcagtacaa ctacgacaag agcattgtgg 19980 acagtgggac caccaacctt cgcttgccca agaaagtatt tgaagctgcc gtcaagtcca 20040 tcaaggcagc ctcctcggtc agtgacagtc acactcctca gcaagcccta aagatacgta 20100 gatccttccc atcaggcgct tacaaaccac tgctctgttg acttggcatt gagatgcctt 20160 tttttttttt tttttttttt ttgagatggg gtcatgaacg tagcccaggt tagcattgaa 20220 cttgacaact tcatgcctta gccttctaag ttctgggacc acaggcagtg tgagccacca 20280 tgcccactca cagctttatg aggccagtat ctcactttct cctcctgatc cgtagacttc 20340 agtttacctt tgctactttg gaaaattccc tggaaaagca cacagtggta ctgtcactgt 20400 ggttctgaac acagagaaga catggtggga taggccgagg cttcacattc ctctcttgtc 20460 aacagacgga gaagttcccg gatggctttt ggctagggga gcagctggtg tgctggcaag 20520 caggcacgac cccttggaac attttcccag tcatttcact ttacctcatg ggtgaagtca 20580 ccaatcagtc cttccgcatc accatccttc ctcaggtatg tccccaagct gagacagtgg 20640 ggtgccaagc agcagacaaa gtatgtaaag agacatagct tgctacaact tgaaagatga 20700 aacgaatgtg tggtggtaaa ggggtctcag aagaatccac atcttggggc ttgaggaaga 20760 gtgtaggaga ctaaaaggca gaggaaggca ggaagaatca gacatgctct caccctgtga 20820 ctgtttaccc ctagcaatac ctacggccgg tggaggacgt ggccacgtcc caagacgact 20880 gttacaagtt cgctgtctca cagtcatcca cgggcactgt tatgggagcc gtcatcatgg 20940 aaggtttcta tgtcgtcttc gatcgagccc gaaagcgaat tggctttgct gtcagcgctt 21000 gccatggtga ggagggtgtg gggtcagtca gctcatgttc ttgctgtcag ctgctaatac 21060 ccagatctct ttgtttggga ggtagtagta gctgcctctg gctaatcaac catgtgtgag 21120 agctagggct aaggcttgct ctggtgaaca cgcctggtgg ggaaagcact attggaaact 21180 gatagcagct cgagcttccc aggcacttgg aaatactagc tgcaagggtg gctgcccgcc 21240 cacagtaaag ctgagtacta ctgcagaaaa gggccagggc tttgtggttg cattttcatg 21300 aagacaggta agtctgctat gtgtggaagg agctagattt tggtcaactt ggaactgttg 21360 actttgatat cacctctact ggatgtgaga atgggagagg cttgcagtcc agttcccgag 21420 gccctggaag gagaaacagc ctccctcagt actagcttcc ctgcctactg ctcgctctgt 21 480 ccaagttggg ctctgcagtc tcacttgggt ggatcccaag gggcccctgt caggaagctc 21540 ttttaattgt ataacctaag ctactctatg acagtaagag ctaccggttc atctccaccc 21600 tctctccagt gcacgatgag ttcaggacgg cggcagtgga aggtccgttt gttacggcag 21660 acatggaaga ctgtggctac aacattcccc agacagatga gtcaacactt atgaccatag 21720 cctatgtcat ggcggccatc tgcgccctct tcatgttgcc actctgcctc atggtatgtc 21780 agtggcgctg cctgcgttgc ctgcgccacc agcacgatga ctttgctgat gacatctccc 21840 tgctcaagta aggaggcccg tgggcagatg atggagacgc ccctggacca catctgggtg 21900 gttccctttg gtcacatgag ttggagctat ggatggtacc tgtggccaga gcacctcagg 21960 accctcacca acctgccaat gcttctggcg tgacagaaca gagaaatcag gcaagctgga 22020 ttacagggct tgcacctgta ggacacagga gagggaagga agcagcgttc tggtggcagg 22080 aatatcctta gacaccacaa acttgagttg gaaattttgc tgcttgaagc ttcagccctg 22140 accctctgcc cagcatcctt tagagtctcc aacctaaagt attctttatg tccttccaga 22200 agtactggcg tcatactcag gctacccggc atgtgtccct gtggtaccct ggcagagaaa 22260 gggccaatct cattccctgc tggccaaagt cagcagaaga aagtgaagtt tgccagttgc 22320 tttagtgata gggactgcag actcaagcct acactggtac aaagactgcg tcttgagata 22380 aacaagaacc tatgcgatgc gaatgtttat actcctgggg gcagtcaaga tgaggagaca 22440 ggataggata gagacaggaa ggagatggta gcaaaactgg gaaaggcaga actctgatca 22500 ctttctagtt ccaagtttag actcatctcc aagacagaag cccatctgga ctaagaggta 22560 tcattcccca atgtgcctgt ggttgtagtc tgaactgaaa tgaaatgggg gaaaaagggc 22620 ttattagcca aagagctctt tttaacactc ttagaggaac agtgctcatg agaaaagtcc 22680 cactggacag atgaattcct atcttgttaa ttctgtctct ctctgcttct tcaacatgct 22740 aagtggcacc aaaatgaccc aaccccaagg tcttaggtgc cctatgggac aacagttaga 22800 atattgtagg gctagggatg gtcttcccag cataggttca ctccaaccaa ggtgctaaaa 22860 ggaacagaca ggagagtcct cctctctgat ccacaaaggc agagccctca agattcatcc 22920 agcagggtta gggctgatgc atttgcctct gcctggattt tgtttttatt ttctttcttt 22980 ttgcccagtg gtacaaaacg ataagctctt tatggaatac tgagtgggtt cattcctctc 23040 ttgccctctc caatggcccc tctatttatc tggctaagga aacaccacgc attggctagt 23100 attaaacagc aactgtaaga tagagggctt tctgttctat gtcattgcct tcagtatcaa 23160 ggctgcctgg agaaaggatg gcagcctcag ggcttcctta ctttcttctc ctttcctgac 23220 agagcagcct ttctgtcctg ctctctgctg cccctcccaa tataatccat gggtacccag 23280 gctggttctt gggctaggtt gtgggggcca cactcacctc ttccctgcca gttctaacac 23340 gacagacatg aagccagtgt tagtgggaag agctgggttt tcccaggatg accactgcat 23400 cctctcctgg tacgctctac actgctttca ggctggggac ctgccaagtg tgggacagtt 23460 gatgaggaag agacattagc agggcctctg gagttgctgg cccagccagc tgcccacaag 23520 ccataaacca ataaaataag aatcctgcgt cacagtttcc agctgggtcc tcttccttgc 23580 cctcgcactg gtgctgctct ggctgagtag gaatacaccc acagactgcc aggaagatgg 23640 agactgtccg cttccggctc agaactacag tgtaattaag cttccaggat cactaccatg 23700 aaaacgccgc attctgcttt atcatttcta cccatgttgg gaaaaactgg ctttttcccc 23760 atttctttac agggcaaaaa aaaaaaaaaa aaaagggaga gagagagaga actcaaccta 23820 gttgttattt accctagtaa ctggtgttct attttttttt aaagggggaa aatttgcatt 23880 tatttttctt ttgatggtta actcctttgt atcataaaat tatgaactct gatatgtaaa 23940 acagaaaaaa atcttgacaa cagcttctcg cttgtaaaaa tatgtattat acagctctat 24000 tttcaaagtc tcctgaaaaa tgactgacct atctccactg tatgtggggc tttgctaccg 24060 tctgtatgaa cggaggccca gtgggcagag gtgatacaaa caggcatggc agactctgag 24120 tgtttttctg actgtttctc 24140 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens tyrosine hydroxylase (TH) fragment <400> 6 ctggccagga agtcccggg 19 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mus musculus tyrosine hydroxylase (Th) fragment <400> 7 cccgtgattt tctggccag 19 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> BACE1 exon7 <400> 8 acggagaagt tcccggatgg cttttggcta ggg 33

Claims (8)

Cas9 protein where the guide RNA is located on the surface; And an amphiphilic peptide having a micellar structure consisting of the amino acid sequence of SEQ ID NO: 2 bound to the surface thereof.
The nanocomposite of claim 1, wherein the guide RNA and Cas9 protein form a hybrid in a molar ratio of 1: 0.5 to 3. 3.
The nanocomposite of claim 2, wherein the hybrid and the amphipathic peptide are included in the nanocomposite in a molar ratio of 1: 2 to 4. 4.
delete The nanocomposite of claim 1, wherein the guide RNA has a sequence complementary to at least one selected from the group consisting of SEQ ID NOs: 6-8.
Alzheimer's pharmaceutical composition comprising the nanocomposite of claim 5.
delete A kit for cutting target DNA in brain tissue comprising the nanocomposite of any one of claims 1 to 3 and 5.

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