WO2023163562A1 - Composition for preventing or treating alzheimer's disease comprising nucleic acid complex - Google Patents

Abstract

The present invention relates to a nucleic acid complex having a blood-brain barrier penetrating ability and a pharmaceutical composition containing same as an active ingredient for preventing or treating degenerative brain disease and, more specifically, to a nucleic acid complex in which a bioactive nucleic acid targeting BACE1 gene is complementarily bound to a carrier peptide nucleic acid, and to a pharmaceutical composition containing same as an active ingredient for preventing or treating degenerative brain disease. The nucleic acid complex in which a bioactive nucleic acid targeting BACE1 is complementarily bound to a carrier peptide nucleic acid according to the present invention has a blood-brain barrier penetrating ability and can efficiently inhibit the expression of BACE1, and thus is useful for the prevention or treatment of a degenerative brain disease, especially, Alzheimer's disease.

Description

Composition for preventing or treating Alzheimer's disease comprising a nucleic acid complex

The present invention relates to a nucleic acid complex having blood-brain barrier penetrating ability and a pharmaceutical composition for preventing or treating degenerative brain disease containing the complex as an active ingredient, and more specifically, to a bioactive nucleic acid targeting the BACE1 gene; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) relates to a complementary nucleic acid complex and a pharmaceutical composition for preventing or treating degenerative brain disease containing the same as an active ingredient.

Alzheimer's disease (AD) is a degenerative brain disease, and it is the most common disease among degenerative brain diseases, accounting for about 50 to 70% of all dementia, and the main symptom is cognitive impairment. Most of the clinical symptoms of Alzheimer's disease appear as memory decline, and it is known that not only short-term memory but also long-term memory is gradually damaged, and various other cognitive functions are also deteriorated (Justin M. Long, David M Holtzman, Cell. 2019 October 03;179(2): 312-339).

When the brains of patients with Alzheimer's disease were confirmed through imaging tests and autopsies, senile plaques and tau protein, which are caused by the accumulation of amyloid-β (Aβ) protein in the brain tissue and the smaller brain size compared to normal people, It can be seen that the formation of a neurofibrillary tangle formed when (tau proteins, τ proteins) are abnormally entangled. Based on these characteristics, several studies using the brain tissue of Alzheimer's disease patients have shown that senile plaques and nerve fiber bundles gradually accumulate in the brain over time, and the resulting neuronal death leads to Alzheimer's disease. (Rafi U. Haque and Allan I. Levey, PNAS December 26, 2019, 116 (52) 26224-26229).

Among the various causes of Alzheimer's disease, the accumulation of beta-amyloid protein is the oldest hypothesis and the most likely pathogenesis mechanism, but most of the treatments that directly target beta-amyloid protein and the senile plaques produced by their accumulation have repeatedly failed in clinical practice. The therapeutic effect of improving disease lesions was also not significantly shown (David S. Knopman, et al., Nature Reviews disease Primers; 7(1);33, 2021). After several failures, researchers recognized that removing the already increased beta-amyloid protein or senile plaques was not suitable as a target for treatment, and studied the mechanism of the previous step that could suppress the increase of beta-amyloid protein.

BACE1 (beta(β)-secretase 1, beta-site amyloid precursor protein (APP) cleaving enzyme 1) is an enzyme that cleaves the precursor of beta-amyloid protein and is a direct action factor that releases beta-amyloid into the cell. Abnormal aggregation occurs when beta-amyloid, which is cleaved by the BACE1 enzyme, accumulates, resulting in the formation of senile plaques commonly found in Alzheimer's disease. Therefore, this enzyme has attracted attention as a target that may act as a direct cause in relation to Alzheimer's disease (Sarah L Cole and Robert Vassar, Molecular Neurodegeneration 2007, 2:22).

Multinational pharmaceutical companies have developed new Alzheimer's disease treatments targeting this, but in many cases, development has been stopped because side effects or clear effects were not confirmed during clinical trials. As a representative cause of this, since BACE1 is an enzyme expressed not only in the brain but also in the liver, unexpected side effects occur when used as a target in the systemic circulation (Judite R. M. Coimbra, et al., Front. Chem. 2018; 6 :178). However, the enzymatic properties of BACE1, which is currently expressed in the brain, are being revealed by many researchers, and many efforts have been made to increase the brain transmission rate to overcome the low blood-brain barrier permeability of existing treatments. (Harald Hampel, et al, Biological Psychiatry. 2021 April 15; 89(8): 745-756).

Accordingly, the present inventors have made intensive efforts to apply a nucleic acid complex having excellent blood-brain barrier penetration ability to the treatment of degenerative brain diseases, particularly Alzheimer's disease. As a result, bioactive nucleic acids targeting the BACE1 gene and carrier peptide nucleic acids are complementaryly bound It was found that the nucleic acid complex exhibits excellent effects in preventing or treating degenerative brain diseases, and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be

Summary of Invention

An object of the present invention is to provide a nucleic acid complex having blood-brain barrier penetrating ability and a pharmaceutical composition for preventing or treating degenerative brain disease containing the same as an active ingredient.

In order to achieve the above object, the present invention is a bioactive nucleic acid targeting the BACE1 gene (Bioactive Nucleic Acid); And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) provides a complementary nucleic acid complex.

The present invention also provides a pharmaceutical composition for preventing or treating degenerative brain disease containing the nucleic acid complex as an active ingredient.

The present invention also provides a method for preventing or treating a degenerative brain disease comprising administering the nucleic acid complex.

The present invention also provides the use of the nucleic acid complex for the prevention or treatment of degenerative brain diseases.

The present invention also provides the use of the nucleic acid complex for the preparation of a drug for preventing or treating degenerative brain disease.

1 is a diagram confirming the ability of a nucleic acid complex to inhibit target gene expression in a mouse or human-derived neuronal cell model.

Figure 2 is a graph confirming the ability to inhibit the enzyme activity generated by the target gene of the nucleic acid complex in a mouse or human-derived neural cell model.

Figure 3a is a diagram confirming the intracellular expression inhibition ability of a nucleic acid complex target gene in a mouse-derived neural cell model by fluorescence.

Figure 3b is a graph quantifying the fluorescence expression value of the target gene according to the nucleic acid complex treatment in the mouse-derived neural cell model.

Figure 3c is a diagram confirming the ability to suppress intracellular expression of a nucleic acid complex target gene in a human-derived neural cell model by fluorescence.

3D is a graph quantifying fluorescence expression values of target genes according to nucleic acid complex treatment in a human-derived neural cell model.

4 is a view confirming the ability to suppress expression of the final product of a target gene according to the treatment of a nucleic acid complex in a mouse or human-derived neural cell model.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

BACE1, used as a target gene in the present invention, is an enzyme that functions to cleave beta-amyloid protein, one of the causes of Alzheimer's disease, from the cytoplasm and release it to the outside (Brati Das and Riqiang Yan, Translational Neurodegeneration (2017) 6: 23). The expression of the corresponding gene is high in the actual patient group, and when the gene is selectively suppressed in the brain in animal models, there are effects such as improving cognitive impairment and behavioral patterns or reducing aggregates of beta-amyloid protein, which is the cause of Alzheimer's disease. It is attracting attention as a new therapeutic target (Wenting Zhang, et al., Experimental and Therapeutic Medicine 16: 2080-2086, 2018).

Accordingly, in the present invention, it was confirmed that a nucleic acid complex in which a bioactive nucleic acid targeting the BACE1 gene and a carrier peptide nucleic acid are complementaryly bound can be used for the prevention and treatment of Alzheimer's disease.

In one embodiment of the present invention, based on Korean Patent Registration No. 10-1963885 of the present inventors, a nucleic acid complex in which a bioactive nucleic acid targeting the BACE1 gene and a carrier peptide nucleic acid were complementaryly coupled was prepared. When the neuroblastoma cell line was treated with these nucleic acid complexes, it was confirmed that the expression of the target gene, BACE1, was effectively suppressed, and the expression of secreted APP protein (sAPP), which is cleaved and released by BACE1, was reduced. It was confirmed that there is an effect of preventing/treating Alzheimer's disease by inhibiting the production of beta amyloid, which is one of the causes of the disease, in the previous stage.

Accordingly, in one aspect, the present invention provides a bioactive nucleic acid targeting the BACE1 gene; and a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

In the present invention, a nucleic acid complex in which a bioactive nucleic acid targeting the BACE1 gene and a carrier peptide are complementaryly bound may have a structure of the following structural formula (1).

structural formula (1)

[ A ≡ C ⁽⁺⁾ ]

In the structural formula (1),

A is a bioactive nucleic acid having a sequence capable of binding to a gene of interest,

C is a carrier peptide nucleic acid capable of binding to a bioactive nucleic acid;

'≡' means a complementary bond between a bioactive nucleic acid and a carrier peptide nucleic acid,

The bioactive nucleic acid represented by A has an overall negative charge or neutral charge,

C ⁽⁺⁾ means that the carrier peptide nucleic acid has an overall positive charge,

The carrier peptide nucleic acid includes one or more peptide nucleic acid monomers modified to have a positive charge throughout the carrier peptide nucleic acid.

The bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex according to the present invention may have anti-parallel binding or parallel binding. In the present invention, the complementary binding form of the nucleic acid can be separated in the presence of a target sequence of the bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).

In the present invention, "Bioactive Nucleic Acid" binds to a target gene and a nucleotide sequence containing the target gene in vitro or in vivo to determine the unique function of the gene (e.g., For example, activating or inhibiting transcript expression or protein expression), or regulating pre-mRNA splicing (eg, exon skipping), etc. , The nucleotide sequence may be characterized as a gene regulatory sequence, a gene coding sequence, or a splicing regulatory sequence. Preferably, the bioactive nucleic acid is expressed A nucleic acid having a complementary sequence capable of binding to a target gene of interest to be reduced, in particular, a complementary sequence capable of binding to the mRNA of such a target gene of interest, and suppressing the expression of the gene. It means a nucleic acid involved in regulation, and may be a nucleic acid having a sequence complementary to a target gene whose expression is to be reduced.

Therefore, the bioactive nucleic acid in the present invention is preferably an antisense peptide nucleic acid of the BACE1 (beta-secretase 1, beta-site amyloid precursor protein cleaving enzyme 1) gene, which is an Alzheimer's disease-related target gene, and more preferably SEQ ID NO: 2 It may include a nucleotide sequence represented by the sequence of, but is not limited thereto.

The bioactive nucleic acids include DNA, RNA, or modified nucleic acids such as PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid), antisense It may be selected from the group consisting of oligonucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme and DNAzyme, preferably the bioactive The nucleic acid is selected from the group consisting of DNA, RNA, or modified nucleic acid PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid) it may be

In the present invention, "Carrier Peptide Nucleic Acid" refers to a nucleic acid to which a bioactive nucleic acid and some or all bases are complementaryly combined to impart functionality, and the carrier peptide nucleic acid used in the present invention is In addition to peptide nucleic acid (PNA), modified nucleic acids similar thereto may be used, and peptide nucleic acids are preferred, but are not limited thereto.

In the present invention, the carrier peptide nucleic acid preferably includes one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so that the entire carrier peptide nucleic acid is positively charged, and the gamma- or alpha-backbone modified peptide nucleic acid It is more preferable that monomers having amino acids having a positive charge are included more than monomers having amino acids having a negative charge so that the overall charge of the carrier peptide nucleic acid is positive.

In particular, in the present invention, the carrier peptide nucleic acid preferably includes a nucleotide sequence represented by any one sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 7, but is not limited thereto.

In the present invention, the “nucleic acid complex” is capable of infiltrating bioactive substances into the body and ultimately into cells through extracellular processing, and specifically, has the ability to deliver a bioactive nucleic acid targeting the BACE1 gene into cells. .

In the present invention, the nucleic acid complex is a bioactive nucleic acid represented by the sequence of SEQ ID NO: 2; and a carrier peptide nucleic acid represented by any one of SEQ ID NOs: 4 to 7, but is not limited thereto. More preferably, the nucleic acid complex comprises a bioactive nucleic acid represented by the sequence of SEQ ID NO: 2; And it may be characterized by comprising a carrier peptide nucleic acid represented by the sequence of SEQ ID NO: 5 or 7, but is not limited thereto.

In addition, the binding ability (melting temperature, Tm) of the bioactive nucleic acid targeting the BACE1 gene and the carrier peptide nucleic acid may be characterized as being lower than the binding ability between the bioactive nucleic acid and the target BACE1 gene of the bioactive nucleic acid. there is.

The binding force is obtained by parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid according to the 5'-direction and the 3'-direction of each nucleic acid, so that the bioactive nucleic acid and the carrier peptide nucleic acid The binding force (Tm) of may be lower than the binding force between the bioactive nucleic acid and the target gene of the bioactive nucleic acid.

In the present invention, the bioactive nucleic acid or carrier peptide nucleic acid may be characterized by additionally binding a substance that helps endosome escape to the 5'-end or 3'-end of each nucleic acid. That is, it may be characterized in that it has a structure of the following Structural Formula (2) by further including a material that helps the endosome escape of the bioactive nucleic acid and the carrier peptide nucleic acid.

structural formula (2)

[ mA ≡ mC ⁽⁺⁾ ]

In the structural formula (2),

'm' means a substance that helps endosome escape of bioactive nucleic acid and carrier peptide nucleic acid.

In the present invention, "substances that help endosomes escape" can be characterized in that they help the escape of bioactive nucleic acids from endosomes by increasing the osmotic pressure inside the endosomes or by destabilizing the membranes of endosomes. there is. It means that bioactive nucleic acids move more efficiently and rapidly to the nucleus or cytoplasm to meet and act on target genes (Daniel W Pack, et al., Nat Rev Drug Discov. 2005 Jul;4(7):581-93 ).

In the present invention, the material that helps the endosome escape is a peptide, lipid nanoparticles, conjugate nanoparticles (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanomaterials (inorganic nanoparticles), cationic lipids It may be characterized in that at least one selected from the group consisting of nanomaterials (cationic lipid-based nanoparticles), cationic polymers (cationic polymers) and pH sensitive polymers (pH sensitive polymers).

In the present invention, a peptide (GLFDIIKKIAESF, SEQ ID NO: 8) may be linked to the bioactive nucleic acid as a material that helps the endosome escape through a linker, and Histidine (10) to the carrier peptide nucleic acid as a linker. It may be characterized by combining, but is not limited thereto.

In the present invention, the lipid nanoparticles may be selected from the group consisting of Lipid, phospholipids, acetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride and Tween 80.

In the present invention, the polyplex nanoparticles may be poly(amidoamine) or polyethylenimine (PEI).

In the present invention, the polymer nanospheres are selected from the group consisting of polycaprolactone, poly(lactide-co-glycolide, polylactide, polyglycolide, poly(d,l-lactide), chitosan, and PLGA-polyethylene glycol. can be characterized.

In the present invention, the inorganic nanoparticles may be selected from the group consisting of Fe2O3, Fe3O4, WO3 and WO2.9.

In the present invention, the cationic lipid-based nanoparticles are 1- (aminoethyl) iminobis [N- (oleicylcysteinyl-1-amino-ethyl) propionamide], N-alkylated derivative of PTA and 3, 5- It may be characterized in that it is selected from the group consisting of didodecyloxybenzamidine.

In the present invention, the cationic polymer may be selected from the group consisting of vinylpyrrolidone-N, N-dimethylaminoethyl methacrylate acid copolymer diethyl sulphate, polyisobutylene and poly(N-vinylcarbazole).

In the present invention, the pH sensitive polymers may be selected from the group consisting of polyacids, poly(acrylic acid), poly(methacrylic acid), and hydrolyzed polyacrylamide.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid may each contain 2 to 50, preferably 2 to 30, more preferably 5 to 20 nucleic acid monomers, but are limited thereto. it is not going to be

The bioactive nucleic acid may be characterized in that it consists of natural nucleic acid bases and/or modified nucleic acid monomers.

In the present invention, when the monomer used for the bioactive nucleic acid is PNA, it is referred to as a bioactive peptide nucleic acid, and when other monomers are used, it is referred to in the same way.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid are phosphodiester, 2' O-methyl, 2' methoxy-ethyl, phosphor It may be characterized by further comprising at least one functional group selected from the group consisting of amidate, methylphosphonate, and phosphorothioate.

In the present invention, the carrier peptide nucleic acid may be characterized in that a part or all of the base sequence of the bioactive nucleic acid is composed of a complementary sequence. In particular, the carrier peptide nucleic acid may include one or more universal bases, and all of the carrier peptide nucleic acids may consist of universal bases.

In the present invention, each of the bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex may be a complex characterized by having a positive charge (positive), negative charge (negative) or neutral charge as a whole.

In the expression of the electrical properties, the meaning of "overall" means the electrical properties of the entire bioactive nucleic acid or carrier peptide nucleic acid as a whole, not the electrical properties of individual bases, when viewed from the outside. Even if some of the monomers in the sexual nucleic acid have a positive charge, if there are more monomers with a negative charge, the bioactive nucleic acid will have a negative charge when looking at the electrical properties “as a whole”, and some bases and/or Even if the backbone has a negative charge, when a larger number of bases and/or backbones having a positive charge are present, the carrier peptide nucleic acid has a positive charge when looking at the electrical characteristics “as a whole”.

From this point of view, the nucleic acid complex of the present invention may be characterized as having a positive charge as a whole. In the nucleic acid complex, preferably, the bioactive nucleic acid has negative or neutral electrical characteristics as a whole, and the carrier peptide nucleic acid has positive electrical characteristics as a whole. It is not limited thereto.

In the present invention, the electrical properties of the bioactive nucleic acid and the carrier peptide nucleic acid can be imparted using a modified peptide nucleic acid monomer, and the modified peptide nucleic acid monomer is a carrier peptide nucleic acid having a positive charge, such as lysine (Lysine, Lys, K) , arginine (Arg, R), histidine (Histidine, His, H), diamino butyric acid (DAB), ornithine (Orn), and any one or more positive charges selected from the group consisting of amino acid analogs It is a carrier peptide nucleic acid that includes amino acids of and has a negative charge, and may be characterized in that it includes a negatively charged amino acid such as glutamic acid (Glu, E) or an amino acid analog that is negatively charged.

In the present invention, the carrier peptide nucleic acid may be characterized by including one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so as to have a positive charge as a whole.

The gamma- or alpha-backbone modified peptide nucleic acid monomer has lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (His, H), diamino butyric acid (Diamino butyric acid) to have an electrical positivity. acid, DAB), ornithine (Orn), and amino acids having at least one positive charge selected from the group consisting of amino acid analogs.

In the present invention, the modification of the peptide nucleic acid monomer for charge imparting may use a peptide nucleic acid monomer having a modified nucleobase in addition to the backbone modification. Preferably, an amine, triazole, or imidazole moiety may be included in the nucleobase to have an electronegative property, or a carboxylic acid may be included in the base to have an electronegative property.

In the present invention, the modified peptide nucleic acid monomer of the carrier peptide nucleic acid may further contain a negative charge in the backbone or nucleobase, but the modified peptide nucleic acid monomer contains more positively charged monomers than monomers having negative charges, so that the carrier peptide as a whole is formed. It is preferred that the charge of the nucleic acid be positive.

Preferably, the nucleic acid complex according to the present invention has a positive charge as a whole.

In the nucleic acid complex according to the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, or a fluorescent/luminescent marker is a bioactive nucleic acid And / or may be characterized in that it is bound to a carrier peptide nucleic acid, preferably the hydrophobic moiety, the hydrophilic moiety, a target antigen-specific antibody, an aptamer, and a fluorescent / luminescent marker for imaging One or more substances selected from the group consisting of and the like may be bound to the carrier peptide nucleic acid.

In the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, a quencher, a fluorescent marker, and a luminescent marker, and a bioactive nucleic acid and/or The binding of the carrier peptide nucleic acid may be characterized as a simple covalent bond or a covalent bond mediated by a linker, but is not limited thereto. Preferably, substances related to cell permeation, solubility, stability, delivery and imaging (eg, hydrophobic residues, etc.) bound to the nucleic acid carrier exist independently of the bioactive nucleic acid that regulates the expression of the target gene.

In the present invention, as described above, the complementary binding form of the bioactive nucleic acid and the carrier peptide nucleic acid may be largely characterized by having the form of antiparallel binding and parallel binding. . The complementary binding form has a structure that separates in the presence of a target sequence of a bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).

The antiparallel bond and parallel bond are determined according to the 5'-direction and the 3'-direction in the binding method of DNA-DNA or DNA-PNA. Antiparallel coupling is a general DNA-DNA or DNA-PNA coupling method. Taking the nucleic acid complex according to the present invention as an example, the bioactive nucleic acid is in the 5' to 3' direction and the carrier peptide nucleic acid is in the 3' to 5' direction. It means that the shape is connected to each other in the direction. Parallel binding is a form in which binding force is somewhat lower than that of anti-parallel binding, and means that both the bioactive nucleic acid and the carrier peptide nucleic acid are bonded to each other in the 5' to 3' direction or the 3' to 5' direction.

In the nucleic acid complex according to the present invention, preferably, the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is lower than the binding force of the bioactive nucleic acid and the target gene, particularly the mRNA of the target gene. . The binding force is determined by melting temperature, melting temperature or Tm.

As an example of a specific method for making the binding strength (melting temperature, Tm) of the bioactive nucleic acid and the carrier peptide nucleic acid lower than the binding strength of the bioactive nucleic acid and the bioactive nucleic acid to the target gene, in particular, the mRNA of the target gene, the above Parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized, but is not limited thereto.

As another example, the carrier peptide nucleic acid has at least one peptide nucleic acid base selected from the group consisting of a linker, a universal base, and a peptide nucleic acid base having a base that is not complementary to a corresponding base of a bioactive nucleic acid. It can be, but is not limited thereto.

In the present invention, the universal base binds to natural bases such as adenine, guanine, cytosine, thymine, and uracil without selectivity, and is complementary to One or more bases selected from the group consisting of inosine PNA, indole PNA, nitroindole PNA, and abasic can be used as a base having a lower binding force than the binding force, preferably. It can be characterized by using inosine PNA.

In the present invention, a combination of the binding form and electrical properties of nucleic acids for function control of the nucleic acid complex is provided, and the combination of the binding form and electrical properties of the nucleic acids controls the particle size and the time point of action, cell permeability, solubility and specificity It can be characterized as improving the degree.

In the present invention, the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is controlled, in the presence of the target gene, when the bioactive nucleic acid binds to the target sequence (when the bioactive nucleic acid is substituted with the target sequence, the target specific It is possible to adjust the timing of enemy separation and joining).

In the nucleic acid complex according to the present invention, the control of the strand displacement time point and the target specific release and bind time point of the bioactive nucleic acid into the target gene is a carrier peptide for non-specific binding of the complex It can be characterized in that it can be controlled by the presence, number, and location of non-specific bases, universal bases, and linkers in nucleic acids. It may be characterized in that control is possible by a combination of the above conditions, such as a parallel or antiparallel bond, which is a form of complementary bond of the peptide complex.

In the present invention, the particle size of the nucleic acid complex may be characterized in that it is controlled by adjusting the charge balance between the bioactive nucleic acid and the carrier peptide nucleic acid. Specifically, when the positive charge of the carrier peptide nucleic acid increases, the particle size decreases, but when the positive charge of the carrier peptide nucleic acid exceeds a certain level, the particle size increases. In addition, the particle size is determined by an appropriate charge balance with the overall carrier peptide nucleic acid according to the charge of the bioactive nucleic acid forming the complex as another important factor determining the particle size.

The positive charge of the carrier peptide nucleic acid according to the present invention is 1 to 7 (meaning that 1 to 7 monomers having a positive charge are included), preferably 2 to 5, most preferably 2 to 3 And, the charge of the bioactive nucleic acid may be characterized in that the net charge of the charge balance is 0 to 5 negative charges, preferably 0 to 3.

In the present invention, the nucleic acid complex can be prepared by hybridizing a bioactive nucleic acid and a carrier peptide nucleic acid under appropriate conditions.

"Hybridization" in the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (perfect match) or even when some mismatch bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and may be particularly controlled by binding temperature.

In the present invention, the nucleic acid complex may have blood-brain barrier penetrating ability.

In the present invention, the term "blood-brain barrier" or "BBB (Blood-Brain Barrier)" is used interchangeably herein, which closely regulates and severely restricts the exchange between blood and brain tissue and is circulated through brain tissue. Therefore, it is used to refer to the permeability barrier present in the blood. Components of the blood brain barrier include the endothelial cells that form the deepest lining of all blood vessels, the dense junctions between adjacent endothelial cells that are structurally correlated with the BBB, the basement membrane of endothelial cells, and almost all of the exposed outer surface of blood vessels. An enlarged foot process of the overlying astrocytes is included. The BBB prevents most substances in the blood, including most large molecules, such as Ig, antibodies, complement, albumin, and drugs and small molecules, from entering brain tissue.

The “target gene” of the present invention refers to a nucleic acid sequence (base sequence) to be activated, inhibited, or labeled, and is not different from the term “target nucleic acid”, and is used interchangeably herein.

In the present invention, when a target nucleic acid (base sequence) containing a target gene is contacted (bound) with the complex in vitro or in vivo , bioactive nucleic acid is separated from the carrier peptide nucleic acid and exhibit biological activity.

In the present invention, diseases that can be prevented or treated using the nucleic acid complex may be determined according to the target gene of the bioactive nucleic acid in the nucleic acid complex. In the present invention, the target gene of the bioactive nucleic acid may be characterized in that BACE1.

Therefore, from another aspect, the present invention provides a bioactive nucleic acid targeting the BACE1 gene; And a pharmaceutical composition for preventing or treating degenerative brain disease containing a nucleic acid complex complementary to a carrier peptide nucleic acid as an active ingredient.

In the present invention, the disease that can be prevented and treated using the nucleic acid complex may preferably be a degenerative brain disease, and the degenerative brain disease may include Alzheimer's disease, Parkinson's disease, Niemann-Pick disease, Creutzfeldt-Jakob disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis or dementia It may be characterized as, but is not limited thereto.

As used herein, the term "prevention" refers to any action that prevents the onset of a disease or delays its progression by administering a composition containing the nucleic acid complex. In addition, the term “treatment” used in the present invention refers to all activities in which symptoms of a disease are improved, symptoms are alleviated, or cured by administration of a composition containing the nucleic acid complex.

A pharmaceutical composition comprising a nucleic acid complex according to the present invention may include a pharmaceutically effective amount of the nucleic acid complex alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents. In the above, the pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat symptoms of degenerative brain disease.

The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives.

The term "carrier" is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

The term “diluent” is defined as a compound that is diluted in water which will dissolve the compound as well as stabilize the biologically active form of the subject compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

A substance containing a nucleic acid complex in the present invention can be administered to a patient by itself or as a mixed pharmaceutical composition together with other active ingredients, such as in combination therapy, or with suitable carriers or excipients.

Compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration. The dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.

The composition of the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on various factors such as the route of administration, age, sex, weight and severity of the patient. can be selected appropriately.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in effective amounts to achieve their intended purpose. More specifically, a therapeutically effective amount refers to an amount of a compound effective to prolong the survival of the subject being treated or to prevent, alleviate or ameliorate symptoms of a disease. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For administration of the nucleic acid complexes of the present invention, any nucleic acid delivery method known in the art may be used. Although not limited thereto, suitable delivery reagents include, for example, Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, polycations (eg, polylysine), atelocollagen, nanoplexes, and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules has been described by Minakuchi et al. Nucleic Acids Res., 32(13):e109 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008). Exemplary interfering nucleic acid delivery systems are provided in U.S. Patent Nos. 8,283,461, 8,313,772, and 8,501,930.

In the present invention, the nucleic acid complex may be administered using a delivery system such as a liposome. The liposome can help target the complex to a specific tissue, such as lymphoid tissue, or selectively target infected cells, and can also help increase the half-life of a composition containing the complex. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In such formulations, the complex to be delivered, alone or to specific cells, in combination with a molecule that binds to a receptor prevalent in lymphocytes, such as a monoclonal antibody that binds to the CD45 antigen, or in combination with other therapeutic compositions, is a liposome. incorporated as part of Thus, liposomes filled or decorated with a given complex of the present invention to deliver the nucleic acid complex composition can be directed to the site of lymphocytes.

Liposomes for use in accordance with the present invention are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols such as cholesterol. In general, lipids are selected in consideration of, for example, stability of liposomes in the blood stream, acid lability, and size of liposomes. A variety of methods can be used to prepare liposomes. See, eg, Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467, 1980), and US Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

In another aspect, the present invention provides a bioactive nucleic acid targeting the BACE1 gene; And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) It relates to a method for preventing or treating degenerative brain disease comprising administering to a subject a nucleic acid complex complementary thereto.

In another aspect, the present invention provides a bioactive nucleic acid targeting the BACE1 gene for the prevention or treatment of degenerative brain diseases; And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

In another aspect, the present invention provides a bioactive nucleic acid targeting the BACE1 gene for the preparation of drugs for the prevention or treatment of degenerative brain diseases (Bioactive Nucleic Acid); And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementaryly bound.

In the present invention, “subject” means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering a nucleic acid complex according to the present invention, and preferably means a human.

In addition, the dose of the nucleic acid complex of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity.

Toxicity and therapeutic efficacy of compositions comprising the nucleic acid complexes described herein can be measured by, for example, the LD50 (lethal dose to 50% of the population), the ED50 (the dose that is therapeutically effective in 50% of the population), the IC50 It can be estimated by standard pharmaceutical procedures in cell culture or laboratory animals to determine (the dose that has a therapeutically inhibitory effect on 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between the LD50 and the ED50 (or IC50). Compounds exhibiting high therapeutic indices are preferred. Data obtained from these cell culture assays can be used to estimate a range of human doses. The dosage or applied amount of such compounds lies preferably within a range of circulating concentrations that include the ED50 (or IC50) with little or no toxicity.

The term "administration" of the present invention refers to the act of introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration may be administered through various oral or parenteral routes as long as it can reach the target tissue. there is.

The administration route of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or intrarectally, as desired. In addition, the composition may be administered by any device capable of transporting an active substance to a target cell.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of bioactive peptide nucleic acids and carrier peptide nucleic acids, and complexes using them

In the present invention, BACE1 was used as a target gene to test the effect of the nucleic acid complex on Alzheimer's disease, and to confirm the therapeutic effect of Alzheimer's disease, antisense peptide nucleic acid (BACE1) was used as a bioactive peptide nucleic acid (BACE1). antisense PNA) was used.

The bioactive nucleic acid (antisense PNA) used as a control of the present invention includes the sequence represented by SEQ ID NO: 1, and the bioactive peptide nucleic acid (antisense PNA) used to confirm the therapeutic effect of Alzheimer's disease has the sequence represented by SEQ ID NO: 2 includes Carrier peptide nucleic acids used in the examples of the present invention include sequences represented by SEQ ID NOs: 3 to 7 (Table 1). All peptide nucleic acids used in the present invention were synthesized by HPLC purification method at PANAGENE (Korea).

Modification of the monomers converts the backbone of the peptide nucleic acid to electrically positive lysine (represented by Lysine, Lys, K, ⁽⁺⁾ ) and electronegatively to glutamic acid (Glu, E, ^(-)) to impart electrical properties. notation) was constructed to have a modified peptide backbone.

Each combination of the bioactive nucleic acid and the carrier peptide nucleic acid was hybridized in the presence of DMSO, and as a result, a complex composed of the bioactive nucleic acid and the carrier peptide nucleic acid was prepared.

Example 2: Analysis of the therapeutic effect of Alzheimer's disease using nucleic acid complexes

According to Example 1, the treatment effect of Alzheimer's disease was analyzed using a nucleic acid complex comprising a carrier peptide nucleic acid and a bioactive peptide nucleic acid having BACE1 as a target gene prepared with the structure shown in Table 2 below.

Example 2-1: Cell culture

A mouse-derived neuroblastoma cell line (Neuro-2a) obtained from ATCC (CCL-131, American Type Culture Collection, USA) and a human-derived neuroblastoma cell line (SH-2a) obtained from KCLB (#22266, Korean Cell Line Bank, Korea) SY5Y) was added to DMEM culture medium (Dulbecco Modified Eagle Medium, Wellgene, Korea) with 10% (v/v) fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin, and 37°C, 5% (v/v). /v) were cultured under conditions of CO ₂ .

Example 2-2: Analysis of gene expression through Western blot assay

Two cell lines (Neuro-2a, SH-SY5Y) were seeded at 1x10 ⁵ in a 6-well plate and cultured for 24 hours, and the experiment was conducted under the conditions of Example 2-1 to obtain bioactive peptide nucleic acid and carrier peptide nucleic acid. The complex containing was treated and incubated for 24, 48, 72, 96, and 120 hours, respectively, and 30 μL of RIPA buffer was added to each well to obtain protein lysate. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane. Then, the primary antibody, BACE1 (Abcam, USA) was used. was treated at 1:1000 and left at 4°C for one day. It was washed with 1X TBS-T, treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000, and left at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated, and the expression inhibition efficiency of the target gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 1, in the mouse-derived cell line, the inhibition efficiency of the target gene, BACE1, was the highest in the group treated with the nucleic acid complex combination of SEQ ID NOs: 2 and 7 (combination 5), and SEQ ID NO: 2 and In the combination of 5 nucleic acid complexes (combination 3), the inhibition efficiency of the target gene was also confirmed from day 3 onwards (Fig. 1a). In the SH-SY5Y cell line, a human-derived cell line, the group treated with the nucleic acid complex combination of SEQ ID NOs: 2 and 5 (combination 3) consistently confirmed the inhibition efficiency of the target gene until the 5th day, and the nucleic acid complex combination of SEQ ID NOs: 2 and 7 ( Combination 5) also confirmed that the target gene was suppressed by day 3 (Fig. 1b).

Example 2-3: Enzyme activity assay analysis of target genes

Two types of neuroblastoma cell lines (Neuro-2a, SH-SY5Y) were seeded in 1x10 ⁵ in a 6-well plate, cultured for 24 hours, and experiments were conducted under the conditions of Example 2-1 to obtain bioactive peptide nucleic acids and carriers. Complexes containing peptide nucleic acids were treated and incubated for 24, 48, 72, 96, and 120 hours, respectively, and protein lysate was obtained by adding 100 μL of extraction buffer to each well. The enzyme activity was analyzed using the BACE1 enzyme activity assay kit (Abcam, USA), and the reaction was induced for 30 minutes in a 37°C incubator after adding the substrate and reaction solvent to the protein lysate. Thereafter, fluorescence values were analyzed under Ex/Em=335/495nm conditions using a HIDEX microplate leader (Finland).

As a result, the enzymatic activity produced by the target gene by day 5 in the group treated with the nucleic acid complex combination of SEQ ID NOs: 2 and 5 (combination 3) and the nucleic acid complex combination of SEQ ID NOs: 2 and 7 (combination 5) in both cell lines It was confirmed that it was reduced by 30-40% or more compared to the control group (FIG. 2). In the mouse-derived cell line (Neuro-2a), the activity of the nucleic acid complex of Combination 3 decreased from day 2 (Fig. 2a), and in the human-derived cell line (SH-SY5Y), the nucleic acid complex of Combination 3 and Combination 5 showed a pattern of decreasing activity. From the first day, it was confirmed that the enzyme activity decreased compared to the control group (Fig. 2b).

Example 3: Analysis of inhibition efficiency of Alzheimer's disease lesion gene using selected nucleic acid complexes

BACE1, used as a target gene of the nucleic acid complex, is known to function to cleave and release Amyloid precursor protein (APP), which is involved in the formation of beta-amyloid plaques, one of the causes of Alzheimer's disease, into the extracellular matrix (M. Sathya, et al., Clinica Chimica Acta 414 (2012) 171-178). Therefore, it has been attracting attention as a target having an expected effect that the expression of Alzheimer's disease onset genes will be reduced when BACE1 is inhibited.

In this example, after selecting nucleic acid combinations whose effects were verified in Example 2, their efficacy on the Alzheimer's disease lesion gene (APP) generated by immunofluorescence staining and the target gene in the same cell line was analyzed.

Nucleic acid complex combinations used are shown in Table 3 below.

Example 3-1: Cell culture

Mouse-derived neuroblastoma cells (Neuro-2a) obtained from ATCC (American Type Culture Collection, USA) and human-derived neuroblastoma cells (SH-SY5Y) obtained from KCLB (Korean Cell Line Bank, Korea) were mixed in DMEM culture medium ( 10% (v/v) fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin were added to Dulbecco Modified Eagle Medium, Wellgene, Korea), and conditions of 37°C and 5% (v/v) CO ₂ cultured under

Example 3-2: Analysis of gene expression using immunofluorescence staining

Cells of the two species were seeded in 3x10 ³ cells in an 8-well plate, cultured for 24 hours, and then the experiment was performed under the conditions of Example 3-1 to treat the complex containing the selected bioactive peptide nucleic acid and the carrier peptide nucleic acid, Incubated for 24, 72, and 120 hours, respectively, and cells were fixed with 4% paraformaldehyde (Sigma, USA). The fixed cells were permeabilized with 0.1% Triton X-100 (Sigma, USA) dissolved in PBS for 10 minutes, blocked with 1% bovine serum albumin (BSA, Sigma, USA) for 1 hour, and then the first The antibody, BACE1 (abcam, USA) was treated at 1:100 and left at 4°C for one day. Washed with 1X PBS, treated with secondary antibody Alexa 488-goat Anti-Rabbit (Abcam Technology, USA) at a ratio of 1:200, and left at room temperature for 1 hour. After diluting the DAPI solution (Sigma, USA) to 1x and leaving it for 10 minutes, the expression inhibition efficiency of the target gene was analyzed using a fluorescence microscope (Leica, Germany).

First, in the mouse-derived cell line (Neuro-2a), as shown in FIG. 3a, a combination of SEQ ID NOs: 2 and 5 (combination 3; PNA 3) and a nucleic acid complex of a combination of SEQ ID NOs: 2 and 7 (combination 5; PNA 5) It was confirmed that the fluorescence expression of the target gene decreased over time compared to the control PNA (combination of SEQ ID NOs: 1 and 3; combination 1; PNA 1) (FIG. 3a). As a result of quantifying the fluorescence intensity using the ImageJ program, it was confirmed that the fluorescence levels of the nucleic acid complexes of Combinations 3 and 5, compared to the control PNA sequence, decreased by about 40% on Days 1, 3, and 5 (FIG. 3B).

The same results were confirmed in the human-derived cell line (SH-SY5Y), and the combination of SEQ ID NOs: 2 and 5 (combination 3; PNA 3) and the nucleic acid complex of SEQ ID NOs: 2 and 7 (combination 5; PNA 5) were treated. In one group, it was confirmed that the fluorescence expression of the target gene was decreased compared to the nucleic acid complex of the combination of SEQ ID NOs: 1 and 3 (combination 1; PNA 1) as a control group (FIG. 3c). Similarly, as a result of quantifying the fluorescence intensity using the ImageJ program, it was confirmed that the fluorescence expression levels of the nucleic acid complexes of Combinations 3 and 5 decreased by about 40% on the 1st, 3rd and 5th days compared to the negative control and control PNA (combination 1). (Fig. 3d).

Example 3-3: Analysis of Alzheimer's disease onset gene expression through Western blot assay

Two cell lines (Neuro-2a, SH-SY5Y) were seeded in 1x10 ⁵ cells in a 6-well plate and cultured for 24 hours, and the experiment was conducted under the conditions of Example 3-1 to obtain bioactive peptide nucleic acid and carrier peptide nucleic acid. The complex containing . The amount of protein in the obtained supernatant was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane, and then the primary antibody, sAPP-beta (BioLegend, USA) at a ratio of 1:1000 and left at 4°C for one day. It was washed with 1X TBS-T, treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000, and left at room temperature for 1 hour. Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) was treated and the expression inhibition efficiency of the Alzheimer's disease gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, in the mouse-derived neuroblastoma cell line (Neuro-2a) and human-derived neuroblastoma cell line (SH-SY5Y), the combination of SEQ ID NOs: 2 and 7 compared to the NC or control nucleic acid complex (combination of SEQ ID NOs: 1 and 3, PNA 1) In the group treated with the nucleic acid complex of (combination 5; PNA 5), it was confirmed that the gene expression was suppressed on the 1st and 3rd day, but the expression pattern was recovered on the 2nd day, whereas the combination of SEQ ID NOs: 2 and 5 In the group treated with the nucleic acid complex of (combination 3; PNA 3), it was confirmed that the expression of the secreted APP protein (sAPP), which is cleaved and released by the target gene BACE1, decreased steadily from day 1 to day 3 (FIG. 4). ).

The nucleic acid complex of the present invention, in which a bioactive nucleic acid targeting BACE1 and a carrier peptide nucleic acid are complementaryly bound, has the ability to penetrate the blood-brain barrier and can efficiently inhibit the expression of BACE1, thereby preventing degenerative brain diseases, particularly Alzheimer's disease. Useful for prevention or treatment.

As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (12)

1. Bioactive Nucleic Acid targeting the BACE1 gene; and a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) complementary nucleic acid complex.
2. The nucleic acid complex according to claim 1, wherein the bioactive nucleic acid targeting the BACE1 gene comprises the nucleotide sequence represented by SEQ ID NO: 2.
3. The nucleic acid complex according to claim 1, wherein the carrier peptide nucleic acid comprises a nucleotide sequence represented by any one sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 7.
4. The method of claim 1, wherein the bioactive nucleic acid or carrier peptide nucleic acid targeting the BACE1 gene is further coupled with a substance that helps endosome escape to the 5'-end or 3'-end of each nucleic acid. nucleic acid complexes.
5. The method of claim 4, wherein the material that helps the endosomes escape is a peptide, lipid nanoparticles, conjugate nanomaterials (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanomaterials (organic nanoparticles, cations) A nucleic acid complex, characterized in that at least one selected from the group consisting of cationic lipid-based nanoparticles, cationic polymers and pH sensitive polymers.
6. The nucleic acid complex according to claim 5, wherein the peptide is GLFDIIKKIAESF (SEQ ID NO: 8) or Histidine (10).
7. The nucleic acid complex according to claim 1, wherein the bioactive nucleic acid targeting the BACE1 gene has a negative charge or neutral charge as a whole.
8. The nucleic acid complex according to claim 1, wherein the carrier peptide nucleic acid has an overall positive charge.
9. The nucleic acid complex according to claim 1, wherein the nucleic acid complex has a positive charge as a whole.
10. The nucleic acid complex according to claim 1, wherein the nucleic acid complex has blood-brain barrier penetrating ability.
11. A pharmaceutical composition for preventing or treating degenerative brain disease, containing the nucleic acid complex according to any one of claims 1 to 10 as an active ingredient.
12. The pharmaceutical composition according to claim 11, wherein the degenerative brain disease is Alzheimer's disease, Parkinson's disease, Niemann-Pick disease, Creutzfeldt-Jakob disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis or dementia.

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