Treatment of tauopathy

Abstract

A method for treating a subject with tauopathy by administering either full-length p38γ isoform 2 or its constitutively active mutant p38γ D179A, which promotes phosphorylation of tau at threonine in the sequence SSPGSPGTPGSRSR, which corresponds to amino acid position threonine 205 (T205) in human tau. The method also includes introducing a phosphomimetic of phosphorylated tau, wherein the phosphorylated tau is phosphorylated at threonine in the sequence SSPGSPGTPGSRSR. The treatment method results in improved cognitive performance and reduced tau aggregates and neurofibrillary tangles in a subject suffering from cognitive impairment associated with a tauopathy.

Description

Technical Field

[0001] The present invention relates to methods for treating tauopathy in a subject, methods for improving cognitive ability in a subject suffering from cognitive impairment associated with tauopathy, methods for reducing tau aggregates and neurofibrillary changes, and compositions and agents for such methods.

Background Art

[0002] Tau protein (also referred to herein as tau) is a highly soluble protein that is normally abundant in the central nervous system. Under healthy conditions, tau protein is associated with microtubules, particularly those of nerve cells, and stabilizes them.

[0003] Tauopathy is a class of progressive neurodegenerative disorders that are pathologically defined by the presence of abnormal aggregation of hyperphosphorylated tau in brain nerve cells. Abnormal aggregation occurs when tau protein is hyperphosphorylated, dissociates from microtubules, and forms insoluble aggregates. When tau aggregates accumulate in nerve cells, they form neurofibrillary changes (NFTs), ultimately leading to nerve cell death and cognitive decline.

[0004] Aggregates of hyperphosphorylated tau are involved in the etiology of neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia, and other tauopathies. The progression of NFT pathology throughout the brain correlates with the disease progression of degenerative diseases. However, to date, the mechanism by which these neurofibrillary changes cause disease is unknown.

[0005] Early diagnosis of tauopathy is usually impossible. Current clinical diagnosis depends on the medical history and progression of symptoms. As a result, the diagnosis of tauopathy is often made only after the onset of symptoms, usually when the disease has progressed to a stage where significant aggregation of tauopathy has occurred.

[0006] To date, there are limited treatment options for tauopathy after tau aggregation has occurred, such as in the advanced stage of Alzheimer's disease (i.e., with tau aggregates) or tauopathy associated solely with tau aggregation.

[0007] There is a need for methods to treat tauopathy in which tau aggregation and NFT formation have already occurred, and / or in which tau aggregation is the sole causative factor. [Overview of the project]

[0008] The inventors have found that promoting the phosphorylation of tau with threonine at the tau sequence SSPGSPGTPGSRSR (SEQ ID NO: 7), such as threonine (T205) corresponding to position 205 of full-length wild-type human tau, and / or introducing a phosphomimetic of tau protein phosphorylated with threonine at the tau sequence SSPGSPGTPGSRSR (SEQ ID NO: 7), such as threonine (T205) corresponding to position 205 of full-length wild-type human tau (SEQ ID NO: 1), into target neurons can reduce cognitive decline and other symptoms associated with tauopathy.

[0009] The first aspect is a method for treating or preventing tauopathy in a subject, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR (SEQ ID NO: 7); and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR (SEQ ID NO: 7). The present invention provides a method that includes the step of administering a drug.

[0010] The first alternative is a drug for use in the treatment or prevention of tauopathy in a subject, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a drug for treating or preventing tauopathy in a subject, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0011] The second aspect is a method for treating or preventing tauopathy in a subject, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. The present invention provides a method that includes the step of administering a drug.

[0012] A second alternative embodiment is a drug for use in the treatment or prevention of tauopathy in a subject, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Drugs; or The use of a drug in the manufacture of a drug for treating or preventing tauopathy in a subject, wherein the drug is (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Provides usage.

[0013] A third aspect is a method for treating or preventing tauopathy in a subject, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at the tau sequence SSPGSPGTPGSRSR and / or with threonine at position 205 of human tau. The present invention provides a method that includes the step of administering a drug.

[0014] A third alternative embodiment is a drug for use in the treatment or prevention of tauopathy in a subject, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at the tau sequence SSPGSPGTPGSRSR and / or with threonine at position 205 of human tau. Drugs; or The use of a drug in the manufacture of a drug for treating or preventing tauopathy in a subject, wherein the drug is (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at the tau sequence SSPGSPGTPGSRSR and / or with threonine at position 205 of human tau. Provides usage.

[0015] A fourth aspect provides a method for treating or preventing tauopathy in a subject, comprising the step of administering a drug that increases p38γ activity or the activity of a variant of p38γ in the target neuron.

[0016] An alternative fourth aspect provides for use in the treatment or prevention of tauopathy in a subject, an agent that increases p38γ activity or the activity of a p38γ variant in neurons; and / or for use in the manufacture of a pharmaceutical product for the treatment or prevention of tauopathy in a subject.

[0017] A fifth aspect provides a method for treating or preventing tauopathy in a subject, comprising the step of administering a drug that modifies the nucleotide sequence encoding tau in a neuron of the subject so that a phosphomimetic of phosphorylated tau is expressed, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of human tau.

[0018] An alternative fifth aspect provides a drug for use in the treatment or prevention of tauopathy in a subject, wherein the drug modifies the nucleotide sequence encoding tau in a neuron of the subject so as to express the phosphometic of phosphorylated tau, wherein the phosphorylated tau is phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of human tau; or a use of the drug in the manufacture of a pharmaceutical for the treatment or prevention of tauopathy in a subject, wherein the drug modifies the nucleotide sequence encoding tau in a neuron of the subject so as to express the phosphometic of phosphorylated tau, wherein the phosphorylated tau is phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR.

[0019] The sixth aspect is a method for treating or preventing tauopathy, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; or (b) A nucleic acid in which the nucleotide sequence encoding tau can be modified to be a phosphomimetic of phosphorylated tau, wherein the phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of human tau. This includes the step of introducing the substance into the target neuron.

[0020] An alternative sixth aspect is for use in the treatment or prevention of tauopathy, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; or (b) A nucleic acid whose nucleotide sequence encoding tau can be modified to be a phosphomimetic of phosphorylated tau, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of human tau; or In the manufacture of pharmaceuticals for the treatment or prevention of tauopathy (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; or (b) A nucleic acid in which the nucleotide sequence encoding tau can be modified to be a phosphomimetic of phosphorylated tau, wherein the phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of human tau. Provides the use of.

[0021] The seventh aspect is a method for improving the cognitive abilities of a subject suffering from cognitive impairment related to tauopathy, wherein the subject (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce a phosphometic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0022] A seventh alternative embodiment is a drug for use in improving the cognitive abilities of subjects suffering from cognitive impairment associated with tauopathy, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a drug for improving the cognitive abilities of a person suffering from cognitive impairment associated with tauopathy, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0023] The eighth aspect is a method for improving the cognitive abilities of a subject suffering from cognitive impairment related to tauopathy, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where phosphorylated human tau is human tau phosphorylated with threonine at position 205. The present invention provides a method that includes the step of administering a drug.

[0024] An alternative eighth aspect is a drug for use in improving cognitive abilities in subjects suffering from cognitive impairment associated with tauopathy, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Drugs; or The use of a drug in the manufacture of a drug for improving the cognitive abilities of a person suffering from cognitive impairment associated with tauopathy, wherein the drug is (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Provides usage.

[0025] The ninth aspect is a method for improving the cognitive abilities of a subject suffering from cognitive impairment related to tauopathy, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. The present invention provides a method that includes the step of administering a drug.

[0026] The ninth alternative aspect is for use in improving cognitive abilities in subjects suffering from cognitive impairment associated with tauopathy, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Drugs; or The use of a drug in the manufacture of a drug for improving the cognitive abilities of a person suffering from cognitive impairment associated with tauopathy, wherein the drug is (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Provides usage.

[0027] The tenth aspect provides a method for improving the cognitive abilities of a subject suffering from tauopathy-related cognitive impairment, comprising the step of administering a drug that increases p38γ activity or the activity of a variant of p38γ in the target neurons.

[0028] An alternative tenth aspect provides a drug that increases p38γ activity or the activity of a p38γ variant in neurons for use in improving the cognitive abilities of subjects suffering from tauopathy-related cognitive impairment; or the use of a drug that increases p38γ activity or the activity of a p38γ variant in neurons in the manufacture of a pharmaceutical product for improving the cognitive abilities of subjects suffering from tauopathy-related cognitive impairment.

[0029] The eleventh aspect provides a method for improving cognitive ability in a subject suffering from tauopathy-related cognitive impairment, comprising the step of administering a drug that modifies the nucleotide sequence encoding tau in a neuron of the subject so that a phosphomimetic of phosphorylated tau is expressed. Herein, phosphorylated tau is tau that is phosphorylated with threonine at the tau sequence SSPGSPGTPGSRSR and / or with threonine at position 205 of full-length human tau.

[0030] An alternative eleventh aspect provides a drug for use in improving the cognitive abilities of a subject suffering from cognitive impairment associated with tauopathy, wherein the drug modifies the nucleotide sequence encoding tau of a neuron in the subject so as to express the phosphometic of phosphorylated tau, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or threonine corresponding to position 205 of full-length human tau; or a use of the drug in the manufacture of a pharmaceutical for improving the cognitive abilities of a subject suffering from cognitive impairment associated with tauopathy, wherein the drug modifies the nucleotide sequence encoding tau of a neuron in the subject so as to express the phosphometic of phosphorylated tau, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or threonine corresponding to position 205 of full-length human tau.

[0031] The twelfth aspect is a method for improving the cognitive abilities of a subject suffering from cognitive impairment associated with tauopathy, wherein the neurons of the subject... (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) A gene editing system capable of modifying a nucleotide sequence encoding tau so that a phosphomimetic of phosphorylated tau is expressed, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of full-length human tau. The present invention provides a method that includes the process of introducing [a certain feature].

[0032] The twelfth alternative aspect is for use in improving cognitive abilities in subjects suffering from cognitive impairment associated with tauopathy. (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) A gene editing system capable of modifying a nucleotide sequence encoding tau so that a phosphomimetic of phosphorylated tau is expressed, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of full-length human tau; or In the manufacture of pharmaceuticals for improving cognitive abilities in subjects suffering from cognitive impairment associated with tauopathy (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) A gene editing system capable of modifying a nucleotide sequence encoding tau so that a phosphomimetic of phosphorylated tau is expressed, wherein the phosphorylated tau is tau phosphorylated with threonine of the tau sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of full-length human tau. Provides the use of.

[0033] The thirteenth aspect is a method for reducing or preventing tau aggregation in neurons, wherein the neurons (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce a phosphometic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes a step of introducing a drug.

[0034] A thirteenth alternative aspect is an agent for use in reducing or preventing tau aggregation in neurons, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing tau aggregation in target neurons, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0035] The fourteenth aspect is a method for reducing or preventing tau aggregation in neurons, wherein the neurons (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. The present invention provides a method that includes a step of introducing a drug.

[0036] An alternative fourteenth aspect is an agent for use in reducing or preventing tau aggregation in neurons, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing tau aggregation in target neurons, wherein the drug is (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Provides usage.

[0037] The fifteenth aspect is a method for reducing or preventing tau aggregation in neurons, wherein the neurons (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. The present invention provides a method that includes a step of introducing a drug.

[0038] An alternative fifteenth aspect is an agent for use in reducing or preventing tau aggregation in neurons, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing tau aggregation in target neurons, wherein the drug is (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Provides usage.

[0039] The sixteenth aspect is a method for reducing or preventing tau aggregation in neurons, wherein the target neuron (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) Nucleic acids in which the nucleotide sequence encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of human tau. The present invention provides a method that includes the process of introducing [a certain feature].

[0040] An alternative sixteenth aspect is for use in reducing or preventing tau aggregation in target neurons, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) A nucleic acid in which the nucleotide sequence encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or with the threonine corresponding to position 205 of human tau; or In the manufacture of pharmaceuticals for reducing or preventing tau aggregation in target neurons, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) Nucleic acids in which the nucleotide sequence encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of human tau. Provides the use of.

[0041] The seventeenth aspect is a method for reducing or preventing neurofibrillary tangles in a target neuron, wherein an effective amount (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce a phosphometic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0042] An alternative seventeenth aspect is for use in reducing or preventing neurofibrillary tangles in target neurons, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing neurofibrillary tangles in target neurons, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0043] The eighteenth aspect is a method for reducing or preventing neurofibrillary tangles in a target neuron, wherein an effective amount (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. The present invention provides a method that includes the step of administering a drug.

[0044] An alternative eighteenth aspect is for use in reducing or preventing neurofibrillary tangles in target neurons, (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing neurofibrillary tangles in target neurons, wherein the drug is (a) Promote the phosphorylation of tau at the threonine corresponding to position 205 of human tau; and / or (b) Introduce the phosphomimetic form of phosphorylated human tau into the target neuron, where the phosphorylated human tau is phosphorylated with threonine at position 205 of tau. Provides usage.

[0045] The nineteenth aspect is a method for reducing or preventing neurofibrillary tangles in a target neuron, wherein the neuron (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. The present invention provides a method that includes a step of introducing a drug.

[0046] The nineteenth alternative aspect is for use in reducing or preventing neurofibrillary tangles in target neurons, (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Drugs; or The use of a drug in the manufacture of a drug for reducing or preventing neurofibrillary tangles in target neurons, wherein the drug is (a) Increase p38γ activity or the activity of a variant of p38γ in the target neuron; and / or (b) Introduce the target neuron a nucleotide sequence encoding the phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of full-length human tau. Provides usage.

[0047] The twentieth aspect is a method for reducing or preventing neurofibrillary tangles in a target neuron, wherein the target neuron (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) Nucleic acids in which the nucleotide sequence encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or the threonine corresponding to position 205 of human tau. The present invention provides a method that includes the process of introducing [a certain feature].

[0048] An alternative 20th aspect is for use in reducing or preventing neurofibrillary tangles in target neurons, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) A nucleic acid in which the nucleotide sequence encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or with the threonine corresponding to position 205 of human tau; or In the manufacture of pharmaceuticals for reducing or preventing neurofibrillary tangles in target neurons, (a) p38γ, or a variant thereof, or a nucleic acid capable of expressing p38γ, or a variant thereof; and / or (b) Nucleic acids whose nucleotide sequences encoding tau can be modified so that a phosphomimetic of phosphorylated tau is expressed, which is tau that is phosphorylated with the threonine of the tau sequence SSPGSPGTPGSRSR and / or with the threonine corresponding to position 205 of human tau. Provides the use of.

[0049] The 21st aspect is a method for reducing the phosphorylation of serine at position 422 of human tau in a subject suffering from tauopathy, wherein an effective amount (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce a phosphometic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0050] The 21st alternative embodiment is a drug for reducing serine phosphorylation at position 422 of human tau in subjects suffering from tauopathy, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a pharmaceutical product for reducing the phosphorylation of serine at position 422 of human tau in subjects suffering from tauopathy, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0051] The 22nd aspect is a method for treating or preventing tauopathy associated with the phosphorylation of serine at position 422 of human tau in a subject, wherein an effective amount (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce a phosphometic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0052] The 22 alternative embodiments are agents for the treatment or prevention of tauopathy related to the phosphorylation of serine at position 422 of human tau in a subject, (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Drugs; or The use of a drug in the manufacture of a pharmaceutical product for the treatment or prevention of tauopathy associated with the phosphorylation of serine at position 422 of human tau in a subject, wherein the drug is (a) Promote the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRSR; and / or (b) Introduce the phosphomimetic form of phosphorylated tau into the target neuron, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRSR. Provides usage.

[0053] The twenty-third aspect provides a drug comprising a tau gene (Mapt gene) editing system or a part thereof, or a nucleic acid encoding a tau gene (Mapt gene) editing system or a part thereof, or a gene editing system or a part thereof comprising one or more nucleic acids that introduce a mutation into the wild-type tau gene to cause phosphomimetic expression of phosphorylated tau, wherein the phosphorylated tau is phosphorylated with threonine of the sequence SSPGSPGTPGSRSR and / or with threonine corresponding to position 205 of full-length human tau. [Brief explanation of the drawing]

[0054] [Figure 1-1]Delivery of active p38γMAP kinase improves cognitive ability in mice with advanced Alzheimer's disease. Figure 1(a) is a schematic diagram of an experiment showing the timeline of delivery (intravenous (iv), 100 μl) of AAV9 / PHP.Bsyn-p38γCA and control syn-eGFP to achieve neuronal expression in 13-month-old APP23 (and non-transgenic control) mice. Two different AAV titers (10¹¹ or 10¹³ virion particles / ml) were used in different experiments. Cognitive ability and histological and biochemical data were assessed 2 months after AAV delivery. Figure 1(b) is an image showing immunofluorescence analysis of the cortex and hippocampus of APP23 mice, confirming AAV-mediated expression of active p38γCA(HA) and transgenic expression of APP(6E10) in neurons. Scale bar, 50 μm. [Figure 1-2] Figures 1(c-g) show the memory assessment of 13-month-old APP23 mice two months after delivery of AAV9 / PHP.Bsyn-p38γCA and control syn-eGFP (iv, 100 μl; 10¹¹ viral genomes per μl) in a Morris water maze (n=10-11). Figure 1(c) shows the acquisition stages: a representative swimming path trace on day 6. Figure 1(d) is a graph showing escape latency during the acquisition stages (days 1-6) of the AAV-treated AAP23 mice. Figure 1(e) is a graph showing the linear regression of the acquisition curve in (c). Figure 1(f) is a graph showing a comparison of learning curves. Figure 1(g) is a graph showing the occupancy rate of the water maze quadrant during probe trials (day 7). Q1, target quadrant. Dashed line, threshold for random occupancy. [Figure 1-3]Figure (hl) shows Morris water maze latency (iv, 100 μl; 10¹³ viral genomes per μl) in 13-month-old APP23 mice two months after the delivery of the indicated AAV. (n=9 for APP23AAVeGFP and APP23AAVp38γCA, n=5-6 for control AAVeGFP and AAVp38γCA). Figure 1(h) shows the acquisition stage: a trace of a typical swimming path on day 6. Figure 1(i) is a graph showing escape latency during the acquisition stage. Figure 1(j) is a graph showing linear regression of the acquisition curve in (i). Figure 1(k) is a graph showing a comparison of learning curves. Figure (l) is a graph showing the occupancy rate of the water maze quadrant during probe trials (day 7). Q1, target quadrant. Data are expressed as mean ± SEM. **p<0.01 *p<0.05 ns, no significant difference. (For d and i, two-way ANOVA; for f, g, k, and l, ANOVA). [Figure 2-1] Tau toxicity is regulated by the phosphorylation levels of endogenous p38γ and threonine-205 (T205). Figure 2(a) is a schematic diagram showing that human wild-type tau transgenic mice (Alz17) were crossed with p38γ knockout (p38γ- / -) mice to achieve reduced tau phosphorylation levels at T205 tau and its analogue kinase p38γ. Figure 2(b) is an image showing immunofluorescence staining of tau and p38γ cortical sections from Alz17.p38γ+ / + and Alz17.p38γ- / - mice. Scale bar, 10 μm. [Figure 2-2]Figures 2(c-e) show the memory / cognitive abilities in the Morris water maze of 10-month-old Alz17.p38γ+ / +, Alz17.p38γ- / -, p38γ+ / +, and p38γ- / - control mice (n=10). Figure 2(c) shows the stages of acquisition: a representative swimming path trace on day 6. Figure 2(d) is a graph showing escape latency from day 1 to 6 in mice of the indicated genotypes. Figure 2(e) is a graph showing a comparison of learning curves using linear regression gradients. Figure 2(f) is a graph showing the Morris water maze quadrant occupancy rate during the probe test on day 7 in mice of the indicated genotypes. Q1, target quadrant. Dashed line, threshold for random occupancy. Data are expressed as mean ± SEM. **p<0.01 *p<0.05 ns, no significant difference. (two-way ANOVA for d; ANOVA for e and f). [Figure 3-1] Phosphorylation of endogenous tau at T205 regulates excitotoxic signaling. Figure 3(a) is a schematic diagram showing the generation of tauT205A and tauT205E mice by genome editing. Figure 3(b) is a DNA sequencing chromatogram showing the success of codon editing of the tau (Mapt) gene encoding the T205 residue in tauT205A / A and tauT205E / E mice compared to the wild type (tauT205T / T). [Figure 3-2] Figure 3(c) shows immunoprecipitation of endogenous tau from cortical lysates of tauT205T / T and tauT205A / A mice using anti-tau (tau5) antibody detected with p-T205-specific tau antibody. Figures 3(d-e) show that tauT205E / E mice are protected from excitotoxic seizures. Figure 3(d) is a graph showing seizure latency. Figure 3(e) is a graph showing the mean seizure severity in pentylenetetrazole (PTZ; 50 mg / kg, ip)-induced tauT205T / T and tauT205E / E mice (n=20-22). [Figure 3-3]Figures 3(f-g) show that tauT205A / A mice are more susceptible to excitotoxic seizures. Figure 3(f) is a graph showing seizure latency. Figure 3(g) is a graph showing the mean seizure severity in tauT205T / T and tauT205A / A mice induced by pentylenetetrazole (PTZ; 30 mg / kg, ip) (n=9-12). Data are expressed as mean ± SEM. ***p<0.001 **p<0.01 *p<0.05 ns, no significant difference. (e is two-way ANOVA; f is ANOVA). [Figure 4-1] Phosphorylation of endogenous tau at T205 modulates cognitive impairment in APP23 mice. Figure 4(a) is a schematic diagram showing the acquisition of APP23.tauT205A / A and APP23.tauT205E / E mice by crossing APP23 mice with tauT205A / A or tauT205E / E mice, respectively. Figure 4(b) is an image showing immunofluorescence staining of phospho-T205 tau and human APP(6E10) in the cortex of APP23.tauT205T / T and APP23.tauT205A / A mice. DAPI, nuclear marker. Scale bar, 50 μm. [Figure 4-2] Figure 4(c) is a graph showing the survival curves for APP23.tauT205T / T (n=72), APP23.tauT205A / A (n=55), and APP23.tauT205E / E (n=38). [Figure 4-3] Figures 4(e-h) show the results of the Morris water maze in APP23.tauT205T / T and APP23.tauT205A / A mice, as well as tauT205T / T and tauT205A / A mice (n=6-10). Figure 4(e) shows the acquisition stages: a typical swimming path trace on day 4. Figure 4(f) is a graph showing escape latency during learning from days 1 to 6. Figure 4(g) is a graph showing a comparison of escape latency curves. Figure 4(h) is a graph showing the Morris water maze quadrant occupancy rate during the probe trial (day 7). Q1, target quadrant. Dashed line, random occupancy threshold. [Figure 4-4]Figure 4(il) shows the results of the Morris water maze in APP23.tauT205T / T and APP23.tauT205E / E mice, as well as tauT205T / T and tauT205E / E mice (n=6~10). Figure 4(i) shows the acquisition stages: trace on day 6. Figure 4(j) is a graph showing the escape latency during learning from day 1 to 6 in APP23.tauT205T / T and APP23.tauT205E / E mice, as well as tauT205T / T and tauT205E / E mice. Figure 4(k) is a graph showing a comparison of the escape latency curves. Figure 4(l) is a graph showing the Morris water maze quadrant occupancy rate during the 7-day probe trial in APP23.tauT205T / T and APP23.tauT205E / E mice, as well as tauT205T / T and tauT205E / E mice. Q1, target quadrant. Dashed line, random occupancy threshold. Data are expressed as mean ± SEM. ***p<0.001 **p<0.01 *p<0.05 ns, no significant difference. (Mantel-Cox test for c and d; two-way ANOVA for f and j; ANOVA for g, h, k, and l). [Figure 5-1] Tau T205 is required to mediate the protective effect of p38γ activity in a mouse model of Alzheimer's disease. Figure 5(a) is a schematic diagram showing the cross to obtain APP23.tau- / -.p38γCA mice to test the requirement of T205 for the protective effect of p38γ in APP23 mice. APP23 mice were crossed with p38γCA mice and tau- / - mice. Figure 5(b) is an experimental schematic: In APP23.tau- / -.p38γCA mice, AAV was injected intracranially at postnatal day 0 (P0) to achieve neuronal expression of tauWT or tauT205A or eGFP control. AAV-mediated tau expression and learning / memory performance in a Morris water maze were addressed at 6 months. Figure 5(c) shows images of immunofluorescence staining for p38γCA(HA) and human tau in cortical sections of APP23.tau- / -.p38γCA mice injected with the indicated AAV vector. DAPI, nuclear marker. Scale bar, 50 μm. (n=5). [Figure 5-2] Figures 5(d-f) show the results of memory / cognitive abilities assessed using the Morris water maze in APP23.tau- / -.p38γCA mice with neuronal expression of tauWT or tauT205A. (n is the number shown in the label legend). Figure 5(d) is a graph showing the acquisition stage: a representative swimming path trace on day 4. Figure 5(e) is a graph showing the acquisition stage: escape latency. Figure 5(f) is a graph showing the Morris water maze: results of probe trials on day 8. Q1, target quadrant. Dashed line, random occupancy threshold. Data are expressed as mean ± SEM. **p<0.01 *p<0.05 ns, no significant difference. (two-way ANOVA for d; ANOVA for e and f). [Figure 6] Control parameters for cognitive tests in APP23 treated with AAVp38γCA. Figure 6(a, b) are images of immunofluorescence analysis of the cortex (a) and hippocampus (b) of APP23 mice, confirming AAV-mediated expression of active p38γ in neurons. Scale bar, 50 μm. [Figure 7-1] In tau transgenic mice lacking p38γ, tau pathology is not exacerbated. Figure 7(a) shows immunofluorescence staining of tau and p38γ cortical sections from Alz17.p38γ+ / + and Alz17.p38γ- / - mice. Scale bar, 10 μm. [Figure 7-2] Figure 7(b) is a graph showing the Morris water maze visual stimulation test at day 8 in 10-month-old Alz17.p38γ+ / +, Alz17.p38γ- / -, p38γ+ / +, and p38γ- / - control mice (n=10). Figure 7(c) is a graph showing the Morris water maze quadrant occupancy rate during the probe trial at day 7 in mice with the indicated genotypes. Q1, target quadrant. Dashed line, random occupancy threshold. [Figure 7-3]Figure 7(d) shows images of silver-stained cortical sections of 20-month-old Alz17.p38γ+ / + and Alz17.p38γ- / - mice. Scale bar, 10 μm. Figure 7(e) shows images of immunofluorescence staining of phospho-S214 tau (pS214 tau) and neuronal filaments (NF) in cortical sections of Alz17.p38γ+ / + and Alz17.p38γ- / - mice. Scale bar, 50 μm. [Figure 8-1] Increased levels of p38γ and tau T205 phosphorylation in neurons of p38γCA mice did not increase human tau toxicity. Figure 8(a) is a schematic diagram: Human wild-type tau transgenic mice (Alz17) were crossed with p38γCA(γCA) mice to increase levels of active T205 tau kinase p38γ in neurons. Figure 8(b) is an image showing immunofluorescence of tissue sections of Alz17 (10 months old) with HA(p38γCA) and human tau. DAPI, nuclear marker. Scale bar, 10 μm. (n=4). Figure 8(c) shows immunoblot images of lysates derived from cortical crude synaptosomes (CS) of Alz17.p38γ+ / +, Alz17.p38γ- / -, and Alz17.p38γCA for pT205 tau, PSD-95, p38γ, HA (p38γCA), and SNAP25. Whole brain lysates (W) and non-synaptosome fractions (NS) derived from Alz17.p38γ+ / + cortex are shown as unenriched control samples. #, nonspecific band. (n=3~4). Figure 8(d) shows immunofluorescence images of cortical tissue sections of Alz17 and Alz17.p38γCA brain (10 months old) for phospho-T205 tau, p38γ, and human tau. Scale bar, 10 μm. (n=4). [Figure 8-2]Figure 8(e) shows images of silver-stained cortical sections of 20-month-old Alz17 and Alz17.p38γCA mice. Scale bar, 100 μm. (n=4). Figures 8(f~h) show memory / cognitive abilities assessed using the Morris water maze in 10-month-old Alz17, Alz17.p38γCA, and p38γCA, non-transgenic control mice. (n=10). Figure 8(f) is a graph showing the Morris water maze acquisition stages: escape latency from days 1 to 7 for mice with the indicated genotypes. Figure 8(g) is a graph showing a comparison of learning curves by linear regression gradient. Figure 8(h) is a graph showing Morris water maze quadrant occupancy during probe trials on day 8 in mice with the indicated genotypes. Data are expressed as mean ± SEM. **p<0.01 *p<0.05 ns, no significant difference. (If e, it's two-way ANOVA; if f, it's ANOVA). [Figure 9-1] Amyloid load and control parameters for cognitive testing in APP23 mice with the genome-edited tauT205 allele. Figure 9(a) is an image showing immunofluorescence staining of phospho-T205 tau and human APP(6E10) in the cortex of APP23.tauT205T / T and APP23.tauT205A / A mice. DAPI, nuclear marker. Scale bar, 50 μm. (n=6). Figure 9(b) is a graph showing survival curves for APP23.tauT205T / T (n=40), APP23.tauT205T / A (n=62), and APP23.tauT205A / A (n=55). Figure 9(c) is a graph showing the survival curves for APP23.tauT205T / T (n = 32), APP23.tauT205T / E (n = 71), and APP23.tauT205E / E (n = 38). [Figure 9-2]Figure 9(d) is a graph showing the Morris water maze acquisition stages: visual stimulation trials in APP23.tauT205T / T and APP23.tauT205A / A, and tauT205T / T and tauT205A / A mice. Figure 9(e) is a graph showing the Morris water maze visual stimulation trials in APP23.tauT205T / T and APP23.tauT205E / E, and tauT205T / T and tauT205E / E mice. Figure 9(f) is a graph showing the Morris water maze retrieval test: swimming speed during probe trials in APP23.tauT205T / T and APP23.tauT205E / E, and tauT205T / T and tauT205E / E mice. Data are expressed as mean ± SEM. [Figure 10-1] Control parameters for immunodetection and cognitive testing of viral transgenes in AAP23.tau- / -.p38γCA mice with AAV expressing tauWT or tauT205A in neurons. Figure 10(a) shows immunofluorescence staining of p38γCA(HA) and human tau in cortical sections of AAP23.tau- / -.p38γCA mice injected with the indicated AAV. DAPI, nuclear marker. Scale bar, 50 μm. (n=5). (b) shows immunofluorescence staining of phospho-T205 tau and human APP(6E10) in the cortex of AAP23.tau- / -.p38γCA mice injected with the indicated AAV. DAPI, nuclear marker. Scale bar, 50 μm. (n=5). [Figure 10-2]Figure 10(c-g) shows memory / cognitive ability assessed using the Morris Water Maze in AAP23.tau- / -.p38γCA mice with neuronal expression of tauWT or tauT205A. (n is shown in the label legend). Figure 10(c, d, e) is a graph showing the acquisition stages: escape latency for all experimental groups (c), non-transgenic tau- / - and tau- / -.p38γCA mice (d), and APP23.tau- / - and APP23.tau- / -.p38γCA mice (e), with tauWT or tauT205A expression via AAV. Figure 10(f) is a graph showing the acquisition stages: comparison of learning curves for all experimental groups. Figure 10(g) is a graph showing the Morris Water Maze: results of probe trials on day 8. Q1, target quadrant. Dashed line, random occupancy threshold. Data are expressed as mean ± SEM. **p<0.01 *p<0.05 ns, no significant difference. (Two-way ANOVA for c, d, e; ANOVA for f, g). [Figure 11] Figure 11 is a graph showing the time spent in the open arm of a mouse maze by wild-type mice, GFP-expressing Tau58 mutant mice, or constitutively active p38γ (Cap38γ)-expressing Tau58 mutant mice. [Figure 12] Figure 12 is a graph showing the time spent in the closed arm of the mouse maze and the number of entries in the closed arm for wild-type mice, GFP-expressing Tau58 mutant mice, or constitutively active p38γ (Cap38γ)-expressing Tau58 mutant mice. [Figure 13] Figure 13 is a graph showing the time spent in the center of the mouse maze, and the distance traveled by mice, wild-type mice, GFP-expressing Tau58 mutant mice, or constitutively active p38γ (Cap38γ)-expressing Tau58 mutant mice within the maze. [Figure 14] Figure 14 shows the amino acid sequence of mature wild-type human p38γ. [Figure 15] Figure 15 shows an example of a nucleotide sequence encoding mature human p38γ. [Figure 16]Figure 16 shows the amino acid sequence of an example of a constitutively active mutant of p38γ(D179A)(p38γCA). [Figure 17] Figure 17 shows the amino acid sequence of mature, wild-type human tau. The amino acid sequence SSPGSPGTPGSRSR within tau is shown in bold, and T205 and S422 are underlined. [Figure 18] Figure 18 shows the amino acid sequence of an example of a phosphomimetic tau in which the threonine at position 205 of wild-type tau is replaced with glutamic acid (T205E). [Figure 19] Figure 19 shows Western blot images of brain extracts from TAU58 / 2 mice probed with the antibody shown and treated with AAV-p38gCA and control (AAV-GFP). [Modes for carrying out the invention]

[0055] Detailed explanation This invention relates to a method for treating tauopathy. Tauopathy is a condition associated with the aggregation of tau protein in neurons of the brain, and is typically associated with neurofibrillary deposits such as neurofibrillary tangles formed from tau protein. In tauopathy, it is thought that tau protein aggregates over time, forming deposits of tau-containing neurofibrils, ultimately leading to neuronal cell death. Tauopathy is typically a neurodegenerative disease associated with cognitive decline. Examples of tauopathy include Alzheimer's disease, frontotemporal lobar dementia, corticobasal degeneration, progressive supranuclear palsy, primary age-related tauopathy, chronic traumatic encephalopathy, frontotemporal dementia with parkinsonism linked to chromosome 17, Pick's disease, globular glial tauopathy, and Parkinson's disease.

[0056] In the early stages of Alzheimer's disease (AD) and other neurodegenerative diseases, it is believed that stimulation of tau-dependent signaling complexes, such as the PSD-95 / tau / FYN receptor complex, induces neuronal excitotoxicity. We have previously shown that phosphorylation of tau at specific sites causes disruption of the PSD-95 / tau / FYN receptor complex, thereby preventing excitotoxicity and further progression of neurodegenerative states mediated by the PSD-95 / tau / FYN signaling complex.

[0057] However, in advanced AD and other tauopathies, tau toxicity is thought to be independent of signaling via tau-dependent signaling complexes such as the PSD-95 / tau / FYN receptor complex, and neuronal toxicity and death are mediated by aggregated hyperphosphorylated tau. Prior to this invention, it was believed that once tau aggregation occurred, its effects could not be stopped or reversed, and as a result, neuronal damage and death were unavoidable once tau aggregation occurred, making advanced AD and other tauopathies incurable.

[0058] The inventors have found that promoting the phosphorylation of threonine in the tau sequence SSPGSPGTPGSRSR (SEQ ID NO: 7), such as the threonine residue at position 205 of the longest human isoform of tau (T205), or mutating the target neurons to express the phosphomimetic form of T205-phosphorylated tau from wild-type tau, improves cognitive function in tauopathy. The amino acid numbering used for tau as used herein is based on the amino acid numbering of the longest human isoform of tau, which has 441 amino acids and is commonly called 2N4R tau (and also referred to herein as full-length human tau). The amino acid sequence of the longest human isoform of tau (2N4R) is shown in Figure 17 and is denoted by SEQ ID NO: 1. The amino acid numbering of tau is based on the full-length human tau isoform (SEQ ID NO: 1) containing 441 amino acids.

[0059] As described in the examples, the inventors found that phosphorylation of human wild-type tau at position 205 (threonine) improves cognitive function in a mouse model of advanced AD and, in some cases, reverses the effects of tauopathy.

[0060] As further described in the examples, the inventors have found that even when the symptoms of tauopathy are caused solely by tau aggregation, the symptoms of tauopathy can be alleviated or reversed. In this regard, the inventors have shown that in a mouse model in which tau aggregation is the sole contributing factor to disease progression, phosphorylation of T205 can reverse or reduce the effects caused by tau aggregation.

[0061] Therefore, in one embodiment, a method for treating or preventing tauopathy in a subject, wherein an effective amount of (a) Promote the phosphorylation of tau at the threonine of the tau amino acid sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphomimetic form of phosphorylated tau, where phosphorylated tau promotes the phosphorylation of tau at the threonine of the tau amino acid sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau. A method is provided that includes the step of administering a drug.

[0062] In another embodiment, a method for improving cognitive ability in a subject suffering from cognitive impairment associated with tauopathy, wherein the subject receives an effective amount (a) Promote the phosphorylation of tau at the threonine of the tau sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphomimetic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at position 205 of full-length human tau, such as the threonine in the tau sequence SSPGSPGTPGSRSR. A method is provided that includes the step of administering a drug.

[0063] Cognitive ability is the brain's ability to process, acquire, and / or store information. An example of cognitive ability is memory. One embodiment is a method for improving the memory of a subject suffering from cognitive impairment associated with tauopathy, wherein the subject is given an effective amount (a) Promote the phosphorylation of tau at the threonine of the tau sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphometic of the tau protein phosphorylated with threonine at position 205 of full-length human tau, such as the threonine of the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0064] Improvement in cognitive ability is an improvement, increase, or enhancement of the subject's cognitive ability compared to the subject's cognitive ability before treatment by the method described herein. Improvement in memory is an improvement, increase, or enhancement of the subject's memory compared to the subject's memory before treatment by the method described herein.

[0065] As described in the examples, the inventors further found that the level of phosphorylated serine 422 (pS422) in insoluble or aggregated tau was reduced in mice treated with activated p38γ (which promotes phosphorylation of T205 in full-length human tau) compared with untreated mice in a mouse model of tauopathy.

[0066] Therefore, one embodiment is a method for reducing the phosphorylation of serine at position 422 of human tau in a subject suffering from tauopathy, wherein an effective amount is given to the subject. (a) Promote the phosphorylation of tau at the threonine of the tau sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphomimetic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at position 205 of full-length human tau, such as the threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0067] Another aspect is a method for treating or preventing a disease or condition related to the phosphorylation of serine at position 422 of human tau in a subject, wherein the subject is given an effective amount (a) Promote the phosphorylation of tau at the threonine of the tau sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphomimetic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at position 205 of full-length human tau, such as the threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0068] Serine phosphorylation at position 422 of human tau is associated with the formation of neurofibrillary tangles.

[0069] Therefore, another aspect is a method for reducing or preventing neurofibrillary tangles in a target neuron, wherein an effective amount (a) Promote the phosphorylation of tau at the threonine of the tau sequence SSPGSPGTPGSRSR, such as the threonine at position 205 of full-length human tau; and / or (b) Introduce a phosphomimetic form of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine at position 205 of full-length human tau, such as the threonine in the tau sequence SSPGSPGTPGSRSR. The present invention provides a method that includes the step of administering a drug.

[0070] In one embodiment, the method includes administering an effective amount of a drug to a target that promotes the phosphorylation of tau at threonine in the tau amino acid sequence SSPGSPGTPGSRSR. In one embodiment, the threonine in the tau sequence SSPGSPGTPGSRSR is the threonine at position 205 of full-length human tau (T205).

[0071] In one embodiment, the method includes administering to a target neuron, typically a neuron in the target brain, an effective amount of a drug that promotes the phosphorylation of tau at the threonine of the tau amino acid sequence SSPGSPGTPGSRSR, for example, at the threonine at position 205 of full-length human tau.

[0072] In one embodiment, the subject is treated by administering an effective amount of a drug that increases tau phosphorylated with T205.

[0073] In one embodiment, the subject is treated by administering a drug that induces phosphomimetic expression of tau pT205.

[0074] Tau phosphorylated at the 205th position of full-length human tau is also referred to as tau pT205 in this specification.

[0075] In one embodiment, the subject is treated by administering an effective amount of a drug that converts one or more genes encoding wild-type tau, typically endogenous wild-type tau, to a gene encoding the phosphomimetic of tau pT205. In one embodiment, the phosphomimetic of tau pT205 is tau comprising the amino acid sequence SSPGSPGXPGSRSR (SEQ ID NO: 8) where X is E or D. In one embodiment, the phosphomimetic of tau pT205 is T205E or T205D.

[0076] As used herein, a phosphomimetic of phosphorylated tau is a variant of tau that functions identically or substantially the same as phosphorylated wild-type tau. Thus, a phosphomimetic of pT205 is a variant of tau that exhibits the same or similar effects as full-length wild-type tau in which threonine is phosphorylated at position 205. As used herein, a variant of tau is a tau protein that contains one or more amino acid substitutions, total tau substitutions, or deletions of wild-type tau, typically full-length wild-type tau (e.g., SEQ ID NO: 1).

[0077] It is understood that the phosphomimetic mutation of tau pT205 does not necessarily involve a mutation at T205, but may involve the substitution, deletion, or insertion of one or more amino acid residues at other sites in tau, and as a result, the mutated tau may have the same or similar effect as T205E.

[0078] The drug may include, for example, nucleic acids, nucleic acid analogs, proteins, peptides, or small molecules, or combinations thereof. Typically, drug administration introduces the drug into the target neurons. More typically, drug administration introduces the drug into the target neurons in the brain.

[0079] In some embodiments, the drug comprises a nucleic acid that is introduced into the target neuron. In some embodiments, the nucleic acid is transcribed in the neuron. In some embodiments, the nucleic acid is transcribed and translated in the neuron. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA.

[0080] In some embodiments, the drug can cross the blood-brain barrier, or can be formulated to cross the blood-brain barrier.

[0081] In one embodiment, tauopathy is an Alzheimer's disease mediated by tau aggregation.

[0082] In one embodiment, tauopathy is frontotemporal lobar dementia mediated by tau aggregation.

[0083] In one embodiment, tauopathy is corticobasal degeneration.

[0084] In one embodiment, tauopathy is progressive supranuclear palsy.

[0085] In one embodiment, the tauopathy is primary age-related tauopathy.

[0086] In one embodiment, tauopathy is chronic traumatic encephalopathy.

[0087] In one embodiment, tauopathy is frontotemporal dementia with parkinsonism linked to chromosome 17.

[0088] In one embodiment, tauopathy is Pick's disease.

[0089] In one embodiment, the tauopathy is globular glial tauopathy.

[0090] In one embodiment, tauopathy is Parkinson's disease.

[0091] As used herein, “Subject” is a mammal. A mammal may be a human, a non-human primate, a sheep, a mouse, a rat, a dog, a cat, a horse, a cattle, a pig, or any other mammal that may be susceptible to tauopathy. Typically, the subject is a human.

[0092] In one embodiment, the subject is treated by administering a drug that increases the activity of an effective amount of p38γ activity or a variant of p38γ in the target neurons. p38γ, also known as ERK6, SAPK3, and MAPK12, is a mitogen-activated protein kinase (MAP kinase). In one embodiment, p38γ is of mammalian origin. For example, p38γ may be derived from humans, mice, dogs, cats, pigs, cattle, rats, non-human primates, goats, or sheep. Typically, p38γ is human p38γ. Wild-type p38γ is activated by phosphorylation of tyrosine and threonine residues of the motif TGY. After activation, wild-type p38γ phosphorylates tau. Activation of p38γ is carried out by MAP kinase kinases MKK3 and MKK6, and is activated by phosphorylation by MAPK kinase MAP3K.

[0093] As described in the examples, the inventors found that phosphorylation of wild-type human tau at T205 by p38γ resulted in improved cognitive function in a mouse model of progressive Alzheimer's disease. The inventors also showed that introducing p38γ, or a constitutively active variant of p38γ, into neurons of mice suffering from progressive AD improved memory.

[0094] One embodiment provides a method for treating or preventing a target tauopathy, comprising the step of administering a drug that increases an effective amount of p38γ activity or the activity of a p38γ variant in a target neuron.

[0095] One embodiment provides a method for improving the cognitive abilities of a subject suffering from cognitive impairment associated with tauopathy, comprising the step of administering a drug that increases an effective amount of p38γ activity or the activity of a p38γ variant in the neurons of the subject.

[0096] One embodiment provides a method for reducing serine phosphorylation at position 422 of human tau in a subject suffering from tauopathy, comprising the step of administering an effective amount of a drug that increases p38γ activity or the activity of a p38γ variant in the subject's neurons.

[0097] One embodiment provides a method for treating or preventing a tauopathy associated with the phosphorylation of serine at position 422 of human tau in a subject, comprising the step of administering an effective amount of a drug that increases p38γ activity or the activity of a p38γ variant in the neuron of the subject.

[0098] One embodiment provides a method for reducing or preventing tau aggregation in a target neuron, comprising administering an effective amount of a drug that increases p38γ activity or the activity of a p38γ variant in the target neuron.

[0099] One embodiment provides a method for reducing or preventing neurofibrillary tangles in a target neuron, comprising administering an effective amount of a drug that increases p38γ activity or the activity of a p38γ variant in the target neuron.

[0100] Drugs that increase p38γ activity or the activity of p38γ variants in neurons may include: (a) increasing the amount of p38γ, usually the amount of active p38γ in neurons; and / or (b) increasing the amount of p38γ variants, usually the amount of active p38γ variants in neurons; and / or (c) increasing the amount of p38γ activation in neurons; and / or (d) increasing the amount of p38γ variant activation in neurons if the variant is not an active variant. As used herein, “p38γ activity” refers to the activity of activated p38γ that phosphorylates tau with threonine in the tau sequence SSPGSPGTPGSRSR, and in the case of wild-type full-length human tau, at T205. “Activity of p38γ variants” refers to the activity of p38γ variants, which is identical or substantially similar to the activity of p38γ. P38γ activity may be observed without activation (for example, active variants such as constitutively active variants), or it may be observed after activation. Increased neuronal p38γ activity occurs when the amount of post-treatment neuronal p38γ activity increases compared to the amount of pre-treatment neuronal p38γ activity. Increased neuronal p38γ variant activity occurs when the amount of post-treatment neuronal variant activity increases compared to the amount of pre-treatment neuronal variant activity. P38γ activity, or the activity of p38γ variants, may be increased by administering an effective amount of a drug that increases the following: (a) Increased expression (transcription and / or translation) of endogenous p38γ, etc., of endogenous p38γ in neurons; and / or (b) the amount of exogenous p38γ in neurons; and / or (c) the amount of p38γ variant in the neuron; and / or (d) Activation of endogenous p38γ, exogenous p38γ, and / or variants of p38γ within neurons.

[0101] In one embodiment, p38γ activity, or the activity of a p38γ variant, is increased by administering an effective amount of a drug that increases the amount of exogenous p38γ or its variant in neurons. The amount of exogenous p38γ or its variant can be increased by introducing p38γ or its variant into neurons, or by introducing nucleic acids capable of expressing p38γ or its variant into neurons.

[0102] Therefore, in one embodiment, a drug that increases p38γ activity or the activity of a variant of p38γ in a target neuron may include the p38γ protein or a variant thereof, or a nucleic acid capable of expressing p38γ or a variant thereof in a target neuron. The nucleic acid sequences encoding full-length wild-type human p38γ used in the examples described herein and the amino acid sequences of full-length wild-type human p38γ are shown in Figure 15 (SEQ ID NO: 2) and Figure 14 (SEQ ID NO: 3). Naturally occurring isoforms and variants of human p38γ are also known (e.g., Genbank accession numbers NP_001290181, CR456515). It is envisioned that natural isoforms or variants of p38γ that phosphorylate tau at T205 may be used in the methods described herein.

[0103] In one embodiment, a drug that enhances p38γ activity, or the activity of a variant of p38γ, comprises a nucleic acid encoding p38γ or its variant. Those skilled in the art will be able to determine a suitable nucleic acid sequence encoding the amino acid sequence of p38γ or its variant. For example, the nucleic acid encoding p38γ may contain a nucleic acid sequence that is approximately 60% to 100% identical to the wild-type coding sequence of human p38γ (SEQ ID NO: 3). For example, a nucleic acid encoding p38γ may have a sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the wild-type coding sequence of p38γ, using one of the alignment programs described herein with standard parameters. Those skilled in the art will recognize that by appropriately adjusting these values ​​and taking into account codon degeneracy, reading frame positioning, etc., the corresponding identity of proteins encoded by nucleotide sequences can be determined.

[0104] In one embodiment, a drug that increases p38γ activity, or the activity of a p38γ variant, comprises a p38γ variant. In one embodiment, a drug that increases p38γ activity, or the activity of a p38γ variant, comprises a nucleic acid encoding a p38γ variant. As used herein, a p38γ variant is a protein that differs from the wild-type human p38γ protein by one or more amino acid substitutions, additions, or deletions, and is capable of phosphorylating tau at the threonine of the sequence SSPGSPGTPSRSR, for example, being capable of phosphorylating wild-type human tau at the threonine residue T205. The p38γ variant phosphorylates wild-type human tau at residue T205. In one embodiment, a p38γ variant comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of wild-type human p38γ. In one embodiment, the p38γ variant includes an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence represented by SEQ ID NO: 3.

[0105] As used herein, “% identity” with respect to a polypeptide, or “% identical to the amino acid sequence” of a polypeptide, means the percentage of residues in two sequences that are identical when aligned for maximum correspondence across a specific comparison window, as measured by a sequence comparison algorithm or visual inspection.

[0106] Sequence comparison algorithms for determining the percentage identity between two polypeptides are known in the art. Examples of such algorithms include the algorithm by Myers and Miller (1988); the local homology algorithm by Smith et al. (1981); the homology algorithm by Needleman and Wunsch (1970); the search-for-similarity method by Pearson and Lipman (1988); and the algorithm by Karlin and Altschul (1990), as modified by Karlin and Altschul (1993). Computer implementations of these algorithms for determining % identity between two polypeptides include, for example, CLUSTAL (Intelligenetics, available from Mountain View, California) (Pearson et al. (1994)); the ALIGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, version 8 (Genetics Computer Group (GCG), available from 575 Science Drive, Madison, Wisconsin, USA).

[0107] In some embodiments, a variant of p38γ may include a portion of p38γ.

[0108] In some embodiments, a variant of p38γ may contain a portion of p38γ, but otherwise it will be different from wild-type p38γ. In this regard, we envision that a variant of p38γ may include a chimeric p38γ protein in which the interaction motif of p38γ is fused to a portion of another kinase, such as MAP kinase or other serine / threonine kinases, or a variant of another kinase having mutations to modify their activity. For example, a variant of p38γ may contain the carboxy-terminal portion of p38γ fused to the N-terminal portion of a kinase selected from the group consisting of p38α, p38β, and p38δ, or variants of p38α, p38β, and p38δ having mutations to modify their activity. In one embodiment, a variant of p38γ is a chimeric p38γ. In various embodiments, the chimeric p38γ includes an amino acid sequence selected from the group consisting of: ETPL (SEQ ID NO: 9), KETPL (SEQ ID NO: 10), SKETPL (SEQ ID NO: 11), VSKETPL (SEQ ID NO: 12), RVSKETPL (SEQ ID NO: 13), ARVSKETPL (SEQ ID NO: 14), GARVSKETPL (SEQ ID NO: 15), LGARVSKETPL (SEQ ID NO: 16), QLGARVSKETPL (SEQ ID NO: 17), RQLGARVSKETPL (SEQ ID NO: 18), PRQLGARVSKETPL (SEQ ID NO: 19), PPRQLGARVSKETPL (SEQ ID NO: 20), KPPRQLGARVSKETPL (SEQ ID NO: 21), FKPPRQLGARVSKETPL (SEQ ID NO: 22), SFKPPRQLGARVSKETPL (SEQ ID NO: 22) Number 23), LSFKPPRQLGARVSKETPL (Sequence ID 24), VLSFKPPRQLGARVSKETPL (Sequence ID 25), EVLSFKPPRQLGARVSKETPL (Sequence ID 26), KEVLSFKPPRQLGARVSKETPL (Sequence ID 27), YKEVLSFKPPRQLGARVSKETPL (Sequence ID 28), TYKEVLSFKPPRQLGARVSKETPL (Sequence ID 29), VTYKEVLSFKPPRQLGARVSKETPL (Sequence ID 30), RVTYKEVLSFKPPRQLGARVSKETPL (Sequence ID 31), KRVTYKEVLSFKPPRQLGARVSKETPL (Sequence ID 32),ETAL (SEQ ID NO: 33), KETAL (SEQ ID NO: 34), PKETAL (SEQ ID NO: 35), VPKETAL (SEQ ID NO: 36), RVPKETAL (SEQ ID NO: 37), ARVPKETAL (SEQ ID NO: 38), GARVPKETAL (SEQ ID NO: 39), LGARVPKETAL (SEQ ID NO: 40), QLGARVPKETAL (SEQ ID NO: 41), RQLGARVPKETAL (SEQ ID NO: 42), PRQLGARVPKETAL (SEQ ID NO: 43), PPRQLGARVPKETAL (SEQ ID NO: 44), KPPRQLGARVPKETAL (SEQ ID NO: 45), FKPPRQLGARVPKETAL (SEQ ID NO: 46), SFKPPRQLGARVP Sequence number 47), LSFKPPRQLGARVPKETAL (Sequence number 48), VLSFKPPRQLGARVPKETAL (Sequence number 49), EVLSFKPPRQLGARVPKETAL (Sequence number 50), KEVLSFKPPRQLGARVPKETAL (Sequence number 51), YKEVLSFKPPRQLGARVPKETAL (Sequence number 52), TYKEVLSFKPPRQLGARVPKETAL (Sequence number 53), VTYKEVLSFKPPRQLGARVPKETAL (Sequence number 54), RVTYKEVLSFKPPRQLGARVPKETAL (Sequence number 55), and KRVTYKEVLSFKPPRQLGARVPKETAL (Sequence number 56).

[0109] In one embodiment, a variant of p38γ is an active variant of p38γ. An active variant of p38γ is a variant that does not require activation by MAP kinase kinases MKK3 and MKK6 to exhibit p38γ activity. In one embodiment, an active variant of p38γ is a constitutively active variant of p38γ. A constitutively active variant of p38γ is a variant of p38γ that is sequentially active and therefore does not require activation by MAP kinase kinases MKK3 and MKK6. Typically, a constitutively active variant contains one or more amino acid substitutions that result in sequential activity. In various embodiments, a constitutively active variant of p38γ includes the following amino acid sequence: (a) ARQAASEMTGY (Sequence ID 57); (b) LARQAASEMTGYV (Sequence ID 58) (c) DFGLARQAASEMTGYVVTRW (Sequence ID 59) (d) VNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNW (Sequence ID 60) (e) HRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIW (Sequence ID 61); (f) LRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMI (Sequence ID 62); (g) QFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSD (Sequence ID 63); (h) KHEKLGEDRIQFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSDHLDQLKEIMK (Sequence ID 64); (i) FMGTDLGKLMKHEKLGEDRIQFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSDHLDQLKEIMKVTGTPPAEFV (Sequence ID 65); (j) DFTDFYLVMPFMGTDLGKLMKHEKLGEDRIQFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSDHLDQLKEIMKVTGTPPAEFVQRLQSDEAKN (Sequence ID 66); (k) DVFTPDETLDDFTDFYLVMPFMGTDLGKLMKHEKLGEDRIQFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAASEMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSDHLDQLKEIMKVTGTPPAEFVQRLQSDEAKNYMKGLPELEK (Sequence ID 67); (l) MRHENVIGLLDVFTPDETLDDFTDFYLVMPFMGTDLGKLMKHEKLGEDRIQFLVYQMLKGLRYIHAAGIIHRDLKPGNLAVNEDCELKILDFGLARQAAS EMTGYVVTRWYRAPEVILNWMRYTQTVDIWSVGCIMAEMITGKTLFKGSDHLDQLKEIMKVTGTPPAEFVQRLQSDEAKNYMKGLPELEKKDFASILTNA (SEQ ID NO: 68).

[0110] In one embodiment, a constitutively active variant of p38γ contains the D179A amino acid substitution of wild-type human p38γ. The amino acid sequence of an example of a constitutively active variant of p38γ is shown in Figure 16 (SEQ ID NO: 4).

[0111] In one embodiment, a method is provided for treating or preventing a target tauopathy, comprising the step of administering an effective amount of a nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the target neurons. Typically, tauopathy is associated with the phosphorylation of serine at position 422 of human tau. In one embodiment, a method is provided for improving cognitive abilities, such as memory, in a subject suffering from cognitive impairment associated with tauopathy, comprising the step of administering an effective amount of a nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the target neurons. Typically, tauopathy is associated with the phosphorylation of serine at position 422 of human tau.

[0112] In one embodiment, a method is provided for treating progressive Alzheimer's disease in a subject, comprising the step of administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in neurons of the subject. Progressive Alzheimer's disease is typically associated with the phosphorylation of serine at position 422 of human tau.

[0113] In one embodiment, a method is provided for treating a target progressive frontotemporal dementia, comprising the step of administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the target neurons. Frontotemporal dementia is typically associated with the phosphorylation of serine at position 422 of human tau.

[0114] In one embodiment, a method is provided for reducing serine phosphorylation at position 422 of human tau in subjects suffering from tauopathy, the method comprising administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the neurons of the subject.

[0115] In one embodiment, a method is provided for treating or preventing a tauopathy associated with the phosphorylation of serine at position 422 of human tau in a subject, the method comprising administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the neurons of the subject.

[0116] Another embodiment provides a method for reducing or preventing tau aggregation in a neuron of interest, the method comprising administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the neuron of interest.

[0117] Another embodiment provides a method for reducing or preventing neurofibrillary tangles in a neuron of interest, the method comprising administering an effective amount of nucleic acid expressing p38γ or a variant thereof, typically a constitutively active variant of p38γ, in the neuron of interest.

[0118] In another embodiment, the subject is treated by administering an agent that introduces a phosphomimetic of tau pT205 into the neurons of the subject. In one embodiment, the agent that introduces a phosphomimetic of tau pT205 is an agent that introduces a phosphomimetic mutation into wild-type tau, typically endogenous wild-type tau. As used herein, a phosphomimetic of tau pT205 is a variant of tau comprising one or more amino acid substitutions, insertions, or deletions, which functions in the same or substantially the same manner as unsubstituted wild-type human tau after phosphorylation of unsubstituted tau at threonine 205. In one embodiment, the phosphomimetic comprises a phosphomimetic substitution.

[0119] As described in the examples, the inventors have shown that expression of the T205E variant of tau in neurons improves cognitive function and survival in an AD mouse model. The T205E variant of tau is a phosphomimetic of tau phosphorylated at T205 (pT205). A phosphomimetic substitution is an amino acid substitution of a protein that results in the protein functioning in the same or substantially the same way as the unsubstituted protein after phosphorylation of the unsubstituted protein. A phosphomimetic substitution of phosphorylated tau is an amino acid substitution at a site in tau that results in a tau protein that functions in the same or substantially the same way as wild-type tau after phosphorylation at a specific site in wild-type tau.

[0120] In one embodiment, the method includes the step of treating a subject to introduce a phosphomimetic of tau, including phosphomimetic substitution of tau at T205.

[0121] In one embodiment, the phosphomimetic substitution of tau is a substitution of threonine to glutamic acid or aspartic acid at position 205 of tau (T205E or T205D), and the amino acid number is based on the longest human tau isoform containing 441 amino acids. The amino acid sequences of full-length wild-type human tau (SEQ ID NO: 1) and tau T205E (SEQ ID NO: 5) are shown in Figures 17 and 18.

[0122] Typically, the tau variant is a human tau variant. In other embodiments, the tau variant may be a tau variant derived from a non-human mammal. For example, the tau variant may be a tau variant derived from a mouse, dog, cat, pig, cattle, rat, non-human primate, goat, or sheep.

[0123] A method for treating or preventing taupathy in a subject is provided, comprising the step of administering a nucleic acid to the neuron of the subject that contains a nucleotide sequence that results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0124] In one embodiment, a method is provided for improving cognitive ability in a subject suffering from cognitive impairment associated with tauopathy, the method comprising administering a nucleic acid containing a nucleotide sequence that results in the production of tau different from wild-type human tau in the subject's neurons due to an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0125] In one embodiment, a method is provided for treating progressive Alzheimer's disease in a subject, comprising the step of administering a nucleic acid to the neurons of the subject, which, when expressed, results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate.

[0126] In one embodiment, a method is provided for treating advanced frontotemporal dementia in a subject, comprising the step of administering a nucleic acid to the neurons of the subject, which, when expressed, results in the production of tau different from wild-type human tau at an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate.

[0127] In one embodiment, a method is provided for reducing serine phosphorylation at position 422 of human tau in a subject suffering from tauopathy, the method comprising administering a nucleic acid containing a nucleotide sequence that, when expressed in the target neurons, results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0128] In one embodiment, a method is provided for treating or preventing a tauopathy associated with the phosphorylation of serine at position 422 of human tau in a subject, comprising the step of administering a nucleic acid to the neuron of the subject, which, when expressed, results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0129] Another embodiment provides a method for reducing or preventing tau aggregation in a neuron of interest, comprising the step of administering to the neuron of interest a nucleic acid comprising a nucleotide sequence that, when expressed, results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0130] Another embodiment provides a method for reducing or preventing neurofibrillary tangles in a neuron of interest, comprising the step of administering to the neuron of interest a nucleic acid comprising a nucleotide sequence that, when expressed, results in the production of tau different from wild-type human tau in an amino acid substitution (T205E or T205D) of threonine to glutamate or aspartate at position 205.

[0131] In one embodiment, the nucleic acid includes a nucleotide sequence encoding tau T205E or T205D.

[0132] In some embodiments, phosphomimetic phosphorylated tau can be introduced into a neuron by introducing a mutation into the nucleotide sequence encoding wild-type tau in the neuron. Typically, the mutation is introduced into the neuron's endogenous tau gene. Thus, in one embodiment, the nucleic acid is a gene editing system, or part of a gene editing system, or encoding a phosphomimetic mutation for introducing a phosphomimetic mutation into the nucleotide sequence encoding wild-type tau, typically the endogenous tau gene, in the neuron of interest. In one embodiment, the nucleic acid comprises a gene editing system, or part of a tau gene editing system, for introducing a phosphomimetic mutation into the nucleotide sequence encoding wild-type tau, typically the endogenous tau gene, in the neuron of interest. In one embodiment, the nucleic acid comprises a nucleotide sequence encoding a tau gene editing system or part of it, which introduces a mutation into the wild-type tau gene to cause the expression of phosphomimetic tau. Typically, phosphomimetic tau is T205E or T205D. In one embodiment, the gene editing system is a CRISPR / Cas complex, typically a CRISPR / Cas9 complex, which is located at position 205 of human tau. From threonine Glutamic acid or aspartic acid to The substitution is introduced into the tau gene. Typically, the nucleic acid encoding a gene editing system or part thereof contains a guide RNA, or a nucleic acid encoding a guide RNA (gRNA). Usually, a gRNA is a single guide RNA or a pair of guide RNAs.

[0133] As used herein, the term "tau gene" has the same meaning as "Mapt gene."

[0134] In embodiments in which the drug comprises a nucleic acid capable of expressing p38γ or a variant thereof, or a tau variant, or a gene editing system in target neurons, the nucleic acid sequence encoding p38γ or a variant thereof, or a tau variant, or a gene editing system is typically operably ligated to a regulatory sequence to direct the expression of p38γ or a variant thereof, or a tau variant, or a gene editing system in the target neurons. The nucleic acid capable of expressing p38γ or a variant thereof, or a tau variant, or a gene editing system in target neurons may comprise an expression cassette containing the coding sequence of p38γ or a variant thereof, or a tau variant, or a gene editing system. The expression cassette is a nucleic acid comprising a coding sequence and a regulatory sequence that work together to express a protein encoded by the coding sequence within the cell. "Coded sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence. The coding sequence may constitute an "uninterrupted coding sequence," that is, it may be free of introns, such as cDNA, or may contain one or more introns surrounded by appropriate splice junctions.

[0135] Expression cassettes typically contain regulatory sequences. “Regulatory sequences” are nucleotide sequences located upstream (5' non-coding), internally, or downstream (3' non-coding) of a coding sequence and affect the transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences are known in the art and may include, for example, transcriptional regulatory sequences such as promoters, enhancers, translational reader sequences, introns, and polyadenylation signal sequences. The coding sequence is typically operably ligated to a promoter. A promoter is a DNA region that, under certain conditions, binds to RNA polymerase and initiates transcription of a coding sequence, typically located downstream (3' direction) of the promoter. The coding sequence may also be operably ligated to a termination signal. Expression cassettes may also contain sequences necessary for proper translation of the coding sequence. An expression cassette containing a coding sequence may be a chimera. As used herein, “chimeric” vector or expression cassette means a vector or cassette containing nucleic acid sequences from at least two different species, or having nucleic acid sequences from the same species that are bound or associated in a manner not occurring in the “natural” or wild type of the species. The coding sequence of an expression cassette is under the control of a constitutive promoter or a moduloable promoter that initiates transcription only in specific tissues or cell types, or can be controlled when a host cell is exposed to a specific stimulus. For example, in an expression cassette containing a nucleic acid encoding p38γ, the coding sequence may be operably linked to a non-native promoter of the p38γ gene, such as a promoter that expresses the coding sequence in neurons or is induced in neurons. In one embodiment, the promoter is a neuronal promoter.Examples of suitable neural promoters include any of the above in combination with enhancers such as synapsin (SYN), calcium / calmodulin-dependent protein kinase (CaMKII), tubulin alpha I (Ta1), neuron-specific enolase (NSE), platelet-derived growth factor beta chain (PDGF), MfP, dox, GFAP, preproenkephalin, dopamine β-hydroxylase (dβH), prolactin, chicken β-actin, prion protein, mouse Thy1.2, myelin basic promoter, or partial cytomegaly virus promoter. Other examples of promoters that can be used to express nucleic acid sequences in neurons include the SV40 early promoter, the mouse mammary cancer virus terminal repeat sequence (LTR) promoter; the adenovirus major late promoter (Ad MLP); the herpes simplex virus (HSV) promoter; cytomegalovirus (CMV) promoters such as the CMV pre-early promoter region (CMVIE); the Roussarcoma virus (RSV) promoter; synthetic promoters; and hybrid promoters. Inducible or controllable promoters include, for example, promoters whose transcriptional activity is modified in the presence or absence of mifepristone, doxycycline, tetracycline, or tamoxifen.

[0136] Nucleic acids that code for a protein (coding sequence) are operably ligated to a regulatory sequence when they are positioned to enable protein expression within a cell. For example, a promoter is operably ligated to a coding region when it assists in initiating the transcription of a coding sequence.

[0137] As used herein, “expression” of a nucleic acid sequence means the transcription and / or translation of a nucleic acid sequence that includes a coding sequence for producing a polypeptide encoded by the coding sequence, or a sequence that encodes a gene editing sequence such as a CRISPR / Cas sequence.

[0138] In one embodiment, the drug is a vector. In such a vector, a nucleic acid sequence encoding p38γ or a variant thereof, or a variant of tau, or a tau gene editing system, or an expression cassette containing such a sequence, is inserted into a suitable vector sequence. The term “vector” refers to a nucleic acid suitable for transferring a gene into a host cell, such as a neuron. The term “vector” includes plasmids, cosmids, naked DNA, viral vectors, etc. In one embodiment, the vector is a plasmid vector. A plasmid vector is a double-stranded circular DNA molecule into which additional sequences can be inserted. A plasmid may also be an expression vector. Plasmids and expression vectors are known in the art and are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual, 4th Ed. Vol. 1-3, Cold Spring Harbor, NY (2012).

[0139] In some embodiments, the vector is a viral vector. Depending on the viral vector, the viral vector includes a viral sequence that enables the production of viral particles and / or their integration into the host cell genome and / or viral replication. Viral vectors that can be used with the methods and compositions described herein include any viral vector that can introduce nucleic acids into neurons, typically neurons of the brain. Examples of viral vectors include adenovirus vectors; lentivirus vectors; adeno-associated virus vectors; rabies virus vectors; herpes simplex virus vectors; SV40; polyomavirus vectors; and poxvirus vectors.

[0140] In one embodiment, the viral vector is an adeno-associated virus (AAV) vector for packaging adeno-associated virus (AAV). In one embodiment, the AAV vector is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh20, AAVrh39, AAVrh43, and AAVcy5 vectors, or their variants. In one embodiment, the viral vector is serotype AAV1, AAV9, AAVrh10, or AAVcy5. In one embodiment, the serotype of the AAV vector is AAV1. In another embodiment, the serotype of the AAV vector is AAV9. In another embodiment, the serotype of the AAV vector is AAVrh10. In another embodiment, the serotype of the AAV vector is AAVcy5. The use of recombinant AAV for introducing nucleic acids into cells is known in the art, e.g., US20160038613; Grieger and Samulski (2005) Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications, Advances in Biochemical Engineering / Biotechnology 99:119-145; methods for producing recombinant AAV are known in the art, e.g., Harasta et al (2015) Neuropsychopharmacology 40: 1969-1978. In nerve cells, p38γ and p38γ CA An example of an adeno-associated virus vector for expressing this gene is described in International Publication No. 2017 / 147654.

[0141] In another embodiment, methods for producing and using lentiviral vectors are known in the art, and are described, for example, in Naldini et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science, 272:263-267; Lois et al. (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors, Science, 295:868-872; and Vogel et al (2004) A single lentivirus vector mediates doxycycline-regulated expression of transgenes in the brain. Hum Gene Ther. 2004;15(2):157-165.

[0142] Adenoviruses are also intended for use in the delivery of nucleic acid agents. Therefore, in another embodiment, the viral vector is an adenovirus vector. Adenovirus vectors are known in the art and are described, for example, in Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993); Southgate et al. (2008) Gene transfer into neural cells in vitro using adenoviral vectors, Current Protocols in Neuroscience, Unit 4 23, Chapter 4; and Akli et al. (1993) Transfer of a foreign gene into the brain using adenovirus vectors. Nature Genetics, 3(3): 224-228.

[0143] Another embodiment provides a vector described herein, typically a viral vector described herein.

[0144] Viral vectors are typically packaged into viral particles using methods known in the art. These viral particles can then be used to transfer cell lines, including nerve cell lines, or nerve tissue, either in vitro or in vivo. Therefore, another embodiment provides viral particles containing the vector described herein.

[0145] Further embodiments provide a drug comprising a tau gene editing system or a part thereof, or a nucleic acid encoding a tau gene editing system or a part thereof, as described herein. In one embodiment, the tau gene editing system or a part thereof introduces a mutation into the wild-type tau gene, typically an endogenous tau gene, to induce the expression of phosphomimetic tau. Typically, phosphomimetic tau is T205E or T205D. In one embodiment, the tau gene editing system comprises a CRISPR / Cas, typically CRISPR / Cas9, targeting the wild-type tau gene, typically an endogenous wild-type tau gene, in combination with a donor nucleic acid that introduces threonine in place of glutamic acid or aspartic acid in the tau sequence SSPGSPGTPGSRSR, typically threonine at position 205 of wild-type human tau.

[0146] Typically, a tau gene editing system or part thereof includes a guide RNA (gRNA), or nucleic acid encoding the guide RNA, which is complementary to a portion of the coding region of the tau gene sequence, typically the sequence encoding tau T205 or nearby. Usually, the gRNA is a single guide RNA or a pair of guide RNAs. Examples of suitable pairs of guide RNA sequences for targeting mouse and human tau include the sequences shown below: Guide RNA1: Mouse: CGAGCGACTGCCAGGCGTTC (Sequence ID 69) Human: GGAGCGGCTGCCGGGAGTGC (Sequence ID 70) Guide RNA2: Mouse: CCCGGCTCTCCCGGAACGCC (Sequence ID 71) Human: CCCGGCTCCCCAGGCACTCC (Sequence No. 72)

[0147] Typically, tau gene editing systems include a CRISPR / Cas9 complex combined with a donor nucleic acid.

[0148] Typically, guide RNA further includes a sequence that binds to an endonuclease, such as a Cas protein, typically the Cas9 protein. Guide RNA usually includes a protospacer-adjacent motif (PAM) sequence and a fusion of bacterial crRNA and tracrRNA for binding to the Cas protein. Thus, guide RNA provides both target specificity and scaffold / binding ability for the Cas endonuclease. In this regard, the guide RNA sequence directs the endonuclease, such as Cas, to a target site where the nuclease creates a double-strand break in the target DNA. To introduce mutations using tau gene editing systems, gRNA and a Cas protein, or nucleic acid encoding a Cas protein, are introduced into cells along with a donor sequence containing a mutant tau sequence for integration into the endogenous wild-type tau gene. The donor nucleic acid typically integrates the mutant tau sequence into the tau allele by homology-directed repair. Examples of donor sequences for mutating the mouse and human tau gene (MAPT gene) are as follows: mouse: CCAGGTGAACCACCAAAATCCGGAGAACGAAGCGGCTACAGCAGCCCCGGCTCTCCCGGAGAGCCTGGCAGTCGCTCGCGCACCCCATCCCTACCAACACCGCCCACCCGGGAGCCCAA (SEQ ID NO: 73) Human: TCTGGTGAACCTCCAAAATCAGGGGATCGCAGCGCTACAGCAGCCCCGGCTCCCCAGGCGAGCCCGGCAGCCGCTCCCGCACCCCGTCCCTTCCAACCCCACCCACCCGGGAGCCCAAG (SEQ ID NO: 6)

[0149] In one embodiment, the donor sequences for mutating the human tau gene are SPGSPGXPGSRSR (SEQ ID NO: 74), SSPGSPGXPGSRSRT (SEQ ID NO: 75), SSPGSPGXPGSRSRT (SEQ ID NO: 76), YSSPGSPGXPGSRSRTP (SEQ ID NO: 77), GYSSPGSPGXPGSRSRTPS (SEQ ID NO: 78), SGYSSPGSPGXPGSRSRTPSL (SEQ ID NO: 79), RSGYSSPGSPGXPGSRSRTPSLP (SEQ ID NO: 80), DRSGYSSPGSPGXPGSRSRTPSLPT (SEQ ID NO: 81), GDRSGYSSPGSPGXPGSRSRTPSLPTP (SEQ ID NO: 82), SGDRSGYSSPGSPGXPGSRSRTPSLPTPP (SEQ ID NO: 83), KSGDRSGYSSPGSPGXPGSRSRTPSLPTPPT (SEQ ID NO: 84), PKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTR It includes a nucleotide sequence encoding an amino acid sequence selected from the group consisting of (SEQ ID NO: 85), PPKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTRE (SEQ ID NO: 86), EPPKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTREP (SEQ ID NO: 87), GEPPKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTREPK (SEQ ID NO: 88), and SGEPPKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTREPK (SEQ ID NO: 89), Here, X is either E or D.

[0150] In one embodiment, the donor sequence for mutating the human tau gene comprises a nucleotide sequence that is at least 60%, more typically at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to Sequence ID No. 6.

[0151] In one embodiment, a donor sequence for mutating the human tau gene comprises a nucleotide sequence that is at least 60%, more typically at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to Sequence ID No. 6, and codes for the amino acid sequence SGEPPKSGDRSGYSSPGSPGXPGSRSRTPSLPTPPTREPK, where X is E or D.

[0152] Methods of genome editing using CRISPR / Cas9 are known in the art and are described, for example, in U.S. Patent No. 10,240,145; and U.S. 201180127783;

[0153] In various embodiments: (i) The gRNA is administered together with the RNA encoding the Cas protein and ds or ss donor DNA; (ii) The gRNA is administered together with the Cas protein and ds or ss donor DNA; (iii) The DNA encoding the gRNA is administered together with the DNA encoding the Cas protein and the ds or ss donor DNA; (iv) The DNA encoding the gRNA is administered along with the Cas protein and ds or ss donor DNA; (v) The DNA encoding the gRNA is administered together with the RNA encoding the Cas protein and the ds or ss donor DNA.

[0154] It will be understood that the DNA encoding gRNA or the DNA encoding Cas protein includes a gRNA sequence or Cas coding sequence operably ligated to a suitable regulatory sequence as described herein.

[0155] It will also be understood that each component of the tau editing system (e.g., gRNA, endonuclease, and donor sequence) can be administered together or separately.

[0156] In some embodiments, the tau gene editing system is introduced into the target neuron via a vector. For example, the tau gene editing system can be introduced into the target neuron in an AAV vector system.

[0157] The agents described herein can be formulated as pharmaceutical compositions. Therefore, in another embodiment, a pharmaceutical composition comprising the agents described herein is provided. The composition comprises the agent in a pharmaceutically acceptable carrier. Methods for formulating agents using pharmaceutical carriers are known in the art and are described, for example, in Remington's Pharmaceutical Science, (17th ed. Mack Publishing Company, Easton, Pa. 1985); and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th Edition, McGraw-Hill Professional, 2005).

[0158] Acceptable carriers, diluents, and adjuvants are nontoxic to the recipient and preferably inactive at the dosage and concentration used, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions that form salts such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0159] The administration of the drug to the target may be by intracranial, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, or intrathecal injection. Compositions suitable for intracranial, intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, or intrathecal use include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Pharmaceutically acceptable carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride.

[0160] In embodiments in which the drug is packaged in viral particles, the pharmaceutical composition may contain viral particles at any concentration that allows the drug to be effective. In such embodiments, the pharmaceutical composition may contain viral particles in amounts from 0.1% to 99.9% by weight. Pharmaceutically acceptable carriers include water, buffer water, saline solutions such as ordinary physiological saline or equilibrium saline such as Hanks or Earl's equilibrium solution, glycine, hyaluronic acid, and the like.

[0161] The titer of the administered viral particles varies depending, for example, the specific vector used, the mode of administration, the degree of the condition, and the individual, and can be determined by methods standard in the art.

[0162] The drugs described herein can be formulated for delivery to nerve cells by non-viral methods such as microinjection, electroporation, particle gun (microparticle bombardment), liposome incorporation, and nanoparticle-based delivery.

[0163] In one embodiment, the agents described herein can be formulated into one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, the agents described herein are formulated into liposomes. Liposomes are monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. The design of the liposome may include, for example, an opsonin or ligand to improve the adhesion of the liposome to tissue or to activate an event such as endocytosis.

[0164] Liposome formation may depend on the physicochemical properties of the drug and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the drug, additional processes involved during administration and / or vesicle delivery, optimized size, polydispersity and shelf life of the vesicles for intended use, as well as batch-to-batch reproducibility and the potential for large-scale production of safe and efficient liposome products.

[0165] Methods for producing liposomes and lipid nanoparticles for drug delivery are known in the art and are described, for example, in U.S. Patent No. 5,264,221.

[0166] The term "administer" should be understood to mean providing a compound or agent to a subject in need of treatment.

[0167] The term "effective amount" refers to the amount of an agent required to elicit the desired biological response of a system, tissue, or subject.

[0168] The specific dosage levels and frequencies of administration for any particular subject may vary and will depend, for example, on the activity of the specific compound or agent being used, the metabolic stability and length of action of that compound or agent, age, body weight, general health, sex, diet, mode and time of administration, drug combinations, the severity of the particular condition, and various factors including the subject being treated.

[0169] A kit is also provided that includes a container containing a drug. The container may be a simple bottle containing the drug in a parenteral dosage form, and each dosage form contains a unit dose of the drug. The kit further includes printed instructions. The manufactured product includes labels, etc. indicating the treatment of the subject by the method of the present invention. In one form, the manufactured product may be a container containing the drug in a form for parenteral administration. For example, the drug may be in the form of an injectable solution in a disposable container.

[0170] As used herein, "treatment" means affecting a subject, tissue, or cell to obtain the desired pharmacological and / or physiological effect, including inhibiting a condition, i.e., preventing its progression; or alleviating or improving the effects of a condition, i.e., reversing the effects of the condition or causing regression.

[0171] As used herein, "prevent" means to prevent the occurrence of a condition in a cell or subject that has a risk of having the condition, but does not necessarily mean that the condition will not ultimately develop, or that the subject will not ultimately develop the condition. Prevention includes delaying the onset of a condition in a cell or subject.

[0172] In the following claims and the foregoing description of the invention, except where the context requires otherwise for express language or essential meaning, the word "comprise", or variations such as "comprises" or "comprising", are used in an inclusive sense. That is, it specifies the presence of the stated features, but does not preclude the presence or addition of further features in various embodiments of the invention.

[0173] All publications referred to herein are hereby incorporated by reference. It will be understood by those skilled in the art that numerous variations and / or modifications can be made to the invention without departing from the broad spirit or scope of the invention as described. Accordingly, this embodiment should be considered illustrative in all respects and not restrictive.

[0174] This application claims priority to Australian Provisional Application No. 2019903530, which is hereby incorporated by reference in its entirety.

[0175] To illustrate the nature of the invention more clearly for understanding, the following non-limiting examples are provided.

Examples

[0176] Materials and Methods Mouse APP23 mice expressing human K670N / M671L mutant APP in neurons (Sturchler-Pierrat et al., 1997), ALZ17 mice expressing human unmutated tau in neurons (Probst et al., 2000), tau - / - (Tucker et al., 2001), p38γ - / - {Perdiguero, 2007#164} Constitutively active p38γ in mouse neurons CA Transgenic mice expressing the P301S mutant human tau (Ittner et al., 2016) and transgenic Tau58 mice expressing the P301S mutant human tau in the brain (van Eersel et al., 2015) have been previously described. All strains were maintained in a C57Bl / 6 background. Animal experiments were approved by the Animal Ethics Committee of the University of New South Wales. Mouse genotyping was determined by polymerase chain reaction using isopropanol-precipitated DNA from tail biopsies as a template. Oligonucleotide primers for target allele and transgene genotyping by polymerase chain reaction (PCR) are shown in Table 1.

[0177] Genome editing The mouse Mapt gene was targeted using CRISPR / Cas9 as previously described (Delerue and Ittner, 2017; Yang et al., 2014). Briefly, two guides targeting codon T194 (homologous to human codon T205) of the endogenous mouse Mapt gene were designed using the computational tool {Ran, 2013#417}(http: / / crispr.mit.edu). These single guide RNAs (sgRNAs) were generated using a non-cloning method, and a linear template was generated by PCR using T7 conjugate forward primers. The pX330 sgRNA scaffold (Addgene #42230, donated by Dr. Feng Zhang) was used as the template. The resulting linear DNA was transcribed in vitro to sgRNA using the T7 Quick High Yield RNA Synthesis Kit, following the manufacturer's instructions (NEB #E2050S). sgRNA was purified using a NucAway Spin column (ThermoFisher #AM10070). Pronuclear injection of Cas9 protein (NEB #M0646T), sgRNA, and donor single-strand oligonucleotides (ssOligos) into C57Bl / 6 zygotes resulted in the birth of live pups. Initial identification of the founding line was performed by sequencing the individual alleles of each pup through cloning. Briefly, the sequence of Mapt exon 9 (ENSEMBL ENSMUST00000106989.2) was amplified from mouse DNA by PCR and cloned into pBluescript (Stratagene) using a HiFi Assembly (New England Biolabs). At least five clones per pup were sequenced to identify potential founding lines exhibiting correct codon exchange at threonine-194. The established lines in which codon exchange was confirmed were crossed with C57Bl / 6 mice to establish the tauT205A and tauT205E lines, respectively.The genotypes of tauT205A and tauT205E mice were determined using DNA precipitated with isopropanol from tail biopsies as templates for tetraprimer ARMS-PCR {Ye, 2001 #395}. Oligonucleotide primers for tetraprimer ARMS PCR were designed using Primer1 (http: / / primer1.soton.ac.uk / primer1.html). Table 1 shows the sequences of the guide RNA, the homologous repair template for codon exchange at T205 in Mapt, and the oligonucleotide primers for tetraprimer ARMS PCR.

[0178] [Table 1]

[0179] Memory testing Spatial learning / memory was tested using the Morris Water Maze (MWM) paradigm {Vorhees, 2006#35; Ittner, 2016#217; Tan, 2018#390}. Briefly, in a room with low-light indirect lighting, a custom-made water tank for a mouse MWM (122 cm in diameter, 50 cm high) with a white, non-reflective inner surface was filled with water (19-22°C) containing diluted, non-irritating white dye. Four different peripheral cues were placed around the tank in the vertical positions of four quadrants. In the target quadrant (Q1), a platform (10 cm) was used. 2The mice were submerged 1 cm below the water surface. Video was recorded with a CCD camera and analyzed using AnyMaze software. For spatial acquisition, four trials were performed at 60-second intervals per session. The starting position was randomized along the outer edge of the starting quadrant in all trials. To test reference memory, probe trials without the platform were performed for 60-second trial periods, and the time spent within each quadrant was recorded and analyzed. For visually-cued control acquisition (to exclude visual impairment), markers were attached to the top of the platform, and four trials (60 seconds) were performed per session. All mice were age and sex matched and tested at 4 months of age. Mice exhibiting persistent floating behavior were excluded. Genotype was not disclosed to the staff recording the tests and analyzing the video tracks. Swimming paths were tracked using AnyMaze software (Stolting). Average swimming speed was determined to exclude motor impairment.

[0180] Behavioral testing Mice were tested in an elevated cusp maze for 10 minutes to assess disinhibition and anxiety using a previously published standard protocol (Ke et al., 2015).

[0181] seizure As previously described (Ittner et al., 2010), seizures were induced with pentylenetetrazole (PTZ, Sigma-Aldrich). Briefly, PTZ was administered intraperitoneally at 30 or 50 mg / kg body weight. Seizures were evaluated as follows: 0, no seizure; 1, immobility; 2, tail extension; 3, forelimb clonus; 4, generalized clonus; 5, bouncing seizure; 6, complete extension; 7, status epilepticus.

[0182] Plasmid construct Table 2 shows oligonucleotide primers for generating plasmid constructs using PCR. The construct for generating AAV particles is pAAV-hsyn1-eGFP-WPRE (Addgene #58867) based. The eGFP coding sequence was generated using HiFi Assembly (New England Biolabs) as described in Ittner et al., 2016, for P38γ CA The coding sequence (p38γAsp179Ala), tauWT (human tau40 441 amino acid), or tauT205A was substituted. Tau constructs with codon exchange T205A have been previously described (Ittner et al., 2016). All plasmids were amplified with E. coli DH5α or XL-1blue. To avoid recombination events, the AAV vector was grown with E. coli Stbl3. All constructs were validated by sequencing.

[0183] [Table 2]

[0184] Production and administration of adeno-associated virus The packaging of AAV vectors was performed as described {Harasta, 2015 #80}. Briefly, to package AAV particles, 293T cells were seeded at 70 - 80% confluence in complete DMEM (Sigma) containing 10% FBS. Three hours before transfection, the medium was changed to IMDM (Sigma) containing 5% FBS. Using polyethyleneimine-Max (PEI-Max, Polysciences) as the transfection reagent, cells were transfected with a plasmid containing the viral genome, pFdelta6 as the helper plasmid, and the AAV-PHP.B plasmid containing the rep and cap sequences. Cells and supernatant were harvested 72 hours after transfection. The supernatant was clarified by adding 40% PEG8000 / 2.5M NaCl to a final concentration of 8% PEG8000 / 0.5M NaCl and incubated at 4°C for at least 2 hours. The clarified supernatant was centrifuged at 2000g for 30 minutes. The combined precipitate from the clarified supernatant and cell pellet was treated with sodium deoxycholate (final concentration 0.5%) and benzonase (~500 U) at 37°C for 40 minutes. NaCl was added, incubated at 56°C for 40 minutes, freeze-thawed, and then the solution was centrifuged at 50000g at 4°C for 30 minutes. The supernatant was purified using an iodixanol gradient by ultracentrifugation (475,900g, 2 hours at 18°C). AAV particles were concentrated and exchanged into PBS at 5000g at 4°C using an Amicon 100kDa 15 mL concentrator. After titration by qPCR, aliquots were stored at -80°C. Titers were determined by quantitative polymerase chain reaction (qPCR). The AAV titers (in viral genomes per ml) were as follows: AAV-PHP.B-syn1-eGFP(1.46×10 14 ), AAV-PHP.B-syn1-p38γCA(1.37×10 14 ), AAV-PHP.B-syn1-tauWT(8.42×10 13 ), AAV-PHP.B-syn1-tauT205A(2.80×10 13). On day 0 (P0) after birth, administer undiluted AAV or intravenously (iv) diluted with sterile saline (0.9% NaCl). For P0 injection, administer 1 μl (1 × 10⁻⁶). 9 AAV particles (viral particles) were injected into three sites on both sides of the brain of cryo-anesthetized neonatal mice, as described (Ittner et al., 2016). For systemic delivery, 100 μl of AAV particle solution (10 11 or 10 13 Either virion particles / ml or one of these was injected into the tail vein of a mouse.

[0185] Mouse brain lysates and immunoblotting Mouse cortical tissue was extracted after transcardiac perfusion with phosphate-buffered saline (PBS pH 7.4). The cortical tissue was homogenized on ice using a Downs homogenizer (Heidolph) with RIPA buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM glycerophosphate, 2.5 mM Na2H2P2O7, 1 mM PMSF, protease inhibitor (Complete, Roche), 1% NP-40 substitute (Sigma-Aldrich), 0.1% SDS, 0.5% sodium deoxycholate). The lysate was removed by centrifugation (16,000 × g / 10 min / 4°C). Protein concentrations were measured (DC protein assay, BioRad). Western blotting was performed as previously described (Ittner et al., 2012). The bands were visualized by chemiluminescence using X-ray film or a digital imaging system (ChemiDoc MP, Biorad). Concentration quantification of Western blot results was performed using ImageJ 2.0.0-rc-49 / 1.51d (NIH). The antibodies used in this study were as follows: anti-PSD95 (Millipore), anti-Aβ (6E10), anti-tau (DAKO), anti-tau (tau-1, Millipore), anti-tau (tau-13, Abcam), anti-phosphothreonine 205 tau (Abcam), anti-phosphoserine 214 tau (Millipore), anti-glyceraldehyde dehydrogenase (anti-GAPDH, Millipore), anti-p38γ (R&D), anti-HA7 (Sigma), anti-HA (Cell Signaling Technologies), anti-SNAP25 (Millipore), and anti-neuronal filament (NF200, Abcam).

[0186] Immunoprecipitation As previously described (Ittner et al., 2010), immunoprecipitation was performed from tissue lysates. Briefly, cortical or hippocampal tissue was homogenized on ice in RIPA buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM glycerophosphate, 2.5 mM Na2H2P2O7, 1 mM PMSF, protease inhibitor (Complete, Roche), 1% NP-40 substitute (Sigma-Aldrich), 0.1% SDS, 0.5% sodium deoxycholate). Lysates were cleared by centrifugation (16,000×g / 10 min / 4 °C). Protein concentration was measured (DC protein assay, BioRad), and 200 μg of lysate was incubated with antibody (1:400) on a rotor at 4 °C for 3 h. Equilibrated and blocked protein G beads (New England Biolabs) were incubated with the lysate on a rotor at 4 °C for 45 min. Next, the beads were washed three times and incubated in sample buffer at 95 °C for 5 min prior to SDS-PAGE. Quantitative densitometric analysis was performed using ImageJ 2.0.0-rc-49 / 1.51d (NIH).

[0187] Tissue Sections and Staining Mice were perfused transcardially with phosphate-buffered saline, followed by perfusion with 4% paraformaldehyde (PFA), and fixed overnight with 4% PFA. Tissues were processed with Excelsior tissue processor (Thermo) for paraffin embedding. Silver staining (Gallyas) was performed to visualize tau aggregates and NFT-like structures in the brains of tau transgenic mice as previously described (Ittner et al., 2015). Brain sections of AAV-injected mice were stained with primary antibodies against tau (Tau13; Abcam) or HA tag (HA-7; Sigma-Aldrich) to visualize the expression of the viral transgene. All tissue sections were imaged using a BX51 bright-field / epifluorescence microscope equipped with a DP70 color camera (Olympus) (UPlanFL N lenses [∞ / 0.17 / FN26.5]: 10× / 0.3, 20× / 0.5, 40× / 0.75, 60× / 1.25 oil, and 100× / 1.3 oil).

[0188] Immunofluorescence Immunofluorescence staining of histological tissue sections was performed as previously described (Ittner et al., 2010). Briefly, tissue sections were deparaffinized, rehydrated, washed with phosphate-buffered saline (PBS), treated with 0.02% NP-40 in PBS, and blocked with blocking buffer (3% horse serum / 1% bovine albumin in PBS). Primary antibodies diluted in blocking buffer were incubated overnight at 4°C or at room temperature for 1 hour. After washing with PBS, secondary antibodies diluted in blocking buffer, with or without the addition of DAPI to visualize cell nuclei, were incubated at room temperature for 1 hour. Cells were then washed and mounted using a bleed-preventing mounting medium (Prolong Gold, Life Technologies). The secondary antibodies used were conjugated to Alexa 488, 555, 568, or 647 dyes (Molecular Probes). Epifluorescence imaging was performed using CellSens software (Olympus) with a BX51 brightfield / epifluorescence microscope equipped with a DP70 color camera (Olympus) (UPlanFL N lenses [∞ / 0.17 / FN26.5]: 10× / 0.3, 20× / 0.5, 40× / 0.75, 60× / 1.25 oil, and 100× / 1.3 oil). Silver staining of histological mouse brain sections (8 μm) was performed as previously described (Ittner et al., 2015). The number and size of amyloid plaques were determined by micrographs of cortical sections with immunofluorescence staining for amyloid-β(6E10) using Image J (https: / / imagej.nih.gov / ij / ). Plaque counts were normalized against tissue surface area.

[0189] statistical analysis All statistical analyses were performed using GraphpadPrism version 6.0. Student's t-tests were performed for pairwise comparisons, and multiple data groups were analyzed by ANOVA. Linear regression and correlation analyses were performed by minimizing the sum of squares. Survival data were analyzed by the Mantel-Cox test of log-rank. All values ​​are expressed as mean ± standard error of the mean (SEM).

[0190] result In one example, the inventors used an APP23 mouse model of Alzheimer's disease and performed transgenic expression of human mutant amyloid-β precursor protein (APP) at 13 months of age. In this model, the mice developed severe memory impairment, and constitutive activity p38γ was used as a control. CA They were treated with different titers of AAV for neuronal expression of either (10) or enhanced green fluorescent protein (eGFP) (Figure 1a). Memory ability was tested in 15-month-old treated mice (Figure 1a). Low (10 10 AAV particles) or high (10 12 p38γ in the brains of APP23 mice treated with AAV particles (AAV titer) CA The expression of p38γ CA The HA tag fused to the Aβ was visualized by staining (Figure 1b). The staining pattern of Aβ visualized with the 6E10 antibody was p38γ CA The expression of p38γ did not change. This indicates that p38γ was not altered in the brains of APP23 mice upon systemic AAV delivery. CA The successful expression of [the substance] was confirmed.

[0191] Memory assessment of 15-month-old mice in Morris water maze (MWM) showed superior memory impairment compared to AAV-p38γ treated APP23 littermates. CA The memory performance of APP23 mice treated with a low titer of AAV-p38γ was significantly improved (Figures 1c-g). CA APP23 mice treated with AAV-p38γ showed similar memory formation ages to those of non-transgenic control mice. In summary, these results suggest that low titer AAV-p38γ CA The study shows that treating AD mice resulted in efficient recovery of memory impairment as they aged.

[0192] Similarly, AAV-p38γ with higher titers CAAlternatively, memory assessments of mice treated with AAV-eGFP showed an efficient reversal of pre-existing memory impairment in APP23 mice (Figures 1h-l). Therefore, high titer AAV-p38γ CA Treatment of aged APP23 mice with AAV-p38γ CA Alternatively, APP23 mice treated with AAV-eGFP showed comparable memory formation and enhancement to non-transgenic mice, but APP23 mice treated with AAV-eGFP showed a persistent decline in memory performance during the MWM test. In summary, these results suggest that high titer AAV-p38γ may not be suitable. CA The study shows that treating AD mice resulted in efficient recovery of memory impairment as they aged.

[0193] In another example, the inventors developed a human non-mutant tau transgenic Alz17 mouse with p38γ-deficient p38γ - / - It was crossbred with a mouse (Figure 2a). This resulted in Alz17.p38γ + / + Compared to mice, Alz17.p38γ - / - As visualized by immunohistochemical staining for endogenous p38γ and tau in the cortex, Alz17.p38γ - / - p38γ was absent from the mouse brain (Figure 2b). As a result, Alz17.p38γ - / - Successful depletion of p38γ in the mouse brain was confirmed.

[0194] Alz17.p38γ in MWM - / - and Alz17.p38γ + / + Memory evaluation in mice revealed Alz17.p38γ + / + It was confirmed that the mice did not have memory impairment, p38γ + / + and p38γ - / - Memory formation and reinforcement were observed to be comparable to that of littermates (Figures 2c-f). In contrast, Alz17.p38γ - / - The mouse is Alz17.p38γ + / +Compared to mice, the mice showed significantly delayed memory formation and significantly reduced memory integration. This indicates that p38γ limits the toxic effects of increased levels of human hyperphosphorylated tau in the absence of Aβ in a mouse model of tauopathy.

[0195] In another example, the inventors modified the endogenous mouse Mapt gene encoding tau using CRSPR / Cas9 gene editing technology to introduce T205E or T205A mutations into different mouse strains (Figure 3a). The resulting strains were homozygous for T205E / E and T205A / A mice, respectively. The success of genome editing was confirmed by sequencing of genomic DNA obtained from both strains and compared with non-mutant T205T / T mice (Figure 3b). The success of genome editing of T205A / A, involving amino acid exchange in translated mouse protein, was confirmed by immunoprecipitation using an antibody specific to tau phosphorylated with T205 (Figure 3c). In summary, the inventors generated two novel mouse strains, T205E / E and T205A / A, expressing mutant tau.

[0196] To determine the functional effects of the T205 mutation in endogenous tau protein, both T205E / E and T205A / A mice were subjected to an established excitotoxicity test paradigm for seizure induction. Compared to heterozygous T205T / E and non-mutant T205T / T controls, which rapidly developed and experienced more severe seizures upon administration of 50 mg / kg pentylenetetrazole, T205E / E mice showed a longer latency to the onset of more severe seizures and a reduced mean seizure severity (Figures 3d-e). Conversely, T205A / A mice showed a more rapid onset of more severe seizures and a significantly increased mean seizure severity compared to T205T / T littermates after injection of 30 mg / kg pentylenetetrazole (Figures 3f-g). In summary, these data indicate that the presence of T205E / E mutations that mimic phosphorylation reduces excitotoxic seizures, while T205A / A mutations that prevent phosphorylation enhance and increase the likelihood of triggering excitotoxic seizures. Therefore, these data demonstrate the protective effect of T205 taur phosphorylation against epilepsy in vivo.

[0197] In another example, the inventors crossed T205E / E and T205A / A mice with APP23 mice to determine the role of T205 taurination in AD-related memory impairment (Figure 4a). Immunostaining of mouse brains using antibodies against tau phosphorylated with T205 (pT205) and Aβ (6E10) confirmed the absence of T205 taurination in APP23.T205A / A mice (Figure 4b). APP23.T205E / E mice showed significantly improved survival rates compared to APP23.T205T / T animals, while APP23.T205A / A mice tended to have an even more accelerated mortality rate (Figure 4c). This data demonstrates the important role of T205 taurination in regulating survival in APP23 mice.

[0198] Memory tests using MWM revealed that memory formation and consolidation were even worse in APP23.T205A / A mice compared to memory impairment in APP23.T205T / T mice (Figures 4e-h). Conversely, APP23.T205E / E mice were prevented from developing the memory impairment seen in APP23.T205T / T mice (Figures 4i-l). Notably, T205A / A and T205E / E mice showed normal memory formation in MWM. This data suggests that tau phosphorylation at T205 limits memory impairment in APP23 mice, while it does not contribute to test paradigm memory in naive mice.

[0199] In another example, the inventors crossed APP23 mice with a tau-deficient background and further obtained APP23.tau - / - mice were transgenicized with p38γ in neurons. CA Cross with a line that expresses APP23.tau - / - .p38γ CA A mouse was created (Figure 5a). Next, APP23.tau - / - .p38γ CATau expression in mouse brains was reconstituted by injecting AAV into the brain at birth using tau variants, either non-mutant tau (tauWT) or antiphosphorylated T205A tau (tauT205A), and memory tests were subsequently performed at 6 months of age (Figure 5b). p38γ CA Immunofluorescence staining using HA tags and antibodies against human tau in the brain revealed that APP23.tau injected with AAV was detected. - / - .p38γ CA p38γ in mice CA The expression of and the successful rearrangement of tau were confirmed (Figure 5c).

[0200] APP23.tau injected with AAV - / - .p38γ CA Memory tests were performed on mice injected with AAV-tauWT. - / - .p38γ CA While improvements in mouse memory capacity were demonstrated, reconstruction using AAV-tauT205A was performed on APP23.tau - / - .p38γ CA We were unable to prevent memory impairment in mice (Figures 5d-f). For comparison, see APP23.tau - / - Neither AAV-tauWT nor AAV-tauT205A injection in mice (in the absence of p38γ) improved memory ability. - / - Mice were used as a baseline for performance on standard memory tasks. In summary, this data suggests that tau phosphorylation at T205 is associated with p38γ in AD mice. CA This indicates that it is necessary for the therapeutic effect.

[0201] In another example, the inventors have shown that human non-mutant tau transgenic Alz17 mice can develop p38γ in neurons. CA By crossing transgenic mice expressing Alz17.p38γ, CA The mice were brought in (Figure 8a). p38γ CA Immunofluorescence staining of the brain using antibodies against human tau HA tags revealed tau and p38γ in brain neurons. CACo-expression of Alz17.p38γ was observed (Figure 8b). Western blotting of synaptosome preparations from mouse brains showed Alz17.p38γ + / + Compared to the control, Alz17.p38γ CA We observed increased tau phosphorylation at postsynaptic T205 in mice (Figure 8c). In contrast, tau phosphorylation at T205 was also observed in Alz17.p38γ, which lacks postsynaptic p38γ. - / - It was no longer detectable in mice (Figure 8c). Detection of postsynaptic thickening protein 95 (PSD-95) and synaptosome-associated protein 25 (SNAP25) confirmed that synaptosomes were equally enriched during preparation. p38γ was Alz17.p38γ + / + and Alz17.p38γ CA It was present in mouse synaptosomes, but Alz17.p38γ - / - It was not present in mouse synaptosomes and was consistent with their genotypes. Immunofluorescence staining of the brain using antibodies against tau and tau phosphorylated with T205 showed Alz17.p38γ compared to Alz17 mice. CA We revealed increased T205 taur phosphorylation in mice (Figure 8d). However, silver staining of the brains of aged mice showed Alz17.p38γ compared to Alz17 littermates. CA No significant increase in neurofibrillary tangles (NFTs) was observed in the mouse brain (Figure 8e). Alz17.p38γ in MWM CA Memory tests in mice showed no memory impairment, and Alz17 or p38γ were observed. CA Alternatively, performance indistinguishable from non-transgenic controls was observed (Figures 8f-h). In summary, these data demonstrate p38γ in Alz17 mice. CA The study shows that expression increased T205 tau phosphorylation, including postsynaptic levels, but did not accelerate tau pathology or cause memory impairment.

[0202] In another example, the inventors developed human P301S mutant tau transgenic Tau58 mice at 3 months of age to administer low and high titer therapeutic AAV-p38γ. CAAlternatively, they were treated with control AAV-GFP. Tau58 mice showed a disinhibition phenotype (= increased open-arm time) during the elevated cusp maze (EPM) test at 3 months of age, which worsened further over time. High titer AAV-p38γ CA Tau58 mice treated with AAV-GFP showed a reduction in open-arm time compared to 5-month-old Tau58 mice treated with AAV-GFP (Figure 11). Furthermore, AAV-p38γ CA Tau58 mice treated with p38γ showed a titer-dependent improvement in hyperactivity compared to Tau58 controls treated with AAV-GFP (Figures 12-13). In summary, this data suggests that p38γ CA We have shown that the expression of this gene efficiently reverses tau pathology-related disorders in a mouse model of tauopathy.

[0203] Effect of p38γ on tau phosphorylation and aggregation To investigate the effects of tau phosphorylation at T205 on tau phosphorylation and aggregation, TAU58 / 2 mice were subjected to AAV-p38γ CA Alternatively, they were treated with a control (AAV-GFP).

[0204] AAV-p38γ CA Serial brain extracts from TAU58 / 2 mice treated with p38γ and control (AAV-GFP) showed a clear lack of taur phosphorylation and insolubility upon treatment with p38γ.

[0205] TAU58 / 2 mice, which express the P301S mutant tau in brain neurons and develop a progressive tau pathology similar to human Alzheimer's disease and frontotemporal dementia, were given p38γ for neuronal expression at 3 months of age. CAIntravenous injection was administered either AAV (operably linked to the human synapsin promoter) encoding or green fluorescent protein (GFP) as a control. At 11 months of age, all brains were analyzed by serial extraction using stringency-enhancing buffers (RAB (0.1M Mes / 1mM EGTA / 0.5mM MgSO4, 750mM NaCl, 20mM NaF, 1mM Na3VO4, 0.1% Roche protease inhibitor, pH 7.4), RIPA (50mM Tris / 150mM NaCl, 1% nonidet P-40 / 5mM EDTA / 0.5% sodium deoxycholate / 0.1% SDS, pH 8.0), and 70% formic acid) (RAB>RIPA>FA) to obtain soluble, intermediately soluble, and insoluble proteins, respectively. The extracts were analyzed by Western blotting using HA, GTP, Tau13, GADPH, and pTauS422 antibodies. p38γ was analyzed using the HA antibody. CA The expression of p38γ was confirmed, and controls were identified using GFP antibodies. The results are shown in Figure 19. Whole human transgenic tau was detected with Tau13 antibody, and phosphorylated tau was detected with site-specific antibodies (pTauT205 and pTauS422). Changes in tau levels (i.e., increases), p38γ CA No phosphorylation or insolubility of tau was observed during expression, confirming that AAV-p38gCA treatment does not accelerate the progression of tau pathology. In fact, phosphorylation of insoluble tau at the late-state disease epitope pTau S422 was observed with AAV-p38γ. CA It was found that the insoluble fraction of TAU58 / 2 mice treated with this drug was reduced.

[0206] Phosphorylation of tau at serine-422 (pS422) is a disease-related modification, and tau has been found to be increasedly phosphorylated at serine-422 in Alzheimer's disease and other tauopathies. The appearance of the pS422 epitope is strongly correlated with the formation of neurofibrillary tangles and cognitive decline. Therefore, given the close association between pS422 and cognitive decline, the ability of P38γ to reduce pTauS422 in the insoluble fraction of tau from TAU58 / 2 mouse brains suggests that p38γ can reverse or reduce tauopathies associated with or mediated by serine-422 phosphorylation of tau in brain neurons, as well as cognitive decline.

[0207] In summary, this data is p38γ CA The expression of this gene has been shown to prevent or reduce the progression of tau disease.

[0208] References Delerue, F., and Ittner, LM (2017). Generation of Genetically Modified Mice through the Microinjection of Oocytes. J Vis Exp. Ittner, A., Bertz, J., Suh, LS, Stevens, CH, Gotz, J., and Ittner, LM (2015). Tau-targeting passive immunization modulates aspects of pathology in tau transgenic mice. J Neurochem 132, 135-145. Ittner, A., Block, H., Reichel, C.A., Varjosalo, M., Gehart, H., Sumara, G., Gstaiger, M., Krombach, F., Zarbock, A., and Ricci, R. (2012). Regulation of PTEN activity by p38delta-PKD1 signaling in neutrophils confers inflammatory responses in the lung. J Exp Med 209, 2229-2246. Ittner, A., Chua, S.W., Bertz, J., Volkerling, A., van der Hoven, J., Gladbach, A., Przybyla, M., Bi, M., van Hummel, A., Stevens, C.H., et al. (2016). Site-specific phosphorylation of tau inhibits amyloid-beta toxicity in Alzheimer's mice. Science 354, 904-908. Ittner, L.M., Ke, Y.D., Delerue, F., Bi, M., Gladbach, A., van Eersel, J., Wolfing, H., Chieng, B.C., Christie, M.J., Napier, I.A., et al. (2010). Dendritic Function of Tau Mediates Amyloid-beta Toxicity in Alzheimer's Disease Mouse Models. Cell 142, 387-397. Ke, Y.D., van Hummel, A., Stevens, C.H., Gladbach, A., Ippati, S., Bi, M., Lee, W.S., Kruger, S., van der Hoven, J., Volkerling, A., et al. (2015). Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS. Acta Neuropathol 130, 661-678. Probst, A., Gotz, J., Wiederhold, K.H., Tolnay, M., Mistl, C., Jaton, A.L., Hong, M., Ishihara, T., Lee, V.M., Trojanowski, J.Q., et al. (2000). Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein. Acta Neuropathol (Berl) 99, 469-481. Sturchler-Pierrat, C., Abramowski, D., Duke, M., Wiederhold, K.H., Mistl, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P.A., et al. (1997). Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci U S A 94, 13287-13292. Tucker, K.L., Meyer, M., and Barde, Y.A. (2001). Neurotrophins are required for nerve growth during development. Nat Neurosci 4, 29-37. van Eersel, J., Stevens, C.H., Przybyla, M., Gladbach, A., Stefanoska, K., Chan, C.K., Ong, W.Y., Hodges, J.R., Sutherland, G.T., Kril, J.J., et al. (2015). Early-onset axonal pathology in a novel P301S-Tau transgenic mouse model of frontotemporal lobar degeneration. Neuropathology and applied neurobiology 41, 906-925. Yang, H., Wang, H., and Jaenisch, R. (2014). Generating genetically modified mice using CRISPR / Cas-mediated genome engineering. Nat Protoc 9, 1956-1968.

Claims

1. A pharmaceutical composition for treating a disease mediated by the aggregation of excessive phosphorylated tau in the brain of a target disease, the disease being selected from progressive Alzheimer's disease, progressive frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, primary age-related tauopathy, chronic traumatic encephalopathy, frontotemporal dementia with parkinsonism linked to chromosome 17, Pick's disease, globular glial tauopathy, or Parkinson's disease, wherein the pharmaceutical composition comprises a drug, the drug is (a) Promoting the phosphorylation of tau at threonine of the tau sequence SSPGSPGTPGSRR, wherein the agent comprises p38γ or a variant thereof, or a nucleic acid capable of expressing p38γ or a variant thereof in the target neuron, wherein the variant of p38γ comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3; and / or (b) Introducing a phosphomimetic of phosphorylated tau, where phosphorylated tau is tau phosphorylated with threonine in the tau sequence SSPGSPGTPGSRR, where the phosphomimetic includes tau in which the threonine at position 205 of human tau is substituted with glutamic acid, and The agent used to introduce the phosphomimetic form of phosphorylated tau is a tau gene editing system that introduces a substitution from threonine to glutamate at position 205 of human tau within the tau gene in the target neuron. Pharmaceutical composition.

2. A pharmaceutical composition according to claim 1, wherein the cognitive ability of the subject is improved by the treatment.

3. The pharmaceutical composition according to claim 2, wherein the improvement in cognitive ability is an improvement in memory.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the disease is related to the phosphorylation of serine at position 422 of human tau.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein p38γ comprises the amino acid sequence of SEQ ID NO:

3.

6. The variants of p38γ are ETPL, KETPL, SKETPL, VSKETPL, RVSKETPL, ARVSKETPL, GARVSKETPL, LGARVSKETPL, QLGARVSKETPL, RQLGARVSKETPL, PRQLGARVSKETPL, PPRQLGARVSKETPL, KPPRQLGARVSKETPL, FKPPRQLGARVSKETPL, SFKPPRQLGARVSKETPL, LSFKPPRQLGARVSKETPL, VLSFKPPRQLGARVSKETPL, EVLSFKPPRQLGARVSKETPL, KEVLSFKPPRQLGARVSKETPL, YKEVLSFKPPRQLGARVSKETPL, TYKEVLSFKPPRQLGA RVSKETPL, VTYKEVLSFKPPRQLGARVSKETPL, RVTYKEVLSFKPPRQLGARVSKETPL, KRVTYKEVLSFKPPRQLGARVSKETPL, ETAL, KETAL, PKETAL A pharmaceutical composition according to any one of claims 1 to 5, comprising an amino acid sequence selected from the group consisting of VPKETAL, RVPKETAL, ARVPKETAL, GARVPKETAL, LGARVPKETAL, QLGARVPKETAL, RQLGARVPKETAL, PRQLGARVPKETAL, PPRQLGARVPKETAL, KPPRQLGARVPKETAL, FKPPRQLGARVPKETAL, SFKPPRQLGARVPKETAL, LSFKPPRQLGARVPKETAL, VLSFKPPRQLGARVPKETAL, EVLSFKPPRQLGARVPKETAL, KEVLSFKPPRQLGARVPKETAL, YKEVLSFKPPRQLGARVPKETAL, TYKEVLSFKPPRQLGARVPKETAL, VTYKEVLSFKPPRQLGARVPKETAL, RVTYKEVLSFKPPRQLGARVPKETAL, and KRVTYKEVLSFKPPRQLGARVPKETAL.

7. The pharmaceutical composition according to claim 6, wherein the p38γ variant comprises the amino acid sequence KRVTYKEVLSFKPPRQLGARVSKETPL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the variant of p38γ is the constitutively active form of p38γ.

9. The constitutive active form of p38γ (p38γ CA The pharmaceutical composition according to claim 8, comprising Sequence ID No.

4.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the gene editing system is a CRISPR / Cas complex or a part thereof.

11. The pharmaceutical composition according to claim 10, wherein the CRISPR / Cas complex is a CRISPR / Cas9 complex.

Images