WO2024211430A2 - Methods and compositions for treating neurodegenerative conditions - Google Patents

Abstract

The present disclosure generally relates to compositions and methods for treating neurodegenerative disease or brain injury or providing protection from neural degeneration and/or neural injury. Aspects of the method comprise administering a MAP4K inhibitor, wherein the MAP4K inhibitor is a Citron homology domain (CNH) or a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, a gRNA comprising a target sequence of MAP4K4, MAP4K6, or MAP4K7, a shRNA comprising a target sequence of MAP4K4, MAP4K6, or MAP4K7, or a chemical inhibitor. Vectors and isolated nucleic acids comprising the MAP4K inhibitors are also described.

Description

TITLE

METHODS AND COMPOSITIONS FOR TREATING NEURODEGENERATIVE CONDITIONS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/493,937, filed April 3, 2023, and titled “METHODS AND COMPOSITIONS FOR TREATING NEURODEGENERATIVE CONDITIONS,” which is incorporated by reference herein in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] This present application contains a Sequence Listing that which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. Said WIPO Sequence Listing was created on March 22, 2024, XML copy is named “106546-792780_Sequence_Listing.xml,” and is 71000 bytes in size.

BACKGROUND

[0003] 1. Field

[0004] The present disclosure relates generally to methods and compositions associated with neurodegenerative conditions or traumatic brain injury.

[0005] 2. Discussion of Related Art

[0006] 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. Several neurodegenerative diseases feature progressive death of motor neurons (MNs), loss of the ability to walk, talk, swallow and breathe, and reduced life expectancy.

[0007] Traumatic brain injury (TBI) 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. 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. [0008] More effective treatment neurodegenerative diseases and TBI are required, and thus there remains a need for discovery of potential therapeutic targets.

SUMMARY

[0009] In some aspects, 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. [0010] In some aspects, 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.

[0011] In some aspects, provided herein is a method of providing protection to a subject in need thereof from neural degeneration and/or neural injury. 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.

[0012] In some aspects of the disclosed method, 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.

[0013] In further aspects, the disclosure provided 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. In some aspects of the composition, the CNH or CNH- containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from SEQ ID NOs:3-6.

[0014] In some aspects, further provided is 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. In some aspects, 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.

[0015] In some aspects, 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.

[0016] In further aspects, 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.

[0017] 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.

[0018] In some aspects, 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.

[0019] In some aspects, further provided in the disclosure is 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. [0020] In some aspects, further provided herein is 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.

[0021] In some aspects, 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.

[0022] In some aspects, further provided herein is 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.

[0023] In some aspects, 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.

[0024] In further aspects, 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.

[0025] In some aspects of the method, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia. In some aspects of the method, the brain injury comprises traumatic brain injury or stroke. [0026] In some aspects, in the disclosed method the inhibitor is administered as a recombinant adeno-associated virus (rAAV) vector encoding the said inhibitor. In further aspects, the rAAV vector comprises AAV9 or AAV-PHP.eB capsid. In some aspects, the AAV vector comprises a human synapsin I promoter (hSYN1 ).

[0027] In some aspects of the method, the inhibitor is expressed ectopically in neuron or motor neuron cells of the subject. In further aspects, 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. In some aspects, the inhibitor is administered parenterally. In some aspects, the inhibitor is administered intrathecally.

[0028] In some aspects of the composition the vector is a rAAV. In some aspects, the vector is a rAAV comprising AAV9 or AAV-PHP.eB capsid. In some aspects, the vector comprises sequence encoding a human synapsin I promoter (hSYN1 ) operably linked to the inhibitor. In some aspects, the composition is administered parenterally. In some aspects, the composition is administered intrathecally.

[0029] In some aspects the disclosure further comprises a host cell transduced with the any of the disclosed nucleic acid sequence provided herein.

[0030] In some aspects, 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.

[0031] In some aspects, 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.

[0032] In some aspects of the disclosure, 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.

[0033] In some aspects of the isolated nucleic acid is administered parenterally. In some aspects, the isolated nucleic acid is administered intrathecally.

[0034] In some aspects, provided herein is a cell based platform for screening compounds with neuroprotective effect, the cell based platform comprising a host cell disclosed herein.

[0035] 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. In another aspect, provided herein is 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. In yet another aspect, 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. In some aspects, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia. In further aspects, the brain injury comprises traumatic brain injury or stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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. Each point represents an individual compound assayed at 2.5 pM (final concentration). 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.

[0037] 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)

[0038] 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. FIG. 3D shows quantification of neuronal soma size, (mean ± SEM; n = 3 independent samples with >70 neurons analyzed for each group; **p < 0.01 and ***p < 0.001). 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.

[0039] 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.

[0040] 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. 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. EV, empty vector, (mean ± SEM; n = 4 independent samples; ***p < 0.001 when compared to the control EV-Veh group). FIG. 5F shows westernblotting analysis of ectopic MAP4K mutants in ALS-hiMNs. EV, empty vector. FIG. 5G illustrates MINK1 mutant dose-dependently improves survival of ALS-hiMNs, comparable to Hit3 treatment (mean ± SEM; n = 4 independent samples; **p < 0.01 and ***p < 0.001 when compared to the control EV-Veh group). FIGs. 5H-5I depict MINK1 mutant, comparable to Hit3, promotes survival of ALS-hiMNs from diverse human patients. Cells were examined at 1 week or 3 weeks after replating on astrocytes. FIG. 5J shows a schematic showing how Hit3 improves survival of ALS-hiMNs.

[0041] 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. FIGs. 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.

[0042] 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.

[0043] 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. 8H shows MINKmt improves nuclear/cytoplasmic (Nuc/Cyt) ratios of the indicated proteins in ALS-hiMNs (mean ± SEM; n = 30 neurons per group; ***p < 0.001). FIG. 8I illustrates MINKmt improves soma sizes of ALS-hiMNs (mean ± SEM; n = 30 neurons per group; ***p < 0.001).

[0044] FIG. 9A-9I illustrate Hit3 improves nucleocytoplasmic transport in ALS-hiMNs. FIGs. 9A-9B depict confocal images of the indicated proteins in NL- or ALS-hiMNs. Cytoplasmic foci of RANGAP1 are indicated by arrows. Scale bar, 10 pm. FIG. 9C shows Hit3 improves nuclear localization of RANGAP1 in hiMNs (n = 30 neurons per group; *p < 0.05 and ***p < 0.001). FIG. 9D shows that Hit3 improves nuclear localization of RAN in hiMNs (n = 30 neurons per group; *p < 0.05). FIG. 9E depicts confocal images of FUS in ALS-hiMNs. Scale bar, 10 pm. FIG. 9F shows that Hit3 improves nuclear fraction of FUS in ALS-hiMNs (n = 25 neurons per group; *p < 0.05, **p < 0.01 and ***p < 0.001). 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).

[0045] 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. 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).

[0046] 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.

[0047] 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. 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 ).

[0048] 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. 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 ).

[0049] 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. 14C depicts body weight changes (n = 12-13 mice per group). FIG. 14D illustrates rotarod tests (n = 12-13 mice per group). FIG. 14E depicts a Kaplan-Meier survival curve. Mice treated with MAP4Ki survived significantly longer than mice with the vehicle control (mean ± SEM; n = 12-13 mice per group; Median survival: Veh = 129 days and MAP4Ki = 139 days; **p = 0.0027 by log-rank Mantel-Cox test). FIG. 14F exhibits confocal images of CHAT+ neurons in the ventral horn of the lumbar spinal cord. Scale bar, 20 pm. FIG. 14G shows MAP4Ki preserves more CHAT+ cells (mean ± SEM; n = 4-5 mice per group; *p < 0.05 and ***p < 0.001 ). FIG. 14H illustrates confocal images of RANGAP1 localization in CHAT+ neurons. Scale bar, 20pm. FIG. 141 shows MAP4Ki preserves CHAT+ neurons with intact RANGAP1 (mean ± SEM; n = 3-4 mice per group; **p < 0.01 and ***p < 0.001 ). FIG. 14J shows confocal images of TDP-43 localization in CHAT+ neurons. Scale bar, 20pm. FIG. 14K shows MAP4Ki preserves CHAT+ neurons with nuclear TDP-43 (mean ± SEM; n = 3-5 mice per group; *p < 0.05, **p < 0.01 , and ***p < 0.001 ; n.s., not significant; n.d., not detected).

[0050] FIG. 15A-15D show MAP4Ki has no effect on reactive gliosis in SOD1^G93A mice. FIG. 15A depicts confocal images showing GFAP expression in the ventral horn of the gray matter of the lumbar spinal cord. Scale bar, 50 pm. FIG. 15B shows quantification of GFAP relative intensity (mean ± SEM; n = 4-5 mice per group; ***p < 0.001 ; n.s., not significant). FIG. 15C illustrates confocal images showing IBA1 expression in the ventral horn of the gray matter of the lumbar spinal cord. Scale bar, 50 pm. FIG. 15D exhibits quantification of IBA1 relative intensity (mean ± SEM; n = 4-5 mice per group; *p < 0.05; n.s., not significant). [0051] 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. 16C shows images of phosphorylated tau (p-tau) stained with the antibodies AT8 (for pS202/T205), AT100 (for pT212/S214), and AT180 (for pT231 ) illustrating TBI over time. FIG. 16D depicts induction of p-tau expression with CCI and gradual reduction to the basal level after a month later.

[0052] 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.

[0053] 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.

[0054] 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. 190 shows specific targeting of brain astrocytes by the AAV5-hGFAP-GFP vector. Astrocytes and neurons are identified with staining for GFAP and NeuN, respectively. Scale bar, 50 pm. 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.

[0055] 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.

[0056] 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. 21 D shows quantification of fluorescence intensity of the indicated markers (mean ± SEM; n = 4 mice per group; ****p < 0.0001 for GFAP, *p = 0.0192 for NG2, and **p = 0.0089 for CD45). FIG. 21E shows representative confocal images for the indicated neuronal damage markers. Scale bar, 50 pm. FIG. 21 F shows quantification of fluorescence intensity of the indicated marker (mean ± SEM; n = 4 mice per group; *p = 0.0193 for SMI32, *p = 0.0118 for APP, and *p = 0.0351 for AT8). FIG. 21G shows representative images showing brain sections for GFAP+ scars. Scale bar, 1 mm. FIG. 21 H shows quantifications of the scar volumes (mean ± SEM; n = 4 mice per group; **p = 0.0092). FIG. 211 shows representative images of brain sections for lesion quantification (yellow outlined). Scale bar, 1 mm. FIG 21J shows quantifications of cortical volumes post injury (mean ± SEM; n = 4 mice per group; *p = 0.0486).

[0057] 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. 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 22G shows STRING analysis of protein association networks in the KEGG category of Neurodegeneration. Major functional enrichments in the network include proteins involved in Wnt signaling (red, p = 2.15e-14), Alzheimer’s disease (blue, p = 7.39e- 16), Proteasome (yellow, p = 2.64e-06), and Oxidative phosphorylation (Green, p = 3.54e-07).

[0058] 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. 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.

[0059] 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. Cells were treated with okadaic acid (OA) for potential stimulation of kinase activity. ACTB, loading control. FIG. 24B shows suppression of the p- catenin-responsive TOPFIash luciferase reporter by MAP4Ks (mean ± SEM; n = 3 per group; *p = 0.0171 for HGK, **p = 0.0015 for MINK1 , and ***p = 0.0003 for TNIK). 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. 24D shows enhanced activity of the p-catenin- responsive TOPFIash luciferase reporter by downregulation of MAP4Ks (mean ± SEM; n = 3 per group; **p = 0.0022 for shHGK, ***p = 0.0004 for shMINKI , and ***p = 0.0002 for shTNIK). 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.

[0060] The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.

DETAILED DESCRIPTION [0061] The following detailed description references the accompanying drawings that illustrate various aspects of the present inventive concept. The drawings and description are intended to describe aspects of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized, and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense.

[0062] Provided herein are methods and compositions of treating a neurodegenerative disease or a brain injury in a subject. Also described herein are methods and compositions of providing protection to a subject from neural degeneration and/or neural injury. The present disclosure is based on the surprising discovery that 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.

I. Terminology

[0063] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0064] As used in the specification, 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. By way of example, “an element” means at least one element and can include more than one element.

[0065] “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.

[0066] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements, or steps but not the exclusion of any other integer or step or group of integers or steps. [0067] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0068] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristics )” of the disclosure. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0069] Moreover, the present disclosure also contemplates that in some aspects, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0070] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0071] As used herein, “treatment,” “therapy” and/or “therapy regimen” refer 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.

[0072] As used herein, “prevent” or “prevention” 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.

[0073] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results. [0074] As used herein, “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. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “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. In some aspects, the subject can be a human. In other aspects, the subject can be a human in need of treating or protection from a from neurodegenerative disease or brain injury.

[0075] As used herein “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. In some aspects, a mammal is a human. In some aspects, a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like). In some aspects, 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. In some aspects, a mammal can be an animal disease model.

[0076] As used herein “MAP4K” refers to Mitogen-activated protein 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). Generally, MAP4Ks have three domains, kinase domain, intermediate domain, and citron homology domain (CNH).

[0077] As used herein “MAP4K4” refers to mitogen-activated protein kinase kinase kinase kinase 4 is an enzyme, specifically a serine/threonine (S/T) kinase encoded by the MAP4K4 gene. 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). As described herein, 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. [0078] As used herein “MAP4K6” refers to mitogen-activated protein 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. As described herein, 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.

[0079] As used herein “MAP4K7” refers to mitogen-activated protein 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. As described herein, 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.

[0080] As used herein “neurodegenerative disease”, “neurodegenerative disorder”, or neurogenerative disorder” 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.

[0081] As used herein “"amyotrophic lateral sclerosis" or "ALS" also known as Lou Gehrig's disease, refers to a disease of the nerve cells in the brain and spinal cord that control voluntary muscle movement. In ALS, 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. There are no known 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. Persons with ALS have a loss of muscle strength and coordination that eventually gets worse and makes it impossible to do routine tasks such as going up steps, getting out of a chair, or swallowing. Breathing or swallowing muscles may be the first muscles affected. As the disease gets worse, more muscle groups develop problems. 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.

[0082] As used herein “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.

[0083] As used herein “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. Almost everyone with Huntington's Disease eventually exhibits similar physical symptoms, but the onset, progression and extent of cognitive and psychiatric symptoms vary significantly between individuals. The most characteristic initial physical symptoms are jerky, random, and uncontrollable movements called chorea. Chorea may be initially exhibited as general restlessness, small unintentionally initiated or uncompleted motions, lack of coordination, or slowed saccadic eye movements. These minor motor abnormalities usually precede more obvious signs of motor dysfunction by at least three years. The clear appearance of symptoms such as rigidity, writhing motions or abnormal posturing appear as the disorder progresses. These are signs that the system in the brain that is responsible for movement has been affected. Psychomotor functions become increasingly impaired, such that any action that requires muscle control is affected. Common consequences are physical instability, abnormal facial expression, and difficulties chewing, swallowing, and speaking. Eating difficulties commonly cause weight loss and may lead to malnutrition. Sleep disturbances are also associated symptoms.

[0084] As used herein the term “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.

[0085] As used herein “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).

[0086] As used herein “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.

[0087] As used herein “Multiple System Atrophy” or “MSA” refers to a neurodegenerative disorder in which degeneration in brain regions leads to impaired control of movement, balance, blood pressure and sexual and urinary tract function. 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. For patients with predominant Parkinsonism symptoms, the term 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.

[0088] As used herein “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. 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 about the expected clinical course.

[0089] 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 (LB) 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.

[0090] As used herein “Spinal muscular atrophy” or “SMA” refers to a collection of inherited and acquired central nervous system (CNS) diseases characterized by progressive motor neuron loss in the spinal cord and brainstem causing muscle weakness and muscle atrophy. The most common form of SMA is caused by a mutation of the survival motor neuron gene 1 (SMN1 ), which is typically responsible for the production of a protein essential to motor neurons. The different types of SMA inlcude Type 0 SMA (In Utero SMA), Type I (also known as Werdnig-Hoffman disease or infantile SMA), SMA Type II, SMA Type III (also known as Kugelberg-Welander disease), and SMA Type IV. Less common 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.

[0091] As used herein “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. 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.

[0092] In some aspect, 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. There are several forms of stroke, including 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).

[0093] As used herein “citron homology domain” or “CNH” refers to conserved domain in MAP4K and other proteins. CNH is often found after cysteine rich and pleckstrin homology (PH) domains at the C-terminal end of the proteins.

[0094] As used herein “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.

[0095] As used herein “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, rAAV2/1 , rAAV2/8, rAAV2/9 and variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. In some aspects, the rAAV vector is a AAV9 vector. In some aspects, the rAAV vector is an AAV-PHP.eB vector.

[0096] As used herein “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.

[0097] As used herein “capsid” refers to the protein shell of a virus particle.

[0098] As used herein “neurotrophic capsid” refers to a capsid of a neurotropic virus.

[0099] 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. For e.g., 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.

[0100] As used herein “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). In one aspect, HIV based vector backbones {i.e., HIV cis-acting sequence elements) are preferred.

[0101] 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. For e.g., nucleotide sequence for Addgene plasmid #90214 is available at Addgene under pCSC-NGN2-IRES-GFP-T2A-Sox1 1 and Addgene plasmid #90215 is available at Addgene under pCSC-ISL1-T2A-LHX3.

[0102] As used herein “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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, 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 .

[0103] As used herein “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. As used herein, the term “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. As used herein, the term “tracrRNA” or “tracrRNA segment” 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). The term “guide RNA” encompasses a single guide RNA (sgRNA), where the crRNA segment and the tracrRNA segment are located in the same RNA molecule. The term “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.

[0104] 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. For example, 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. Similarly, 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.

[0105] 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. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include sequence that has a single occurrence in the gene. In some aspects, 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.

[0106] As used herein “short hairpin RNA” or “shRNA” is an RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, MAP4K4, MAP4K6 and/or MAP4K7. A “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.

[0107] In general, 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.

[0108] As used herein, “ectopic expression” refers to expression of the transgene in a tissue or cell where it is not normally expressed. [0109] As used herein “excipient” refers to an inert substance added to a composition to further facilitate administration of an active ingredient.

[0110] As used herein “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. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.). In some aspects, 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. For example, 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. If it is active in 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. Preferably, the promoter is specific for cells in particular regions of the brain, for example, the cortex, striatum, nigra, and hippocampus.

[0111] As used herein “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, 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. Thus, for example, 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.

[0112] As used herein “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. In some aspects, 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. For example, in various aspects, 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. However, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.

II. Methods of Treatment

[0113] 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. In some aspects, 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. In some aspects, the inhibitor of MAP4K can be any of those disclosed herein.

[0114] In some aspects of the method, 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. In some aspects, the neurodegenerative disease comprise ALS. In some aspects, the neurodegenerative disease comprise multiple sclerosis. In some aspects, the neurodegenerative disease comprise ALS. In some aspects, the neurodegenerative disease comprise Parkinson's disease. In some aspects, the neurodegenerative disease comprise Alzheimer's disease. In some aspects, the neurodegenerative disease comprise Huntington's disease. In some aspects, the neurodegenerative disease comprise multiple system atrophy. In some aspects, 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.

[0115] In some aspects, 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.

[0116] In some aspects, 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.

[0117] In further aspects, 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. In some aspects, 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.

[0118] In some aspects, administering the composition treats or reduces one or more symptoms associated with neurodegenerative disease or brain injury in a subject. In some aspects, 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. In some aspects, 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.

[0119] In some aspects, 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.

[0120] In some aspects, administering the composition protects a subject from neurodegeneration and/or neural injury. In some aspects, 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. In some aspects, 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.

[0121] In further aspects, 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. In some aspects, 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.

[0122] Memory, cognitive performances, and motor functions can be tested using well established tests, such as evaluation of motor-spatial skills or memory recall testing. Nonlimiting examples of 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. Other tests known in the art, such as positron emission tomography (PET), magnetic resonance imaging (MRI), and 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.

[0123] In some aspects of the present disclosure, 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. In some aspects, 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. In some aspects, 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)

[0124] In some aspects, 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. In some aspects, 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.

[0125] In some aspects, provided herein is a method of treating a neurodegenerative disease or brain injury in a subject, the method 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0126] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0127] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0128] In some aspects, 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. In some aspects, 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%.

[0129] In some aspects, 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. In some aspects, 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.

[0130] In further aspects, 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. In some aspects, 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.

[0131] In some aspects, 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. In some aspects, 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.

[0132] In further aspects, 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. In some aspects, 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.

[0133] In some aspects, 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.

[0134] In some aspects, 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. In some aspects, said vector engineered to express gRNA may be any of those disclosed herein. For example, the vector may be a lentivirus vector or rAAV vector. [0135] In some aspects, 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.

[0136] In some aspects, 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 1X10² genome copies (GC)/ml to about 2X10¹⁵ GC/ml. In some aspects, the lentivirus vector or rAAV engineered to express CNH domain and/or a CNH- containing truncation, is administered at a concentration of about 1X10² GC/ml, about 1X10³ GC/ml, about 1X10⁴ GC/ml, about 1X10⁵ GC/ml, about 1X10⁶ GC/ml, about

1X10⁷ GC/ml, about 1X10⁸ GC/ml, about 1X10⁹ GC/ml, about 1X10¹° GC/ml, about

1X10¹¹ GC/ml, about 1X10¹² GC/ml, about 1X10¹³ GC/ml, about 1X10¹⁴ GC/ml, about

1X10¹⁵ GC/ml, about 2X10² GC/ml, about 2X10³ GC/ml, about 2X10⁴ GC/ml, about 2X10⁵ GC/ml, about 2X10⁶ GC/ml, about 2X10⁷ GC/ml, about 2X10⁸ GC/ml, about

2X10⁹ GC/ml, about 2X10¹° GC/ml, about 2X10¹¹ GC/ml, about 2X10¹² GC/ml, about

2X10¹³ GC/ml, about 2X10¹⁴ GC/ml, or about 2X10¹⁵ GC/ml. In some aspects, lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 1X10¹³GC/mL. In some aspects, lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 2.0X10¹² GC/mL In some aspects, rAAV engineered to express CNH domain, and/or a CNH- containing truncation is administered at a concentration of 1X10¹³GC/mL. In some aspects, rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at a concentration of 2.0X10¹² GC/mL.

[0137] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, lentivirus vector engineered to express CNH domain, and/or a CNH- containing truncation is administered at about 1.0 pl. In some aspects, lentivirus vector engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 8 pl - about 10 pl. In some aspects, rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 1.0 pl. In some aspects, rAAV engineered to express CNH domain, and/or a CNH-containing truncation is administered at about 8 pl - about 10 pl.

[0138] In some aspects, 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. In some aspects, the dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). qRNAs comprising a target sequence of MAP4K

[0139] In some aspects, 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. In some aspects, 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.

[0140] In some aspects, provided herein is a method of treating a neurodegenerative disease or brain injury in a subject, the method 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0141] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0142] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0143] In some aspects, 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. In some aspects, the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1% to about 100%. For example, 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%. In some aspects, the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.

[0144] In some aspects, 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. In some aspects, 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.

[0145] In further aspects, 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. In some aspects, 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.

[0146] In some aspects, 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. In some aspects, 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.

[0147] In further aspects, 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. In some aspects, 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.

[0148] In some aspects, gRNAs can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof. In some aspects, gRNA can be engineered and produced using primers disclosed in Table 2. In certain aspects, the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop. In some aspects, 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. [0149] In some aspects, 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. In some aspects, said vector engineered to express gRNA may be any of those disclosed herein. For example, the vector may be a lentivirus vector or rAAV vector.

[0150] In some aspects, 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.

[0151] In some aspects, 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 1X10² genome copies (GC)/ml to about 2X10¹⁵ GC/ml. In some aspects, 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 1X10² GC/ml, about 1X10³ GC/ml, about 1X10⁴ GC/ml, about 1X10⁵ GC/ml, about 1X10⁶ GC/ml, about

1X10⁷ GC/ml, about 1X10⁸ GC/ml, about 1X10⁹ GC/ml, about 1X10¹° GC/ml, about

1X10¹¹ GC/ml, about 1X10¹² GC/ml, about 1X10¹³ GC/ml, about 1X10¹⁴ GC/ml, about

1X10¹⁵ GC/ml, about 2X10² GC/ml, about 2X10³ GC/ml, about 2X10⁴ GC/ml, about 2X10⁵ GC/ml, about 2X10⁶ GC/ml, about 2X10⁷ GC/ml, about 2X10⁸ GC/ml, about

2X10⁹ GC/ml, about 2X10¹° GC/ml, about 2X10¹¹ GC/ml, about 2X10¹² GC/ml, about

2X10¹³ GC/ml, about 2X10¹⁴ GC/ml, or about 2X10¹⁵ GC/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 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 2.0X10¹² 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 1X10¹³GC/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 2.0X10¹² GC/mL.

[0152] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. 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 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.

[0153] In some aspects, 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. In some aspects, the dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). shRNAs comprising a target sequence of MAP4K

[0154] In some aspects, 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. In some aspects, 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. [0155] In some aspects, provided herein is a method of treating a neurodegenerative disease or brain injury in a subject, the method 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0156] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0157] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0158] In some aspects, 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. In some aspects, the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1% to about 100%. For example, 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%. In some aspects, the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%. [0159] In some aspects, 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. In some aspects, 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.

[0160] In further aspects, 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. In some aspects, 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.

[0161] In some aspects, 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. In some aspects, 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.

[0162] In further aspects, 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. In some aspects, 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.

[0163] In some aspects, shRNAs can be engineered using known methods in the art, to comprise any target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof. In some aspects, shRNA can be engineered and produced using primers disclosed in Table 2. In some aspects, the length of the duplex of shRNAs is less than 30 base pairs. In some embodiments, 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. In some aspects, the length of the duplex is 19 to 25 base pairs in length. In certain aspects, 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. In addition to the duplex portion, 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.

[0164] In some aspects, 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.

[0165] In some aspects, 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. In some aspects, said vector engineered to express shRNA may be any of those disclosed herein. For example, the vector may be a lentivirus vector or rAAV vector.

[0166] In some aspects, 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.

[0167] In some aspects, 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 1X10² genome copies (GC)/ml to about 2X10¹⁵ GC/ml. In some aspects, 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 1X10² GC/ml, about 1X10³ GC/ml, about 1X10⁴ GC/ml, about 1X10⁵ GC/ml, about

1X10⁶ GC/ml, about 1X10⁷ GC/ml, about 1X10⁸ GC/ml, about 1X10⁹ GC/ml, about

1X10¹° GC/ml, about 1X10¹¹ GC/ml, about 1X10¹² GC/ml, about 1X10¹³ GC/ml, about

1X10¹⁴ GC/ml, about 1X10¹⁵ GC/ml, about 2X10² GC/ml, about 2X10³ GC/ml, about 2X10⁴ GC/ml, about 2X10⁵ GC/ml, about 2X10⁶ GC/ml, about 2X10⁷ GC/ml, about

2X10⁸ GC/ml, about 2X10⁹ GC/ml, about 2X10¹° GC/ml, about 2X10¹¹ GC/ml, about 2X10¹² GC/ml, about 2X10¹³ GC/ml, about 2X10¹⁴ GC/ml, or about 2X10¹⁵ GC/ml. In some aspects, 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. In some aspects, 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. In some aspects, rAAV 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. In some aspects, rAAV engineered to express a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof, is administered at a concentration of 2.0X10¹² GC/mL.

[0168] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. 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 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.

[0169] In some aspects, 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. In some aspects, the dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg).

[0170] In some aspects, 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. In some aspects, 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.

Chemical inhibitor

[0171] In some aspects, the MAP4K inhibitor can comprise a chemical inhibitor. In some aspects, the chemical inhibitor can comprise a small molecule, or a large molecule. In some aspects, the chemical inhibitor can be K02288.

[0172] Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, 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. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration. [0173] 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.

[0174] 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. In general, however, 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. For example, 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 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, 2300 mg/kg, 2400 mg/kg, 2500 mg/kg, 2600 mg/kg, 2700 mg/kg, 2800 mg/kg, 2900 mg/kg, 3000 mg/kg, 3500 mg/kg, 4000 mg/kg, or 5000 mg/kg.

[0175] In further aspects, other therapies can be used in conjunction with MAP4K inhibitor according to the present disclosure. In such aspects, 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), CX717, Cyclophosphamate, DCB-AD1 , Dextroamphetamine, DHA (Docosahexaenoic Acid), Digoxin, Dimebon (Latrepirdine), Divalproex, DMXB-A, Donepezil, Doxycycline, Egb 761 , EHT 0202 tazolate, ELND005 (scyllo-inositol), EPAX 1050TG, Ergoloid mesylate, Epigallocatechin- Gallate, Escitalopram, Estradiol, Estrogen, Etanercept, EVP-6124, EVT101 , Exelon, Fish oil, FK962, florpiramine F 18, Folate+Vitamin B6+ Vitamin B21 , Gabapentin, Galantamine, Gemfibrozil, Ginkgo biloba extracts (for example EGb 761 or CP401 ), improved extracts of Ginkgo biloba (for example enriched in active ingredients or lessened in contaminant) or drug containing Ginkgo biloba extracts (for example Tanakan or Gingkor fort), Glucose, L- Glutamic Acid, GSI 136, GSI-953, GSK239512, GSK933776A, Haloperidol, HF0220, Huperzine A, hydrocodone/APAP, Ibuprofen, IFN-alpha2A, Indomethacin, Insulin, Intravenous Immunoglobulin, Ketasyn, Lecozotan, Leuprolide, Levodopa, Lipoic Acid, Lithium, Lorazepam, Lovostatin, Lutein, LY2062430 (solanezumab), LY2811376, LY450139, LY451395, MABT5102A, Malate, Masitinib (AB1010), Medroxyprogesterone, Melatonin, MEM 1003, MEM 3454, Memantine, Methylene blue, Methylphenidate, Mifepristone, MK0249, MK0677, MK0952, MK0952, MK3328, Modafinil, MPC-7869, NADH, Naproxen, Nefiracetam, Neptune Krill Oil, Neramexane, NIC5-15, Nicoderm Patch, Nicotinamide (vitamin B3), Novasoy, NPO31 112, NS 2330, NSA-789, NSAIDs, Olanzapine, omega-3 polyunsaturated fatty acids (EPA+DHA), ONO-2506PO, Oxybate, Panax Ginseng, PAZ-417, PBT2, Perphenazine, PF-04360365, PF-04447943, PF-04494700, Phenserine, Phosphatidylserine, Pitavastatin, Posiphen, PPI-1019 (APAN), Pravastatin, Prazosin, Prednisone, Progesterone, PRX-03140, PYM50028, Quetiapine, R1450, Raloxifene, Ramipril, Rasagiline, Razadyne, resveratrol, rifampicin, risperidone, Rivastigmine, RN1219, R05313534, Rofecoxib, Rosiglitazone, Salvia officinalis (sage), SAM-315, SAM-531 , SAM-760, SB-742457, Selenium, Sertraline, SGS-742, Simvastatin, SK-PC-B70M, Solanezumab, SR57667B, SRA- 333, SRA-444, SSR18071 1c, ST101 , T-817MA, Tacrine, Tarenflurbil, Testosterone, Tramiprosate (3APS), Trazodone, TRx0014 (methylthioninium chloride), Tryptophan, V950, Valproate, Varenicline, Vitamin C, Vitamin E, VP4896, Xaliproden, Zeaxanthin, Zolpidem, and ZT-1 (DEBIO-9902 SR).

III. Compositions

[0176] The present disclosure provides a MAP4K inhibitor. In some aspects, 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. In some aspects, 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. In some aspects, the inhibitor of MAP4K comprises a CNH of MAP4K4, and/or a CNH-containing truncation of MAP4K4. In some aspects, 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.

[0177] In some aspects, the disclosure provides a composition comprising an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof.

[0178] In some aspects, 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.

[0179] According to some aspects of the method, the inhibitor of MAP4K signaling or activity, is an inhibitor of MAP4K4 (HGK). The human MAP4K4 comprises an amino acid sequence of:

MANDSPAKSLVDIDLSSLRDPAGIFELVEVVGNGTYGQVYKGRHVKTGQLAAIKVMDVTED EEEEIKLEINMLKKYSHHRNIATYYGAFIKKSPPGHDDQLWLVMEFCGAGSITDLVKNTKGN TLKEDWIAYISREILRGLAHLHIHHVIHRDIKGQNVLLTENAEVKLVDFGVSAQLDRTVGRRN TFIGTPYWMAPEVIACDENPDATYDYRSDLWSCGITAIEMAEGAPPLCDMHPMRALFLIPRN PPPRLKSKKWSKKFFSFIEGCLVKNYMQRPSTEQLLKHPFIRDQPNERQVRIQLKDHIDRTR

KKRGEKDETEYEYSGSEEEEEEVPEQEGEPSSIVNVPGESTLRRDFLRLQQENKERSEALR RQQLLQEQQLREQEEYKRQLLAERQKRIEQQKEQRRRLEEQQRREREARRQQEREQRRR EQEEKRRLEELERRRKEEEERRRAEEEKRRVEREQEYIRRQLEEEQRHLEVLQQQLLQEQ AMLLECRWREMEEHRQAERLQRQLQQEQAYLLSLQHDHRRPHPQHSQQPPPPQQERSK PSFHAPEPKAHYEPADRAREVEDRFRKTNHSSPEAQSKQTGRVLEPPVPSRSESFSNGNS

ESVHPALQRPAEPQVPVRTTSRSPVLSRRDSPLQGSGQQNSQAGQRNSTSIEPRLLWERV EKLVPRPGSGSSSGSSNSGSQPGSHPGSQSGSGERFRVRSSSKSEGSPSQRLENAVKKP EDKKEVFRPLKPADLTALAKELRAVEDVRPPHKVTDYSSSSEESGTTDEEDDDVEQEGADE STSGPEDTRAASSLNLSNGETESVKTMIVHDDVESEPAMTPSKEGTLIVRQTQSASSTLQK HKSSSSFTPFIDPRLLQISPSSGTTVTSVVGFSCDGMRPEAIRQDPTRKGSWNVNPTNTRP

QSDTPEIRKYKKRFNSEILCAALWGVNLLVGTESGLMLLDRSGQGKVYPLINRRRFQQMD VLEGLNVLVTISGKKDKLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDLEGCVHYKWKY ERIKFLVIALKSSVEVYAWAPKPYHKFMAFKSFGELVHKPLLVDLTVEEGQRLKVIYGSCA GFHAVDVDSGSVYDIYLPTHIQCSIKPHAIIILPNTDGMELLVCYEDEGVYVNTYGRITKDW LQWGEMPTSVAYIRSNQTMGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKVF

FASVRSGGSSQVYFMTLGRTSLLSW (SEQ ID NO: 1 )

[0180] In some aspects, 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.

[0181] In some aspects, the inhibitor of MAP4K signaling or activity, is an inhibitor of MAP4K6 (MINK1). The human MAP4K6 comprises an amino acid sequence of:

MGDPAPARSLDDIDLSALRDPAGIFELVEVVGNGTYGQVYKGRHVKTGQLAAIKVMDVTED EEEEIKQEINMLKKYSHHRNIATYYGAFIKKSPPGNDDQLWLVMEFCGAGSVTDLVKNTKG NALKEDCIAYICREILRGLAHLHAHKVIHRDIKGQNVLLTENAEVKLVDFGVSAQLDRTVGRR NTFIGTPYWMAPEVIACDENPDATYDYRSDIWSLGITAIEMAEGAPPLCDMHPMRALFLIPR

NPPPRLKSKKWSKKFIDFIDTCLIKTYLSRPPTEQLLKFPFIRDQPTERQVRIQLKDHIDRSRK

KRGEKEETEYEYSGSEEEDDSHGEEGEPSSIMNVPGESTLRREFLRLQQENKSNSEALKQ

QQQLQQQQQRDPEAHIKHLLHQRQRRIEEQKEERRRVEEQQRREREQRKLQEKEQQRRL

EDMQALRREEERRQAEREQEYKRKQLEEQRQSERLQRQLQQEHAYLKSLQQQQQQQQL

QKQQQQQLLPGDRKPLYHYGRGMNPADKPAWAREVEERTRMNKQQNSPLAKSKPGSTG

PEPPIPQASPGPPGPLSQTPPMQRPVEPQEGPHKSLVAHRVPLKPYAAPVPRSQSLQDQP

TRNLAAFPASHDPDPAIPAPTATPSARGAVIRQNSDPTSEGPGPSPNPPAWVRPDNEAPPK

VPQRTSSIATALNTSGAGGSRPAQAVRARPRSNSAWQIYLQRRAERGTPKPPGPPAQPPG

PPNASSNPDLRRSDPGWERSDSVLPASHGHLPQAGSLERNRVGVSSKPDSSPVLSPGNK

AKPDDHRSRPGRPADFVLLKERTLDEAPRPPKKAMDYSSSSEEVESSEDDEEEGEGGPAE

GSRDTPGGRSDGDTDSVSTMVVHDVEEITGTQPPYGGGTMVVQRTPEEERNLLHADSNG

YTNLPDVVQPSHSPTENSKGQSPPSKDGSGDYQSRGLVKAPGKSSFTMFVDLGIYQPGGS

GDSIPITALVGGEGTRLDQLQYDVRKGSVVNVNPTNTRAHSETPEIRKYKKRFNSEILCAAL

WGVNLLVGTENGLMLLDRSGQGKVYGLIGRRRFQQMDVLEGLNLLITISGKRNKLRVYYL

SWLRNKILHNDPEVEKKQGWTTVGDMEGCGHYRWKYERIKFLVIALKSSVEVYAWAPK

PYHKFMAFKSFADLPHRPLLVDLTVEEGQRLKVIYGSSAGFHAVDVDSGNSYDIYIPVHIQ SQITPHAIIFLPNTDGMEMLLCYEDEGVYVNTYGRIIKDWLQWGEMPTSVAYICSNQIMGW GEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKVFFASVRSGGSSQVYFMTLNRNCI

MNW (SEQ ID NO: 2)

[0182] In some aspects, 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.

[0183] In some aspects, the inhibitor of MAP4K signaling or activity, is an inhibitor of MAP4K7 (TNIK). The human MAP4K7 comprises an amino acid sequence of:

MASDSPARSLDEIDLSALRDPAGIFELVELVGNGTYGQVYKGRHVKTGQLAAIKVMDVTGD EEEEIKQEINMLKKYSHHRNIATYYGAFIKKNPPGMDDQLWLVMEFCGAGSVTDLIKNTKGN TLKEEWIAYICREILRGLSHLHQHKVIHRDIKGQNVLLTENAEVKLVDFGVSAQLDRTVGRRN TFIGTPYWMAPEVIACDENPDATYDFKSDLWSLGITAIEMAEGAPPLCDMHPMRALFLIPRN PAPRLKSKKWSKKFQSFIESCLVKNHSQRPATEQLMKHPFIRDQPNERQVRIQLKDHIDRTK KKRGEKDETEYEYSGSEEEEEENDSGEPSSILNLPGESTLRRDFLRLQLANKERSEALRRQ QLEQQQRENEEHKRQLLAERQKRIEEQKEQRRRLEEQQRREKELRKQQEREQRRHYEEQ MRREEERRRAEHEQEYIRRQLEEEQRQLEILQQQLLHEQALLLEYKRKQLEEQRQAERLQ RQLKQERDYLVSLQHQRQEQRPVEKKPLYHYKEGMSPSEKPAWAKEVEERSRLNRQSSP AMPHKVANRISDPNLPPRSESFSISGVQPARTPPMLRPVDPQIPHLVAVKSQGPALTASQS VHEQPTKGLSGFQEALNVTSHRVEMPRQNSDPTSENPPLPTRIEKFDRSSWLRQEEDIPPK VPQRTTSISPALARKNSPGNGSALGPRLGSQPIRASNPDLRRTEPILESPLQRTSSGSSSSS STPSSQPSSQGGSQPGSQAGSSERTRVRANSKSEGSPVLPHEPAKVKPEESRDITRPSRP ASYKKAIDEDLTALAKELRELRIEETNRPMKKVTDYSSSSEESESSEEEEEDGESETHDGTV AVSDIPRLIPTGAPGSNEQYNVGMVGTHGLETSHADSFSGSISREGTLMIRETSGEKKRSG HSDSNGFAGHINLPDLVQQSHSPAGTPTEGLGRVSTHSQEMDSGTEYGMGSSTKASFTPF VDPRVYQTSPTDEDEEDEESSAAALFTSELLRQEQAKLNEARKISVVNVNPTNIRPHSDTPE IRKYKKRFNSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYNLINRRRFQQMDVLEGLN VLVTISGKKNKLRVYYLSWLRNRILHNDPEVEKKQGWITVGDLEGCIHYKWKYERIKFLVI ALKNAVEIYAWAPKPYHKFMAFKSFADLQHKPLLVDLTVEEGQRLKVIFGSHTGFHVIDVD SGNSYDIYIPSHIQGNITPHAIVILPKTDGMEMLVCYEDEGVYVNTYGRITKDWLQWGEMP TSVAYIHSNQIMGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKVFFASVRSGG

SSQVFFMTLNRNSMMNW (SEQ ID NO: 3) [0184] In some aspects, 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)

[0185] In some aspects, 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. In some aspects, the CNH domain of human MAP4K4 comprises an amino acid sequence of:

NSEILCAALWGVNLLVGTESGLMLLDRSGQGKVYPLINRRRFQQMDVLEGLNVLVTISGKK DKLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDLEGCVHYKVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFGELVHKPLLVDLTVEEGQRLKVIYGSCAGFHAVDVDSGSVYDIYL PTHIQCSIKPHAIIILPNTDGMELLVCYEDEGVYVNTYGRITKDWLQWGEMPTSVAYIRSNQ TMGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 4)

[0186] In some aspects, 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.

[0187] In some aspects of the disclosure, 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. In some aspects, 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 ).

[0188] In some aspects, 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. In some aspects, the CNH domain of human MAP4K6 comprises an amino acid sequence:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYGLIGRRRFQQMDVLEGLNLLITISGKRN KLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDMEGCGHYRVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFADLPHRPLLVDLTVEEGQRLKVIYGSSAGFHAVDVDSGNSYDIYIP VHIQSQITPHAIIFLPNTDGMEMLLCYEDEGVYVNTYGRIIKDWLQWGEMPTSVAYICSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 5)

[0189] In some aspects, 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.

[0190] In some aspects of the disclosure, 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. In some aspects, 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).

[0191] In some aspects, 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. In some aspects, the CNH domain of human MAP4K7 comprises an amino acid sequence of:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYNLINRRRFQQMDVLEGLNVLVTISGKK NKLRVYYLSWLRNRILHNDPEVEKKQGWITVGDLEGCIHYKWKYERIKFLVIALKNAVEIYA WAPKPYHKFMAFKSFADLQHKPLLVDLTVEEGQRLKVIFGSHTGFHVIDVDSGNSYDIYIPS HIQGNITPHAIVILPKTDGMEMLVCYEDEGVYVNTYGRITKDVVLQWGEMPTSVAYIHSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 6)

[0192] In some aspects, 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.

[0193] In some aspects of the disclosure, 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. In some aspects, 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

[0194] In some aspects, 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. 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 MAP4K4. 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 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.

[0195] In some aspects, the gRNA comprises a target sequence of MAP4K4, for example CAGGACATGATGACCAACTC (SEQ ID NO: 13) or GGGCGGAGAAATACGTTCAT (SEQ ID NO: 14). In some aspects, the gRNA comprises a target sequence of MAP4K6, for example CGGACAGGTCGATGTCGTCC (SEQ ID NO: 15) or AGGGTCGGCATGTCAAGACG (SEQ ID NO: 16). In some aspects, the gRNA comprises a target sequence of MAP4K7, for example CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18).

[0196] In some aspects, 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.

[0197] In some aspects, 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. In some aspects, gRNA can be engineered and produced using primers disclosed in Table 2. In certain aspects, the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop. In some aspects, 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

[0198] In some aspects, 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. 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 shRNA comprising a target sequence of MAP4K4. 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 shRNA 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 shRNA comprising a target sequence of MAP4K7.

[0199] In some aspects of the disclosed 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). In some aspects, 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). In some aspects, the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor 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.

[0200] In some aspects of the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor, a target sequence of MAP4K6 for inhibition by shRNA is a nucleotide sequence comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9). In some aspects, 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). In some aspects, 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.

[0201] In some aspects of the MAP4K inhibitor and/or composition comprising a MAP4K inhibitor, a target sequence of MAP4K7 for inhibition by shRNA is a nucleotide comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 1 1 ). In some aspects, 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). In some aspects, 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.

Vectors

[0202] The present disclosure further provides a vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof. In some aspects, the vector encoding an inhibitor of MAP4K signaling or activity, or a biologically active fragment thereof, is part of a composition. In some aspects, 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. In some aspects, the disclosed vector and/or composition comprising said vector can be administered in a subject for treating neurodegenerative disease or brain injury. In some aspects, 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.

[0203] In some aspects of the disclosed composition, 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. In some aspects, the neurodegenerative disease comprise ALS. In some aspects, the neurodegenerative disease comprise multiple sclerosis. In some aspects, the neurodegenerative disease comprise ALS. In some aspects, the neurodegenerative disease comprise Parkinson's disease. In some aspects, the neurodegenerative disease comprise Alzheimer's disease. In some aspects, the neurodegenerative disease comprise Huntington's disease. In some aspects, the neurodegenerative disease comprise multiple system atrophy. In some aspects, 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.

[0204] According to some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0205] In some aspects, the vector comprises a CNH of MAP4K4, and/or a CNH- containing truncation of MAP4K4, or any combination thereof. In some aspects, the CNH domain of human MAP4K4 comprises an amino acid sequence:

NSEILCAALWGVNLLVGTESGLMLLDRSGQGKVYPLINRRRFQQMDVLEGLNVLVTISGKK DKLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDLEGCVHYKVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFGELVHKPLLVDLTVEEGQRLKVIYGSCAGFHAVDVDSGSVYDIYL PTHIQCSIKPHAIIILPNTDGMELLVCYEDEGVYVNTYGRITKDWLQWGEMPTSVAYIRSNQ TMGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 4)

[0206] In some aspects, 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.

[0207] In some aspects of the disclosed vector, 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. In some aspects, 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 ).

[0208] In some aspects, the vector comprises a CNH of MAP4K6, and/or a CNH- containing truncation of MAP4K6, or any combination thereof. In some aspects, the CNH domain of human MAP4K6 comprises an amino acid sequence:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYGLIGRRRFQQMDVLEGLNLLITISGKRN KLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDMEGCGHYRVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFADLPHRPLLVDLTVEEGQRLKVIYGSSAGFHAVDVDSGNSYDIYIP VHIQSQITPHAIIFLPNTDGMEMLLCYEDEGVYVNTYGRIIKDWLQWGEMPTSVAYICSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 5)

[0209] In some aspects, 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.

[0210] In some aspects of the vector, 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. In some aspects, 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).

[0211] In some aspects, the vector comprises a CNH of MAP4K7, and/or a CNH- containing truncation of MAP4K7, or any combination thereof. In some aspects, the CNH domain of human MAP4K7 comprises an amino acid sequence:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYNLINRRRFQQMDVLEGLNVLVTISGKK NKLRVYYLSWLRNRILHNDPEVEKKQGWITVGDLEGCIHYKWKYERIKFLVIALKNAVEIYA WAPKPYHKFMAFKSFADLQHKPLLVDLTVEEGQRLKVIFGSHTGFHVIDVDSGNSYDIYIPS HIQGNITPHAIVILPKTDGMEMLVCYEDEGVYVNTYGRITKDVVLQWGEMPTSVAYIHSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 6)

[0212] In some aspects, 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.

[0213] In some aspects of the vector, 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. In some aspects, 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).

[0214] In some aspects, 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. In some aspects, 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%.

[0215] According to some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0216] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0217] In some aspects, 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. In some aspects, gRNA can be engineered and produced using primers disclosed in Table 2. In certain aspects, the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop. In some aspects, 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.

[0218] In some aspects, 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.

[0219] In some aspects, 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. In some aspects, the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1 % to about 100%. For example, 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%. In some aspects, the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.

[0220] According to some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0221] In some aspects of the disclosed vector, a target sequence of MAP4K4 for inhibition by shRNA is nucleotide sequence comprising AACCGAAGACGATTTCAACAAA (SEQ ID NO: 7). In some aspects, the vector comprises a shRNA comprising a target sequence of MAP4K4 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCACCGAAGACGATTTCAACAAATAGTGAAGCCACAGATGTA TTTGTTGAAATCGTCTTCGGTTTGCCTACTGCCTCGGA (SEQ ID NO: 8). In some aspects, the 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.

[0222] In some aspects of the disclosed vector, a target sequence of MAP4K6 for inhibition by shRNA is a nucleotide sequence comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9). In some aspects, the vector comprises a shRNA comprising a target sequence of MAP4K6 which is a nucleotide sequence comprising TGCTGTTGACAGTGAGCGCCCGGAACAAGATTCTGCACAATAGTGAAGCCACAGATGT ATTGTGCAGAATCTTGTTCCGGATGCCTACTGCCTCGGA (SEQ ID NO: 10). In some aspects, the 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.

[0223] In some aspects of the vector, a target sequence of MAP4K7 for inhibition by shRNA is a nucleotide comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 11 ). In some aspects, the composition comprises a shRNA comprising a target sequence of MAP4K7 which is a nucleotide sequence comprising

TGCTGTTGACAGTGAGCGAAAGGTCAAAGATTAAAGGTTATAGTGAAGCCACAGATGTA TAACCTTTAATCTTTGACCTTCTGCCTACTGCCTCGGA (SEQ ID NO: 12). In some aspects, 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.

[0224] In some aspects, 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. In some aspects, the MAP4K4, MAP4K6, and/or MAP4K7 gene expression is lowered by about 1 to about 100%. For example, 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%. In some aspects, the expression of MAP4K4, MAP4K6, and/or MAP4K7 is reduced by at least about 90%.

[0225] In some aspects, the composition comprises a vector engineered to express the inhibitor of MAP4K. Vectors engineered to carry inhibitors of MAP4K can be lentiviral plasmids. In some aspects, lentiviral plasmids can be lentiviral plasmids for motor neurons (e.g., Addgene plasmid #90214 or Addgene plasmid #90215). In some aspects of the composition, the lentivirus vector is Addgene plasmid #90214. In some aspects of the composition, the lentivirus vector is Addgene plasmid #90215.

[0226] In some aspects, the composition comprises a lentivirus vector engineered to express a CNH of MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof. In some aspects, the composition comprises lentivirus vector is engineered to express a CNH of MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof. In some aspects, the composition comprises lentivirus vector is engineered to express a CNH of MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.

[0227] In some aspects, 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). In some aspects, the composition comprises lentivirus vector is engineered to express a gRNA comprising a target sequence of MAP4K6, for example

CGGACAGGTCGATGTCGTCC (SEQ ID NO: 15) or AGGGTCGGCATGTCAAGACG (SEQ ID NO: 16). In some aspects, the composition comprises lentivirus vector is engineered to express a gRNA comprising a target sequence of MAP4K7, for example

CGACTCCCCGGCTCGAAGCC (SEQ ID NO: 17) or TTCATCCAGGCTTCGAGCCG (SEQ ID NO: 18).

[0228] In some aspects, 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.

[0229] In some aspects, the composition comprises a lentivirus vector engineered to express a shRNA comprising a target sequence of MAP4K4. In some aspects, the composition comprises lentivirus vector is engineered to express a shRNA comprising a target sequence of MAP4K6. In some aspects, the composition comprises lentivirus vector is engineered to express a shRNA comprising a target sequence of MAP4K7.

[0230] In some aspects, 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. In some aspects, 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.

[0231] In some aspects, the composition comprises recombinant adeno-associated virus (rAAV) vector system engineered to express an inhibitor of MAP4K. In some aspects, 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. In some aspects of the composition, the rAAV vector is a AAV9 vector. In some aspects of the composition, the rAAV vector is an AAV-PHP.eB vector. [0232] In some aspects, the composition comprises a AAV9 vector engineered to express a CNH of MAP4K4, a CNH-containing truncation of MAP4K4, or any combination thereof. In some aspects, the composition comprises a AAV9 vector engineered to express a CNH of MAP4K6, a CNH-containing truncation of MAP4K6, or any combination thereof. In some aspects, the composition comprises a AAV9 vector engineered to express a CNH of MAP4K7, a CNH-containing truncation of MAP4K7, or any combination thereof.

[0233] In some aspects, 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.

[0234] In some aspects, 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.

[0235] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0236] In some aspects, 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.

[0237] In some aspects, 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.

[0238] In some aspects, 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. 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.

[0239] In some aspects, 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 1X10² genome copies (GC)/ml to about 2X10¹⁵ GC/ml. In some aspects, the composition is administered at a concentration of about 1X10² GC/ml, about 1X10³ GC/ml, about 1X10⁴ GC/ml, about 1X10⁵ GC/ml, about 1X10⁶ GC/ml, about

1X10⁷ GC/ml, about 1X10⁸ GC/ml, about 1X10⁹ GC/ml, about 1X10¹° GC/ml, about

1X10¹¹ GC/ml, about 1X10¹² GC/ml, about 1X10¹³ GC/ml, about 1X10¹⁴ GC/ml, about

1X10¹⁵ GC/ml, about 2X10² GC/ml, about 2X10³ GC/ml, about 2X10⁴ GC/ml, about 2X10⁵ GC/ml, about 2X10⁶ GC/ml, about 2X10⁷ GC/ml, about 2X10⁸ GC/ml, about

2X10⁹ GC/ml, about 2X10¹° GC/ml, about 2X10¹¹ GC/ml, about 2X10¹² GC/ml, about

2X10¹³ GC/ml, about 2X10¹⁴ GC/ml, or about 2X10¹⁵ GC/ml. In some aspects, the 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 1X10¹³GC/mL. In some aspects, composition comprising an rAAV engineered to express an inhibitor of MAP4K, is administered at a concentration of 2.0X10¹² GC/mL.

[0240] In some aspects, the 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. In some aspects, 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. In some aspects, 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.

[0241] In some aspects, 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. In some aspects, the dosage of the composition is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg).

[0242] In further aspects, the vectors disclosed herein is engineered to express MAP4K inhibitor comprises a promoter operably linked to the MAP4K inhibitor. In some aspects, 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, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, EAAT2 promoter, myelin basic protein (MBP) promoter, dopamine- b-hydroxylase gene promoter, L7 Purkinje cell protein promoter, human hypoxanthine phosphoribosyltransferase promoter, SCG10 promoter, Ta1 a-tubulin promoter, aldolase C promoter, beta-tubulin gene promoter, GnRH gene enhancer and promoter, glutamate decarboxylase 65 gene promoter, beta-galactoside alpha 1 ,2-fucosyltransferase gene promoter, neuronal nicotinic acetylcholine receptor beta3 gene promoter, GABA(A) receptor delta subunit gene promoter, neuron-specific FE65 gene promoter, N-type calcium channel alphal B subunit gene promoter, S100 promoter, glutamine synthase promoter, microtubule- associated protein 1 B gene promoter, tyrosine hydroxylase promoter, acetylcholinesterase promoter, choline acetyltransferase promoter, dopamine receptor I and II promoters, dopamine transporter promoter, vesicular monoamine transporter promoter, neuopsin promoter, hybrid cytomegalovirus/chicken beta-actin (CBA, also known as the CAG promoter) promoter, or vesicular acetylcholine transporter promoter. 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. For e.g., nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956. In some aspects, vectors encoding neuron specific promoter (e.g., human synapsin 1 ) is available at publicly accessible 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.

[0243] In some aspects, the disclosed vectors comprise a neuron specific 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.

[0244] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0245] In some aspects, 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. In some aspects, rAAV disclosed herein comprises a hSYN1 promoter operably linked to a gRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof. In some aspects, rAAV disclosed herein comprises a hSYN1 promoter operably linked to a shRNA comprising a target sequence of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.

Chemical inhibitor

[0246] In some aspects, the composition can comprise a chemical inhibitor of MAP4K. In some aspects, the chemical inhibitor can comprise a small molecule, or a large molecule. In some aspects, the chemical inhibitor can be K02288.

Pharmaceutical Compositions

[0247] In certain aspects, the 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. Examples, without limitation, of excipients 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.

[0248] In certain aspects, 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. In some aspects, any of the well-known techniques, carriers, and excipients may be used as suitable and/or as understood in the art.

[0249] In certain aspects, compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, 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. In some aspects, 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.

[0250] In certain aspects, compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents. In some aspects, 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. In some aspects, 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. In some aspects, pharmaceutical 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. In some aspects, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, 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, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVM/MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof.

[0251] In certain aspects, 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. In some aspects, 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. In some aspects, pharmaceutical 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. In some aspects, 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. In some aspects, additives may be biocompatible, without being harsh, abrasive, and/or allergenic. [0252] In certain aspects, compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In some aspects, pharmaceutical 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.

[0253] In certain aspects, compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “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. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, pharmaceutical 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.

[0254] In certain aspects, compositions disclosed herein may comprise one or more antioxidants. As used herein, “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. In some aspects, 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.

[0255] In certain aspects, compositions disclosed herein may comprise a buffer system. As used herein, 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. In some aspects, 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.

[0256] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some aspects, the compositions disclosed herein may have a pH of about 6 to about 9. In some aspects, 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.

[0257] In certain aspects, compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, 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. 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 preservatives by total weight of the composition.

[0258] In certain aspects, compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In some aspects, 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. In some aspects, 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.

[0259] In certain aspects, compositions disclosed herein may comprise one or more stabilizers. As used herein, 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. In some aspects, 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.

[0260] In some aspects, compositions disclosed herein may comprise one or more tonicity agents. As used herein, 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. In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0261] The 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.

[0262] In some aspects, 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. For such injectable compositions, sterile aqueous or oleaginous suspensions may be formulated according to the known technique using suitable dispersing agents, wetting agents and/or suspending agents. In some aspects of the disclosure the aqueous injectable composition is formulated for administration to said subject by bolus administration. In other aspects, 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.

[0263] In some aspect, the composition may comprise one or more active agents in addition to the inhibitor of MAP4K, and vectors, provided herein. Non limiting examples of 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.

[0264] In some aspects, 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. In some aspects, 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.

IV. Isolated nucleic acids

[0265] In some aspects, the disclosure provides an isolated nucleic acid sequence comprising a nucleic acid sequence encoding a nucleic acid sequence encoding MAP4K inhibitor. In some aspects, the isolated nucleic acid can be formulated into a composition. In some aspects, the composition is a pharmaceutical composition comprising the isolated nucleic acid and one or more pharmaceutically acceptable excipients.

[0266] In some aspects, provided herein is 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.

[0267] In some aspects, 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.

[0268] In some aspects, 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:

NSEILCAALWGVNLLVGTESGLMLLDRSGQGKVYPLINRRRFQQMDVLEGLNVLVTISGKK DKLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDLEGCVHYKVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFGELVHKPLLVDLTVEEGQRLKVIYGSCAGFHAVDVDSGSVYDIYL PTHIQCSIKPHAIIILPNTDGMELLVCYEDEGVYVNTYGRITKDWLQWGEMPTSVAYIRSNQ TMGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 4).

[0269] In some aspects, 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.

[0270] In some aspects, 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. In some aspects, 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 ).

[0271] In some aspects, 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:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYGLIGRRRFQQMDVLEGLNLLITISGKRN KLRVYYLSWLRNKILHNDPEVEKKQGWTTVGDMEGCGHYRVVKYERIKFLVIALKSSVEVY AWAPKPYHKFMAFKSFADLPHRPLLVDLTVEEGQRLKVIYGSSAGFHAVDVDSGNSYDIYIP VHIQSQITPHAIIFLPNTDGMEMLLCYEDEGVYVNTYGRIIKDWLQWGEMPTSVAYICSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 5).

[0272] In some aspects, 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.

[0273] In some aspects, 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. In some aspects, 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 (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).

[0274] In some aspects, 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:

NSEILCAALWGVNLLVGTENGLMLLDRSGQGKVYNLINRRRFQQMDVLEGLNVLVTISGKK NKLRVYYLSWLRNRILHNDPEVEKKQGWITVGDLEGCIHYKWKYERIKFLVIALKNAVEIYA WAPKPYHKFMAFKSFADLQHKPLLVDLTVEEGQRLKVIFGSHTGFHVIDVDSGNSYDIYIPS HIQGNITPHAIVILPKTDGMEMLVCYEDEGVYVNTYGRITKDVVLQWGEMPTSVAYIHSNQI MGWGEKAIEIRSVETGHLDGVFMHKRAQRLKFLCERNDKV (SEQ ID NO: 6).

[0275] In some aspects, 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.

[0276] In some aspects, 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. In some aspects, 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:

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).

[0277] In some aspects, 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.

[0278] In some aspects, provided herein is 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.

[0279] In some aspects, 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). In some aspects, 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). In some aspects, 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).

[0280] In some aspects, 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.

[0281] In some aspects, 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. In some aspects, gRNA can be engineered and produced using primers disclosed in Table 2. In certain aspects, the guide RNA is a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are linked through a loop. In some aspects, 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.

[0282] In some aspects, 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.

[0283] In some aspects, 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.

[0284] In some aspects, the isolated nucleic acid sequence comprises a target sequence of MAP4K4 for inhibition by shRNA comprising AACCGAAGACGATTTCAACAAA (SEQ ID NO: 7). In some aspects, 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). In some aspects, 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.

[0285] In some aspects, the isolated nucleic acid sequence comprises a target sequence of MAP4K6 for inhibition by shRNA comprising TCCGGAACAAGATTCTGCACAA (SEQ ID NO: 9). In some aspects, 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). In some aspects, 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. [0286] In some aspects, the isolated nucleic acid sequence comprises a target sequence of MAP4K7 for inhibition by shRNA comprising GAAGGTCAAAGATTAAAGGTTA (SEQ ID NO: 11 ). In some aspects, 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). In some aspects, 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.

[0287] In some aspects, 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.

[0288] As disclosed herein, 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. In some aspects, the capsid is an AAV9 capsid. In some aspects, AAV9 is AAV9, AAV9.9, AAV9.11 , AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61 , AAV9.68, or AAV9.84. In some aspects In some aspects the capsid is an AAV- PHP.eB capsid.

[0289] In some aspects, 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.

[0290] In some aspects, 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.

[0291] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0292] In some aspects, 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. In some aspects, 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.

[0293] In some aspects, 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. In some aspects, 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.

[0294] In some aspects, 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. In some aspects, 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.

[0295] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0296] In some aspects, 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. In some aspects, 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.

[0297] In some aspects, 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. In some aspects, 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.

[0298] In some aspects, 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. In some aspects, 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.

[0299] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0300] In some aspects, 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. In some aspects, 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.

[0301] In some aspects, 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. In some aspects, 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.

[0302] In some aspects, 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. In some aspects, 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.

[0303] In some aspects, 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 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, EAAT2 promoter, myelin basic protein (MBP) promoter, dopamine- b-hydroxylase gene promoter, L7 Purkinje cell protein promoter, human hypoxanthine phosphoribosyltransferase promoter, SCG10 promoter, Ta1 a-tubulin promoter, aldolase C promoter, beta-tubulin gene promoter, GnRH gene enhancer and promoter, glutamate decarboxylase 65 gene promoter, beta-galactoside alpha 1 ,2-fucosyltransferase gene promoter, neuronal nicotinic acetylcholine receptor beta3 gene promoter, GABA(A) receptor delta subunit gene promoter, neuron-specific FE65 gene promoter, N-type calcium channel alphal B subunit gene promoter, S100 promoter, glutamine synthase promoter, microtubule- associated protein 1 B gene promoter, tyrosine hydroxylase promoter, acetylcholinesterase promoter, choline acetyltransferase promoter, dopamine receptor I and II promoters, dopamine transporter promoter, vesicular monoamine transporter promoter, neuopsin promoter, hybrid cytomegalovirus/chicken beta-actin (CBA, also known as the CAG promoter) promoter, or vesicular acetylcholine transporter promoter. These 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. For e.g., nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956. In some aspects, vectors encoding neuron specific promoter (e.g., human synapsin 1 ) is available at publicly accessible 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.

[0304] In some aspects, 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.

[0305] In some aspects, 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.

[0306] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0307] In some aspects, 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. In some aspects, 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.

[0308] In some aspects, 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. In some aspects, 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.

[0309] In some aspects, 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. In some aspects, 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.

[0310] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0311] In some aspects, 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. In some aspects, 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.

[0312] In some aspects, 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. In some aspects, 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.

[0313] In some aspects, 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. In some aspects, 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.

[0314] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0315] In some aspects, 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. In some aspects, 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.

[0316] In some aspects, 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. In some aspects, 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.

[0317] In some aspects, 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. In some aspects, 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.

[0318] In some aspects, 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.

[0319] As disclosed herein, 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.

[0320] 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 neuron specific promoter; and a nucleic acid sequence encoding MAP4K inhibitor. In some aspects, 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. In some aspects, 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.

[0321] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0322] In some aspects, 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. In some aspects, 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.

[0323] In some aspects, 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. In some aspects, 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.

[0324] In some aspects, 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. In some aspects, 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.

[0325] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0326] In some aspects, 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. In some aspects, 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.

[0327] In some aspects, 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. In some aspects, 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.

[0328] In some aspects, 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. In some aspects, 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.

[0329] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0330] In some aspects, 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. In some aspects, 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.

[0331] In some aspects, 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. In some aspects, 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.

[0332] In some aspects, 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. In some aspects, 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.

[0333] In some aspects, 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 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, EAAT2 promoter, myelin basic protein (MBP) promoter, dopamine- b-hydroxylase gene promoter, L7 Purkinje cell protein promoter, human hypoxanthine phosphoribosyltransferase promoter, SCG10 promoter, Ta1 a-tubulin promoter, aldolase C promoter, beta-tubulin gene promoter, GnRH gene enhancer and promoter, glutamate decarboxylase 65 gene promoter, beta-galactoside alpha 1 ,2-fucosyltransferase gene promoter, neuronal nicotinic acetylcholine receptor beta3 gene promoter, GABA(A) receptor delta subunit gene promoter, neuron-specific FE65 gene promoter, N-type calcium channel alphal B subunit gene promoter, S100 promoter, glutamine synthase promoter, microtubule- associated protein 1 B gene promoter, tyrosine hydroxylase promoter, acetylcholinesterase promoter, choline acetyltransferase promoter, dopamine receptor I and II promoters, dopamine transporter promoter, vesicular monoamine transporter promoter, neuopsin promoter, hybrid cytomegalovirus/chicken beta-actin (CBA, also known as the CAG promoter) promoter, or vesicular acetylcholine transporter promoter. These 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. For e.g., nucleotide sequence for neuron specific enolase (NSE) is available at NCBI database under GenBank Accession No: X51956. In some aspects, vectors encoding neuron specific promoter (e.g., human synapsin 1 ) is available at publicly accessible 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.

[0334] In some aspects, 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.

[0336] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0337] In some aspects, 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. In some aspects, 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.

[0338] In some aspects, 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. In some aspects, 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.

[0339] In some aspects, 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. In some aspects, 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.

[0340] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0341] In some aspects, 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. In some aspects, 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.

[0342] In some aspects, 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. In some aspects, 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.

[0343] In some aspects, 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. In some aspects, 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.

[0344] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0345] In some aspects, 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. In some aspects, 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.

[0346] In some aspects, 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. In some aspects, 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.

[0347] In some aspects, 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. In some aspects, 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.

[0348] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0349] In further aspects, 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. In some aspects, 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.

[0350] In some aspects, 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. In some aspects, 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.

[0351] In further aspects, 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. In some aspects, 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.

[0352] In some aspects, 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. For example, 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.

[0353] In some aspects, 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. In some aspects, 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.

[0354] In some aspects, 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. In some aspects, 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.

[0355] In some aspects, 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. For example, 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.

V. Kits

[0356] 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. For example, 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.

[0357] In some aspects, 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. In many cases, 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. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In some aspects, 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.

[0358] In addition to the inhibitor or a vector engineered to express the inhibitor, 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. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the inhibitor. In such aspects, 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.

[0359] The inhibitor or a vector engineered to express the inhibitor, can be provided in any form, e.g., liquid, dried or lyophilized form. In some aspects, the inhibitor or a vector engineered to express the inhibitor, is substantially pure and/or sterile. When the inhibitor or a vector engineered to express the inhibitor, is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution. When the inhibitor or a vector engineered to express the inhibitor, is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. In some aspects, the solvent, e.g., sterile water or buffer, can be provided in the kit.

[0360] The kit can include one or more containers for the composition containing the inhibitor or a vector engineered to express the inhibitor. In some aspects, 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. For example, the inhibitor or a vector engineered to express the inhibitor (e.g., in a composition) can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other aspects, the separate elements of the kit are contained within a single, undivided container. For example, the inhibitor or a vector engineered to express the inhibitor (e.g., in a composition) is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some aspects, 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). For example, 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.

[0361] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.

[0362] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.

EXAMPLES

[0363] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Materials and Methods

Animals

[0364] 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.

Human fibroblasts

[0365] 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.

Chemicals

[0366] 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.

Plasmids and virus production

[0367] 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 ). To monitor the transduction rate, mCherry was inserted to replace the Puro fragment to construct pCSC-hU6-Filler-EFS- SpCas9-FLAG-P2A-mCherry. Then 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

Table 3A: shRNA sequences

Table 3B: shRNA target site

Table 4: qRT-PCR primers

Fibroblast-derived human induced motor neurons (hiMNs)

[0368] 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. They were then switched into 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). Medium was half-changed every other day until replating at 14 days post virus infection (dpi). The replating procedure was performed to isolate hiMNs from nonconverted fibroblasts. 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.

Chemical screens

[0369] For primary and secondary screens, 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. As previously reported, assay quality for each plate was evaluated by the Z-prime value [=1-3*((STDEVKen+STDEWeh)/ABS(MeanKen-MeanVeh))], whereas relative survival for each chemical was calculated by (Sample- MeanVeh)/((MeanKen-MeanVeh)*100). Chemicals were then ranked based on their effects on relative survival of ALS-hiMNs. Top hits with a greater than 1 .5x standard deviations above the mean in primary screens were selected for secondary screens and those with greater than 3x standard deviations above the mean in secondary screens were selected for subsequent studies. All top hits from secondary screens were further assessed in dose-response assays (7-points in triplicates). Relative survival for each dosage was calculated via normalization to the vehicle controls.

[0370] For long-term chemical treatments, 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.

Primary astrocytes

[0371] Primary 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.

Neuromuscular junctions

[0372] Primary myoblasts were isolated from skeletal muscles of P0.5 mouse pups and differentiated into myotubes as previously described. Myotubes were resuspended in neuronal culture medium and then plated onto coverslips with co-cultured astrocytes and hiMNs at 40 to 50 dpi. After another 4 to 7 days, these sandwich cultures of myotubes, hiMNs, and astrocytes were live-stained with rhodamine-conjugated a-BTX (Invitrogen, 1 :10,000) for 1 hour at 37°C. 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.

Immunocytochemistry

[0373] Cells were processed for immunocytochemistry as previously described. Antibodies used in this work were listed in Table 5. Nuclei were counterstained with Hoechst 33342 or DAPI. Images were obtained with a NIKON A1 R confocal microscope, a Cytation3 imaging reader, or an EVOS fluorescence microscope. Confocal images were used for quantification of subcellular distribution of RANGAP1 , RAN, TDP-43, or FUS, as well as the number of RANGAP1+ foci and NMJ+ myotubes. Fluorescence intensity was quantified as previously described with minor modifications. In brief, ImageJ with a plugin of Bio-Formats was used to measure total fluorescence intensity separately in the soma and the nucleus. Cytoplasmic intensity was obtained by subtracting the intensity in the nucleus from that in the soma. Subcellular distribution was then represented by the nuclear/cytoplasmic ratio.

Table 5: Antibodies used in the examples

Gene overexpression or knockdown in hiMNs

[0374] 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. After neuronal induction, 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.

[0375] Doxycycline (Dox) inducible system was used for shRNA-mediated knockdowns. To determine knockdown efficiency, 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. For knockdowns in hiMNs, the lentiviruses FUW-M2rtTA and TRE3G-miRE-shRNAwere combined with the reprogramming factors. Cells were replated onto astrocyte-coated and Matrigel-treated coverslips. Dox (0.5 pg/ml) was added daily for the first week after replating and weekly thereafter. Hit3 (10 pM) was added twice in the first week after replating, then once per week. At the indicated time-points, cells were fixed for immunocytochemistry. Images of single confocal plane across the center of the nucleus were obtained on the NIKON A1 confocal microscope under a 100x objective with a pinhole setting at 1.2 under 488 nm laser. Because of the complexity of neuron-astrocyte co-cultures, neuronal nucleus and soma were manually defined by using the ImageJ program. 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.

Co-immunoprecipitation

[0376] Co-immunoprecipitation experiments were carried out as previously described with modifications. Briefly, cells were harvested and lysed in five volumes of lysis buffer (10 mM Tris-HCI, 150 mM NaCI, 1% NP-40, 1% Triton X-100, 1 mM dithiothreitol, 1% protein inhibitors (Pierce), 1x PhosSTOP (Roche), pH 7.5) for 20 min with rotations at 4°C. Cell lysates were cleared by centrifugation at 21 ,000 g for 30 min. Equal amounts of proteins were incubated overnight with the anti-FLAG M2 (Sigma M8823) or anti-HA (Pierce #88836) magnetic beads at 4°C. 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). 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-HCI buffer (pH 3.0). The elutes were balanced by adding 10 pl solution consisting of 0.5 M Tris-HCI, 1.5 M NaCI, pH 7.4.

Western blotting

[0377] Cells were collected in lysis buffer (50 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.01% NaN3, protease inhibitors (Pierce), and Phos-STOP (Roche). Protein concentration was determined by the Bradford assay. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking in 5% nonfat milk in PBST for 1 h at RT, membranes were incubated overnight with primary antibodies at 4 °C. 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.

Proximity-labeling proteomics

[0378] 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. Samples were injected onto a 75 pm i.d., 75-cm long EasySpray column (Thermo) and eluted with a gradient from 0-28% buffer B over 90 min. Buffer A contained 2% (v/v) AON and 0.1 % formic acid in water, and buffer B contained 80% (v/v) AON, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.8 kV and an ion transfer tube temperature of 275°C. 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.

Quantitative RT-PCR (qRT-PCR)

[0379] 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.

In vitro kinase assay

[0380] 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. Cell lysates were added with 1% Triton X-100, incubated for 15 min on ice, and cleared by centrifugation at 21 ,000 g for 15 min at 4 °C. Supernatant was transferred to a new tube and incubated overnight with glutathione magnetic beads at 4°C. After washing four times with ice-cold PBS, bead-bound proteins were eluted with buffer composed of 100 mM Tris, 300 mM NaCI, 20 mM glutathione, pH 8.0. Glutathione was removed by multiple times of exchanging buffer and concentration. Concentration of purified proteins was measured with Nanodrop. HA-HGK or HA-HGKmt was expressed in HEK293T cells after transient transfections. Two days post transfection, cells were collected in 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).

Pharmacokinetics

[0381] CD1 mice (21 females for Hit3 and 10 males for MAP4Ki) were injected IP with 10 mg/kg Hit3 (formulated as 5% DMSO, 5% Pharmasolve, 10% Tween 80, and 90% of 50 mM Citrate Buffer pH 4.6) or MAP4Ki (formulated in ddH2O). At the indicated timepoints after dosing, 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. Tissues were weighed and homogenized in a 4-fold volume of 1xPBS (4 x weight of brain in g = vol PBS in ml; total homogenate volume (in ml) = 5 X weight of tissue). 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.

MAP4Ki treatments of SOD1^G93A mice

[0382] MAP4Ki (100 mM stock soultuion in DMSO) was diluted into 200 pM in saline. 2% DMSO in saline was prepared as vehicle. SOD1^G93A female mice were randomly assigned to two groups (n = 12-13/group) at 40 days of age. Mice were anesthetized with isoflurane and injected intrathecally with 0.5 pl vehicle or MAP4Ki/g body weight.

Body weight and rotarod test

[0383] 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.

Survival curve

[0384] Disease progression was monitored by body weight, hind limb weakness and paralysis. The end stage was determined as the time when the mouse was unable to right itself within 15 s when placed on its back.

Immunohistochemistry

[0385] 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.

The Traumatic Brain injury model/CCI

[0386] The controlled cortical impact (CCI) model was employed as previously described. For the present study, 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 preparation and administration

[0387] All the 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. 72 hours post infection, cells were collected by cell scraper and centrifuged at 2000g rpm for 10 minutes at 4°C. The AAV virus in the supernatant medium precipitated with PEG8K (polyethylene glycol 8,000) at 4°C for overnight, then concentrated by centrifugation, the white pellet was resuspended in GC buffer (1 mM Tris-HCI, pH 7.6, 15mM NaCI, 1 mM MgCI2). The cell pellet also was resuspended in GC buffer, lysis the cell the via Bioruptor sonication and concentrated by centrifugation. Mix the medium/PEG supernatant and cell lysate together, Benzonase was added to the mixture which was then incubated in a 37°C water bath for 45 minutes. The mixed supernatant was added over gradient of 15%, 25%, 40% and 58% iodixanol solution and centrifuged using AH-629 rotor, spun at 29,000 rpm for 4 hours. The viral solution was washed with 1X PBS three times and concentrated with 100K PES concentrator (PierceTM, Thermo Scientific). Aliquots were stored at -80°C until use. 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). For AAV-PHP.eB virus, 8-10 ul of AAV-PHP.eB virus(2.0X10¹² 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. For AAV5s and AAV9s, a total of 2 stereotaxic intracerebral injections (1X10¹³GC/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. At each distance, 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.

Drug administration

[0388] PF06260933 (10mpk. b.i.d.) was delivered into mice at predetermined time points after CCI injury by intraperitoneal injection. K02288(20pM stock solution, 8-1 Opl) was delivered into mice daily following CCI via intrathecal injection for 7 days. The control group of mice were administrated with 0.9% Saline.

Tissue preparation and immunohistochemistry

[0389] 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. The following primary antibodies were used: mouse 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). Appropriate 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.

Behavior tests

[0390] All behavior experiments were conducted in a separate institution in a randomized and blinded fashion. Animals were randomized into groups and treated with letter-coded viruses.

[0391] 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.

[0392] The tail suspension test: 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.

[0393] 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.

[0394] 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.

[0395] 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.

In vivo BiolD2 and Affinity Purification for MS [0396] Adult Wild-type C57BL/6J mice were intracranially infused with the AAV-BiolD2 probe viruses predominately into the cortex, then the mice received daily 24 mg/kg biotin intraperitoneal injection for consecutive 7 days. After biotin injection, the cortexes were quickly dissected and homogenized (25mM HEPES pH 7.5, 150 mM NaCI, 1 mM EDTA, 1% NP-40, freshly added with Protease and Phosphatase Inhibitor Cocktails). Western blot analysis validated the approach for in vivo biotinylation efficiency. Biotinylated protein purification was performed according to previously established protocols. Briefly, A total of three purification rounds were performed independently. 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.

Proximity-labeling proteomics

[0397] 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. Briefly, 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. STRING analysis of association networks of proteins was carried out with the online software ( ttps://stri g-db.or /) and the following settings: high confidence (0.700), 1^st shell with query proteins only and no 2^nd shell, and hided disconnected nodes in the network.

Co-immunoprecipitation and western blotting

[0398] Co-immunoprecipitation and western blotting experiments were carried out essentially as described previously. Briefly, cells were harvested and lysed with lysis buffer (50 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1% Triton X-100, 1 mM EDTA, protein inhibitors and PhosSTOP was freshly added) for 20 mins. Cell lysates were centrifugated at 14,000 g for 15 mins. Equal amounts of proteins were incubated overnight with magnetic beads in the cold room. The beads then washed for 3 times with the lysis buffer. Bead-bound proteins were eluted with 1 *SDS loading buffer. Then elutes were used for the western blot. The following are primary antibodies were used for western blot: FLAG (1 :5000; Proteintech, 20543-1-AP), HA (1 :5000; Biolegend, MMS-101 P), pThr (1 :1000; Cell signaling technology, 9386), ACTB (1 :10000; Proteintech, HRP-66009), b-Catenin(1 :3000; Cell signaling technology, 9386), p- JNK(1 :2000; Cell signaling technology, 9251 ), JNK(1 :3000; Selleckchem, A5005). All the HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch.

Dual Luciferase Assays

[0399] HEK293T cells or HepG2 cells were seeded in 24 well plate at 1X10⁵ cells/well in 0.5 ml culture medium, after 12-16h, cells were transfected with MAP4Ks expression or shRNA plasmid using transfection reagent. 24h later, TOPFIash/pRL-SV40P vectors (DNA ratio 10= 1 ) were transfected into the cells. After an additional 24 h, change the media including with Wnt3A. After 4h, dual luciferase assays were done with the Promega Dual Luciferase Assay System (Madison, Wl, USA) according to manufacturer’s protocols. 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.

Statistical analysis

[0400] Data are presented as mean ± SEM. Statistical significance of differences between groups was determined using a two-tailed unpaired student’s t-test. Histological data was analyzed by one-way ANOVA and Tukey’s post hoc test. Lifespan were analyzed by Kaplan- Meier survival curve and log-rank Mantel-Cox test. One-way ANOVA followed by the Tukey’s multiple comparison post hoc test were applied for group analysis of behavioral data. Significant differences are indicated by *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001 . All statistical tests were performed using Prism software (version 8.0, GraphPad).

Example 1. A compound screening platform with ALS-hiMNs.

[0401] Using ALS-hiMNs, prior pilot screen had identified the GSK3B inhibitor kenpaullone (Ken) as a chemical compound with neuroprotective effect. This cell-based platform was then modified for screening additional chemical compounds. Since reprogrammed cells were initially smaller in size and slower to attach to the plastic plates than the non-reprogrammed fibroblasts, a replating step and passing through a 20-pm strainer enriched highly pure (> 85%, based on the co-expressed GFP) ALS-hiMNs for cell viability assays (FIG. 1A). 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.

[0402] 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. Most GSK3 inhibitors (Hit1, 4, 5, 6, and 9) could promote neuronal survival but unanticipatedly inhibited neurite outgrowth (FIG. 11, FIG. 2A- 2B). In contrast, the 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.

[0403] The above 15 chemicals were further examined through a tertiary screen, which was conducted with ALS-hiMNs cocultured with primary astrocytes from wildtype mice. Cells were treated with these chemicals at their optimal dosage and were quantified by automated imaging of GFP+ hiMNs through a time-course (FIG. 1J). Unexpectedly, some chemicals no longer exhibited protections of ALS-hiMNs, indicating that this coculture system was very useful to exclude false-positives (FIG. 1 J). The targets of some of those chemicals showed neuronal protections known to be associated with ALS, such as GSK3 (Ken, Hit1 , 4, 5, 6, and 9) 22, 23 and TGFp/BMP (Hit3). Of note, several other TGFp/BMP inhibitors, such as A83, AC (LDN-214117), LDN-193189 (ALKil) and LDN-212854 (ALKi2), were less effective (FIG. 1D-1H, FIG. 1J). Overall, 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

[0404] Using the neuron-glia coculture system, Hit3, along with Ken and two TGFp/BMP inhibitors (A83 and ALKi), was examined on hiMNs derived from fibroblasts of healthy control patients (NL) or patients with mutations on FUS, SOD1 or C9ORF72 (FIG. 3A; Table 1). Hit3 treatments dosage-dependently improved survival of all these hiMNs (FIG. 3A), while the control TGFp/BMP inhibitor either had no effect (A83) or was toxic (ALKi). Hit3 outperformed Ken, with the latter showing toxicity at higher concentrations (FIG. 3B).

[0405] It was previously showed that ALS-hiMNs exhibited poor survival but also smaller soma and less complex neurites, all of which could be much improved by Ken. Such morphological improvements of ALS-hiMNs could also be observed for cells treated with Hit3 (FIG. 3C). Compared to the vehicle controls, Hit3 robustly increased neurite complexity via promoting outgrowth and branching of neuronal processes when examined at 52 days. Additionally, Hit3-treated ALS-hiMNs showed larger somas, comparable to those treated with Ken (FIG. 3D).

[0406] To further examine the functional effect of Hit3, a co-culture system was employed including ALS-hiMNs and primary mouse skeletal myotubes on top of primary astrocytes. NMJs were detected by a-BTX staining, whereas hiMNs and skeletal muscles were revealed by staining of SYN1 and MHC, respectively (FIG. 3E-3F). Under the vehicle control condition, both FUS-hiMNs and SOD1-hiMNs rarely formed NMJs, consistent with our previous observation. Remarkably, Hit3-treated ALS-hiMNs could robustly form NMJs on the cocultured myotubes, comparable to those treated with the positive control Ken (FIG. 3E-3F). Together, these data indicated that Hit3 could remarkably rescue both the morphological and functional deficits of ALS-hiMNs.

Example 3: MAP4Ks are major targets of Hit3 for protecting ALS-hiMNs

[0407] 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.

[0408] In addition to ALK inhibition, kinome-wide analysis showed that Hit3 also potently inhibited HGK (MAP4K4), MINK1 (MAP4K6) and TNIK (MAP4K7). These three MAP4Ks 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). To examine a potential role of MAP4Ks in ALS-hiMNs, another chemical inhibitor, PF6260933 (designated as MAP4Ki), an HGK-specific inhibitor but also potently inhibiting MINK1 and TNIK, was tested. Interestingly, 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.

[0409] A genetic approach was undertaken by knocking down each of the three MAP4Ks. 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.

[0410] To further examine the role of MAP4Ks, a dominant-negative approach was undertaken through ectopic expression of kinase-dead mutants, including HGK-K54R (HGKmt), MINK1-K54R (MINKI mt) and TNIK-K54R (TNIKmt). These mutants and their wild type controls were introduced during reprogramming of patient fibroblasts to ALS-hiMNs (FIG. 5D-5E). Compared to the empty virus control, wild type kinases showed toxicity during the early reprogramming process; however, they had not much effect once the reprogrammed ALS-hiMNs were replated onto astrocytes (FIG. 5E). Interestingly, the kinase-dead MINKI mt or TNIKmt improved survival of ALS-hiMNs (FIG. 5E). Further analysis showed that MINKI mt dosage-dependently enhanced survival of ALS-hiMNs, with an effect at the highest dosage comparable to those treated with Hit3 (FIG. 5F-5G). In contrast, the HGKmt was less effective at all the examined dosages. In comparison to Hit3, examination of MINKI mt on additional ALS-hiMNs including those derived from patients with mutations on FUS, SOD1 , TDP43, and C9ORF72 was undertaken. MINKI mt and Hit3 showed comparable effect on survival of all these ALS-hiMNs, although the latter was slightly more effective potentially due to more efficient inhibition of MAP4Ks (FIG. 5H-5I). 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. Mechanistically, 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; HDAC6, 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. Altogether, these results indicated that Hit3-mediated inhibition of MAP4Ks improved survival of ALS-hiMNs.

Example 4: CNH domain of MAP4Ks mimics the effect of Hit3

[0411] MINK1 consists of three highly conserved domains: a kinase domain, an intermediate domain, and a citron homology domain (CNH). To map out the functional domain for MINKI mt-mediated neuroprotection, a series of HA-tagged truncations of MINKI mt were constructed (FIG. 6A). Expression of these constructs in ALS-hiMNs was confirmed by western blots (FIG. 6B). When examined at 1 week or 3 weeks post replating and compared to the empty virus controls, truncation mutants lacking the CNH domain had no effect (1-320, 1-959, and 296-959; FIG. 6C-6G). In contrast, 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

[0412] To determine how inhibition of MAP4Ks regulates hiMNs, the role of p38 and JNK were examined since they were reported to be the downstream signaling targets and shown to be involved in neuronal degeneration. However, their inhibition through a panel of small molecules appeared to be either non-effective or toxic to the survival of ALS-hiMNs (FIG. 7A- 7B), suggesting that neither p38 nor JNK mediated the effect of MAP4Ks in ALS-hiMNs.

[0413] An unbiased approach to identify MAP4K-associated targets was undertaken through BiolD2-mediated protein proximity labeling. ALS-hiMNs were transduced with lentivirus expressing either BiolD2-MINK1mt or MINKI mt alone and treated with biotin. Biotinylated proteins were efficiently pulled down by streptavidin Dynabeads (FIG. 8A). Two batches of samples were analyzed by mass spectrometry. With a cutoff of 2.5-fold enrichment, 443 proteins were common to these two sets of data (FIG. 8B), including those known MINK1 interactors such as NCK2, STRN, STRN3, STRN4, HGK, TANC1 , and TRIM25. These 443 proteins were subjected to KEGG pathway analysis. The top 5 pathways were Proteasome, Ribosome, Pathogenic E. coli Infection, RNA Transport, and Aminoacyl-tRNA Biosynthesis (FIG. 8C-8D). Dysfunctions of proteasome, ribosome, RNA transport, and tRNA biogenesis are known to be associated with neurodegeneration including ALS. Several tubulins are included in the Pathogenic E. coli Infection pathway, which involves in microtubule destabilization. Among these tubulins, TUBA4A mutations were recently identified in familial ALS. Similarly, several nuclear pore proteins in the RNA Transport pathway, including RANBP2, RANGAP1 , and NUP214, are also closely involved in ALS. Such data further implicated MAP4Ks in the regulation of this type of neurodegeneration.

[0414] A few top candidate interactors were examined through co-immunoprecipitation and western blotting. Both strong (PRKAR2A and DVL3) and weak (PCBP3, PSMD1 , CRYAB) interactions could be identified (FIG. 8D), consistent with the principle of BiolD2-mediated protein proximity labeling that identifies both interacting proteins and those in the vicinity. Coimmunoprecipitation assays also showed that MINK1 interacted with endogenous RANGAP1 and RAN (FIG. 8F). Domain mapping of MINK1 indicated that both its intermediate domain (aa296-959) and CNH domain (aa866-1312) could mediate interactions with RANGAP1 or RAN (FIG. 8F). Functionally, 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

[0415] The above data from proteomics prompted to examine the role of Hit3 on subcellular distribution of nucleopore proteins and those involved in ALS. hiMNs from five ALS patients and three healthy controls were compared. RANGAPI-containing cytoplasmic foci could be observed in all these hiMNs; however, quantifications showed that the Nuc/Cyt ratio of RANGAP1 was significantly lower in ALS-hiMNs than in the control hiMNs (FIG. 9A-9C). Interestingly, Hit3 treatments greatly improved the Nuc/Cyt ratio of RANGAP1 in all these hiMNs, with largely disappeared cytoplasmic RANGAPI-containing foci (FIG. 9A-9C). Hit3 also markedly improved the Nuc/Cyt ratio of RAN, despite a lack of difference between NL- and ALS-hiMNs under the untreated condition (FIG. 9A-9B, FIG. 9D).

[0416] It has been previously shown that FUS protein is mislocalized in the cytoplasm of ALS-hiMNs with mutated FUS gene. Hit3 treatments significantly improved its nuclear localization, indicated by the increased total nuclear protein and the Nuc/Cyt ratio of FUS (FIG. 9E-9F). A lower Nuc/Cyt ratio of TDP-43 was also observed in ALS-hiMNs when compared to NL-hiMNs (FIG. 9G-9I). Hit3 treatments similarly enhanced nuclear localization of TDP-43 in all these samples (FIG. 9I).

[0417] To confirm a role of MAP4Ks in these above functions of Hit3, shRNA-mediated knockdown approach was undertaken. Knockdown efficiency of HGK, MINK1 , or TNIK was individually validated through qRT-PCR (FIG. 10A). These shRNAs and a control shRNA were then examined in ALS-hiMNs. A mixture of shRNAs against all three MAP4Ks was most efficient in improving the Nuc/Cyt ratio of RANGAP1 , with a result comparable to those Hit3- treated ALS-hiMNs (FIG. 10B). This mixture of shRNAs also significantly increased the Nuc/Cyt ratio of RAN or TDP-43 (FIG. 10C). All together, these results indicated that Hit3, through inhibition of MAP4Ks, significantly enhanced nuclear localization of proteins controlling nucleopore function and those involved in ALS.

Example 7: MAP4Ks regulate RANGAP1 distribution through a HDAC6-TUBA4A axis

[0418] Previous studies showed that the 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). Unexpectedly, neither HGK nor MINK1 induced phosphorylation changes of RAN GAP 1 (FIG. 11 A).

[0419] It was previously shown that microtubule-dependent transport of AL-localized pore complexes regulated biogenesis of nuclear pores. Interestingly, proximity-labeling proteomics identified many tubulins that are associated with MAP4Ks (FIG. 8D). Among these, TUBA4A is especially interesting due to its genetic association with familial ALS. Coimmunoprecipitation assays confirmed that MAP4Ks and TUBA4A were within the same protein complex (FIG. 11B). Through an shRNA approach, it was found that downregulation of TUBA4A significantly reduced nuclear localization of RANGAP1 in hiMNs (FIG. 12A-12C). Once again, however, MAP4K-induced phosphorylation of TUBA4Awas failed to be detected, indicating alternative regulations of TUBA4A by MAP4Ks (FIG. 11C-11F).

[0420] 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).

[0421] Tubulin acetylation is governed by two opposing enzymes: 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. Knockdown of HDAC6 significantly was found to enhance the levels of ac- TUBA4A in the somas of hiMNs (FIG. 12G-12H, FIG. 12J, FIG. 13A). Accordingly, HDAC6 knockdown markedly increased RANGAP1 localization to the nucleus (FIG. 121, FIG. 12K), indicating an inverse correlation of HDAC6 activity and RANGAP1 nuclear/nuclear membrane distribution. HDAC6 knockdown also promoted nuclear localization of TDP-43 (FIG. 13B-C).

[0422] 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). Together, 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. These results are consistent with previous reports demonstrating that the alpha-tubulin deacetylase activity of HDAC6 was stimulated through phosphorylation by multiple kinases.

[0423] To further determine the functional relationship of MAP4Ks and HDAC6, 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). Collectively, 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

[0424] To examine a potential role of MAP4K inhibition in vivo, the SOD1^G93A mouse model of ALS was used. Pharmacokinetics showed that the MAP4K inhibitor PF-6260933 (MAP4Ki) exhibited higher concentrations than Hit3 in both the plasma and the central nervous system after intraperitoneal injections (Table 7, Table 8, FIG. 14A-14B). As such, the MAP4Ki was used for subsequent in vivo studies. To further increase drug accessibility to the nervous system, daily intrathecal injections were performed starting at 40 days of age and until the end stage. When compared to the vehicle control, MAP4Ki had no significant effects on either body weights or performances on rotarod tests (FIG. 14C-14D). Very interestingly, Kaplan-Meier survival curves showed that MAP4Ki significantly though modestly prolonged the median life span of SOD1G93A mice (FIG. 14E; median survival: vehicle = 129 d, MAP4Ki = 139 d; x2 = 8.975, ***p = 0.0027, log-rank Mantel-Cox test).

Table 7: Concentrations of MAP4K inhibitor PF-6260933 (MAP4Ki) after IP injection

Table 8: Concentrations of Hit3 (K02288) after IP injection

[0425] The effect of MAP4Ki on motor neurons was examined through immunohistochemistry by using another cohort of mice at about 126 days of age, a time point around the median survival of vehicle-treated mice. A dramatic reduction of CHAT+ motor neurons was observed in the ventral horns of SOD1G93A mice when compared to their wildtype counterparts (FIG. 14F-14G; group effect: F(2, 11 ) = 47.61 , ****p < 0.0001 , one-way ANOVA), consistent with known pathological features of this ALS mouse model. Importantly, more CHAT+ motor neurons were preserved in mice treated with MAP4Ki than the vehicle control (FIG. 14F-14G; *p = 0.0206, one-way ANOVA and Tukey’s post hoc test).

[0426] RANGAP1 expression in motor neurons was examined by immunostaining. It was mainly localized in the ring-like nuclear membrane in CHAT+ cells of wildtype mice (FIG. 14H). Such a localization was rarely observed in survived CHAT+ motor neurons of vehicle-treated SOD1 G93A mice. Interestingly, MAP4Ki treatments maintained a significantly larger number of motor neurons with intact RANGAP1 expression (FIG. 14H-14I; group effect: F(2, 7) = 57.46, ****p < 0.0001 ; MAP4Ki vs. Veh: **p = 0.0055; one-way ANOVA and Tukey’s post hoc test). TDP-43 expression in CHAT+ cells was examined in the ventral horn of the lumbar spinal cord. It was predominantly detected in the nucleus in wildtype mice, whereas a large fraction of CHAT+ cells showed cytoplasmic localization of TDP-43 in vehicle-treated SOD1G93A mice (FIG. 14J-14K; group effect: F(2, 9) = 27.82, ***p = 0.0001 , one-way ANOVA). In contrast, a significant number of CHAT+ cells maintained nuclear TDP-43 in SOD1 G93A mice when treated with MAP4Ki (FIG. 14J, I; *p = 0.0452, one-way ANOVA and Tukey’s post hoc test). On the other hand, 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.

Summary of Examples 1-8 [0427] The proof-of-concept high-throughput screens identified a lead compound, Hit3, which significantly promoted survival, growth, and function of aging-relevant ALS-hiMNs. Mechanistically, results from the above-described examples demonstrated that Hit3 mainly functions as an inhibitor of the GCK-IV subfamily of MAP4Ks. It was further shown that the MAP4Ks-HDAC6-TUBA4A pathway regulated the subcellular distribution of RANGAP1 , a key component of the nuclear pore complexes (NPCs), and TDP-43, an RNA/DNA binding protein involved in human ALS. Importantly, MAP4Ki treatment of SOD1^G93A mice preserved motor neurons and improved animal survival.

[0428] The above-described examples apply aging-relevant human neurons in screens for therapeutic small molecules. Unlike iPSC-derived neurons with age-rejuvenation, directly reprogrammed neurons from skin fibroblasts of adult human patients reserve aging-associate features and are therefore advantageous in modeling adult-onset neurodegenerative diseases. However, these aging-relevant human neurons have not been previously utilized for high-throughput chemical screens, partly due to limited neuronal purity and yield. With improved conversion efficiency and simple enrichment procedures, the current disclosure showed the feasibility of employing these neurons in screens for small molecules and pathways that may be therapeutically relevant. Such a screening strategy may be applied to studying other neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease by using directly reprogrammed human neurons.

[0429] The results disclosed herein showed that MAP4Ks were the major targets of the lead compound Hit3. Hit3, formally known as K02288, was initially discovered as a selective inhibitor of the bone morphogenetic protein (BMP) type I receptor kinases. Nonetheless, the examination of multiple additional inhibitors targeting the BMP/TGF-beta pathway failed to show a comparable effect on promoting survival of ALS-hiMNs, suggesting alternative pathways targeted by Hit3 in these neurons. Indeed, kinome-wide analysis clearly showed that Hit3 potently inhibited the GCK-IV family of the STE20 group kinases, including HGK (MAP4K4), MINK1 (MAP4K6) and TNIK (MAP4K7). Accordingly, 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. Interestingly, 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. Thus, the role of MAP4Ks were consistent with previous studies implicating MAP4Ks in neural degeneration. One major difference was that 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. [0430] A key downstream effect of MAP4Ks, that was revealed, was the subcellular localization of RANGAP1. Previously, the JNK signaling pathway was identified as a target of MAP4Ks in controlling degeneration and apoptosis of embryonic or iPSC-derived neurons under stress. However, 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. In contrast, the unbiased proteomics disclosed herein uncovered that 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. Although the co-immunoprecipitation assays indicated that MAP4Ks and RANGAP1 were in the same protein complex, multiple efforts failed to show RANGAP1 being phosphorylated by MAP4Ks, indicating alternative regulation of RANGAP1 subcellular localization.

[0431] 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. Instead of a direct regulation of TUBA4A through phosphorylation, the results indicated that 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.

[0432] 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.

[0433] In summary, by using aging-relevant human neurons chemical screens identified MAP4Ks as potential therapeutic targets for treating neurodegenerative diseases. The studies described above revealed redundancy of highly homologous enzymes and novel molecular mechanisms by which they control cellular function.

Example 9: Traumatic brain injury causes gliosis, neurodegeneration, and tau pathology

[0434] The cellular responses to controlled cortical impact (CCI), a widely employed and highly reproducible mouse model of traumatic brain injury was examined. When the injured cortical area was analyzed at 7 days post injury (dpi) and compared to the sham controls (FIG. 16A), CCI resulted in severe reactive gliosis indicated by dramatic increases of expression of GFAP (astrocytes), NG2 (NG2 glia), and IBA1 (microglia) (FIG. 16B). CCI caused a remarkable upregulation of markers for axonal damage including SMI32 (a monoclonal antibody for detecting dephosphorylated neurofilaments) and amyloid precursor protein (APP) (FIG. 16B). Phosphorylated tau (p-tau) was also examined with the antibodies AT8 (for pS202/T205), AT100 (for pT212/S214), and AT180 (for pT231 ) as biomarkers of TBI. In contrast to the sham controls, 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). Together, these results confirmed that TBI caused robust reactive gliosis, neurodegeneration, and tau pathology.

Example 10: The CNH domain ameliorates brain injury-induced pathology

[0435] To determine whether TBI-induced pathology could be molecularly modulated, the Ste20 family kinases MAP4Ks were focused on due to their broad brain expression and their critical roles in neurodegeneration. Furthermore, the CNH domain of MINK1 could serve as a dominant-negative form blocking MAP4Ks to promote survival and function of human patient- derived neurons. This result raised a possibility of using the CNH domain as a potential gene therapy for neural injury or degeneration. To test this possibility, 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. Adult wild type mice were intrathecally injected with AAVs and subjected to CCI 7 days post virus (dpv) (FIG. 17A-17B). When examined at 7 dpi and compared to the GFP control, the CNH group showed a significant reduction of reactive gliosis, measured by the relative expression of GFAP, NG2, and IBA1 surrounding the cortical injury (FIG. 17C-17D). Injury-induced glial scars, quantified by the volume of GFAP+ area, were also greatly reduced in the CNH group (FIG. 17E-17F). Similarly, CNH expression led to significant reductions on markers of neuron damage (SMI32) and tau pathology (AT8, AT100 and AT180) (FIG. 17G-17H). 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. In summary, these results indicated that 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

[0436] Supported by these above results, whether the CNH domain had any functional impact on animal behaviors after TBI was next examined. The functional impact of CNH on animals was then determined by behaviors after TBI. A cohort of adult wild type mice were injected with AAVs, subjected to severe CCI, and examined by a battery of behavioral tests (FIG. 18A). Since impaired locomotion is a sequela of TBI, animals were evaluated through a time-course with the grid walking test, which quantified foot-faults made by the affected side (contralateral to the injury) when animals were allowed to walk on an elevated wire grid. This type of test is especially useful for models of unilateral TBI. When compared to sham animals at one dpi, both the CNH group and the GFP control exhibited near identical impairments indicating relatively uniform injuries among these mice (FIG. 18B; p = 0.8662). However, the CNH group improved their motor function in a significantly faster pace than the GFP control in subsequent days (FIG. 18B; **p = 0.0082 at 8 dpi and *p = 0.0491 at 21 dpi). Another TBI- induced impairment of brain function is depressive-like behavior, which was monitored by tailsuspension test at 28 dpi (FIG. 18A). When compared to the GFP control, the CNH group spent significantly less time in immobility indicating much-reduced depressive-like behavior (FIG. 18C; *p<0.05 and **p<0.01). Together, these results showed that the CNH domain can improve functional recovery after TBI.

Example 12: The CNH domain exerts its neuroprotective function in neurons

[0437] Since PHP.eB-serotyped AAVs can efficiently transduce both neurons and astrocytes, the cell type in which the CNH domain exerted its function was examined. To target astrocytes, 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. 19A), however, 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.

[0438] To target neurons, the human synapsin I promoter (hSYN1 ) was used to drive gene expression and the AAV9 capsid to package AAVs (FIG. 19H). Immunohistochemistry confirmed that about 94% of GFP+ cells were detected in NeuN+ neurons in the control AAV9- hSYN1-GFP virus-transduced cortex (FIG.19P). When examined at 7 dpi, neuronal expression of the CNH domain led to a significant reduction of TBI-associated reactive gliosis and tau pathology (FIG. 19J-19I). It also resulted in greatly reduced glial scars and the lesioned cortical size (FIG. 19K-19N). Together, these results indicated that the CNH domain exerted its neuroprotective function predominantly in neurons but not in astrocytes after brain injury.

Example 13: The CNH domain alleviates tau pathology in a mouse model of Alzheimer’s disease

[0439] Brain injury is a well-known risk factor for Alzheimer’s disease (AD), of which 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. Consistent with the above findings in TBI, a significant reduction of AT8+ staining was observed in both the cortex and the hippocampal CA1 region of mice injected with the CNH virus (FIG. 20A-20B; ***p = 0.0003 for the cortex and *p = 0.0333 for the CA1 ). The staining intensity of AT100 was also similarly reduced in the CNH group, although it did not reach a statistically significant level in the CA1 possibly due to its weaker signal at this early age (FIG. 20C-20D; *p = 0.0198 for the cortex and p = 0.0639 for the CA1 ). Tauopathy-induced neuroinflammation was determined by IBA1 staining for microglia, which showed that the CNH domain caused a significant reduction of the number of IBA1+ cells in the cortex of rTg4510 mice (FIG. 20E-20F).

[0440] Since AD is an age-dependent neurodegenerative disease, whether the CNH domain exerted a protective function in older rTg4510 mice was also determined. 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. Together, 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

[0441] 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). However, four more months later rTg4510 mice with the CNH virus performed markedly better than those injected with the GFP control, evidenced by the reduced total traveled distances and limb clasping scores (FIG. 20G-20H). Upon completion of behavioral tests, brain tissues were collected for western blot analyses of tauopathy by using the antibodies AT8, AT100, and AT180. Consistent with the previous results, the CNH domain reduced the accumulation of p-tau to different levels in the cortex and the hippocampus of rTg4510 mice (FIG. 20I-20J). Together, these results implicated that the CNH domain slowed behavioral deficits of AD mice potentially through reducing tauopathy.

Example 15: Pharmacological inhibition of MAP4Ks ameliorates brain injury-induced pathology

[0442] The results indicated that CNH worked as a dominant-negative form to block MAP4Ks for improved survival and functions of human patient-derived neurons. To determine whether blocking MAP4Ks could exert a similar protective effect after TBI, a pharmacological approach was undertaken. 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). When compared to vehicle-treated mice, K02288 treatments markedly lowered reactive gliosis indicated by substantial reductions of the markers for astrocytes (GFAP+), NG2 glia (NG2+), and reactive microglia/macrophages (CD45+) after CCI (FIG. 21C-21D; p < 0.0001 for GFAP, p = 0.0192 for NG2, p = 0.0089 for CD45). 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. 21 E- 21 F; p = 0.0193 for SMI32, p = 0.0118 for APP, p = 0.0351 for AT8). Consequently, K02288- treated mice exhibited much reduced scar volume and lesion size (FIG. 21G-21 J; p = 0.0092 and p = 0.0486, respectively). Together, these results indicated that pharmacological inhibition of MAP4Ks was neuroprotective, consistent with the functions of AAV-CNH in vivo.

Example 16: CNH self-associates and binds to MAP4Ks

[0443] To understand how CNH works, its association with MAP4Ks was examined since the results indicated that it may function as a dominant-negative form in improving survival of human patient-derived neurons. Co-immunoprecipitation assays showed that the CNH domain of MINK1 could efficiently bind to the GCK sub-family of MAP4Ks, including HGK (MAP4K4), MINK1 (MAP4K6) and TNIK (MAP4K7) (FIG. 22A). To map the protein regions mediating such interactions, a series of MINK1 deletion mutants were analyzed, including the kinase domain (1-320 aa), the intermediate domain (296-959 aa), and the CNH domain (960- 1312 aa) (FIG. 22B). While no interaction was found with the kinase domain, the CNH domain could self-associate as well as bind to the intermediate domain (FIG. 22B-22C).

Example 17: Proteomic analysis of the CNH-associated network

[0444] To further tease out the CNH-regulated networks, an unbiased, BiolD2-based proximity-labeling proteomics was conducted in the adult mouse brain. An adeno-associated virus (AAV) expressing BiolD2-CNH as the bait was injected into the adult mouse cortex (FIG. 22D). One week later, mice were treated with biotin for another week and processed for brain lysates. The biotinylated proteins were then affinity-purified with streptavidin beads and identified by mass spectrometry. Among these, 907 proteins showed more than 10-fold enrichments for association with the CNH domain versus the PBS control (Table S1 ). It was observed many of the endogenous MAP4Ks were isolated, including MAP4K2 (GCK), MAK4K4 (HGK), MAP4K6 (MINK1 ) and 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). On the other hand, 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). Major functional enrichments in the network include proteins involved in Wnt signaling (red colored, p = 2.15e-14), Alzheimer’s disease (blue colored, p = 7.39e-16), Proteasome (yellow colored, p = 2.64e-06), and Oxidative phosphorylation (Green colored, p = 3.54e-07).

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. 23B) and that the PDZ domain-containing region of DVL3 may mediate such interactions (FIG. 23C). The PDZ domain may also mediate the interactions of CNH with many other PDZ domain-containing proteins such as DLG4 and NOS1 (FIG. 22G).

[0446] From the western blots and when compared to the GFP controls, it was noticed that overexpression of MAP4Ks caused an apparent mobility shift of the DVL3 protein band (FIG. 23D). Such a phenomenon was not observed when we overexpressed the kinase-dead mutants of MAP4Ks, including HGK-K54R, MINK1-K54R, and TNIK-K54R (FIG. 23D). These results indicate that MAP4Ks might regulate DVL3 in a kinase activity-dependent manner. We therefore examined phosphorylation of DVL3 by using an antibody specific to phosphothreonine (pThr). Ectopic expression of either HGK, MINK1 or TNIK could enhance the phosphorylation of a DVL3 fragment, and such phosphorylation could be largely abrogated by treatment with the lambda protein phosphatase (APP) (FIG. 23E).

[0447] Protein phosphorylation with Phos-tag gels were also examined and found that overexpression of HGK markedly upper-shifted the DVL3 band (FIG. 23F). Interestingly, such phosphorylation could be largely blocked by the co-expressed CNH, confirming a dominantnegative effect of CNH on the activity of MAP4Ks (FIG. 23F). To identify the MAP4K-induced phosphorylation sites on DVL3, we conducted phospho-proteomics of immunoprecipitated DVL3 in the absence or presence of HGK. Such an analysis confirmed and further showed that DVL3 could be phosphorylated at many sites by HGK (FIG. 23G), consistent with the many upper-shifted bands detected in the Phos-tag gels (FIG. 23F). Initial mutagenesis analyses, nonetheless, failed to pinpoint the critical phosphorylation site(s) in DVL3.

Example 19: MAP4K4s negatively regulate Wnt/B-catenin signaling

[0448] Since DVLs are a principal component of the Wnt signaling pathway, we determined the effect of MAP4Ks on its activation measured by the stabilization of the downstream target p-catenin (CTTNB1 ). Indeed, overexpression of HGK, MINK1 or TNIK, but not their kinase-dead mutants (KM), reduced the protein levels of endogenous CTTNB1 (FIG. 24A) with orwithout the treatment of okadaic acid, a PP2A inhibitor that may activate MAP4Ks. Such a result was further supported by using the p-catenin-responsive TOPFIash luciferase reporter, which was inhibited when MAP4Ks were ectopically expressed (FIG. 24B). Conversely, shRNA-mediated downregulation of endogenous HGK, MINK1 or TNIK enhanced the expression level of CTTNB1 (FIG. 24C) and increased the TOPflash reporter activity (FIG. 24D). Stabilization of CTTNB1 could also be observed when cells were treated with the MAP4K inhibitor, K02288 (FIG. 24E). Notably, phosphorylation of JNK, an indicator of the non- canonical Wnt signaling pathway, was not significantly altered by MAP4K knockdown (FIG. 24C) or chemical inhibition (FIG. 24E).

[0449] Since 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).

Summary of Examples 9-19

[0450] After injury, 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.

[0451] In addition to AAV-PHP.eB-GFP-CNH, 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. However, when the function of AAV5-GFAP-CNH was examined in astrocytes, no effect was found of CNH on TBI as has been observed in neurons. These results suggested that CNH domain biased to play neuroprotective role in neurons which may depend on the MAP4Ks’s localization and activity after TBI. [0452] Moreover, drugging MAP4Ks with K02288 or PF-06260933 protected neuron from damage and reduce tau pathology after TBI. Although the therapeutic potential of MAP4Ks inhibitors has been discussed in various disease contexts, it is important to recognize the different effects of K02288 and PF-06260933. The disclosed study has shown that both K02288 and PF-06260933 reduced the neuron damage and tau pathology significantly but the former also inhibited gliosis. Although the possibility that systemic MAP4Ks inhibition with K02288, may also ameliorate neuron damage and tau pathology via its effects on additional cell types such as microglia, macrophages and vascular cells cannot be ruled out, there is still a compelling support for a common mechanism with AAV gene-therapy that inhibition of MAP4Ks reduced tau phosphorylation and protect neuron from damage after brain injury.

[0453] To understand the mechanisms by which MAP4Ks promoted 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.

[0454] It is interested to note, that the validation studies not only revealed several universal MAP4K-associated proteins, such as STRN4, HSP70, and CTTN, but also discovered DVL3 as a specific binding partner for MAP4Ks, which is an essential component of the Wnt signaling cascades under various biological processes. Although MAP4Ks signaling have connected with Wnt signaling pathways in previous studies, their architectural interplay and functional regulation have not been elucidated. Interestingly, the disclosed CoIP assay analysis uncovered MAP4Ks involved in the Wnt signaling pathway regulation via directly interacted with DVL3. In the present study, it was shown that MAP4Ks phosphorylates DVL3 246-496 at the region between PDZ domain and DEP domain. The MAP4Ks-based regulatory mechanism is very similar to the one involved in the Prickle-1 mediated phosphorylation of Dvl3 which negatively regulates Wnt/p-catenin signaling.

[0455] Growing evidence suggested that deregulation of the Wnt canonical pathway could be involved in the pathogenesis of neurodegenerative diseases, including synaptic decline and Tau Tangles. Interestingly, it was found that overexpression DVL3 reduced tau phosphorylation significantly but has less effect on the expression level or activity of GSK3p, which is a ubiquitously expressed serine/threonine kinase that plays a key role in the pathogenesis of Alzheimer's disease (AD) and Wnt signaling pathway. These reports suggested the possibility that MAP4Ks kinases might be involved in tau pathology more directly via inhibiting the DVL3-suppressing functions on Wnt signaling pathway. Although it is unclear how DVL3 reduces tau phosphorylation in mammalian cell. [0456] In summary, the data generated from the methods of gene silencing and pharmacological inhibition identified 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.

[0457] The results showed that AAV-mediated delivery of CNH could ameliorate in vivo histological and behavioral pathology, implicating a therapeutic potential of CNH or its associated pathways in treating traumatic brain injury or associated neurodegeneration.

Claims

CLAIMS What is claimed is:

1 . 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 citron homology domain (CNH) of MAP4K4, MAP4K6, or MAP4K7, a CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7, or any combination thereof.

2. 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 CNH of MAP4K4, MAP4K6, MAP4K7, a CNH- containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.

3. 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, MAP4K7, a CNH- containing truncation of MAP4K4, MAP4K6, MAP4K7, or any combination thereof.

4. The method of any one of the preceding claims, wherein 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.

5. 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.

6. The composition of claim 5, wherein the CNH or CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from SEQ ID NOs:3-6.

7. 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.

8. The isolated nucleic acid sequence of claim 7, wherein the CNH or CNH-containing truncation of MAP4K4, MAP4K6, or MAP4K7 comprises a sequence selected from SEQ ID NOs:3-6.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. The method of any one of claims 1 , 2, 9, 10, 14, or 15, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia.

20. The method of any one of claim 1 , 2, 9, 10, 14, or 15 wherein the brain injury comprises traumatic brain injury or stroke.

21. The method of any one of claims 1-4, 9-11 , 14-16, 19, or 20, wherein the inhibitor is administered as a recombinant adeno-associated virus (rAAV) vector encoding the said inhibitor.

22. The method of claim 21, wherein the rAAV vector comprises AAV9 or AAV-PHP.eB capsid.

23. The method of any one of claims 21 or 22, wherein the AAV vector comprises a human synapsin I promoter (hSYN1).

24. The method of any one of claims 1-4, 9-11 , 14-16, or 19-23, wherein the inhibitor is expressed ectopically in neuron or motor neuron cells of the subject.

25. The method of any one of claims 1-4, 9-11 , 14-16, or 19-24, wherein 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.

26. The method of any one of claims 1-4, 9-11 , 14-16, or 19-25 wherein the inhibitor is administered parenterally.

27. The method of any one of claims 1-4, 9-11 , 14-16, or 19-25, wherein the inhibitor is administered intrathecally.

28. The composition of any one of claims 5, 6, 12, or 17, wherein the vector is a rAAV.

29. The composition of claim 28, wherein the vector is a rAAV comprising AAV9 or AAV- PHP.eB capsid.

30. The composition of any one of claims 28-29, wherein the vector comprises sequence encoding a human synapsin I promoter (hSYN1) operably linked to the inhibitor.

31. The composition of any one of claims 5, 6, 12, 17, or 28-30, wherein the composition is administered parenterally.

32. The composition of any one of claims 5, 6, 12, 17, or 28-30, wherein the composition is administered intrathecally.

33. A host cell transduced with the nucleic acid sequence according to any one of claims 7, 8, 13, or 18.

34. 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 of any one of claims 7, 8, 13, or 18, to the subject in need thereof.

35. 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 the isolated nucleic acid of any one of claims 7, 8, 13, or 18, to the subject in need thereof.

36. 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 isolated nucleic acid of any one of claims 7, 8, 13, or 18, to the subject in need thereof.

37. The method of any one of claims 7, 8, 13, 18, or 33-36, wherein the isolated nucleic acid is administered parenterally.

38. The method of any one of the claims 7, 8, 13, 18, or 33-36, wherein the isolated nucleic acid is administered intrathecally.

39. A cell based platform for screening compounds with neuroprotective effect, the cell based platform comprising a host cell of claim 33.

40. 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.

41. 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.

42. 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.

43. The method of any one of claims 40-42, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, or Friedreich ataxia.

44. The method of any one of claims 40-42 wherein the brain injury comprises traumatic brain injury or stroke.