JP2014144933A - REMOVAL OF AMYLOID β - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide pharmaceutical products and methods for preventing or treating diseases caused by amyloid β.SOLUTION: The invention provides pharmaceutical products for preventing or treating diseases caused by amyloid β containing, as an active ingredient, Nla (nuclear inclusion a) protease or a gene transporter including a nucleotide sequence encoding the Nla protease. The Nla protease cleaves intracellular or extracellular amyloid β, or amyloid β present in Citosol.

Description

The present invention relates to a pharmaceutical composition and method for preventing or treating diseases caused by various amyloid βs.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects approximately 24,000,000 people worldwide and is the most common form of senile dementia. AD is characterized by progressive memory deficits and cognitive impairment. A prominent feature of AD is the accumulation of amyloid spots whose main component is amyloid β (Aβ), which is 40, 42 and 43 amino acids in length. Aβ is produced by gradually cleaving amyloid β precursor metabolizing protein (APP) by β- and γ-secretase ^{1, 2} . Aβ may be present in other forms such as monomers, oligomers (dimers, trimers and tetramers), protofibrils and fibrils, the state of these other forms being related to their toxicity. Oligomeric A [beta] was found often are about 10-fold and 40-fold cytotoxic than fibrils A [beta] and monomer A [beta] ^3. A recent report also found that dimer Aβ was three times more toxic than monomeric Aβ, and trimer Aβ and tetramer Aβ were 13 times more toxic ⁴ .

Even though Aβ clearly plays a role in causing AD, the underlying mechanisms that contribute to the pathogenesis of this disease remain controversial. It is widely accepted that Aβ exerts extracellular pathological activity. From the brain of pathological AD patients, Aβ is secreted into the space of cells that form amyloid spots ⁵ . When added to the culture medium, Aβ can induce in vitro cell killing in a variety of cell types ^{3, 4, 6} .

However, accumulated evidence suggests that intracellular Aβ activity is important for the pathogenesis of AD. Several authors have reported the intracellular localization of Aβ from brain tissue of post-mortem AD patients and transplanted gene AD mice ^1,7,8 . Closer investigations using electron microscopy and immunocytochemistry have shown that Aβ is an early endosome, trans-Golgi network, coarse endoplasmic reticulum, outer mitochondrial membrane and outer mitochondrial membrane and neuronal differentiated P19 cells from has been reported to be present in the cell organizations of various subcellular ^9. In the triple-transgene AD mouse model, early cognitive impairment was associated with intracellular Aβ accumulation in the hippocampus and tonsils without overt amyloid spot accumulation or neurofibrillary tangles ¹⁰ .

Intracellular Aβ was also found to induce death of p53-dependent neurons ^11,12 through mitochondrial function ¹³ . Infusion of antibodies into the hippocampus towards Aβ not only reduces cellular Aβ precipitate, but also reduces intracellular Aβ accumulation. When this antibody disappeared, intracellular Aβ accumulation occurred before the cell precipitate reappeared. Such observations indicate that there is a dynamic exchange between intracellular Aβ and extracellular Aβ, suggesting a role for intracellular Aβ in the pathogenesis of AD, with intracellular Aβ acting as a source of extracellular amyloid precipitate. ^{Present 14, 15} .

Since the accumulation of Aβ is considered to be one of the most important factors in the pathogenesis of AD, degrading and removing Aβ from the brain should be a valuable therapeutic strategy. Neprilysin (NEP), insulin-degrading enzyme, some proteases, including endothelin converting enzyme and uPA / tPA-plasmin has the ability to degrade A [beta], NEP was found to have the highest resolution among the ¹⁶ . Pharmacologic suppression or genetic removal of NEP from mice has been found to increase Aβ accumulation, with synaptic plasticity deficits and hippocampal dependent memory impairment ^17,18 , whereas NEP viruses or transplants Gene-arbitration and overexpression reduced Aβ accumulation and associated cytopathology ^19,20 . However, it has recently been found that overexpression of NEP does not reduce oligomeric Aβ levels or improve learning and memory impairment. These results appeared by presenting that NEP-dependent degradation of Aβ affects the spots more efficiently than oligomeric Aβ ²¹ . In such applications, various patents and publications are referred to, and quotations are presented as references. In order to more fully describe the present invention and the state of the art relating to the present invention, the disclosures of these patents and publications are incorporated herein by reference.

  The inventors have made intensive efforts to develop a proteolytic treatment for amyloid β-induced diseases, particularly Alzheimer's disease. As a result, the inventors have found that the nucleus-containing a (Nla) protease of turnip yellow mosaic virus (TuMV) cleaves monomeric and oligomeric amyloid β (Aβ) in an effective and specific manner, and Aβ is It has been discovered that induced cell killing and a substantial prevention of Aβ-related lesions in gene-transplanted AD (Alzheimer's) mice. Thus, Nla can provide a new strategy for removing toxic oligomeric Aβ from the brain of AD patients.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by amyloid β. Another object of the present invention is to provide a method for preventing or treating a disease caused by amyloid β.
Other objects and advantages of the present invention will become apparent from the detailed description provided in conjunction with the appended claims and drawings.

It is a figure which shows the cutting | disconnection of A (beta) by Nla. It is a figure which shows the cutting | disconnection of A (beta) by Nla. It is a figure which shows the cutting | disconnection of A (beta) by Nla. It is a figure which shows the cutting | disconnection of A (beta) by Nla. (A) The amino acid sequence of Aβ is aligned with the common cleavage site of Nla, Val-Xaa-His-Gin. (B) Nla was formulated from E. coli and separated by SDS-PAGE. Rain 1, molecular size marker; Rain 2, Nla (10 pg). (1C) Monomer Aβ (2.5 μm) was incubated with Nla (1.5 μm) at 25 ° C. for 3 hours in the presence or absence of NEM. The reaction mixture was separated on a Tris-Tricine gel, blotted and examined with anti-Aβ antibody, 6E10. The density of each Aβ band was quantified by a densitometer. After 3 hours of incubation, the band intensity (Rain 2, 4 and 6) floated relative to the band intensity (Rain 1, 3 and 5) of each sample at 0 hour. n = 4 (D) oligomer Aβ (2.5 μm) was incubated with Nla (1.5 μm) for 3 hours at 25 ° C. in the presence or absence of NEM. The reaction mixture was isolated and immunoblotted with anti-Aβ antibody, 6E10. The density of the oligomeric Aβ band was quantified by a densitometer. After incubation for 3 hours, when the oligomeric Aβ band intensity (Rain 2, 4 and 6) was incubated for 3 hours, it floated with respect to the band intensity of the Aβ sample (Rain 2). n = 4 Error bars indicate SD. ^* P <0.05 and ^** P <0.01 FIG. 3 shows a mass spectrum of monomer Aβ incubated with Nla. FIG. 3 shows a mass spectrum of monomer Aβ incubated with Nla. (A) The calculated molecular mass of the expected cleavage product was shown. (B) Monomer Aβ (2.5 μm) was incubated with Nla (1.5 μm) at 25 ° C. for 3 hours and analyzed using a MALDI-TOF / TOF mass spectrometer. It should be noted that not only the peak corresponding to Aβ, but two peaks corresponding to the Aβ cleavage product were detected. As symmetry, Nla and Aβ were analyzed separately. Three small peaks displayed separately indicate contamination of the Nla formulation. FIG. 5 shows localization of Nla subcells from B103 neuronal pheochromocytoma cells. FIG. 5 shows localization of Nla subcells from B103 neuronal pheochromocytoma cells. (A) B103 neuronal subtype cells transformed into pcDNA-HA-Nla were immunostained with anti-HA antibody and observed with a confocal microscope. (B) B103 cells transformed into blank plasmid (Mock) or pcDNA-HA-Nla (Nla) were classified into microparticles (P) and soluble (S) fragments by a deviation centrifuge. The two fragments were separated by SDS-PAGE and examined with blots and antibodies against Octl (nuclear), VDAC2 (mitochondrial), catepsin D (lysosomal), o-tubulin (cytosolic) and HA (Nla). It is a figure which shows degradation of intracellular A (beta) by Nla, and suppression of the cell death induced by intracellular A (beta). It is a figure which shows degradation of intracellular A (beta) by Nla, and suppression of the cell death induced by intracellular A (beta). It is a figure which shows degradation of intracellular A (beta) by Nla, and suppression of the cell death induced by intracellular A (beta). It is a figure which shows degradation of intracellular A (beta) by Nla, and suppression of the cell death induced by intracellular A (beta). (A) B103 neuroblastoma cells were infected together with pGFPUb-Aβ and empty vector (Mock), pcDNA-HA-Nla (Nla) or pcDNA-HA-mNla (mNla). After 48 hours incubation, cells were immunostained with anti-Aβ antibody, 6E10. (B) The numbers of Aβ-positive cells (red) and GFP-expressing cells (green) were counted under a microscope, and their ratio was calculated. Cell killing induced by n = 6 (C) intracellular Aβ peptides was measured by morphology and MTT assay. n = 6 Error bars indicate SD. ^** P <0.01 It is a figure which shows suppression of the cell death induced by the exogenously added A (beta) by Nla. B103 neuroblastoma cells infected with empty vector (Mock), pcDNA-HA-Nla (Nla) or pcDNA-HA-mNla (mNla) were treated with Aβ (5 μm) for 48 hours in culture medium. Cell killing was measured with a microscope and MTTassay. n = 6 Error bars indicate SD. ^** P <0.01 It is a figure which shows the expression of Nla which the lentivirus arbitrated. It is a figure which shows the expression of Nla which the lentivirus arbitrated. It is a figure which shows the expression of Nla which the lentivirus arbitrated. It is a figure which shows the expression of Nla which the lentivirus arbitrated. (A) Lentiviral structure for expression of HA-Nla and GFP. (B) Western blotting performed with anti-HA antibody showed Nla expression levels in 293T cells infected with lenti-Nla. (C) Nla expression from mouse brain was detected by immunohistochemistry with anti-HA antibody and anti-mouse-FUC secondary antibody. The frontal lobe portion of the brains of mice injected with wrench-Nla was compared with the brain portion of non-injected control mice. (D) Brains injected with lenti-GFP and lenti-Nla received PT-PCR treatment. A PCR product corresponding to Nla was detected. GAPDH was used as a control group. CMV, Cytomegalovirus RNA polymerase II promoter; WPRE, Woodchuck post-hepatitis regulatory elements; U5, HIV5'-long terminal repeat; ΔU3, 3 'self-inactive long terminal repeat; There is a region of viral RNA; PRE, the binding site for the Rev protein that helps transport unlinked RNA from the nucleus to the cytoplasm. FIG. 4 is a graph showing the decrease in Aβ levels and Aβ spots that Nla mediates in the brain of APPsw / PSldE9 mice. FIG. 4 is a graph showing the decrease in Aβ levels and Aβ spots that Nla mediates in the brain of APPsw / PSldE9 mice. FIG. 4 is a graph showing the decrease in Aβ levels and Aβ spots that Nla mediates in the brain of APPsw / PSldE9 mice. (A) Lenti-GFP and Lenti-Nla were injected into the APPsw / PSldE9 brain in both directions, and the amounts of Aβ _1-40 and Aβ _1-42 were measured by ELISA. The amount of soluble and insoluble Aβ _1-40 was indicated (upper rain). The amount of soluble and insoluble Aβ _1-42 was indicated (lower rain). (B) Frontal lobe, parietal lobe, hippocampus, and piriform cortex of APPsw / PSldE9 male mice injected with Lenti-GFP and Lenti-Nla were stained with anti-Aβ antibody (Bam-10). (C) The number of spots was counted from frontal lobe, parietal lobe, hippocampus and piriform cortex of APPsw / PSldE9 male mice injected with Lenti-GFP and Lenti-Nla. For males injected with Lenti-GFP, n = 5 and for females, n = 5. For males injected with Lenti-Nla, n = 6 and for females, n = 3. Error bars indicate SD. ^* P <0.05 and ^** P <0.01

  In one embodiment of the present invention, pharmacology for the prevention or treatment of a disease caused by amyloid β containing Nla (nuclear content a) protease or a gene carrier containing a nucleotide sequence encoding the Nla protease as an active ingredient An artificial product is provided.

  In another aspect of the invention, a method is provided for preventing or treating a disease caused by amyloid β comprising administering to a mammalian subject a pharmaceutical composition as described above.

  The inventors have made intensive efforts to develop a proteolytic treatment for amyloid β-induced diseases, particularly Alzheimer's disease. As a result, the inventors have found that the nucleus-containing a (Nla) protease of turnip yellow mosaic virus (TuMV) cleaves monomeric and oligomeric amyloid β (Aβ) in an effective and specific manner, and Aβ is It has been found that induced cell death and a substantial portion of Aβ-related lesions are prevented in gene-transplanted AD (Alzheimer's) mice. Thus, Nla could provide a new strategy for removing toxic oligomeric Aβ from the brains of AD patients.

Turnip yellow mosaic virus (TuMV) nuclear a (Nla) protease is responsible for processing viral multiproteins with functional proteins. Nla previously preferred Val-Xaa-His-Gln and was found to have a relatively stringent temperament specificity with a ready-to-cleave bond located after Gln. The presence of the same common sequence, Val ¹² -His-His-Gln ¹⁵ , near the putative a-secretase cleavage site of amyloid-β (Aβ) suggests to the inventors that “Nla will have activity against Aβ. "The hypothesis was to be made. According to a preferred embodiment, Nla protease has the activity of cleaving not only monomeric Aβ but also oligomeric Aβ. As is well known in the art, oligomer Aβ was found to be about 10 and 40 times more cytotoxic than fibril Aβ and monomer Aβ, respectively. Dimer Aβ is 3 times more toxic than monomeric Aβ, and trimer Aβ and tetramer Aβ are 13 times more toxic. In this respect, the Nla protease used in the present invention is very advantageous for treating AD by cleaving the more toxic Aβ molecule.

  According to a preferred embodiment, Nla protease cleaves intracellular or extracellular Aβ. More preferably, the Nla protease used in the present invention cleaves Aβ present in cytosol. As shown in the Examples, Nla expression in neurons suppresses cell death induced by both Aβ expressed intracellularly and Aβ added extracellularly. And overexpression of Nla, which was mediated by lentivirus from the brains of AD gene-transplanted mice, reduces the level of Aβ and spot formation. These data provide additional evidence that supports an important role for intracellular Aβ in the pathogenesis of AD. In this respect, Nla could be used as a new tool to study the molecular events underlying the induction of cell death by intracellular Aβ. Furthermore, the present invention provides proof of concept that removal of intracellular Aβ by cytosolic protease can be a viable strategy for the treatment of AD.

  According to a preferred embodiment, Nla protease inhibits amyloid β-induced cell death.

According to a preferred embodiment, the Nla protease has activity to cleave amyloid β _1-40 and amyloid β _1-42 .

  According to a preferred embodiment, the Nla protease has the activity of cleaving the peptide bond between amino acids 15 and 16 of sequence number 3.

  The Nla protease used in the present invention may be provided as a protein or gene carrier comprising a nucleotide sequence encoding the Nla protease. If Nla protease is provided in the form of a protein, it can instead be fused to a protein delivery domain (PTD) so as to effectively penetrate into the cell. A preferred amino acid sequence of Nla protease is described in sequence number 1.

  Protein transport domains useful in the present invention are HIV-TAT, VP-22, growth factor signal peptide sequence, Pep-1, Pep-7, Drosophila Antp peptide, oligoarginnie, Transportant, Buforin II, MAP (model amphipophite). -Including but not limited to FGF, ku70, pVEC, SynBl and HN-1.

If Nla protease is provided as a gene carrier for removing Aβ, the Nla protease can be configured as a gene carrier by conventional techniques known to those with ordinary knowledge in the art.
As used herein, the term “gene carrying” refers to the transfer of a gene into a cell and has the same meaning as gene transfer.

  In order to constitute the gene carrier, it is preferred that the Nla protease encoding the nucleotide sequence is included in the adapted expression structure. According to the expression structure, the Nla protease encoding nucleotide sequence is operably linked to a promoter. The term “Operatively linked” refers to a functional link between a nucleic acid expression regulatory sequence (such as an array of promoters, signal sequences or transcription factor binding sites) and a second nucleic acid sequence. The expression regulation sequence affects the transcription and / or translation of the nucleic acid corresponding to the second sequence. According to the present invention, the promoter linked to the Nla protease gene is preferably operable in an animal, more preferably a mammalian cell, such as a mammalian cell genome or a mammalian virus, such as a CMV (cytomegalovirus) promoter. , Adenovirus late promoter, cowpox virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, β-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter and human GM It regulates transcription of Nla protease gene comprising a promoter derived from CSF gene promoter. Most preferably, the promoter is a CMV promoter.

  Preferably, the expression structure used in the present invention comprises an adenylate polymerization reaction sequence (eg, an adenylate polymerization reaction sequence derived from bovine growth hormone termination and SV40).

According to a preferred embodiment, the expression structure for the Nla protease encoding nucleotide sequence has the structure “Nla protease-adenylate polymerization reaction sequence encoding promoter-nucleotide sequence”.
The gene carrier of the present invention can be in various forms, preferably (i) a naked recombinant DNA molecule, (II) a plasmid, (iii) a viral vector or (iv) a liposome comprising a naked recombinant DNA molecule and a plasmid or Consists of neosomes.

Nla Protease-encoded nucleotide sequences are a number of gene carriers useful for gene therapy, preferably plasmids, adenovirus ²² , adeno-associated viruses (AAV, Lashford LS, etc., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retrovirus ²³ , herpes simplex virus ²⁴ , cowpox virus ²⁵ , liposomes (Methods in Molecular Biology, Vol 199, SC Basu and M. Basu (Eds.), Human Pres. 200, Human Pres. Or it can be applied to neosomes. Most preferably, the gene carrier of the present invention is constructed by binding a Nla protease-encoded nucleotide sequence to a retrovirus or lentivirus.

(I) Adenovirus Adenovirus has generally been adopted as a gene carrier because of its intermediate size genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 bp UR (Inverted Terminal Repeats), which are cis elements necessary for viral DNA replication and packaging. The E1 region (E1A and E1B) encodes proteins responsible for transcription of the viral genome and a few cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication.

Of the adenoviral vectors developed so far, incompetent adenoviral replications with deleted E1 regions are commonly used. The E3 region deleted from the adenovirus vector can provide an insertion site for the transplanted gene ^25,26 . Thus, the Nla protease-encoded nucleotide sequence is more preferably deleted in either one of the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or the deleted E3 region. Inserted into the E3 area. The promoter-Nla protease gene-polyA sequence is preferably any of the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or the deleted E3 region, more preferably the deleted E3 region. Or more preferably in the deleted E3 region.

  In effect, adenovirus can package about 105% of the wild-type genome, with a capacity for about 2 extra kb of DNA. In this respect, the external sequence inserted into the adenovirus described above can be further inserted into the adenovirus wild type genome.

  The adenovirus can be composed of any one of 42 other known serotypes or subgroups AF. Subgroup C adenovirus type 5 is the most preferred starting material for constructing the adenovirus gene carrier of the present invention. Numerous biochemical and genetic information for adenovirus type 5 is known.

  Since the external gene transmitted by this adenovirus gene carrier is episomal, it has low genotoxicity to the host cell. Therefore, it can be said that gene therapy using the adenovirus gene carrier of the present invention is quite safe.

(Ii) Retroviruses or lentiviruses Retroviruses or lentiviruses ²³ that can carry relatively large exogenous genes are used as viral gene transfer vectors in that their genomes are integrated into the host genome and have a broad host spectrum. It has been.

To construct a retroviral vector or a lentiviral vector, the transported Nla protease-encoded nucleotide sequence is inserted as a viral genome instead of a certain viral sequence to produce a replication defective virus. A packaged cell line is constructed that contains the gag, pol and env genes to produce virions, but lacks the long terminal repeat (LTR) and Ψ components ²⁸ . When a recombinant plasmid containing the Nla protease-encoding sequence, LTR and ψ is introduced into this cell column, the ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged with viral particles, The particles are then secreted into the culture medium Nicolas and Rubinstein “Retroviral vectors” In: Vectors: A survey of molecular cloning vectors and the theaters, Rodriguez and Denhardt ²⁹ . The medium containing the recombinant retrovirus is then collected, selectively enriched and used for gene transfer.

Successful gene transfer using two-generation retroviral vectors has been shown to be the result of the introduction of Eryotropin (EPO) sequences in place of the coat region, resulting in a chimeric protein with a new binding property. ³⁰ reported by Kasahara et al., Who produced variants. Perhaps the gene carrier can be attacked by a construction strategy for a two generation retroviral vector.

(Iii) AAV vectors Adeno-associated viruses can infect undivided cells and various types of cells, and can be used effectively in constructing the gene carrier of the present invention. A detailed description of the use and manufacture of AAV vectors is set forth in US Pat. Nos. 5,139,941 and 4,797,368.

Research results on AAV as a gene carrier are disclosed in LaFace et al. ³¹ , Zhou et al. ³² , Walsh et al. ^33, and Flotte et al. ³⁴ . Recently, AAV vectors have been approved for Phase 1 human clinical trials to treat cystic fibrosis.

Typically, recombinant AAV viruses are produced by plasmids ^{35, 36} containing the Nla protease gene with two AAV terminal repeats, etc. and an expression plasmid ³⁷ containing the wild type AAV coding sequence without terminal repeats.

(Iv) Other viral vectors Other viral vectors can be employed as gene carriers in the present invention. Cowpox virus ^{25 (Ridgeway,, In: Vectors} : "Mammalian expression vectors." A survey of molecular cloning vectors and their uses.Rodriguez and Denhardt 29; Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes. In: Kucherlapati R, ed. Gene transfer ³⁸ (New York: Plenum Press, 117-148 (1986)) and herpes simplex Vectors derived from viruses such as Ruth ²⁴ can be used in the present delivery system to deliver the Nla protease gene into cells.

(V) Liposomes Liposomes are spontaneously formed when phospholipids are suspended in a large amount of aqueous medium. Transfer of a nucleic acid liposome has arbitration, Nicolau and Sene ^39, and was very successful, as described in Nicolau like ^40. An example of a commercially available reagent for infecting animal cells utilizing liposomes includes Lipofectamine (Gibco BRL). Liposomes that capture the Nla protease gene interact with the cell by instruments such as endocytosis, adsorption and fusion, and then sequential transmission into the cell.

  In yet another aspect of the present invention, there is provided a gene transfer method comprising contacting the gene carrier of the present invention as described above with a biosample containing cells.

  When the gene carrier is constructed based on a viral vector structure, the contacting is performed as a conventional infection method known in the art. Host infection using viral vectors is described in detail in the publications cited above.

If the gene carrier is a naked (naked) recombinant DNA molecule or plasmid, Nla protease is transmitted - coded hierarchy and Nyukureochido hierarchy is microinjection ^{(microinjection) 41 and 42,} calcium phosphate co-precipitation ( Calcium phosphate co-precipitation ^43,44 , electroporation ^45,46 , liposome-mediated transformation ^47,39,40 , DEAE-dextran treatment ⁴⁸ and particle collision ⁴⁹ Is done.

  According to a preferred embodiment, the Nla protease gene (eg, sequence number 2) is a viral vector, more preferably a retroviral vector or a lentiviral vector.

  In the pharmaceutical composition of the present invention, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium, phosphate, alginate, Contains gelatin, potassium silicate, microcrystalline cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and magnesium mineral oils. It may be a conventional component for form, but is not limited thereto. The pharmaceutical composition according to the present invention may further comprise lubricants, humectants, sweeteners, fragrances, emulsifiers, suspending agents and preservatives. For detailed contents of compatible and pharmaceutically acceptable vehicles and dosage forms, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995), which is included herein by reference.

  The pharmaceutical composition of the present invention may be preferably administered parenterally. Minimal injection, spinal injection, intravenous injection or intraperitoneal injection may be employed for non-oral administration.

  The suitable dosage of the pharmaceutical composition of the present invention is as follows: pharmaceutical dosage form method, administration method, patient age, weight, sex, disease severity, eating habits, administration time, administration route, excretion It can vary depending on the rate and sensitivity to the pharmaceutical composition used. A physician with ordinary knowledge in the art may determine the effective amount of the pharmacological composition for the treatment desired. Preferably, the pharmaceutical composition of the present invention is administered in an amount of 0.001 to 1000 mg / kg (body weight) daily.

  According to the prior art known to those having ordinary skill in the art, the pharmacological composition is formulated with a pharmaceutically acceptable carrier and / or carrier as described above. Finally, a variety of forms may be provided, including single dose forms and multiple dose forms. Non-limiting examples of dosage forms include, but are not limited to, solutions, suspensions or emulsions in oil or aqueous media, extracts, elixirs, powders, granules, formulations and capsules. An agent or stabilizer may further be included.

  The pharmaceutical composition of the present invention includes Alzheimer's disease, mild cognitive impairment (MCI), mild to moderate cognitive impairment, vascular dementia, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, senile dementia, It can be employed to prevent or treat a variety of diseases or disorders including those associated with Down's syndrome, inclusion body myositis, age related macular degeneration and Alzheimer's disease. In particular, the pharmaceutical composition of the present invention is a promising therapeutic agent for Alzheimer's disease.

  Hereinafter, the present invention will be described in more detail through examples. These examples are for the purpose of illustrating the present invention more specifically, and it is usual in the art that the scope of the present invention described in the appended claims is not limited by these examples. It is self-evident for those who have knowledge of.

Materials and Methods Antibodies and Reagents Cell culture reagents were purchased from GIBCO-BRL (Invitrogen, Carlsbad, CA, USA). Synthetic Aβ ^1-42 peptides were purchased from Sigma (St Louis, MO, USA) and Anygen (Gwangju, Korea). The 6E10 antibody recognition residue of the Aβ peptide was purchased from Signet ^™ (Dedham, MA, USA). Antibodies against HA, a-tubulin, VDAC2, Octl and Catepsin D were purchased from Abeam (Cambridge, MA, UK). Chitin beads were purchased from New England BioLabs (Ipswich, MA, USA). All other reagents were purchased from Sigma.

Purification of Nla protease To produce recombinant Nla protein from E. coli, the Nla gene was replicated in pTYB12 (New England BioLabs) via the EcoRI and Xhol sites. The pTYB12 vector contains an N-terminal intein tag. pTYB12-Nla vector E. coli variant BL21 (DE3) was transformed and grown in LB medium at 37 ° C. 400 μm IPTG was added overnight at 20 ° C. to induce Nla protein. Cells were harvested, resuspended in column buffer (20 mM HEPES [pH 7.9], 500 mM NaCl, ImM EDTA) and lysed by sonication. The degradation product was centrifuged, and the resulting supernatant was loaded onto a chitin column maintained parallel with column buffer. After washing for a long time, the Nla protein is dissolved and separated from the column using a column buffer containing 50 mM DTT and stored in a storage buffer (50 mM HEPES [pH 7.6], 1 mM EDTA, 1 mM DTT, 10% glycerol). Dialyze and concentrate with Amicon Centriprep (Millipore, Billerica, MA, USA). The protein concentration was determined by BCA method and analyzed on a 12% SDS-PAGE gel.

To produce a preparation A [beta] solution of A [beta], we followed the like ^50, a method of Dahlgren like ^3-written Yan. Synthetic human Aβ _1-42 peptide (> 95% purity (high performance chromatography and mass spectrometry)) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM. In the case of monomeric Aβ, the Aβ solution in DMSO was diluted in PBS immediately before use to a final concentration of 25 μm. For oligomeric Aβ, the Aβ solution in DMSO was diluted in PBS to a final concentration of 100 μm and incubated at 4 ° C. for 36 hours. The physical state of Aβ was confirmed by PAGE on a 10-20% Tris-Tricine gel (Bio-Rad, Hercules, CA, USA).

Cleavage analysis and mass spectrometry 1.5 μm of recombinant Nla protease was incubated with analysis buffer (HEPES [pH 7.4], 20 mM KCL, 20 mM MgCl ₂ ) in a 2.5 μm Aβ formulation at 25 ° C. for 3 hours. As a control, Nla protease was preincubated with cysteine protease inhibitor, N-ethylmaleimide (NEM) for 10 minutes at 4 ° C. After incubation, the mixture was subjected to Western blotting using PAGE using 10-20% Tris-Tricine gel and anti-Aβ antibody 6E10. The reaction mixture was analyzed with a MALDI-TOF / TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Carlsbad, California, USA) for further analysis of the cleavage products. As symmetry, Nla and Aβ were analyzed separately.

Cell culture, transfusion and Aβ treatment B103 murine neuronal subcellular tumor cells were cultured in DMEM supplemented with 10% (vol / vol) fetal bovine serum ⁵¹ . A variant Nla gene in which Asp ⁸¹ in the catalytic trivalent element was changed to Ala was purified by PCR mutation purification. In order to express wild-type and variant Nla from B103 cells, the gene of interest was subcloned into pcDNA3 (invitrogen) containing an N-terminal HA tag. According to the manufacturer's protocol, the cells were transfected using Lipofectamine reagent (invitrogen). The cytosol Aβ _1-42 expression vector has already been described above ⁵² . To treat Aβ, Aβ solution (100 μm) was added to a final concentration of 5 μm.

Assessment of cell killing Cell viability was assessed by MTT analysis and cytomorphological methods. 3- [4,5-dimethylthiazole-2-yl] -2,5-diphenyltetrazole bromide (MTT) was solubilized with PBS to 5 mg / ml. The same volume of MTT solution as the volume of 10% culture medium was added at 37 ° C. for 3 hours. Add the same volume of solubilization solution (10% Triton X-100 in anhydrous isopropanol and 0.1 NHCL) to the volume of the culture medium and continue at 37 ° C. until the resulting formazan crystals are completely dissolved. Incubated. The absorbance of the material was measured at 570 nm, and the absorbance of the background of the nuclear well was measured at 690 nm. To assess cell morphology, cultured cells were transformed with experimental and GFP plasmids, and the shape of GFP-positive cells was examined with a fluorescence microscope 42 (Olympus, Shinjuku, Tokyo, Japan).

Immunofluorescence and confocal microscopy B103 murine neuronal subcellular tumor cells were washed with PBS containing 1 mM CaCl ₂ and 1 mM MgCl ₂ and fixed with 3.5% paraformaldehyde (Paraformaldehyde) for 10 minutes. Cells were permeabilized by incubation with 0.2% Triton X-100 in PBS for 10 minutes, blocked with 5% BSA in PBS for 1 hour, and incubated with anti-6E10 monoclonal antibody or HA monoclonal antibody for 1 hour. .

The fixed cells were then rinsed with PBS and incubated with Alexa 488 fluor-conjugated secondary antibody (invitrogen) and TRITC-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) for 1 hour. For immunofluorescence microscopy, immunoreactivity was captured with a fluorescence microscope (Olympus) equipped with a ProgRes C10 ^plus camera (JENOPT1K, Goeschewitzer Strasse, Jena, Germany). Color coding was performed using IMT-i solution software (IMTi-solution Inc., Vancouver, BC, Canada). In order to determine the level of Aβ aggregation among GFP positive cells, the number of Aβ positive cells versus GFP positive cells was counted in 20 arbitrary fields per culture. For confocal microscopy analysis, the fluorescence signal was visualized using a confocal microscope (TCS SP2, LEICA, Ernst-Leitz-Strasse, Wetzlar, Germany).

To determine the subcellular localization of taxonomy Nla protein subcellular were classified Nla- expressing cells using the above-described protocol ^53. Briefly, cells are collected by scraping into a homogeneous buffer (200 mM sucrose, 20 mM Tris [pH 7.4], 1 mM EGTA, 1 mM EDTA, 1X complete protease inhibitor cocktail) and injected with a syringe with a 26 gauge needle. Separated by several transfers and ultracentrifuged at 70,000 xg for 30 minutes at 4 ° C. The pellet (crude membrane fragment) was resuspended in homogeneous buffer containing 0.5% Triton X-100 and sonicated for 1 minute. A partial sample (50 pg) from each fragment was analyzed by Western blotting.

Electrophoresis and Western Blotting After washing 3 times with PBS, cells were collected, resuspended in RIPA buffer containing IX protease inhibitor nuclear and sonicated briefly. After centrifugation at 10,000 × g, soluble protein fragments were recovered and separated by SDS-PAGE. The protein concentration was determined by the BCA method. In order to analyze Aβ peptides, the materials were separated by electrophoresis using 10-20% Tris-Tricine gel. The protein was then transferred onto a PVDF membrane soaked with 50 mM Tris, 192 mM glycine and 20% methanol. Membranes were blocked with 5% non-fat milk and incubated with antibodies against 6E10, HA, a-tubulin, VDAC2, Octl and CathesinD. Bands were visualized using ECL reagent (GE Healthcare / Amersham Bioscience, Piscataway, NJ, USA) and the intensity of each band was quantified with a densitometer (Bio-Rad).

Lentivirus production cDNA fragments encoding Nla and GFP were sub-replicated into the pLEX-MCS lentiviral vector (Openbiosystems, Huntsville, AL, USA). The resulting recombinant plasmid was transformed into 293T together with the packing plasmid, and the supernatant was collected. Lentivirus was collected and concentrated in the ultracentrifuge ^19,54 described above. Nla and GFP titers were estimated by measuring the amount of HIV p24 antigen using PCR.

AD murine model and surgical procedure As described above, gene-transplanted AD model mice overexpressing human mutant APP and PSI (APPswe / PSldE9), Tg-APPswe / PSldE9 maintained in C57BL6xC3H F1 hybrid mice did. These mice were left in a regular plastic cage with free access to food and drink in a temperature and humidity controlled environment under a 12 hour light / dark cycle (lights up at 7 am). 6.5 months old Tg-APPswe / PSldE9 mice were arbitrarily extracted from the Lenti-Nla (n = 9) and Lenti-GFP (n = 10) groups. These mice were injected with 3 μm Lenti-Nla virus (1 × 10 8 TU) or Lenti-GFP lentivirus at the same titer in the ventricle (icv) on both sides of these mice. One month later, the injected mice were sacrificed and perfused with 0.9% saline. The left and right hemispheres of the brain were used for histology and biochemical analysis, respectively. All animals were handled in accordance with the Ewha Womans University Medical School Animal Care Guidelines.

RP-PCR
Total RNA was isolated from frontal cerebral cortex tissue with TRI reagent (Sigma). Reverse transcription was performed using ImPromII reverse transcriptase (Promega, Madison, Wisconsin, USA) with oligo-dT priming. To detect Nla expression, PCR was performed using Nla specific primer sets (5′-ACG AAA GAC GGC CAA TGC GGA-3 ′ and 5′-ACC CGA CGG TTG CGA TGC TT-3 ′). went. For control experiments, PCR was performed using GAPDH specific primer sets (5′-TCC GTG TTC CTA CCC CCA ATG-3 ′ and 5′-GGG AGT TGC TGT TGA AGT CGC-3 ′).

Immunohistochemistry The right hemisphere was post-fixed with 0.1% phosphate buffer (pH 7.4) with 4% paraformaldehyde overnight at 4 ° C. and Vibratome (Leica VT 1000S; Leica, (Germany) was cut into a tube having a thickness of 40 pm. The free-floating part was blocked with 5% normal goat serum, 2% BSA and 2% FBS. A biotinylated HRP system was used for color development. Anti-Aβ antibody Bam-10 (A5213) was purchased from Sigma (USA). Microscopy was performed using an Olympus BX51 microscope equipped with a DP71 camera and DP-B software (Olympus, Japan). In order to quantify the speckle level, the number of speckles in each region was measured using the TOMORO ScopeEye 3.6 program (Techsan Community, Seoul, Korea).

Assessment of Aβ levels ELISA analysis for Aβ (1-42) and Aβ (1-40) levels has been described in a previous study ⁴⁶ . Briefly, frontal cerebral cortex was homogenized with Tris-buffered saline (20 mM Tris and 137 mM NaCl, [pH 7.6]) in the presence of a protease inhibitor mixture (Complete Mini; Roche, USA). The homogenate was centrifuged at 100,000 × g for 1 hour at 4 ° C. and the supernatant was used to determine the level of Aβ Tris buffer-soluble form. The pellet was sonicated with 70% formic acid and centrifuged as described above. The resulting supernatant was considered to be Aβ from which formic acid can be extracted and was collected for further analysis. As stated, the formic acid extract was neutralized with 1 M Tris-Cl buffer (pH 11) at a 1:20 dilution prior to use. A final analysis was performed using human Aβ (1-40) or Aβ (1-42) colorimetric sandwich ELISA kits (BioSource, Invitrogen) according to the manufacturer's instructions.

Statistical analysis Two materials are compared using an unpaired Student'st-test with Unequal Variance, and multiple comparisons are performed in one-way analysis of variance before doing a Newman-Keuls multiple range test. ANOVA). A p value of less than 0.05 was accepted as statistically significant. Data were provided as mean ± SD.

Results Cleavage of Monomeric and Oligomeric Aβ by Nla Previously, we have conserved sequence element Val-Xaa-His-Gln where Nla has very stringent temperament specificity and a bond that can be easily cleaved is located behind Gln. With its cleavage site defined by. Interestingly, the sequence Val-Xaa-His-Gln is present in Aβ near the putative a-secretase cleavage site (FIG. 1A). Based on such findings, the inventors aimed to determine whether Nla can specifically cleave Aβ. For this purpose, recombinant Nla protein is obtained from E. coli. It was expressed in E. coli and purified to homogeneity on a chitin bead column (FIG. 1B). Nla was then incubated for 3 hours with the monomeric Aβ formulation in the presence or absence of NEM, a cysteine protease inhibitor. Analysis by Western blotting showed that monomeric Aβ levels were greatly reduced by Nla (FIG. 1C, Lane 2 vs. 4), which was partially reversed in the presence of NEM (FIG. 1C, Rain 6) Densitometer analysis showed that Nla reduced Aβ levels by about 64% in the absence of NEM and reduced Aβ levels by about 33% in the presence of NEM, which is a monomer by Nla. Presents specific cleavage of Aβ.

  Our findings indicate that NEM was unable to completely suppress Nla activity, which indicated that mutation of a cysteine residue in Nla's catalytic trivalent element completely eliminated its proteolytic activity. Consistent with previous reports showing that it did not expire. The inventors then tested whether Nla can cleave oligomer Aβ, which is known to be more toxic than monomeric Aβ. The Aβ peptide solution was incubated at 4 ° C. for 36 hours to produce oligomer Aβ. As assessed by SDS-PAGE, the oligomeric Aβ formulation contains approximately the same amount of monomer and oligomeric Aβ (FIG. 1D, Lanes 1, 3 and 5), and after an additional incubation at 25 ° C. for 3 hours, A balance was achieved by changing the formation of oligomeric Aβ at the expense of monomeric Aβ. Our findings are consistent with previous reports showing that Aβ oligomers were accelerated by increasing incubation time and temperature. Under the same conditions, the remaining amount of oligomeric Aβ was greatly reduced by Nla (Rain 4) as quantified by densitometer evaluation showing that only 19% of oligomeric Aβ remained. This phenomenon of oligomeric Aβ by Nla arbitration was markedly blocked by NEM (Rain 6), implying that Nla specifically cleaves Aβ.

To further analyze the specific cleavage of Aβ by Nla, the cleavage products were analyzed by MALDI-TOF / TOF mass spectrometry (FIG. 2). The monomer Aβ formulation was uncontaminated and produced a single peak, while Nla produced 3 contaminated peepes. In the reaction mixture containing Aβ and Nla, the peaks corresponding to Aβ are greatly reduced, and two new peaks with molecular weights of 1,826 Da and 2,704 Da corresponding to amino acids 1-15 and 16-42 of Aβ are detected. (FIG. 2B). This result indicates that, as expected, Nla cleaves the peptide bond located behind Gln ¹⁵ .

Localization of Nla subcells B103 neurocytoma cells were transformed with the HA-tagged Nla expression vector and stained with anti-HA antibody. Examination using a confocal microscope shows that Nla was predominantly expressed in the cytoplasm (FIG. 3A). The transformed cells were classified into microparticles (P) and soluble (S) fragments and subjected to Western blotting (FIG. 3B). Octl (nuclear marker), VDAC2 (mitochondrial marker) and cathepsin D (lysosomal marker) were found in the microparticle fragment, and HA was present in the same cells with a-tubulin (cytosolic marker) exclusively in the soluble fragment. These data suggest that Nla is predominantly present in cytosol.

Nla prevents Aβ-induced cell death In order to test whether Nla has activity against Aβ in cells, we have triple fusion of green fluorescent protein (GFP), ubiquitin (Ub) and Aβ Aβ was purified intracellularly using the plasmid pGFPUb-A encoding the protein. ⁵² ub Bae Puchido bond between the A [beta], which is quickly transmitted by the raw deubiquitination (deubiquitinating) enzyme inner generating GFP-Ub and equimolar ratio of A [beta] a (equimolar ratio) to Chitozoru. B103 cells were transformed with pGFPUb-Aβ and empty plasmid, Nla-expression plasmid, pcDNA-HA-Nla or Nla expression plasmid, pcDNA-HA-mNla. The Nla mutation is composed of Asp to Ala substitution in the catalytic trivalent element. Thereafter, the cells were immunostained with anti-Aβ antibody, 6E10 (FIGS. 4A and B). The results show that the proportion of Aβ positive cells is 56% of GFP-positive cells from such cells providing the pGFPUb-Aβ empty plasmid (MocK), while that proportion is pGFPUb-Aβ and pcDNA. -It was shown that cells that provided residence for HA-Nla (Nla) decreased rapidly to 14%. The percentage observed from cells expressing the variant Nla protease plasmid (mNla) was 42%, which was significantly different from that obtained with the empty plasmid. These data indicate that Nla can specifically degrade intracellular Aβ.

  To assess whether Nla prevents Aβ-induced cell death, we used two different methods: morphological access and MTT cell viability analysis (FIGS. 4B and C). ). Intracellular expression of Aβ via pGFPUb-Aβ resulted in a significant increase in cell death (62% for morphological analysis and 55% for MTT analysis). Such intracellular Aβ-induced cell death was almost completely blocked by co-transformation with pcDNA-HA-Nla, but was not affected by cells co-expressing pcDNA-HA-mNla. When B103 cells were treated with exogenous Aβ, there was a significant proportion of cell death (40% by morphological analysis and 38% by MTT analysis) but not by pcDNA-HA-mNla co-expression but pcDNA-HA -Suppressed by co-transformation with Nla. First, extracellular Aβ is made by itself by cell surface receptors and is detected from subcellular cell organs such as lysosomes, mitochondria and cytosol, and causes cell death through dysfunction of those organs. I understood. It was found that even if Nla and internalized Aβ were co-localized, cytosolic Nla could cleave internalized Aβ. Nevertheless, the inventors' data show that Nla can prevent cell death induced by both Aβ expressed intracellularly and exogenously added Aβ.

Overexpression of Nla lentivirus arbitration Lentiviral vectors expressing Nla and GFP were purified (FIG. 6A). As assessed by Western blotting using anti-HA antibodies, human 293T cells infected with Lenti-Nla showed strong Nla expression. Stereotaxically 3 μm Lenti-Nla (1 × 108 TU) was injected into the bilateral ventricles of double-transplanted mice (APPswe / PSldE9). In order to evaluate the expression of Nla, immunohistochemistry was performed one month after the injection. Nla expression was detected in the brain portion of mice injected with Lenti-Nla, compared to the brain portion of control mice that were not injected. Nla expression pattern showed a wide distribution across the cerebral cortex, hippocampus, tonsils and thalamus (data not shown).

RT-PC also showed the presence of Nla transcription from Lenti-Nla infected brains. The GAPDH signal that served as a control was also expressed from all samples (FIG. 6D).
To assess whether reduced Aβ levels Nla from the brains of APPsw / PSl transplanted mice injected with Lenti-Nla cause a decrease in Aβ from the mouse brain, APPsw / PSldE9 at 6 and 5 months of age. Lenti-Nla was injected into the ventricles on both sides of the mouse brain. As a control, the same amount of Lenti-GFP was injected in the same manner. One month after injection, brains were removed and Aβ levels were measured by ELISA from both soluble (extractable with Tris-buffer) and insoluble (extractable with FA-buffer) fragments. The inventors found that the levels of both Aβ _1-40 and Aβ _1-42 were significantly reduced compared to brains injected with Lenti-GFP (FIG. 7A). Lenti-Nla injection reduced soluble Aβ _1-40 by 33% in males and 36% in females, decreased insoluble Aβ _1-40 by 24% in males and 21% in females (FIG. 7A, upper rain) Nla Also reduced soluble Aβ _{1-42 in} males by 38%, females by 28%, males by 33% insoluble Aβ _1-42 and females by 36% (FIG. 7A, lower rain) male brain. The decrease in Aβ _1-42 levels at 5 was not statistically significant.

Decreased Aβ accumulation from brains of APPsw / PSl transplanted mice injected with Lenti-Nla Immunohistochemical analysis revealed that Aβ accumulation was injected from Lenti-GFP from frontal lobe, parietal lobe, hippocampus, and piriform cortex It was found that there was a marked decrease in the brain injected with Lenti-Nla compared to the brain (FIG. 7B). An abolished assessment of Aβ levels showed that Lenti-Nla infusion reduced the spots by 57% in the frontal lobe, 62% in the parietal lobe, and 59% in the piriform cortex (Figure 7C).

Discussion The production and accumulation of Aβ is the most important event in the pathogenesis of AD, and we present that removal of Aβ may provide a valuable strategy for the treatment of AD. Although Aβ exists in several assembled and assembled forms, oligomeric Aβ is known to be the most toxic form. Aβ is oligomerized within the cell immediately after it is produced, and these molecules are then secreted from the cell. Some of the distributed Aβ oligomers result in dysfunction of subcellular cellular machinery after entering cells via selective absorption, which is associated with the memory and cognitive decline typically observed in AD patients There are ⁶⁰ . Aβ is detected on both the cells in the AD brain neurons and the extracellular space. Recent studies have shown that intracellular Aβ levels decrease as the number of extracellular spots increases in AD patients and AD gene-transplanted mouse models ^10,61 . These results suggest that the accumulation of intracellular Aβ during disease progression precedes the formation of extracellular Aβ deposits. Interestingly, it can be seen that Aβ is maintained intracellularly in cells expressing AD-related variant APP, whereas Aβ is largely secreted in cells expressing wild-type APP ³² . And, in senile mice carrying the variant presenilin (from presenilin) 1, although Aβ aggregation can be detected in neurons, not detected in the extracellular fluid ^62. Inhibition of proteasome activity results in higher Aβ levels both in vivo and in vitro, suggesting that proteasomes are responsible for Aβ treatment with ^{cytosole 52,63,64} . Excessive production of Aβ leads to an excessive increase in proteasome, ultimately leading to abnormal proteasome activity characteristic of AD ^65,66 .

These reports support a central role for intracellular Aβ in the pathogenesis of AD. Increased proteosome activity brought about by the plant polyphenol resveratrol was found to reduce not only extracellular Aβ levels but also intracellular Aβ levels, preventing neurodegenerative disease ⁶⁷ . Parkin is an E3 ligase that participates in ubiquitination of Aβ expressed in cells. Parkin overexpression resulted in a decrease ⁶⁸ in proteasome arbitration Aβ levels, whereas parkin knockout was found to result in accumulation of Aβ deposits ^68,69 . Thus, an increase in the removal rate of intracellular Aβ can prevent spot formation, secondary pathological abnormalities and early death.

  In this study, we found that Nla, a plant viral protease, specifically cleaves not only monomeric Aβ but also oligomeric Aβ in vitro, and that neurons are predominantly localized to cytosol. Indicates.

  In neurons, Nla expression suppresses cell death induced by both Aβ expressed intracellularly and Aβ added extracellularly. In addition, overexpression of Nla, which was mediated by lentivirus from the brains of AD transgenic mice, was found to reduce the level of Aβ and spot formation. These data provide additional evidence that supports an important role for intracellular Aβ in the pathogenesis of AD. In this regard, Nla could be used as a new tool to study the molecular events underlying the induction of cell death by intracellular Aβ. Finally, our results provide proof of concept that removal of intracellular Aβ by cytosolic protease can be a viable strategy for the treatment of AD.

Acknowledgment This study was supported by the System Biology Infrastructure Facility Grant provided by the Korea Research Foundation (NRF) and the Gwangju Science and Technology Fund, funded by the Korean government (MEST) (No. 2009-0085747).

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While the preferred embodiment of the present invention has been described, modifications and variations that do not depart from the scope of the invention will be apparent to those of ordinary skill in the art of the invention, the scope of the invention being claimed. It should be understood that it is determined by the terms and their equivalents.

Sequence listing
<110> Gwangju Institute of Science and Technology

<120> AMYLOID-BETA CLEARANCE

<160> 3

<170> KopatentIn 1.71

<210> 1
<211> 243
<212> PRT
<213> Turnip mosaic virus

<400> 1
Ser Asn Ser Met Phe Arg Gly Leu Arg Asp Tyr Asn Pro Ile Ser Asn
1 5 10 15

Asn Ile Cys His Leu Thr Asn Val Ser Asp Gly Ala Ser Asn Ser Leu
20 25 30

Tyr Gly Val Gly Phe Gly Pro Leu Ile Leu Thr Asn Arg His Leu Phe
35 40 45

Glu Arg Asn Asn Gly Glu Leu Val Ile Lys Ser Arg His Gly Glu Phe
50 55 60

Val Ile Lys Asn Thr Thr Gln Leu His Leu Leu Pro Ile Pro Asp Arg
65 70 75 80

Asp Leu Leu Leu Ile Arg Leu Pro Lys Asp Ile Pro Pro Phe Pro Gln
85 90 95

Lys Leu Gly Phe Arg Gln Pro Glu Lys Gly Glu Arg Ile Cys Met Val
100 105 110

Gly Ser Asn Phe Gln Thr Lys Ser Ile Thr Ser Val Val Ser Glu Thr
115 120 125

Ser Thr Ile Met Pro Val Glu Asn Ser Gln Phe Trp Lys His Trp Ile
130 135 140

Ser Thr Lys Asp Gly Gln Cys Gly Ser Pro Met Val Ser Thr Lys Asp
145 150 155 160

Gly Lys Ile Leu Gly Leu His Ser Leu Ala Asn Phe Gln Asn Ser Ile
165 170 175

Asn Tyr Phe Ala Ala Phe Pro Asp Asp Phe Ala Glu Lys Tyr Leu His
180 185 190

Thr Ile Glu Ala His Glu Trp Val Lys His Trp Lys Tyr Asn Thr Ser
195 200 205

Ala Ile Ser Trp Gly Ser Leu Asn Ile Gln Ala Ser Gln Pro Ser Gly
210 215 220

Leu Phe Lys Val Ser Lys Leu Ile Ser Asp Leu Asp Ser Thr Ala Val
225 230 235 240

Tyr Ala Gln



<210> 2
<211> 732
<212> DNA
<213> Turnip mosaic virus

<400> 2
gagagtaact ccatgttcag agggttgcgt gattacaacc caatatcaaa caacatttgt 60

catctcacaa atgtttcaga tggagcatca aactcgttat atggagtcgg tttcggacca 120

ctcatattaa cgaaccgaca cctctttgag cggaataacg gtgaactcgt aataaaatca 180

cgacatggtg agttcgtgat taaaaacaca actcagctac atttgctacc gattccagac 240

agagatctcc tgctaatccg gttaccaaag gacatcccac cctttccaca gaaattgggt 300

ttcaggcaac ctgagaaggg tgagcgaatc tgcatggtgg ggtccaactt ccaaactaag 360

agcataacga gtgtagtctc tgagactagc acaataatgc cagtggaaaa cagtcagttt 420

tggaaacact ggattagcac gaaagacggc caatgcggaa gtccaatggt gagcacgaaa 480

gacgggaaaa tacttggact acacagccta gcaaacttcc agaattccat taattacttt 540

gctgctttcc cagatgattt tgccgagaag tatctccata ccattgaagc acacgagtgg 600

gtcaagcatt ggaaatataa tactagtgcc atcagctggg gctctttgaa tatacaagca 660

tcgcaaccgt caggtttgtt caaagtaagc aaactaatct cagacctcga cagcacggca 720

gtctacgcac aa 732


<210> 3
<211> 42
<212> PRT
<213> Homo sapiens

<400> 3
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys
1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile
20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala
35 40

Claims (12)

  A pharmaceutical composition for the prevention or treatment of a disease caused by amyloid β comprising Nla (nuclear content a) protease or a gene carrier containing a nucleotide sequence encoding the Nla protease as an active ingredient.   The pharmaceutical composition according to claim 1, wherein the Nla protease cleaves intracellular or extracellular amyloid β.   The pharmaceutical composition according to claim 2, wherein the Nla protease cleaves amyloid β present in cytosol.   The pharmaceutical composition according to claim 2, wherein the Nla protease suppresses cell death induced by the amyloid β.   The pharmaceutical composition according to claim 1, wherein the Nla protease has an activity of cleaving oligomeric amyloid β. The pharmaceutical composition according to claim 1, wherein the Nla protease has an activity of cleaving amyloid β _1-40 and amyloid β _1-42 .   The pharmaceutical composition according to claim 1, wherein the Nla protease has an activity of cleaving a peptide bond between amino acids 15 and 16 of the sequence number 3.   The pharmacological composition according to claim 1, wherein the gene carrier is a virus vector.   The pharmacological composition according to claim 8, wherein the gene carrier is a lentiviral vector.   The diseases caused by the amyloid β include Alzheimer's disease, mild cognitive impairment (MCI), mild to moderate cognitive dysfunction, vascular dementia, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, senile The pharmaceutical composition according to claim 1, which is a disease selected from a group consisting of diseases related to dementia, Down's syndrome, inclusion body myositis, age-related macular degeneration, and Alzheimer's disease.   The pharmaceutical composition according to claim 10, wherein the disease caused by amyloid β is Alzheimer's disease.   A method for preventing or treating a disease caused by amyloid β, comprising administering the pharmaceutical composition according to any one of claims 1 to 11 to a subject who is a mammal.

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