Modified carboxyl terminus of heat shock 70-interacting proteins and uses thereof

Abstract

This invention relates to a modified Carboxyl Terminus of Heat Shock Cognate 70-interacting protein (CHIP) or functional fragment thereof wherein the modified CHIP or functional fragment thereof comprises at least two amino acid modifications that reduce the activity of a U-Box domain and a tetratricopeptide repeat (TPR) domain. The modified CHIP or functional fragment thereof may be used to treat neurological diseases and diseases associated with tau protein aggregates and tau protein phosphorylation, such as neurodegenerative diseases, neuroinflammation, traumatic brain injury (TBI), and cerebrovascular disease (CVD).

Description

MODIFIED CARBOXYL TERMINUS OF HEAT SHOCK 70-INTERACTINGPROTEINS AND USES THEREOFRELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 554,583, filed February 16, 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5470-965WO_ST26.xml, 20,772 bytes in size, generated on February 14, 2025, and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant Nos. AG061188 and AG072826 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0004] This invention relates to a modified Carboxyl Terminus of Heat Shock Cognate 70- interacting protein (CHIP) or functional fragment thereof wherein the modified CHIP or functional fragment thereof comprises at least two amino acid modifications that reduce the activity of a U-Box domain and a tetratricopeptide repeat (TPR) domain. The modified CHIP or functional fragment thereof may be used to treat neurological diseases and diseases associated with tau protein aggregates and tau protein phosphorylation, such as neurodegenerative diseases, neuroinflammation, traumatic brain injury (TBI), and cerebrovascular disease (CVD).BACKGROUND OF THE INVENTION

[0005] The prevalence of Alzheimer’s disease (AD) in our population is staggering, affecting over 5 million people nationwide, a looming epidemic. 1 in 9 people over the age of 65 are diagnosed with AD, and this is more prevalent in those who experience traumatic brain injuries,post-traumatic stress disorder, and / or depression. An enormous amount of effort has been spent on the neuropathology present in these patient’s brains that defines their dementia, which has caused many research groups to focus primarily on the tau protein that accumulates in AD, for the following reasons: (1) tau forms the hallmark pathology in all AD and related dementia patients; (2) strong evidence indicates that tau aggregation drives synaptic dysfunction, neurodegeneration, and disease progression; and (3) depleting tau leads to cognitive improvements both in neurons and AD model mice, and it is being targeted in current human clinical trials with antisense oligos (ASO) and immunotherapies among other modalities. These data collectively point towards the targeting of tau proteins as an appealing approach to either prevent or treat AD dementia patients.

[0006] Tau is normally a microtubule (MT)-associated protein (MAP) that regulates the vast MT network in neurons. Given the enormous and complex neuronal cytoskeleton, tau is estimated to represent 0.025-0.25% of total protein in the brain. Adding further complexity, the dynamic nature of neuronal cytoskeleton requires tau-MT binding to be in constant flux. Therefore, large pools of tau are constantly evaluated, or “triaged”, for either refolding or degradation to avoid tau accumulation and the emergence of AD pathology. If this fails, tau becomes hyper-phosphorylated and aggregates into neurofibrillary tangles (NFTs) to form a defining neuropathological lesion in AD that correlates with neurodegeneration and cognitive decline.

[0007] Little is known about early events that detect and target tau aggregation, a time frame in which chaperone-dependent refolding acts in a compensatory manner to refold or degrade tau. Obviously, targeting tau in an earlier window of pathogenesis is highly desirable, particularly in asymptomatic individuals that are years to decades from overt symptoms. Thus, there is an urgent need to understand how tau aggregates are initially detected and targeted so therapies in this time-window can be developed.SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, on the discovery that a modified CHIP or a functional fragment thereof that comprises reduced activity of a U-Box domain a TPR domain, and / or a coiled-coil domain is able to reduce and / or prevent tau protein aggregation and pathologies associated therewith.

[0009] Accordingly, one aspect of the invention relates to a modified CHIP or a functional fragment thereof, wherein the modified CHIP or functional fragment thereof comprises at least two amino acid modifications (e.g., 2, 3, 4, 5, or more amino acid modifications) that reducethe activity of a U-Box domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%), reduce the activity of a TPR domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%), and / or reduce the activity of a coiled-coil domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%). In some embodiments, the modified CHIP or functional fragment thereof is devoid of E3 ubiquitin ligase activity, heat shock protein 70 (Hsp70) chaperone activity, heat shock cognate 70 (Hsc70) chaperone activity, and / or heat-shock protein 90 (Hsp90) chaperone activity.

[0010] Another aspect of the invention relates to a nucleic acid molecule encoding any one of the modified CHIPs or functional fragments thereof of the invention.

[0011] An additional aspect of the invention relates to a vector comprising any one of the nucleic acid molecules of the invention.

[0012] A further aspect of the invention relates to a pharmaceutical composition comprising any one of the modified CHIPs or functional fragments thereof, nucleic acid molecules, or vectors of the invention, and a pharmaceutically acceptable carrier.

[0013] Another aspect of the invention relates to a method of preventing and / or reducing tau protein aggregation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein aggregation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0014] Another aspect of the invention relates to a method of inhibiting formation of protein inclusions comprising tau protein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby inhibiting formation of protein inclusions comprising tau protein in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0015] Another aspect of the invention relates to a method of preventing and / or reducing tau protein phosphorylation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein phosphorylation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0016] Another aspect of the invention relates to a method of reducing neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby reducing neuroinflammation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0017] Another aspect of the invention relates to a method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a neurodegenerative disease in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0018] Another aspect of the invention relates to a method of treating or preventing a traumatic brain injury (TBI) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a TBI in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0019] Another aspect of the invention relates to a method of treating or preventing a cerebrovascular disease (CVD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a CVD in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0020] Another aspect of the invention relates to a use of CHIP for preventing and / or reducing tau protein aggregation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein aggregation in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for preventing and / or reducing tau protein aggregation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein aggregation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0021] Another aspect of the invention relates to a use of CHIP for inhibiting formation of protein inclusions comprising tau protein in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby inhibiting formation of protein inclusions comprising tau protein in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for inhibiting formation of protein inclusions comprising tau protein in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby inhibiting formation of protein inclusions comprising tau protein in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0022] Another aspect of the invention relates to a use of CHIP for preventing and / or reducing tau protein phosphorylation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein phosphorylation in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for preventing and / or reducing tau protein phosphorylation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein phosphorylation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0023] Another aspect of the invention relates to a use of CHIP for reducing neuroinflammation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby reducing neuroinflammation in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for reducing neuroinflammation in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby reducing neuroinflammation in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0024] Another aspect of the invention relates to a use of CHIP for treating or preventing a neurodegenerative disease in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a neurodegenerative disease in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for treating or preventing a neurodegenerative disease in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a neurodegenerative disease in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0025] Another aspect of the invention relates to a use of CHIP for treating or preventing a TBI in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a TBI in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for treating or preventing a TBI in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a TBI in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0026] Another aspect of the invention relates to a use of CHIP for treating or preventing a CVD in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a CVD in the subject. Another aspect of the invention relates to a use of CHIP in the manufacture of a medicament for treating or preventing a CVD in a subject in need thereof, the use comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a CVD in the subject. In some embodiments, the CHIP is the modified CHIP or functional fragment thereof of the invention.

[0027] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1 is an immunoblot showing the control and Braak Stage VI AD samples (N=5) as analyzed with the indicated antibodies. Quantitative measurements demonstrate that CHIP levels are reduced in AD brain by 75% (N=5, p-value < 0.001 by Student’s t-test).

[0029] Fig. 2 is a schematic of CHIP variants and the associated domains.

[0030] Fig. 3 is a western blot of primary neurons that were transduced with the indicated CHIP variants followed by immunoprecipitation. The boxes labeled "A" indicate the loss of chaperone activity in the CHIP variants with a TPR mutation, and the box labeled "B" indicates the expected molecular weight of the CHIP variant with the U-box deletion.

[0031] Fig. 4, panel A is a schematic of the P301L and S320F familial mutations (PL-SF) in the human tau gene (mutation sites are indicated by arrows (SEQ ID NOS: 5 and 6)). Fig. 4, panel B is a series of 20X confocal microscopy images of PL-SF pathology in primary mouse neurons with staining for of AT8 (phosphorylated tau), MCI (pre-tangle tau), and Thioflavin S (aggregated tau). Insets show human AD brain as a comparison (Moloney et al 2021).

[0032] Fig. 5, panel A is a western blot of WT or PL-SF tau expressed with each CHIP variant in CHIP KO neurons. AT8, p-262 (p-tau), and total tau were probed in both RIPA soluble and SDS soluble (aggregated) cellular fractions. The box labeled "A" indicates a >90% reduction of PL-SF tau phosphorylation due to a CHIP variant of the present invention named CHIP- neuroprotection (CHIP-NP); the box labeled "B" indicates a reduction in the SDS-insoluble tau aggregates due to CHIP-NP; the box labeled "C" indicates that WT tau does not aggregate; the box labeled "D" indicates that CHIP-NP also reduced soluble PL-SF tau by -50%; the box labeled "E" indicates that none of the CHIP -variants altered WT tau. Fig. 5, panel B is a bargraph showing the quantification of AT8 in PLSF RIP A, WT RIP A, and PL-SF in the SDS soluble fraction (N=3; *= significant by one-way ANOVA compared to control lentivirus).

[0033] Fig. 6, panel A is a series of representative 20X confocal microscopy images of mouse primary neurons after 11 days of culture in vitro after dissection (DIV11) co-expressing PL-SF and either WT CHIP or CHIP NP. Top row: AT 8 (GFP) and Total Tau (RFP). Bottom row: MCI (GFP) and CHIP-myc (RFP). White arrows indicate regions of accumulation of pathogenic tau species p-Tau (AT8) and conformational tau (MCI). Fig. 6, panel B shows a box and dot plot indicating the quantification comparing control lentivirus and CHIP-NP transfection efficiency as indicated by the number of neurons positive for AT8 or MCI (n=15). *=significant by t-test.

[0034] Fig. 7 is a series of microscopy images showing the tau pathology in PS 19 transgenic mice. Panel A shows the injection of short tau seeds (MT-repeat region) for one month into the hippocampus of 4-month PS 19 mice led to accelerated phospho-tau (AT8) pathology at 5 months. Panel B shows a comparison of the tau pathology between 12-month-old control and late-stage non-injected PS 19 mice.

[0035] Fig. 8, panel A is a series of immunocytochemistry images stained for CHIP-myc show the AAV-PHP.eb (AAV9) expression in primary mouse neurons. Fig. 8, panel B is an immunoblot showing the CHIP-myc molecular weights for all four indicated CHIP mutants in AAV-PHP.eb (AAV9).

[0036] Fig. 9, panel A is a series of microscopy images showing the AAV-PHP.eB peripheral delivery in mice via control GFP-expressing Synapsin-driven AAV-PHP.eB. Fig. 9, panel B is a series of microscopy images showing the GFP expression in cortex and hippocampus that was specific to neurons.

[0037] Fig. 10 is a series of bar graphs showing the behavior analysis in PS19 tau Tg mice. Consecutive days of water maze testing in 8-month-old WT vs. PS 19 mice shows significant learning deficits at 2 and 3 days by latency to escape (left panel) and quadrant preference (middle panel, # - within genotype comparison, * - comparison to WT). N=8 animals per genotype, * p <0.05, *** p <0.0001 by repeated measures ANOVA. There were no differences in general motility (swimming) at 8-months old between WT and PS 19 mice (right panel).

[0038] Fig. 11, panel A is a gel image showing WT or aggregate-prone (P301L) tau proteins that were aggregated in vitro using heparin to generate pelleted (P) tau fibrils and a supernatant (S) fraction. Fig. 11, panel B is a bar graph showing tau fibrils were incubated with thioflavin- T (ThT) and monitored by fluorescence (** indicates p-value < 0.01). Fig. 11, panel C is acircular dichroism graph showing the fibrillar 0-structure of P301L tau as assessed at 0-2 hr. Fig. 11, panel D is a transmission electron microscopy (TEM) image of P301L tau aggregates.

[0039] Fig. 12 is a series of confocal microscopy images showing staining for tau WT tau (Ms Tau via mouse-tau specific T49 antibody) or imaging of GFP-tagged PL-SF tau (PLSF-GFP), and the colocalization of the two (Merge). PL-SF tau recruits endogenous WT mouse tau to inclusions in primary neurons.

[0040] Fig. 13 is a series of graphs showing the neuronal connectivity and synchrony in primary mouse neurons. The neurons were pre-recorded at DIV18 then subsequently lentiviral infected with vehicle lentivirus or PLSF. Neurons were recorded daily until DIV48. Quantification of number of network bursts (left- a measure of connectivity) and area under normalized cross-correlation (right- a measure of synchrony) reveal drastic deficits in PL-SF neurons after DIV30.

[0041] Fig. 14 is an interactome map showing the CEUP / STUB 1 interactome revealed by mass- spectrometry confirms known CHIP interactors, potential tau interactors, and other new candidates. CHIP is in a circle labeled "A", known CHIP interactors are in circles labeled "B", MAPT is in a circle labeled "C", and known tau-interacting proteins are in circles labeled "D".

[0042] Fig. 15 is a schematic showing known ataxia-related variants in the coiled-coiled (C- C) domain of CHIP (STUB1) that are associated with severe dementia. Missense mutations associated with impaired cognition in humans are underlined.

[0043] Fig. 16, panel A is a series of immunocytochemistry images showing the neuronal expression of CHIP-myc across different CHIP variants. Fig. 16, panel B is an immunoblot showing the molecular weight for the different CHIP variants.

[0044] Fig. 17, panel A is a series of immunocytochemistry images showing the accumulation of Huntingtinin-99Q (HTT-99Q) aggregates after 1% Triton fixation after treatment with different CHIP variants. Fig. 17, panel B is a bar graph showing the cell viability as measured by CellTiterBlue in HTT-99Q cells after treatment with different CHIP variants.

[0045] Fig. 18, panel A is a series of immunocytochemistry images showing the reduced expression of TDP-43 protein levels after treatment with different CHIP variants; the magnified inset boxes show the diffuse TDP-43 signal as a result of CHIP K30A / AU-box treatment. Fig. 18, panel B is two immunoblot images showing the soluble (RIP A) or aggregated (SDS) levels of TDP-43 protein after treatment with different CHIP variants.

[0046] Fig. 19, panel A is a graph showing the in vitro fluorescent activity of Thioflavin T over time for samples comprising PL-SF, and PL-SF with a CHIP variant comprising the aminoacids as indicated in the parenthetical. For example, CHIP (1-303) comprises amino acids 1 through 303 of e.g., wild-type CHIP. Fig. 19, panel B is a graph showing the in vitro fluorescent activity of Thioflavin T over time for samples comprising PL-SF; PL-SF with wildtype CHIP (CHIP WT); PL-SF with the U-box domain of wild-type CHIP (CHIP U-box); PL- SF with CHIP AU-box; or PL-SF, CHIP AU-box, and the stand-alone U-box domain of wildtype CHIP (CHIP AU-box + Ubox). Fig. 19, panel C is a graph showing fraction of PL-SF tau that aggregates in vitro with a Congo Red assay when combined with wild-type or AU-box CHIP. Significance was analyzed using a 1-way ANOVA test.DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0049] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0050] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying, and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g. , Sambrook et al. , Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0051] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0052] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0053] 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.Definitions

[0054] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0055] Also 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 when interpreted in the alternative (“or”).

[0056] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0057] 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 characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0058] The term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C- terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total numberof additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g. , chaperone and / or ubiquitin ligase activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0059] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) refers to a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term “carrier” encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0060] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g, an increase) or inhibition (e.g, a decrease) in the specified level or activity.

[0061] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0062] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0063] A “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of Alzheimer’s disease, improved memory, prevention of seizures, improved attention span, or increase in survival time). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0064] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / ortherapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human. In some embodiments a human subject is a neonate, child, adult, or geriatric subject. In some embodiments a human subject is at least 40, 50, 60, 70, 80, or 90 years old.

[0065] A “subject in need” of the methods of the invention can be any subject known or suspected of having or having increased risk of developing a tauopathy, neurodegenerative disease, neuroinflammation, a traumatic brain injury (TBI), cerebrovascular disease (CVD), and / or other disease / disorder to which administering a CHIP protein of the present invention may provide beneficial health effects.

[0066] “ Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome, or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, and / or reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have beenidentified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0067] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intracerebroventricular, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, retro-orbital, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subj ect an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0068] “Prevent” or “preventing” or “prevention” refer to prevention or delay of the onset of the disorder and / or a decrease in the severity of the disorder in a subject relative to the severity that would develop in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of tauopathy in a subject. The prevention can also be partial, such that the occurrence or severity of tauopathy in a subject is less than that which would have occurred without the present invention.

[0069] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g. , chemically synthesized) DNA or RNA and chimeras of RNA and DNA.The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or singlestranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.

[0070] An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.

[0071] The term “isolated” can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and / or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be foundin the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0072] An “isolated cell” refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier of this invention. Thus, an isolated cell can be delivered to and / or introduced into a subject. In some embodiments, an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.

[0073] The term “fragment,” as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and / or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0074] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. The term “functional fragment,” when applied to a polypeptide, will be understood to mean a fragment of a reference or wild-type polypeptide where the functional fragment has at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, or 95%) of the biological activity of the reference or wild-type polypeptide sequence.

[0075] A “vector” is any nucleic acid molecule for the cloning of and / or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may beattached to allow for replication of the attached nucleotide sequence. A “replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and / or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and / or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and / or selection of host cells that incorporate and express the proteins encoded by the marker. A “recombinant” vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences. In some embodiments, a vector of the present invention may be a neurotropic vector.

[0076] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and / or adenovirus vectors. Non-viral vectors include, but are not limited to, plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acidprotein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0077] As used herein, the term "adeno-associated virus" (AAV) includes but is not limited to, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Recently, a number of putative new AAV serotypes and clades have been identified (see, e.g.,Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383; and Table 1). One example of an AAV9 serotype variant that may be used in the present invention is AAV9 variant PHP.eB (AAV-PHP.eB).

[0078] Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g. , Wu et al. , J. Biol. Chem. 267963 ( 1992); Wu et al. , J. Biol. Chem. 263'.14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0079] In some embodiments, a polynucleotide of this invention can be delivered to a cell in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al., Proc. Natl. Acad. Sci. USA 84 1413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 55:8027 (1988); and Ulmer et al., Science 259'.Y1 5 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95 / 18863 and WO96 / 17823, and in U.S. Patent No. 5,459,127. The use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey, et al., 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

[0080] In various embodiments, other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95 / 21931), peptides derived from nucleic acid binding proteins (e.g., WO96 / 25508), and / or a cationic polymer (e.g., WO95 / 21931).

[0081] It is also possible to introduce a vector in vivo as naked nucleic acid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3 N1 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0082] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0083] A "native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, protein, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a "wild type protein" is a protein that is naturally occurring in or endogenous to a reference organism. A "homologous" nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.

[0084] A “modified” nucleic acid, nucleotide sequence, polypeptide, protein, or amino acid sequence refers to a nucleic acid, nucleotide sequence, polypeptide or amino acid sequence that has one or more modifications or mutations made to its wild-type sequence. The term "mutation" or “modification” refers to point mutations (e.g., missense, or nonsense, or insertions or deletions of single base pairs that result in frame shifts), insertions, deletions, and / or truncations. When the mutation is a substitution of a residue within an amino acid sequence with another residue, or a deletion or insertion of one or more residues within a sequence, the mutations are typically described by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. In some embodiments, a modification to a polypeptide, protein, or amino acid sequence includes a post-translational modification of one or more residues (e.g., phosphorylation, acetylation, amidation, hydroxylation, methylation, acylation, alkylation, glycosylation, biotinylation, pegylation, ubiquitination, SUMOylation, etc.). A protein of the present invention may be modified from its wild-type sequence. Such modifications include, but are not limited to, a substitution of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications); a deletion of at least one amino acid (e.g., a deletion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more amino acids); an insertion of at least one amino acid (e.g., an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more amino acids); the addition of one or more post-translational modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more post-translational modifications); the removal of one or more post-translational modifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more post-translational modifications); and / or any combination thereof.

[0085] A “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. Illustrativefusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to all or a portion of glutathione-S-transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, P-glucuronidase, P-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0086] By the term “express” or “expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.

[0087] As used herein, the term “over-expression” or “over-expressing” refers to increased levels of a polypeptide being produced and / or increased time of expression (e.g., constitutively expressed) compared to a wild-type cell.

[0088] As used herein, the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5’ and 3’ untranslated regions). A gene may be “isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid.

[0089] “Introducing” in the context of a cell or organism means presenting the nucleic acid molecule to the organism and / or cell in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events. Thus, the term “transformation” as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient.

[0090] “ Transient transformation” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

[0091] By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell, it is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0092] “ Stable transformation” or “stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein includes the nuclear and mitochondrial genome, and therefore includes integration of the nucleic acid into, for example, the mitochondrial genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a mini chromosome.

[0093] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0094] Embodiments of the invention are directed to expression cassettes designed to express the nucleic acids of the present invention. As used herein, “expression cassette” means a nucleic acid molecule having at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable interaction with the nucleotide sequences for the polypeptides of the invention are provided in expression cassettes for expression in an organism or cell.

[0095] As used herein, the term “promoter” refers to a region of a nucleotide sequence that incorporates the necessary signals for the efficient expression of a coding sequence. This may include sequences to which an RNA polymerase binds, but is not limited to such sequencesand can include regions to which other regulatory proteins bind together with regions involved in the control of protein translation and can also include coding sequences.

[0096] Furthermore, a “promoter” of this invention is a promoter capable of initiating transcription in a cell of an organism. Such promoters include those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner, as these various types of promoters are known in the art.

[0097] For purposes of the invention, the regulatory regions (z.e., promoters, transcriptional regulatory regions, and translational termination regions) can be native / analogous to the organism or cell and / or the regulatory regions can be native / analogous to the other regulatory regions. Alternatively, the regulatory regions may be heterologous to the organism or cell and / or to each other (z.e., the regulatory regions). Thus, for example, a promoter can be heterologous when it is operably linked to a polynucleotide from a species different from the species from which the polynucleotide was derived. Alternatively, a promoter can also be heterologous to a selected nucleotide sequence if the promoter is from the same / analogous species from which the polynucleotide is derived, but one or both (z.e., promoter and polynucleotide) are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0098] The choice of promoters to be used depends upon several factors, including, but not limited to, cell- or tissue-specific expression, desired expression level, efficiency, inducibility and selectability. For example, where expression in a specific tissue or organ is desired, a tissue-specific promoter (e.g., a neuron-specific or neuron-preferred promoter) can be used, such as camkll and synapsin for delivery to neurons. In contrast, where expression in response to a stimulus is desired, an inducible promoter can be used. Where continuous expression is desired throughout the cells of an organism, a constitutive promoter can be used. It is a routine matter for one of skill in the art to modulate the expression of a nucleotide sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence.

[0099] In addition to the promoters described above, the expression cassette also can include other regulatory sequences. As used herein, “regulatory sequences” means nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, butare not limited to, enhancers, introns, translation leader sequences and polyadenylation signal sequences.

[0100] The expression cassette also can optionally include a transcriptional and / or translational termination region (z.e., termination region) that is functional in the organism. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host, or may be derived from another source (z.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host, or any combination thereof).

[0101] A signal sequence can be operably linked to nucleic acids of the present invention to direct the nucleotide sequence into a cellular compartment or to be secreted from the cell. In this manner, the expression cassette will comprise a nucleotide sequence encoding the monoclonal antibodies or an antigen binding fragment thereof operably linked to a nucleic acid sequence for the signal sequence. The signal sequence may be operably linked at the N- or C- terminus of the monoclonal antibodies or an antigen binding fragment thereof.

[0102] Regardless of the type of regulatory sequence(s) used, they can be operably linked to the nucleotide sequence of the polypeptides of the invention. As used herein, “operably linked” means that elements of a nucleic acid construct such as an expression cassette are configured so as to perform their usual function. Thus, regulatory or control sequences (e.g., promoters) operably linked to a nucleotide sequence of interest are capable of effecting expression of the nucleotide sequence of interest. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence. A nucleotide sequence of the present invention (z.e., encoding a polypeptide of the invention) can be operably linked to a regulatory sequence, thereby allowing its expression in a cell and / or subject.

[0103] The expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed organism or cell. As used herein, “selectable marker” means a nucleic acid that when expressed imparts a distinct phenotype to the organism or cell expressing the marker and thus allows such transformed organisms or cells to be distinguished from those that do not have the marker. Such a nucleic acid may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for bychemical means, such as by using a selective agent (e.g. , an antibiotic or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0104] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W ., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0105] As used herein, the term “substantially identical” or “corresponding to” means that two nucleic acid sequences have at least 60%, 70%, 80% or 90% sequence identity. In some embodiments, the two nucleic acid sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

[0106] An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.

[0107] As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.

[0108] Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA availableas part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.). Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0109] The percent of sequence identity can be determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFif ’ performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482-489, 1981, Smith etal., Nucleic Acids Res. 11 :2205-2220, 1983).

[0110] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., (Applied Math 48: 1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.Modified CHIP

[0111] Chaperone proteins, which play critical roles in tau refolding and degradation, have been classically thought to play a neuroprotective role in Alzheimer's disease and other neurodegenerative diseases. One of the most common chaperones implicated in tau pathology is CHIP, which plays a central role in orchestrating protein quality control. Prior studies pointed to either CHIP’S E3 ubiquitin ligase activity as a mediator of substrate degradation, or to its intrinsic chaperone function independent of its E3 activity. However, the inventors show herethe surprising discovery that expression of a CHIP protein without E3 ubiquitin ligase or chaperone activity can potently combat tau pathology in a highly aggressive, tangle-producing variant of tau which models late-stage AD in neuronal cell culture. Accordingly, one aspect of the invention relates to a modified CHIP or functional fragment thereof, wherein the modified CHIP or functional fragment thereof comprises at least two amino acid modifications (e.g., 2, 3, 4, 5, or more amino acid modifications) that reduce the activity of a U-Box domain (e.g., amino acid residues 231 to 303 based on SEQ ID NO: 1) by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%) reduce the activity of a TPR domain (e.g., amino acid residues 27 to 127 based on SEQ ID NO: 1) by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%), and / or reduce the activity of a coiled-coil domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%).

[0112] In some embodiments, the at least two amino acid modifications comprise at least one amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the U-Box domain, at least one amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the TPR domain, at least 1 amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the coiled-coil domain, and / or at least one amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the N-terminus domain (e.g., the region immediately N-terminal ("upstream") of the TPR domain, e.g., amino acids 1- 26 numbered according to SEQ ID NO: 1). In some embodiments, the at least two amino acid modifications comprise one or more substitutions, deletions, insertions, or any combination thereof. In some embodiments, the at least one amino acid modification in the U-Box domain comprises a deletion of about 10 to about 150 amino acid residues (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residues) from the C-terminus relative to a wild-type CHIP. In some embodiments, the at least one amino acid modification in the U-Box domain comprises a deletion of about 73, 79, 110, or about 117 amino acid residues from the C-terminus relative to a wild-type CHIP. In some embodiments, the deletion of about 10 to about 100 amino acid residues from the C-terminus is a deletion of an E3 ligase domain (e.g., the U-Box domain). In some embodiments, the modified CHIP or functional fragment thereof is devoid of E3 ubiquitin ligase activity (e.g., the CHIP or functional fragment thereof comprises a U-Box domain deletion). In some embodiments, the at least two amino acid modifications comprise a deletion of about 1 to about 100 amino acid residues (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues) from the N-terminus relative to a wild-type CHIP. In some embodiments, the at least two amino acid modifications comprise a deletion of about 10 or about 19 amino acid residues from the N-terminus relative to a wildtype CHIP. In some embodiments, the at least two amino acid modifications comprise at least one amino acid modification in the TPR domain. In some embodiments, the at least two amino acid modifications comprise a lysine to alanine substitution at residue 30 (K30A) (numbering based on SEQ ID NO: 1) in the TPR domain. In some embodiments, the at least two amino acid modifications comprise a glutamine to alanine substitution at residue 127 (Q127A) and an arginine to alanine substitution at residue 128 (R128A) (numbering based on SEQ ID NO: 1). In some embodiments, the at least two amino acid modifications comprise a phenylalanine to alanine substitution at residue 131 (F131A) (numbering based on SEQ ID NO: 1).

[0113] One of skill in the art would understand that the amino acid modifications as described herein can be combined with one another to achieve the modified CHIP as described herein. In some embodiments, the combination of at least two amino acid modifications has a synergistic effect on altering (e.g., reducing) the activity of the U-box domain, the TPR domain, and / or the coiled-coil domain. In some embodiments, the combination of at least two amino acid modifications have a synergistic effect on altering the anti-tau activity (e.g., preventing and / or reducing tau protein aggregation, inhibiting formation of protein inclusions comprising tau protein, preventing and / or reducing tau protein phosphorylation, reducing neuroinflammation, treating and / or preventing a neurodegenerative disease, treating and / or preventing a TBI, and / or treating and / or preventing a CVD) of the modified CHIP protein in comparison to a wild-type CHIP protein that is devoid of the at least two amino acid modifications.

[0114] In some embodiments, the at least two amino acid modifications comprise a deletion of about 10 to about 150 amino acid residues from the C-terminus relative to a wild-type CHIP, a deletion of about 1 to about 100 amino acid residues from the N-terminus relative to a wildtype CHIP, a K30A mutation according to SEQ ID NO: 1, a QI 27 A mutation according to SEQ ID NO: 1, an R128A mutation according to SEQ ID NO: 1, an F131A mutation according to SEQ ID NO: 1, or any combination thereof.

[0115] In some embodiments, the wild-type TPR domain of the wild-type CHIP is responsible for the Hsp70 chaperone activity, Hsc70 chaperone activity, and / or Hsp90 chaperone activity. In some embodiments, the at least one amino acid modification in the TPR domain reduces the Hsp70 chaperone activity, Hsc70 chaperone activity, and / or Hsp90 chaperone activity of the CHIP or functional fragment thereof. In some embodiments, the modified CHIP or functional fragment thereof is devoid of heat shock protein 70 (Hsp70) chaperone activity, heat shock cognate 70 (Hsc70) chaperone activity, and / or heat-shock protein 90 (Hsp90) chaperoneactivity. In some embodiments, the at least two amino acid modifications comprise a deletion of about 10 to about 100 amino acid residues from the C-terminus relative to a wild-type CHIP and K30A mutation (numbering based on SEQ ID NO: 1). In some embodiments, the modified CHIP or functional fragment thereof comprises an amino acid sequence at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:2. In some embodiments, the modified CHIP or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO:2. In some embodiments, the at least one amino acid modification in the U-box domain comprises a histidine to glutamine substitution at residue 260 (H260Q) (numbering based on SEQ ID NO: 1). In some embodiments, the modified CHIP is a human CHIP. In some embodiments, the at least one amino acid modification in the U-box domain comprises a C232G, S236T, E238X, M240T, R241W, P243L, C244Y, T246M, H260E, H260Q, V264G, P269A, P274A, L275V, L275D, or a I294F mutation (numbering based on SEQ ID NO: 1). In some embodiments, the at least one amino acid modification in the TPR domain comprises a E28K, G33S, R35S, F37L, I53L, P57L, N65S, A67T, A79T, A79D, or a L123 V mutation (numbering based on SEQ ID NO: 1).

[0116] In some embodiments, the modified CHIP or functional fragment thereof binds to a tau protein and / or tau protein aggregates. In some embodiments, the modified CHIP or functional fragment thereof dephosphorylates the tau protein. In some embodiments, the modified CHIP or functional fragment thereof prevents aggregation of the tau protein.SEQ ID NO:1 Wild-type human CHIPMKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRN PLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYD EAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIA AERERELEECQRNHEGDEDDSHVRAQQACIEAI<HDI<YMADMDELFSQVDEI<RI<I<R DIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLA MKEVIDAFISENGWVEDYSED ID NO:2 CHIP NPMKGKEEKEGGARLGAGGGSPEKSPSAQELAEQGNRLFVGRKYPEAAACYGRAITRN PLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYD EAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIA AERERELEECQRNHEGDEDDSHVSEQ ID NO:3 K30A CHIPMKGKEEKEGGARLGAGGGSPEKSPSAQELAEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDYSEQ ID NO:4 AU-Box CHIP 1-230MKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKRDIPDYSEQ ID NO:5 Region of the microtubule-associated protein tau (e.g., Tau) comprising the P301L familial mutationKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSEQ ID NO:6 Region of Tau comprising the S320F familial mutationSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGSEQ ID NO:7 Region of Tau comprising the PL-SF double mutationsKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGSEQ ID NO:8 Full Tau sequence comprising the P301L familial mutationMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRS S AKTLKNRPCL SPKHPTPGS SDPLIQPS SP AVCPEPP S SPK YVS S VTSRTGS SGAKEM KLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVK SKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVD LSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATL ADEVSASLAKQGLSEQ ID NO:9 Full Tau sequence comprising the S320F familial mutationMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTED GSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTP SLEDEAAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQ PSGTGPEDTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVD ESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPD GPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSL GEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTR S S AKTLKNRPCL SPKHPTPGS SDPLIQPS SP AVCPEPP S SPK YVS S VTSRTGS SGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVK SKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVD LSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKI ETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATL ADEVSASLAKQGLSEQ ID NO: 10 Full Tau sequence comprising the PL-SF double mutationsMAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTED GSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTP SLEDEAAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQ PSGTGPEDTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVD ESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPD GPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSL GEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTR S S AKTLKNRPCL SPKHPTPGS SDPLIQPS SP AVCPEPP S SPK YVS S VTSRTGS SGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLSEQ ID NO: 11 AU-Box CHIP 1-224MKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKSEQ ID NO: 12 AU-Box CHIP 1-193MKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDEDDSHVSEQ ID NO: 13 AU-Box CHIP 1-186MKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGSEQ ID NO: 14 AU-Box CHIP 10-224GARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEE CQRNHEGDEDD SHVRAQQ ACIEAKHDK YMADMDELF SQ VDEKRKKSEQ ID NO: 15 AU-Box CHIP 19-224SPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYDEAIANLQRAYSLAKEQRLNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIAAERERELEECQRNHEGDED DSHVRAQQACIEAKHDKYMADMDELFSQVDEKRKKSEQ ID NO: 16 Q127A:R128A CHIPMKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYD EAIANLQRAYSLAKEAALNFGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIA AERERELEECQRNHEGDEDDSHVRAQQACIEAI<HDI<YMADMDELFSQVDEI<RI<I<R DIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDYSEQ ID NO: 17 F131A CHIPMKGKEEKEGGARLGAGGGSPEKSPSAQELKEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCYLKMQQHEQALADCRRALELDGQSVKAHFFLGQCQLEMESYD EAIANLQRAYSLAKEQRLNAGDDIPSALRIAKKKRWNSIEERRIHQESELHSYLSRLIA AERERELEECQRNHEGDEDDSHVRAQQACTEAI<HDI<YMADMDELFSQVDEI<RI<I<R DIPDYLCGKISFELMREPCITPSGITYDRKDIEEHLQRVGHFDPVTRSPLTQEQLIPNLAMKEVIDAFISENGWVEDY

[0117] The amino acid sequence and nucleic acid sequence of human wild-type CHIP is well- known in the art and can be found, e.g., in UniProt Accession No. Q9UNE7, incorporated by reference herein in its entirety. Other CHIP sequences relevant to this invention can be found in Table 1 below.Table 1: CHIP UniProt reference numbers

[0118] A functional fragment of the modified CHIP may comprise one or more amino acid sequences and / or domains that are known to have a specific biological activity. In some embodiments, the functional fragment may comprise the TPR domain of the modified CHIP, e.g., amino acid residues 27 to 127 of SEQ ID NO: 1, which is known to have Hsp70 chaperone activity, Hsc70 chaperone activity, and Hsp90 chaperone activity. In some embodiments, the functional fragment may comprise the coiled-coil domain of the modified CHIP, e.g., amino acid residues 128 to 230 of SEQ ID NO: 1, which is known to function in dimerization of the CHIP. In some embodiments, the functional fragment may comprise the U-box domain of the modified CHIP, e.g., amino acid residues 231 to 303 of SEQ ID NO: 1, which is known to have E3 ubiquitin ligase activity. In some embodiments, the functional fragment comprises the modified TPR domain and the coiled-coil domain. In some embodiments, the functional fragment comprises the modified TPR domain, the coiled-coil domain, and the modified U- box domain.

[0119] The modified CHIP or functional fragment thereof can be produced by any suitable method. In some embodiments, the modified CHIP or functional fragment thereof is produced recombinantly using methods well known in the art and as described herein.

[0120] Another aspect of the invention is a nucleic acid encoding the modified CHIP or functional fragment thereof. In some embodiments, the nucleic acid may be part of a vector, such as a neurotropic vector. In some embodiments, the vector is a non-viral (e.g., a plasmid) or a viral vector (e.g., adeno-associated virus (AAV), retrovirus, lentivirus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, or adenovirus vector). In some embodiments, the AAV vector is a serotype 9 AAV vector (e.g., AAV9). In some embodiments, the AAV9 vector is a PHP.eB variant vector (e.g., AAV-PHP.eB). In some embodiments, the nucleic acid encoding the modified CHIP or functional fragment thereof is operably linked to a promoter, e.g., a constitutive or regulatable promoter. In some embodiments, the promoter is a neuron-specific or neuron preferred promoter. In someembodiments, the vector may be one that expresses the modified CHIP or functional fragment thereof for a limited time, e.g., 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, or 6 months. In some embodiments, the promoter is a regulatable promoter and expression of the modified CHIP or functional fragment thereof protein is regulated to occur for a specific amount of time, e.g., 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, or 6 months.

[0121] Another aspect of the invention is a composition comprising one or more of the modified CHIP or functional fragment thereof, nucleic acid molecules, or vectors of the invention. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0122] In some embodiments, if not present in an amino acid sequence described herein (e.g., a CHIP and / or modified CHIP) and / or encoded in a nucleotide sequence described herein, the amino acid sequence and / or the nucleotide sequence may further include 1, 2, 3, 4, or 5 additional amino acid(s) or corresponding nucleotides such as a methionine at amino acid residue 1 of the amino acid sequence or the corresponding nucleotides and / or 1, 2, 3, or 4 additional amino acid(s) such as glycine or alanine or the corresponding nucleotides, which may aid in expression or enable the construction or cloning as needed for construction of a plasmid or construct or vector. In some embodiments, if present in an amino acid sequence described herein (e.g., a CHIP and / or modified CHIP) and / or encoded in a nucleotide sequence described herein, the amino acid sequence and / or the nucleotide sequence may be devoid of 1, 2, 3, 4, or 5 amino acid(s) present at amino acid residues 1-5 at the N-terminus of the amino acid sequence or corresponding nucleotides such as a methionine at amino acid residue 1 of the amino acid sequence or the corresponding nucleotides and / or 1, 2, 3, or 4 additional amino acid(s) such as glycine or alanine or the corresponding nucleotides, which may aid in expression or enable the construction or cloning as needed for construction of a plasmid or construct or vector.

[0123] In some embodiments, an amino acid sequence as described herein (e.g., a CHIP and / or modified CHIP) and / or encoded in a nucleotide sequence described herein may further comprise a linker and / or a peptide tag. The term "linker" is art-recognized and refers to a chemical group, or a molecule linking two molecules or moi eties, e.g., linking two polypeptides or domains of a fusion protein, such as, for example, a modified CHIP and a peptide tag. A linker may be comprised of a single linking molecule (e.g., a single amino acid) or may comprise more than one linking molecule. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety such as a bivalent organic moiety. In some embodiments, the linker may be an amino acid or it may be a peptide. In some embodiments,the linker is a peptide (e.g., a peptide linker). In some embodiments, the linker has an amino acid sequence comprising SLDLE. A peptide tag (e.g., epitope) useful with this invention may include, but is not limited to, a GCN4 peptide tag (e.g., Sun-Tag), a c-Myc affinity tag, an HA affinity tag, a His affinity tag (e.g., an amino acid sequence comprising four or more histidine residues, e.g., six histidine residues), an S affinity tag, a methionine-His affinity tag, an RGD- His affinity tag, a FLAG octapeptide, a strep tag or strep tag II, a V5 tag, and / or a VSV-G epitope. In some embodiments, a peptide tag may comprise 1 or 2 or more copies of a peptide tag (e.g., repeat unit, multimerized epitope (e.g., tandem repeats)) (e.g., 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 more repeat units.Methods of Use

[0124] Another aspect of the invention is a method of preventing and / or reducing tau protein aggregation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein aggregation. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention.

[0125] Another aspect of the invention is a method of inhibiting formation of protein inclusions comprising tau protein in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby inhibiting formation of protein inclusions comprising tau protein. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention.

[0126] Another aspect of the invention is a method of preventing and / or reducing tau protein phosphorylation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein phosphorylation. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention.

[0127] Another aspect of the invention is a method of reducing neuroinflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby reducing neuroinflammation. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention.

[0128] Another aspect of the invention is a method of treating and / or preventing a neurodegenerative disease in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of CHIP, thereby treating or preventing a neurodegenerative disease. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention. Treating a neurodegenerative disease, as used herein, includes slowing or stopping the progression of the disease or partially or completely reversing the disease. The neurodegenerative disease to be treated may include, but is not limited to, Alzheimer's disease, Parkinson’s disease, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), Down’s syndrome, corticobasal degeneration, Pick’s disease, multisystem atrophy, inclusion body myositis (IBM), cerebral amyloid angiopathy (CAA) (e.g., prion protein CAA), argyrophilic grain disease (AGD), tangle predominant dementia (TPD), or chronic traumatic encephalopathy (CTE). In some embodiments, the neurodegenerative disease to be treated is associated with a change in the tau protein aggregation, the formation of protein inclusions comprising tau protein, the tau protein phosphorylation, and / or neuroinflammation in the subject.

[0129] Another aspect of the invention is a method of treating and / or preventing a TBI in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a TBI. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention. In some embodiments, the TBI is a repetitive TBI.

[0130] Another aspect of the invention is a method of treating and / or preventing a CVD in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a CVD. In some embodiments, the CHIP is one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention. The CVD to be treated may include, but is not limited to, an aneurysm, a vascular malformation (e.g., an arteriovenous malformation or a cerebral cavernous malformation), a fistula (e.g., an arteriovenous fistula or a carotid-cavernous fistula), carotid artery disease (e.g., carotid stenosis), a stroke, a transient ischemic attack (TIA), Moyamoya disease, a hemorrhage (e.g., an intracranial hemorrhage or a subarachnoid hemorrhage), hereditary hemorrhagic telangiectasia, or reversible cerebral vasoconstriction syndrome.

[0131] The methods taught herein can improve a range of physical, mental, and emotional attributes of the treated subject. The subject can show an improvement in one or more symptoms of a tau protein aggregation, protein inclusions comprising tau protein, tau protein phosphorylation, neuroinflammation, a neurodegenerative disease, a TBI, and / or a CVD. Suchimprovements include, but are not limited to, improved physical abilities such as fine motor skills (e.g., writing and typing, grasping small objects, cutting, pointing, etc.), or gross motor skills (e.g., walking, balance, jumping, standing up, throwing); improved sensations such as decreased tingling and / or increased sensitivity in extremities, reduced sensation of muscle weakness or rigidity, and reduced tremors or pain; improved cognitive abilities such as increased alertness, reduced memory loss / improved memory recall, increased cognitive comprehension, improved speech and sleep, improved puzzle-solving abilities, increased focus; and improved behavioral performance such as decreased apathy, depression, agitation, or anxiety, and improved mood and general contentment.

[0132] The methods can generate neuroprotective results when performed in a subject. As used herein, the term “neuroprotective” refers to maintaining or improving existing neurological function in the target neurological organ or tissue (e.g., nerve, spinal cord), or can refer to maintaining or improving the rate or overall amount of neuronal cell death in target neuronal cells. For example, “neuroprotective” can refer to slowing the rate of nerve tissue destruction, deterioration, or malfunction, slowing the rate of neuronal cell death, reducing the rate at which nerve conduction speed slows, etc. In some embodiments, the methods can generate at least 5%, at least 10%, at least 20%, or at least 25% or more neuroprotective improvement, as compared to a control.

[0133] Another aspect of the invention relates to a method of delivering any one of the modified CHIP or functional fragments thereof of the invention, the method comprising administering to the subject one or more of the modified CHIP or functional fragments thereof, nucleic acid molecules, or vectors of the invention, thereby delivering the modified CHIP or functional fragment thereof to the subject, wherein the modified CHIP or functional fragment thereof may be optionally delivered to the brain, spinal cord, and / or muscle tissue of the subject.

[0134] The administering step of any one of the methods described herein can include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten dosages. The administering step can be performed before the subject exhibits disease symptoms (e.g., prophylactically), or during or after disease symptoms occur. The administering step can be performed prior to, concurrent with, or subsequent to administration of other agents to the subject. In some embodiments, the administering step is performed prior to, concurrent with, or subsequent to the administration of one or more additional diagnostic or therapeutic agents. In some embodiments, the methods comprise administering one or more additional modified CHIP or functional fragments thereof, one or more additional nucleic acid molecules, or one or more additional vectors of the invention.

[0135] In some embodiments, the methods comprise administering one or more additional modified CHIP or functional fragments thereof to the brain of the subject.

[0136] In some embodiments, a subsequent administration is provided at least one day after a prior administration, or at least two days, at least three days, at least four days, at least five days, or at least six days after a prior administration. In some embodiments, a subsequent administration is provided at least one week after a prior administration, or at least two weeks, at least three weeks, or at least four weeks after a prior administration. In some embodiments, a subsequent administration is provided at least one month, at least two months, at least three months, at least six months, or at least twelve months after a prior administration.Subjects, Pharmaceutical Formulations, and Modes of Administration

[0137] As a further aspect, the invention provides pharmaceutical formulations and methods of administering the same to achieve any of the therapeutic effects (e.g., preventing and / or reducing tau protein aggregation, inhibiting formation of protein inclusions comprising tau protein, preventing and / or reducing tau protein phosphorylation, reducing neuroinflammation, treating or preventing a neurodegenerative disease, treating or preventing a TBI, and / or treating or preventing a CVD) discussed above. The pharmaceutical formulation may comprise any of the reagents discussed above in a pharmaceutically acceptable carrier.

[0138] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

[0139] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0140] One embodiment of the invention is a composition including a modified CHIP or functional fragment thereof, an isolated polynucleotide sequence encoding the modified CHIP or functional fragment thereof, a plasmid or vector containing the isolated polynucleotide sequence and a suitable carrier, diluent, or excipient, and optionally a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the composition is in a form suitable for parenteral, oral, rectal, systemic, urogenital, topical, intravitreal, intraocular, retro- orbital, otic, intranasal, dermal, sublingual, or buccal administration. In another embodiment, the composition is in a form suitable for delivery to the central nervous system (CNS), e.g., intrathecal delivery, intraparenchymal delivery, intra-ci sterna magna delivery, and / or intracerebroventricular delivery, e.g., intrathecal injection, intraparenchymal injection, intra- cistema magna injection, and / or intracerebroventricular injection.

[0141] The modified CHIP or functional fragment thereof of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (23rd Ed. 2020). In the manufacture of a pharmaceutical formulation according to the invention, the modified CHIP or functional fragment thereof (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is preferably formulated with modified CHIP or functional fragment thereof as a unit-dose formulation, for example, a tablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight of the modified CHIP or functional fragment thereof. One or more modified CHIP or functional fragment thereof can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.

[0142] A further aspect of the invention is a method of treating subjects in vivo, comprising administering to a subject a pharmaceutical composition comprising a modified CHIP or functional fragment thereof of the invention in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the modified CHIP or functional fragment thereof of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering compounds. In some embodiments, administration of the modified CHIP or functional fragment thereof comprises administering a nucleic acid encoding the modified CHIP or functional fragment thereof. In some embodiments, administration of the modified CHIP or functional fragment thereof comprises administering a vector comprising the nucleic acid encoding the modified CHIP or functional fragment thereof.

[0143] Non-limiting examples of formulations of the invention include those suitable for retro- orbital, oral, rectal, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intracranial, intracerebroventricular, intrathecal, intraparenchymal, intra-ci sterna magna, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into a limb, into the brain or spinal cord for delivery to the central nervous system, into the pancreas, or into a tumor or the tissue surrounding a tumor). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular compound which is being used. In an embodiment, administration may be direct delivery to the cerebrospinal fluid (CSF) via intrathecal delivery,or administration utilizing delivery systems that can cross the blood brain barriers, e.g., via AAV vectors such as AAV9. In some embodiments, it may be desirable to deliver the formulation locally to avoid any side effects associated with systemic administration. For example, local administration can be accomplished by direct injection at the desired treatment site, by introduction intravenously at a site near a desired treatment site e.g., into a vessel that feeds a treatment site, or CNS administration with a neuron-specific or neuron-preferred promoter). In some embodiments, the formulation can be delivered locally to ischemic tissue. In certain embodiments, the formulation can be a slow release formulation, e.g., in the form of a slow release depot.

[0144] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.

[0145] For oral administration, the compound can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Compounds can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0146] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[0147] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can containanti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit / dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-inj ection immediately prior to use.

[0148] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising a compound of the invention, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 1 mg to about 10 grams of the compound or salt (e.g., from about 1 mg to about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 750 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, or about 10 g). When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0149] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0150] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0151] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the compound. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol / water and contain from about 0.1 M to about 5 M of the compound (e.g., from about 0.1 M to about 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.75 M, 1 M, 1.25 M, 1.5 M, 1.75 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M, or about 5 M).

[0152] The compound can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the compound, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles comprising the compound can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the compound can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0153] Alternatively, one can administer the compound in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0154] Further, the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.

[0155] The liposomal formulations containing the compounds disclosed herein or salts thereof, can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0156] In the case of water-insoluble compounds, a pharmaceutical composition can be prepared containing the water-insoluble compound, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound. Particularly useful emulsifying agents include phosphatidyl cholines and lecithin.

[0157] The amount of the disclosed compositions administered to a subject will vary from subject to subject, depending on the nature of the disclosed compositions and / or formulations, the species, gender, age, weight and general condition of the subject, the mode of administration, and the like. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the disclosed compositions are those large enough to produce the desired effect (e.g., to reduce protein inclusions or to improve a symptom of a neurodegenerative disease). The dosage should not be so large as to outweigh benefits by causing extensive or severe adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like, although some adverse side effects may be expected. The dosage can be adjusted by the individual clinician in the event of any counterindications. Generally, the disclosed compositions and / or formulations are administered to the subject at a dosage of active component(s) ranging from 0.1 mg / kg body weight to 100 g / kg body weight. In some embodiments, the disclosed compositions and / or formulations are administered to the subject at a dosage of active component(s) ranging from 1 mg / kg to 10 g / kg, from 10 mg / kg to 1 g / kg, from 10 mg / kg to 500 mg / kg, from 10 mg / kg to 100 mg / kg, from 10 mg / kg to 10 mg / kg, from 10 mg / kg to 1 mg / kg, from 10 mg / kg to 500 mg / kg, or from 10 mg / kg to 100 mg / kg body weight. Dosages above or below the range cited above may be administered to the individual subject if desired. The compositions can be administered in any herein disclosed pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0158] Suitable carriers include, but are not limited to, salts, diluents, (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, sorbents, solvents, pH modifying agents, antioxidants, anti-infective agents, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and other components and combinations thereof. Suitable pharmaceutically acceptable carriers are preferably selected from materials which are generally recognized as safe (GRAS) and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

[0159] Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, 23rd ed. 2020, Academic Press. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions. Typically, the components of the formulation are dissolved or suspended in a suitable solvent such as, for example, water, Ringer's solution, phosphate buffered saline (PBS), or isotonic sodium chloride. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3 -butanediol. In some cases, formulations can include one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes. In some cases, the formulations can be buffered with an effective amount of buffer necessary to maintain a pH suitable for parenteral administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers. In some embodiments, the formulation can be distributed or packaged in a liquid form, or alternatively, as a solid, obtained, for example by lyophilization of a suitable liquid formulation, which can be reconstituted with an appropriate carrier or diluent prior to administration. The pharmaceutical compositions comprise the modified CHIP or functional fragment thereof as taught herein, any one of the nucleic acid molecules as taught herein, or any one of the vectors as taught herein. The pharmaceutical compositions can be formulated for medical and / or veterinary use.

[0160] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLESExample 1: Development of Anti-Tau CHIP.

[0161] Background.

[0162] Aggregated proteins that accumulate in the brain are detected and “detoxified” by a specialized cellular surveillance system that monitors protein integrity by (1) identifying unfolded or damaged proteins, (2) repairing them, or (3) targeting them for degradation. The protein CHIP, also referred to as STUB1, plays a central role in this process by maintainingprotein quality control. Since the discovery of CHIP in 19999, numerous reports detailing CHIP’S cellular activities have been published10'16. Traditionally, as a ubiquitin E3 ligase, CHIP ubiquitinates defective proteins and targets them for degradation by the Ubiquitin Proteasome System (UPS). The importance of CHIP function is illustrated by human genetics — numerous CHIP mutations were identified as causative for autosomal recessive spinocerebellar ataxia 16 and 48 (SCAR16 / SCAR48)17'19, a rare neurodegenerative disorder characterized by neuronal loss and cognitive deficits20. In addition to rare ataxias, our unpublished findings show that CHIP levels drop drastically in AD patient postmortem brain (FIG. 1), suggesting a gradual loss in CHIP-mediated neuroprotection in the vast number of patients with AD.

[0163] CHIP was initially implicated in the ubiquitination of tau within tau’s MT -binding region21,22, leading to tau degradation12,23'25, consistent with the detection of tau within and surrounding proteasomes26'28. However, in complete contrast, several reports have proposed ubiquitin-independent roles for CHIP, and even suggested a paradoxically limited role for CHIP in tau degradation29,30. Indeed, analysis of CHIP knock-out (KO) in the brain did not show altered total tau levels, as one might expect in the absence of CHIP12. Without wishing to be bound by any particular theory, these findings support a new concept that CHIP likely regulates tau via some poorly understood mechanism, which is strongly supported by our new preliminary data showing that E3 -deficient CHIP can still lead to tau degradation. Without wishing to be bound by any particular theory, we considered the interesting new possibility that CHIP may chaperone tau completely independently of tau degradation. In fact, recent publications have shown that CHIP, in some cases, can directly chaperone its clients including AMPK31, for example, in the absence of heat shock proteins (HSPs). In addition, CHIP can operate as a co-chaperone by interacting with HSP -bound substrates to aid in their stabilization, refolding, or degradation32. Regardless, without wishing to be bound by any particular theory, we hypothesize that enhancing this novel protective CHIP function could reduce pathological tau that is so intimately linked to cognitive decline.

[0164] Overall, CHIP’S role in the brain, certainly in regulating tau, is still a mystery. The notion that CHIP may represent a plausible therapeutic target or for consideration as a legitimate gene therapy in AD or TBI patients is very appealing yet has not been previously considered.

[0165] Engineering “potentiated” versions of CHIP with heightened anti-tau activity (CHIP- NP).

[0166] Given the discrepancies in the literature surrounding how CHIP regulates tau, we sought to analyze different CHIP variants containing or lacking chaperone or E3 ligase activity. We generated four genetic constructs encoding wild-type (WT) CHIP, chaperone-deficient CHIP that cannot associate with its chaperones (Hsp70 or Hsp90) via mutation of the TPR domain (e.g., a K30A mutation), a catalytically inactive CHIP in which the E3 ligase domain has been deleted (e.g., a AUbox CHIP mutation), or a novel CHIP variant that we developed in which both chaperone-binding and E3-ligase activity have been completely abolished. We term this latter variant CHIP-NP (which stands for CHIP -neuroprotection), since it displays several beneficial properties discussed below (FIG. 2). Lentiviral expression of CHIP variants alongside full-length human tau (2N4R isoform) in primary neurons confirmed the expression and proper molecular weights for all CHIP variants (including the expected smaller molecular weights for AUbox and CHIP-NP corresponding to the deletion of the U-box domain (box labeled "B"; FIG. 3). We also observed the expected loss of CHIP binding to chaperones (Hsp70, Hsc70, and Hsp90) upon immunoprecipitation of CHIP-K30A or CHIP-NP followed by blotting of bound chaperones (boxes labeled "A"; FIG. 3). All variants were coimmunoprecipitated with tau, as expected (FIG. 3).

[0167] To examine the anti-tau potential of these CHIP constructs, we had to decide which tau aggregation model to employ in these studies. We needed a reliable model that could develop mature tau pathology, such that we could monitor the suppression of aggregation in neurons (not cell lines) and in a somewhat high-throughput manner that is rapid, easily accessible, and with very robust readouts. Tau is challenging to aggregate in many different cell culture models, each of which have their pros and cons. We chose to use a recent model of full-length human tau harboring a combination of both P301L and S320F familial mutations, termed PL- SF, which enhances P-sheet structure and exhibits robust tau phosphorylation and aggregation in vitro35'37. PL-SF even generates robust tau pathology in primary neurons that is comparable to late-stage human AD. One simply cannot achieve the same tau pathology without addition of exogenous tau seeds, for example. Thus, the PL-SF variant represents a reliable model, even more so than seeding, to test the anti-tau properties of different CHIP variants. Indeed, we confirmed that one week of PL-SF expression resulted in tau pathology which closely resembles human AD pathology in terms of AT8 (phosphorylated tau), MCI (pre-tangle tau), and Thioflavin S (aggregated tau)-positive pathology (FIG. 4)38.

[0168] Having established the model, we expressed each individual CHIP variant with either wild-type tau or PL-SF in primary cortical neurons for 7 days up until DIV 10. Strikingly,CHIP NP, which completely lacks chaperone and E3 ligase activity, exhibited the highest degree of anti-tau activity compared to the other CHIP variants and dramatically reduced tau phosphorylation and aggregation in this model of mature tau pathology (FIGS. 5 and 6). We even performed this experiment in CHIP KO neurons, to provide a more physiologically relevant system to ectopically deliver CHIP in the absence of endogenous CHIP. Even in this scenario, CHIP-NP showed >90% reduction of PL-SF tau phosphorylation (box labeled "A"; FIG. 5) and reduction in SDS-insoluble tau aggregates (box labeled "B"; FIG. 5). CHIP-NP was significantly more effective at reducing tau aggregation compared to wild-type CHIP. As expected, WT-tau does not aggregate whatsoever, highlighting the need for an aggregate-prone PL-SF tau in these assays (box labeled "C"; FIG. 5). Without wishing to be bound by any particular theory, CHIP-NP also reduced soluble PL-SF tau by -50% (box labeled "D"; FIG. 5) suggesting CHIP-NP may function, in part, by reducing the soluble tau pool. CHIP-NP also ameliorated tau pathology in neurons expressing endogenous CHIP (FIG. 6) and therefore the use of CHIP KO neurons is not required to observe this suppression.

[0169] We were also quite surprised to observe that none of the CHIP variants, including CHIP-NP, altered WT-tau in any way including its levels or phosphorylation state (box labeled "E", FIG. 5). Without wishing to be bound by any particular theory, this is also an important consideration since it suggests that CHIP-NP preferentially targets aggregate-prone tau but not normal tau. This was exactly the type of therapeutic we sought, since only aberrant tau species would be targeted while normal tau is left intact. These data highlight CHIP-NP as an unanticipated, highly desirable anti-tau potentiated variant, to be deployed as a suppressor of protein aggregation.

[0170] After 25 years since the discovery of CHIP, and after much debate on its true function, we believe we have homed in on a surprising property of CHIP in the nervous system that may confer neuroprotection. Without wishing to be bound by any particular theory, we hypothesize that CHIP-NP affords neuroprotection via a novel gain-of-function mechanism through CHIP’ s coiled-coil (C-C) domain. Previously, this domain was thought to primarily stabilize CHIP dimerization with no other known functions noted39. This is the first indication of an anti-tau function of this domain, which will certainly advance our understanding of its therapeutic potential. Finally, we note that the CHIP-NP variant is only -25 kD, much smaller than full- length CHIP (-35 kD), which is more amenable for packaging and delivery to the brain via AAV-based gene therapy approaches to treat AD, TBI, and other tauopathies. A CHIP gene therapy based on AAV technology is appealing for neurodegenerative diseases where drugbased pharmacology is challenging, as is the case with CHIP. AAVs will allow high-efficiencyperipheral delivery to the brain and long-term, sustained effects. In fact, an AAV-based gene therapy for spinal muscular atrophy (SMA), a crippling motor disease in children, is now FDA approved. Similar AAV-based approaches are in phase 1 / 2 clinical trials for AD, Huntington’s disease (HD), and Parkinson’s disease (PD)40. Without wishing to be bound by any particular theory, we expect that this powerful therapeutic technology will also be applicable for AD, and other similar diseases or disorders, with CHIP-NP.Example 2: CHIP-NP as a protective factor that alleviates tau-mediated neurodegeneration.

[0171] Without wishing to be bound by any particular theory, our data above suggest that CHIP-NP should be considered as an anti-tau therapeutic, and we hypothesize that CHIP-NP will alter tau’s conformation, reduce tau phosphorylation, and alleviate AD progression in mice, more so than any other known CHIP variant. To assess its therapeutic impact, we will deliver CHIP-NP (or its WT counterpart) to PS 19 tau model mice using a brain-specific AAV- targeting approach and determine the extent of neuroprotection by cognitive testing.

[0172] Tau Tg Mouse Model.

[0173] While PL-SF is needed in cultured neurons to generate mature tau pathology, in mice only a single mutation is needed over the course of a ~ 12-month lifespan. PS19 mice harbor a tau-P301S mutant transgene and recapitulate many AD / TBI hallmarks including tangle pathology (FIG. 7), neuronal loss, and cognitive deficits (41). These mice are viable and fertile as heterozygotes. We will deliver control (GFP-expressing) or CHIP variants (wild-type, AUbox, K30A, or the AUbox / K30A) by AAV-mediated viral transduction to their brains. We note that a CHIP AAV-based approach is feasible, at least in a mouse model of brain ischemia (42). Given our extensive data showing that CHIP-NP dramatically depletes mutant tau, we expect this particular variant to show the most robust reduction of pathological tau and alleviate memory deficits in AD model mice. Transduced PS 19 mice will be analyzed by biochemical, histological, and behavioral readouts below to assess the extent of neurodegeneration.

[0174] CHIP-AAV vector.

[0175] To facilitate this proof-of-concept preclinical study, designed as a translational aim, we will generate AAVs suitable for peripheral intravenous (i.v.) injection. Among the different AAV serotypes, AAV9 shows the best tropism for neurons when delivered directly to the brain. To optimize AAV9’s properties, a variant known as AAV-PHP.eB43’44showed the highest affinity for the brain after peripheral injection into mice. We therefore engineered a synapsin promoter into AAV-PHP.eB to drive pan-neuronal CHIP expression in the cortex andhippocampus where tau is highly expressed in PS 19 mice, similar to tau pathology in human AD brain. The new CHIP-expressing AAVs have been generated in-house and validated in cultured neurons (FIG. 8). Using the GFP-alone expressing AAV, we injected this peripherally by tail -vein injection to pilot its neuronal expression and found robust GFP expression in the hippocampus that was restricted to neurons as indicated by neuronal nuclear protein (NeuN; e.g., NeuN positive) rather than microglia, astrocytes, or oligodendrocytes (FIG. 9). Therefore, AAV-PHP.eB can be administered i.v. to achieve pan-neuronal expression, thus avoiding any confounding issues of intracerebroventricular (i.c.v.) injections (e.g., needle damage). We will perform the same pilot with our CHIP-expressing AAV-PHP.eB to ensure proper expression and localization.

[0176] CHIP- AAV peripheral delivery to PS 19 mice.

[0177] Cohorts of PS19 mice will be injected i.v. with CHIP-expressing AAVs at two different ages. One cohort of mice will be injected at 6 months of age prior to the emergence of tau pathology (preventative paradigm). Another set of mice will be injected at 9 months of age once symptoms have emerged (treatment paradigm). Both paradigms are important since one is disease-preventative, and the other is more translational. Since we currently cannot identify at-risk patients until symptoms emerge, post-symptomatic treatment is the most relevant to populations with known risk factors, such as those with a family history, who have experienced chronic brain trauma, as well as those who have been diagnosed with depression and / or post- traumatic stress disorder. Based on published reports45, we will inject AAVs at a concentration of 1-5 x 1012vector genomes (vg). Mice will be analyzed 1- or 3-months later. Therefore, the oldest mice would be 12 months old, which coincides with late-stage cognitive impairments and accelerated mortality (~ 50% survival). There are 1 control and 4 CHIP variants to be tested (control GFP, WT CHIP, AUBox, K30A, CHIP-NP).

[0178] Tau phosphorylation in vivo.

[0179] A sampling of mouse brain regions (hippocampus, cortex, brainstem, spinal cord) will be analyzed by immunohistochemistry using specific antibodies to assess markers of tau pathology. These include acetylated tau (ac-K280 antibody), phosphorylated tau (AT8, PHF-1 epitopes), and pathological tau conformation (MC-1 antibody), all of which correlate with different stages of tau pathology. Verification of tau pathology will be confirmed biochemically by immunoblotting. For example, more mature tau pathology accumulates in the insoluble SDS-extractable brain fractions (referred to as insoluble AD tau). We expect CHIP-NP to preferentially target and dephosphorylate tau. Although our focus is on tau, we also feel it isimportant to examine CHIP’S activity towards non-tau targets, and we will therefore use Kinexus antibody arrays to globally monitor the phospho-proteome in vivo, simply to determine whether other phospho-proteins are also targeted by the CHIP variants.

[0180] Neuroinflammation.

[0181] Neuroinflammation is commonly observed in AD48. Microgliosis will be assessed histologically by monitoring resting (ramified) and activated (amoeboid) microglia49. We will stain sections with NeuN (neurons), glial fibrillary acidic protein (GFAP; astrocytes), ionized calcium -binding adaptor molecule 1 (IB Al; marker of resting and reactive microglia) and image brains by confocal microscopy. We will quantify the number of resting versus activated microglia relative to total cell number in each region. We will similarly quantify neurons (NeuN+) and astrocytes (GFAP+).

[0182] Neurodeseneration.

[0183] We will determine body and brain weight and cortical atrophy using standard hematoxylin and eosin (H&E) staining. The presence of neuronal damage and apoptosis will be monitored by histological methods: tunnel staining, cytochrome C, and cleaved caspase-3 detection are routinely used to assess neuronal death, which is increased in AD brains50. Additionally, ROS accumulation, DNA damage, and oxidative damage are reportedly early biochemical events in AD progression51, and will be monitored by 8-hydroxyguanine (8-OHG) and phospho-H2AX immunoreactivity, which parallels neuronal loss in AD.

[0184] Behavior and Co nition.

[0185] We note there are no reports of survival, behavior, or other pathophysiology that link CHIP function to tau pathology in AD or other tauopathies. We will analyze transduced PS 19 mice expressing the CHIP variants described herein. She will test mice in the following assays for AD-like behavior. A 4-point measure of ledge, clasp, gait, and kyphosis will assess general features of motor function and coordination53. Since hippocampal loss is prominent in PS19 mice, we will measure spatial learning using the Morris Water Maze, including a measure of cognitive flexibility in which the hidden platform is moved to a new location; the Barnes maze, a land version of the water maze for confirmation of impaired spatial learning; and the conditioned fear response for context and cue. To reiterate, 6 month and 9-month-old mice will be tested at two different timepoints post injection: 1 month and 3 months post-injection. Since AD-like behavior is prominent in PS 19 mice41,54, this line is ideal to interrogate CHIP’S role in cognition. Our preliminary data show significant Morris water maze deficits in PS 19 mice with little impact on motor function, as assessed by swim speed (FIG. 10). Therefore, cognition and motor function are separable in the PS19 model. Our goal here is to highlight CHIP-NP as agene therapy capable of suppressing the cognitive dysfunction in AD, TBI, and other tauopathies.

[0186] Survival.

[0187] To detect differences in survival in CHIP-expressing PS 19 mice compared to controls, we will perform long-term survival analysis using separate aged cohorts of mice since the mice described above will be sacrificed for tissue analysis at the indicated timepoints. In this scenario, control GFP or the CHIP variants will be injected at either 6- or 9-months of age and mice will be allowed to age until criteria are met for humane endpoint. As a reminder, the PS 19 line rarely survives beyond 12-14 months and therefore this analysis will fit nicely within the project period.

[0188] Statistical Analysis.

[0189] We will achieve a statistical power of 0.80-90 for these assays. PS 19 mice show a narrow window of 50% survival at ~12 months old. Our power analysis is structured to detect 8% differences in the phenotypes described, assuming signal / noise ratio of 2.0. By this calculation, for example, we should reliably detect + / - 1 -month differences in survival in CHIP- transduced PS 19 mice. Similar changes in life expectancy have been shown, for example prior study in which an AP-model was crossed to PS 19 mice showed a 2-month increase in mortality55. To provide sufficient animals for the pathology, biochemistry, and behavior assays, we require n=20 mice per group, n=5 groups (control GFP, WT CHIP, AUBox, K30A, CHIP- NP), n=2 ages (6- and 9-months), and n=2 timepoints (1- and 3-months post-infection). This equates to a generous total of - 400 PS 19 injected mice to provide adequate mouse numbers for tissue analysis including backups. Survival will require a separate aged cohort — n=20 mice per group, n=3 groups (control GFP, WT CHIP, CHIP-NP), and n=2 ages (6- and 9- months). This equates to an additional 120 PS19 mice needed for survival. Cohorts for both cognition and survival are very important to the long-term goals of this study. We note that injecting this number of mice is feasible and readily managed at UNC’s behavior core, since i.v. injection allows much higher throughput analysis than other methods. Survival differences will be determined using the log rank test to generate chi-squared (X2) values.Example 3: Mechanism of CHIP-NP Tau Suppression and Neuroprotection.

[0190] These experiments will help determine exactly how the CHIP-NP variant achieves such a remarkable suppression of tau aggregation. Without wishing to be bound by any particular theory, we hypothesize that CHIP-NP may act to target and remove toxic tau species througha combination of the following: tau dephosphorylation, disaggregation, proteasome and autophagy pathways, or gain-of-function interactions with unknown cytoplasmic factor(s). Therefore, there are three distinct goals to evaluate CHIP’S mode of action: (1) evaluate the direct impact on tau using molecular, biophysical, and functional assays; (2) evaluate the activation of proteasome and autophagy pathways; and (3) define the CHIP interactome and determine whether protective CHIP variants might take advantage of a unique gain-of-function interactions to facilitate tau removal.

[0191] We will employ three full-length, human 2N4R tau variants for the studies in this example: (1) WT tau which is soluble, has no observed pathological phenotypes, and no impact on neuronal function; (3) P301L tau, which has single missense mutation commonly used to generate pathological tau over long timeframes, causes dissociation from microtubules, and is moderately aggregate-prone; and (3) PL-SF tau, which has a combination double mutant harboring P301L and S320F missense mutations that creates a more highly aggregate-prone phenotype over 10+ days in culture, generating AD-relevant pathology.

[0192] We will test how these three different versions of tau respond to any of the four CHIP variants that we described above (WT, K30A, AU-box, and CHIP NP). All tau and CHIP constructs were created on the same lentiviral expression system. WT tau represents the normal non-aggregate-prone control tau. On the other hand, PL-SF is the only known cell autonomous model for mature tau aggregation in cells without the need of exogenous seeding or AD fibrils. Thus, it is an ideal model to examine the effects of CHIP variants on AD-like pathology. In addition to PL-SF, we will include the P301L mutant, which behaves identical to P301S. This is the most common genetic model to mimic some aspects of pathological tau, as in PS 19 mice. While this mutant exhibits some AD-like hallmarks in mouse models, it does not spontaneously aggregate in cultured neurons, limiting its utility in examining the anti-aggregation properties of CHIP. Our description of tau variants is particularly important, as we found that CHIP-NP exclusively acts on aggregate-prone PL-SF tau while sparing the non-aggregated WT tau. The specificity for tau aggregates is an important consideration when evaluating therapeutic potential. Lentiviral expression of these variants is highly reliable and reproducible. Coexpression of tau variants with each CHIP variant (CHIP+Tau) will be performed in primary cortical mouse neurons unless otherwise stated.

[0193] Assessing CHIP’S function to prevent tau phosphorylation, tau aggregation, tau spreading.

[0194] CHIP-mediated tau dephosphorylation.

[0195] Tau hyperphosphorylation is a hallmark of tauopathies, including TBI. While we already assessed the effect of CHIP-NP on several tau phosphorylation sites via western blot (e.g., Ser202 / Thr205 and Ser262), tau contains >50 other phosphorylation sites. We will assess the extent to which all CHIP variants (e.g., CHIP-NP) promote tau dephosphorylation in lentiviral transduced primary mouse cortical neurons. Rather than focus on individual sites of interest, we optimized a mass spectrometry (MS) approach to simultaneously monitor all tau post-translational modifications (PTMs) by purifying large amounts of tau (approx. 10 pg) using two tau monoclonal antibodies (T46 and T14). After purification, we applied high- resolution MS that achieves ~ 90% tau sequence coverage33, allowing screening of nearly all of tau’s Ser / Thr / Tyr sites of interest. Follow-up confirmation will be assessed by standard immunoblotting in lentiviral transduced primary neurons using commercially available antibodies. Without wishing to be bound by any particular theory, we expect that CHIP-NP exerts broad tau dephosphorylation at the majority of pathology-relevant phosphorylation sites to a greater extent than other CHIP variants. Finally, we will also monitor oligomeric tau species that can track with tau hyperphosphorylation. Immunocytochemistry and dot blotting will be performed to assess MCI (pre-tangle conformational tau), as well as TOC1 and T22 (oligomeric tau.)

[0196] Pathological tau aggregation.

[0197] Tau phosphorylation accelerates its aggregation in vitro and in diseased AD brain57'59, in a similar manner to TBI and blast-injury patients. Thus, we will also consider whether CHIP variants can alter tau’s aggregation propensity using a series of very well-characterized in vitro and neuronal assays that we have optimized in our laboratory over the last 10 years (FIG. 11). Since tau aggregation assays in vitro employ purified proteins in the absence of cellular degradation machinery (e.g., ubiquitin, proteasome, or autophagy components), the impact of CHIP-NP’ s chaperone activity on tau aggregates can be evaluated via these methods. Recombinant purified tau variants will be incubated with purified CHIP variants to determine the extent of tau aggregate suppression. We will perform sedimentation assays and thioflavin- T (ThT) assays (FIG. 11, panels A and B) to measure tau filament formation after incubation with CHIP variants. Comparable assays for aggregation will be performed in lentiviral- transduced primary neurons to confirm that CHIP variants reduce tau aggregation in PL-SF tau-expressing neurons. We will evaluate the extent of tau aggregation in neurons by SDS insoluble levels of tau and thioflavin-S (ThS) positive neurons.

[0198] Pathological tau conformation.

[0199] To further assess changes in tau conformation, circular dichroism (CD) will determine the extent of P-sheet-rich structure (P- structure). A shift in minimum wavelength of the CD spectra from random coil structure at 200 nm up to 218 nm suggests transition to anti-parallel P-sheet observed with tau aggregates. CD spectra of CHIP alone, tau alone, or all CHIP+Tau variants will be read at 190-250 nm wavelengths. Our analysis of an aggregate-prone P301L mutant showed transition from monomer (random coil) to P-sheet structure by 1-hr post aggregation (FIG. 11, panel C). In parallel, to visualize how CHIP impacts tau conformation, we will employ transmission electron microscopy (TEM) on tau aggregation reactions and assess tau filament morphology and length. Fibril reactions will be adhered to mesh copper grids and then stained with 2% uranyl acetate followed by analysis with a LEO EM910 TEM. As an example, aggregate-prone P301L mutant showed accelerated filament formation in vitro (FIG. 11, panel D)

[0200] Tau spreading suppressed by CHIP-NP.

[0201] Tau seeding and propagation are thought to be causal in the spread of tau pathology across the brain during tauopathies. In our preliminary data, we found that PL-SF recruits endogenous WT mouse tau into inclusions, highly suggestive of PL-SF recruiting and seeding normal tau (FIG. 12, the T49 antibody exclusively detects mouse tau only). Here, we will test whether CHIP-NP can suppress seeding.

[0202] First, we will introduce CHIP variants into the experiment shown in FIG. 12 and determine whether they suppress the recruitment and seeding of mouse tau by immunofluorescence in mouse neurons. We will also test whether CHIP variants suppress tau seeding in the well characterized HEK293 tau biosensor cells60. CHIP variants will be expressed in the biosensor cells and then seeding will occur by introducing either recombinant tau fibrils, as we have generated61, or by introduction of human AD-extracted brain material, for which we and others recently developed homogenization protocols47,62. Finally, we will determine whether tau transfer between neurons is impacted by CHIP-NP. The propagation of tau occurs via tau release and uptake in neighboring neurons. We will express a GFP-tagged version of PL-SF (FIG. 12) in tau KO mouse neurons using a 3-chamber microfluidic device that separates neuronal cell bodies from axons using isolated compartments. In this manner, we can monitor the movement of PL-SF aggregates between inter-connected neurons by analyzing the different compartments. Since we will be using tau KO neurons, there is no endogenous mouse tau to be seeded, but rather we can simply monitor the movement of tau aggregates from one neuron to the other. We will then introduce CHIP-NP and determine whether the movement of tau aggregates is suppressed.

[0203] Neuronal activity and synaptic integrity.

[0204] To assess how neuronal function changes with CHIP suppression of the pathological tau phenotypes described above, our lab set up an in-house multi-electrode array (MEA) system (Maestro Edge). We published a recent study documenting the MEA’s potential to monitor tau- mediated changes in neuronal activity63. MEAs can rapidly assess changes in neuronal activity and network communication (activity, synchrony, and oscillations) in real time in a 24-well plate format64. As preliminary data, we performed daily MEA recordings beginning at DIV18 for ~2 months and found that PL-SF expressing neurons showed progressive deficits in neuronal activity, connectivity, and synchrony beginning at DIV30. Thus, this experiment provides a very reliable, high-throughput model to monitor deficits in neuronal function in response to tau aggregates (FIG. 13) Thus, we will perform similar analysis in neurons expressing CHIP variants in parallel with tau and record them daily. We will evaluate changes in firing rates within bursts, number of bursts, burst durations and synchrony and can readily assess dozens of other metrics post-hoc. This approach provides an excellent strategy to evaluate neuronal function, rather than just markers of neuronal death or dysfunction. Nonetheless, in parallel, we will confirm any improvement in MEA metrics by monitoring changes in synaptic integrity by co-staining neuron cultures with postsynaptic density protein 95 (PSD95), and the presynaptic glycoprotein synaptophysin. Colocalization of the two synaptic markers will be used as a measure of synapse density65.

[0205] Assessing the mechanism of CHIP-NP promoted tau degradation.

[0206] CHIP can function as a molecular switch between proteasomal or autophagy -mediated degradation of pathological proteins14. As CHIP-NP reduced tau pathology quite robustly (FIGS. 5 and 6), we therefore will test whether known degradation pathways (e.g., proteosome and / or autophagy) mediate this effect.

[0207] Proteasomal degradation.

[0208] We will first examine whether CHIP variants accelerate proteosome activity using a well-known UbG76V-GFP fusion system66, an unstable ubiquitin variant that becomes stabilized (e.g., GFP-positive) when proteasomes are inhibited. We will deliver tau alone, CHIP alone, or CHIP+Tau variants followed by measurement of UbG76V-GFP abundance as a readout of UPS activity. In parallel, we will measure changes in three major proteasome activities: chymotrypsin-like, trypsin-like, and caspase-like. These can be assayed using commercially available peptide-based fluorometric assays and we can correlate proteasome dysfunction with PL-SF tau aggregation. If CHIP increases proteasomal function, we anticipate reduced UbG76V- GFP abundance and increased proteosome activity. Lastly, using proteosome inhibitors(MG132), we will inhibit the proteosome and assess the extent of tau pathology in the presence of each CHIP variant.

[0209] Autophagy.

[0210] To complement our proteosome analysis, we will evaluate whether CHIP or its variants can influence autophagy to clear tau. Autophagy is strongly implicated in age-related neurodegeneration and brain injury67'69. A role for CHIP in this process is consistent with its known role in regulating the expression and turnover of autophagic proteins including the transcription factor TFEB70’71. First, we will measure the levels of the autophagic markers p62, an autophagy adaptor protein, and LC3, which initiates autophagosome formation72, and will determine the ratio between mature lipidated LC3-II and immature unlipidated LC3-I, a measure of autophagic flux72. We will then determine whether autophagy activation or flux is altered by introduction of PL-SF and then restored by CHIP-NP. Second, we will use the tandem LC3B-mCherry-EGFP autophagic flux reporter73to quantify autophagosomelysosome fusion. Upon fusion with the lysosome, the GFP signal is quenched. If flux is impaired, however, we expect more yellow / green puncta. Third, we will assess the number of lysosomes using LysoTracker dye, since the number of lysosomes can influence autophagic flux and capacity. Finally, if we detect appreciable upregulation of autophagy, we will investigate whether CHIP variants increase tau internalization into autophagosomes or lysosomes by immuno-gold cryo-electron microscopy, the gold standard for autophagic internalization. Without wishing to be bound by any particular theory, and given that CHIP is known to interact with components of both the proteasome and autophagy, we expect that CHIP-NP may preferentially engage both pathways to reduce tau pathology.

[0211] Assessing CHIP-NP’ s interaction profile.

[0212] While CHIP-NP is effective at preventing tau pathology, we do not know which, if any, CHIP-associated factors that may help to mediate this effect. However, without wishing to be bound by any particular theory, we hypothesize that gain-of-function interactions within CHIP- NP’ s coiled-coil (C-C) domain are responsible for its potentiated neuroprotection.

[0213] CHIP regulation of Heat Shock Factor 1 (HSF1).

[0214] CHIP can directly interact with the N-terminal domain of HSF1 and facilitate its nuclear translocation74. In turn, HSF1 is known to upregulate a transcriptional profile that includes critical chaperones and anti-apoptotic factors. Here, we will determine whether CHIP-NP and other variants show differential abilities to activate HSF1 and its downstream targets, and whether HSF1 activation is a dominant mechanism by which CHIP-NP exerts its anti -tau properties.

[0215] First, we will determine whether CHIP-NP causes nuclear accumulation and activation of HSF-1, as determined by immunofluorescence and immunoblotting in cytoplasmic vs. nuclear fractions with pan and phospho-HSF-1 antibodies, an indicator of its nuclear accumulation, dimerization, and activation75. Then, we will use quantitative RT-PCR (qRT- PCR) to determine if CHIP variants activate an HSF1 -dependent transcriptional program by focusing on downstream HSF-1 targets of interest. We will infect primary neurons with CHIP variants in the presence of absence of tau variants (WT vs. PL-SF) and use a multiplexed RT- PCR array (SA-Biosciences) that prioritizes expression of well-known chaperones to identify all HSF-1 dependent chaperones that are induced by CHIP-NP. The induction of any chaperone hits will be further validated by manual quantitative qRT-PCR with specific primers and immunoblotting.

[0216] To extend these findings and determine whether any identified chaperones act downstream of CHIP-NP, we will then use shRNA knockdown of HSF-1 or identified chaperones in the presence of CHIP-NP to determine whether any anti-tau neuroprotection afforded by CHIP-NP has now been abrogated.

[0217] Mass spectrometry to identify CHIP -interactins partners.

[0218] As an unbiased approach, we will use high-resolution MS to build a comprehensive CHIP-NP interactome map. To accomplish this, we will immunopurify control GFP vs. CHIP- WT vs. CHIP-NP in the absence or present of PL-SF tau. First, we will perform this experiment in lentiviral expressing primary neurons simply to provide the sensitivity and resolution to detect robust changes, for which we provide strong preliminary data below. Subsequently, we will perform this analysis in the AAV-infected PS19 mice described above. The latter approach is especially appealing since we can correlate any neuroprotection that we observe in vivo with a distinct CHIP-interactome profile.

[0219] Methods.

[0220] Immunoprecipitated purified CHIP isolated from transduced primary neurons or AAV- infected PS 19 mice will be subjected to mass spectrometry analysis using the following parameters and safeguards. Three separate biological sets, separate dissection and transduction, of primary neurons will be used. We employ a stringent bioinformatics approach that ensures identification of modified peptides. Samples will be processed with both biological (N>3) and technical (N>3) replicates using single-shot liquid chromatography mass spectrometry (LC- MS). Peptides are quantified using false discovery rates (FDR) of 0.01 for peptides and 0.05 for total protein. All identified correlations between different replicates are confirmed with a Pearson correlation analysis (score >0.7). Label-free quantification (LFQ) of all identifiedpeptides is determined by the given peak areas using specialized MaxQuant software, as we have previously done in a recent publication76. We will focus on interaction partners with the highest p-values and mass accuracy scores.

[0221] In vivo considerations for mass spectrometry.

[0222] CHIP will be immunoprecipitated from 6-12-month-old CHIP-AAV-injected PS19 mice using isolated hippocampus, a region with robust tau pathology in the PS 19 model (FIG. 7). The exact ages of the mice to be used will be partly determined by results from our earlier experiments. For example, if we find that three months post-CHIP-AAV infection is needed for maximum neuroprotection, then the longer timepoints will be prioritized. An approximate total of n=6 timepoints, n=3 variants (GFP, CHIP-WT, CHIP-NP), and n=5 mice / time point means that 90 total mice needed for in vivo mass spectrometry analysis.

[0223] Preliminary mass spectrometry analysis of a CHIP interactome.

[0224] In our preliminary analysis of transduced CHIP-WT expressing primary neurons (FIG. 14), we found several known CHIP interactors (e.g., HSP and DNAJ chaperones in the circles labeled "B") in addition to tau (MAPT in the circle labeled "C"). However, we also uncovered several tau interactors including VPS35, which regulates tau phosphorylation77, RAB7, which regulates tau secretion78, and NSF, which regulates tau-mediated AMP A receptor trafficking79(circles labeled "D"). Therefore, our preliminary data has already uncovered three tau-relevant targets of CHIP which may play critical roles in CHIP-NP’ s neuroprotective function towards tau.

[0225] Validation of CHIP -inter actins partners that suppress tau pathology.

[0226] Using both our primary neuron and in vivo mass spectrometry data, we will prioritize hits that show the strongest statistical interactions with CHIP and those that are enriched in CHIP-NP interactome compared to CHIP-WT or control GFP. We expect to identify baseline CHIP interactors that may be present at low levels in CHIP-WT samples but increase substantially in CHIP-NP samples. We might also expect completely new gain of function interactions that are specific to CHIP-NP, given its enhanced activity. We will validate our top hits by performing immunoprecipitation experiments to detect their interaction with CHIP and will include the three targets identified in FIG. 14 (in the circles labeled "D"). We will perform shRNA knockdown of these hits, and subsequently assess whether CHIP-NP is no longer capable of promoting tau dephosphorylation and disaggregation. Without wishing to be bound by any particular theory, we anticipate that knockdown of specific CHIP-interacting proteins will negate CHIP-NP’ s anti -tau properties and begin to uncover its mechanism of neuroprotection.

[0227] CHIP-NP ’s Coiled-Coil (C-C) domain.

[0228] We will also focus on CHIP’S C-C domain, which is poorly characterized, but surprisingly could be at the heart of why CHIP-NP exerts neuroprotection. After all, it is the only domain left once the E3 ligase and TPR domains have been inactivated. The only known function of the C-C domain is to stabilize CHIP dimerization. While this function is critical to CHIP’S function, it is not clear how this alone could suppress tau pathology. In fact, we have not observed that CHIP-NP shows any differences in dimerization compared to CHIP-WT, as assessed by native-PAGE gels.

[0229] However, we made a very interesting observation when analyzing known SCAR16- causing familial CHIP mutations when overlaid onto CHIP’S molecular domains. The C-C domain and the closely adjacent regions harbor several mutations that are associated with more severe cognitive impairment in SCAR16 patients (FIG. 15, underlined)80. Without wishing to be bound by any particular theory, we hypothesize that these specific mutations in the C-C domain may abrogate CHIP-NP’s protective function. Here, we will create these same patient mutations inserted into the CHIP-NP construct and determine whether these new variants now abrogate CHIP-NP’s ability to suppress the tau phenotypes described above. Furthermore, one might expect these mutations to abrogate any protective gain-of-function interactions identified above. Alternatively, if these mutations have no effect on CHIP-NP’s protective function, we will create serial deletions within the remaining 5’ TPR and 3’ C-C domain to determine the critical elements within these two remaining domains that may confer protection.Example 4: CHIP variants have in vitro activity.

[0230] CHIP variants as described above were tested in vitro for their activity in preventing tau protein aggregation. PL-SF mutant tau and different CHIP variants were incubated in vitro and aggregation was measured using Thioflavin T (ThT) fluorescence after induction of tau aggregates using Congo Red (FIG. 19). Progressive deletion of CHIP’S U-box domain improves its ability to inhibit heparin-induced PL-SF tau aggregation, while adding back CHIP’S U-box alone can restore its aggregation by impeding the chaperone function of CHIP AU-box (FIG. 19, panels A-B). CHIP AU-box has improved chaperone function compared to CHIP WT in an orthogonal system using Mass Photometry (FIG. 19, panel C). Tau aggregates were induced via the addition of Congo Red and quantified. Tau (10 pM) aggregation, in the presence or absence of CHIP WT (10 pM) or variants, was induced by adding Congo Red (50 pM) and incubating at 37 °C for 2 hours. The reactions were stopped by placing them on iceand then diluted to 20 nM Tau for measurement on the mass photometer. Tau was considered to be in an aggregated form if the size was larger than 200 kDa.Example 5: Testing combinatorial CHIP variants for enhanced anti-tau activity.

[0231] Several CHIP variants have been discovered that could amplify the anti-tau activity, when used in combination with each other and / or with the K30A and / or AU-box mutations (e g., CHIP AU-box 1-230, CHIP AU-box 1-224, CHIP AU-box 1-193, and / or CHIP AU-box 1-186) described above. In some embodiments, the combinatorial mutants comprise a deletion of 10 or 19 amino acids from the N-terminus of the CHIP; QI 27 A and R128A mutations; and / or a F131A mutation. These variants can be tested for anti-tau activity using the in vivo and in vitro experiments as described above.

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Claims

WHAT IS CLAIMED:

1. A modified Carboxyl Terminus of Heat Shock Cognate 70-interacting protein (CHIP) or functional fragment thereof, wherein the modified CHIP or functional fragment thereof comprises at least two amino acid modifications (e.g., 2, 3, 4, 5, or more amino acid modifications) that reduce the activity of a U-Box domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%) and reduce the activity of a tetratricopeptide repeat (TPR) domain by at least about 50% (e.g., by about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or by about 100%).

2. The modified CHIP or functional fragment thereof of claim 1, wherein the at least two amino acid modifications comprise at least one amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the U-Box domain, at least one amino acid modification (e.g., 1, 2, 3, 4, 5, or more amino acid modifications) in the TPR domain, at least one amino acid modification in the coiled-coil domain, and / or at least one amino acid modification in the N-terminus domain (e.g., amino acids 1-26 numbered according to SEQ ID NO: 1).

3. The modified CHIP or functional fragment thereof of claim 1 or 2, wherein the at least two amino acid modifications comprise a substitution of at least one amino acid (e.g., a substitution of 1, 2, 3, 4, 5, or more amino acids), a deletion of at least one amino acid (e.g., a deletion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, or more amino acids), an insertion of at least one amino acid (e.g., an insertion of 1, 2, 3, 4, 5, or more amino acids), and / or any combination thereof.

4. The modified CHIP or functional fragment thereof of any one of claims 1-3, wherein the at least one amino acid modification in the U-Box domain comprises a deletion of about 10 to about 150 amino acid residues from the C-terminus relative to a wild-type CHIP.

5. The modified CHIP or functional fragment thereof of claim 4, wherein the deletion of about 10 to about 150 amino acid residues from the C-terminus is a deletion of an E3 ligase domain.

6. The modified CHIP or functional fragment thereof of claim 5, wherein the modified CHIP or functional fragment thereof is devoid of E3 ubiquitin ligase activity.

7. The modified CHIP or functional fragment thereof of any one of claims 1-6, wherein the at least two amino acid modifications comprise a lysine to alanine substitution at residue 30 (K30A) corresponding to SEQ ID NO: 1.

8. The modified CHIP or functional fragment thereof of claim 7, wherein the modified CHIP or functional fragment thereof is devoid of heat shock protein 70 (Hsp70) chaperone activity, heat shock cognate 70 (Hsc70) chaperone activity, and / or heat-shock protein 90 (Hsp90) chaperone activity.

9. The modified CHIP or functional fragment thereof of any one of claims 1-8, wherein the at least two amino acid modifications comprise a deletion of about 10 to about 150 amino acid residues from the C-terminus relative to a wild-type CHIP and a lysine to alanine substitution at residue 30 (K30A) corresponding to SEQ ID NO: 1.

10. The modified CHIP or functional fragment thereof of claim 9, wherein the modified CHIP or functional fragment thereof comprises an amino acid sequence at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO:2.

11. The modified CHIP or functional fragment thereof of claim 10, wherein the modified CHIP or functional fragment thereof comprises the nucleotide sequence of SEQ ID NO:2.

12. The modified CHIP or functional fragment thereof of any one of claims 1-11, wherein the at least two amino acid modifications comprise a histidine to glutamine substitution at residue 260 (H260Q) corresponding to SEQ ID NO: 1; a glutamine to alanine substitution at residue 127 (Q127A), an arginine to alanine substitution at residue 128 (R128A) corresponding to SEQ ID NO: 1; a phenylalanine to alanine substitution at residue 131 (F131 A) corresponding to SEQ ID NO: 1; a deletion of about 1 to about 20 amino acids from the N-terminus relative to a wild-type CHIP; or any combination thereof.

13. The modified CHIP or functional fragment thereof of any one of claims 1-12, wherein the modified CHIP is a human CHIP.

14. The modified CHIP or functional fragment thereof of any one of claims 1-13, wherein the modified CHIP or functional fragment thereof binds to tau protein.

15. The modified CHIP or functional fragment thereof of claim 14, wherein the modified CHIP or functional fragment thereof dephosphorylates the tau protein.

16. The modified CHIP of claim 14 or 15, wherein the modified CHIP prevents aggregation of the tau protein.

17. A nucleic acid molecule encoding the modified CHIP or functional fragment of any one of claims 1-16.

18. The nucleic acid molecule of claim 17, operably linked to a promoter.

19. The nucleic acid molecule of claim 18, wherein the promoter is a neuron-specific or neuron-preferred promoter.

20. A vector comprising the nucleic acid molecule of any one of claims 17-19.

21. The vector of claim 20, wherein the vector is a neurotropic vector.

22. The vector of claim 20 or 21, wherein the vector is a viral vector.

23. The vector of claim 22, wherein the viral vector is a lentiviral vector.

24. The vector of claim 22, wherein the viral vector is an adeno-associated viral (AAV) vector.

25. The vector of claim 24, wherein the AAV is serotype 9 (AAV9).

26. The vector of claim 25, wherein the AAV9 is variant PHP.eB (AAV-PHP.eB).

27. A pharmaceutical composition comprising the modified CHIP or functional fragment thereof of any one of claims 1-16, the nucleic acid of any one of claims 17-19, or the vector of any one of claims 20-26 and a pharmaceutically acceptable carrier, diluent, or excipient.

28. A method of preventing and / or reducing tau protein aggregation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein aggregation in the subject.

29. The method of claim 28, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

30. A method of inhibiting formation of protein inclusions comprising tau protein in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby inhibiting formation of protein inclusions comprising tau protein in the subject.

31. The method of claim 30, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

32. A method of preventing and / or reducing tau protein phosphorylation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby preventing and / or reducing tau protein phosphorylation in the subject.

33. The method of claim 32, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

34. A method of reducing neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP thereby reducing neuroinflammation in the subject.

35. The method of claim 34, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

36. A method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a neurodegenerative disease in the subject.

37. The method of claim 36, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

38. The method of claim 36 or 37, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson’s disease, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), Down’s syndrome, corticobasal degeneration, Pick’s disease, multisystem atrophy, inclusion body myositis (IBM), cerebral amyloid angiopathy (CAA) (e.g., prion protein CAA), argyrophilic grain disease (AGD), tangle predominant dementia (TPD), or chronic traumatic encephalopathy (CTE).

39. A method of treating or preventing a traumatic brain injury (TBI) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a TBI in the subject.

40. The method of claim 39, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

41. The method of claim 39 or 40, wherein the TBI is a repetitive TBI.

42. A method of treating or preventing a cerebrovascular disease (CVD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of CHIP, thereby treating or preventing a CVD in the subject.

43. The method of claim 42, wherein the CHIP is the modified CHIP or functional fragment thereof of any one of claims 1-16.

44. The method of claim 42 or 43, wherein the CVD is an aneurysm, a vascular malformation (e.g., an arteriovenous malformation or a cerebral cavernous malformation), a fistula (e.g., an arteriovenous fistula or a carotid-cavernous fistula), carotid artery disease (e.g., carotid stenosis), a stroke, a transient ischemic attack (TIA), Moyamoya disease, a hemorrhage(e.g., an intracranial hemorrhage or a subarachnoid hemorrhage), hereditary hemorrhagic telangiectasia, or reversible cerebral vasoconstriction syndrome.

45. The method of any one of claims 28-44, wherein the administering comprises administering the nucleic acid of any one of claims 17-19 or the vector of any one of claims 20-2646. The method of any one of claims 28-45, wherein the administering comprises administration to the brain of the subject.

47. The method of any one of claims 28-46, wherein the administering comprises an intracerebroventricular injection or intrathecal injection.

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