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WO2024211430A2 - Méthodes et compositions pour traiter des états neurodégénératifs - Google Patents

Méthodes et compositions pour traiter des états neurodégénératifs Download PDF

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Publication number
WO2024211430A2
WO2024211430A2 PCT/US2024/022860 US2024022860W WO2024211430A2 WO 2024211430 A2 WO2024211430 A2 WO 2024211430A2 US 2024022860 W US2024022860 W US 2024022860W WO 2024211430 A2 WO2024211430 A2 WO 2024211430A2
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inhibitor
aspects
map4k
map4k4
map4k7
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WO2024211430A3 (fr
Inventor
Chun-li ZHANG
Meng-lu LIU
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders

Definitions

  • the present disclosure relates generally to methods and compositions associated with neurodegenerative conditions or traumatic brain injury.
  • Neurodegenerative disease encompasses several distinct conditions characterized by progressive neuropathy and loss of neurons in motor, sensory, and/or cognitive systems.
  • Neurodegenerative diseases can include Amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Friedreich ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, Spinal muscular atrophy.
  • ALS Amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • Friedreich ataxia Huntington's disease
  • Lewy body disease Lewy body disease
  • Parkinson's disease Spinal muscular atrophy.
  • MNs motor neurons
  • Traumatic brain injury is a major cause of death and disability and is a complex disease process. TBI causes structural damage and functional deficits due to the damage of Blood-Brain Barrier, neuronal injury, neuroinflammation and tau pathology.
  • TBI Traumatic brain injury
  • Several studies indicate that TBI seems to be a risk factor for tauopathies, which has been described in the onset of Alzheimer disease and chronic traumatic encephalopathy, and there is a relationship of TBI severity and propensity to the development of these tauopathies.
  • More effective treatment neurodegenerative diseases and TBI are required, and thus there remains a need for discovery of potential therapeutic targets.
  • the disclosure provides a method of treating a neurodegenerative disease or brain injury in a subject in need thereof.
  • the method comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a citron homology domain (CNH) of MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the disclosure further provides a method of reducing a symptom associated with neurodegenerative disease or brain injury in a subject in need thereof.
  • the method comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the CNH or CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from a group consisting of SEQ ID NOs: 3-6.
  • the disclosure provided a composition
  • a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, and a pharmaceutically acceptable excipient.
  • the CNH or CNH- containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from SEQ ID NOs:3-6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding hSYN1 promoter and a nucleic acid sequence encoding a CNH, wherein the CNH is from MAP4K4, MAP4K6, MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the isolated nucleic acid sequence comprises the CNH or CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from SEQ ID NOs:3-6.
  • the current disclosure encompasses a method of treating a neurodegenerative disease or brain injury in a subject in need thereof comprising, administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a guide RNA (gRNA) comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the gRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 13-18.
  • gRNA guide RNA
  • the disclosure provides a method of reducing a symptom associated with neurodegenerative disease or brain injury in a subject in need thereof comprising, administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the gRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 13-18.
  • the current disclosure further encompasses a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising, administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the gRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 13-18.
  • the disclosure further provides a composition comprising, a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, wherein the gRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 13-18, and a pharmaceutically acceptable excipient.
  • an isolated nucleic acid sequence comprising, a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding hSYN1 promoter; and a nucleic acid sequence encoding gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the gRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 13-18.
  • a method of treating a neurodegenerative disease or brain injury comprising administering to a subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the shRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 8, 10, or 12.
  • the disclosure encompasses a method of reducing a symptom associated with neurodegenerative disease or brain injury comprising, administering to a subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the shRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 8, 10, or 12.
  • a method of providing protection to a subject from neural degeneration and/or neural injury comprising, administering to a subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the shRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 8, 10, or 12.
  • the disclosure provides a composition comprising, a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, wherein the shRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 8, 10, or 12, and a pharmaceutically acceptable excipient.
  • the disclosure provides an isolated nucleic acid sequence comprising, a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding hSYN1 , and a nucleic acid sequence encoding shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, and wherein the shRNA comprises a sequence selected from a group consisting of SEQ ID NOs: 8, 10, or 12.
  • the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia.
  • the brain injury comprises traumatic brain injury or stroke.
  • the inhibitor is administered as a recombinant adeno-associated virus (rAAV) vector encoding the said inhibitor.
  • the rAAV vector comprises AAV9 or AAV-PHP.eB capsid.
  • the AAV vector comprises a human synapsin I promoter (hSYN1 ).
  • the inhibitor is expressed ectopically in neuron or motor neuron cells of the subject.
  • the administration of the inhibitor reduces traumatic brain-induced tau phosphorylation, reactive gliosis, lesion size, behavioral deficits, and/or severity or progression of the neurodegenerative disease or brain injury, or improve brain tissue damage, improve memory and/or cognitive performance, improve motor function, improve neuronal survival and neurite outgrowth, and/or improve the life span of the subject.
  • the inhibitor is administered parenterally. In some aspects, the inhibitor is administered intrathecally.
  • the vector is a rAAV.
  • the vector is a rAAV comprising AAV9 or AAV-PHP.eB capsid.
  • the vector comprises sequence encoding a human synapsin I promoter (hSYN1 ) operably linked to the inhibitor.
  • the composition is administered parenterally. In some aspects, the composition is administered intrathecally.
  • the disclosure further comprises a host cell transduced with the any of the disclosed nucleic acid sequence provided herein.
  • the disclosure encompasses a method of treating a neurodegenerative disease or brain injury in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the isolated nucleic acid provided herein, to the subject in need thereof.
  • provided herein is a method of reducing a symptom associated with neurodegenerative disease or brain injury in a subject in need thereof, the method comprising, administering a therapeutically effective amount of any of the disclosed isolated nucleic acid, to the subject in need thereof.
  • a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising, the method comprising: administering a therapeutically effective amount of the disclosed isolated nucleic acid, to the subject in need thereof, is provided.
  • the isolated nucleic acid is administered parenterally. In some aspects, the isolated nucleic acid is administered intrathecally.
  • a cell based platform for screening compounds with neuroprotective effect comprising a host cell disclosed herein.
  • the disclosure further encompasses a method of treating a neurodegenerative disease or brain injury in a subject in need thereof comprising: administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, wherein the inhibitor of MAP4K is K02288.
  • a method of reducing a symptom associated with neurodegenerative disease or brain injury in a subject in need thereof comprising: administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, wherein the inhibitor of MAP4K is K02288.
  • the disclosure encompasses a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, wherein the inhibitor of MAP4K is K02288.
  • the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia.
  • the brain injury comprises traumatic brain injury or stroke.
  • FIG. 1A-1J illustrate screens for chemicals improving survival of ALS-hiMNs.
  • FIG. 1A depicts flowchart of the screening procedure. Highly pure (>85%) ALS-hiMNs could be obtained after a replating procedure and passing through a cell strainer based on differential plate-attachment and cell size. The non-converted fibroblasts (indicated by an arrow or arrowhead) were larger and attached faster than the converted cells (indicated by a white arrow). An ATP-based assay was used to measure cell numbers. Veh (DMSO) and Ken (Kenpaullone) served as negative and positive controls, respectively. Scale bar, 100 pm.
  • FIG. 1 B illustrates scatter plots of primary screens.
  • FIG. 1C shows scatter plots of secondary screens. Top 65 hits from primary screens, together with 23 additional inhibitors targeting ALK, TGFp, and GSK3, were examined at four different concentrations.
  • FIG. 1D-1H illustrate doseresponse curves. Top 15 hits from secondary screens, together with the indicated additional chemicals, were examined at 7 concentrations.
  • FIG. 11 depicts typical morphology of ALS- hiMNs treated with the indicated chemicals. Hit3 promoted neurite outgrowth when compared to the other chemicals. Scale bar, 100 pm.
  • FIG. 1J illustrates survival of ALS-hiMNs when cocultured with astrocytes and treated with the indicated chemicals. Red line indicates the effect of the positive control Ken.
  • FIG. 2A-2C exhibit survival effects of the selected chemicals on ALS-hiMNs.
  • FIG. 2A illustrates low magnification images of ALS-hiMNs treated with the indicated chemicals. Scale bar, 100 pm.
  • FIG. 2B shows the effect of GSK-3 inhibitors on survival of ALS-hiMNs.
  • FIG. 2C illustrates survival effect on ALS-hiMNs by top15 hits, including six inhibitors of GSK- 3 (Hit1 , 1-Azakenpaullone; Hit4, AZD1080; Hit5, AZD2858; Hit6, SB216763; Hit10, CHIR- 99021 ; Hit13, BIO)
  • FIG. 3A-3F illustrate Hit3 improves survival and function of hiMNs from diverse human patients.
  • FIG. 3A depicts dose-dependent effects of the indicated chemicals on diverse hiMNs.
  • FIG. 3B illustrates Hit3 outperforms the positive control Ken, in promoting survival of diverse hiMNs co-cultured with astrocytes.
  • FIG. 3C illustrates Hit3 and the positive control Ken increased neuronal soma size and complexity of FUS-hiMNs and SOD1-hiMNs. Scale bar, 50 pm.
  • FIGs. 3E- 3F illustrate Hit3 and the positive control Ken rescued the ability of ALS-hiMNs (E, FUS-hiMNs; F, SOD1-hiMNs) to form neuromuscular junctions (NMJs, indicated by arrows) on co-cultured myotubes. NMJ frequencies were indicated as percentage values over 100 hiMN network- associated myotubes counted for each group. Scale bar, 10 pm.
  • FIG. 4A-4D depict MAP4K inhibition promotes survival of ALS-hiMNs.
  • FIG. 4A illustrates a lack of survival effect on ALS-hiMNs by ALK inhibitors.
  • FIGs. 4B-4C exhibit dosedependent effect of the indicated chemicals on survival of ALS-hiMNs with or without cocultured astrocytes. Ken, kenpaullone; MAP4Ki, PF-06260933.
  • FIG. 4D depicts morphological changes of ALS-hiMNs under the indicated conditions. Genes were downregulated via sgRNAs and CRISPR-Cas9. H, HGK; M, MINK1 ; T, TNIK. Scale bar, 100 pm.
  • FIG. 5A-5J show MAP4Ks are targets of Hit3 for improving ALS-hiMNs.
  • FIG. 5A depicts western-blotting analysis of knocking down MAP4Ks. HA-tagged MAP4Ks were coexpressed with sgRNAs and Cas9 in human fibroblasts and examined 10 days later.
  • FIGs. 5B-5C depict knockdowns of MAP4Ks improve survival of ALS-hiMNs, examined at 1 week or 3 weeks post replating on astrocytes (mean ⁇ SEM; n 4 independent samples; *p ⁇ 0.05, **p ⁇ 0.01 , and ***p ⁇ 0.001 when compared to the control sgLacZA/eh group).
  • FIG. 5A depicts western-blotting analysis of knocking down MAP4Ks. HA-tagged MAP4Ks were coexpressed with sgRNAs and Cas9 in human fibroblasts and examined 10 days later.
  • FIG. 5D exhibits western-blotting analysis of ectopic MAP4Ks or their kinase-dead mutants in ALS- hiMNs.
  • TUJ1 and GAPDH are loading controls.
  • FIG. 5E illustrates kinase-dead mutants of MAP4Ks improve survival of ALS-hiMNs.
  • FIG. 5F shows westernblotting analysis of ectopic MAP4K mutants in ALS-hiMNs. EV, empty vector.
  • FIG. 5J shows a schematic showing how Hit3 improves survival of ALS-hiMNs.
  • FIG. 6A-6G illustrate CNH domain of MINK1 improves survival of ALS-hiMNs.
  • FIG. 6A depicts schematic diagrams of MINK1 protein and its truncations. The point mutation, K54R, renders MINK1 inactive.
  • FIG. 6B illustrates western blotting analysis of ectopic HA- tagged proteins in ALS-hiMNs purified at 14 dpi.
  • FIGs. 6C-6D show the CNH-containing domain (866C: aa866-1312) is sufficient to improve survival of ALS-hiMNs.
  • FIG. 6E-6F illustrate the CNH domain (960C: aa960-1312) mimics Hit3’s effect on survival of ALS-hiMNs.
  • FIG. 6G depicts the kinase-dead MINK mutant and its functional domains recapitulated the effect of Hit3 on morphology of ALS-hiMNs 1-week post replating. Scale bar, 100 pm.
  • FIG. 7A-7B show a lack of protective effect of p38 or JNK inhibitors on ALS-hiMNs.
  • FIG. 7A depicts survival assays of ALS-hiMNs treated with the indicated inhibitors.
  • FIG. 7B illustrates effect of the selected chemicals on morphology of ALS-hiMNs.
  • MAP4Ki PF- 06260933
  • p38i p38 inhibitor SB203580
  • JNKi JNK inhibitor SP600125.
  • FIG. 8A-8I show MAP4K interactome and the effect on RANGAP1 subcellular distribution.
  • FIG. 8A depicts western-blotting analysis of proteins after proximity labeling in ALS-hiMNs at 10 dpi.
  • FIG. 8B shows venn diagram of proteins with >2.5-fold enrichment in two biological repeats.
  • FIG. 8C illustrates the top 5 KEGG pathways.
  • FIG. 8D exhibits STRING analysis of association networks of proteins in the top 4 KEGG pathways.
  • FIG. 8E shows validation of protein associations by co-immunoprecipitations (co-IP) and western blots.
  • FIG. 8F depicts co-IP assays showing association of MINKI mt with RANGAP1 or RAN.
  • FIG. 8G illustrates confocal images showing subcellular distribution of RANGAP1 or RAN in ALS- hiMNs. Arrows indicate aggregated cytoplasmic RANGAP1 foci. Scale bar, 10 pm.
  • FIG. 9A-9I illustrate Hit3 improves nucleocytoplasmic transport in ALS-hiMNs.
  • FIG. 9C shows Hit3 improves nuclear localization of RANGAP1 in hiMNs (n 30 neurons per group; *p ⁇ 0.05 and ***p ⁇ 0.001).
  • FIG. 9E depicts confocal images of FUS in ALS-hiMNs. Scale bar, 10 pm.
  • FIGs. 9G-9H illustrates confocal images of TDP-43 expression in NL- or ALS-hiMNs. Scale bar, 10 pm.
  • FIG. 9I shows Hit3 improves nuclear fraction of TDP-43 in hiMNs (n > 28 neurons per group; *p ⁇ 0.05 and ***p ⁇ 0.001).
  • FIG. 10A-10C illustrate downregulation of MAP4Ks, resembling Hit3 treatments, promotes nuclear localization of key proteins in ALS-hiMNs.
  • FIG. 10A depicts qRT-PCR analysis of shRNA-mediated knockdowns in human fibroblasts.
  • FIG. 10B shows confocal images of RANGAP1 and TDP-43 in ALS-hiMNs cocultured with astrocytes at 51 dpi. The soma and nucleus are outlined. Scale bar, 10 pm.
  • FIG. 10A-10C illustrate downregulation of MAP4Ks, resembling Hit3 treatments, promotes nuclear localization of key proteins in ALS-hiMNs.
  • FIG. 10A depicts qRT-PCR analysis of shRNA-mediated knockdowns in human fibroblasts.
  • FIG. 10B shows confocal images of RANGAP1 and TDP-43 in ALS-hiMNs cocultured with astrocytes at 51 d
  • 10C shows downregulation of MAP4Ks, like Hit3 treatments, improves nuclear localization of the indicated proteins in ALS- hiMNs (mean ⁇ SEM; **p ⁇ 0.01 , ***p ⁇ 0.001 , and ****p ⁇ 0.0001).
  • FIG. 11A-11F shows neither RANGAP1 nor TUBA4A is phosphorylated by MAP4Ks.
  • FIG. 11A shows Phos-tag SDS-PAGE and western blots failed to detect phosphorylation of RANGAP1 by MAP4Ks.
  • FIG. 11B depicts Co-IP results showing interactions between TUBA4A and HGK or MINK1 or their kinase-dead mutants.
  • FIGs. 11C- 11D show pIMAGO kit or pThr phospho-antibody failed to detect phosphorylation of TUBA4A by HGK.
  • FIGs. 11E-11F illustrate pIMAGO kit or pThr phospho-antibody failed to detect phosphorylation of TUBA4A by MINK1.
  • FIG. 12A-12O illustrate a role of the MAP4K-HDAC6-TUBA4A axis in subcellular distribution of RANGAP1.
  • FIG. 12A illustrates qRT-PCR analysis of shRNA-mediated knockdown of endogenous TUBA4A in human fibroblasts.
  • FIG. 12B depicts confocal images of RANGAP1 distribution in hiMNs co-cultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 12C illustrates TUBA4A knockdown increases cytoplasmic fraction of RANGAP1 in hiMNs (mean ⁇ SEM; *p ⁇ 0.05).
  • FIG. 12A illustrates qRT-PCR analysis of shRNA-mediated knockdown of endogenous TUBA4A in human fibroblasts.
  • FIG. 12B depicts confocal images of RANGAP1 distribution in hiMNs co-cultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 12D depicts western blots showing enhanced acetylation of TUBA4A by Hit3 in hiMNs.
  • FIG. 12E depicts confocal images of ac-TUBA4A in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 12F illustrates knockdown of MAP4Ks, comparable to Hit3 treatments, enhances TUBA4A acetylation in somas of hiMNs (mean ⁇ SEM; *p ⁇ 0.05).
  • FIG. 12G shows qRT-PCR analysis of shRNA-mediated knockdown of endogenous HDAC6 in human fibroblasts.
  • FIG. 12H illustrates confocal images of ac- TUBA4A in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 50 pm.
  • FIG. 121 shows confocal images of RANGAP1 distribution in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 12J depicts knockdown of HDAC6 promotes TUBA4A acetylation in somas of hiMNs (mean ⁇ SEM; ****p ⁇ 0.0001).
  • FIG. 12K shows knockdown of HDAC6 promotes nuclear localization of RANGAP1 in hiMNs (mean ⁇ SEM; *p ⁇ 0.05 and ****p ⁇ 0.0001).
  • FIG. 12L illustrates in vitro kinase assay and western blotting showing phosphorylation of purified GST-HDAC6 by HGK.
  • FIG. 12M shows confocal images of RANGAP1 distribution in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 12N illustrates knockdown of MAP4Ks, as well as Hit3 treatments, reduces hiMNs with HDAC6-induced abnormal cytoplasmic distribution of RANGAP1.
  • FIG. 120 depicts knockdown of MAP4Ks, as well as Hit3 treatments, promotes nuclear localization of RANGAP1 even in the presence of HDAC6 (mean ⁇ SEM; ****p ⁇ 0.0001 ).
  • FIG. 13A-13E illustrate HDAC6 regulates TUBA4A acetylation and TDP-43 subcellular distribution.
  • FIG. 13A shows confocal images of ac-TUBA4A in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 50 pm.
  • FIG. 13B shows confocal images of ac-TUBA4A and TDP-43 in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 13C depict HDAC6 knockdown, like Hit3 treatments, improves nuclear localization of TDP-43 (mean ⁇ SEM; **p ⁇ 0.01 and ****p ⁇ 0.0001 ).
  • FIG. 13A shows confocal images of ac-TUBA4A in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 50 pm.
  • FIG. 13B shows confocal images of ac-TUBA4A
  • FIG. 13D shows confocal images of TDP-43 distribution in hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 pm.
  • FIG. 13E illustrates inhibition or knockdown of MAP4Ks reverses HDAC6’s effect on the subcellular distribution of TDP-43 (mean ⁇ SEM; *p ⁇ 0.05 and ****p ⁇ 0.0001 ).
  • FIG. 14A-14K show MAP4Ki is neuroprotective in SOD1 G93A mice.
  • FIG. 14A illustrates plasma, brain and spinal cord concentration of Hit3 after IP injection.
  • FIG. 14B illustrates plasma, brain and spinal cord concentration of MAP4Ki (PF-6260933) after IP injection.
  • FIG. 14E depicts a Kaplan-Meier survival curve.
  • FIG. 14F exhibits confocal images of CHAT+ neurons in the ventral horn of the lumbar spinal cord. Scale bar, 20 pm.
  • FIG. 14H illustrates confocal images of RANGAP1 localization in CHAT+ neurons. Scale bar, 20pm.
  • FIG. 14J shows confocal images of TDP-43 localization in CHAT+ neurons. Scale bar, 20pm.
  • FIG. 16A-16D show traumatic brain injury causes gliosis, neurodegeneration and tau pathology.
  • FIG. 16A depicts injured cortical area 7 days post injury and the sham controls.
  • FIG. 16B illustrates severe reactive gliosis indicated by dramatic increases of expression of GFAP (astrocytes), NG2 (NG2 glia), and IBA1 (microglia).
  • FIG. 16A shows traumatic brain injury causes gliosis, neurodegeneration and tau pathology.
  • FIG. 16A depicts injured cortical area 7 days post injury and the sham controls.
  • FIG. 16B illustrates severe reactive gliosis indicated by dramatic increases of expression of GFAP (astrocytes), NG2 (NG2 glia), and IBA1 (microglia).
  • FIG. 17A-17J show the amelioration of brain injury-induced pathology by CNH domain.
  • FIG. 17A shows the schematic of GFP-CNH.
  • FIG. 17B shows the schematic of the time course of the method used.
  • FIGs. 17C-17D depict relative expression of GFAP, NG2, and IBA1 surrounding the cortical injury.
  • FIGs. 17E-17F illustrate injury-induced glial scars, quantified by the volume of GFAP+ area.
  • FIGs. 17G-17H show markers of neuron damage (SMI32) and tau pathology (AT8, AT100 and AT180).
  • FIGs. 17I-17J depict lesion size measured by the ratio of tissue area in the ipsilateral cortex to the contralateral cortex.
  • FIG. 18A-18C show the promotion of functional recovery after brain injury by CNH.
  • FIG. 18A is the schematic of the time course of the grid walking test.
  • FIG. 18B depicts motor functions in CNH group and the GFP control group.
  • FIG. 18C illustrates immobility time in CNH group and the GFP control group.
  • FIG. 19A-19P show the CNH domain exerts its neuroprotective function in neurons.
  • FIG. 19A shows a schematic of the time course of administration of AAV2/5 packaged CNH.
  • FIGs. 19B-19C illustrate the astrocyte-expressed CNH domain on tau pathology.
  • FIGs. 19D-19E show glial scars indicated by the GFAP+ cortical area after TBI.
  • FIGs. 19F-19G show brain lesion size indicated by the relative remaining cortical tissues after TBI.
  • FIG. 19H shows a schematic of the time course of of AAV2/9 packaged CNH.
  • FIGs. 191- 19J illustrate TBI-associated tau pathology.
  • FIGs. 19K-19N depict glial scars and lesioned cortical size.
  • FIG. 19K-19N depict glial scars and lesioned cortical size.
  • FIG. 19P shows specific targeting of brain neurons by the AAV9-hSYN1- GFP vector. Astrocytes and neurons are identified with staining for GFAP and NeuN, respectively. Scale bar, 50 pm.
  • FIG. 20A-20J show CNH domain alleviates tau pathology and improves behaviors of AD mice.
  • FIGs. 20A-20B depict AT8 staining and relative intensity in the cortex and the hippocampal CA1 region of mice injected with the CNH or GFP control.
  • FIGs. 20C-20D depict AT100 staining and relative intensity in the cortex and the hippocampal CA1 region of mice injected with the CNH or GFP control.
  • FIG. 20E-20F depict tauopathy-induced neuroinflammation determined by IBA1 staining for microglia.
  • FIGs. 20G-20H illustrate behavioral differences observed between mice injected with the CNH or the GFP control measured as total traveled distances and limb clasping scores.
  • FIGs. 20I-20J illustrate accumulation of p-tau in the cortex and the hippocampus of rTg4510 mice.
  • FIG. 21A-21J show pharmacological inhibition of MAP4Ks was neuroprotective.
  • FIG. 21A shows chemical structure of the MAP4K inhibitor K02288.
  • FIG. 21 B shows experimental design. IHC, immunohistochemistry; Br, bregma.
  • FIG. 21C shows representative confocal images for the indicated reactive gliosis markers. Scale bar, 50 pm.
  • FIG. 21E shows representative confocal images for the indicated neuronal damage markers. Scale bar, 50 pm.
  • FIG. 21G shows representative images showing brain sections for GFAP+ scars. Scale bar, 1 mm.
  • FIG. 211 shows representative images of brain sections for lesion quantification (yellow outlined). Scale bar, 1 mm.
  • FIG. 22A-22G show functional interactions of CNH with MAP4Ks and proteomic analysis.
  • FIG. 22A shows validation of the interaction between CNH and MAP4Ks by coimmunoprecipitation (co-IP). The HA-tagged MAP4Ks were pulled down and the associated CNH was examined by western blotting.
  • FIG. 22B shows schematic diagram of the MINK1 protein and its truncations. Their binding to CNH is also summarized as “+” (binding) or (no binding).
  • KD kinase domain; IM, intermediate domain; CNH, citron homology domain.
  • FIG. 22C shows co-IP results showing the interaction of CNH with MINK1 or its truncations.
  • FIG. 22A shows validation of the interaction between CNH and MAP4Ks by coimmunoprecipitation (co-IP). The HA-tagged MAP4Ks were pulled down and the associated CNH was examined by western blotting.
  • FIG. 22D shows experimental design for BiolD2-mediated proximity-labeling proteomics in the mouse brain.
  • FIG. 22E-22F show Gene Ontology (GO) and KEGG pathways of proteins enriched in the BiolD2-CNH group. The number of proteins in each category is indicated in the parenthesis.
  • FIG. 23A-23G show CNH inhibits MAP4K-induced phosphorylation of DVL3.
  • FIG. 23A shows co-IP results showing the interaction of CNH with DVL3.
  • FIG. 23B shows co-IP results showing the interaction of DVL3 with HGK, MINK1 or TNIK.
  • FIG. 23C shows the PDZ domain of DVL3 mediates its interaction with MINK1. DIX, homology region between Dishevelled and aXin; PDZ, homology region shared by PSD95, Dig 1 , and Zo-1 ; DEP, shared homology region between Dishevelled, Egl-10 and Pleckstrin domain.
  • DIX homology region between Dishevelled and aXin
  • PDZ homology region shared by PSD95, Dig 1 , and Zo-1
  • DEP shared homology region between Dishevelled, Egl-10 and Pleckstrin domain.
  • FIG. 23D shows mobility shift of DVL3 induced by wildtype but not kinase-dead MAP4Ks. The migration front of the indicated protein bands is marked by a red line.
  • FIG. 23E shows western blots showing MAP4K-induced threonine phosphorylation of DLV3, which could be largely abolished after treatment with lambda protein phosphatase (APP). p-Thr, antibody specific for phosphorylated threonine.
  • FIG. 23F shows phos-tag SDS-PAGE and western blotting to show suppression of HGK-mediated DVL3 phosphorylation by CNH.
  • FIG. 23G shows schematic representation of HGK-induced phosphorylation of DVL3. The sites were identified by phospho proteomics. Different color represents relative enrichment of phosphorylation when compared to the control without ectopic HGK.
  • FIG. 24A-24G show MAP4Ks negatively regulate Wnt/p-catenin signaling.
  • FIG. 24A shows western blots showing p-catenin (CTNNB1 ) destabilization by wildtype (WT) but not kinase-dead (KM) MAP4Ks.
  • WT wildtype
  • KM kinase-dead
  • OA okadaic acid
  • ACTB loading control.
  • FIG. 24C shows western blots showing CTNNB1 stabilization through downregulation of MAP4Ks. JNK phosphorylation, a target of the non-canonical Wnt signaling, is not obviously altered.
  • shLuc a control for shRNA-mediated knockdown.
  • FIG. 24E shows western blots showing CTNNB1 stabilization by K02288-mediated inhibition of MAP4Ks. Cells treated with Wnt3A-conditioned medium (Wnt3A-CM) were used as positive controls. JNK phosphorylation was not obviously altered by K02288.
  • FIG. 24F shows co-IP results showing a lack of interaction between MAP4Ks with CTTNB1.
  • FIG. 24G shows a schematic diagram summarizing the functional regulation of the crosstalk between MAP4Ks and Wnt signaling by CNH.
  • MAP4Ks signaling is associated with brain injury induced gliosis, neuron damage, and tau pathology, and that suppression of MAP4Ks reduce gliosis and tau phosphorylation and facilitates brain tissue remolding and behavioral recovery.
  • the MAP4Ks inhibitors described herein can be used to as a therapeutic agent for treating, reducing one or more symptoms and/or providing protection from neurodegenerative disease or brain injury.
  • articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article.
  • an element means at least one element and can include more than one element.
  • “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
  • the term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • treatment refers to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible.
  • the aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder, or condition.
  • prevent refers to eliminating or delaying the onset of a particular disease, disorder, or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and/or degree of onset or severity in the absence of intervention.
  • an effective amount refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, pets, livestock, horses or other animals.
  • the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals.
  • nonhuman animals of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like.
  • the subject can be a human.
  • the subject can be a human in need of treating or protection from a from neurodegenerative disease or brain injury.
  • mammal include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). Any mammal can be treated by a method or composition described herein.
  • a mammal is a human.
  • a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like).
  • a non-rodent mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a mammal can be an animal disease model.
  • MAP4K refers to Mitogen-activated protein kinase kinase kinase kinase kinase and is a family of proteins involved in cellular signaling.
  • MAP4Ks are serine/threonine (S/T) protein kinases that belong to the mammalian STE20-like family, and includes MAP4K1 (also referred as HPK1 ), MAP4K2 (also referred as GCK), MAP4K3 (also referred as GLK), MAP4K4 (also referred as HGK), MAP4K5 (also referred as KHS), MAP4K6 (also referred as MINK or MINK1 ) and MAP4K7 (also referred as TNIK).
  • MAP4Ks have three domains, kinase domain, intermediate domain, and citron homology domain (CNH).
  • MAP4K4 refers to mitogen-activated protein kinase kinase kinase kinase kinase 4 is an enzyme, specifically a serine/threonine (S/T) kinase encoded by the MAP4K4 gene.
  • S/T serine/threonine
  • MAP4K4 is alternatively known as hepatocyte progenitor kinase-like/germinal center kinase-like kinase (HGK), FLH21957, HEL-S-31 , MEKKK4, or Nck-interacting kinase (NIK).
  • MAP4K4 can be from vertebrates, including bovine, ovine, porcine, chicken, and human MAP4K4.
  • the wild type sequences of MAP4K4 are well known in the art and may be obtained from publicly available databases. For e.g., nucleotide sequence for human MAP4K4 is available at NCBI database under accession number NM 001024937.4 and the protein sequence under accession number NP 001020108.1.
  • MAP4K6 refers to mitogen-activated protein kinase kinase kinase kinase kinase 6 is an enzyme, specifically a serine/threonine (S/T) kinase encoded by the Misshapen- like kinase 1 (MINK1 ) gene.
  • MAP4K6 is alternatively known as MINK1 , B55, MINK, MEKKK6, YSK2, or ZC3.
  • MAP4K6 can be from vertebrates, including bovine, ovine, porcine, chicken, and human MAP4K6.
  • the wild type sequences of MAP4K6 are well known in the art and may be obtained from publicly available databases. For e.g., nucleotide sequence for human MAP4K4 is available at NCBI database under accession number NM 001242559.2 and the protein sequence under accession number NP 001229488.1.
  • MAP4K7 refers to mitogen-activated protein kinase kinase kinase kinase kinase 7 is an enzyme, specifically a serine/threonine (S/T) kinase encoded by the TRAF2 and NCK-interacting protein kinase (TNIK) gene.
  • MAP4K7 is alternatively known as TNIK or MRT54.
  • MAP4K7 can be from vertebrates, including bovine, ovine, porcine, chicken, and human MAP4K7.
  • the wild type sequences of MAP4K7 are well known in the art and may be obtained from publicly available databases. For e.g., nucleotide sequence for human MAP4K4 is available at NCBI database under accession number NM 001 161560.3 and the protein sequence under accession number NP 001 155032.1.
  • neurodegenerative disease refers to a disease, disorder, or condition caused by the progressive loss of structure or function of neurons, including death of neurons, in a process known as neurodegeneration.
  • Non-limiting examples of neurodegenerative diseases include Amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, Friedreich ataxia, Lewy body dementia, or Spinal muscular atrophy. These examples of neurodegenerative diseases and their symptoms are well-known in the art.
  • Subjects can be diagnosed as having a neurodegenerative disease by a health care provider, medical caregiver, physician, nurse, family member, or acquaintance, who recognizes, appreciates, acknowledges, determines, concludes, opines, or decides that the subject has a neurodegenerative disease.
  • ALS myotrophic lateral sclerosis
  • ALS also known as Lou Gehrig's disease
  • neurons waste away or die, and can no longer send messages to muscles. This eventually leads to muscle weakening, twitching, and an inability to move the arms, legs, and body. The condition slowly gets worse. When the muscles in the chest area stop working, it becomes hard or impossible to breathe on one's own.
  • risk factors for ALS except for having a family member who has a hereditary form of the disease. Symptoms usually do not develop until after age 50, but they can start in younger people.
  • ALS does not affect the senses (sight, smell, taste, hearing, touch). It only rarely affects bladder or bowel function, or a person's ability to think or reason.
  • Alzheimer's Disease refers to a progressive mental deterioration manifested by memory loss, confusion, and disorientation beginning in late middle life and typically resulting in death in five to ten years. Pathologically, Alzheimer's Disease can be characterized by thickening, conglutination, and distortion of the intracellular neurofibrils, neurofibrillary tangles and senile plaques composed of granular or filamentous argentophilic masses with an amyloid core.
  • Huntington's Disease refers to a neurodegenerative genetic disorder that affects muscle coordination and leads to cognitive decline and psychiatric problems. It typically becomes noticeable in mid-adult life. Huntington's Disease is the most common genetic cause of abnormal involuntary writhing movements called chorea. Symptoms of Huntington's disease commonly become noticeable between the ages of 35 and 44 years, but they can begin at any age from infancy to old age. In the early stages, there are subtle changes in personality, cognition, and physical skills. The physical symptoms are usually the first to be noticed, as cognitive and psychiatric symptoms are generally not severe enough to be recognized on their own at the earlier stages.
  • Parkinson's Disease refers to a disorder of the brain that leads to shaking (tremors) and difficulty with walking, movement, and coordination. Parkinson's Disease most often develops after age 50. It is one of the most common nervous system disorders of the elderly. It affects both men and women. In some cases, Parkinson's Disease runs in families. When a young person is affected, it is usually because of a form of the disease that runs in families. There are currently no known cures for Parkinson's Disease. The goal of treatment is to control symptoms. Nerve cells use a brain chemical called dopamine to help control muscle movement. Parkinson's Disease occurs when the nerve cells in the brain that make dopamine are slowly destroyed. Without dopamine, the nerve cells in that part of the brain cannot properly send messages and leads to the loss of muscle function.
  • Friedreich's ataxia refers to an inherited disease that causes progressive damage to the nervous system, resulting in symptoms ranging from gait disturbance to speech problems; it can also lead to heart disease and diabetes.
  • the ataxia of Friedreich's ataxia results from the degeneration of nerve tissue in the spinal cord, in particular sensory neurons essential (through connections with the cerebellum) for directing muscle movement of the arms and legs.
  • the spinal cord becomes thinner and nerve cells lose some of their myelin sheath (the insulating covering on some nerve cells that helps conduct nerve impulses).
  • multiple sclerosis refers to a disease caused by damage to the myelin sheath, the protective covering that surrounds neurons. When this nerve covering is damaged, nerve signals slow down or stop. The nerve damage is caused by inflammation. Inflammation occurs when the body's own immune cells attack the nervous system. This can occur along any area of the brain, optic nerve, and spinal cord. It is unknown what exactly causes this to happen. The most common thought is that a virus or gene defect, or both, are to blame. Environmental factors may play a role. Symptoms vary because the location and severity of each attack can be different. Episodes can last for days, weeks, or months. These episodes alternate with periods of reduced or no symptoms (remissions).
  • Fever hot baths, sun exposure, and stress can trigger or worsen attacks. It is common for the disease to return (relapse). However, the disease may continue to get worse without periods of remission. Because nerves in any part of the brain or spinal cord may be damaged, patients with multiple sclerosis can have symptoms in many parts of the body.
  • MSA Multiple System Atrophy
  • the term MSA comprises a chronic degenerative disorder producing different combinations of symptoms from the basal ganglia, pyramidal pathways, cerebellum, brainstem, and autonomic nervous system.
  • the nomenclature of the different manifestations of MSA has been variable and has probably delayed the awareness of the disease.
  • MSA-SND has been suggested, whereas MSA-OPCA could be used when cerebellar predominance is found.
  • MSA-P and MSA-C are also terms that have been proposed as description of the various expressions of the disease with Parkinsonism and cerebellar predominance, respectively.
  • the symptoms of MSA includes tremor, muscular rigidity, hypokinesia, impaired balance, impaired speech, impaired swallowing, ataxia, orthostatic hypotension, impotence, urinary incontinence or urinary retention.
  • Prion diseases refers to disease when prion protein, found throughout the body, begins folding into an abnormal three-dimensional shape and the damaged prion protein destroys brain cells, leading to a rapid decline in thinking and reasoning. Prion disease is generally manifested with cognitive difficulties, ataxia, and myoclonus (abrupt jerking movements of muscle groups and/or entire limbs. The order and/or predominance of these features and associated neurologic and psychiatric findings vary with prion disease subtype and/or PRNP mutation. Death generally results from infection, either by pneumonia (typically from aspiration) or urosepsis.
  • Prion diseases can include Creutzfeldt- Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straussler- Scheinker Syndrome (GSS), Fatal Familial Insomnia (FFI) or Kuru.
  • CJD Creutzfeldt- Jakob Disease
  • vCJD Variant Creutzfeldt-Jakob Disease
  • GSS Gerstmann-Straussler- Scheinker Syndrome
  • FFI Fatal Familial Insomnia
  • Kuru The three phenotypes classically associated with genetic prion disease (fCJD, GSS, and FFI), were defined by clinical and neuropathologic findings long before the molecular basis of this group of disorders was discovered. Although it is now recognized that these three phenotypes are part of a continuum and have overlapping features, it can be helpful to think of genetic human prion disease at least in part in terms of these phenotypes when providing individuals and families with information
  • Lewy body dementia As used herein “Lewy body dementia”, “LBD”, “Dementia with Lewy Bodies” or “DLB” refers to a disease associated with abnormal deposits of a protein called alpha- synuclein in the brain. These deposits, called Lewy bodies, affect chemicals in the brain whose changes, in turn, can lead to problems with thinking, movement, behavior, and mood.
  • Lewy bodies are intracytoplasmic, spherical, eosinophilic neuronal inclusion bodies. The areas of predilection for LB are brainstem, subcortical nuclei, limbic cortex, and neocortex. Their accumulation results in a loss of functional dopaminergic neuron terminals in the striatum.
  • SMA spinal muscular atrophy
  • CNS central nervous system
  • SMA central nervous system
  • CNS1 central nervous system
  • SMA Type 0 SMA In Utero SMA
  • Type I also known as Werdnig-Hoffman disease or infantile SMA
  • SMA Type II also known as Kugelberg-Welander disease
  • SMA Type IV also known as Kugelberg-Welander disease
  • SMA forms are caused by changes in other genes including the:VAPB gene on chromosome 20, DYNC1 H1 gene on chromosome 14, BICD2 gene on chromosome 9 or UBA1 gene on the X chromosome.
  • Symptoms of SMA include muscle weakness, poor muscle tone, weak cry, limpness, or a tendency to flop, difficulty sucking or swallowing, accumulation of secretions in the lungs or throat, feeding difficulties, and increased susceptibility to respiratory tract infections.
  • the legs tend to be weaker than the arms and developmental milestones, such as lifting the head or sitting up, cannot be reached. In general, the earlier the symptoms appear, the shorter the lifespan.
  • brain injury refers to any heady injury and can include traumatic brain injury caused by trauma to the brain, including, but not limited to, striking of the head with solid objects, falls, contusions, concussions, including brain injury caused by repeated concussions, such as those that may be suffered by those participating in sports, such as football, baseball, basketball, wrestling, skiing, horse racing, auto racing, and hockey, and brain injuries caused by explosions resulting from explosive devices including, but not limited to, incendiary explosive devices (lEDs) and accidents. There may or may not be penetration of the head or brain. TBI may also be caused by blunt injury, motor vehicle accident (MVA) , falling, or high velocity-bullet wound.
  • MVA motor vehicle accident
  • TBI also include, but are not limited to, any brain injury resulting from diseases or disorders of the brain, including, but not limited to, stroke, Parkinson's Disease, autoimmune encephalitis, amyotrophic lateral sclerosis (Lou Gehrig's Disease or ALS), for example.
  • TBI can comprise a primary injury, which can be focal or diffuse, caused by mechanical impact, that results in primary pathological events such as hemorrhage and ischemia, tearing of tissue and axonal injuries and a secondary injury such as diffuse inflammation, cell death and gliosis, which is a consequence of the primary one.
  • This secondary injury starts immediately after injury and can continue for weeks and is thought to involve an active inhibition of neural stem cell activity. Collectively, these events lead to neurodegeneration.
  • TBI can be graded as mild, moderate, or severe.
  • brain injury comprises stroke.
  • a stroke occurs when the blood supply to part of the brain is suddenly interrupted or when a blood vessel in the brain bursts, spilling blood into the spaces surrounding brain cells, or when the brain or a portion of the brain is deprived of oxygen or oxygenation is impaired by exogenous substances such as carbon monoxide, hemorrhage, or hypoperfusion. Brain cells die when they no longer receive adequate oxygen and nutrients from the blood or there is sudden bleeding into or around the brain.
  • the symptoms of a stroke include sudden numbness or weakness, especially on one side of the body; sudden confusion or trouble speaking or understanding speech; sudden trouble seeing in one or both eyes; sudden trouble with walking, dizziness, or loss of balance or coordination; or sudden severe headache with no known cause.
  • ischemic blockage of a blood vessel supplying the brain due to thrombosis or embolus, and hemorrhagic bleeding into the brain tissue (intracerebral hemorrhage), or into the subarachnoid space (subarachnoid hemorrhage).
  • CNH citron homology domain
  • vector refers to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA.
  • Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors.
  • Useful viral vectors include, e.g., adeno-associated virus, replication defective retroviruses and lentiviruses.
  • adeno-associated virus or “AAV” refers to members of the dependovirus genus comprising any particle, sequence, gene, protein, or component derived from the dozens of naturally occurring and available adeno-associated viruses, as well as artificial AAVs.
  • An adeno-associated virus (AAV) viral vector is an AAV DNase-resistant particle having an AAV protein capsid into which is packaged nucleic acid sequences for delivery to target cells.
  • An AAV capsid is composed of 60 capsid (cap) protein subunits, VP1 , VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1 :1 :10 to 1 :1 :20, depending upon the selected AAV.
  • Various AAVs may be selected as sources for capsids of AAV viral vectors as identified above.
  • the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1 , AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12, AAV13, AAV8 bp, AAV7M8, AAVAnc80, AAVrhl 0, AAVPHP.B, AAV type rh32.33, AAV type rh8, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV-PHP.eB, AAV-TT, AAVv66,
  • recombinant AAV or “rAAV” refers to AAV produced recombinantly and may be based on AAV parent or reference sequences.
  • An rAAV may comprise a non-naturally occurring capsid protein.
  • Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vp1 capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source.
  • the methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.
  • capsid refers to the protein shell of a virus particle.
  • neurotrophic capsid refers to a capsid of a neurotropic virus.
  • AAV capsid Methods of generating AAV capsid, coding sequences therefore, and methods for production of rAAV viral vectors are well known in the art.
  • the sequences of several AAVs are well known in the art and may be obtained from publicly available databases.
  • nucleotide sequence for AAV9 is available at NCBI database under Genbank accession number MB442163.1.
  • Nucleotide sequence of AAV-PHP.eB is available at NCBI database under Genbank accession number MF187357.1.
  • lentivirus refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritisencephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV based vector backbones ⁇ i.e., HIV cis-acting sequence elements) are preferred.
  • Methods of generating lentiviral vectors are well known in the art.
  • the sequences of several lentivirus vectors are well known in the art and may be obtained from publicly available databases.
  • nucleotide sequence for Addgene plasmid #90214 is available at Addgene under pCSC-NGN2-IRES-GFP-T2A-Sox1 1
  • Addgene plasmid #90215 is available at Addgene under pCSC-ISL1-T2A-LHX3.
  • Cas9 or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 nuclease is also referred to sometimes as a casn 1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease.
  • CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids).
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • tracrRNA trans-encoded small RNA
  • rnc endogenous ribonuclease 3
  • Cas9 protein serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically.
  • DNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs (“sgRNA” or “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species.
  • the sequences of Cas9 and Cas9 variants are well known in the art and may be obtained from publicly available databases. For e.g., the nucleotide sequence for wild type Cas9 from Streptococcus pyogenes is available at NCBI database under Genbank ID No. 69900935 and the protein sequence for wild type Cas9 from Streptococcus pyogenes is available at NCBI Accession No. WP_038431314.1 .
  • gRNA or “guide RNA” refers to refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas protein and aid in targeting the Cas protein to a specific location within a target polynucleotide (e.g., a DNA).
  • a guide RNA can comprise a crRNA segment and a tracrRNA segment.
  • crRNA or “crRNA segment” refers to an RNA molecule or portion thereof that includes a polynucleotide-targeting guide sequence, a stem sequence, and, optionally, a 5'-overhang sequence.
  • tracrRNA refers to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as a Cas9).
  • guide RNA encompasses a single guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule.
  • guide RNA also encompasses, collectively, a group of two or more RNA molecules, where the crRNA segment and the tracrRNA segment are located in separate RNA molecules, guide RNAs, including single guide RNAs can be produced by chemical synthesis or enzymatic synthesis, using methods known in the art.
  • a guide RNA can comprise any ribonucleotide, namely A, C, G, and U, unnatural or natural, such as a pseudouridine, inosine, or a deoxynucleotide, and/or can possess a chemical modification or substitution.
  • the ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a guide sequence may be selected to target any target sequence in MAP4Ks, including MAP4K4, MAP4K6 and/or MAP4K7.
  • Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in a genome may include sequence that has a single occurrence in the gene.
  • a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.
  • RNA duplex refers to the structure formed by the complementary pairing between two regions of a RNA molecule.
  • shRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the shRNA is complementary to a nucleotide sequence of the targeted gene. In certain embodiments, the shRNAs are targeted to the sequence encoding MAP4K4, MAP4K6 and/or MAP4K7.
  • shRNAs consist of a stem-loop structure which consists of a stem portion that comprises a double stranded sequence.
  • the double stranded stem portion comprises a guide strand on one side of the stem, and a passenger strand on the other side of the stem.
  • the stem-loop structure further comprises a single stranded loop portion at one end of the stem.
  • the stem-loop structures of the shRNA molecules described herein may be about 40 to 100 nucleotides long or, about 50 to 75 nucleotides long.
  • the stem region may be about 19-45 nucleotides in length (or more), or about 20-30 nucleotides in length.
  • the stem may comprise a perfectly complementary duplex (but for any 3' tail), however, bulges or interior loops may be present, on either arm of the stem.
  • the number of such bulges and asymmetric interior loops are preferably few in number (e.g., 1 , 2 or 3) and are about 3 nucleotides or less in size.
  • the terminal loop portion may comprise about 4 or more nucleotides, but preferably not more than about 25. More particularly, the loop portion will preferably be 6-15 nucleotides in size.
  • ectopic expression refers to expression of the transgene in a tissue or cell where it is not normally expressed.
  • excipient refers to an inert substance added to a composition to further facilitate administration of an active ingredient.
  • promoter refers to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 ’-direction) coding sequence.
  • Transcription promoters can include “inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “constitutive promoters” and “tissue specific promoters” (direct expression primarily in a desired tissue of interest). Promoters can also be synthetic, chimeric and/or hybrid promoters.
  • Non-limiting examples of promoters include, neuron-specific enolase promoter, a GFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, and the like.
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • tissue specific promoter can be neuron specific promoter, which directs expression primarily in neuronal tissues, and is essentially not active outside the central nervous system, or the activity of the promoter is higher in the central nervous system that in other systems.
  • a promoter specific for the spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus striatum, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), or a combination thereof may be selected.
  • the promoter may be specific for particular cell types, such as neurons or glial cells in the CNS.
  • glial cells it may be specific for astrocytes, oligodendrocytes, ependymal cells, Schwann cells, or microglia. If it is active in neurons, it may be specific for particular types of neurons, e.g., motor neurons, sensory neurons, or interneurons. Additionally, it may be specific for neurons with a specific phenotype, e.g., dopamine -producing neurons, serotonin-producing neurons, etc.
  • the promoter is specific for cells in particular regions of the brain, for example, the cortex, striatum, nigra, and hippocampus.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • isolated nucleic acid refers to a nucleic acid sequence, wherein the indicated molecule is present in the substantial absence of other biological macromolecules of the same type or substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an “isolated nucleic acid” is further free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
  • the present disclosure provides methods of treating a neurodegenerative disease or brain injury in a subject.
  • the method comprises administering to the subject in need thereof, an effective amount of a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof.
  • the inhibitor of MAP4K signaling or activity can be selected from the group consisting of citron homology domain (CNH) of an MAP4K, a CNH- containing truncation of an MAP4K, a guide RNA (gRNA) comprising a target sequence of MAP4K, a shRNA comprising a target sequence of MAP4K, or any combination thereof.
  • CNH citron homology domain
  • gRNA guide RNA
  • the composition may be a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • the inhibitor of MAP4K can be any of those disclosed herein.
  • the neurodegenerative disease comprises amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, Friedreich ataxia, Lewy body dementia, or Spinal muscular atrophy.
  • the neurodegenerative disease comprise ALS.
  • the neurodegenerative disease comprise multiple sclerosis.
  • the neurodegenerative disease comprise ALS.
  • the neurodegenerative disease comprise Parkinson's disease.
  • the neurodegenerative disease comprise Alzheimer's disease.
  • the neurodegenerative disease comprise Huntington's disease.
  • the neurodegenerative disease comprise multiple system atrophy.
  • the neurodegenerative disease comprise prion diseases. In some aspects, the neurodegenerative disease comprise Friedreich ataxia. In some aspects, the neurodegenerative disease comprise Lewy body dementia. In some aspects, the neurodegenerative disease comprise Spinal muscular atrophy. In some aspects, brain injury comprises traumatic brain injury (TBI). In some aspects, brain injury comprises stroke.
  • TBI traumatic brain injury
  • the method of treatment comprises a method of treating a neurodegenerative disease or brain injury in a subject in need thereof comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • the method of treatment comprises a method of treating or reducing one or more symptom associated with neurodegenerative disease or brain injury in a subject in need thereof comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administering a disclosed composition comprises, improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • treating or reducing one or more symptoms comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • administering the composition treats or reduces one or more symptoms associated with neurodegenerative disease or brain injury in a subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • the method of treatment comprises a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • administering the composition protects a subject from neurodegeneration and/or neural injury.
  • protection of a subject from neurodegeneration and/or neural injury comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administering a disclosed composition comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • providing protection comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • Memory, cognitive performances, and motor functions can be tested using well established tests, such as evaluation of motor-spatial skills or memory recall testing.
  • tests include test for rodents such as Morris Water Maze, Radial Maze, T Maze, and Fear Conditioning, test for primates such as Eye Blink, Delayed Recall, Cued Recall, and Face Recognition, and test for humans, such as using various mazes, pattern recognition tests, condition tasks, etc.
  • Diseases specific tests can also be administered such as Minimental and ADAS-Cog tests used for cognitive assessment of subjects with Alzheimer’s disease.
  • PET positron emission tomography
  • MRI magnetic resonance imaging
  • biomarker-based assay may be used to detect and track changes in brain function and structure, and/or tau pathology in a subject, before and/or after treatment.
  • the inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof is a CNH of MAP4K4, MAP4K6 or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • Citron homology domain (CNH)
  • the method of the present disclosure comprises administering to the subject in need thereof, an effective amount of a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the composition may be a pharmaceutical composition comprising a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof, and one or more pharmaceutically acceptable excipients.
  • a method of treating a neurodegenerative disease or brain injury in a subject comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, or any combination thereof.
  • treating a neurodegenerative disease or brain injury in a subject comprises a method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the disclosure provides a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the CNH of MAP4K4, MAP4K6, or MAP4K7, and/or the CNH- containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof reduces the activity of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, in the subject as compared to the in the subject activity prior to the administration of the CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the activity of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is reduced by about 1% to about 100%. In some aspects, the activity of MAP4K4, MAP4K6, MAP4K7, or any combination thereof may be reduced by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. In some aspects, the activity of MAP4K4, MAP4K6 or MAP4K7 is reduced by about 90%.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • treating or reducing one or more symptoms comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH- containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • providing protection comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • the CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH- containing truncation of MAP4K4, MAP4K6, MAP4K7, used in the methods of this disclosure may comprise any of those disclosed herein, including any composition, pharmaceutical composition, and/or kit comprising said CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7.
  • one or more of the CNH domain of MAP4K4, MAP4K6, and/or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, and/or MAP4K7 can be administered to a subject in need thereof, using a vector engineered to express gRNA.
  • said vector engineered to express gRNA may be any of those disclosed herein.
  • the vector may be a lentivirus vector or rAAV vector.
  • lentivirus vector or rAAV vector engineered to express CNH domain of MAP4K4, MAP4K6, and/or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, and/or MAP4K7 can be administered intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v.), or direct administration into the cerebrospinal fluid (CSF), e.g., via intrathecal and/or intracerebral injection.
  • CSF cerebrospinal fluid
  • an effective amount of the lentivirus vector or rAAV engineered to express CNH domain and/or a CNH-containing truncation is administered in a subject, at a concentration of about 1X10 2 genome copies (GC)/ml to about 2X10 15 GC/ml.
  • the lentivirus vector or rAAV engineered to express CNH domain and/or a CNH- containing truncation is administered at a concentration of about 1X10 2 GC/ml, about 1X10 3 GC/ml, about 1X10 4 GC/ml, about 1X10 5 GC/ml, about 1X10 6 GC/ml, about
  • lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 1X10 13 GC/mL. In some aspects, lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 2.0X10 12 GC/mL. In some aspects, rAAV engineered to express CNH domain, and/or a CNH- containing truncation is administered at a concentration of 1X10 13 GC/mL. In some aspects, rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 2.0X10 12 GC/mL.
  • the lentivirus vector or rAAV engineered to express CNH domain, and/or a CNH-containing truncation is formulated for administration as a liquid with a volume in a range of about 1 pl to about 1 ml.
  • the dose of the rAAV for administration is formulated as a liquid a volume of about 1 pl, about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 85 pl, about 90 pl, about 95 pl, about 100 pl, about 125 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl or about 1 ml.
  • the lentivirus vector or rAAV engineered to express CNH domain, and/or a CNH- containing truncation, for administration is formulated as a liquid of a volume of 1 ml or more.
  • lentivirus vector engineered to express CNH domain, and/or a CNH- containing truncation is administered at about 1.0 pl.
  • lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 8 pl - about 10 pl.
  • rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 1.0 pl.
  • rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 8 pl - about 10 pl.
  • the lentivirus vector or rAAV engineered to express CNH domain and/or a CNH-containing truncation is formulated for administration at a dose from 1 pg/kg to 100 mg/kg, 1 pg/kg to 50 mg/kg, 1 pg/kg to 20 mg/kg, 1 pg/kg to 10 mg/kg, 1 pg/kg to 1 mg/kg, 100 pg/kg to 100 mg/kg, 100 pg/kg to 50 mg/kg, 100 pg/kg to 20 mg/kg, 100 pg/kg to 10 mg/kg, 100 pg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg.
  • the dosage is 0.1 mg/kg of body
  • the method of the present disclosure comprises administering to the subject in need thereof, an effective amount of a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a guide RNA (gRNA) comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the composition may be a pharmaceutical composition comprising a guide RNA (gRNA) comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, and one or more pharmaceutically acceptable excipients.
  • a method of treating a neurodegenerative disease or brain injury in a subject comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K6.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K7.
  • treating a neurodegenerative disease or brain injury in a subject comprises a method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K6.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K7.
  • the disclosure provides a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K4.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K6.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a gRNA comprising a target sequence of MAP4K7.
  • the gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof reduces the expression of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, as compared to the gene expression prior to the introduction of the gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, which can lead to the inhibition of production of the MAP4K4, MAP4K6, MAP4K7 gene product.
  • the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1% to about 100%.
  • the amount of MAP4K4, MAP4K6, and/or MAP4K7 expression may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%.
  • the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • treating or reducing one or more symptoms comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • providing protection comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • gRNAs can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • gRNA can be engineered and produced using primers disclosed in Table 2.
  • the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop.
  • the sgRNA can be between 50-220 (e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60- 130, and 60-120) nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • the gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof can be administered to a subject in need thereof, using a vector engineered to express gRNA.
  • said vector engineered to express gRNA may be any of those disclosed herein.
  • the vector may be a lentivirus vector or rAAV vector.
  • lentivirus vector or rAAV vector engineered to express gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof can be administered intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v. ), or direct administration into the cerebrospinal fluid (CSF), e.g., via intrathecal and/or intracerebral injection.
  • CSF cerebrospinal fluid
  • an effective amount of the lentivirus vector or rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered in a subject, at a concentration of about 1X10 2 genome copies (GC)/ml to about 2X10 15 GC/ml.
  • the lentivirus vector or rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of about 1X10 2 GC/ml, about 1X10 3 GC/ml, about 1X10 4 GC/ml, about 1X10 5 GC/ml, about 1X10 6 GC/ml, about
  • lentivirus engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 1X1013GC/mL. In some aspects, lentivirus engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is administered at a concentration of 2.0X10 12 GC/mL.
  • rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 1X10 13 GC/mL. In some aspects, rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is administered at a concentration of 2.0X10 12 GC/mL.
  • an effective amount of the lentivirus vector or rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration as a liquid with a volume in a range of about 1 pl to about 1 ml.
  • the dose of the lentivirus or rAAV for administration is formulated as a liquid a volume of about 1 pl, about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 85 pl, about 90 pl, about 95 pl, about 100 pl, about 125 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl or about 1 ml.
  • the lentivirus or rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration as a liquid of a volume of 1 ml or more.
  • lentivirus engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 1.0 pl.
  • lentivirus engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 8 pl - about 10 pl.
  • rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 1.0 pl. In some aspects, rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is administered at about 8 pl - about 10 pl.
  • the lentivirus or rAAV engineered to express a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration at a dose from 1 pg/kg to 100 mg/kg, 1 pg/kg to 50 mg/kg, 1 pg/kg to 20 mg/kg, 1 pg/kg to 10 mg/kg, 1 pg/kg to 1 mg/kg, 100 pg/kg to 100 mg/kg, 100 pg/kg to 50 mg/kg, 100 pg/kg to 20 mg/kg, 100 pg/kg to 10 mg/kg, 100 pg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg
  • the method of the present disclosure comprises administering to the subject in need thereof, an effective amount of a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the composition may be a pharmaceutical composition comprising a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, and one or more pharmaceutically acceptable excipients.
  • a method of treating a neurodegenerative disease or brain injury in a subject comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K6.
  • the method of treating a neurodegenerative disease or brain injury in a subject comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K7.
  • treating a neurodegenerative disease or brain injury in a subject comprises a method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K6.
  • the method of reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject in need thereof comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K7.
  • the disclosure provides a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprising administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K4.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K6.
  • the method of providing protection to a subject in need thereof from neural degeneration and/or neural injury comprises administering to the subject in need thereof, an effective amount of a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a shRNA comprising a target sequence of MAP4K7.
  • the shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof reduces the expression of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, as compared to the gene expression prior to the introduction of the shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, which can lead to the inhibition of production of the MAP4K4, MAP4K6, MAP4K7 gene product.
  • the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1% to about 100%.
  • the amount of MAP4K4, MAP4K6, and/or MAP4K7 expression may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%.
  • the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • treating or reducing one or more symptoms comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • providing protection to a subject comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • shRNAs can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • shRNA can be engineered and produced using primers disclosed in Table 2.
  • the length of the duplex of shRNAs is less than 30 base pairs.
  • the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length.
  • the length of the duplex is 19 to 25 base pairs in length.
  • the length of the duplex is 19 or 21 base pairs in length.
  • the RNA duplex portion of the shRNA can be part of a hairpin structure.
  • the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex.
  • the loop can vary in length. In some aspects the loop is 5, 6, 7, 8, 9, 10, 11 , 12 or 13 nucleotides in length.
  • the hairpin structure can also contain 3' or 5' overhang portions. In some aspects, the overhang is a 3' or a 5' overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length.
  • the shRNA used in the methods of this disclosure may comprise any of those disclosed herein, including any composition, pharmaceutical composition, and/or kit comprising said shRNA.
  • the shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof can be administered to a subject in need thereof, using a vector engineered to express shRNA.
  • said vector engineered to express shRNA may be any of those disclosed herein.
  • the vector may be a lentivirus vector or rAAV vector.
  • lentiviral vector or rAAV vector engineered to express shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof can be administered intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v. ), or direct administration into the cerebrospinal fluid (CSF), e.g., via intrathecal and/or intracerebral injection.
  • CSF cerebrospinal fluid
  • an effective amount of the lentivirus vector or rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered in a subject, at a concentration of about 1X10 2 genome copies (GC)/ml to about 2X10 15 GC/ml.
  • the lentivirus vector or rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of about 1X10 2 GC/ml, about 1X10 3 GC/ml, about 1X10 4 GC/ml, about 1X10 5 GC/ml, about
  • 1X10 14 GC/ml, about 1X10 15 GC/ml, about 2X10 2 GC/ml, about 2X10 3 GC/ml, about 2X10 4 GC/ml, about 2X10 5 GC/ml, about 2X10 6 GC/ml, about 2X10 7 GC/ml, about
  • lentivirus engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 1X1013GC/mL.
  • lentivirus engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 2.0X1012 GC/mL.
  • rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 1X10 13 GC/mL.
  • rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at a concentration of 2.0X10 12 GC/mL.
  • an effective amount of the lentivirus vector or rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration as a liquid with a volume in a range of about 1 pl to about 1 ml.
  • the dose of the lentivirus or rAAV for administration is formulated as a liquid a volume of about 1 pl, about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 85 pl, about 90 pl, about 95 pl, about 100 pl, about 125 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl or about 1 ml.
  • the lentivirus or rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration as a liquid of a volume of 1 ml or more.
  • lentivirus engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 1.0 pl.
  • lentivirus engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 8 pl - about 10 pl.
  • rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is administered at about 1.0 pl. In some aspects, rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is administered at about 8 pl - about 10 pl.
  • the lentivirus or rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof is formulated for administration at a dose from 1 pg/kg to 100 mg/kg, 1 pg/kg to 50 mg/kg, 1 pg/kg to 20 mg/kg, 1 pg/kg to 10 mg/kg, 1 pg/kg to 1 mg/kg, 100 pg/kg to 100 mg/kg, 100 pg/kg to 50 mg/kg, 100 pg/kg to 20 mg/kg, 100 pg/kg to 10 mg/kg, 100 pg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg.
  • administration of disclosed lentivirus vector or rAAV results in MAP4K inhibitor expressed ectopically in neuron or motor neuron cells of the subject.
  • the ectopically expressed MAP4K inhibitor lead to an altered phenotype or physiology of the neuron or motor neuron cells of the subject.
  • ectopic expression of MAP4K inhibitor leads to treatment of neurodegenerative disease or brain injury, reduction of one or more symptoms associated with neurodegenerative disease or brain injury, or provide protection from neural degeneration and/or neural injury, in the subject.
  • the MAP4K inhibitor can comprise a chemical inhibitor.
  • the chemical inhibitor can comprise a small molecule, or a large molecule.
  • the chemical inhibitor can be K02288.
  • Effective dosages may be estimated initially from in vitro activity and metabolism assays.
  • an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay.
  • Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans.
  • Initial dosages of compound can also be estimated from in vivo data, such as animal models.
  • Dosage amounts can be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, its metabolism kinetics, and other pharmacokinetic properties, the mode of administration and various other factors, discussed above.
  • a suitable, non-limiting example of a dosage of a disclosed compound according to the present disclosure may be from about 1 ng/kg to about 5000 mg/kg.
  • doses employed for adult human treatment typically may be in the range of 0.0001 mg/kg/day to 0.0010 mg/kg/day, 0.0010 mg/kg/day to 0.010 mg/kg/day, 0.010 mg/kg/day to 0.10 mg/kg/day, 0.10 mg/kg/day to 1 .0 mg/kg/day, 1 .00 mg/kg/day to about 200 mg/kg/day, 200 mg/kg/day to about 5000 mg/kg/day.
  • the dosage may be about 1 mg/kg/day to about 100 mg/kg/day, such as, e.g., 2-10 mg/kg/day, 10-50 mg/kg/day, or 50-100 mg/kg/day.
  • the dosage can also be selected from about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800
  • MAP4K inhibitor can be used in conjunction with MAP4K inhibitor according to the present disclosure.
  • the method can comprise administering MAP4K inhibitor disclosed herein simultaneously, separately, or sequentially to a subject in need thereof with other drugs or therapies.
  • Non-limiting examples of drugs can be selected from 3APS, AAB-001 , ABT-089, ABT-126, AC-3933, ACC-001 , Acetaminophen, AFFITOPE AD01 , AFFITOPE AD02, alpha-lipoic acid, alpha-tocopherol, AN 1792, anti-Abeta, AQW051 , Aripiprazole, Atomoxetine, Atorvastatin, AVE1625, AVP-923, AZD0328, AZD3480, Bapineuzumab, BAY94-9172 (ZK 6013443), Bifeprunox, Bioperine, BMS-708163, BRL- 049653, Bryostatin, CAD106, Celecoxib, CERE-110, Cerebrolysin, CHF 5074, Choline, Circadin, Citalopram, Coenzyme Q, Copper, CTS21 166, Curcumin, CX516 (Ampalex
  • the present disclosure provides a MAP4K inhibitor.
  • the MAP4K inhibitor comprises a CNH of MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof, or a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, or a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof.
  • the MAP4K inhibitor comprises a CNH of MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof, or a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof, or a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combinations thereof.
  • the inhibitor of MAP4K comprises a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4.
  • the inhibitor of MAP4K comprises a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6. In some aspects, the inhibitor of MAP4K comprises a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7.
  • the disclosure provides a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof.
  • the composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof is a pharmaceutical composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • the inhibitor of MAP4K signaling or activity is an inhibitor of MAP4K4 (HGK).
  • the human MAP4K4 comprises an amino acid sequence of:
  • the MAP4K4 comprises an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 1.
  • the inhibitor of MAP4K signaling or activity is an inhibitor of MAP4K6 (MINK1).
  • MINK1 is an inhibitor of MAP4K6 (MINK1).
  • the human MAP4K6 comprises an amino acid sequence of:
  • the MAP4K6 comprises an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 2.
  • the inhibitor of MAP4K signaling or activity is an inhibitor of MAP4K7 (TNIK).
  • the human MAP4K7 comprises an amino acid sequence of:
  • the MAP4K7 comprises an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 3.
  • Citron homology domain (CNH)
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the CNH domain of human MAP4K4 comprises an amino acid sequence of:
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 4.
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH-containing truncation of MAP4K4, wherein the CNH-containing truncations of MAP4K4 can comprise 1-700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K4 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO:1 ) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 1 ) and 95 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO:1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 1 ).
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the CNH domain of human MAP4K6 comprises an amino acid sequence:
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 5.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH-containing truncation of MAP4K6, wherein the CNH-containing truncations of MAP4K6 can comprise 1-700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K6 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 2) and 95 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 2).
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the CNH domain of human MAP4K7 comprises an amino acid sequence of:
  • the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 6.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprise a CNH-containing truncations of MAP4K7, wherein the CNH-containing truncations of MAP4K7 can comprise 1-700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K7 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 3) and 95 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 3).
  • qRNAs comprising a target sequence of MAP4K
  • the disclosure further provides a MAP4K inhibitor and/or composition comprising a MAP4K inhibitor, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K6. In some aspects, the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K7.
  • the gRNA comprises a target sequence of MAP4K4, for example CAGGACATGATGACCAACTC (SEQ ID NO: 13) or GGGCGGAGAAATACGTTCAT (SEQ ID NO: 14).
  • the gRNA comprises a target sequence of MAP4K6, for example CGGACAGGTCGATGTCGTCC (SEQ ID NO: 15) or AGGGTCGGCATGTCAAGACG (SEQ ID NO: 16).
  • the gRNA comprises a target sequence of MAP4K7, for example CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18).
  • the gRNA comprises a target sequence, comprising a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • gRNAs used in the disclosed MAP4K inhibitor and/or composition comprising a MAP4K inhibitor can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • gRNA can be engineered and produced using primers disclosed in Table 2.
  • the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop.
  • the sgRNA can be between 50-220 (e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • shRNAs comprising a target sequence of MAP4K
  • the disclosure further provides a MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K6.
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K7.
  • MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a target sequence of MAP4K4 for inhibition by shRNA is nucleotide sequence comprising AACCGAAGACGATTTCAACAAA (SEQ ID NO: 7).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a shRNA comprising a target sequence of MAP4K4 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCACCGAAGACGATTTCAACAAATAGTGAAGCCACAGATGTA TTTGTTGAAATCGTCTTCGGTTTGCCTACTGCCTCGGA (SEQ ID NO: 8).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprising the target sequence of MAP4K4 for inhibition by shRNA or shRNA targeting MAP4K comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 7 or SEQ ID NO: 8.
  • a target sequence of MAP4K6 for inhibition by shRNA is a nucleotide sequence comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a shRNA comprising a target sequence of MAP4K6 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCCCGGAACAAGATTCTGCACAATAGTGAAGCCACAGATGT ATTGTGCAGAATCTTGTTCCGGATGCCTACTGCCTCGGA (SEQ ID NO: 10).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprising the target sequence of MAP4K6 for inhibition by shRNA or the shRNA targeting MAP4K7 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • a target sequence of MAP4K7 for inhibition by shRNA is a nucleotide comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 1 1 ).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprises a shRNA comprising a target sequence of MAP4K7 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGAAAGGTCAAAGATTAAAGGTTATAGTGAAGCCACAGATGTA TAACCTTTAATCTTTGACCTTCTGCCTACTGCCTCGGA (SEQ ID NO: 12).
  • the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor comprising the target sequence of MAP4K7 for inhibition by shRNA or the shRNA targeting MAP4K7 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 11 or SEQ ID NO: 12.
  • the present disclosure further provides a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof.
  • the vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof is part of a composition.
  • the composition is a pharmaceutical composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, and one or more pharmaceutically acceptable excipients.
  • the disclosed vector and/or composition comprising said vector can be administered in a subject for treating neurodegenerative disease or brain injury.
  • the disclosed vector and/or composition comprising said vector can be administered in a subject for reducing a symptom associated with neurodegenerative disease or brain injury. In some aspects, the disclosed vector and/or composition comprising said vector can be administered in a subject providing protection to a subject from neural degeneration and/or neural injury.
  • the neurodegenerative disease comprise ALS, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, Friedreich ataxia, Lewy body dementia, or Spinal muscular atrophy.
  • the neurodegenerative disease comprise ALS.
  • the neurodegenerative disease comprise multiple sclerosis.
  • the neurodegenerative disease comprise ALS.
  • the neurodegenerative disease comprise Parkinson's disease.
  • the neurodegenerative disease comprise Alzheimer's disease.
  • the neurodegenerative disease comprise Huntington's disease.
  • the neurodegenerative disease comprise multiple system atrophy.
  • the neurodegenerative disease comprise prion diseases.
  • the neurodegenerative disease comprise Friedreich ataxia. In some aspects, the neurodegenerative disease comprise Lewy body dementia. In some aspects, the neurodegenerative disease comprise Spinal muscular atrophy. In some aspects, brain injury comprises traumatic brain injury (TBI). In some aspects, brain injury comprises stroke.
  • TBI traumatic brain injury
  • the disclosure provides a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, MAP4K6, or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, and a pharmaceutically acceptable excipient.
  • the vector comprises a CNH of MAP4K4, and/or a CNH- containing truncation of MAP4K4, or any combination thereof.
  • the CNH domain of human MAP4K4 comprises an amino acid sequence:
  • the vector comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 4.
  • the CNH-containing truncations of MAP4K4 can comprise 1-700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • CNH- containing truncations of MAP4K4 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 1 ) and 95 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 1 ).
  • the vector comprises a CNH of MAP4K6, and/or a CNH- containing truncation of MAP4K6, or any combination thereof.
  • the CNH domain of human MAP4K6 comprises an amino acid sequence:
  • the vector comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 5.
  • the CNH-containing truncations of MAP4K6 can comprise 1-700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K6 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 2) and 95 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 2).
  • the vector comprises a CNH of MAP4K7, and/or a CNH- containing truncation of MAP4K7, or any combination thereof.
  • the CNH domain of human MAP4K7 comprises an amino acid sequence:
  • the vector comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 6.
  • CNH-containing truncations of MAP4K7 can comprise 1-700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K7 can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 3) and 95 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 3).
  • the vector comprising CNH of MAP4K4, MAP4K6 or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof reduces the activity of a MAP4K4, MAP4K6, or MAP4K7, or any combination thereof, as compared to the expression prior to the administration of the CNH of MAP4K4, MAP4K6 or MAP4K7, and/or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • the activity of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof is reduced by about 1% to about 100%. In some aspects, the activity of MAP4K4, MAP4K6, MAP4K7, or any combination thereof may be reduced by 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. In some aspects, the activity of MAP4K4, MAP4K6 or MAP4K7 is reduced by about 90%.
  • the disclosure further provides a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the disclosure further provides a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, and one or more pharmaceutically acceptable excipients.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K4, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K6, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is gRNA comprising a target sequence of MAP4K7, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding a gRNA that comprises a target sequence of MAP4K4, for example CAGGACATGATGACCAACTC (SEQ ID NO: 13) or GGGCGGAGAAATACGTTCAT (SEQ ID NO: 14), and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding a gRNA that comprises a target sequence of MAP4K6, for example CGGACAGGTCGATGTCGTCC (SEQ ID NO: 15) or AGGGTCGGCATGTCAAGACG (SEQ ID NO: 16), and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding a gRNA that comprises a target sequence of MAP4K7, for example CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18), and a pharmaceutically acceptable excipient.
  • a gRNA that comprises a target sequence of MAP4K7, for example CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18), and a pharmaceutically acceptable excipient.
  • gRNAs used in the vector can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • gRNA can be engineered and produced using primers disclosed in Table 2.
  • the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop.
  • the sgRNA can be between 50-220 (e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • 50-220 e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120 nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • the composition comprising a vector engineered to express a gRNA comprising the target sequence, comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, and a pharmaceutical excipient.
  • the vector comprising a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof reduces the expression of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, as compared to the gene expression prior to the introduction of the gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, which can lead to the inhibition of production of the MAP4K4, MAP4K6, MAP4K7 gene product.
  • the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1 % to about 100%.
  • the amount of MAP4K4, MAP4K6, and/or MAP4K7 expression may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%.
  • the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.
  • the disclosure further provides a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the disclosure further provides a composition comprising a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K4, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K6, and a pharmaceutically acceptable excipient.
  • the composition comprises a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, wherein the inhibitor of MAP4K is shRNA comprising a target sequence of MAP4K7, and a pharmaceutically acceptable excipient.
  • a target sequence of MAP4K4 for inhibition by shRNA is nucleotide sequence comprising AACCGAAGACGATTTCAACAAA (SEQ ID NO: 7).
  • the vector comprises a shRNA comprising a target sequence of MAP4K4 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCACCGAAGACGATTTCAACAAATAGTGAAGCCACAGATGTA TTTGTTGAAATCGTCTTCGGTTTGCCTACTGCCTCGGA (SEQ ID NO: 8).
  • composition comprising the target sequence of MAP4K4 for inhibition by shRNA or shRNA targeting MAP4K4, comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 7 or SEQ ID NO: 8.
  • a target sequence of MAP4K6 for inhibition by shRNA is a nucleotide sequence comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9).
  • the vector comprises a shRNA comprising a target sequence of MAP4K6 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCCCGGAACAAGATTCTGCACAATAGTGAAGCCACAGATGT ATTGTGCAGAATCTTGTTCCGGATGCCTACTGCCTCGGA (SEQ ID NO: 10).
  • composition comprising the target sequence of MAP4K6 for inhibition by shRNA or the shRNA targeting MAP4K7, comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • a target sequence of MAP4K7 for inhibition by shRNA is a nucleotide comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 11 ).
  • the composition comprises a shRNA comprising a target sequence of MAP4K7 which is a nucleotide sequence comprising
  • the composition comprising the target sequence of MAP4K7 for inhibition by shRNA or the shRNA targeting MAP4K7 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 1 1 or SEQ ID NO: 12.
  • the vector comprising a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof reduces the expression of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, as compared to the gene expression prior to the introduction of the shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, which can lead to the inhibition of production of the MAP4K4, MAP4K6, MAP4K7 gene product.
  • the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1 to about 100%.
  • the amount of MAP4K4, MAP4K6, and/or MAP4K7 expression may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%.
  • the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.
  • the composition comprises a vector engineered to express the inhibitor of MAP4K.
  • Vectors engineered to carry inhibitors of MAP4K can be lentiviral plasmids.
  • lentiviral plasmids can be lentiviral plasmids for motor neurons (e.g., Addgene plasmid #90214 or Addgene plasmid #90215).
  • the lentivirus vector is Addgene plasmid #90214.
  • the lentivirus vector is Addgene plasmid #90215.
  • the composition comprises a lentivirus vector engineered to express a CNH of MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the composition comprises lentivirus vector is engineered to express a CNH of MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the composition comprises lentivirus vector is engineered to express a CNH of MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the composition comprises a lentivirus vector engineered to express a gRNA comprising a target sequence of MAP4K4, for example CAGGACATGATGACCAACTC (SEQ ID NO: 13) or GGGCGGAGAAATACGTTCAT (SEQ ID NO: 14).
  • the composition comprises lentivirus vector is engineered to express a gRNA comprising a target sequence of MAP4K6, for example
  • composition comprises lentivirus vector is engineered to express a gRNA comprising a target sequence of MAP4K7, for example
  • CGACTCCCCGGCTCGAAGCC SEQ ID NO: 17
  • TTCATCCAGGCTTCGAGCCG SEQ ID NO: 18
  • the composition comprising a lentivirus vector engineered to express a gRNA comprising the target sequence comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the composition comprises a lentivirus vector engineered to express a shRNA comprising a target sequence of MAP4K4.
  • the composition comprises lentivirus vector is engineered to express a shRNA comprising a target sequence of MAP4K6.
  • the composition comprises lentivirus vector is engineered to express a shRNA comprising a target sequence of MAP4K7.
  • the lentiviral vectors disclosed herein further comprises elements such as 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components, which are polypeptides required for the assembly of viral particles, rev and rev response element (RRE) sequences, cloning sites, promoters, regulatory elements, or heterologous nucleic acids.
  • RRE rev and rev response element
  • at least part of one or more protein coding regions essential for replication may be removed from the virus, which makes the viral vector replication defective.
  • the sequences the disclosed elements are well known in the art. Further, the assembly of such elements into the vector can be performed using methods well known in the art.
  • the composition comprises recombinant adeno-associated virus (rAAV) vector system engineered to express an inhibitor of MAP4K.
  • rAAV vector comprises a AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-rh10, AAV-hu11 , AAV-PHP.B, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2/1 , rAAV2/8, or rAAV2/9.
  • the rAAV vector is a AAV9 vector.
  • the rAAV vector is an AAV-PHP.eB vector.
  • the composition comprises a AAV9 vector engineered to express a CNH of MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the composition comprises a AAV9 vector engineered to express a CNH of MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the composition comprises a AAV9 vector engineered to express a CNH of MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the composition comprises a AAV9 vector engineered to express a gRNA comprising a target sequence of MAP4K4. In some aspects, the composition comprises a AAV9 vector engineered to express a gRNA comprising a target sequence of MAP4K6. In some aspects, the composition comprises a AAV9 vector engineered to express a gRNA comprising a target sequence of MAP4K7.
  • the composition comprises a AAV9 vector engineered to express a shRNA comprising a target sequence of MAP4K4. In some aspects, the composition comprises a AAV9 vector engineered to express a shRNA comprising a target sequence of MAP4K6. In some aspects, the composition comprises a AAV9 vector engineered to express a shRNA comprising a target sequence of MAP4K7.
  • the composition comprises an AAV-PHP.eB vector engineered to express a CNH of MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • the composition comprises an AAV-PHP.eB vector engineered to express a CNH of MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • the composition comprises an AAV-PHP.eB vector engineered to express a CNH of MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the composition comprises an AAV-PHP.eB vector engineered to express a gRNA comprising a target sequence of MAP4K4. In some aspects, the composition comprises an AAV-PHP.eB vector engineered to express a gRNA comprising a target sequence of MAP4K6. In some aspects, the composition comprises an AAV-PHP.eB vector engineered to express a gRNA comprising a target sequence of MAP4K7.
  • the composition comprises an AAV-PHP.eB vector engineered to express a shRNA comprising a target sequence of MAP4K4. In some aspects, the composition comprises a AAV-PHP.eB vector engineered to express a shRNA comprising a target sequence of MAP4K6. In some aspects, the composition comprises an AAV-PHP.eB vector engineered to express a shRNA comprising a target sequence of MAP4K7.
  • rAAV vector further comprises elements such as a 5' ITR, a promoter, an enhancer, Kozak sequence, a polyadenylation signal, intronic sequence, and/or a 3' ITR.
  • elements such as a 5' ITR, a promoter, an enhancer, Kozak sequence, a polyadenylation signal, intronic sequence, and/or a 3' ITR.
  • the sequences such elements are well known in the art. Further, the assembly of such elements into the vector can be performed using methods well known in the art.
  • an effective amount of the composition comprising a lentivirus vector or rAAV engineered to express an inhibitor of MAP4K, administered in a subject is at a concentration of about 1X10 2 genome copies (GC)/ml to about 2X10 15 GC/ml.
  • the composition is administered at a concentration of about 1X10 2 GC/ml, about 1X10 3 GC/ml, about 1X10 4 GC/ml, about 1X10 5 GC/ml, about 1X10 6 GC/ml, about
  • composition comprising a lentivirus engineered to express an inhibitor of MAP4K is administered at a concentration of 1X1013GC/mL. In some aspects, the composition comprising a lentivirus engineered to express an inhibitor of MAP4K, is administered at a concentration of 2.0X1012 GC/mL. In some aspects, the composition comprising rAAV engineered to express an inhibitor of MAP4K, is administered at a concentration of 1X10 13 GC/mL. In some aspects, composition comprising an rAAV engineered to express an inhibitor of MAP4K, is administered at a concentration of 2.0X10 12 GC/mL.
  • composition comprising an effective amount of the lentivirus vector or rAAV engineered to express an inhibitor of MAP4K, is formulated for administration as a liquid with a volume in a range of about 1 pl to about 1 ml.
  • the dose of the composition comprising lentivirus or rAAV for administration formulated as a liquid a volume of about 1 pl, about 2 pl, about 3 pl, about 4 pl, about 5 pl, about 6 pl, about 7 pl, about 8 pl, about 9 pl, about 10 pl, about 15 pl, about 20 pl, about 25 pl, about 30 pl, about 35 pl, about 40 pl, about 45 pl, about 50 pl, about 55 pl, about 60 pl, about 65 pl, about 70 pl, about 75 pl, about 80 pl, about 85 pl, about 90 pl, about 95 pl, about 100 pl, about 125 pl, about 150 pl, about 200 pl, about 250 pl, about 300 pl, about 350 pl, about 400 pl, about 450 pl, about 500 pl, about 550 pl, about 600 pl, about 650 pl, about 700 pl, about 750 pl, about 800 pl, about 850 pl, about 900 pl, about 950 pl or about 1 ml.
  • the composition comprising lentivirus or rAAV engineered to express an inhibitor of MAP4K is formulated for administration as a liquid of a volume of 1 ml or more. In some aspects, the composition comprising a lentivirus engineered to express an inhibitor of MAP4K, is administered at about 1.0 pl. In some aspects, the composition comprising a lentivirus engineered to express an inhibitor of MAP4K, is administered at about 8 pl - about 10 pl. In some aspects, the composition comprising an rAAV engineered to express an inhibitor of MAP4K, is administered at about 1.0 pl. In some aspects, the composition comprising an rAAV engineered to express an inhibitor of MAP4K, is administered at about 8 pl - about 10 pl.
  • the composition comprising a lentivirus or rAAV engineered to express an inhibitor of MAP4K, is formulated for administration at a dose from 1 pg/kg to 100 mg/kg, 1 pg/kg to 50 mg/kg, 1 pg/kg to 20 mg/kg, 1 pg/kg to 10 mg/kg, 1 pg/kg to 1 mg/kg, 100 pg/kg to 100 mg/kg, 100 pg/kg to 50 mg/kg, 100 pg/kg to 20 mg/kg, 100 pg/kg to 10 mg/kg, 100 pg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg.
  • the dosage of the composition is 0.1 mg/kg of body weight (
  • the vectors disclosed herein is engineered to express MAP4K inhibitor comprises a promoter operably linked to the MAP4K inhibitor.
  • promoter is a neuron specific promoter, wherein the neuron specific promoter is neuronspecific enolase (NSE) promoter, platelet-derived growth factor (PDGF) promoter, platelet- derived growth factor B-chain (PDGF-p) promoter, synapsin (Syn) promoter, Synapsin 1 (Syn1 ) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+/calmodulin- dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, Neuropeptide Y promoter, neurofilament light (NFL) promoter, heavy (NFH) promoter, p-globin minigene np2 promoter, preproenkephalin (PPE) promoter, enke
  • NSE neuronspecific en
  • the promoter can be a human, mouse, rat, or a synthetically engineered promoter.
  • the sequences of these promoters are well known in the art and may be obtained from publicly available databases.
  • nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956.
  • vectors encoding neuron specific promoter e.g., human synapsin 1
  • Addgene database nonlimiting examples include plasmid #22907 encoding pAAV-hSyn-RFP, plasmid #177810 encoding pLV-hSyn1-GFP. Using these vectors human synapsin 1 can be subcloned into a desired vector, using known methods in the art.
  • the disclosed vectors comprise a neuron specific synapsin promoter.
  • the neuron specific promoter is a synapsin 1 promoter.
  • the neuron specific promoter is a human synapsin 1 (hSYN1 ) promoter.
  • lentivirus vector disclosed herein comprises a hSYN1 promoter operably linked to a CNH of MAP4K4, MAP4K6 or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • lentivirus vector disclosed herein comprises a hSYN1 promoter operably linked to a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • lentivirus vector disclosed herein comprises a hSYN1 promoter operably linked to a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • rAAV disclosed herein comprises a hSYN1 promoter operably linked to a CNH of MAP4K4, MAP4K6 or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • rAAV disclosed herein comprises a hSYN1 promoter operably linked to a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • rAAV disclosed herein comprises a hSYN1 promoter operably linked to a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the composition can comprise a chemical inhibitor of MAP4K.
  • the chemical inhibitor can comprise a small molecule, or a large molecule.
  • the chemical inhibitor can be K02288.
  • compositions disclosed herein may be pharmaceutical compositions.
  • Pharmaceutical compositions disclosed herein may comprise one or more pharmaceutically acceptable diluent, excipient, and/or carrier.
  • Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.
  • Pharmaceutically acceptable carriers include any and all solvents, adjuvants, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, colorants, other medicinal or pharmaceutical agents, wetting agents, emulsifying agents, solution promoters, solubilizers, antifoaming agents, and such like materials and any combinations thereof, as would be known to one of ordinary skill in the art.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may also be found for example in Remington’s Pharma. Sci. 18th ed. 1990. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of engineered vectors into preparations which can be used pharmaceutically.
  • physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of engineered vectors into preparations which can be used pharmaceutically.
  • any of the well-known techniques, carriers, and excipients may be used as suitable and/or as understood in the art.
  • compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents.
  • polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof.
  • water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose
  • water-insoluble polymers such as cross-linked carboxyl-containing polymers
  • mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylme
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.
  • compositions disclosed herein may comprise a viscous formulation.
  • viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents.
  • compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of gelling or thickening agent(s) by total weight of the composition.
  • compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition.
  • suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate.
  • viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethyl hydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose
  • compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of one or more agents by total weight of the composition.
  • compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition.
  • one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and/or other property of the muscarinic antagonist composition of the present disclosure.
  • additives may be biocompatible, without being harsh, abrasive, and/or allergenic.
  • compositions disclosed herein may comprise one or more acidifying agents.
  • acidifying agents refers to compounds used to provide an acidic medium.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.
  • compositions disclosed herein may comprise one or more alkalizing agents.
  • alkalizing agents are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art.
  • any pharmaceutically acceptable organic or inorganic base can be used.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.
  • compositions disclosed herein may comprise one or more antioxidants.
  • antioxidants are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process.
  • Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition.
  • compositions disclosed herein may comprise a buffer system.
  • a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.
  • the amount of one or more buffering agents may depend on the desired pH level of a composition.
  • the compositions disclosed herein may have a pH of about 6 to about 9.
  • the compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.
  • compositions disclosed herein may comprise one or more preservatives.
  • preservatives refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form.
  • preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof.
  • any pharmaceutically acceptable preservative can be used.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition.
  • compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.
  • compositions disclosed herein may comprise one or more surface-acting reagents or detergents.
  • surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic.
  • compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.
  • compositions disclosed herein may comprise one or more stabilizers.
  • a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent.
  • Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.
  • compositions disclosed herein may comprise one or more tonicity agents.
  • a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation.
  • Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art.
  • Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm/L). Osmolarity may be measured using methods commonly known in the art.
  • a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein.
  • the amount of one or more tonicity agents comprising a composition disclosed herein may result in a composition osmolarity of about 150 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370 mOsm/L or about 250 mOsm/L to about 320 mOsm/L.
  • a composition herein may have an osmolality ranging from about 100 mOsm/kg to about 1000 mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320 mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsm/kg.
  • a composition described herein may have an osmolarity of about 100 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L.
  • compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.
  • composition disclosed herein can be formulated for enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal) administration.
  • enteral e.g., oral
  • parenteral e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal
  • the composition described herein can be formulated for administration intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v.), or direct administration into the cerebrospinal fluid (CSF), e.g., via intrathecal and/or intracerebral injection.
  • sterile aqueous or oleaginous suspensions may be formulated according to the known technique using suitable dispersing agents, wetting agents and/or suspending agents.
  • the aqueous injectable composition is formulated for administration to said subject by bolus administration.
  • the said aqueous injectable composition is formulated for administration to the subject by infusion.
  • Non-limiting acceptable carriers or excipients that can be used include water, Ringer's solution, isotonic sodium chloride solution, lactose, sucrose, organic solvents and polyethylene glycol. Sterile oils are also conventionally used as solvents or suspending media.
  • the compositions may include additional excipients such as binders, disintegrants, lubricants, surface active agents (surfactants), emulsifiers, preservatives and favoring agents.
  • the compositions may be prepared by any method known in the art.
  • the composition may comprise one or more active agents in addition to the inhibitor of MAP4K, and vectors, provided herein.
  • additional active agents include but are not limited to anti-inflammatories, analgesics, cholinesterase inhibitors, antipsychotics, dopamine agonists, anti-depressants, anti-epileptic agents, L-dopamine, or any other known agents used for treating neurodegenerative diseases or brain injury.
  • administration of disclosed composition results in MAP4K inhibitor expressed ectopically in neuron or motor neuron cells of the subject.
  • the ectopically expressed MAP4K inhibitor leads to an altered phenotype or physiology of the neuron or motor neuron cells of the subject.
  • ectopic expression of MAP4K inhibitor leads to treatment of neurodegenerative disease or brain injury, reduction of one or more symptoms associated with neurodegenerative disease or brain injury, or provide protection from neural degeneration and/or neural injury, in the subject.
  • the disclosure provides an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a nucleic acid sequence encoding MAP4K inhibitor.
  • the isolated nucleic acid can be formulated into a composition.
  • the composition is a pharmaceutical composition comprising the isolated nucleic acid and one or more pharmaceutically acceptable excipients.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprises a nucleic acid sequence encoding a CNH from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof. In some aspects, an isolated nucleic acid sequence comprises a nucleic acid sequence encoding a CNH from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof. In some aspects, an isolated nucleic acid sequence comprises a nucleic acid sequence encoding a CNH from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • the isolated nucleic acid sequence comprises a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4, or any combination thereof, comprising an amino acid sequence:
  • the isolated nucleic acid sequence comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 4.
  • the CNH-containing truncations of MAP4K4 in the isolated nucleic acid sequence can comprise 1-700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K4 in the isolated nucleic acid sequence can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO:1 ) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 1 ) and 95 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO:1 ) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K4 sequence (SEQ ID NO: 1 ) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 1 ).
  • the isolated nucleic acid sequence comprises a CNH of MAP4K6, and/or a CNH-containing truncation of MAP4K6, or any combination thereof, comprising an amino acid sequence:
  • the isolated nucleic acid sequence comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 5.
  • the CNH-containing truncations of MAP4K6 in the isolated nucleic acid sequence can comprise 1-700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • CNH-containing truncations of MAP4K6 in the isolated nucleic acid sequence can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the N terminus of the CNH and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 amino acids of the disclosed MAP4K6 sequence (SEQ ID NO: 2) flanking the C terminus of the CNH.
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K6 sequence (
  • the isolated nucleic acid sequence comprises a CNH of MAP4K7, and/or a CNH-containing truncation of MAP4K7, or any combination thereof, comprising an amino acid sequence:
  • the isolated nucleic acid sequence comprises a CNH with an amino acid sequence having at least 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% identity or similarity to SEQ ID NO: 6.
  • CNH-containing truncations of MAP4K7 in the isolated nucleic acid sequence can comprise 1-700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and/or 1-26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH.
  • CNH- containing truncations of MAP4K7 in the isolated nucleic acid sequence can comprise 1 , 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO:
  • Non-limiting examples of truncation comprises 700 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 296 to 1312 of SEQ ID NO: 3) and 95 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the N terminus of the CNH and 26 amino acids of the disclosed MAP4K7 sequence (SEQ ID NO: 3) flanking the C terminus of the CNH (amino acids 866 to 1312 of SEQ ID NO: 3).
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is provided herein.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4. In some aspects, provided herein is an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6. In some aspects, provided herein is an isolated nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • the isolated nucleic acid is a gRNA that comprises a target sequence of MAP4K4, for example CAGGACATGATGACCAACTC (SEQ ID NO: 13) or GGGCGGAGAAATACGTTCAT (SEQ ID NO: 14).
  • the isolated nucleic acid is a gRNA that comprises a target sequence of MAP4K6, for example CGGACAGGTCGATGTCGTCC (SEQ ID NO: 15) or AGGGTCGGCATGTCAAGACG (SEQ ID NO: 16).
  • the isolated nucleic acid is a gRNA that comprises a target sequence of MAP4K7, for example CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18).
  • the isolated nucleic acid sequence comprising the target sequence gRNA comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • gRNAs in the isolated nucleic acid sequence can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • gRNA can be engineered and produced using primers disclosed in Table 2.
  • the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop.
  • the sgRNA can be between 50-220 (e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • 50-220 e.g., 55-200, 60-190, 60-180, 60-170, 60-160, 60-150, 60-140, 60-130, and 60-120 nucleotides in length, such as 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 220 nucleotides in length.
  • an isolated nucleic acid sequence comprises a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • the isolated nucleic acid sequence comprises a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4. In some aspects, the isolated nucleic acid sequence comprises a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6. In some aspects, the isolated nucleic acid sequence comprises a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • the isolated nucleic acid sequence comprises a target sequence of MAP4K4 for inhibition by shRNA comprising AACCGAAGACGATTTCAACAAA (SEQ ID NO: 7).
  • the isolated nucleic acid sequence comprises a shRNA comprising a target sequence of MAP4K4 comprising the nucleotide sequence of TGCTGTTGACAGTGAGCGCACCGAAGACGATTTCAACAAATAGTGAAGCCACAGATGTA TTTGTTGAAATCGTCTTCGGTTTGCCTACTGCCTCGGA (SEQ ID NO: 8).
  • the isolated nucleic acid sequence comprising the target sequence of MAP4K4 for inhibition by shRNA or shRNA targeting MAP4K4 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 7 or SEQ ID NO: 8.
  • the isolated nucleic acid sequence comprises a target sequence of MAP4K6 for inhibition by shRNA comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9).
  • the isolated nucleic acid sequence comprises a shRNA comprising a target sequence of MAP4K6 comprising the nucleic acid sequence of TGCTGTTGACAGTGAGCGCCCGGAACAAGATTCTGCACAATAGTGAAGCCACAGATGT ATTGTGCAGAATCTTGTTCCGGATGCCTACTGCCTCGGA (SEQ ID NO: 10).
  • the isolated nucleic acid sequence comprising the target sequence of MAP4K6 for inhibition by shRNA or the shRNA targeting MAP4K7 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • the isolated nucleic acid sequence comprises a target sequence of MAP4K7 for inhibition by shRNA comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 11 ).
  • the isolated nucleic acid sequence comprises a shRNA comprising a target sequence of MAP4K7 comprising the nucleic acid sequence TGCTGTTGACAGTGAGCGAAAGGTCAAAGATTAAAGGTTATAGTGAAGCCACAGATGTA TAACCTTTAATCTTTGACCTTCTGCCTACTGCCTCGGA (SEQ ID NO: 12).
  • the isolated nucleic acid sequence comprising the target sequence of MAP4K7 for inhibition by shRNA or the shRNA targeting MAP4K7 comprise a nucleotide sequence having at least 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% identity or similarity to SEQ ID NO: 11 or SEQ ID NO: 12.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a neurotropic capsid; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • a neurotropic capsid is a capsid of AAV1 , AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-rh10, AAV-hu1 1 , AAV-PHP.B, AAV-PHP.eB, AAV-TT, AAVv66, rAAV2/1 , rAAV2/8, or rAAV2/9.
  • the capsid is an AAV9 capsid.
  • AAV9 is AAV9, AAV9.9, AAV9.11 , AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61 , AAV9.68, or AAV9.84.
  • the capsid is an AAV- PHP.eB capsid.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH- containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • the isolated nucleic acid sequence comprises a nucleic acid sequence encoding a neuron specific promoter, wherein the neuron specific promoter is neuron-specific enolase (NSE) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B-chain (PDGF-p) promoter, synapsin (Syn) promoter, Synapsin 1 (Syn1 ) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+/calmodulin- dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, Neuropeptide Y promoter, neurofilament light (NFL) promoter, heavy (NFH) promoter, p-globin minigene np2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT) promoter, neuron-
  • promoters can be from human, mouse, rat, or synthetically engineered promoter.
  • the sequences of these promoters are well known in the art and may be obtained from publicly available databases.
  • nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956.
  • vectors encoding neuron specific promoter e.g., human synapsin 1
  • Addgene database nonlimiting examples include plasmid #22907 encoding pAAV-hSyn-RFP, plasmid #177810 encoding pLV-hSyn1-GFP. Using these vectors human synapsin 1 can be subcloned into a desired vector, using known methods in the art.
  • the neuron specific promoter is a synapsin promoter. In some aspects, the neuron specific promoter is a synapsin 1 promoter. In some aspects, the neuron specific promoter is a human synapsin 1 (hSYN1 ) promoter.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid; a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid; a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid or an AAV-PHP.eB capsid a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV9 capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an AAV-PHP.eB capsid, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding lentivirus vector; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • a lentivirus vector is Addgene #90214 or Addgene #90215. In some aspects, the lentivirus vector is Addgene #90214. In some aspects, the lentivirus vector is Addgene #90215.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215; a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH- containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a CNH, from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, or MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215 a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a neuron specific promoter; and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a neuron specific promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • the isolated nucleic acid sequence comprises a nucleic acid sequence encoding a neuron specific promoter, wherein the neuron specific promoter is neuron-specific enolase (NSE) promoter, platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B-chain (PDGF-p) promoter, synapsin (Syn) promoter, Synapsin 1 (Syn1 ) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+/calmodulin- dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, Neuropeptide Y promoter, neurofilament light (NFL) promoter, heavy (NFH) promoter, p-globin minigene np2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT) promoter, neuron-
  • promoters can be from human, mouse, rat, or synthetically engineered promoter.
  • the sequences of these promoters are well known in the art and may be obtained from publicly available databases.
  • nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956.
  • vectors encoding neuron specific promoter e.g., human synapsin 1
  • Addgene database nonlimiting examples include plasmid #22907 encoding pAAV-hSyn-RFP, plasmid #177810 encoding pLV-hSyn1-GFP. Using these vectors human synapsin 1 can be subcloned into a desired vector, using known methods in the art.
  • the neuron specific promoter is a synapsin promoter. In some aspects, the neuron specific promoter is a synapsin 1 promoter. In some aspects, the neuron specific promoter is a human synapsin 1 (hSYN1 ) promoter. [0335] In some aspects, the disclosure further provides, an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a CNH, wherein the CNH is from MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a CNH from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding a CNH, from MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a gRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214 or an Addgene #90215 a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter; and a nucleic acid sequence encoding MAP4K inhibitor, wherein the MAP4K inhibitor is a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K4.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K6.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90214, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • an isolated nucleic acid sequence comprising a nucleic acid sequence encoding an Addgene #90215, a nucleic acid sequence encoding a hSYN1 promoter, and a nucleic acid sequence encoding a shRNA comprising a target sequence of MAP4K7.
  • the disclosed isolated nucleic acids or inhibitor can treat or reduce one or more symptoms associated with neurodegenerative disease or brain injury in a subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject comprises reducing traumatic brain- induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • treating or reducing one or more symptoms associated with neurodegenerative disease or brain injury in a subject by administration of disclosed isolated nucleic acids or inhibitor comprises, improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • treating or reducing one or more symptoms comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • the administration of the disclosed isolated nucleic acids or inhibitor can provide protection to a subject from neurodegeneration and/or neural injury.
  • Providing protection to a subject from neurodegeneration and/or neural injury comprises reducing traumatic brain-induced tau phosphorylation, reducing reactive gliosis, reducing lesion size, reducing behavioral deficits, and/or reducing severity or progression of the neurodegenerative disease or brain injury.
  • the rate of severity or progression, tau phosphorylation, reactive gliosis, lesion size, and/or behavioral deficits is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • providing protection to a subject from neurodegeneration and/or neural injury by administration of disclosed isolated nucleic acids or inhibitor can comprise improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject.
  • providing protection comprises improving brain tissue damage, improving memory and/or cognitive performance, improving motor function, improving neuronal survival and neurite outgrowth, and/or improving the life span of the subject by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to baseline assessment in the subject, before treatment, or a control subject.
  • the disclosed isolated nucleic acids can be introduced into cells using either in vivo or in vitro (also termed ex vivo) transduction techniques. If transduced in vitro, the desired recipient cell, can be removed from the subject, transduced with isolated nucleic acids and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject. Suitable methods for the delivery and introduction of transduced cells into a subject have been described.
  • cells can be transduced in vitro by combining the isolated nucleic acids disclosed herein, with cells to be transduced in appropriate media, and screening transduced cells using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into compositions, and the composition introduced into the subject by various techniques as described herein.
  • the isolated nucleic acids disclosed herein can be formulated for enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal) administration.
  • the isolated nucleic acids described herein can be formulated for administration intraperitoneally (i.p.), intramuscularly (i.m.), intravenously (i.v.), or direct administration into the cerebrospinal fluid (CSF), e.g., via intrathecal and/or intracerebral injection.
  • CSF cerebrospinal fluid
  • administration of disclosed isolated nucleic acid results in MAP4K inhibitor expressed ectopically in neuron or motor neuron cells of the subject.
  • the ectopically expressed MAP4K inhibitor lead to an altered phenotype or physiology of the neuron or motor neuron cells of the subject.
  • ectopic expression of MAP4K inhibitor leads to treatment of neurodegenerative disease or brain injury, reduction of one or more symptoms associated with neurodegenerative disease or brain injury, or provide protection from neural degeneration and/or neural injury, in the subject.
  • the transduced cells can be used as a platform used for evaluating, screening or monitoring effects of drugs, or compounds or understanding biological mechanisms underlying neurodegeneration or nerve injury.
  • transduced cells can be exposed to a candidate drug or compound. After being cultured under suitable conditions for a suitable period, the phenotypic or physiological changes in cells can be compared with a control cells that does not contain the candidate molecule. If the phenotype or physiology of the cells change in the presence of the candidate drug or compound as compared to that in the absence of the candidate drug or compound, it indicates that the candidate drug or compound may affect development, differentiation, or growth of cells.
  • An inhibitor of MAP4K described herein can be provided in a kit.
  • the kit includes the inhibitor, e.g., a composition that includes the inhibitor or a vector engineered to express the inhibitor, and informational material.
  • the informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of the inhibitor or a vector engineered to express the inhibitor, for the methods described herein.
  • the informational material describes methods for administering the inhibitor or a vector engineered to express the inhibitor, to treat or protect a subject from the development of a neurodegenerative disease or brain injury, or at least one symptom of the neurodegenerative disease or brain injury.
  • the informational material can include instructions to administer the inhibitor or a vector engineered to express the inhibitor, in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein).
  • the informational material of the kits is not limited in its form.
  • the informational material, e.g., instructions is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet.
  • the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording.
  • the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the inhibitor or a vector engineered to express the inhibitor, and/or its use in the methods described herein.
  • a link or contact information e.g., a physical address, email address, hyperlink, website, or telephone number
  • the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative, and/or a second agent for treating a neurodegenerative disease or brain injury, described herein.
  • the other ingredients can be included in the kit, but in different compositions or containers than the inhibitor.
  • the kit can include instructions for admixing the inhibitor or a vector engineered to express the inhibitor, and the other ingredients, or for using the inhibitor or a vector engineered to express the inhibitor, together with the other ingredients.
  • the inhibitor or a vector engineered to express the inhibitor can be provided in any form, e.g., liquid, dried or lyophilized form.
  • the inhibitor or a vector engineered to express the inhibitor is substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution.
  • the solvent e.g., sterile water or buffer, can be provided in the kit.
  • the kit can include one or more containers for the composition containing the inhibitor or a vector engineered to express the inhibitor.
  • the kit contains separate containers, dividers or compartments for the inhibitor or a vector engineered to express the inhibitor (e.g., in a composition) and informational material.
  • the inhibitor or a vector engineered to express the inhibitor can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the inhibitor or a vector engineered to express the inhibitor is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agent (e.g., in a composition).
  • the kit can include a plurality of syringes, ampules, or foil packets, each containing a single unit dose of the inhibitor, or a vector engineered to express the inhibitor.
  • the containers of the kits can be airtight and/or waterproof.
  • C57BL/6J (Jax #000664), B6SJLF1 (Jax #100012), and SOD1 G93A (Jax #002726) were purchased from the Jackson Laboratory.
  • the SOD1 G93A strain was maintained by breeding male hemizygous carriers to B6SJLF1 hybrids. Standard PCRs were used for genotyping.
  • the Wild-type C57BL/6J mice and the rTg4510 mice were derived from The Jackson Laboratory. All mice were housed under a controlled temperature and a 12-h light/dark cycle with free access to water and food in a barrier animal facility. Sample sizes were empirically determined. Animal procedures and protocols were approved by the Institutional Animal Care and Use Committee at UT Southwestern.
  • the human fibroblast line C9-4 and C9-5 were gifts of Dr. Corey lab. Other fibroblast lines were obtained from Coriell, ATCC, or Cedars-Sinai (Table 1). All fibroblasts were maintained in DMEM-high glucose supplemented with 15% fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO2.
  • Table 1 Sources of fibroblast lines examined in this work.
  • the L1700 bioactive compound library was purchased from Selleck Chemicals. Kenpaullone (Ken) and A83-1 were obtained from Tocris. Forskolin (FSK), LDN193189.2HCI (LDN), PF-6260933 (MAP4Ki), and other individual chemicals were ordered from Sigma or Selleck.
  • Lentiviral plasmids for hiMNs are available from Addgene (#90214 and #90215). cDNAs for HA-tagged HGK, MINK1 , TNIK, and their kinase-dead mutants were individually subcloned into a third-generation lentiviral vector, pCSC-SP-PW-IRES-GFP. The same vector was also used to express BiolD2-myc-HA-MINK1 mt.
  • a single vector CRISPR/Cas9 system was developed by replacing CMV-SP-PW-IRES-GFP with the hU6-Filler-EFS-SpCas9-FLAG- P2A-Puro cassette from lentiCRISPRv2 (Addgene #52961 ).
  • mCherry was inserted to replace the Puro fragment to construct pCSC-hU6-Filler-EFS- SpCas9-FLAG-P2A-mCherry.
  • each individual sgRNA was subcloned into this new vector by replacing the Filler fragment. All primers used for PCR and/or subcloning of cDNA or sgRNA are listed in Table 2.
  • Table 3A discloses shRNA sequences and Table 3B discloses shRNA target sites.
  • Table 4 discloses qRT-PCR primers. Replication-incompetent lentiviruses were generated in HEK293T cells (ATCC) via co-transfections of lentiviral vectors, pREV, pMDL and pVSV-G. They were stored at 4°C before cell transductions.
  • Table 2 Primer sequences
  • NL- and ALS-hiMNs were converted from adult human skin fibroblasts as previously described with modifications. Briefly, fibroblasts were plated onto Matrigel-coated 10-cm dishes at 1.5x104 cells per cm2. The next day cells were transduced with lentiviral supernatants containing 6 pg/ml polybrene. After overnight transduction, fibroblasts were cultured in fresh fibroblast culture media for one more day.
  • C2 medium consisting of DMEM:F12:neurobasal (2:2:1 ), 0.8% N2 (Invitrogen), 0.8% B27 (Invitrogen), and supplemented with 10 pM FSK, 0.5 pM LDN, and 10 ng/ml FGF2 (PeproTech).
  • dpi post virus infection
  • hiMNs were further enriched by passing through a 20-pm cell strainer and were then seeded into culture vessels coated with Matrigel or co-cultured with primary mouse cortical astrocytes, using C2 medium supplemented with 5 pM FSK and 10 ng/ml each of BDNF, GDNF, and NT3 (PeproTech). The medium was half-changed weekly until further analysis.
  • hiMNs were plated into Matrigel-coated 96- well plates. They quickly attached to the surface and outgrew processes within 2-4 hours. Individual chemical was then added into each well at 2.5 pM for primary screens and at 0.5, 1.0, 2.5, or 5 pM for secondary screens. Vehicle (DMSO) and Ken in quadruplicates served as the negative and positive control, respectively. Immediately prior to survival assays at about 72-hour post treatments, cells in each well were quickly imaged for morphology under an EVOS fluorescence microscope. Viable cells were then determined by the CellTiter-Glo Luminescent Cell Viability Assay.
  • hiMNs were co-cultured with mouse primary cortical astrocytes seeded onto 96-well plates or coverslips in 24-well plates.
  • One 96-well plate was used to determine the number of seeded GFP+ hiMNs 4-hour post plating.
  • the rest plates were treated with the top 15 hits, vehicle, Ken, and other selected chemicals in pentaplicates at a proper concentration determined by the dose-response assays. Chemical treatments were repeated weekly until analysis at 1 , 3, or 5 weeks later.
  • hiMNs were then fixed and stained with antibodies for GFP and the neuronal marker TUJ1.
  • Cells in each well were imaged and quantified with a Cytation3 imaging reader and software (BioTek). As all GFP+ cells were also TUJ1+, viable neurons in each well were further confirmed by manual counting GFP+ cells. Relative survival was calculated by first normalizing to the number of seeded GFP+ cells, followed by normalization to the vehicle control.
  • astrocytes were prepared from the cerebral cortices of postnatal day (P)1 - P3 mouse pups as previously described. Contaminating neurons and microglia were removed via vigorous shaking and a few cycles of passaging, freezing, thawing, and replating. For coculture with neurons, proliferating astrocytes were inhibited via treatments with 2 pM ara-C, a mitotic inhibitor, for at least 48 hours.
  • a-BTX labelled cells were then processed for immunostaining with antibodies of SYN1 (Cell Signaling Technology, 1 :500) and MHC (Sigma, 1 :1 ,000).
  • NMJ formation frequency was presented as the percentage of NMJs on myotubes associated with hiMNs networks.
  • Lentivirus carrying cDNA or sgRNA/Cas9 was co-transduced with the reprogramming lentiviruses in fibroblasts, using empty vector (EV) or sgLacZ/Cas9 as the respective control.
  • the replated hiMNs at 14 dpi were directly used for western blotting or seeded onto mouse astrocytes-coated 96-well plates or coverslips in 24-well plates.
  • the seeding density of GFP+ hiMNs in 96-well plates was determined 4-hour post plating as described above. These cultures were then processed for immunocytochemistry and analyzed for survival, morphology, soma size, and other features at the indicated time-points.
  • Doxycycline (Dox) inducible system was used for shRNA-mediated knockdowns.
  • the lentiviruses FUW-M2rtTA and TRE3G-miRE-shRNA were applied to cultured fibroblasts 2 ⁇ 6 hours after seeding.
  • shRNA expression was induced by daily addition of Dox (0.5 pg/ml) into the medium.
  • Cells were collected for qRT-PCR or western blotting 4 days post virus transduction.
  • the lentiviruses FUW-M2rtTA and TRE3G-miRE-shRNA were combined with the reprogramming factors.
  • the mean fluorescence intensity of acetylated TUBA4A was measured in the cytoplasm of hiMNs.
  • the mean fluorescence intensity of RANGAP1 , RAN, TDP-43 were separately measured in the cytoplasm or nucleus of hiMNs.
  • the Nuc/Cyt ratios of different proteins were calculated by Microsoft Excel and analyzed by GraphPad Prism 9.
  • the beads were then sequentially washed for 3 min with gentle shaking with the following buffers: buffer 1 (10 mM Tris-HCI, 150 mM NaCI, 1% NP-40, pH 7.4), buffer 2 (10 mM Tris-HCI, 500 mM NaCI, 1 mM PMSF, pH 7.4), buffer 3 (10 mM Tris-HCI, 150 mM NaCI, pH 7.4), and buffer 4 (10 mM, pH 7.4).
  • buffer 1 (10 mM Tris-HCI, 150 mM NaCI, 1% NP-40, pH 7.4
  • buffer 2 (10 mM Tris-HCI, 500 mM NaCI, 1 mM PMSF, pH 7.4)
  • buffer 3 10 mM Tris-HCI, 150 mM NaCI, pH 7.4
  • buffer 4 10 mM, pH 7.4).
  • Bead-bound proteins were twice (5 min each) eluted with 50 pl of 2 x SDS loading buffer, or 20 pl of 0.1 M Glycine-
  • Horseradish peroxidase-conjugated secondary antibodies (Jackson) were applied, and the blots were developed with Pierce ECL Western Blotting Substrate (Thermo-Fisher) or Immobilon Western Chemiluminescent HRP substrate (Millipore-Sigma). Antibodies are listed in Table 5.
  • Lentivirus expressing BiolD2-myc-HA-MINK1 mt or HA-MINK1 mt was introduced during fibroblast reprogramming. After neuronal induction, the replated hiMNs at 10 dpi were seeded onto Matrigel-coated 6-well plates in neuronal culture medium containing 50 pM biotin. After 24 hours, biotin-treated cells were collected into lysis buffer composed of 50 mM Tris- HCI (pH 7.4), 500 mM NaCI, 0.2% SDS, and protease inhibitors (Pierce). Biotinylated proteins were pulled down with streptavidin beads following a published procedure 78.
  • Samples were run 5—10 mm into a 4 - 15% gradient precast protein gel and stained with G250 Coomassie brilliant blue. Each gel lane with protein bands was cut out and chopped into ⁇ 1 mm3 cubes. Proteins were in-gel digested overnight with trypsin (Pierce), followed by reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). Samples were then undergone solidphase extraction cleanup with an Oasis HLB plate (Waters) and the resulting samples were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system.
  • MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HOD) for ions with charges 2-7.
  • Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.
  • Raw MS data were analyzed using Proteome Discoverer v2.2 (Thermo), with peptide identification performed using Sequest HT searching against the human protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed.
  • Carbamidomethylation of Cys was set as a fixed modification, with oxidation of Met set as a variable modification.
  • the false-discovery rate (FDR) cutoff was 1% for all peptides.
  • qRT-PCR was conducted as previously described with modifications. After removing medium, cells were collected in TRIzol reagent (Invitrogen) and total RNA was isolated by using a commercial kit (RNA Clean & Concentrator kits, ZYMO Research). Total RNA (500 ng each) was used for cDNA synthesis with the SuperScriptill First-Strand Synthesis kit (Invitrogen). Real-time PCR was performed with the SYBR GreenER SuperMix (Invitrogen) on the QuantStudio 5 Real-Time PCR System (Thermo Fisher). Primer sequences are listed in Table 4 and their quality was assessed by the dissociation curve. Relative gene expression was determined by using the 2 -AACt method after normalization to the loading control GAPDH.
  • GST or GST fusion proteins were expressed in BL21 (DE3) E. coli. Cells were harvested and washed with ice-cold 1xPBS (137 mM NaCI, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCI, pH 7.2). Cell pellet was resuspended in ice-cold lysis buffer (1xPBS, 2 mM PMSF, 2 mM DTT, 1 x PI) and sonicated (Bioruptor Pico, Diagenode) at 4 °C with alternating 10 s burst/10 s break: 10 min under high frequency and 5 min under super high frequency.
  • 1xPBS 137 mM NaCI, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCI, pH 7.2. Cell pellet was resuspended in ice-cold lysis buffer (1xPBS, 2 mM PMSF, 2 mM DTT, 1 x
  • lysis buffer 50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1% Triton X-100, 1 mM MgCI2, 1 mM PMSF, and 1 x protease inhibitor. After incubation for 15 min on ice, cell lysates were cleared by centrifugation at 21 ,000 g for 20 min. 20 pl anti-HA beads (Pierce PI88836) were added to the supernatants and incubated overnight at 4 °C.
  • Beads were washed three times with 1 ml lysis buffer and twice with 1 ml kinase assay buffer (50 mM HEPES, pH7.4, 5 mM MgCI2, and 1 mM DTT). Beads in a final volume of 20 pl kinase assay buffer were supplemented with 1 pg purified GST or GST- HDAC6 proteins and 2 mM ATP. After 1 hour incubation at 37 °C, samples were separated by SDS-PAGE (10%). Protein phosphorylation was analyzed by western blotting with antibodies for phospho-Ser (pSer) or phospho-Thr (pThr).
  • pSer phospho-Thr
  • CD1 mice 21 females for Hit3 and 10 males for MAP4Ki
  • Hit3 formulated as 5% DMSO, 5% Pharmasolve, 10% Tween 80, and 90% of 50 mM Citrate Buffer pH 4.6
  • MAP4Ki formulated in ddH2O
  • mice were sacrificed and whole blood, brain, and spinal cord were isolated. Blood samples were separated by centrifugation for 10' at 10,000 rpm and the plasma supernatant was saved. Tissue samples were snap frozen in liquid nitrogen.
  • Standards were made by spiking 50 pl blank plasma or brain homogenate with 1 pl of varying concentrations of compound and processed like samples. Brain standard curve was used to quantitate both brain and spinal cord.
  • 50 pl of each plasma or tissue homogenate sample was crashed with 150 pl acetonitrile + 0.1% final concentration formic acid + 25 ng/ml final concentration tolbutamide IS, vortexed for 15 seconds, incubated at RT for 10 min and spun in a tabletop, chilled centrifuge for 5 minutes at 13,200 rpms. Supernatant (200 pl) was then transferred to an Eppendorf tube and spun again. Supernatant (180 pl) was analyzed by HPLC/MS.
  • mice were weighed weekly beginning at the age of 40 days. All behavior experiments were conducted in a randomized and blinded fashion. Mice were initially trained at 38 days of age on an accelerating rotarod from Touchscreen Rota Rod (Panlab). Training was performed by placing mice on the rotarod moving at 5 rpm for 300 s. Mice were trained to stay on the rotarod for the entire 300 s. If the mouse fell from the rotarod, it was placed back until total 300 s was completed. Mice were trained for two consecutive days. At 40 days of age, accelerating rotarod test was conducted. The rotarod began at 4 rpm and accelerated to 40 rpm over 600 s. The time to fall was automatically recorded.
  • mice were tested for four consecutive trials with a resting time of 20 min between trials. Mice were examined weekly until the time at which they were unable to stay on the rotarod for more than 10 s during the four trials. Mean of the four trials was calculated as the final value.
  • mice were sacrificed and sequentially perfused with ice-cold PBS and 4% (w/v) paraformaldehyde (PFA) in PBS.
  • Whole spinal cords were carefully dissected out, post-fixed overnight with 4% PFA at 4°C, and cryoprotected with 30% sucrose in PBS for 48 h at 4°C.
  • Frozen sections of the lumbar segments were collected at 20-pm thickness by using a cryostat (Leica).
  • Antibodies used for immunofluorescence are listed in Table 5. Nuclei were counterstained with Hoechst 33342 (Hst).
  • CHAT+ neurons were analyzed in the ventral horn of the gray matter of the lumbar spinal cord segments using a Zeiss LSM 700 confocal microscope. CHAT+ neurons with a minimum size of 200 pm2 were counted as motoneurons 80. Reactive gliosis was evaluated by measuring the fluorescence intensity of GFAP or IBA1 staining in the gray matter of the spinal cords using ImageJ program. Cell counting and morphological analyses were performed in a blinded manner.
  • CCI controlled cortical impact
  • an impact velocity of 3.0 m/sec, an impact depth of 1.0 mm, a 2.0 or 3.0-mm-diameter impounder tip, and a dwell time of 0.1 sec were used.
  • Adult 2-month-old male C57BL/6J mice were anesthetized with Ketamine/Xylazine. The mice were placed on a stereotaxic apparatus. A 10-mm midline incision was made over the skull, and the skin and fascia were reflected to make a 3 or 4-mm craniotomy on the central aspect of the right parietal bone. Drill the skull to make a hole.
  • the impact tip of the injury device was lowered to the surface of the exposed tissue until contact was made, and the impact tip was retracted and lowered by the desired injury depth (1 .0 mm) before inducing impact. After injury, the incision was closed with suture, and the animal was placed in a heated cage post-injury until recovery.
  • AAV helper plasmid pAd-deltaF6 (Addgene #1 12867) and packaging plasmids pUCmini-iCAP-PHP.eB (Addgene #103005), pAAV2/5 (Addgene #104964) and pAAV2/9 (Addgene #112865) were obtained from Addgene.
  • AAV expressing constructs pAAV-CAG-GFP-CNH, pAAV-GFAP-CNH, pAAV-Syn1-CNH were made by subcloning cDNA into the Plasmid pAAV-CAG-GFP (Addgene #37825), pAAV-GFAP and pAAV-Syn1 , respectively. All the vectors were verified via restriction enzyme digestions and DNA sequencing. For AAV virus production purification were done according to previously described protocols.
  • HEK293T cells were plated one day before transfection. Next day, cells were transfected with ‘packaging’ plasmids and ‘customer’ plasmid with PEI. 12 hours later, medium was replaced.
  • AAV virus titers were determined by quantitative PCR with ITR primers (forward primer: 5-GGAACCCCTAGTGATGGAGTT-3 (SEQ ID NO: 74); reverse primer: 5- CGGCCTCAGTGAGCGA-3 (SEQ ID NO: 75).
  • forward primer 5-GGAACCCCTAGTGATGGAGTT-3
  • reverse primer 5- CGGCCTCAGTGAGCGA-3
  • AAV-PHP.eB virus 8-10 ul of AAV-PHP.eB virus(2.0X10 12 GC/mL) was manually injected with a Hamilton syringe and needle into the spinal parenchyma at the indicated spinal cord levels by intrathecal injection, which was performed as previously described.
  • a total of 2 stereotaxic intracerebral injections (1X10 13 GC/mL, 1.0 ul/site) were made at 0.5 mm rostral and caudal to the cortex at the edge of the predetermined lesion site.
  • bilateral injections were made according to the following coordinates: mediolateral (ML): 1.0 mm to the midline, and dorsoventral (DV): two injections at 0.6 mm and 1.0 mm each from the dorsal surface of the cortex.
  • PF06260933 (10mpk. b.i.d.) was delivered into mice at predetermined time points after CCI injury by intraperitoneal injection.
  • mice were euthanized with CO2 overdose and intracardially perfused with ice-cold PBS and 4% paraformaldehyde (PFA) in PBS. Brains were collected and post-fixed with 4% PFA overnight at 4 °C. Then the brain samples were cryoprotected with 30% sucrose solution in PBS for 24 hours and cut into 40-pm-thick sections. Sections were serially collected and stored in anti-freezing solution at -20 °C. The immunostaining procedure was conducted as previously described.
  • PFA paraformaldehyde
  • mice anti-GFAP (1 :1000; Sigma, G3893
  • rabbit anti-NG2 (1 :500; Millipore, AB5320
  • rabbit anti-IBA1 (1 :200; Wako#019-19741)
  • rat anti-CD45 (1 :500; BD550539)
  • mouse anti-SMI32 (1 :1500; Biolegend #801701
  • mouse anti Phospho-Tau Ser202, Thr205) (AT8) (1 :500; Thermofisher, MN1020
  • mouse anti Phospho-Tau Thr212, Ser214
  • AT100 (1 :500; Thermofisher, MN1060
  • mouse anti Phospho-Tau Thr231
  • AT180 (1 :500; Thermofisher, MN 1040
  • rabbit anti-NeuN (1 :1000; abeam, ab177487)
  • mouse anti-APP (1 :200; Millipore, MABN380.
  • Alexa Fluor Secondary antibodies were purchased from Invitrogen. Cell nuclei were counterstained with Hoechst 33342 when appropriate. Images were taken using a Zeiss LSM700 confocal microscope. The Image J program was used to measure fluorescence intensity and count cells. Data were obtained from 12 random sections from four to six mice in each group.
  • the grid-walk test An assessment of sensorimotor deficits after TBI, the mice were subjected to explore on an elevated grid surface (40 L x 30 W x 30 H cm with grid spacing of 1.0 cm) for 5 mins at predetermined experimental time points after CCI which is a unilateral TBI model. The number of left hindfoot drops below the grid plane was calculated from the Noldus recorded videos.
  • mice Four week after CCI, the mice were subjected to the tail suspension test, performed as previously described. Briefly, mice were individually suspended by the tail using adhesive tape for 6 mins and recorded by a video camera. The immobility time is considered as depression-like behavior.
  • the elevate plus maze (EPM): The EPM test was performed according to previously established protocols. Briefly, the EPM apparatus consists of a plus shaped maze elevated above the floor. As animals freely explored the maze for 5 mins, their behavior was recorded by a video camera mounted above the maze and analyzed using the Noldus video tracking system. The preference for being in open arms over closed arms (expressed as a percentage of time spent in the open arms) was calculated to measure anxiety-like behavior.
  • Open field test An open field maze is one of the most commonly platforms that used for measure locomotor ability and anxiety-related emotional behaviors in animal models (Carter and Shieh, 2015).
  • the apparatus used was a chamber consisting of a wall-enclosed area (40 L x 40 W x 30 H cm), theTg4510 mice was placed in the middle of the open field maze while concurrently start to track mouse movement by EthoVision XT Video Tracking software for 5 mins. The distance traveled in the field was used to measure the locomotor ability of the mice.
  • Limb clasping test Significant limb clasping in rTg4510 mice was observed, thus functional motor tests were performed to guantify the mice deficits in corticospinal function. The mice’s hind and forepaws were recorded while suspended by the tail for 10 s, then were assigned a score based on the previously described criteria.
  • Lysates were incubated with Streptavidin-coupled Dynabeads (invirogene: Cat No.65002) overnight followed by 6 washes with the 3 different buffers (Wash buffer I: 2%SDS; Wash buffer II: HEPES pH 7.5, 500mM NaCI, 0.1% dexycholic acid, 1%Triton X-100,1mM EDTA; Wash buffer III: 10mM Tris. Cl, pH 7.4, 0.5% dexycholic acid, 0.5% NP-40, 1 mM EDTA, 250mM LiCI). After additional washes with 50 mM Tris. Cl, Pulldown-samples were directly eluted with sample buffer and subjected to MS analysis.
  • Wash buffer II HEPES pH 7.5, 500mM NaCI, 0.1% dexycholic acid, 1%Triton X-100,1mM EDTA
  • Wash buffer III 10mM Tris. Cl, pH 7.4, 0.5% dexycholic acid, 0.5% NP
  • AAV-BiolD2-CNH virus was infused into the cortices of adult wildtype mice. 7 days later, these mice received daily intraperitoneal injections of biotin (24 mg/kg) for 7 consecutive days. The cortices were then quickly dissected and homogenized in an ice-cold solution (25 mM HEPES, pH7.5, 150 mM NaCI, 1 mM EDTA, 1% NP-40, with freshly added Protease and Phosphatase Inhibitor Cocktails). Purification of biotinylated proteins was performed according to a previously established protocol.
  • brain lysates were incubated overnight with streptavidin-coupled Dynabeads (Invitrogen, Cat No.65002). They were sequentially washed twice in each of the following buffers: buffer I (2%SDS), buffer II (50 mM HEPES, pH7.5, 500 mM NaCI, 0.1% dexycholic acid, 1% Triton X-100,1 mM EDTA), and buffer III (10 mM Tris- HCI, pH7.4, 0.5% dexycholic acid, 0.5% NP-40, 1 mM EDTA, 250 mM LiCI). After an additional wash with 50 mM Tris-CI (pH 7.4), the samples were processed for mass spectrometry and protein identifications.
  • buffer I 20%SDS
  • buffer II 50 mM HEPES, pH7.5, 500 mM NaCI, 0.1% dexycholic acid, 1% Triton X-100,1 mM EDTA
  • buffer III 10 mM Tris- H
  • Luciferase activity was measured in BioTek Cytation 3 Cell Imaging Multi-Mode Reader. Briefly, the cells were washed once with sterile PBS, and lysed in 1 * lysis buffer (Promega) (0.1 ml/well) for 15 min at room temperature. 20 pl of crude cell lysate (prepared according to Promega Protocol) was added to 100 pl of luciferase assay LARI I substrate in a black 96-well plate to measure the firefly luciferase activity, followed by addition of 100 pl of Stop and Gio substrate to measure the Renilla luciferase activity. The measured Renilla luciferase activity was used to normalize the measured firefly luciferase activity. All transfections were performed in duplicate and repeated at least two times.
  • Example 1 A compound screening platform with ALS-hiMNs.
  • the ATP- based CellTiter-Glo luminescent assays exhibited good linearity over low ( ⁇ 1 ,000 cells/well) to high (> 10,000 cells/well) seeding density of ALS-hiMNs in 96-well plates. Each screen cycle could be finished three days post a single treatment of individual compound. Using Ken as a positive control, a Z-prime value > 0.5 could be obtained for each assay.
  • a library of about 2,000 compounds consisting of FDA-approved drugs and bioactive chemicals was screened. The primary screens were conducted at 2.5 pM (final concentration) for each compound. Riluzole and Radicava, two medications approved by FDA for ALS therapy, showed only subtle effect on ALS-hiMNs.
  • the top 65 chemicals (>1.5 standard deviations above the mean of all tested compounds; FIG. 1B), were chosen together with 23 additional inhibitors targeting ALK, TGF-p, or GSK3, for secondary screens at four concentrations. From secondary screens, the top 15 chemicals (Table 6) were further assessed in dose-response assays (FIG. 1D-1H). Their effects on neuronal morphology were also examined under an inverted microscope.
  • GSK3 inhibitors Hit1, 4, 5, 6, and 9 could promote neuronal survival but unanticipatedly inhibited neurite outgrowth (FIG. 11, FIG. 2A- 2B).
  • ALK inhibitor K02288 Hit3 greatly enhanced neuronal survival and neurite outgrowth at all tested concentrations (FIG. 1D-1I, FIG. 2A, FIG. 2C).
  • Table 6 Chemical information of Top 15 hits and controls.
  • TGFp/BMP inhibitors such as A83, AC (LDN-214117), LDN-193189 (ALKil) and LDN-212854 (ALKi2), were less effective (FIG. 1D-1H, FIG. 1J).
  • Hit3 showed the most protective effect on ALS-hiMNs and was selected for subsequent analyses.
  • Example 2 Hit3 broadly protects ALS-hiMNs and improves their function
  • Example 3 MAP4Ks are major targets of Hit3 for protecting ALS-hiMNs
  • Baseline Hit3 (K02288) was initially identified as a potent inhibitor of BMP/ALK signaling. To determine whether ALK inhibition was involved in ALS-hiMN protection, a list of 12 ALK inhibitors was further evaluated. While Hit3 consistently showed robust protection, the other ALK inhibitors including the widely used LDN-193189 and LDN-212854 had only minor or no protection (FIG. 4A), indicating that ALK inhibition might not be a mechanism for Hit3- mediated neuroprotection.
  • MAP4K4 also potently inhibited HGK
  • MINK1 MAP4K6
  • TNIK MAP4K7
  • MAP4K4Ks belong to the GCK-IV family of the STE20 group kinases. They share similar protein structures and exhibit very high homology within the kinase domain (>90% amino acid identity).
  • PF6260933 designated as MAP4Ki
  • MAP4Ki treatments also greatly improved survival of ALS- hiMNs (FIG. 4B), resembling the effect of Hit3 and indicating that MAP4K inhibition might be an underlying mechanism for Hit3’s action.
  • sgRNA-mediated knockdowns were confirmed through western blotting analysis of HA-tagged proteins (FIG. 5A). These sgRNAs individually or in combinations were then tested on ALS- hiMNs. While knocking down individual MAP4K had not much effect, their combinations especially triple knockdowns significantly improved survival of ALS-hiMNs when compared to the control sgRNA (sgLacZ) at both 1 week and 3 weeks post cell replating (FIG. 5B-5C). Neuronal survival was accompanied with enhanced neurite outgrowth and branching, as well as bigger somas after MAP4K triple knockdowns when compared to the control (FIG. 4D). Of note, sgRNAs were less effective than Hit3 treatment, which might be due to a lower efficiency of targeting these kinases through genetic knockdowns than chemical inhibition.
  • HGK-K54R HGKmt
  • MINK1-K54R MINKI mt
  • TNIK-K54R TNIK-K54R
  • FIG. 5J shows a schematic showing how Hit3 improves survival of ALS-hiMNs.
  • hiMNs derived from ALS patient’ skin fibroblasts degenerate with time in culture. Such degeneration could be rescued by the small molecule Hit3.
  • Hit3 functions as an inhibitor of MAP4Ks to block phosphorylation of HDAC6, resulting in enhanced acetylation of TUBA4A, stabilized microtubules, and nuclear localization of RANGAP1 and TDP-43.
  • ALS amyotrophic lateral sclerosis
  • HDAC histone deacetylase 6
  • hiMNs human-induced motor neurons directly converted from adult skin fibroblasts
  • MAP4Ks MAP kinase kinase kinase kinases
  • RANGAP1 Ran GTPase-activating protein 1
  • TDP-43 TAR DNA-binding protein 43
  • TUBA4A tubulin alpha-4A.
  • Example 4 CNH domain of MAP4Ks mimics the effect of Hit3
  • MINK1 consists of three highly conserved domains: a kinase domain, an intermediate domain, and a citron homology domain (CNH).
  • CNH citron homology domain
  • CNH-containing truncations (296-1312 and 866-1312) improved survival and morphology of ALS-hiMNs, comparable to those treated with Hit3 or full-length MINK1 mt (FIG. 6C-6F).
  • Further truncation mutants showed that the flanking sequences surrounding the CNH domain were also important, as the mutant 994-1312 or 960- 1292 largely failed to promote survival of ALS-hiMNs (FIG. 6E-6F). Together, these results indicate that CNH domain of MINK1 may play a dominant-negative function in improving ALS- hiMNs.
  • Example 5 MAP4Ks interact with RANGAP1 and affect its subcellular distribution
  • ectopic MINK1 mt significantly increased the nuclear/cytoplasmic (Nuc/Cyt) ratio of RANGAP1 or RAN in ALS-hiMNs (FIG. 8G-8H). It also greatly promoted soma size of these neurons (FIG. 8I). Together, these results indicated that MAP4Ks regulated nuclear pore function and their inhibition protected ALS-hiMNs.
  • Example 6 MAP4K inhibition improves nuclear pore proteins and those involved in ALS
  • Example 7 MAP4Ks regulate RANGAP1 distribution through a HDAC6-TUBA4A axis
  • cytoplasmic RANGAP1 + foci are pore complexes associated with annulate lamellae (AL), membrane sheets of the endoplasmic reticulum.
  • MAP4K- mediated phosphorylation could cause redistribution of RANGAP1 between AL and nuclear membrane.
  • Regulation of the subcellular distribution of RNAGAP1 by MAP4K was examined by Phos-tag gels and western blots with antibodies broadly recognizing phosphoserine (pSer) or phospho-threonine (pThr).
  • pSer phosphoserine
  • pThr phospho-threonine
  • Tubulin acetylation enhances microtubule stability and intracellular transport. It was examined whether acetylation status of TUBA4A could be regulated by MAP4Ks. When cells were treated with Hit3, western blotting showed that ac-TUBA4A was greatly increased indicating a negative regulation by MAP4Ks (FIG. 12D). Immunocytochemistry also showed that the level of ac-TUBA4A was enhanced in the somas of hiMNs when treated with Hit3 or after shRNA-mediated knockdown of endogenous MAP4Ks (FIG. 12E-12F).
  • HDAC6 alpha-tubulin acetyltransferase 1 (ATAT1 ) and histone deacetylase 6 (HDAC6).
  • HDAC6 was previously shown to play a role in protein aggregation, axonal transport, and pathological phenotypes of ALS models.
  • HDAC6 knockdown also promoted nuclear localization of TDP-43 (FIG. 13B-C).
  • a motif scan revealed that HDAC6 harbors five predicted MAP4K phosphorylation sites.
  • a phosphorylation assay was then performed by using purified GST-HDAC6 as the substrate and immunoprecipitated HA-HGK as the kinase. Both full-length and some degraded forms of GST-HDAC6 could be phosphorylated by HGK when examined by western blotting with antibodies for pSer or pThr (FIG. 12L).
  • the findings showed an increased level of ac-TUBA4A when MAP4Ks or HDAC6 was inhibited (FIG. 12F, FIG. 12J), which indicated that MAP4K-mediated phosphorylation may enhance HDAC6 activity.
  • HDAC6 was overexpressed in hiMNs with or without Hit3 treatments or MAP4K knockdowns.
  • Ectopic HDAC6 resulted in a higher percentage of cells with diffused cytoplasmic RANGAP1 and a much-reduced Nuc/Cyt ratio of TDP-43 (FIG. 12M-12O, FIG. 13D-13E).
  • Such cellular phenotypes could be reversed by Hit3 treatments or MAP4K knockdowns, indicating requirements of endogenous MAP4Ks (FIG. 12M-12O, FIG. 13D-13E).
  • the results suggested that MAP4Ks phosphorylated and stimulated HDAC6 activity, which led to a reduction of ac-TUBA4A and nuclear pore-localized RANGAP1.
  • Example 8 Inhibition of MAP4Ks is neuroprotective in vivo
  • MAP4Ki treatments had no effect on reactive gliosis, indicated by the expression of GFAP or IBA1 (FIG. 15A-15D). Together, these results showed that inhibition of MAP4Ks was neuroprotective and prolongs survival of SOD1G93A mice, potentially mediated through improved subcellular localization of proteins involved in motor neuron function.
  • MAP4Ks were the major targets of the lead compound Hit3.
  • HGK HGK
  • MINK1 MINK1
  • TNIK MAP4K7
  • PF6260933 a structural analog of Hit3 and a selective inhibitor of HGK as well as MINK1 and TNIK, exhibited a similar protective effect on ALS-hiMNs.
  • the individual and combinatorial knockdown experiments also confirmed a critical and redundant role of these three highly homologous kinases in neuronal survival.
  • the kinase-dead MINK1 or its CNH domain can function as a dominant-negative mutant improving survival of hiMNs that are derived from diverse ALS patients.
  • MAP4Ks were consistent with previous studies implicating MAP4Ks in neural degeneration.
  • HGK alone was identified to regulate death of iPSC-derived motor neurons under stress, a result that may reflect the differential response of embryonic versus aging-relevant neurons.
  • the JNK signaling pathway was identified as a target of MAP4Ks in controlling degeneration and apoptosis of embryonic or iPSC-derived neurons under stress.
  • its inhibition with multiple specific inhibitors was either toxic or non- effective to ALS-hiMNs, once again revealing a differential response of embryonic versus aging-relevant neurons.
  • MAP4Ks regulated multiple biological processes, such as proteosome, ribosome, and RNA transport. Dysfunction of these processes is well-known to be involved in neurodegeneration including ALS.
  • RANGAP1 due to its abnormal subcellular distribution in ALS-hiMNs and whether such abnormality significantly improved by either Hit3 or a dominant-negative MINK1 mutant were tested.
  • RANGAP1 a core component of the NPCs, is the GTPase converting RAN-GTP to RAN-GDP, an essential step for RAN-mediated nuclear import and export of proteins and RNAs.
  • RANGAP1 -associated pore proteins not only reside within the nuclear membrane but are also found in annulate lamellae (AL), membrane sheets of the rough endoplasmic reticulum. AL are highly abundant in neurons and are the storage compartment for nuclear pore proteins that can later be transported to the NPCs. Such transport requires stable microtubules since their disruption leads to increased levels of AL and AL-localized nuclear pore proteins. Consistently, the above disclosed data showed enhanced RANGAP1 localization in cytoplasmic foci after down-regulation of the MAP4K-associated tubulin TUBA4A, mutations of which destabilized the microtubule network and are implicated in familial ALS.
  • MAP4Ks inhibited TUBA4A acetylation, a posttranslational modification that increased microtubule stability. This is likely due to altered HDAC6 activity through phosphorylation by MAP4Ks, as indicated by in vitro kinase assays.
  • HDAC6 is the major a-tubulin deacetylase in the nervous system. Its activity is regulated by multiple posttranslational modifications including phosphorylation. Phosphorylation of HDAC6 increased its enzymatic activity or protein stability. MAP4Ks also regulated HDAC6 in a similar fashion since their inhibition by Hit3 or shRNA-mediated knockdowns promoted acetylation of TUBA4A. At the functional level, the disclosed results demonstrated that HDAC6 downregulation enhanced nuclear localization of RANGAP1 and TDP-43 in hiMNs. These results were consistent with previous studies showing that HDAC6 inhibition restored subcellular mislocalization of TDP-43 and improved NMJ morphology in iPSC-derived MNs. Further supporting a functional interaction of MAP4Ks and HDAC6 was that either MAP4Ki or HDAC6 deletion significantly promoted survival of the SOD G93A ALS mice.
  • Example 9 Traumatic brain injury causes gliosis, neurodegeneration, and tau pathology
  • CCI controlled cortical impact
  • Phosphorylated tau was also examined with the antibodies AT8 (for pS202/T205), AT100 (for pT212/S214), and AT180 (for pT231 ) as biomarkers of TBI.
  • CCI induced very extensive but dynamic p-tau expression, with the highest level at 4 dpi and gradual reduction to the basal level one month later (FIG. 16D).
  • Example 10 The CNH domain ameliorates brain injury-induced pathology
  • MAP4Ks Ste20 family kinases
  • MINK1 the CNH domain of MINK1 could serve as a dominant-negative form blocking MAP4Ks to promote survival and function of human patient- derived neurons.
  • adeno-associated viruses AAVs were prepared to express GFP-CNH under the constitutively active CAG promoter. GFP alone was used as a control.
  • AAVs were packaged with the PHP.eB capsid for efficient and brain-wide distributions.
  • the CNH group When compared to the GFP control after CCI, the CNH group also showed a much-reduced lesion size that was measured by the ratio of tissue area in the ipsilateral cortex to the contralateral cortex (FIG. 17I-17J). The injury-induced hyperphosphorylated tau was also markedly dampened by the CNH expression when compared to the control.
  • AAV-mediated expression of the CNH domain of MINK1 could broadly suppressed brain injury-induced pathology including gliosis, neuronal damage, and tau pathology.
  • Example 11 The CNH promotes functional recovery after brain injury
  • Example 12 The CNH domain exerts its neuroprotective function in neurons
  • PHP.eB-serotyped AAVs can efficiently transduce both neurons and astrocytes, the cell type in which the CNH domain exerted its function was examined.
  • the human GFAP (hGFAP) promoter was used to drive gene expression and the AAV5 capsid to package AAVs.
  • Immunohistochemistry showed about 92% of the GFP+ cells were GFAP+ astrocytes in the mouse cortex that were transduced with the AAV5-hGFAP- GFP virus (FIG. 190). When analyzed at 7 dpi and compared to the GFP control (FIG.
  • FIG. 19A a significant effect of the astrocyte-expressed CNH domain on either reactive gliosis (indicated by the relative GFAP expression; FIG. 19B-19C) or tau pathology (indicated by the AT8 staining; FIG. 19A) was failed to be detected.
  • the astrocyte-expressed CNH domain was also unable to reduce glial scars (indicated by the GFAP+ cortical area; FIG. 19D-19E) or brain lesion size (indicated by the relative remaining cortical tissues; FIG. 19F-19G) after TBI.
  • Example 13 The CNH domain alleviates tau pathology in a mouse model of Alzheimer’s disease
  • AD Alzheimer’s disease
  • tau hyperphosphorylation is a key hallmark.
  • the ability of the CNH domain to suppress TBI- induced tau pathology prompted the examination of its role in rTg4510 mouse, a transgenic mouse line expressing tauP301 L in forebrain neurons as a model of AD-associated tauopathy. Since hyperphosphorylated tau is normally observed between 3.5 and 5.5 months of age in rTg4510 mice, AAVs through intrathecal injections were delivered at 3 months of age and immunohistochemistry was conducted 3 more months later.
  • AD is an age-dependent neurodegenerative disease
  • CNH domain exerted a protective function in older rTg4510 mice
  • AAVs were injected into the cortex of rTg4510 mouse at 4 or 6 months of age and were analyzed 2 months later.
  • the CNH domain-induced reduction of AT8 staining was only detected at the early time point, whereas AT100 staining did not show a difference at both age points.
  • these results indicated that the CNH domain prevents the development of tauopathy but may not be able to clear p-tau once it is formed in AD.
  • Example 14 The CNH domain improves behaviors of AD mice
  • mice In addition to cognitive impairments, the rTg4510 AD mice exhibited age- and transgenic tau-dependent hyperactivity and limb clasping, two behavioral deficits that can be assessed straightforwardly. Therefore, AAVs were intrathecally injected into a cohort of rTg4510 mice at 2 months of age. Their wild type littermates were used as controls. Locomotor and exploratory activity were assessed by the open field test, while the limb clasping behavior was evaluated by the tail suspension test. When examined at 8 months of age, no significant behavioral differences were observed between mice injected with the CNH virus and those with the GFP control virus (FIG. 20G-20H).
  • Example 15 Pharmacological inhibition of MAP4Ks ameliorates brain injury-induced pathology
  • K02288 (FIG. 21A), also known as Hit3 after chemical screens in human diseased neurons, is a potent inhibitor of MAP4Ks. It was administered daily to mice for 7 days after CCI (FIG. 21 B).
  • Significant reductions were similarly observed for markers of neuronal damage (SMI32+ and APP+) and tau pathology (AT8 and AT100) in mice treated with K02288 after CCI (FIG.
  • Example 16 CNH self-associates and binds to MAP4Ks
  • Example 17 Proteomic analysis of the CNH-associated network
  • MAP4K2 MAP4K2
  • MAK4K4 HGK
  • MAP4K6 MAP4K6
  • MAP4K7 The founding member of the CNH domain, Cit (Citron Rho-lnteracting Kinase), was also pulled down.
  • Other known interactors of MAP4Ks include STRN4, HSP70, and CTTN. Based on Gene Ontology, biological functions of these enriched proteins are mainly involved in Actin cytoskeleton organization, Endocytosis, Synaptic transmission, and Axonogenesis (FIG. 22E).
  • KEGG pathway analysis revealed that the largest number of identified proteins are implicated in Neurodegeneration (57 proteins) and Endocytosis (48 proteins) (FIG. 22F).
  • STRING analysis of proteins involved in the KEGG pathway of Neurodegeneration revealed a complex interaction network (FIG. 22G).
  • Example 18 CNH blocks MAP4K-mediated phosphorylation of the Dishevelled proteins (DVLs) [0445] Since both TNIK and MINK1 were previously implicated in the regulation of Wnt signaling during early Xenopus development, proteins involved in this pathway were further examined. Within this pathway, DVL1 , DVL2, and DVL3 were among the most abundant proteins identified by the proteomics (Table S1). Using DVL3 as an example and through coimmunoprecipitation assays, its association with CNH was confirmed (FIG. 23A). Importantly, co-immunoprecipitations also revealed that DVL3 could bind to full-length MAP4Ks (FIG.
  • the PDZ domain may also mediate the interactions of CNH with many other PDZ domain-containing proteins such as DLG4 and NOS1 (FIG. 22G).
  • Example 19 MAP4K4s negatively regulate Wnt/B-catenin signaling
  • CTNNB1 was also identified in the proteomics dataset (FIG. 22G), it was examined but failed to detect an interaction between CTTNB1 and MAP4Ks through the coimmunoprecipitation assay (FIG. 24F). Such a result indicated either the interaction was relatively weak, or the two proteins were just in proximity. This was consistent with the detection principle of BiolD-mediated proximity-labeling proteomics. Taken together, it was likely that CNH works as a dominant-negative form blocking MAP4K- mediated suppression of DVLs within the Wnt/p-catenin pathway (FIG. 24G).
  • MAP4Ks signaling pathway can be activated leads to stress-mediated pathology and subsequent inflammation-induced tau pathology.
  • the data disclosed herein indicated inhibiting this pathway by AAVs or pharmacological inhibitor prolongs survival of neuron and alleviates tau pathology.
  • Previous finding showed that MAP4Ks have similar protein structures and act redundantly.
  • the disclosed examples showed that the Citron-NIK- Homology domain as the dominant negative mutant of MINK1 for gene silencing, and overexpression the CNH domain of MINK1 mimicked the protective effect of MAP4Ks shRNA.
  • AAV-PHP.eB-GFP-CNH was generated and delivered into mice by intrathecal administration, which is a safe and efficient delivery system for DNA for clinical use of gene therapy.
  • AAV- PHP.eB-GFP-CNH reduced gliosis and neuron damage in vivo, importantly, AAV-PHP.eB- GFP-CNH also promoted brain tissue remodeling and behavioral recovery after TBI. Moreover, in vivo studies showed that AAV-PHP.eB-GFP-CNH act to protect against tauopathy in chronic period of Alzheimer's disease, preceding any effects of behavior recovery.
  • AAV9-Syn1-CNH delivered by stereotaxic intracerebral injection also contributed to neuroprotective effect on TBI, and both AAV-PHP.eB and AAV9s have strong neuronal tropism.
  • AAV5-GFAP-CNH was examined in astrocytes, no effect was found of CNH on TBI as has been observed in neurons.
  • MAP4Ks promote neuron inflammation induced tau pathology in vivo
  • BiolD2 approach was utilized and identified many MAP4Ks interacting proteins, including several proteins first identified here, are implicated by tricarboxylic acid cycle and/or long-term neuronal synaptic plasticity, locomotory activity or learning.
  • the comprehensive comparative study on pathway analysis revealed the role of MAP4Ks on functional behavior in TBI model and rTg4510 animals.
  • MAP4Ks as novel regulators of neuron inflammation and tau pathology that is required for neurodegeneration after TBI. Understanding the molecular control of MAP4Ks-mediated DVL3 inhibition will advance the knowledge of MAP4Ks integration with Wnt signaling during tau pathology, as well as in such diseases as TBI or Alzheimer disease.

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Abstract

La présente invention concerne de manière générale des compositions et des méthodes pour traiter une maladie neurodégénérative ou une lésion cérébrale ou fournir une protection contre une dégénérescence neuronale et/ou une lésion neuronale. Des aspects de la méthode comprennent l'administration d'un inhibiteur de MAP4K, l'inhibiteur de MAP4K étant un domaine d'homologie Citron (CNH) ou une troncature contenant un CNH de MAP4K4, MAP4K6 ou MAP4K7, un ARNg comprenant une séquence cible de MAP4K4, MAP4K6 ou MAP4K7, un ARNsh comprenant une séquence cible de MAP4K4, MAP4K6, ou MAP4K7, ou un inhibiteur chimique. L'invention concerne également des vecteurs et des acides nucléiques isolés comprenant les inhibiteurs de MAP4K.
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