Methods and compositions for modulation of tau proteins

Abstract

To provide pharmaceutical compositions for synergistically modulating microtubule associated protein tau (MAPT) gene expression in a cell.SOLUTION: A pharmaceutical composition comprises: a first genetic modulator which comprises a first DNA-binding domain that binds to a first target site of at least 12 nucleotides in the MAPT gene, and a first transcriptional regulatory domain or a first nuclease domain; and a second genetic modulator which comprises a second DNA-binding domain that binds to a second target site of at least 12 nucleotides in the MAPT gene, and a second transcriptional regulatory domain or a second nuclease domain; where the binding of the first genetic modulator to the first target site and the binding of the second genetic modulator to the second target site results in synergistic repression of MAPT gene expression.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 740,162, filed October 2, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure is in the field of compositions and methods for modulating tau expression, including for the treatment and / or prevention of tauopathies such as Alzheimer's disease. [Background technology]

[0003] Abnormal levels and / or aggregation of tau protein, a microtubule-associated protein (also called MAPT) that can accumulate in neurofibrillary tangles (NFTs), are involved in several conditions collectively referred to as tauopathies. These include Alzheimer's disease (AD), frontotemporal dementia (FTD, see Benussi et al. (2015) Front Aging Neurosci. 7:171), progressive supranuclear palsy (PSP), intractable genetic epilepsy (e.g., Dravet syndrome, see Gheyara et al. (2014) Ann Neurol 76:443-456), traumatic brain injury (TBI), and corticobasal degeneration (CBD, see Scholz and Bras (2015) Int J. Mol Sci 16(10):24629-24655). Previous studies have shown that reducing tau protein expression in adult mice using antisense oligonucleotides administered directly into the cerebrospinal fluid (CSF) resulted in a complete or partial reduction in tau protein levels and also protected treated mice from chemically induced seizures in terms of seizure intensity (DeVos et al. (2013) J of NeuroSci 33(31):12887). In AD, for example, there is a direct correlation between the presence of tau NFTs in the brain and cognitive decline (Spires-Jones and Hyman (2014) Neuron 82:756).

[0004] It has also been suggested that tau protein may have prion-like properties, as misfolded, hyperphosphorylated tau protein may be more easily taken up by neurons and propagate disease through the brain. This misfolded tau protein isolated from the brains of AD patients can be easily taken up by mouse neurons (Takeda et al. (2015) Nat Comm doi:10.1038 / ncomms9490; Hyman (2014) Neuron 82:1189). Expression of human tau protein, associated with neurofibrillary tangles, restricted to the entorhinal cortex in a transgenic mouse model resulted in misfolding and aggregation of the mouse tau protein in neurons that did not express detectable human tau protein (de Calignon et al. (2012) Neuron 73:685-697). This study suggests that misfolded human protein can "seed" misfolding and cause aggregation of the mouse protein. Furthermore, genetic reduction or loss of endogenous mouse tau is protective against neuropathotoxicity caused by overexpression of a mutant human tau transgene (Wegmann et al. (2015) EMBO J. 34(24):3028-41).

[0005] An estimated 5.3 million Americans have Alzheimer's disease (AD), making it one of the top 10 causes of death in the United States, and it is estimated that by 2050, 106.2 million people worldwide will have the disease (van Dijk et al. (2015) Front Neurosci 9:173). The disease is more prevalent in women (two-thirds of cases), and people of African or Hispanic descent are more likely to develop AD than white people. The cause of AD is thought to be related to genetics (especially for the early-onset form, which accounts for 5% of cases) as well as environmental and lifestyle factors. Typically, the disease is diagnosed in people in their mid-60s. However, by the time a diagnosis is made, the disease has been progressing for years or even decades. The disease progresses over a long period of time, and to date, no therapeutic intervention has been identified to slow or reverse the effects of the disease.

[0006] Repression or activation of disease-associated genes has been achieved through the use of engineered transcription factors. Methods for designing and using engineered zinc finger transcription factors (ZFP-TFs) have been well documented (see, e.g., U.S. Pat. No. 6,534,261), and more recently, both transcription activator-like effector transcription factors (TALE-TFs) and clustered regularly interspaced short palindromic repeats Cas9-based transcription factors (CRISPR-Cas-TFs) have also been described (see review in Kabadi and Gersbach (2014) Methods 69(2):188-197). Non-limiting examples of target genes include phospholamban (Zhang et al. (2012) Mol Ther 20(8):1508-1515), GDNF (Langaniere et al. (2010) J. Neurosci 39(49):16469), and VEGF (Liu et al. (2001) J Biol Chem 276:11323-11334). Furthermore, gene activation has been achieved by the use of CRISPR / Cas-acetyltransferase fusions (Hilton et al. (2015) Nat Biotechnol 33(5):510-517). Engineered TFs (repressors) that suppress gene expression have also been shown to be effective in treating trinucleotide disorders such as Huntington's disease (HD). See, for example, U.S. Patent No. 8,956,828 and U.S. Patent Application Publication No. 2015 / 0335708. US Patent Application Publication No. 2018 / 0153921 discloses tau modulators. Summary of the Invention [Problem to be solved by the invention]

[0007] However, there remains a need for improved compositions and methods for the diagnosis, prevention, and / or treatment of tauopathies. Thus, described herein are compositions and methods for the prevention and / or treatment of tauopathies, including AD. [Means for solving the problem]

[0008] Disclosed herein are methods and compositions for diagnosing, preventing, and / or treating one or more tauopathies, such as Alzheimer's disease (AD). In particular, provided herein are methods and compositions for modifying (e.g., modulating) tau alleles to treat at least one tauopathy, such as AD, comprising engineered transcription factor repressors (which suppress tau protein expression). Furthermore, these methods and compositions can be used to modify MAPT alleles for the treatment and / or prevention of other tauopathies, including AD, FTD, PSP, CBD, and / or seizures. Furthermore, the use of two or more tau repressors provides surprising and unexpected synergistic effects compared with the use of a single repressor. In particular, provided herein are methods and compositions for detecting, reducing, and / or removing tau protein aggregates in vivo in subjects with tauopathies.

[0009] Thus, a gene modulator of the microtubule-associated protein tau (MAPT) gene is described herein for use in modulating tau expression in vivo. The modulator comprises at least one fusion molecule comprising a DNA-binding domain that binds to a target site of at least 12 nucleotides in the MAPT gene and a functional domain (for example, a transcriptional regulatory domain (such as a repression domain or an activation domain) or a nuclease domain). Any DNA-binding domain can be used, including, but not limited to, zinc finger protein (ZFP), TAL-effector domain protein (TALE), single guide RNA (CRISPR system), Argonaute protein, etc. In certain embodiments, the DNA-binding domain is a fusion protein comprising a zinc finger protein DNA-binding domain, for example, a ZFP-TF, i.e., a ZFP that specifically binds to a tau allele, and a transcriptional repression domain (for example, KOX, KRAB, etc.). In certain embodiments, the zinc finger protein DNA-binding domain has a recognition helix in a protein shown in Table 1, including, but not limited to, the ZFPs designated 57890, 65918, and 57930. In any of the compositions and methods described herein, two or more gene modulators are used (e.g., 65918 in combination with 57890). The two or more fusion proteins may bind to different target sites and contain the same or different functional domains. Two or more tau repressors can provide surprising and unexpected synergistic effects compared to the use of a single repressor. Alternatively, the two or more fusion proteins described herein may bind to the same target site but contain different functional domains. In some instances, three or more fusion proteins are used, in other instances, four or more fusion proteins are used, and in other instances, five or more fusion proteins are used. In preferred embodiments, two or more, three or more, four or more, or five or more fusion proteins are delivered to cells as nucleic acids (e.g., rAAV). One or more nucleic acids (e.g., AAV vectors) can be used to deliver the tau repressors described herein.In certain embodiments, the tau repressor comprises two or more tau repressors carried by a single nucleic acid vector (e.g., an AAV vector) in which the repressor-encoding sequences are separated by a 2A (e.g., T2a) sequence. In these embodiments, the sequences encoding the two or more tau repressors can be in any order (e.g., 65918 repressor-T2a-57890 repressor or 57890 repressor-T2A-65918 repressor). In preferred embodiments, the fusion protein causes repression of target gene expression. In some embodiments, the two fusion proteins are administered at doses where each protein is active on its own but where the repression activity is additive when combined. In preferred embodiments, the two fusion proteins are administered at doses where neither protein is active on its own but where the repression activity is synergistic when combined.

[0010] The gene modulators described herein may be provided to a subject in any form, including in the form of polynucleotides and / or proteins, and pharmaceutical compositions comprising such polynucleotides and / or proteins.

[0011] In some aspects, the gene modulator (or a component thereof, e.g., a DNA-binding protein) is provided in polynucleotide form. In certain embodiments, the polynucleotide is a gene delivery vector comprising any of the polynucleotides described herein (e.g., encoding a gene modulator (repressor)). In certain embodiments, the vector is an adenoviral vector (e.g., an Ad5 / F35 vector), a lentiviral vector (LV), including integration-competent or integration-deficient lentiviral vectors, or an adeno-associated viral vector (AAV). In certain embodiments, the gene modulator is carried on at least one AAV vector (or pseudotype or variant thereof), including, but not limited to, one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAVrhlO, pseudotypes of these vectors (e.g., as AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, etc.), such as AAV vector variants known in the art (e.g., U.S. Patent Nos. 9,585,971 and 7,198,951; U.S. Patent Application Publication No. 2017 / 0119906). In some embodiments, the AAV vector is an AAV variant capable of crossing the blood-brain barrier (e.g., U.S. Patent No. 9,585,971).

[0012] In certain embodiments, provided herein are tau gene modulators comprising one or more vectors, including viral and non-viral gene delivery vehicles (e.g., as mRNA, plasmids, AAV vectors, lentiviral vectors, Ad vectors), encoding a gene modulator described herein (or one or more components thereof on the same or different polynucleotides). In certain embodiments, the polynucleotide is mRNA. In some aspects, the mRNA may be chemically modified (see, e.g., Kormann et al. (2011) Nature Biotechnology 29(2):154-157). In other aspects, the mRNA may include a cap (e.g., an ARCA cap (see U.S. Pat. Nos. 7,074,596 and 8,153,773)). In further embodiments, the mRNA may include a mixture of unmodified and modified nucleotides (see U.S. Patent Application Publication No. 2012 / 0195936).

[0013] Pharmaceutical compositions and isolated cells comprising one or more gene modulators, one or more polynucleotides, and / or one or more gene delivery vehicles are also provided. In certain embodiments, a pharmaceutical composition comprises two or more gene modulators. For example, certain compositions comprise a nucleic acid comprising a sequence encoding one of the tau-modulating ZFPs, Cas, or TALEs described herein operably linked to a regulatory sequence, with a pharmaceutically acceptable carrier or diluent, wherein the regulatory sequence enables expression of the nucleic acid in the cell. In certain embodiments, the encoded ZFP, CRISPR / Cas, or TALE is specific for a mutant or wild-type MAPT allele. In some embodiments, a pharmaceutical composition comprises a ZFP, CRISPR / Cas, or TALE that modulates a mutant or wild-type MAPT allele. Protein-based compositions comprise one or more ZFPs, CRISPR / Cas, or TALEs disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0014] In other embodiments, methods and uses are described herein for suppressing MAPT expression in a subject, such as by providing one or more polynucleotides, one or more gene delivery vehicles, and / or pharmaceutical compositions to a subject in need thereof. In certain embodiments, the compositions described herein are used to suppress MAPT expression in a subject (e.g., by reducing the amount of tau in the subject), such as for treating and / or preventing a tauopathy. The compositions described herein reduce tau levels over the long term (4 weeks, 3 months, 6 months to 1 year or more) in the brain (including, but not limited to, the frontal cortex, prefrontal cortex, parietal cortex, occipital cortex, temporal cortex including, but not limited to, the entorhinal cortex, hippocampus, brainstem, striatum, thalamus, midbrain, and cerebellum) and spinal cord (including, but not limited to, the lumbar, thoracic, and cervical regions). The compositions described herein can be provided to a subject by any means of administration, including, but not limited to, intraventricular, intrathecal, intracranial, intravenous, retroorbital (RO), intranasal, and / or intracapsular administration. Kits containing one or more compositions (e.g., gene modulators, polynucleotides, pharmaceutical compositions, and / or cells) described herein and instructions for use of these compositions are also provided.

[0015] Thus, provided herein are methods for treating and / or preventing tauopathies, such as Alzheimer's disease or stroke, using the methods and compositions described herein. In some embodiments, the methods include compositions in which polynucleotides and / or proteins (or pharmaceutical compositions comprising the polynucleotides and / or proteins) can be delivered using viral vectors, non-viral vectors (e.g., plasmids), and / or combinations thereof. Administration of the compositions described herein (proteins, polynucleotides, cells, and / or pharmaceutical compositions comprising these proteins, polynucleotides, and / or cells) results in therapeutic (clinical) effects, including, but not limited to, amelioration or elimination of any clinical symptoms associated with AD, tauopathy, or stroke, and an increase in the function and / or number of CNS cells (e.g., neurons, astrocytes, myelin, etc.). In certain embodiments, the compositions and methods described herein reduce tau gene and / or protein expression (compared to a control not receiving an artificial repressor described herein) by at least 30%, or 40%, preferably at least 50%, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95%, or more than 95%. In some embodiments, a reduction of at least 50% is achieved.

[0016] Delivery can be to any brain region, for example, the hippocampus or entorhinal cortex, by any suitable means, such as by using a cannula or any other delivery technique. Any AAV vector can provide broad delivery of the repressor to the subject's brain, such as by anterograde and retrograde axonal transport to brain regions not directly administered by the vector (e.g., delivery to the nucleus results in delivery to other structures such as the cortex, substantia nigra, and thalamus). In certain embodiments, the subject is a human, and in other embodiments, the subject is a non-human primate (NHP). Administration can be in a single dose, or in a series of doses given simultaneously, or in multiple doses (with any timing between doses).

[0017] Furthermore, in any of the methods described herein, the repressor can be delivered at any concentration (dosage) that provides the desired effect. In a preferred embodiment, the repressor is delivered using an adeno-associated viral (AAV) vector at 10,000 to 500,000 vector genomes / cell (or any value therebetween). In certain embodiments, the repressor is delivered using a lentiviral vector at a multiplicity of infection (MOI) of 250 to 1,000 (or any value therebetween). In other embodiments, the repressor is delivered using a plasmid vector at 0.01 to 1,000 ng / 100,000 cells (or any value therebetween). In other embodiments, the repressor is delivered as mRNA at 0.01-3000 ng / cell (e.g., 50,000-200,000 (e.g., 100,000) cells (or any value in between). In other embodiments, the repressor is delivered using an adeno-associated viral (AAV) vector in a fixed volume of 1-300 μl into the brain parenchyma at 1E10-1E14 VG / ml (or any value in between). In other embodiments, the repressor is delivered using an adeno-associated viral (AAV) vector in a fixed volume of 0.5-10 ml into the CSF at 1E10-1E14 VG / ml (or any value in between).

[0018] Thus, in another aspect, described herein is a method for preventing and / or treating a tauopathy (e.g., AD) in a subject, comprising administering a repressor of a tau allele to the subject using one or more AAV vectors. In certain embodiments, the AAV encoding the repressor is administered to the CNS (brain and / or CSF) by any delivery method, including, but not limited to, intracerebroventricular, intrathecal, intracranial, intravenous, intranasal, retroorbital, or intracapsular delivery. In other embodiments, the AAV encoding the repressor is administered directly into the subject's parenchyma (e.g., hippocampus and / or entorhinal cortex). In other embodiments, the AAV encoding the repressor is administered intravenously (IV). In any of the methods described herein, administration may be performed once (single administration) or multiple times (with any time between administrations) with the same or different doses per administration. When multiple administrations are performed, the same or different dosages and / or delivery vehicles may be used (e.g., different AAV vectors administered by IV and / or ICV).All of the methods include reducing tau aggregation (e.g., reducing NFTs characteristic of tau aggregation) in a subject, for example, in AD neurons of a subject with AD, compared to a subject that has not been administered the method, or compared to the subject before being administered the method;reducing apoptosis in neurons or a population of neurons (e.g., AD neurons or a population of AD neurons);reducing neuronal hyperexcitability;reducing amyloid beta-induced toxicity (e.g., synaptic loss and / or neurite degeneration);and / or reducing the loss of one or more cognitive functions in AD subjects.Thus, the methods described herein result in a reduction in biomarkers and / or symptoms of tauopathies, including one or more of the following: neurotoxicity, pathological tau species (e.g., NFTs or phosphorylated tau), neurofilament light chains (NF1), CSF tau, gliosis, degenerative neurites, spinal cord loss, excitotoxicity, cortical and hippocampal shrinkage, volumetric changes associated with regions affected by particular tauopathies, dendritic tau accumulation, cognitive (e.g., radial arm maze and Morris water maze, fear conditioning, etc. in rodent models) and / or motor deficits.

[0019] In some aspects, methods and compositions are provided for reducing the amount of pathogenic tau species in cells. In some embodiments, the methods result in a reduction of hyperphosphorylated tau. In some examples, the reduction of hyperphosphorylated tau results in a reduction of soluble or particulate tau. In other embodiments, the reduction of pathogenic tau species reduces tau aggregation and causes a reduction in neurofibrillary tangles (NFTs) compared to cells or subjects not treated according to the methods and / or with the compositions described herein. In further embodiments, methods are provided for reversing the amount of NFTs observed in cells. In further embodiments, the methods and compositions of the present invention cause a slowing of the growth of pathogenic tau species (e.g., NFTs, hyperphosphorylated tau) in the brain of a subject. In some embodiments, the growth of pathogenic tau throughout the brain is halted, and in other embodiments, the growth of pathogenic tau throughout the brain is reversed. In further embodiments, the number of degenerated neurites associated with amyloid-β plaques in the brain is reduced. In some embodiments, the number of degenerated neurites is reduced to levels found in age-matched wild-type brains. In further embodiments, provided herein are methods and compositions for reducing hyperphosphorylated tau associated with amyloid-β plaques in the brain of a subject.

[0020] In some embodiments, after administration to a subject, a sequence (e.g., a ZFP-TF, a TALE-TF, or a CRISPR / Cas-TF) encoding a gene modulator (gene repressor) described herein is inserted (integrated) into the genome, while in other embodiments, the sequence encoding the repressor is maintained episomally. In some examples, a nucleic acid encoding a TF fusion is inserted (e.g., by nuclease-mediated integration) into a safe harbor site containing a promoter such that the endogenous promoter drives expression. In other embodiments, a repressor (TF) donor sequence is inserted (by nuclease-mediated integration) into a safe harbor site, where the donor sequence includes a promoter that drives expression of the repressor. In some embodiments, the sequence encoding the gene modulator is maintained extrachromosomally (episomal) after delivery and may include a heterologous promoter. The promoter may be a constitutive or inducible promoter. In some embodiments, the promoter sequence is broadly expressed, while in other embodiments, the promoter is tissue or cell / type specific. In a preferred embodiment, the promoter sequence is specific to neural cells. In another preferred embodiment, the selected promoter is characterized by its low expression. Non-limiting examples of preferred promoters include neurospecific promoters NSE, synapsin, CAMKiia and MECP. Non-limiting examples of ubiquitous promoters include CMV, CAG and Ubc. Further embodiments include the use of the autoregulatory promoter described in US Patent Application Publication No. 2015 / 0267205.

[0021] In any of the methods described herein, the method can result in about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, 98% or more, or 99% or more suppression of tau alleles in one or more AD neurons of the subject.

[0022] In certain aspects, the methods and compositions described herein are used to prevent and / or treat tauopathy.In certain embodiments, the artificial transcription factors (for example, zinc finger proteins (ZFP-TFs), TALEs (TALE-TFs), and / or CRISPR / Cas-TFs, ZFP-TFs including ZFPs designated as 65918, 57890, and / or 57930) that modulate (for example, suppress) MAPT gene expression and / or tau protein level in the central nervous system (CNS) of a subject are used to prevent and / or treat tauopathy (for example, Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear palsy, traumatic brain injury (TBI), seizure disorders and / or corticobasal degeneration), and preferably, the symptoms of tauopathy are reduced or eliminated by reducing the occurrence of neural tangles in the brain of the subject, as appropriate. In any of the methods and uses described herein, the artificial transcription factor is delivered to the CNS (e.g., brain or spinal cord) of a subject by a viral vector such as an AAV vector (e.g., AAV9), for example, intravenously, or to the CNS, optionally to the striatum or hippocampus of one or both hemispheres of the subject's brain.In certain embodiments, the AAV vector comprises a CMV or synapsin (SYN) promoter.In further embodiments, the artificial transcription factor reduces MAPT gene expression and / or tau levels in the brain of a primate subject by 50% or more, optionally 70% or more, and up to 99% compared to untreated subjects.In any of the embodiments described herein, the artificial transcription factor is carried by an AAV vector comprising a CMV or SYN promoter, with 6E11 rAAV vector genomes per hemisphere.

[0023] Kits are also provided that include one or more AAV tau modulators (e.g., repressors) and / or components of the tau modulators described herein and / or polynucleotides encoding the modulators (or components thereof). The kits may further include cells (e.g., neurons), reagents (e.g., for detecting and / or quantifying tau protein in CSF) and / or instructions for use, including methods described herein.

[0024] Thus, provided herein are compositions comprising two or more artificial zinc finger protein transcription factors (ZFP-TFs) that repress MAPT expression (e.g., ZFPs designated 65918 in combination with 57890 or 57930). Compositions comprising two or more ZFP-TFs may repress MAPT expression by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more compared to a single repressor and / or an untreated control (subject). The compositions typically comprise one or more polynucleotides encoding two or more ZFP-TFs, encoded by polynucleotides that can be carried by one or more viral (e.g., AAV, such as AAV9) vectors, e.g., a single AAV vector containing polynucleotides encoding both ZFP-TFs or separate AAV vectors encoding each ZFP-TF. Any promoter can be used to drive expression of the ZFP-TFs, including, but not limited to, the CMV and / or synapsin (SYN) promoters. One or more compositions described herein (e.g., one or more AAV vectors comprising sequences encoding two or more ZFP-TFs) can be used to prevent and / or treat a tauopathy (e.g., Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear palsy, traumatic brain injury (TBI), seizure disorders, and / or corticobasal ganglionic degeneration) in a subject in need thereof, optionally reducing or eliminating the symptoms of the tauopathy by reducing the development of neuronal tangles in the subject's brain. The compositions can be administered to the subject intravenously or to the CNS (e.g., into the striatum or hippocampus of one or both hemispheres of the subject's brain), optionally with one or more AAV vectors administered at approximately 1E10 to 6E11 rAAV vector genomes per hemisphere. Expression of two or more ZFP-TFs of the composition reduces MAPT gene expression and / or tau levels in the brain of a primate subject by 50% or more, optionally 70% or more, and up to 99% compared to an untreated subject. [Brief explanation of the drawings]

[0025] [Figure 1]Figure 1 is a graph depicting tau expression and ZFP transcript levels in an exemplary sample ("Punch 088") taken from the caudal hippocampus of a non-human primate (NHP) treated with a tau repressor described herein. The top plot shows normalized tau expression (%), and the bottom plot shows ZFP mRNA copies (transcripts / ng RNA). The left half of the graph represents data from Punch 088 obtained from the left hemisphere of each animal in the study, and the right half shows data from Punch 088 obtained from the right hemisphere of each animal in the study. The promoters used in the different constructs are indicated along the bottom, with "CMV" indicating use of the CMV promoter and "SYN" indicating use of the synapsin promoter.

[0026] [Figure 2] 2 is a graph depicting tau expression and ZFP levels in exemplary samples taken from NHPs treated with tau repressors described herein ("Punch 088" in the upper panel taken from the caudal hippocampus and "Punch 035" in the lower panel taken from the rostral hippocampus). The upper plot in each panel shows normalized tau suppression (%), and the lower plot in each panel shows ZFP mRNA levels (copies / ng mRNA).

[0027] [Figure 3] Figure 3 shows graphs depicting ZFP levels and unscaled tau protein expression levels (upper panel), tau protein expression levels scaled relative to the average of three vehicle-treated animals (middle panel), or tau protein expression levels scaled relative to the average of vehicle- and ZFP-treated animals that showed no detectable ZFP expression (lower panel) for the indicated NHP brain samples ("Punch 037," "Punch 039," and "Punch 061," derived from the hippocampus). The upper plot in each panel shows normalized tau suppression (%) (left axis), and the lower plot in each panel shows ZFP mRNA levels (copies / ng mRNA) (right axis).

[0028] [Figure 4] Figure 4 is a graph depicting tau expression and ZFP levels from 74 punches taken from various brain slices (5, 6, 7, 8, and 9 in each hemisphere) in rostral to caudal order in an exemplary NHP subject (NHP07) treated with a tau repressor described herein. The top plot in each panel shows normalized tau suppression (%) (left axis), and the bottom plot in each panel shows ZFP mRNA levels (copies / ng mRNA) (right axis).

[0029] [Figure 5A] Figures 5A and 5B depict tau expression and ZFP levels in control and treated NHP subjects and the corresponding MRI images. Figure 5A is a graph showing tau expression and ZFP levels focused on a punch from brain slice 7, showing the cortex and hippocampus in control ("vehicle" shown in the left panel) and NHP subjects treated with tau repressors 65918 ("918") and 57890 ("890"), delivered by an AAV vector (AAV9) in which expression of the repressors (918 and 890) is driven by the synapsin (SYN1) promoter and linked by a T2A cleavage peptide. The upper plot in each panel shows normalized tau suppression (%), and the lower plot in each panel shows ZFP mRNA levels (copies / ng mRNA). Figure 5B depicts MRI scans from the same control (left panel) and treated (right panel) subjects at the level of the injection tract. Co-infused gadolinium tracer is evident in the hippocampal regions of both hemispheres. [Figure 5B] Same as above

[0030] [Figure 6A]Figures 6A-6E are graphs depicting tau expression and ZFP levels in samples taken from control and treated NHP subjects. Figure 6A shows results from control subjects (NHP01, NHP02, and NHP03). Figure 6B shows results from NHP subjects (NHP04, NHP05, and NHP06) treated with gene repressors 65918 ("918") and 57890 ("890") carried by an AAV vector (AAV9) in which expression of the repressors (918 and 890) is driven by the synapsin (SYN1) promoter ("SYN1.918-890"). Figure 6C shows results from subjects (NHP07 and NHP08) treated with gene repressors 65918 ("918") and 57890 ("890") carried by an AAV vector (AAV9) in which expression of the repressors (918 and 890) is driven by a CMV promoter ("CMV.918-890") (left panel), and subjects (NHP09 and NHP10) treated with gene repressor 57930 ("930") carried by an AAV vector (AAV9) in which expression of the repressor (930) is driven by the synapsin (SYN1) promoter ("SYN1.930") (right panel). Figure 6D shows results from subjects (NHP11, NHP12, and NHP13) treated with 57890 ("890"), carried by an AAV vector (AAV9) in which repressor expression is driven by the synapsin (SYN1) promoter ("SYN1.890"). Figure 6E shows results from subjects (NHP14 and NHP15) treated with 65918 ("918"), carried by an AAV vector (AAV9) in which repressor expression is driven by the synapsin (SYN1) promoter ("SYN1.918"). The upper plot in each panel shows normalized tau suppression (%), and the lower plot in each panel shows ZFP mRNA levels (copies / ng mRNA). [Figure 6B] Same as above [Figure 6C] Same as above [Figure 6D] Same as above [Figure 6E] Same as above

[0031] [Figure 7] 7 is a graph depicting a combined analysis of ZFP levels and unscaled tau expression levels (top panel) for the indicated NHP brain samples (punch ID on the X-axis), or tau expression scaled relative to the average of three vehicle-treated animals (middle panel), or tau expression scaled relative to the average of vehicle- and ZFP-treated animals that showed no detectable ZFP expression (bottom panel). The left panel shows control subjects ("vehicle"), and the right panel shows all treated subjects ("all AAV-treated NHPs").

[0032] [Figure 8] Figure 8 is a graph depicting a combined analysis of tau expression (top panel) and ZFP levels (bottom panel) in control (left graph) and treated subjects (right graph) using a method that scales tau expression relative to the average of vehicle-treated and ZFP-treated animals, which show no detectable ZFP expression.

[0033] [Figure 9]FIG. 9 is a graph depicting tau expression in the left (left panel) and right (right panel) hemispheres of the indicated control and treated subjects. "VEH" refers to a control subject administered vehicle only; "SYN918-890" refers to a subject receiving an AAV vector encoding the 65918 and 57890 gene repressors, the expression of which is driven by the synapsin promoter; "CMV918-890" refers to a subject receiving an AAV vector encoding the 65918 and 57890 gene repressors, the expression of which is driven by the CMV promoter; "SYN930" refers to a subject receiving an AAV vector encoding the 57930 gene repressor, the expression of which is driven by the synapsin promoter; "SYN890" refers to a subject receiving an AAV vector encoding the 57890 gene repressor, the expression of which is driven by the synapsin promoter; and "SYN918" refers to a subject receiving an AAV vector encoding the 65918 gene repressor, the expression of which is driven by the synapsin promoter. For this analysis, normalized tau expression was scaled relative to the average of tau levels measured from vehicle-treated and ZFP-treated animals that showed no detectable ZFP expression for each punch.

[0034] [Figure 10] Figure 10 is a graph depicting tau expression levels in the indicated NHP subjects with ZFP transcript levels below 1E4 ZFP transcripts (copies / ng mRNA) (left panel); 1E4-1E5 transcripts (copies / ng mRNA) (middle panel); and above 1E5 transcripts (copies / ng mRNA) (right panel). Levels in the left hemisphere are shown in the upper panel, and levels in the right hemisphere are shown in the lower panel. For this analysis, normalized tau expression for a given punch was scaled relative to the average of tau levels measured from vehicle-treated and ZFP-treated animals that showed no detectable ZFP expression.

[0035] [Figure 11]Figure 11 is a graph depicting tau expression levels in the left and right hemispheres in the indicated NHP subjects, where ZFP transcript levels are shown for ZFP transcript levels (upper left panel), lower than 1E4 ZFP transcripts (copies / ng mRNA) (lower left panel), 1E4-1E5 transcripts (copies / ng mRNA) (upper right panel), and higher than 1E5 transcripts (copies / ng mRNA) (lower right panel). For this analysis, normalized tau expression for a given punch was scaled relative to the average of tau levels measured from vehicle-treated and ZFP-treated animals that showed no detectable ZFP expression.

[0036] [Figure 12] Figure 12 is a graph depicting a correlation plot showing tau expression and ZFP transcript levels in control subjects (left panel—"vehicle") and subjects treated with an AAV vector encoding the 65918 and 57890 gene repressors, whose expression is driven by the synapsin promoter ("hSYN1.5789-65918"). The limit of the ZFP qRT-PCR assay for absolute quantification is approximately 1E2 transcripts / ng mRNA, as indicated by the lower limit of quantification (BLOQ). For this analysis, tau expression is scaled relative to the average of vehicle- and ZFP-treated animals, which show no detectable ZFP expression.

[0037] [Figure 13] Figure 13 is a correlation plot showing tau expression at the indicated ZFP transcript levels for the indicated treatment groups. For this analysis, tau expression levels for each punch are scaled to the average of three vehicle-treated animals. The top panel shows the relationship across all ZFP mRNA levels; the bottom panel shows only the range of ZFP expression associated with tau reduction, approximately 1E4 to 1E6 ZFP transcripts / ng mRNA. Also shown are R-squared and P-values. For this analysis, tau expression is scaled to the average of vehicle- and ZFP-treated animals, which showed no detectable ZFP expression.

[0038] [Figure 14] Figure 14 is a correlation plot showing tau expression and ZFP transcript levels in control subjects (left panel—"vehicle") and subjects treated with an AAV vector encoding the 65918 and 57890 gene repressors, whose expression is driven by the synapsin promoter ("hSYN1.5789-65918"). The limit of the ZFP qRT-PCR assay for absolute quantification is approximately 1E2 transcripts / ng mRNA, as indicated by the lower limit of quantification (BLOQ). In contrast to Figure 12, for this analysis, tau expression levels are scaled relative to the average of three vehicle-treated animals.

[0039] [Figure 15] Figure 15 is a correlation plot showing tau expression at the indicated ZFP transcript levels for the indicated treatment groups. For this analysis, tau expression levels for each punch are scaled relative to the average of three vehicle-treated animals. The top panel shows the relationship across all ZFP mRNA levels; the bottom panel shows only the range of ZFP expression associated with tau reduction, approximately 1E4 to 1E6 ZFP transcripts / ng mRNA. Also shown are R-squared and P values. In contrast to Figure 13, for this analysis, tau expression levels are scaled relative to the average of three vehicle-treated animals.

[0040] [Figure 16] Figure 16 is a correlation plot showing tau expression and ZFP transcript levels in control subjects (left panel - "vehicle") and subjects treated with an AAV vector encoding the 65918 and 57890 gene repressors, whose expression is driven by the synapsin promoter ("hSYN1.5789-65918"). The limit of the ZFP qRT-PCR assay for absolute quantification is approximately 1E2 transcripts / ng mRNA, as indicated by the lower limit of quantification (BLOQ). For this analysis, tau expression levels were not scaled to correct for baseline tau levels in untreated or ZFP-negative animals.

[0041] [Figure 17] Figure 17 is a correlation plot showing tau expression at the indicated ZFP transcript levels for the indicated treatment groups. For this analysis, tau expression levels for each punch are scaled relative to the average of three vehicle-treated animals. The top panel shows the relationship across all ZFP mRNA levels; the bottom panel shows only the range of ZFP expression associated with tau reduction, approximately 1E4 to 1E6 ZFP transcripts / ng mRNA. Also shown are R-squared and P values. For this analysis, tau expression levels were not scaled to correct for baseline tau levels in untreated or ZFP-negative animals.

[0042] [Figure 18A]Figures 18A-18D are bar graphs depicting results from humanized tau mice treated with tau ZFP-TFs described herein. Figure 18A shows relative (left graph) and absolute (right graph) ZFP levels in mice treated with the indicated constructs. As shown, mice treated with the synergistic combination of 65918 and 57890 exhibited expression levels two-fold higher than 57890 alone. Figure 18B shows human tau ("hTAU," left graph), mouse tau ("mTAU," center graph), and GFP (right graph) expression levels in mice treated with the indicated constructs. As shown, human tau mRNA was suppressed by approximately 90% by 65918-T2a-57890, compared to approximately 60% suppression by 57890. Similarly, mouse tau mRNA was suppressed by approximately 87% by 65918-T2a-57890, compared with approximately 81% suppression by 57890 alone. Figure 18C depicts the expression of hTAU (left graph) and human Saitohin ("hSTH," right graph) in humanized tau mice treated under the indicated conditions. Consistent with the fact that STH is packaged within tau and thus the two genes are co-regulated, similar levels of human tau and STH reduction were observed. Figure 18D shows the expression levels of GFAP (astrocyte marker) (left graph), IBA1 (microglia marker) (middle graph), and NeuN (neuronal marker) (right graph) in humanized tau mice treated with the indicated constructs. [Figure 18B] Same as above [Figure 18C] Same as above [Figure 18D] Same as above

[0043] [Figure 19A]Figures 19A-19C are graphs depicting increased expression levels of microglial and astrocyte markers in non-human primates (NHPs) after treatment with the indicated tau-specific ZFP-TFs expressed from ZFP-TF constructs containing either the CMV or synapsin (SYN) promoter. Figures 19A and 19B show levels of ionized calcium-binding adaptor molecule 1 (IBA1), a marker of microglia. Microglial activation is indicative of an inflammatory response. Figure 19C shows astrocyte activation (as measured by GFAP levels). As shown, there was no ZFP-dependent increase in microglial or astrocyte levels for any of the SYN promoter-containing constructs, whereas there was an increase in microglial and astrocyte markers for the CMV promoter-containing constructs. [Figure 19B] Same as above [Figure 19C] Same as above

[0044] [Figure 20A] Figures 20A and 20B are graphs showing bulk levels of IBA1 (Figure 20A) and GFAP (Figure 20B) in the left ("L") and right ("R") hemispheres of primates treated as indicated. As shown, IBA1 and GFAP levels vary among NHPs, but no significant bulk effects were observed for any of the treatment groups. [Figure 20B] Same as above

[0045] [Figure 21] 21 is a graph depicting the normalized expression of the housekeeping gene EIF4a2 relative to ZFP in subjects treated under the indicated conditions. As shown, there was no correlation with the levels of housekeeping gene (EIF4A2) and ZFP expression in either treatment group.

[0046] [Figure 22]Figure 22 is a graph depicting total tau protein levels in human iPSC-derived (IPS) neurons under the indicated conditions. "Mock" refers to cells transfected with an empty AAV vector; "Control" refers to cells transfected with an AAV vector encoding a ZFP that does not target tau; "57930" refers to cells treated with an AAV vector encoding the 57930 repressor; "57890" refers to cells treated with an AAV vector encoding the 57890 repressor; "65918" refers to cells treated with an AAV vector encoding the 65918 repressor; and "65918 / 57890" refers to cells treated with an AAV vector encoding both the 65918 and 57890 repressors. All AAV constructs encoding ZFP repressors contained synapsin promoter (SYN1)-driven expression of the repressor. Tau protein levels were assessed by ELISA 32 days after AAV vector administration. DETAILED DESCRIPTION OF THE INVENTION

[0047] Disclosed herein are compositions and methods for preventing and / or treating tauopathy.In particular, the compositions and methods described herein are used to suppress the expression of MAPT (tau) protein to prevent or treat tauopathy, such as Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear palsy, traumatic brain injury (TBI), seizure disorders and / or corticobasal ganglionic degeneration.MAPT repressors (e.g., MAPT-modulating transcription factors, such as MAPT-modulating transcription factors including zinc finger proteins (ZFP TFs), TALE (TALE-TFs), and / or CRISPR / Cas-TFs) modify the CNS, for example, by reducing tau aggregation and reducing the occurrence of neuronal tangles in the brain of subjects with tauopathy (e.g., AD), thereby reducing or eliminating the effects and / or symptoms of tauopathy. In a preferred embodiment, MAPT-modulating transcription factors are delivered to the brain by viral vectors such as AAV.AAV has been shown to be well suited for delivery to the brain, and therefore the use of these viral vectors to deliver MAPT-modulating transcription factors is particularly useful for treating diseases such as Alzheimer's disease, which are associated with the inappropriate expression and subsequent aggregation of tau protein.

[0048] The microtubule-associated protein tau (MAPT) is closely associated with the development of several neurodegenerative disorders, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While gene- and antisense-based approaches to tau reduction are effective and well tolerated in mice, the development of single-dose, intracellular tau-targeted therapies remains a long-standing goal. Advances in zinc finger protein (ZFP) design and AAV delivery have created new possibilities for single-dose DNA-targeted therapies for neurodegenerative diseases. ZFP repressors targeting mouse, nonhuman primate (NHP), and human tau transcriptional regulatory elements were delivered into mouse and NHP brains using AAV vectors. MAPT-targeting ZFPs reduced mouse and human tau by up to 99%, with no detectable off-target gene regulation in primary and iPSC neurons. Intrahippocampal ZFP delivery to adult mice resulted in >90% tau reduction. Intravenous ZFP administration reduced tau levels throughout the brain by 50–70%. ZFP expression and mouse tau reduction remained stable for at least 6 months in APP / PS1 mice, with no detectable off-target activity, resulting in a greater than 80% reduction in CSF tau and a 50% reduction in degenerating neurites. Using stereotaxic techniques guided by bilateral real-time MRI, ZFPs were delivered to the hippocampus of NHPs. They were well tolerated and resulted in a maximum reduction of greater than 80% in tau in the hippocampus and entorhinal cortex. ZFP levels were strongly correlated with tau reduction. The potency, efficacy, specificity, and tolerability of ZFPs indicate that they can achieve sustained tau downregulation for the treatment of human tauopathies.

[0049] general The practice of the methods, and the preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields that are within the knowledge of those of ordinary skill in the art, and these techniques are fully explained in the literature. See, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and its periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin" (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols." (PB Becker, ed.) Humana Press, Totowa, 1999.

[0050] definition The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in linear or cyclic conformation, in single-stranded or double-stranded form. For purposes of this disclosure, these terms should not be interpreted as limiting the length of the polymer. The terms may encompass known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, analogs of a particular nucleotide have the same base-pairing specificity; i.e., an analog of A will base pair with T.

[0051] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0052] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the overall interaction is sequence-specific. Such interactions generally occur in the presence of a single molecule. -6 M -1 The following dissociation constants (K d "Affinity" refers to the strength of binding, and increased binding affinity is characterized by a lower K d correlates with.

[0053] A "binding protein" is a protein that can non-covalently bind to another molecule. A binding protein can bind, for example, to a DNA molecule (DNA-binding protein), an RNA molecule (RNA-binding protein), and / or a protein molecule (protein-binding protein). In the case of a protein-binding protein, it can bind to itself (forming a homodimer, homotrimer, etc.) and / or to one or more molecules of a different protein or proteins. A binding protein may have more than one type of binding activity. For example, a zinc finger protein has DNA-binding, RNA-binding, and protein-binding activity.

[0054] A "zinc finger DNA-binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0055] A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are responsible for binding of a TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within naturally occurring TALE proteins. See, e.g., U.S. Patent No. 8,586,526.

[0056] "TtAgo" is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus. (See, e.g., Swarts et al. (2014) Nature 507(7491):258-261; G. Sheng et al. (2013) Proc. Natl. Acad. Sci. USA 111, 652.) A "TtAgo system" refers to all the components required, such as guide DNA, for cleavage by the TtAgo enzyme. "Recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, non-homologous end joining (NHEJ) and donor capture via homologous recombination. For purposes of this disclosure, "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells via the homology-directed repair mechanism. This process requires nucleotide sequence homology, uses a "donor" molecule to template repair of a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion" because it results in the transfer of genetic information from the donor to the target. Without being bound by any particular theory, such transfer may involve mismatch correction of heteroduplex DNA formed between the cleaved target and the donor, and / or "synthesis-dependent strand annealing," in which the donor is used to resynthesize the genetic information that will become part of the target, and / or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that some or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0057] DNA-binding domains such as sgRNAs, zinc finger binding domains, or TALE DNA-binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by designing an sgRNA that binds to a selected target site, or by manipulating the recognition helix region of a naturally occurring zinc finger protein (changing one or more amino acids), or by manipulating the RVD of a TALE protein. Thus, engineered zinc finger proteins or TALEs are proteins that do not occur in nature. A non-limiting example of a method for engineering a DNA-binding domain is design and selection. A "designed" zinc finger protein or TALE is a protein that does not occur in nature whose design / composition results primarily from rational criteria. Rational criteria for design include the application of substitution rules and computer algorithms to manipulate information in databases of existing ZFP design and binding data. A "selected" zinc finger protein or TALE is a protein not found in nature whose production results primarily from empirical processes such as phage display, interaction capture, or hybrid selection. See, e.g., U.S. Patent Nos. 8,586,526; 6,140,081; 6,453,242; 6,746,838; 7,241,573; 6,866,997; 7,241,574; and 6,534,261; see also International Patent Application Publication No. WO 03 / 016496.

[0058] The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA, linear, circular, or branched, and either single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence to be inserted into a genome. The donor sequence may be of any length, for example, 2 to 10,000 nucleotides in length (or any integer value therebetween or above), preferably about 100 to 1,000 nucleotides in length (or any integer value therebetween), and more preferably about 200 to 500 nucleotides in length.

[0059] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided conditions sufficient for binding exist.

[0060] An "exogenous" molecule is one that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods. "Normal presence in a cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic muscle development is an exogenous molecule with respect to a mature muscle cell. Similarly, a molecule that is induced by heat shock is an exogenous molecule with respect to a cell that has not been subjected to heat shock. Exogenous molecules may include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0061] Exogenous molecules may be small molecules, particularly those produced by combinatorial chemical processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, and may be single-stranded or double-stranded, linear, branched, or circular, and may be of any length. Nucleic acids include those capable of forming duplexes as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0062] An exogenous molecule may be the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, an exogenous nucleic acid may include an infectious viral genome, a plasmid or episome introduced into a cell, or a chromosome not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated introduction (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated introduction, and viral vector-mediated introduction. An exogenous molecule may also be the same type of molecule as an endogenous molecule, but may be derived from a species different from that from which the cell is derived. For example, a human nucleic acid sequence can be introduced into a cell line originally derived from a mouse or hamster.

[0063] In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, endogenous nucleic acids may include chromosomes, the genomes of mitochondria, chloroplasts, or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules may include proteins, such as transcription factors and enzymes.

[0064] A "fusion" molecule is a molecule in which two or more subunit molecules are preferably covalently linked. The subunit molecules may be of the same chemical type or different chemical types. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (e.g., fusions of a ZFP or TALE DNA binding domain with one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the above-mentioned fusion proteins). Examples of the second type of fusion molecule include, but are not limited to, fusions of a triplex-forming nucleic acid with a polypeptide and a fusion of a minor groove binder with a nucleic acid. This term also includes systems in which a polynucleotide component associates with a polypeptide component to form a functional molecule (e.g., a CRISPR / Cas system in which a single guide RNA associates with a functional domain to modulate gene expression).

[0065] Expression of the fusion protein in a cell may result from delivery of the fusion protein to the cell, or may occur by delivery of a polynucleotide encoding the fusion protein to the cell, where the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation may be involved in expression of the protein in the cell. Methods for delivery of polynucleotides and polypeptides to cells are presented elsewhere in this disclosure.

[0066] A "multimerization domain" (also referred to as a "dimerization domain" or "protein interaction domain") is a domain incorporated into the amino-, carboxy-, or amino- and carboxy-terminal regions of a ZFP TF or TALE TF. These domains enable the multimerization of multiple ZFP TF or TALE TF units, such that larger tracts of trinucleotide repeat domains become selectively bound by the multimerized ZFP TF or TALE TF compared to shorter tracts with wild-type length. Examples of multimerization domains include leucine zippers. Multimerization domains can also be regulated by small molecules that allow the multimerization domain to adopt an appropriate conformation to interact with another multimerization domain only in the presence of a small molecule or external ligand. In this way, exogenous ligands can be used to regulate the activity of these domains.

[0067] "Gene," for purposes of this disclosure, includes a DNA region that encodes a gene product (see above), as well as all DNA regions that regulate the production of the gene product, regardless of whether regulatory sequences flank the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0068] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product may be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

[0069] "Modulation" of gene expression refers to a change in the activity of a gene. Modulation of expression may include, but is not limited to, gene activation and gene repression. Genome editing (e.g., truncation, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any reduction in gene expression compared to cells that do not contain the ZFP or TALE protein described herein. Thus, gene inactivation may be partial or complete.

[0070] "Gene modulator" refers to any molecule that alters the expression and / or sequence of one or more genes. Non-limiting examples of gene modulators include transcription factors (such as the artificial transcription factors described herein) that bind to and alter the expression of a target gene, and nucleases that modify the sequence of a target gene and then alter its expression (e.g., inactivating the target by insertion and / or deletion). Thus, a gene modulator may be a gene repressor (which suppresses and / or inactivates gene expression) or a gene activator.

[0071] A "region of interest" is any region of cellular chromatin, such as a gene or non-coding sequence within or adjacent to a gene, to which it is desired to bind an exogenous molecule. Binding may be for targeted DNA cleavage and / or targeted recombination. A region of interest may be present, for example, in a chromosome, episome, organelle genome (e.g., mitochondria, chloroplasts), or infectious viral genome. A region of interest may be within the coding region of a gene, within a transcribed non-coding region such as a leader sequence, trailer sequence, or intron, or within a non-transcribed region upstream or downstream of a coding region. A region of interest may be as small as a single nucleotide pair or up to 2,000 nucleotide pairs in length, or any integer number of nucleotide pairs.

[0072] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (eg, T cells).

[0073] The terms "operably linked" and "operatively linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) where the components are arranged in a manner that allows for both components to function normally and allows at least one component to mediate a function exerted on at least one of the other components. By way of illustration, a transcriptional regulatory sequence such as a promoter is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even though they are not contiguous.

[0074] With respect to a fusion polypeptide, the term "operably linked" may refer to the fact that each component, in association with the other component, performs the same function that it would perform if it were not so linked. For example, with respect to a fusion molecule in which a ZFP or TALE DNA-binding domain is fused to an activation domain, the ZFP or TALE DNA-binding domain and the activation domain are in operable linkage if, in the fusion polypeptide, the ZFP or TALE DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the activation domain is capable of upregulating gene expression. ZFPs fused to domains capable of regulating gene expression are collectively referred to as "ZFP-TFs" or "zinc finger transcription factors," while TALEs fused to domains capable of regulating gene expression are collectively referred to as "TALE-TFs" or "TALE transcription factors." In the case of a fusion polypeptide in which a ZFP DNA-binding domain is fused to a cleavage domain ("ZFN" or "zinc finger nuclease"), the ZFP DNA-binding domain and the cleavage domain are operably linked if, in the fusion polypeptide, the ZFP DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the cleavage domain is capable of cleaving DNA near the target site. In the case of a fusion polypeptide in which a TALE DNA-binding domain is fused to a cleavage domain ("TALEN" or "TALE nuclease"), the TALE DNA-binding domain and the cleavage domain are operably linked if, in the fusion polypeptide, the TALE DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the cleavage domain is capable of cleaving DNA near the target site. In the case of a fusion molecule in which a Cas DNA-binding domain (e.g., a single guide RNA) is fused to an activation domain, the Cas DNA-binding domain and the activation domain are operably linked if, in the fusion polypeptide, the Cas DNA-binding domain portion is capable of binding to its target site and / or its binding site, while the activation domain is capable of up-regulating gene expression.In the case of a fusion polypeptide in which a Cas DNA-binding domain is fused to a cleavage domain, the Cas DNA-binding domain and the cleavage domain are operably linked if, in the fusion polypeptide, the Cas DNA-binding domain portion is capable of binding to its target site and / or its binding site, but the cleavage domain is capable of cleaving DNA near the target site.

[0075] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to the full-length protein, polypeptide, or nucleic acid, but which still retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment may have more, fewer, or the same number of residues as the corresponding native molecule and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays, or both genetic and biochemical complementation. See, e.g., Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and International Patent Application Publication No. WO 98 / 44350.

[0076] A "vector" is capable of transferring a gene sequence into a target cell. Typically, the terms "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and transferring a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as integration vectors.

[0077] A "reporter gene" or "reporter sequence" preferably, but not necessarily, refers to any sequence that results in a protein product that is easily measured in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored, fluorescent, or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and / or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. An "expression tag" includes a sequence encoding a reporter that may be operably linked to a desired gene sequence to monitor the expression of the gene of interest.

[0078] Throughout this specification and the embodiments, the words "have" and "comprises" or variations such as "has," "having," "comprises," or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. As used herein, the term "approximately" or "about," as applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that are within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or lesser) of a stated reference value, unless otherwise stated or clear from the context.

[0079] Tau and Alzheimer's Disease Tau protein is encoded by the MAPT gene, which contains 16 exons. Interestingly, exons 1, 4, 5, 7, 9, 11, and 12 are constitutively expressed, whereas exons 2, 3, and 10 can exist in tau protein species derived from alternatively spliced ​​variants, resulting in the existence of six distinct tau protein isoforms in the adult brain. Tau binds to microtubules via three or four repeated tubulin-binding motifs in the C-terminal half of the protein, and tau 4R (four tubulin-binding motifs) is thought to interact more strongly with microtubules than tau 3R, stabilizing tubules. The ratio of 3R to 4R is generally stable but can be affected by pathological conditions. Tau forms that interact with microtubules are phosphorylated, and hyperphosphorylation is thought to cause tau detachment from microtubules. Hyperphosphorylated tau can lead to conformational changes and aggregation in the protein after sequestration in cells. These aggregates may be an early step in the formation of pathogenic neurofibrillary tangles (NFTs), but hyperphosphorylated tau can be pathogenic not only when present in neurofibrillary tangles but also in soluble forms (Bodea et al. (2016) J of Neurochem 138(Suppl 1):71-94). NFTs are limited to the entorhinal cortex and medial temporal lobe in the early stages of AD, but by the time severe clinical symptoms of the disease appear, NFTs have spread throughout the brain. Consistent with the presence of abundant NFTs, a widespread distribution of amyloid plaques also occurs. Indeed, amyloid deposition in the cortex is thought to lead to an increased rate of tau propagation and the spread of NFTs to distal regions of the brain. As tau tangles spread, there is a concomitant increase in neuronal loss (Pooler et al. (2015) Acta Neuropathol Commun 3:14, doi:10.1186 / s40478-015-0199-x).

[0080] Tau has 95 amino acid residues that can be phosphorylated, and several kinases that can be responsible for tau phosphorylation have been identified, which may be potential target candidates for new therapeutic agents, such as glycogen synthase-3, cyclin-dependent kinase 5, members of the MAPK family, extracellularly regulated kinase, c-Jun N-terminal kinase, and microtubule affinity-regulating kinase (Bodea (2016) ibid).

[0081] Amyloid beta protein (Aβ) is the primary component of senile plaques and, along with NFTs, is a hallmark of Alzheimer's disease neuropathology. Aβ is a peptide with a chain of 39–42 amino acids; 42 amino acids form more robust aggregates and are thought to be involved in the pathogenesis of the disease, forming the basis of the amyloid hypothesis (the proposal that the accumulation of Aβ in the brain is the primary cause of AD; for a review, see Hardy and Selkoe (2002) Science 297:353). Aβ is the product of proteolytic cleavage of amyloid precursor protein (APP), a ubiquitous, glycosylated, sulfated, and phosphorylated integral membrane protein (Sorrentino et al. (2014) FEBS Lett 588:641–652). However, the pathogenesis leading to AD is highly complex, and it has become clear that the pathogenesis of Aβ accumulation may play a role in abnormal tau behavior (Ando et al. (2016) PLoS Genet 12(3):e1005917).

[0082] Reduction of tau in the brain has been shown to ameliorate AD pathology. Regulated suppression of tau transgene expression in a mouse AD model demonstrated a reduction in transgene-bound tau aggregates and reduced concentrations of hyperphosphorylated tau and NFTs. Indeed, this study also demonstrated a loss of overall NFTs, indicating that NFT accumulation may be reversible (Polydoro et al. (2013) J of Neurosci 33(33):13300-13311). Furthermore, a study using an intracellular anti-tau antibody delivered directly via AAV intrahippocampal administration demonstrated a reduction in insoluble tau species, NFTs, and rescue of hippocampal atrophy observed in untreated mouse models (Liu et al. (2016) J Neurosci 36(49):12425-12435).

[0083] DNA-binding domain The methods described herein utilize compositions, such as tau-modulating transcription factors, that contain DNA-binding domains that specifically bind to target sequences in the tau (MAPT) gene. Any polynucleotide or polypeptide DNA-binding domain, such as a DNA-binding protein (e.g., ZFP or TALE) or a DNA-binding polynucleotide (e.g., single guide RNA), can be used in the compositions and methods disclosed herein. Thus, gene modulators (repressors) of the tau gene are described.

[0084] In certain embodiments, the tau repressor or the DNA-binding domain therein comprises a zinc finger protein. Methods for selecting target sites; designing and constructing ZFPs and fusion proteins (and polynucleotides encoding same) are known to those skilled in the art and are described in U.S. Patent Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; etc. and in International Patent Application Publications WO95 / 19431; WO96 / 06166; WO98 / 53057; WO98 / 54311; WO00 / 27878; WO01 / 60970; WO01 / 88197; WO02 / 099084; WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536; and WO03 / 016496.

[0085] Tau target sites typically contain at least one zinc finger but may contain multiple zinc fingers (e.g., 2, 3, 4, 5, 6, or more fingers). ZFPs typically contain at least three fingers. Certain ZFPs contain 4, 5, or 6 fingers, while some contain 8, 9, 10, 11, or 12 fingers. ZFPs containing three fingers typically recognize target sites containing 9 or 10 nucleotides, ZFPs containing four fingers typically recognize target sites containing 12-14 nucleotides, while ZFPs with six fingers can recognize target sites containing 18-21 nucleotides. ZFPs can also be fusion proteins containing one or more regulatory domains, which may be transcriptional activation or repression domains. In some embodiments, fusion proteins contain two ZFP DNA-binding domains linked together. Thus, these zinc fingers may contain 8, 9, 10, 11, 12, or more fingers. In some embodiments, the two DNA-binding domains are linked by an extendable, flexible linker, such that one DNA-binding domain contains 4, 5, or 6 zinc fingers and the second DNA-binding domain contains an additional 4, 5, or 5 zinc fingers. In some embodiments, the linker is a standard inter-finger linker, such that the finger array contains one DNA-binding domain containing 8, 9, 10, 11, or 12 or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. The DNA-binding domain may be fused to at least one regulatory domain, which may be considered a "ZFP-ZFP-TF" construct. Specific examples of these embodiments may be referred to as "ZFP-ZFP-KOX," which contains two DNA-binding domains linked by a flexible linker and fused to a KOX repressor, and "ZFP-KOX-ZFP-KOX," in which two ZFP-KOX fusion proteins are fused together via a linker.

[0086] The engineered zinc finger binding domain may have new binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, involves using a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence. See, for example, co-owned U.S. Patent Nos. 6,453,242 and 6,534,261, the entire contents of which are incorporated herein by reference.

[0087] Furthermore, as disclosed in these and other references, zinc finger domains and / or multi-finger zinc finger proteins can be linked together using any suitable linker sequence, such as, for example, a linker of 5 or more amino acids in length. See also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences of 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

[0088] ZFPs can be operably linked (linked) to one or more transcriptional regulatory (e.g., repression) domains to form ZF-TFs (e.g., repressors). Methods and compositions can also be used to increase the specificity of a ZFP for its intended target relative to other unintended cleavage sites, known as off-target sites, by mutations to the ZFP backbone, for example, as described in U.S. Patent Application Publication No. 2018 / 0087072. Thus, the tau repressors described herein may contain mutations in one or more of its DNA-binding domain backbone regions and / or one or more mutations in its transcriptional regulatory domain. These ZFPs may contain mutations to amino acids within the ZFP DNA-binding domain ("ZFP backbone") that can nonspecifically interact with phosphates on the DNA backbone, but they do not contain changes in the DNA recognition helix. Thus, the present invention includes mutations of cationic amino acid residues in the ZFP backbone that are not required for nucleotide target specificity. In some embodiments, these mutations in the ZFP backbone include mutating cationic amino acid residues to neutral or anionic amino acid residues. In some embodiments, these mutations in the ZFP backbone include mutating polar amino acid residues to neutral or nonpolar amino acid residues. In preferred embodiments, mutations are introduced at positions (-5), (-9), and / or (-14) relative to the DNA-binding helix. In some embodiments, a zinc finger may contain one or more mutations at positions (-5), (-9), and / or (-14). In further embodiments, one or more zinc fingers in a multi-finger zinc finger protein may contain mutations at positions (-5), (-9), and / or (-14). In some embodiments, the amino acid at positions (-5), (-9), and / or (-14) (e.g., arginine (R) or lysine (K)) is mutated to alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y), and / or glutamine (Q).

[0089] Alternatively, the DNA-binding domain may be derived from a nuclease, for example, recognition sequences for homing endonucleases and meganucleases are known, such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII. See also U.S. Patent No. 5,420,032; U.S. Patent No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22:1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. Furthermore, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites.See, for example, Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Application Publication No. 2007 / 0117128.

[0090] A "two-handed" zinc finger protein is a protein in which two clusters of zinc finger DNA-binding domains are separated by an intervening amino acid, and the two zinc finger domains bind to two discontinuous target sites. An example of a two-handed zinc finger binding protein is SIP1, in which a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three fingers is located at the carboxyl terminus (see Remacle et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins can bind to a unique target sequence, and the spacing between the two target sequences can include many nucleotides. A two-handed ZFP can contain a functional domain, for example, fused to one or both ZFPs. Thus, it will be apparent that a functional domain can be attached to the outside of one or both ZFPs or positioned between the ZFPs (binding to both ZFPs).

[0091] In certain embodiments, the DNA-binding domain comprises a naturally occurring or engineered (non-naturally occurring) TAL effector (TALE) DNA-binding domain. See, for example, U.S. Patent No. 8,586,526, the entire contents of which are incorporated herein by reference. In certain embodiments, the TALE DNA-binding protein binds to 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of the tau target site set forth in U.S. Patent Application Publication No. 2018 / 0153921. The RVD of the TALE DNA-binding protein that binds to the tau target site may be a naturally occurring or non-naturally occurring RVD. See, for example, U.S. Patent Nos. 8,586,526 and 9,458,205.

[0092] Plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important crops. Xanthomonas pathogenicity depends on a conserved type III secretion (T3S) system that injects more than 25 different effector proteins into plant cells. Among these, the injected proteins are transcription activator-like effectors (TALEs), which mimic plant transcription activators and manipulate the plant transcriptome (see Kay et al. (2007) Science 318:648-651). These proteins contain a DNA-binding domain and a transcription activation domain. One of the best-characterized TALEs is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al. (1989) Mol Gen Genet 218: 127-136 and International Patent Application Publication WO2010 / 079430). TALEs contain a central domain of tandem repeats, each containing approximately 34 amino acids that are key to the DNA-binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for review, see Schornack et al. (2006) J Plant Physiol 163(3):256-272). Furthermore, in the pathogenic bacterium Ralstonia solanacearum, two genes, designated brg11 and hpx17, were found to be homologous to the Xanthomonas AvrBs3 family in Ralstonia solanacearum biovar 1 strain GMI1000 and biovar 4 strain RS1000 (see Heuer et al. (2007) Appl and Envir Micro 73(13):4379-4384). These genes are 98.9% identical in nucleotide sequence to each other, except for a 1,575-bp deletion in the repeat domain of hpx17. However, both gene products share less than 40% sequence identity with Xanthomonas AvrBs3 family proteins.

[0093] The specificity of these TALEs depends on the sequences found in the tandem repeats. The repeats contain approximately 102 bp, and the repeats are typically 91–100% homologous to each other (Bonas et al., ibid.). Polymorphisms in the repeats are usually located at positions 12 and 13, and there is thought to be a one-to-one correspondence between the identity of the two hypervariable residues at positions 12 and 13 and the identity of the consecutive nucleotides in the TALE's target sequence (see Moscou and Bogdanove (2009) Science 326:1501 and Boch et al. (2009) Science 326:1509–1512). Experimentally, the codes for DNA recognition of these TALEs have been determined: the HD sequence at positions 12 and 13 results in binding to cytosine (C); NG binds to T; NI binds to A, C, G, or T; NN binds to A or G; and NG binds to T. These DNA-binding repeats have been assembled into proteins with new combinations and numbers of repeats to create artificial transcription factors that can interact with new sequences. Additionally, U.S. Patent No. 8,586,526 and U.S. Patent Application Publication No. 2013 / 0196373, the entire contents of which are incorporated herein by reference, describe TALEs with N-cap polypeptides, C-cap polypeptides (e.g., +63, +231, or +278) and / or novel (atypical) RVDs.

[0094] Exemplary TALEs are described in US Pat. Nos. 8,586,526 and 9,458,205, which are incorporated by reference in their entireties.

[0095] In certain embodiments, the DNA binding domain comprises a dimerization and / or multimerization domain, such as a coiled coil (CC) and a dimerizing zinc finger (DZ). See U.S. Patent Application Publication No. 2013 / 0253040.

[0096] In a further embodiment, the DNA binding domain comprises a single guide RNA of a CRISPR / Cas system, such as the sgRNA disclosed in 20150056705.

[0097] Compelling evidence has recently emerged for the existence of an RNA-mediated genome defense pathway in archaea and many bacteria that is hypothesized to be similar to the eukaryotic RNAi pathway (for reviews, see Godde and Bickerton (2006) J. Mol. Evol. 62:718-729; Lillestol et al. (2006) Archaea 2:59-72; Makarova et al. (2006) Biol. Direct 1:7; Sorek et al. (2008) Nat. Rev. Microbiol. 6:181-186). The pathway known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi) is proposed to arise from two evolutionarily and often physically linked loci: the CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes the RNA component of the system, and the cas (CRISPR-associated) locus, which encodes the protein (Jansen et al. (2002) Mol. Microbiol. 43:1565-1575; Makarova et al. (2002) Nucleic Acids Res. 30:482-496; Makarova et al. (2006) Biol. Direct 1:7; Haft et al. (2005) PLoS Comput. Biol. 1:e60). CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Individual Cas proteins do not share significant sequence similarity with protein components of the eukaryotic RNAi machinery, but have similar predicted functions (e.g., RNA binding, nuclease, helicase, etc.) (Makarova et al. (2006) Biol. Direct 1:7). CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. More than 40 different Cas protein families have been described. Of these protein families, Cas1 is thought to be ubiquitous among different CRISPR / Cas systems.Using specific combinations of cas genes and repeat structures, eight CRISPR subtypes have been defined (E. coli, Y. pest, N. meni, D. vulg, T. neap, H. mari, A. pern, and M. tuber), some of which are associated with additional gene modules encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype can exist in a single genome. The sporadic distribution of CRISPR / Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

[0098] Type II CRISPR, originally described in S. pyogenes, is one of the best-characterized systems and executes targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNA containing individual spacer sequences, where processing occurs by double-strand-specific RNase III in the presence of Cas9 protein. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA by Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Furthermore, tracrRNA must also be present, as it base-pairs with the crRNA at its 3' end, and this binding triggers Cas9 activity. Finally, Cas9 mediates cleavage of the target DNA, generating a double-strand break within the protospacer. The activity of the CRISPR / Cas system involves three steps: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks in a process called "adaptation," (ii) expression of associated proteins, and expression and processing of the array, and then (iii) RNA-mediated inhibition of the foreign nucleic acid. Thus, in bacterial cells, several so-called "Cas" proteins are involved in the natural function of the CRISPR / Cas system.

[0099] Type II CRISPR systems have been found in many different bacteria. Fonfara et al. (2013) Nuc Acid Res 42(4):2377-2590 discovered Cas9 orthologues in 347 bacterial species through a BLAST search against publicly available genomes. Furthermore, this group demonstrated in vitro CRISPR / Cas cleavage of DNA targets using Cas9 orthologues from S. pyogenes, S. mutans, S. thermophilus, C. jejuni, N. meningitides, P. multocida, and F. novicida. Thus, the term "Cas9" refers to an RNA-dependent DNA nuclease containing a DNA-binding domain and two nuclease domains, and the gene encoding Cas9 may be derived from any suitable bacterium.

[0100] The Cas9 protein has at least two nuclease domains: one nuclease domain is similar to an HNH endonuclease, while the other is similar to a Ruv endonuclease domain. The HNH-type domain cleaves the DNA strand complementary to the crRNA, while the Ruv domain is thought to be responsible for cleaving non-complementary strands. Cas9 nucleases can be engineered so that only one nuclease domain is functional, creating a Cas nickase (see Jinek et al. (2012) Science 337:816). Nickases can be generated by specific mutation of amino acids in the catalytic domain of the enzyme or by truncation of part or all of the domain so that it is no longer functional. Because Cas9 contains two nuclease domains, this approach can be performed on either domain. Double-strand breaks can be achieved in target DNA by using two such Cas9 nickases. Nickases will each cleave one strand of DNA, and the use of two will generate a double-strand break.

[0101] The requirement for a crRNA-tracrRNA complex can be circumvented by the use of engineered "single guide RNAs" (sgRNAs) that contain the hairpin normally formed by annealing the crRNA and tracrRNA (see Jinek et al., ibid and Cong et al. (2013) Sciencexpress / 10.1126 / science.1231143). In S. pyrogenes, the engineered tracrRNA:crRNA fusion, or sgRNA, guides Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer forms between the Cas-bound RNA and the target DNA. This system, which includes the Cas9 protein and an engineered sgRNA containing a PAM sequence, has been used for RNA-dependent genome editing (see Ramalingam et al. (2013) Stem Cells and Development 22(4):595-610), and was useful for in vivo zebrafish embryo genome editing with editing efficiency similar to ZFNs and TALENs (Hwang et al. (2013) Nature Biotechnology 31(3):227).

[0102] The primary products of CRISPR loci are thought to be short RNAs containing the invader target sequence, termed guide RNAs or prokaryotic silencing RNAs (psiRNAs) based on their hypothesized role in the pathway (Makarova et al. (2006) Biol. Direct 1:7; Hale et al. (2008) RNA, 14:2572-2579). RNA analysis indicates that CRISPR locus transcripts are cleaved within the repeat sequences, liberating approximately 60-70 nt RNA intermediates containing individual Invader target sequences and flanking repeat fragments (Tang et al. (2002) Proc. Natl. Acad. Sci. 99:7536-7541; Tang et al. (2005) Mol. Microbiol. 55:469-481; Lillestol et al. (2006) Archaea 2:59-72; Brouns et al. (2008) Science 321:960-964; Hale et al. (2008) RNA, 14:2572-2579). In the archaeon Pyrococcus furiosus, these intermediate RNAs are further processed into abundant, stable mature psiRNAs of approximately 35–45 nt (Hale et al. (2008) RNA, 14:2572–2579).

[0103] The requirement for a crRNA-tracrRNA complex can be circumvented by the use of engineered "single guide RNAs" (sgRNAs) that contain the hairpin normally formed by annealing of the crRNA and tracrRNA (see Jinek et al. (2012) Science 337:816 and Cong et al. (2013) Sciencexpress / 10.1126 / science.1231143). In S. pyrogenes, the engineered tracrRNA:crRNA fusion, or sgRNA, guides Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer forms between the Cas-bound RNA and the target DNA. This system, which includes the Cas9 protein and an engineered sgRNA containing a PAM sequence, has been used for RNA-dependent genome editing (see Ramalingam, ibid.), but was useful for in vivo zebrafish embryo genome editing with editing efficiency similar to ZFNs and TALENs (see Hwang et al. (2013) Nature Biotechnology 31 (3):227).

[0104] Chimeras or sgRNAs can be engineered to contain sequences complementary to any desired target.In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or more in length.In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or less.In certain embodiments, the sgRNA comprises a sequence that binds to 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides of the tau target site disclosed in US Patent Application Publication No. 20180153921. In some embodiments, the RNA contains 22 bases of complementarity to a target of the form G[n19] followed by a protospacer adjacent motif (PAM) of the form NGG or NAG for use with the S. pyogenes CRISPR / Cas system. Thus, in one method, an sgRNA can be designed by (i) aligning the recognition sequence of the ZFN heterodimer with a reference sequence of the relevant genome (human, mouse, or specific plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) identifying the location of the motif G[N20]GG closest to the spacer region (if more than one such motif overlaps with the spacer, a motif centered relative to the spacer is selected); and (iv) using that motif as the core of the sgRNA by utilizing a known ZFN target in the gene of interest. This method advantageously relies on a proven nuclease target. Alternatively, sgRNAs can be designed to target any region of interest simply by identifying a suitable target sequence that follows the G[n20]GG formula. Along with the complementary region, the sgRNA may contain additional nucleotides that span the tail region of the tracrRNA portion of the sgRNA (see Hsu et al. (2013) Nature Biotech doi:10.1038 / nbt.2647). The tail may be +68 to +85 nucleotides, or any number in between, with +85 nucleotides being the preferred length.A truncated sgRNA, "tru-gRNA," can also be used (see Fu et al. (2014) Nature Biotech 32(3):279), in which the complementary region is reduced to 17 or 18 nucleotides in length.

[0105] Additionally, alternative PAM sequences can be used, such as NAG as an alternative to NGG using S. pyogenes Cas9 (Hsu 2013, ibid). Additional PAM sequences can also include those lacking the initial G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the Cas9 PAM sequence encoded by S. pyogenes, other PAM sequences specific to Cas9 proteins from other bacterial sources can be used. For example, the PAM sequences shown below (adapted from Sander and Joung, ibid and Esvelt et al. (2013) Nat Meth 10(11):1116) are specific for these Cas9 proteins: Species PAM S. pyogenes NGG S. pyogenes NAG S.mutans NGG S. thermophilius NGGNG S. thermophilius NNAAAW S. thermophilius NNAGAA S. thermophilius NNNGATT C. jejuni NNNNACA N. meningitides NNNGATT P. multocida GNNNCNNA F. novicida NG

[0106] Thus, suitable target sequences for use with the S. pyogenes CRISPR / Cas system can be selected according to the following guidelines: [n17, n18, n19, or n20](G / A)G. Alternatively, the PAM sequence may follow the guideline G[n17, n18, n19, n20](G / A)G. For Cas9 proteins derived from non- S. pyogenes bacteria, the same guidelines can be used, with an alternative PAM substituted for the S. pyogenes PAM sequence.

[0107] It is most preferable to select a target sequence with the highest likelihood of specificity that avoids potential off-target sequences. These unwanted off-target sequences can be identified by considering the following attributes: i) similarity in the target sequence with the Cas9 protein being used, followed by a known functional PAM sequence; ii) a similar target sequence with fewer than three mismatches from the desired target sequence; iii) mismatches all located in the PAM-distal region rather than the PAM-proximal region (there is some evidence that nucleotides 1-5 immediately adjacent to or close to the PAM, sometimes referred to as the "seed" region (Wu et al. (2014) Nature Biotech doi:10.1038 / nbt2889), are most important for recognition; thus, putative off-target sites with mismatches located in the seed region are at least likely to be recognized by the sgRNA); and iv) a similar target sequence where the mismatches are not consecutively spaced or are spaced more than four nucleotides apart (Hsu 2013, ibid.). Thus, using these criteria above, suitable target sequences for sgRNAs can be identified by performing an analysis of the number of potential off-target sites in the genome for any CRISPR / Cas system used.

[0108] In some embodiments, the CRISPR-Cpf1 system is used. Identified in Francisella spp., the CRISPR-Cpf1 system is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. While functionally conserved, Cpf1 and Cas9 differ in many aspects, such as their guide RNA and substrate specificity (see Fagerlund et al. (2015) Genom Bio 16:251). A major difference between Cas9 and Cpf1 proteins is that Cpf1 does not use tracrRNA and thus requires only crRNA. The FnCpf1 crRNA is 42-44 nucleotides long (19-nucleotide repeat and 23-25-nucleotide spacer) and contains a single stem-loop, allowing for sequence changes that preserve secondary structure. Furthermore, the Cpf1 crRNA is significantly shorter than the approximately 100 nucleotide engineered sgRNA required by Cas9, and the PAM requirement for FnCpf1 is 5'-TTN-3' and 5'-CTA-3' on the displaced strand. While both Cas9 and Cpf1 generate double-stranded breaks in target DNA, Cas9 uses its RuvC and HNH-like domains to generate a blunt-end cut within the seed sequence of the guide RNA, while Cpf1 uses its RuvC-like domain to create a staggered cut outside the seed. Because Cpf1 generates a staggered cut away from the critical seed region, NHEJ does not destroy the target site, thus ensuring that Cpf1 can continue to cut at the same site until the desired HDR recombination event occurs. Thus, in the methods and compositions described herein, the term "Cas" is understood to include both Cas9 and Cpf1 proteins. Thus, as used herein, "CRISPR / Cas system" refers to both CRISPR / Cas and / or CRISPR / Cpf1 systems, and includes both nuclease, nickase and / or transcription factor systems.

[0109] In some embodiments, other Cas proteins can be used. Some exemplary Cas proteins include Cas9, Cpf1 (also known as Cas12a), C2c1, C2c2 (also known as Cas13a), C2c3, Cas1, Cas2, Cas4, CasX, and CasY; engineered and naturally occurring variants thereof (Burstein et al. (2017) Nature 542:237-241), such as HF1 / spCas9 (Kleinstiver et al. (2016) Nature 529:490-495; Cebrian-Serrano and Davies (2017) Mamm Genome 28(7):247-261); split Cas9 systems (Zetsche et al. (2015) Nat Biotechnol 33(2):139-142), trans-spliced ​​Cas9 based on an intein-extein system (Troung et al. (2015) Nucl Acid Res 43(13):6450-8); mini-SaCas9 (Ma et al. (2018) ACS Synth Biol 7(4):978-985). Thus, in the methods and compositions described herein, the term "Cas" is understood to include all Cas mutant proteins, both naturally occurring and engineered. Thus, as used herein, "CRISPR / Cas system" refers to any CRISPR / Cas system that includes both nuclease, nickase, and / or transcription factor systems.

[0110] In certain embodiments, a Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a native sequence polypeptide is a compound that shares qualitative biological properties with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of native sequence polypeptides and fragments thereof, provided that they share biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent variants, and fusions thereof. In some aspects, a functional derivative may comprise a single biological property of a naturally occurring Cas protein. In other aspects, a functional derivative may comprise a subset of the biological properties of a naturally occurring Cas protein. Suitable derivatives of a Cas polypeptide or fragments thereof include, but are not limited to, mutants, fusions, and covalent variants of a Cas protein or fragments thereof. Cas proteins, including Cas proteins or fragments thereof, as well as derivatives of Cas proteins or fragments thereof, may be obtained from cells, chemically synthesized, or obtained by a combination of these two procedures. The cells may be cells that naturally produce Cas proteins, or cells that naturally produce Cas proteins and produce endogenous Cas proteins at higher expression levels, or cells that have been genetically engineered to produce Cas proteins from exogenously introduced nucleic acids that encode the same or different Cas as the endogenous Cas. In some cases, the cells do not naturally produce Cas proteins but are genetically engineered to produce Cas proteins.

[0111] Exemplary CRISPR / Cas nuclease systems targeted to specific genes (including safe harbor genes) are disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0056705.

[0112] Thus, the nuclease contains a nuclease domain that cleaves DNA, along with a DNA binding domain that specifically binds to a target site in any gene into which it is desired to insert the donor (transgene).

[0113] Tau gene modulators The tau DNA-binding domain may be fused to or otherwise associated with any additional molecule (e.g., a polypeptide) for use in the methods described herein. In certain embodiments, the methods employ a fusion molecule comprising at least one DNA-binding molecule (e.g., a ZFP, a TALE, or a single guide RNA) and a heterologous regulatory (functional) domain (or a functional fragment thereof).

[0114] In certain embodiments, the functional domain of tau modulator comprises transcriptional regulatory domain.Common domains include, for example, transcription factor domain (activator, repressor, coactivator, corepressor), silencer, oncogene (for example, myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzyme and its related factor and modifier; DNA rearrangement enzyme and its related factor and modifier; chromatin-associated protein and its modifier (for example, kinase, acetylase and deacetylase); and DNA modifying enzyme (for example, methyltransferase, topoisomerase, helicase, ligase, kinase, phosphatase, polymerase, endonuclease), proteolytic modifier (deubiquitinase, ligase, degron) and its related factor and modifier.See, for example, US Patent Application Publication No. 2013 / 0253040, the entire contents of which are incorporated herein by reference.

[0115] Suitable domains for achieving activation include the HSV VP16 activation domain (see, e.g., Hagmann et al. (1997) J. Virol. 71:5952-5962), nuclear hormone receptors (see, e.g., Torchia et al. (1998) Curr. Opin. Cell. Biol. 10:373-383); the p65 subunit of nuclear factor kappa B (Bitko & Barik (1998) J. Virol. 72:5610-5618 and Doyle & Hunt (1997) Neuroreport 8:2937-2942; Liu et al. (1998) Cancer Gene Ther. 5:3-28), or VP64 (Beerli et al. (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degrons (Molinari et al. (1999) EMBO J. 18:6439-6447). Additional exemplary activation domains include Oct1, Oct-2A, Sp1, AP-2, and CTF1 (Seipel et al. (1992) EMBO J. 11:4961-4968), as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A, and ERF-2. For example, Robyr et al. (2000) Mol. Endocrinol. 14:329-347;Collingwood et al. (1999) J. Mol. Endocrinol. 23:255-275;Leo et al. (2000) Gene 245:1-11;Manteuffel-Cymborowska (1999) Acta Biochim. Pol. See McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Further exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1. For example, Ogawa et al. (2000) Gene 245:21-29;Okanami et al. (1996) Genes Cells 1:87-99;Goff et al. (1991) Genes Dev. 5:298-309;Cho et al. (1999) Plant Mol. Biol. 40:419-429;Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849;Sprenger-Haussels et al. (2000) Plant J. 22:1-8;Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA See 96:15,348-15,353.

[0116] Exemplary repression domains that can be used to generate tau repressor include, but are not limited to, KRAB A / B, KOX, TGF-beta inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (such as DNMT1, DNMT3A, DNMT3B), Rb and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; and Robertson et al. (2000) Nature Genet. 25:338-342.Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, e.g., Chem et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

[0117] In some examples, the domain is involved in the epigenetic regulation of chromosomes. In some embodiments, the domain is the A-type nuclear localization domain of histone acetyltransferase (HAT), such as MYST family members MOZ, Ybf2 / Sas3, MOF and Tip60, GNAT family members Gcn5 or pCAF, p300 family members CBP, p300 or Rtt109 (Berndsen and Denu (2008) Curr Opin Struct Biol 18(6):682-689). In other examples, the domain is a histone deacetylase (HDAC), such as class I (HDAC-1, 2, 3, and 8), class II (HDAC IIA (HDAC-4, 5, 7, and 9), HDAC IIB (HDAC6 and 10)), class IV (HDAC-11), or class III (also known as sirtuins (SIRTs); SIRT1-7) (see Mottamal et al. (2015) Molecules 20(3):3898-3941). Another domain used in some embodiments is a histone phosphorylase or kinase, examples of which include MSK1, MSK2, ATR, ATM, DNA-PK, Bub1, VprBP, IKK-α, PKCβ1, Dik / Zip, JAK2, PKC5, WSTF, and CK2. In some embodiments, a methylation domain is used and can be selected from the group including Ezh2, PRMT1 / 6, PRMT5 / 7, PRMT2 / 6, CARM1, set7 / 9, MLL, ALL-1, Suv39h, G9a, SETDB1, Ezh2, Set2, Dot1, PRMT1 / 6, PRMT5 / 7, PR-Set7, and Suv4-20h. In some embodiments, domains involved in sumoylation and biotinylation (Lys9, 13, 4, 18, and 12) can also be used (for review, see Kousarides (2007) Cell 128:693-705).

[0118] Fusion molecules are constructed by cloning and biochemical conjugation methods well known to those skilled in the art. Fusion molecules contain a DNA binding domain and a functional domain (e.g., a transcription activation or repression domain). Fusion molecules may also contain a nuclear localization signal (e.g., derived from SV40 medium T antigen) and an epitope tag (e.g., FLAG and hemagglutinin). Fusion proteins (and the nucleic acids encoding them) are designed so that the reading frame of transcription is preserved between the components of the fusion.

[0119] Fusions between a polypeptide component (or functional fragment thereof) of a functional domain on the one hand and a non-protein DNA-binding domain (e.g., an antibiotic, an intercalator, a minor groove binder, a nucleic acid) on the other hand are constructed by biochemical conjugation methods known to those skilled in the art. See, for example, the Pierce Chemical Company (Rockford, IL) Catalogue. Methods and compositions for making fusions between minor groove binders and polypeptides are described. Mapp et al. (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Similarly, nucleases containing sgRNA nucleic acid components linked to CRISPR / Cas TF and polypeptide component functional domains are also known to those skilled in the art and are described in detail herein.

[0120] Fusion molecules can be formulated with pharmaceutically acceptable carriers as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned International Patent Application Publication No. WO 00 / 42219).

[0121] Once the fusion molecule binds to the target sequence via its DNA-binding domain, the functional component / domain of the fusion molecule can be selected from a variety of different components that can affect gene transcription. Thus, functional components can include various transcription factor domains, such as, but not limited to, activators, repressors, coactivators, corepressors, and silencers.

[0122] In certain embodiments, the fusion molecule comprises a DNA-binding domain and a nuclease domain, creating a functional entity that can recognize its intended nucleic acid target through its engineered (ZFP or TALE or sgRNA) DNA-binding domain, or a nuclease (e.g., zinc finger nuclease, TALE nuclease, or CRISPR / Cas nuclease) whose nuclease activity causes DNA cleavage near the DNA-binding site. This cleavage results in the inactivation (suppression) of the tau gene. Thus, the tau repressor also includes a tau nuclease.

[0123] Thus, the methods and compositions described herein are widely applicable and can include any nuclease of interest.Non-limiting examples of nuclease include meganuclease, TALEN and zinc finger nuclease.Nuclease can include heterologous DNA binding and cleavage domain (for example, zinc finger nuclease; TALEN; meganuclease DNA binding domain containing heterologous cleavage domain, sgRNA combined with nuclease domain), or alternatively, the DNA binding domain of naturally occurring nuclease can be modified to bind to selected target site (for example, meganuclease engineered to bind to a site different from its cognate binding site).

[0124] The nuclease domain may be derived from any nuclease, e.g., any endonuclease or exonuclease. Non-limiting examples of suitable nuclease (cleavage) domains that can be fused to the tau DNA-binding domain described herein include domains derived from any restriction enzyme, e.g., type IIS restriction enzymes (e.g., FokI). In certain embodiments, the cleavage domain is a cleavage half-domain that requires dimerization for cleavage activity. See, e.g., U.S. Patent Nos. 8,586,526, 8,409,861, and 7,888,121, which are incorporated herein by reference in their entireties. Generally, when a fusion protein contains a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein containing two cleavage half-domains can be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). Furthermore, it is preferred that the target sites for the two fusion proteins be positioned with respect to each other so that binding of the two fusion proteins to their corresponding target sites places the cleavage half-domains in a spatial orientation with respect to each other that enables the cleavage half-domains to form a functional cleavage domain, e.g., by dimerization.

[0125] The nuclease domain may also be derived from any meganuclease (homing endonuclease) domain, however, the cleavage activity may also be used with nucleases described herein, including but not limited to, I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.

[0126] In certain embodiments, the nuclease comprises a compact TALEN (cTALEN), which is a single-stranded fusion protein that links a TALE DNA-binding domain to a TevI nuclease domain. The fusion protein can act as a nickase localized by the TALE region, or can generate a double-stranded break depending on where the TALE DNA-binding domain is located relative to the meganuclease (e.g., TevI) nuclease domain (Beurdeley et al. (2013) Nat Comm 4:1762, DOI: 10.1038 / ncomms2782).

[0127] In other embodiments, the TALE-nuclease is a megaTAL. These megaTAL nucleases are fusion proteins comprising a TALE DNA-binding domain and a meganuclease cleavage domain. The meganuclease cleavage domain is active as a monomer and does not require dimerization for activity (see Boissel et al. (2013) Nucl Acid Res:1-13, doi: 10.1093 / nar / gkt1224).

[0128] Furthermore, the nuclease domain of the meganuclease may also exhibit DNA binding function. Any TALEN can be used in conjunction with additional TALENs (e.g., one or more TALENs (cTALENs or FokI-TALENs) together with one or more mega-TALs) and / or ZFNs.

[0129] Additionally, the cleavage domain may contain one or more alterations compared to the wild type, e.g., for essential heterodimer formation that reduces or eliminates off-target cleavage effects (see, e.g., U.S. Patent Nos. 7,914,796, 8,034,598, and 8,623,618, which are incorporated herein by reference in their entireties).

[0130] The nucleases described herein may generate double-stranded or single-stranded breaks in double-stranded targets (e.g., genes). The generation of single-stranded breaks ("nicks") is described, for example, in U.S. Patent Nos. 8,703,489 and 9,200,266, which are incorporated herein by reference, and describe how mutations in the catalytic domain of one nuclease domain result in nickases.

[0131] Thus, a nuclease (cleavage) domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

[0132] Alternatively, nucleases can be assembled in vivo at nucleic acid target sites using so-called "split enzyme" technology (see, e.g., U.S. Patent Application Publication No. 2009 / 0068164). The components of such split enzymes can be expressed on separate expression constructs, or the individual components can be linked in a single open reading frame separated, for example, by a self-cleaving 2A peptide (e.g., T2A) or an IRES sequence. The components can be individual zinc finger domains or domains of meganuclease nucleic acid binding domains.

[0133] Nucleases can be screened for activity prior to use, for example, in the yeast-based chromosomal system described in U.S. Patent Application Publication No. 2009 / 0111119. Nuclease expression constructs can be readily designed using methods known in the art.

[0134] Expression of the fusion protein (or components thereof) may be under the control of a constitutive or inducible promoter, such as a galactokinase promoter, which is activated (derepressed) in the presence of raffinose and / or galactose and repressed in the presence of glucose. Non-limiting examples of preferred promoters include the neuron-specific promoters NSE, synapsin, CAMKiia, and MECP. Non-limiting examples of ubiquitous promoters include CAS and Ubc. Further embodiments include the use of the autoregulatory promoter described in U.S. Patent Application Publication No. 2015 / 0267205 (due to the inclusion of a high-affinity binding site for the tau DNA-binding domain).

[0135] In certain embodiments, a tau modulator for use in a subject comprises a ZFP designated 57890, 65918, and / or 57930. In certain embodiments, two or more such tau modulators (e.g., 57890 and 65918 ZFP repressors; 57890 and 57930 ZFP repressors; 65918 and 57930; 57890, 65918, and 57930) that provide a synergistic effect compared to a single ZFP tau repressor are provided to a subject for the treatment and / or prevention of a tauopathy, such as AD, including the suppression of tau and amelioration of symptoms of the tauopathy.

[0136] delivery Proteins and / or polynucleotides (e.g., tau modulators) and compositions comprising the proteins and / or polynucleotides described herein can be delivered to target cells by any suitable means, such as, for example, injection of the protein via mRNA and / or use of expression constructs (e.g., plasmids, lentiviral vectors, AAV vectors, Ad vectors, etc.). In a preferred embodiment, the repressor is delivered using an AAV vector, such as, but not limited to, AAV9 (see U.S. Patent No. 7,198,951), the AAV vectors described in U.S. Patent No. 9,585,971.

[0137] Methods for delivering proteins, including the zinc finger proteins described herein, are described, for example, in U.S. Patent Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated herein by reference in their entireties.

[0138] Any vector system can be used, including but not limited to, plasmid vector, retrovirus vector, lentivirus vector, adenovirus vector, poxvirus vector; herpesvirus vector and adeno-associated virus vector. Also see U.S. Patent Nos. 8,586,526; 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, all of which are incorporated by reference in their entirety. Furthermore, it is contemplated that any of these vectors may contain one or more DNA binding protein coding sequences. Thus, when one or more tau modulators (e.g., repressors) are introduced into cells, the sequences encoding protein components and / or polynucleotide components can be carried on the same vector or different vectors. When multiple vectors are used, each vector may contain a sequence encoding one or more tau modulators (e.g., repressors) or components thereof. In a preferred embodiment, the vector system is an AAV vector, such as the AAV9 or AAV variants described in U.S. Patent No. 9,585,971 or U.S. Patent Application Publication No. 2017 / 0119906.

[0139] Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids encoding engineered tau modulators into cells (e.g., mammalian cells) and target tissues. Such methods can also be used to administer nucleic acids encoding such repressors (or components thereof) to cells in vitro. In certain embodiments, nucleic acids encoding repressors are administered for use in in vivo or ex vivo gene therapy. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers. Viral vector delivery systems can include DNA and RNA viruses with episomal or integrated genomes after delivery to cells. For reviews of gene therapy procedures, see Anderson (1992) Science 256:808-813; Nabel & Felgner (1993) TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154 (1988); Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al., in Current Topics in Microbiology and Immunology. See Doerfler and Boehm (eds.) (1995); and Yu et al. (1994) Gene Therapy 1:13-26.

[0140] Non-viral nucleic acid delivery methods include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and drug-enhanced uptake of DNA. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids. In a preferred embodiment, one or more nucleic acids are delivered as mRNA. Furthermore, the use of capped mRNA is preferred to increase translation efficiency and / or mRNA stability. Particularly preferred is the ARCA (anti-reverse cap analog) cap or its variants. See U.S. Patent Nos. 7,074,596 and 8,153,773, incorporated herein by reference.

[0141] Further exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc. (see, for example, U.S. Patent No. 6,008,336).Lipofection is described, for example, in U.S. Patent No. 5,049,386; No. 4,946,787; and No. 4,897,355, and lipofection reagents are commercially available (for example, Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX).Cationic lipids and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Felgner's International Patent Application Publications WO91 / 17424 and WO91 / 16024. Delivery may be to cells (ex vivo administration) or to target tissues (in vivo administration).

[0142] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (e.g., Crystal (1995) Science 270:404-410 (1995); Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0143] Another delivery method involves packaging the nucleic acid to be delivered in an EnGeneIC delivery vehicle (EDV). These EDVs are specifically delivered to target tissues using bispecific antibodies, where one arm of the antibody has specificity for the target tissue and the other arm has specificity for the EDV. The antibody then brings the EDV to the surface of the target cell, where it is then taken into the cell by endocytosis. Once inside the cell, its contents are released (see MacDiarmid et al. (2009) Nature Biotechnology 27(7):643).

[0144] The use of RNA or DNA virus-based systems for the delivery of nucleic acids encoding engineered ZFPs, TALEs, or CRISPR / Cas systems takes advantage of the highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells administered to patients (ex vivo). Conventional virus-based systems for the delivery of ZFPs, TALEs, or CRISPR / Cas systems include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, vaccinia virus, and herpes simplex viral vectors for gene transfer. Integration into the host genome can be achieved using retroviral, lentiviral, and adeno-associated viral gene transfer methods, which often result in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0145] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors contain cis-acting long terminal repeats that have packaging capacity for up to 6-10 kb of foreign sequence. A minimal set of cis-acting LTRs is sufficient for replication and packaging of the vector, which is then used to integrate a therapeutic gene into target cells and achieve sustained transgene expression. Widely used retroviral vectors include murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; International Patent Application Publication No. WO 1994 / 026877).

[0146] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors allow for very high transduction efficiency in many cell types and do not require cell division. Using such vectors, high titer and high level expression have been obtained. This vector can be produced in large quantities using a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce target nucleic acids into cells, for example, in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, for example, West et al. (1987) Virology 160:38-47; U.S. Patent No. 4,797,368; International Patent Application Publication WO93 / 24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1351). The construction of recombinant AAV vectors has been described in several publications, including U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260; Tratschin et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat & Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol. 63:03822-3828.

[0147] At least six viral vector approaches are currently available for gene transfer in clinical trials and use approaches that involve complementation of a defective vector with a gene inserted into a helper cell line to produce the transducing agent.

[0148] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94(22):12133-12138). PA317 / pLASN was the first therapeutic vector used in a gene therapy trial (Blaese et al. (1995) Science 270:475-480). Transduction efficiencies of over 50% have been observed with MFG-S-packaged vectors (Ellem et al. (1997) Immunol Immunother. 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2).

[0149] Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery system based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that contain only the 145-bp inverted terminal repeats of AAV flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genome of the transduced cell are key features of this vector system (Wagner et al. (1998) Lancet 351(9117):1702-3, Kearns et al. (1996) Gene Ther. 9:748-55). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9 and AAV rh10, and pseudotyped AAV such as AAV2 / 8, AAV2 / 5, AAV2 / 9 and AAV2 / 6 can also be used according to the present invention.New AAV serotypes that can cross the blood-brain barrier can also be used according to the present invention (see, for example, U.S. Patent No. 9,585,971).In a preferred embodiment, AAV9 vectors (including AAV9 mutants and pseudotypes) are used.

[0150] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titers and easily infect several different cell types. Many adenoviral vectors are engineered so that a transgene replaces the Ad E1a, E1b, and / or E3 genes; the replication-deficient vector is then propagated in human 293 cells, which supply the deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of Ad vectors in clinical trials included polynucleotide therapy for anti-tumor immunization using intramuscular injection (Sterman et al. (1998) Hum. Gene Ther. 7:1083-9). Further examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al. (1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther. 971083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez et al. (1997) Hum. Gene Ther. 5:597-613 (1997); Topf et al. (1998) Gene Ther. 5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

[0151] Packaging cells are used to form viral particles that can infect host cells. Such cells include 293 cells, which package adenovirus, and Ψ2 or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually produced by a producer cell line that packages nucleic acid vectors into viral particles. Typically, the vector contains the minimum viral sequences required for packaging and subsequent integration into the host (if applicable), with other viral sequences replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically contain only the inverted terminal repeat (ITR) sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA is packaged in a cell line that contains a helper plasmid encoding other AAV genes, namely, rep and cap, but lacks ITR sequences. The cell line is also infected with adenovirus as a helper. Helper virus promotes the replication of AAV vector and the expression of AAV gene from helper plasmid.Helper plasmid is not packaged in significant amounts due to the lack of ITR sequence.Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0152] In many gene therapy applications, it is desirable to deliver gene therapy vectors with high specificity to particular tissue types. Therefore, viral vectors can be engineered to have specificity for a given cell type by expressing a ligand as a fusion protein with the viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia virus can be engineered to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein containing a ligand for the cell surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for virtually any selected cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors: such vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

[0153] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intravesical, intraventricular, or intracranial infusion, including direct injection into the brain, including any region of the brain such as the hippocampus, cortex, striatum, etc.) or local application, as described below. Alternatively, vectors can be delivered to ex vivo cells, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy), or to universal donor hematopoietic stem cells, which can then be re-implanted into the patient, typically after selection for cells that have incorporated the vector.

[0154] In certain embodiments, the compositions (e.g., polynucleotides and / or proteins) described herein are delivered directly in vivo. The compositions (cells, polynucleotides, and / or proteins) can be administered directly to the central nervous system (CNS), including, but not limited to, direct injection into the brain or spinal cord. One or more regions of the brain can be targeted, such as, but not limited to, the hippocampus, substantia nigra, nucleus basalis of Meynert (NBM), striatum, and / or cortex. Alternatively, or in addition to CNS delivery, the compositions can be administered systemically (e.g., intravenous, intraperitoneal, intracardiac, intramuscular, subcutaneous, intrathecal, intravesical, intraventricular, and / or intracranial infusion). Methods and compositions for delivering the compositions described herein directly to a subject (including direct delivery to the CNS) include, but are not limited to, direct injection (e.g., stereotaxic injection) using a needle assembly. Such methods are described, for example, in U.S. Pat. Nos. 7,837,668 and 8,092,429 and U.S. Patent Application Publication No. 2006 / 0239966, which relate to delivery of compositions (including expression vectors) to the brain and are incorporated herein by reference in their entireties.

[0155] The effective amount administered will vary from patient to patient and depending on the mode and site of administration. Therefore, the effective amount is best determined by the physician administering the composition, and those skilled in the art can easily determine the appropriate dosage. After allowing a sufficient period of time for incorporation and expression (typically, e.g., 4-15 days), analysis of serum or other tissue levels of the therapeutic polypeptide and comparison with initial levels before administration will determine whether the amount administered is too low, within the correct range, or too high. Suitable regimens for initial and subsequent administrations are also variable, but typically involve an initial administration followed by subsequent administrations as needed. Subsequent administrations can be administered at variable intervals ranging from daily to annually to every few years. In certain embodiments,

[0156] To deliver ZFPs directly to the human brain using adeno-associated virus (AAV) vectors, approximately 1x10 cells per striatum are needed. 10 ~5x10 15 A dose range of 1000 vector genomes (or any value therebetween) can be applied. As mentioned above, the dosage may vary for other brain structures and different delivery protocols. Methods for delivering AAV vectors directly to the brain are known in the art. See, for example, U.S. Patent Nos. 9,089,667; 9,050,299; 8,337,458; 8,309,355; 7,182,944; 6,953,575; and 6,309,634.

[0157] Ex vivo cell transfection for diagnosis, research, or gene therapy (e.g., by reinfusion of transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a target organism, transfected with at least one tau modulator (e.g., a repressor) or a component thereof, and then reinfused back into the target organism (e.g., a patient). In a preferred embodiment, one or more nucleic acids of a tau modulator (e.g., a repressor) are delivered using AAV9. In other embodiments, one or more nucleic acids of a tau modulator (e.g., a repressor) are delivered as mRNA. Also, the use of capped mRNA is preferred to increase translation efficiency and / or mRNA stability. Particularly preferred is the ARCA (anti-reverse cap analog) cap or a variant thereof. See U.S. Patent Nos. 7,074,596 and 8,153,773, the entire contents of which are incorporated herein by reference. Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994) and references cited therein for a discussion of methods for isolating and culturing cells from patients).

[0158] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy.The advantage of using stem cells is that they can be differentiated into other cell types in vitro, or they can be introduced into mammals (such as cell donors) where they engraft into bone marrow.Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ and TNF-α are known (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).

[0159] Stem cells are isolated for transduction and differentiation using known methods.For example, stem cells are isolated from bone marrow cells by panning bone marrow cells with antibodies that bind to unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (pan B cells), GR-1 (granulocytes), and Iad (differentiated antigen-presenting cells) (see Inaba et al. (1992) J. Exp. Med. 176:1693-1702).

[0160] In some embodiments, modified stem cells can also be used.For example, neural stem cells that are made resistant to apoptosis can be used as therapeutic compositions, where the stem cells also contain the ZFP TF of the present invention.Resistance to apoptosis can be achieved, for example, by knocking out BAX and / or BAK in stem cells using BAX or BAK-specific TALEN or ZFN (see U.S. Patent No. 8,597,912), or again, by knocking out those that are destroyed in caspases, for example, using caspase-6-specific ZFN.These cells can be transfected with ZFP TF or TALE TF that is known to regulate tau gene.

[0161] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic ZFP nucleic acids can also be administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA can be administered. Administration can be by any route typically used to introduce a molecule into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while a particular composition can be administered using more than one route, certain routes can often result in a more immediate and effective response than other routes.

[0162] Methods for introducing DNA into hematopoietic stem cells are disclosed, for example, in U.S. Patent No. 5,928,638. + Vectors useful for introduction into cells include adenovirus type 35.

[0163] Suitable vectors for introducing transgenes into immune cells (for example, T cells) include non-integrating lentiviral vectors.See, for example, Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388;Dull et al. (1998) J. Virol. 72:8463-8471;Zuffery et al. (1998) J. Virol. 72:9873-9880;Follenzi et al. (2000) Nature Genetics 25:217-222.

[0164] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of pharmaceutical compositions available, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0165] As noted above, the disclosed methods and compositions can be used in any type of cell, including, but not limited to, prokaryotic cells, fungal cells, archaeal cells, plant cells, insect cells, animal cells, vertebrate cells, mammalian cells, and human cells. Suitable cell lines for protein expression are known to those of skill in the art and include, but are not limited to, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, insect cells such as Spodoptera fugiperda (Sf), and fungal cells such as Saccharomyces, Pischia, and Schizosaccharomyces. Progeny, mutants, and derivatives of these cell lines can also be used. In a preferred embodiment, the methods and compositions are delivered directly to brain cells, for example, to the striatum.

[0166] Models of CNS disorders CNS disorder testing can be performed in non-human primates (e.g., Parkinson's disease (Johnston and Fox (2015) Curr Top Behav Neurosci 22: 221-35); amyotrophic lateral sclerosis (Jackson et al. (2015) J. Med Primatol: 44(2):66-75); Huntington's disease (Yang et al. (2008) Nature 453(7197):921-4); Alzheimer's disease (Park et al. (2015) Int J Mol Sci 16(2):2386-402); seizures (Hsiao et al. (2016) E Bio Med 9:257-77)), canines (e.g., MPS VII (Gurda et al. (2016) Mol Ther 24(2):206-216); Alzheimer's disease (Schutt et al. (2016) J Alzheimers Dis 52(2):433-49; seizures (Varatharajah et al. (2017) Int J Neural Syst 27(1):1650046)) and mice (e.g., seizures (Kadiyala et al. (2015) Epilepsy Res 109:183-96); Alzheimer's disease (Li et al. (2015) J Alzheimers Dis Parkin 5(3) doi 10:4172 / 2161-0460)) (reviewed in: Webster et al. (2014) Front Genet 5 art 88, doi:10.3389f / gene.2014.00088). These models can be useful for probing specific symptom sets of a disease, and can be used even when an animal model that fully recapitulates a CNS disease does not exist. This model can help determine the efficacy and safety profile of the therapeutic methods and compositions (gene repressors) described herein.

[0167] Applicable The tau modulators (e.g., tau repressors) described herein, including the MAPT-binding molecules (e.g., ZFPs, TALEs, CRISPR / Cas systems, Ttago, etc.) described herein, and the nucleic acids encoding them, can be used for a variety of applications. These applications include therapeutic methods in which MAPT-binding molecules (including nucleic acids encoding DNA-binding proteins) are administered to a subject using viral (e.g., AAV) or non-viral vectors to modulate the expression of target genes in the subject. Modulation can be in the form of suppression, e.g., suppression of tau expression that contributes to the AD disease state. Alternatively, modulation can be in the form of activation, where activation or increased expression of an endogenous cellular gene can ameliorate the disease state. In further embodiments, modulation can be suppression by cleavage (e.g., by one or more nucleases) to inactivate the MAPT gene, for example. As described above, for such applications, the MAPT-binding molecules, or more typically, the nucleic acids encoding them, are formulated into pharmaceutical compositions together with a pharmaceutically acceptable carrier.

[0168] MAPT-binding molecules, or vectors encoding them, alone or in combination with other suitable components (e.g., liposomes, nanoparticles, or other components known in the art), can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Suitable formulations for parenteral administration, e.g., by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. For example, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, retroorbitally (RO), intracranially (e.g., to any region of the brain, such as, but not limited to, the hippocampus and / or cortex), intracisternally, or intrathecally. The formulations of the compounds may be presented in single-dose or multi-dose sealed containers, such as ampoules and vials. Injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0169] The dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over an extended period of time. The dose will depend on the potency and K d The size of the dose will be determined by the condition of the target cells and the patient, as well as the body weight or surface area of ​​the patient to be treated. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular compound or vector in a particular patient.

[0170] The following examples relate to exemplary embodiments of the present disclosure in which the MAPT modulator comprises a zinc finger protein. This is for illustrative purposes only, and it will be understood that other MAPT modulators (e.g., repressors) can be used, including, but not limited to, TALE-TFs, CRISPR / Cas systems, additional ZFPs, ZFNs, TALENs, additional CRISPR / Cas systems, and homing endonucleases (meganucleases) with engineered DNA-binding domains. It will be understood that these modulators that bind to target sites as exemplified below can be easily obtained using methods known to those skilled in the art. Similarly, while the following examples relate to exemplary embodiments in which the delivery vehicle is an AAV vector, it will be understood that any viral (e.g., Ad, Lv) or non-viral (e.g., plasmid, mRNA) vector can be used to deliver the tau repressor described herein. [Example]

[0171] Example 1 Suppression of MAPT in vivo A zinc finger protein specific for the MAPT (tau) target site described in US Patent Application Publication No. 2018 / 0153921 was used as follows.

[0172] [Table 1]

[0173] All of the ZFPs described herein were operably linked to a KRAB repression domain to form ZFP-TFs, and all repressed MAPT expression.

[0174] Primate tau-specific ZFP-TFs are tested in cynomolgus monkeys (M. fascicularis) to observe the suppression of tau expression in primates (a non-human primate (NHP) model). Cynomolgus monkeys are housed in stainless steel cages equipped with an automatic water supply system. The study complies with all applicable sections of the Final Rules of the Animal Welfare Act Regulations (Code of Federal Regulations, Title 9) and the current version of the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 8th edition.

[0175] The ZFP-TF repressors in Table 1 were cloned into AAV vectors (AAV2 / 9, or variants thereof) with a SYN1 promoter or a CMV promoter, essentially as described in U.S. Patent Application Publication No. 20180153921. The AAV vectors used included a vector with a SYN1 promoter driving expression of a repressor comprising 65918 and 57890 linked by a T2A peptide (SYN918-890); a vector with a CMV promoter driving expression of a repressor comprising 65918 and 57890 linked by a T2A peptide (CMV918-890); a vector with a SYN1 promoter driving expression of a repressor comprising 57930 (SYN930); a vector with a SYN1 promoter driving expression of a repressor comprising 57890 (SYN890); and a vector with a SYN1 promoter driving expression of a repressor comprising 65918 (SYN918).

[0176] Fifteen NHP subjects were treated as shown in the table below.

[0177] [Table 2]

[0178] In this experiment, AAV9 vectors containing hSYN1 or CMV-induced ZFP TF were delivered at 6E11 vg to the left hemisphere and 6E11 vg to the right hemisphere. Animals received a single dose of test article in a volume of 60 μL in the left hemisphere and a single dose of 60 μL in the right hemisphere. For all test articles, the dose concentration was 1E13 vg / mL.

[0179] After 28 days, the animals were sacrificed, and the brains were removed and placed in a coronal brain matrix in ice-cold PBS. The brains were sliced ​​into 3 mm coronal slices (divided into approximately 17 slices). Some brain slices (right and left hemispheres) were preserved in 10% neutral buffered formalin for histopathology and in situ hybridization analysis. All other brain slices (right and left hemispheres) were placed in RNAlater (Qiagen) and refrigerated for approximately 24 hours, after which 2-3 mm diameter punches were collected according to the predetermined brain template. The punches were processed for qRT-PCR and biodistribution analysis. Additionally, CSF was collected for tau protein analysis.

[0180] Using slices containing the hippocampus and entorhinal cortex, we analyzed the mRNA expression levels of tau, ZFPs, glial and neuronal markers, and housekeeping genes by qRT-PCR. The results show that ZFP-TFs delivered by AAV to the hippocampus resulted in a reduction of tau expression.

[0181] Figures 1 and 2 show exemplary results of tau suppression and ZFP mRNA levels (copies / ng mRNA) for hippocampal punches 088 and 035 using the indicated vectors in the indicated subjects.

[0182] 3 shows the results from the indicated punch samples and three methods evaluated to establish baseline tau levels for a given punch: no scaling (top panel), scaling to the average of three vehicle-treated animals (middle panel), or scaling to the average tau expression of vehicle- and ZFP-treated animals that showed no detectable ZFP expression (bottom panel). As shown, scaling by either method better approximates baseline levels across all 15 animals, and the third method (scaling to NHPs that showed no ZFP expression) is, in some cases (e.g., punch 037), somewhat more representative of the tau baseline across the 15 animals in the study.

[0183] Figure 4 shows tau modulation and ZFP levels in NHP07, a subject treated with AAV CMV918-890. For this analysis, normalized tau expression was scaled relative to the average tau levels measured for each punch from vehicle-treated and ZFP-treated animals that did not show detectable ZFP expression. Data from each punch for NHP07 was then extracted and grouped according to the brain section analyzed. As shown, NHP07 showed tau suppression in certain brain samples.

[0184] Figure 5A shows a comparison of tau expression and ZFP levels in a control subject (vehicle, NHP01) and a subject treated with AAV SYN918-890 (NHP04) (top panel), and Figure 5B shows MRI results from slice 7. For this analysis, normalized tau expression was scaled relative to the average of tau levels measured from vehicle- and ZFP-treated animals, which showed no detectable ZFP expression, for each punch.

[0185] As shown, vehicle-treated NHPs had neither detectable tau reduction nor ZFP expression. In contrast, NHP04 showed detectable ZFP expression in slice 7 in the right hemisphere, which also correlated with the two central hippocampal punches. Increased ZFP coverage and tau reduction were observed in slices 8 and 9 in the right hemisphere, correlating well with MRI data from these levels of the brain.

[0186] Figure 6 shows the results of tau expression and ZFP levels in control subjects (Figure 6A; NHP01, NHP02, NHP03); AAV SYN1.918-890-treated subjects (Figure 6B; NHP04, NHP05, NHP06); AAV CMV.918-890-treated subjects (Figure 6C; NHP07, NHP08); AAV SYN1.930-treated subjects (Figure 6C; NHP09, NHP10); AAV SYN1.890-treated subjects (Figure 6D; NHP11, NHP12, NHP13); and SYN1.918-treated subjects (Figure 6E; NHP14, NHP15).

[0187] Figure 7 shows a combined analysis of all punches across all animals evaluating three methods for establishing baseline tau levels for a given punch: no scaling (top panel), scaling to the average of three vehicle-treated animals (middle panel), or scaling to the average tau expression of vehicle- and ZFP-treated animals that show no ZFP expression (bottom panel). Results from the indicated subject (vehicle NHP subject shown in the left panel) and all AAV-treated subjects (right panel) are shown. As shown, scaling by either method better approximates baseline levels across all 15 animals, while the third method (scaling to vehicle- and ZFP-treated NHPs that show no ZFP expression) somewhat better represents the tau baseline across the three vehicle- and 12 ZFP-treated animals in the study.

[0188] For this analysis, normalized tau expression was scaled relative to the average tau levels measured from vehicle-treated and ZFP-treated animals, which showed no detectable ZFP expression, for each punch. Figure 8 shows results from the indicated subject (vehicle NHP subject shown in the left panel) and all AAV-treated subjects (right panel), both for scaled tau expression levels (top) and absolute ZFP transcript levels (copies / ng mRNA). Figure 9 shows tau expression results from the left and right hemispheres of the indicated subjects. Figures 10 and 11 show results for indicated animals in which fewer than 1E4 ZFP transcripts were present (left panel of Figure 10; bottom left panel of Figure 11); 1E4 to 1E5 ZFP transcripts were present (center panel of Figure 10; top right panel of Figure 11); and more than 1E5 ZFP transcripts were present (right panel of Figure 10; bottom right panel of Figure 11) and all levels of ZFP transcripts (top left panel of Figure 11). As shown, the majority of punches exhibiting low levels of ZFP expression do not have significant tau reduction, however punches with 1E4-1E5 ZFP transcripts / ng mRNA have intermediate tau reduction for some treatments, and those with 1E5 or more transcripts / ng mRNA have even higher tau reduction.

[0189] Figures 13, 15, and 17 show the percent normalized tau expression as a function of ZFP levels (transcripts / ng mRNA) in subjects treated as indicated. Figure 13 shows the results when tau expression is scaled to the average of vehicle-treated and ZFP-treated animals, which show no detectable ZFP expression. Figure 15 shows the results when tau expression is scaled to the average of vehicle-treated animals only; Figure 17 shows the results when tau expression was not scaled to adjust for baseline tau levels. Regardless of the scaling method, the data show a significant correlation between ZFP expression levels and tau reduction for four of the five ZFP treatments: AAV SYN1.65918-57890, AAV CMV.65918-57890, AAV SYN1.57890, and AAV SYN1.65918. The highest degree of tau reduction was achieved with AAV SYN1.65918-57890 and AAV CMV.65918-57890 treatment, with some punches showing greater than 80% tau reduction.

[0190] Figures 12, 14, and 16 show correlation plots between tau expression and ZFP levels for NHP subjects treated with either vehicle (left panel) or AAV vectors encoding the 57890 and 65918 ZFP-TF repressors, whose expression is driven by the synapsin 1 promoter (right panel). The limit of the ZFP qRT-PCR assay for absolute quantity measurements is approximately 1E2 transcripts / ng mRNA, as indicated by the lower limit of quantification (BLOQ). Figure 12 shows results where tau expression is scaled to the average of vehicle-treated and ZFP-treated animals that show no detectable ZFP expression. Figure 14 shows an analysis where tau expression levels are scaled to the average of three vehicle-treated animals. Figure 16 shows an analysis where tau expression levels were not scaled to correct for baseline tau levels. As shown, tau suppression correlated with the amount of ZFP-TF present.

[0191] Example 2 Tau suppression in a humanized tau mouse model We also developed a P301L mutant human tau (P301L) transgenic mouse model (rTg4510, Jackson Labs) and a hTau mouse model (B6.Cg-Mapt) for tauopathy. tml(EGFP)Klt Tau reduction in hTau mice (Tg(MAPT)8cPdav / J, Jackson Labs) was assessed after administration of the gene repressors described herein. hTau mice express the WT human MAPT gene, and in addition, the endogenous (mouse) Mapt gene has been knocked out and replaced with a GFP-expressing construct. Treatment groups were as shown in the table below.

[0192] [Table 3]

[0193] The endpoints measured were: ZFP and tau mRNA expression levels by RT-qPCR; GFAP, Iba1, and NeuN mRNA expression levels by RT-qPCR; Saitohin (STH) mRNA expression levels by RT-qPCR (STH is a protein-coding gene in apes and humans located in an intron between exons 9 and 10 of the human tau gene; see, e.g., Conrad et al. (2002) Proc Natl Acad Sci US A. 99(11):7751-7756); and tau protein levels.

[0194] As shown in Figures 18A-18D, the synergistic pairing of 57890 and 65918 (AAV9.hSYN.65918-T2a-57890) resulted in ZFP expression levels at least two-fold higher than 57890 (AAV9.hSY1.57890) alone. See Figure 18A. Furthermore, in humanized tau mice administered a construct encoding 57890 and 65918, human tau mRNA was suppressed by approximately 90% suppression, compared to approximately 60% suppression in mice administered a construct encoding 57890 alone (mouse tau mRNA expression was suppressed by approximately 87% by the combination of 57890 and 65918 and approximately 81% by 57890). See Figure 18B. Similar results were obtained after RT-qPCR analysis of human Saitohin (hSTH), which was suppressed by approximately 88% by the synergistic combination of 57890 and 65918, compared to approximately 68% with 57890 alone. See Figure 18C. IBA1 and GFAP levels were elevated in both treatment groups compared to controls, whereas NeuN levels did not differ significantly between groups. See Figure 18D.

[0195] Additionally, the clinical and therapeutic efficacy of repressors will be further evaluated in this and other mouse models of AD (e.g., APPswe / PS1d9, Jackson Labs) to determine whether there is a reduction in biomarkers and symptoms of tauopathy, including one or more of the following: RNAscope ISH analysis (single-cell analysis of ZFP, human tau, and mouse tau, see, e.g., Carstens et al. (2016) J Neurosci. 36(23):6312-6320); IHC ZFP / tau analysis (see, e.g., Zeitler et al. (2019) Nature Medicine 25:1131-1142); neurotoxicity, gliosis, degenerative neurites, spinal cord loss, excitotoxicity, cortical and hippocampal shrinkage, dendritic tau accumulation, cognitive (e.g., radial arm maze and Morris water maze, fear conditioning, etc.) and motor deficits. See, e.g., Bryan et al., (2009) Chapter 1: Transgenic Mouse Models of Alzheimer's Disease: Behavioral Testing and Considerations in Methods of Behavior Analysis in Neuroscience. 2nd edition, ed. Buccafusco, Boca Raton (FL): CRC Press / Taylor & Francis. Additionally, wild-type mice treated with chemically induced seizure models, e.g., excitotoxic compounds such as pentylenetetrazole (PTZ, see, e.g., Meyers et al. (1975) Epilepsia 16(2):257-67) or kainate (Ferraro et al. (1997) Mamm Genome 8:200-208), are evaluated 4-8 weeks after administration of a gene repressor described herein to determine whether tau reduction provides a protective effect against seizures, including reducing seizure-associated mortality, prolonging the latency to seizures, and / or reducing seizure severity.

[0196] Example 3 Neuroinflammatory response Primates treated in vivo with the MAPT repressor ZFP-TF were also evaluated for the expression levels of microglial and astrocyte markers. Specifically, punches as described in Example 1 were evaluated for IBA1 and GFAP expression using RT-qPCR reagents. In addition, the levels of the E1F4A housekeeping gene were also evaluated in treated primates. Briefly, brain punches were transferred to a 1.5 mL Eppendorf tube containing 0.6 mL of TRI Reagent (Thermo Fisher) and two 3.2 mm steel beads (BioSpec Products) on ice.

[0197] Tissues were lysed using a Qiagen TissueLyser at 4°C using the following parameters: 5 cycles, 90 s duration, and 25.1 frequency. After brief centrifugation, 70 μL of 1-bromo-3-chloropropane was added to each sample at RT. Samples were vortexed for 10 seconds and centrifuged at 12,000 x g for 10 minutes at 4°C. 120 μL of aqueous phase from each sample was transferred to a well of a 96-well plate. 60 μL of isopropyl alcohol and 12 μL of MagMax magnetic beads (Thermo Scientific) were added to each sample well containing the aqueous phase sample. RNA was isolated from the tissue lysates using a Kingfisher 96 robot (Thermo Scientific) and a MagMax kit (Thermo Fisher) according to the manufacturer's instructions. 100 μL of eluted RNA was separated from the magnetic beads using a magnetic stand.

[0198] RNA yield and quality were assessed using a Nanodrop 8000 instrument (Thermo Scientific). For all samples, cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with 10 μL of RNA and 10 μL of RT Master Mix (10x RT buffer, 10x random primers, 25x dNTP mix, Multiscribe enzyme, and RNAse-free water). Reverse transcription was performed on a C1000 Touch Biorad thermal cycler using the following program: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, and a hold at 4°C. qRT-PCR was performed using a Biorad CFX384 thermal cycler. cDNA was diluted 10-fold in nuclease-free water, and 4 μL of diluted DNA was added to each 10 μL PCR reaction. Each sample was assayed in quadruplicate. Custom TaqMan primer:probe assays were used in this study. 2xFast Multiplex PCR (Qiagen) master mix was used for the tau / EIF4a2 / ATP5b triplex assay, and SsoAdvanced Universal Probes Supermix (Biorad) was used for the other assays. qPCR cycling conditions were as follows: Qiagen Fast Multiplex master mix → 95°C for 5 minutes, 95°C for 45 seconds, 60°C for 45 seconds, plate read, 40 cycles; Biorad SsoAdvanced master mix → 95°C for 90 seconds, 95°C for 12 seconds, 60°C for 40 seconds, plate read, 42 cycles.

[0199] As shown in Figures 19A-C, IBA1 and GFAP analysis did not reveal ZFP-dependent increases in microglial or astrocytic marker levels in any primates treated with AAV constructs containing the synapsin promoter. However, primates treated with AAV constructs containing the CMV promoter showed increased levels of astrocytic markers. Thus, no ZFP-dependent neuroinflammatory responses were observed when primates were administered constructs containing the neuron-specific synapsin promoter.

[0200] Furthermore, as shown in Figures 20A and 20B, no significant bulk effects on IBA1 (Figure 19A) or GFAP (Figure 19B) were observed for either treatment group.

[0201] Furthermore, as shown in Figure 21, there was no correlation between the levels of a housekeeping gene (EIF4A2) and ZFP expression in primates treated with a ZFP MAPT repressor. Any toxicity due to ZFP expression or tau reduction was accompanied by a decrease in E1F4A2 levels as ZFP levels increased.

[0202] Thus, the synergistic tau repressors described herein efficiently suppress tau in vivo (up to 90% or more compared to controls) without eliciting a neuroinflammatory response.

[0203] Example 4 Tau protein levels Tau protein levels were also examined in cells and subjects receiving the repressors described herein.

[0204] Briefly, neurons (derived from induced pluripotent stem cells) are administered with the above-mentioned AAV vector, and the tau protein level in cells or punches taken from treated subjects is evaluated by ELISA using standard techniques.Briefly, neurons derived from human IPSCs are administered with the AAV6 vector encoding single ZFP or synergistic 65918 / 57890 combination at a dose of 1E5 VG / cell (n=4 replicates).Cells are cultured for 32 days, and tau protein level is evaluated by ELISA using standard techniques.

[0205] As shown in Figure 22, the ratio of tau to total protein in human iPSC-derived neurons was significantly reduced in cells treated with the tau repressors described herein. Specifically, the 57930 repressor alone reduced tau protein expression by more than five-fold compared to controls; the 57890 repressor alone reduced tau protein expression by two-fold; the 65918 repressor alone reduced tau expression by more than two-fold; and the synergistic 65918-57890 reduced tau protein expression by more than ten-fold compared to controls. Tau protein levels were also assessed in the CSF and / or brain homogenates of a humanized tau mouse model treated as described in Example 2 above using standard ELISA (Thermo). A 50% reduction in human tau protein was seen for the 65918-57890 synergistic pair compared to controls (vehicle) at 6 weeks. The 65918-57890 pair also reduced human tau protein levels compared to animals that received only 57890. Furthermore, tau protein levels were further reduced at later time points in the animals after treatment, including 8 to 12 weeks or longer.

[0206] Thus, the tau repressors described herein reduce tau protein levels in vitro and in vivo.

[0207] This study demonstrates that tau ZFP-TF reagents suppress tau expression in the primate brain, including at therapeutic levels.

[0208] All patents, patent applications and publications mentioned herein are incorporated by reference in their entirety for all purposes.

[0209] Although the present disclosure has been provided in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the foregoing description and examples should not be construed as limiting.

Claims

1. A composition comprising two or more artificial zinc finger protein transcription factors (ZFP-TFs) that repress MAPT expression.

2. The composition of claim 1, comprising two ZFP-TFs.

3. 3. The composition of claim 1 or 2, wherein the two ZFP-TFs comprise ZFPs designated 65918 together with 57890 or 57930.

4. The composition of claim 3, wherein the two ZFP-TFs comprise ZFPs designated 65918 and 57890.

5. 5. The composition of any one of claims 1 to 4, wherein the two or more ZFP-TFs are encoded by polynucleotides carried by one or more viral vectors.

6. The composition of claim 5 , wherein the one or more viral vectors are AAV vectors.

7. The composition of claim 6, wherein one AAV vector comprises polynucleotides encoding two or more ZFP-TFs.

8. The composition of claim 7, wherein the AAV vector is an AAV9 vector.

9. 9. The composition of any one of claims 5 to 8, wherein one or more viral or AAV vectors comprise a CMV or synapsin (SYN) promoter.

10. 10. Use of one or more compositions according to any one of claims 1 to 9 for the prevention and / or treatment of a tauopathy in a subject in need thereof.

11. 11. The use according to claim 10, wherein the composition prevents and / or treats Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear palsy, traumatic brain injury (TBI), seizure disorders and / or corticobasal ganglionic degeneration, preferably reducing or eliminating the symptoms of a tauopathy by reducing the occurrence of neuronal tangles in the brain of the subject, as appropriate.

12. The use of claim 10 or 11, wherein the composition comprising two or more ZFP-TFs is delivered to the CNS of the subject using one or more AAV vectors comprising one or more polynucleotides encoding the two or more ZFP-TFs.

13. The use according to claim 12, wherein the AAV vector is an AAV9 vector.

14. 14. The use according to claim 12 or 13, wherein the AAV vector is administered intravenously or to the CNS.

15. 15. The use of claim 14, wherein the AAV vector is administered to the striatum or hippocampus in one or both hemispheres of the subject's brain.

16. 16. The use of any one of claims 10 to 15, wherein expression of two or more ZFP-TFs of the composition reduces MAPT gene expression and / or tau levels in the brain of a primate subject by 50% or more, optionally 70% or more, up to 99% compared to an untreated subject.

17. 17. The use according to any one of claims 10 to 16, wherein the artificial transcription factors of the composition are carried by one or more AAV vectors comprising a CMV or SYN promoter, at between about 1E10 and 6E11 rAAV vector genomes per hemisphere.

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