WO2023132673A1 - Composition comprising nucleic acid complex for prevention or treatment of degenerative brain disease - Google Patents

Abstract

The present invention relates to a nucleic acid complex having an ability to penetrate the blood-brain barrier and a pharmaceutical composition comprising same as an active ingredient for prevention or treatment of degenerative brain diseases and, more specifically, to a nucleic acid complex in which a NLRP3 gene-targeting bioactive nucleic acid complementarily binds to a carrier peptide nucleic acid, and a pharmaceutical composition comprising same as an active ingredient for prevention or treatment of degenerative brain diseases. The nucleic acid complex in which the NLRP3-targeting bioactive nucleic acid complementarily binds to the carrier peptide nucleic acid has an ability to penetrate the blood-brain barrier and can efficiently inhibit the expression of NLRP3 and as such, is useful for preventing or treating degenerative brain diseases, especially Parkinson's disease.

Description

Composition for preventing or treating degenerative brain diseases containing nucleic acid complexes

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 NLRP3 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.

Parkinson's disease is a degenerative brain disease caused by a decrease in dopamine, a neurotransmitter, in the striatum and substantia nigra. . Among degenerative brain diseases, the prevalence is second only to Alzheimer's disease, and it is reported that about 1% of the population aged 60 or older suffer from this disease (Ali Samii, et al., Lancet. 2004 May 29; 363(9423):1783-93).

Parkinson's disease is characterized by the loss of dopaminergic neurons due to the accumulation and diffusion of Lewy bodies composed of misfolded α-synuclein. . Lewy bodies, which are the main cause of Parkinson's disease, are neurotoxic substances that propagate to peripheral areas of the brain and cause dopaminergic degeneration (Werner Poewe, et al., Nat Rev Dis Primers. 2017 Mar 23;3:17013). It is known that chronic microglia neuroinflammation occurs in the substantia nigra region of patients with Parkinson's disease in the early stages of onset, and this feature is also known to be prominent in the brains of patients with Parkinson's disease confirmed postmortem (Alexander Gerhard, et al., Neurobiol Dis. 2006 Feb;21(2):404-12). During dopamine degeneration, this chronic neuroinflammation continues to occur and contributes to exacerbation of the disease, but a clear mechanism linking the decrease in alpha-synuclein and dopamine has not yet been identified.

NLRP3 (NLR family pyrin domain containing 3; NACHT, leucine-rich repeat, and pyrin domain (PYD)-containing protein 3 (NALP3)) is a protein-coding gene that binds to nucleotide-binding and oligomerization domain-like receptors (nucleotide- It belongs to the family of binding oligomerization domain-like receptors (NLRs). The inflammasome functions as a sensor that responds to intracellular stress and environment. Among them, the NLRP3 inflammasome is a NLRP3 sensor and signal adapter ASC (apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment) domain)) and pro-caspase-1. When the NLRP3 inflammasome is activated by cellular stress, caspase-1 is activated, triggering the release of interleukin-1β (IL-1β) and IL-18, which are inflammatory cytokines, to initiate an inflammatory response. (Shuo Wang, et al., Int Immunopharmacol. 2019 Feb; 67:458-464).

Recently, it was confirmed that the accumulation of misfolded alpha-synuclein protein and activated microglia could be responsible for the activation of the NLRP3 inflammasome in the brains of patients with Parkinson's disease (Gaia Codolo, et al., PLoS One. 2013 ;8(1):e55375). This can be said to be the result of confirming that there is a direct correlation with the decrease in dopamine due to the activation of microglia, together with the result that insoluble alpha-synuclein, which is mis-aggregated, activates the NLRP3 inflammasome. Through these recent studies, it was confirmed that alpha-synuclein aggregation activates the NLRP3 inflammasome and induces dopamine degeneration due to an increase in the inflammatory response of microglia (Richard Gordon, et al., Sci Transl Med. 2018 Oct. 31;10(465):eaah4066).

A significant number of drugs developed for the treatment of brain diseases have a problem in that they do not pass through the blood-brain barrier well. The penetration mechanism of the blood-brain barrier has not yet been elucidated, and even if a disorder occurs in the central nervous system, it is a reality that drugs cannot reach the target area of the central nervous system, and effective treatment methods have not yet been developed.

On the other hand, unlike traditional drugs, nucleic acid drugs suppress the expression of target-specific messenger RNA (mRNA), so it is possible to address research areas that could not be treated with existing protein-targeting drugs (Ryszard Kole, et al. al., Nat Rev Drug Discov. 2012 Jan 20;11(2):125-40). Due to its performance and advantages as a drug, various clinical trials using nucleic acids are in progress, and despite the increasing use of nucleic acid-based therapeutics, the use of carriers for intracellular introduction or blood-brain barrier penetration is extremely limited.

In this regard, the present inventors have found that a nucleic acid complex in which a bioactive nucleic acid and a carrier peptide nucleic acid modified to have a positive charge as a whole are complementarily coupled have surprisingly improved cell permeability, and using this It was confirmed that the expression of the target gene can be controlled very efficiently, and a patent has been registered for a new structure with low cytotoxicity and improved cell permeability of bioactive nucleic acids and the ability to regulate gene expression (Korean Registered Patent No. 10). -1963885). In addition, the present inventors have continuously conducted research on the function of the construct to develop a new construct having the ability to penetrate the blood-brain barrier with excellent efficiency (Korean Patent Application No. 10-2019-0128465).

Accordingly, the present inventors have made diligent efforts to use nucleic acid complexes with excellent blood-brain barrier penetration ability to apply them to the treatment of degenerative brain diseases. It was found that it exhibits excellent effects in preventing or treating 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 NLRP3 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 nucleic acid complexes to inhibit the expression of target genes and subgenes in an LPS-induced Parkinson's disease-like cell model.

2 is a diagram confirming the ability of a nucleic acid complex to suppress intracellular expression of a target gene in a Parkinson's disease-like cell model induced by LPS.

Figure 3 is a diagram confirming the efficacy of the nucleic acid complex in a primary microglia model isolated from tissue.

Figure 4 is a view confirming the ability to improve animal behavior according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

5 is a diagram confirming the ability to inhibit target gene expression and subgene expression by brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

6a is a view showing changes in expression of Tyrosine Hydroxylase (TH) in the striatum of the brain according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

6B is a diagram showing changes in expression of Tyrosine Hydroxylase (TH) in the substantia nigra of the brain according to administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

Figure 6c is a view confirming the expression suppression ability of α-Synuclein in the striatum (striatum) of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

Figure 6d is a view confirming the expression suppression ability of α-Synuclein in the substantia nigra of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

Figure 6e is a view confirming the expression inhibition ability of ionized calcium-binding adapter molecule 1 (IBA-1) in the striatum (striatum) of the brain region according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal model.

6f is a diagram confirming the expression inhibition ability of ionized calcium-binding adapter molecule 1 (IBA-1) in the substantia nigra of the brain according to the administration of the nucleic acid complex in the MPTP/P Parkinson's disease animal 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.

The causes of Parkinson's disease are very diverse. About 5% of all patients are caused by genetic factors, and external environmental factors such as inflammation and oxidative stress play an important role. Parkinson's disease is caused by abnormal protein aggregation in dopaminergic neurons, which is the aggregation of alpha-synuclein protein. Aggregation of these proteins is further promoted by oxidative stress. Until now, the treatment of Parkinson's disease has been made by supplementing the dopamine neurotransmitter, but this is not a fundamental treatment for Parkinson's disease. For the fundamental treatment of Parkinson's disease, research on biological agents such as small-molecule therapeutic drugs that directly target disease-modifying genes, gene therapy, monoclonal antibodies, immunotherapy targeting related symptoms, and stem cells and induced pluripotent stem cells are actively being conducted. It's getting done.

On the other hand, the number of patients with Parkinson's disease in Korea was 61,565 in 2010 and increased to 85,888 in 2014 with an average annual growth rate of 8.7%. In the case of patients in 2014, the proportion of patients aged 60 years or older was 95.7%, and the prevalence showed a correlation with the patient's age. Parkinson's disease patient and treatment cost increase, young doctors, 2015). Compared to Alzheimer's disease, the onset of Parkinson's disease is very early, around 60 years of age, and because of this, the progression of the disease lasts for more than 20 years, so a treatment that slows the disease progression through early detection is actively required.

In the present invention, it was confirmed that a nucleic acid complex in which a bioactive nucleic acid targeting the NLRP3 gene and a carrier peptide nucleic acid are complementaryly bound can be used for the prevention and treatment of Parkinson's disease.

In one embodiment of the present invention, based on Korean Patent Registration No. 10-1963885 of the present inventors, treatment of a nucleic acid complex in which a bioactive nucleic acid targeting the NLRP3 gene and a carrier peptide nucleic acid are complementaryly coupled to a microglial cell line In one case, it was confirmed that the expression of NLRP3 and sublevel genes were effectively suppressed, and it was confirmed that behavioral disorders, motor function disorders, and sensorimotor disorders were improved in a Parkinson's disease animal model to which the nucleic acid complex was administered.

Therefore, the present invention, in one aspect, a bioactive nucleic acid targeting the NLRP3 gene (Bioactive Nucleic Acid); 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 NLRP3 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 NLRP3 (NLR family pyrin domain containing 3) gene, a target gene related to Parkinson's disease, and more preferably the nucleotide sequence represented by the sequence of SEQ ID NO: 2 It may include, 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 the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5, but is not limited thereto.

In the present invention, the "nucleic acid complex" can penetrate the bioactive substance into the body and ultimately into the cell through extracellular treatment, and specifically has the ability to deliver the bioactive nucleic acid targeting the NLRP3 gene into the cell. .

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 the sequence of SEQ ID NO: 4 or 5, but is not limited thereto.

In addition, the binding ability (melting temperature, Tm) of the bioactive nucleic acid targeting the NLRP3 gene and the carrier peptide nucleic acid is lower than that of the bioactive nucleic acid and the target NLRP3 gene. 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, as a material that helps the endosome escape, a peptide (GLFDIIKKIAESF, SEQ ID NO: 6) may be linked to the bioactive nucleic acid via a linker, and Histidine (10) to the carrier peptide nucleic acid via 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 each comprise 2 to 50, preferably 5 to 30, more preferably 10 to 25, most preferably 15 to 17 nucleic acid monomers. that can be characterized.

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 the 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 refers to a form in which 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.

In one embodiment of the present invention, as a result of administering the nucleic acid complex according to the present invention to the tail vein of a mouse, the ability to inhibit the expression of target genes and subgenes in brain tissue was confirmed, and the effect of improving motor function loss in Parkinson's disease was confirmed As confirmed, the nucleic acid complex suggests that it has BBB penetrating ability.

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 NLRP3.

Therefore, the present invention from another point of view, a bioactive nucleic acid targeting the NLRP3 gene (Bioactive Nucleic Acid); 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 includes Parkinson's disease, Alzheimer's disease, Niemann-Pick disease, Creutzfeldt-Jakob disease, Huntington's disease, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), multiple sclerosis or dementia ( dementia), 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 NLRP3 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 NLRP3 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 NLRP3 gene for the preparation of drugs for preventing or treating degenerative brain diseases; 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, NLRP3 was used as a target gene to test the effect of the nucleic acid complex on Parkinson's disease, and to confirm the therapeutic effect of Parkinson's disease, antisense peptide nucleic acid (Bioactive Peptide Nucleic Acid) for NLRP3 was used. 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 Parkinson's disease has the sequence represented by SEQ ID NO: 2 includes The carrier peptide nucleic acids used in the examples of the present invention are composed of sequences represented by SEQ ID NOs: 3 to 5 (Table 1). All peptide nucleic acids used in the present invention were synthesized by HPLC purification method from PANAGENE (Korea).

Modification of the monomers converts the backbone of the peptide nucleic acid to lysine (indicated by Lysine, Lys, K, ⁽⁺⁾ ) for electrical properties and glutamic acid (Glutamic acid, Glu, E, ^(-)) for electrical negative to impart electrical properties. ) 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 treatment effects of Parkinson's disease using nucleic acid complexes

According to Example 1, the therapeutic effect of Parkinson's disease was analyzed using a nucleic acid complex comprising a carrier peptide nucleic acid and a bioactive peptide nucleic acid targeting NLRP3, which was prepared according to the structure shown in Table 2 below.

Example 2-1: Cell culture

Human-derived microglia (HMC-3) obtained from ATCC (American Type Culture Collection, USA) were mixed with 10% (v/v) fetal bovine serum, penicillin 100 units/ml, and streptomycin in MEM culture medium (Wellgene, Korea). 100 μg/ml was added and cultured under conditions of 37°C and 5% (v/v) CO ₂ .

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

Human-derived microglial cell lines were seeded in 1x10 ⁵ cells in a 6-well plate and cultured for 24 hours, and treated with 1 μg/ml of Lipopolysaccharide (LPS, Sigma, USA) to induce an immune response. After 4 hours of LPS treatment, the experiment was conducted under the conditions of Example 2-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid was treated and cultured for 24, 48, 72, 96, and 120 hours, respectively, and RIPA buffer Protein lysate was obtained by adding 30 μL to each well. 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. Pro Cas-1 (abcam, USA) and Pro IL-1β (abcam, USA) were treated 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 target gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 1, it was confirmed that the expression of target genes and sub-pathway genes was most suppressed in the group treated with the nucleic acid complex of SEQ ID NOs: 2 and 5 (combination 3, PNA 3).

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

Human-derived microglial cell lines were seeded in 3x10 ³ cells in an 8-well plate and cultured for 24 hours, and treated with 1 μg/ml of Lipopolysaccharide (LPS, Sigma, USA) to induce an immune response. After 4 hours of LPS treatment, the experiment was conducted under the conditions of Example 2-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid was treated, cultured for 24 and 72 hours, respectively, and cells were treated with 4% paraformaldehyde (Sigma, USA). was fixed. 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 antibody, NLRP3 (ABclonal, USA) was treated at a ratio of 1:100 and left at 4° C. for one day. After washing with 1X PBS, the secondary antibody, Alexa 488-goat Anti-Mouse (Abcam Technology, USA) was treated at a ratio of 1:200 and allowed to stand 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).

As a result, as shown in Figure 2a, it was confirmed that the fluorescence expression of the target gene, NLRP3, increased in the immune induction group treated with LPS. It was confirmed that the fluorescence expression of one group continuously decreased the most until 72 hours (Fig. 2a). As a result of quantifying the fluorescence intensity using the ImageJ program, the fluorescence intensity quantification of the nucleic acid complex (PNA 3) of the combination of SEQ ID NOs: 2 and 5 compared to the immune induction group and the control nucleic acid complex treatment group (combination 1, PNA 1) It was confirmed that it decreased in a time-dependent manner (Fig. 2b).

Example 3: Analysis of Parkinson's disease treatment effect using nucleic acid complex in primary cultured microglial cells isolated from brain tissue

After selecting the nucleic acid combination whose effect was verified through Example 2, the treatment effect of Parkinson's disease was analyzed using primary microglia isolated from neonatal rats having the most similar characteristics to the body in culture conditions. .

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

Example 3-1: Cell culture

Human-derived neuroblastoma (SH-SY5Y) obtained from KCLB (Korean Cell Line Bank, Korea) was mixed with 10% (v/v) fetal bovine serum, penicillin 100 units/ml, and streptomycin 100 in DMEM culture medium (Wellgene, Korea). [mu]g/ml was added and cultured under the conditions of 37°C and 5% (v/v) CO ₂ .

Example 3-2: Isolation and culture of primary microglia from mouse brain tissue

In order to isolate and culture primary microglia from brain tissues of mice, 1-day-old neonatal SD rats (Nara Biotech, Korea) were purchased and brain tissues were isolated. The cortex from which the meninges and other tissue parts were removed was transferred to a Petri dish containing iced 1x HBSS (gibco, USA), and then the tissue was dissociated using a pipette and centrifuged at 1200 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 10% (v/v) fetal bovine serum, penicillin 100 units/ml, streptomycin 100 μg/ml, GlutaMAX ( After releasing the cells by adding mixed glial cell culture medium containing 0.1% of Gibco, USA), the cells were seeded in a 75T flask coated with PLL (Poly-L-lysine, Sigma, USA) the day before. After 14 days of cell culture, the mixed glial cell culture medium was replaced with the microglia culture medium in which 5 μg/ml of insulin (Sigma, USA) was added, and then the flask was stirred with an orbital shaker for 2 hours at 160 rpm and 37°C. Then, the supernatant containing only floating cells was collected and centrifuged at 1200 rpm for 8 minutes. After centrifugation, the cells were seeded in 1x10 ⁵ in a 6-well plate, and cultured under conditions of 37°C and 5% (v/v) CO ₂ . In order to create a Parkinson's disease-like inflammatory cell model, 100 ng/mL of LPS was treated for 4 hours, followed by nucleic acid complex treatment and culture.

Example 3-3: Analysis of gene expression using Western blot assay

Primary microglia cultured under the culture conditions of Example 3-2 were treated with 100 ng/ml of Lipopolysaccharide (LPS, sigma, USA) to induce an immune response. After 4 hours of LPS treatment, complexes containing bioactive peptide nucleic acids and carrier peptide nucleic acids were treated, and after incubation for 24, 48, and 72 hours, respectively, 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. Pro Cas-1 (abcam, USA) and Pro IL-1β (abcam, USA) were treated 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 target gene was analyzed using Image600 (Amersham, Germany) equipment.

As a result, as shown in FIG. 3a, the group treated with the nucleic acid complex of SEQ ID NOs: 2 and 5 in the Parkinson's disease-like cell model (combination 2, PNA 2) inhibited the expression of target genes and sub-pathway genes the most. It was confirmed (Fig. 3a).

Example 3-4: Analysis of cell viability of neuroblastoma cell lines using MTT assay

Primary microglia cultured under the culture conditions of Example 3-2 were treated with a complex containing a bioactive peptide nucleic acid and a carrier peptide nucleic acid, and 4 hours later, LPS was treated with 100 ng/ml and cultured for 24, 48, and 72 hours, respectively. did After filtering the culture medium obtained at the end of each culture using a syringe filter (Millipore, USA), it was treated with human-derived neuroblastoma. Human-derived neuroblastoma was seeded in 2x10 ⁴ in a 96-well plate, and after 24 hours, the medium was replaced with the medium obtained from primary microglia, and cultured for 24, 48, and 72 hours, respectively. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, sigma, USA) solution was prepared at a concentration of 5 mg/mL in 1X PBS, treated at 20 μL per well and incubated for 4 hours, then the OD (optical density) was measured and analyzed with a spectrophotometer.

In this example, changes in neuroblastoma survival rate by media obtained from primary microglial cell lines treated with LPS, a Parkinson's disease-like inflammatory cell model, were analyzed, and the nucleic acid complex combinations used are the same as those in Table 3 above.

As a result, as shown in FIG. 3B, the cell viability decreased compared to the negative control group (NC) in the group treated with the culture medium of the nucleic acid complex of the immune induction group (LPS) and the control sequence (combination 1, PNA 1). It was confirmed that the cell viability of neuroblastoma was increased up to 72 hours by the culture medium of the primary microglial cell line treated with the nucleic acid complex of 2 and 5 (combination 2, PNA 2) ( FIG. 3B ).

Example 4: Analysis of Parkinson's disease treatment effect using nucleic acid complex in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid-induced mouse model

In this example, in order to evaluate the Parkinson's disease therapeutic effect of the combination of nucleic acid complexes verified based on the results of Example 3 in an animal model, MPTP (1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine) and probenecid, which increase uptake rate, were administered to mice, and in an animal model of Parkinson's disease, it was verified that the decline in motor function due to neuronal cell death was recovered by the nucleic acid complex.

Example 4-1: Production of MPTP/probenecid-induced Parkinson's disease animal model

To verify the efficacy of MPTP/probenecid in an animal model of Parkinson's disease, 7-week-old male C57BL/6 mice (Nara Biotech, Korea) were used, and after acclimatization for 7 days, they were used in experiments. 25 mg/kg of MPTP and 250 mg/kg of probenecid were used to construct a Parkinson's disease-like model, and intraperitoneal injections were performed a total of 10 times at 3.5 day intervals for 5 weeks to create a damage model due to long-term administration. After 4 administrations of MPTP and probenecid, 10 animals per experimental group were randomly selected and used, and the nucleic acid complex was administered to the tail vein of mice twice a week for 4 weeks. At this time, in the positive control group, 250 mg/kg of levodopa (Levodopa, sigma, USA), which is used as a standard treatment for patients with Parkinson's disease, was mixed with 25 mg/kg carbidopa (carbidopa, sigma, USA) and administered orally every day for 4 weeks from the start of nucleic acid complex administration. Dosing proceeded.

Example 4-2: Analysis of motor function change using rotarod test analysis

In this example, a rotarod test was performed to evaluate the effect of improving motor deficits and balance maintenance, which are characteristics of lesions in animal models of Parkinson's disease, by nucleic acid complexes.

For the verification of motility, a rotarod apparatus (Jeongdo B&P, Korea) with a rotatable cylindrical rod composed of 5 compartments with a diameter of 7 cm and a height of 60 cm at 15 cm intervals was used. Before the test, all the experimental animals were sufficiently trained for 3 days, and this experiment was conducted after the administration of the nucleic acid complex was completed. The speed of the rotarod used in the test was set to increase constantly from 0 to 40 rpm, and the latency time taken until the animal was placed on the rotating rod and dropped was measured. The maximum measurement time was limited to 300 seconds, and the therapeutic effect of the nucleic acid complex was analyzed using the average value after three repeated measurements.

As a result, as shown in FIG. 4a, the time spent on the rod in the group treated with the nucleic acid complex of No. 2 (SEQ ID NOs: 2 and 5, PNA 2) compared to the Parkinson's disease induction model that fell without being able to hold on to the rotating rod for a long time. As this increased, it was confirmed that the behavioral disorder was improved (FIG. 4a).

Example 4-3: Analysis of motor function change using pole test analysis

A pole test was performed to evaluate the recovery of akinesia and spastic response using the Parkinson's disease animal disease model prepared under the conditions of Example 4-1.

The pole test was performed by placing the mouse facing the sky using a stick with a width of 0.8 cm and a height of 55 cm, and then rotating 180° to measure the total time required to reach the floor. The same number of training sessions were conducted for all experimental animals before the test, and this experiment was conducted after the administration of the nucleic acid complex was completed. The experiment was repeated three times, and the average value was used for analysis of the results.

As a result, as shown in FIG. 4b, the MPTP/probenecid animal group that induced Parkinson's disease had an increased total time to reach the ground compared to normal mice (NC). On the other hand, in the group treated with the nucleic acid complex of SEQ ID NOs: 2 and 5 (PNA 2), it was confirmed that the time from the directional rotation at the top to the bottom was reduced and motor dysfunction was improved (FIG. 4b).

Example 4-4: Analysis of motor function changes using cylinder test analysis

In order to confirm the effect of sensorimotor function treatment in an animal model induced with Parkinson's disease by the method of Example 4-1, a cylinder test was performed.

When a mouse is placed in a new environment, it is common to lift two forepaws and touch the wall to explore the surroundings, so the number of times the forepaws were used was measured after placing the mouse in a transparent cylinder. The experiment was conducted the day after the end of the nucleic acid complex administration, and the result of measuring the number of times of pressing the cylinder wall with the forepaw for 3 minutes in the cylinder without separate training was used.

As a result, as shown in FIG. 4c, the group of animals exposed to MPTP, a neurotoxin, and administered with only MPTP/probenecid decreased the number of times of using the paws compared to normal mice. The group treated with the complex (PNA 2) increased the number of times of using the forelimbs, confirming the therapeutic efficacy of sensorimotor impairment in an animal model of Parkinson's disease (FIG. 4c).

Example 5: Gene expression analysis in Parkinson's disease induced animal model using MPTP/probenecid

In this example, in the Parkinson's disease animal model induced by the method of Example 4-1, changes in the expression of Parkinson's disease-related genes were confirmed in the striatum and substantia nigra regions of brain tissue where dopamine production and metabolism occur. .

Example 5-1: Analysis of gene expression in Parkinson's disease animal model tissue using Western blot assay

The striatum and substantia nigra were separated from the brain tissue extracted on the end day after the experiment was conducted by the method of Example 4-1, and protein lysate was obtained by adding RIPA buffer. 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. Pro IL-1β (abcam, USA), Tyrosine Hydroxylase (TH, abcam, USA), and alpha-synuclein (α-synuclein, abcam, USA) were treated 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 target gene was analyzed using Image600 (Amersham, Germany) equipment.

In this example, NLRP3 and lower-order gene expression changes and the expression levels of dopaminergic neurons and alpha-synuclein in Parkinson's disease-induced animal models were analyzed, and the nucleic acid complex combinations used are the same as those in Table 3 above.

As a result, as shown in FIG. 5a, when expression analysis was performed using the striatum region of the brain tissue extracted on the end day of the experiment conducted by the method of Example 4-1, the nucleic acid complex of No. 2 combination (sequence It was confirmed that the expression of NLRP3, a target gene, and IL-1β, a sub-pathway gene, in the group treated with the combination of numbers 2 and 5, PNA 2) decreased compared to the diseased animal group (MPTP/p). In addition, dopaminergic neurons (TH, Tyrosine hydroxylase) decreased the most in the MPTP/probenecid-induced model, and increased in the group treated with the nucleic acid complex of No. 2 (PNA 2). The recovery phenomenon was confirmed. In addition, it was confirmed that the expression of alpha-synuclein, the etiological factor of Parkinson's disease, which was increased in the MPTP/probenecid induction group, was suppressed in the group treated with the nucleic acid complex of No. 2 combination (FIG. 5a).

The expression in the substantia nigra region is shown in FIG. 5B, and the expression of the target gene NLRP3 and IL-1β, a sub-pathway gene, increased in the disease induction group (MPTP/p) in the second combination of nucleic acid complexes (PNA). 2), and the decreased dopaminergic neurons in the disease model were recovered with increased expression in the group treated with the nucleic acid complex of No. 2 (combination of SEQ ID NOs: 2 and 5, PNA 2) confirmed that In addition, the expression of alpha-synuclein decreased after treatment with the nucleic acid complex in combination No. 2, confirming the therapeutic efficacy of the nucleic acid complex of SEQ ID NOs: 2 and 5 (FIG. 5b).

Example 5-2: Analysis of expression changes in tissues in Parkinson's disease animal models using immunostaining

After the experiment was conducted under the conditions of Example 4-1, mouse brain tissue was separated on the end day, fixed in 4% formalin solution for one day, immersed in 30% sugar solution, dehydrated for 3 days, and then embedded in OCT (Leica, USA) solution (embedding) was done. The embedded tissue was stored and frozen at -20°C, and then the tissue was sectioned at 20 μm using a cryostat (Cryostat, Leica, USA) and then mounted on a slide glass. The mounted tissues were blocked for 2 hours using 1% BSA solution, and primary antibodies were used to detect Tyrosine Hydroxylase (abcam, USA), alpha-synuclein (α-synuclein, abcam, USA), and IBA1 (wako, Japan). After treatment, it was left at 4° C. for one day. Subsequently, the primary antibody solution was removed, washed with 1X TBS, and then treated with a secondary antibody solution, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000 at room temperature for 2 hours. After washing the secondary antibody with 1X TBS, expression was analyzed by DAB staining.

In this example, the expression levels of dopaminergic neurons, alpha-synuclein, and immune cells, which are onset factors, were analyzed in brain tissues of Parkinson's disease-induced animal models, and comparative analysis was performed in the striatum and substantia nigra, which are brain regions important for onset.

As a result of analyzing changes in dopaminergic neurons, it was confirmed that the expression of tyrosine hydroxylase representing dopaminergic neurons in the striatum was reduced in the MPTP/probenecid Parkinson's disease induction model, as shown in FIG. In the group administered with the nucleic acid complex (SEQ ID NO: 2 and 5 combination, PNA 2), it was confirmed that expression increased (FIG. 6a). In the substantia nigra region, as shown in FIG. 6b, it was confirmed that the level of expression decreased in the group administered with only MPTP/probenecid, and the expression increased in the group administered with nucleic acid complex No. 2 (PNA 2) (FIG. 6b). ).

When the expression of alpha-synuclein, a pathogenic factor, was confirmed, in the case of the striatum, it was confirmed that the brown reaction increased in the Parkinson's disease induction model (MPTP/p) and the expression increased. observed a significant reduction in the brown response of alpha-synuclein. When compared by quantification, it was found that the expression of alpha-synuclein was decreased in the group administered with the nucleic acid complex of No. 2 combination compared to the group treated with the positive control group (FIG. 6c). As a result of comparing the expression level of alpha-synuclein in the substantia nigra, as in the striatum, the brown reaction, which was increased in the MPTP/probenecid disease induction group, was remarkably reduced in the group administered with nucleic acid complex No. 2. In the graph showing this numerically, it was confirmed that the expression level decreased in the group treated with the combined nucleic acid complex No. 2 (FIG. 6d).

Finally, as a result of confirming the expression of a marker (IBA1) of microglia, which is a brain immune cell, as shown in FIG. In the group administered with the complex, the brown reaction was reduced, confirming the reduction of immune cells. It was also shown in the numerical graph that the IBA response was reduced in the group administered with the nucleic acid complex No. 2 (FIG. 6e). As a result of confirming the expression of IBA1 in the substantia nigra shown in FIG. 6f, it was confirmed that the brown reaction in the group treated with the combination nucleic acid complex No. 2 was the most reduced compared to the other disease-induced group (MPTP/p), as in the striatum. It was found to be the most decreased in the graph (FIG. 6f).

The nucleic acid complex of the present invention, in which a bioactive nucleic acid targeting NLRP3 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 NLRP3, thereby preventing degenerative brain diseases, particularly Parkinson'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 NLRP3 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 NLRP3 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 SEQ ID NO: 4 or SEQ ID NO: 5.
4. The method of claim 1, wherein the bioactive nucleic acid or carrier peptide nucleic acid targeting the NLRP3 gene is additionally bound to a material 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: 6) or Histidine (10).
7. The nucleic acid complex according to claim 1, wherein the bioactive nucleic acid targeting the NLRP3 gene has a negative 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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