RU2789459C2 - Tau modulators and methods and compositions for their delivery - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a gene transcription repressor of microtube associated protein tau (hereinafter – MAPT), polynucleotide encoding a transcription repressor, a gene delivery agent, a pharmaceutical composition for suppression of MAPT expression, the use of a transcription repressor, polynucleotide, a gene delivery agent, and/or a pharmaceutical composition for suppression of MAPT expression in a subject, and a kit for suppression of MAPT expression.

EFFECT: invention expands the arsenal of agents for suppression of MAPT expression.

30 cl, 17 dwg, 3 tbl, 11 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/428871, filed December 1, 2016; U.S. Provisional Application No. 62/450895, filed January 26, 2017; U.S. Provisional Application No. 62/466198, filed March 2, 2017; U.S. Provisional Application No. 62/500807, filed May 3, 2017; and U.S. Provisional Application No. 62/584,342, filed November 10, 2017, the disclosure of which is hereby incorporated by reference in their entirety.

FIELD OF TECHNOLOGY

The present invention relates to the field of diagnosis and therapy of taupathies such as Alzheimer's disease.

BACKGROUND OF THE INVENTION

Many, perhaps most, physiological and pathophysiological processes can be accompanied by aberrant up- or down-regulation of gene expression. Examples include inappropriate expression of proinflammatory cytokines in rheumatoid arthritis, underexpression of the liver LDL receptor in hypercholesterolemia, overexpression of proangiogenic factors, and underexpression of antiangiogenic factors in solid tumor growth, to name but a few. In addition, pathogenic organisms such as viruses, bacteria, fungi and protozoa can be controlled by altering gene expression.

The promoter regions of genes typically contain proximal, central, and downstream (3') elements, and transcription can be regulated by a variety of enhancers. These sequences contain multiple binding sites for multiple transcription factors and can activate transcription regardless of location, distance, or orientation relative to the promoter sequence. To achieve regulation of gene expression, enhancer-associated transcription factors loop intermediate sequences and contact the promoter region. In addition, activation of eukaryotic genes may require decompactization of the chromatin structure, which can be done by recruiting histone-modifying enzymes or ATP-dependent chromatin remodeling complexes, so that the chromatin structure is altered and DNA accessibility to other proteins involved in gene expression is increased ( Ong and Corces (2011) Nat Rev Genetics 12:283).

The disruption of chromatin structure can occur through several mechanisms, some of which are gene-specific, while others are ubiquitous throughout the genome and occur during cellular processes such as mitosis that require chromatin condensation. Lysine residues in histones can become acetylated, effectively neutralizing the charge interaction between histone proteins and chromosomal DNA. This has been observed at the hyperacetylated and highly transcribed β-globin locus, which has also been shown to be DNase sensitive, indicative of wide availability. Other types of histone modifications that have been observed include methylation, phosphorylation, deamination, ADP-ribosylation, addition of β-N-acetylglucosamine sugars, ubiquitination, and sumoylation (see Bannister and Kouzarides (2011) Cell Res 21: 381).

The repression or activation of disease-associated genes has been achieved through the use of engineered transcription factors. Methods for the construction and use of engineered zinc finger domain (ZFP-TF) transcription factors have been fully described (see, for example, US Pat. No. 6,534,261) and more recently have also been described as transcription activator-like effectors (TALE) factors transcription factors (TF) (TALE-TF) and transcription factors based on CRISPR-Cas (CRISPR-Cas-TF) (see review by 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). In addition, gene activation has been achieved by using the CRIPSR/Cas-acetyltransferase fusion (Hilton et al., (2015) Nat Biotechnol 33(5):510-517). Engineered transcription factors (TFs) that downregulate gene expression (are repressors) have also been shown to be effective in the treatment of trinucleotide diseases such as Huntington's disease (HD). See, for example, U.S. Patent No. 89568282 and U.S. Patent Application Publication No. 2015/0335708.

Alzheimer's disease (AD) is a complex multifactorial disease characterized by several different disease mechanisms that are not fully understood and may interact with each other in ways that are not well understood. An estimated 5.3 million Americans of all ages have AD, making it one of the top ten causes of death in America, and it is estimated that there will be 106.2 million people worldwide with the disease by 2050 (van Dijk et al ., (2015) Front Neurosci 9, art.173). The disease is more common in women (two-thirds of cases), and people of African or Hispanic descent are more likely to develop AD than white Caucasians. The causes of AD appear to be related to genetics (especially in the case of early onset, 5% of cases), as well as environmental and lifestyle factors. As a rule, the disease is diagnosed in the middle of the seventh decade of a person, although by the time the diagnosis is established, the disease has progressed for years or even decades. The disease continues to progress over time, and to date no therapeutic interventions have been identified that would limit or reverse the effects of the disease.

A symptom of the disease is the loss of synapses in the brain, which leads to a decrease in cognitive abilities. In a healthy brain, synaptic plasticity is believed to be what allows learning and memory formation. During AD, synaptic plasticity changes and many of the mechanisms involved in maintaining this plasticity are deregulated, resulting in synaptic dysfunction and destruction (Spiers-Jones and Hyman (2014) Neuron 82:756).

Although there appear to be many molecular factors that influence the onset and progression of AD, much of the scientific research has focused on two main actors in the disease. The first member is a 40-42 amino acid fragment of the amyloid precursor protein (APP) called amyloid β (Aβ), which is formed by proteolytic cleavage by beta-secretase and gamma-secretase (Olsson et al., (2014) J Biol Chem 289 ( 3):1540-1550). Insoluble fragments of Aβ accumulate in "senile" plaques in the brain, although there does not appear to be a strong correlation between the presence of these plaques and neurodegeneration.

Another member, Tau, also received a lot of attention. Tau is a microtubule-associated protein that was originally thought to stabilize microtubules. In patients with AD and in the elderly in general, but to a lesser extent, tau can accumulate in neurofibrillary tangles (NFTs). As the disease progresses, tau becomes hyperphosphorylated and detaches from microtubules and accumulates in filaments. In contrast to Aβ plaques, there is a direct correlation between the presence of NFTs and cognitive decline (Spiers-Jones and Hyman, ibid.).

Interestingly, both tau and Aβ appear to play important roles in normal synaptic function. Tau appears to play an important role in the transport of mitochondria in the cell to the synapse, and tau overexpression has been shown to inhibit this transport. It is believed that disruption of mitochondrial transport causes synapse loss due to the important role that mitochondria play in ATP production and calcium buffering. Aβ appears to play a role in synapse plasticity in a healthy cell. However, the accumulation of these two proteins in plaques and tangles is associated with the progression of AD. Indeed, it appears that the appearance of amyloid plaques is important in the very early stages of AD, and this leads to the appearance of NFTs, and that these two proteins actually act synergistically with each other to accelerate the progression of the disease (Pooler et al. al., (2015) Acta Neuropath Comm 3(14):1). However, it is becoming increasingly clear that soluble forms of these proteins contribute to toxicity (Spiers-Jones and Hyman, ibid.).

In fact, abnormal levels and/or aggregation of tau have been shown to be involved in a number of conditions that are collectively referred to as taupathies. These include Alzheimer's disease (AD), frontotemporal dementia (FTD, see Benussi et al., (2015) Front Ag Neuro 7, art. 171)), progressive supranuclear paresis (PSP), intractable genetic epilepsies (eg, Dravet's syndrome). , see Gheyara et al., (2014) Ann Neurol 76:443-456) and corticobasal degeneration (CBD, see Scholz and Bras 2015, Int J. Mol. Sci 16(10):24629-24655). Decreased tau expression in adult mice using direct injection of antisense oligonucleotides into the cerebrospinal fluid (CSF) induced a complete or partial reduction in tau levels and also protected treated mice from chemically induced epileptic seizures as measured by seizure severity (DeVos et al. , (2013) J of NeuroSci 33(31):12887).

It has been found that AD proceeds in the brain in a hierarchical manner, starting in the entorhinal cortex, and then spreading through the hippocampal formation, limbic and association cortices, and finally affecting most areas of the brain in the advanced stages of the disease. Interestingly, the progression of AD is marked by the appearance of NFTs, indicating the involvement of tau in the later stages of the disease. It has even been suggested that tau may have prion-like properties, as scientific research shows that misfolded, highly phosphorylated tau protein is more readily taken up by neurons and can spread disease throughout the brain, and that this misfolded tau, isolated from the brains of AD patients, can be readily taken up by mouse neurons (Takeda et al., (2015) Nat Comm doi: 10.1038/ncomms9490; Hyman (2014) Neuron 82:1189). In addition, a scientific study using a transgenic mouse model showed that expression of the human tau mutant, which is associated with glomerular formation only in the mouse entorhinal cortex, resulted in mouse tau misfolding and aggregation of this tau in neurons without any detectable human tau expression (de Calignon et al., (2012) Neuron 73:685-697), suggesting that the human misfolded protein is capable of seeding the misfolding and causing aggregation of mouse proteins. In addition, genetic reduction or loss of endogenous murine tau protects against neuropathological toxicity induced by overexpression of a mutant human tau transgene (Wegmann et al., (2015) EMBO J. 34(24):3028-41).

Thus, there remains a need for methods to prevent and/or treat taupathies, including AD, FTD, PSP CBD, and epileptic seizures; including in modes of action that show almost ubiquitous delivery to the brain.

SUMMARY OF THE PRESENT INVENTION

Disclosed here are methods and compositions for diagnosing, preventing and/or treating one or more taupathies, such as Alzheimer's disease (AD). In particular, provided herein are methods and compositions for modifying (eg, modulating expression) a tau allele for the treatment of at least one tauopathy, such as AD, comprising engineered transcription factor repressors (which repress tau expression). In addition, these methods and compositions can be used to modify the tau allele to treat and/or prevent other taupathies, including AD, FTD, PSP CBD, and/or seizures. In particular, methods and compositions for detecting, reducing and/or eliminating tau aggregates in a subject with tauopathy are provided herein.

Thus, genetic modulators of the microtubule associated protein tau (MAPT) gene are described herein, wherein the modulator includes: a DNA binding domain that binds to a target site of at least 12 nucleotides in the MAPT gene; and a functional domain (eg, a transcription-regulating domain (such as an inhibitory domain or an activating domain) or a nuclease domain). Any DNA binding domain may be used, including, but not limited to, zinc finger protein (ZFP), TAL effector domain protein (TALE), chimeric guide RNA (from CRISPR systems), Argonaute protein, and the like. Also provided are one or more polynucleotides, including viral and non-viral gene delivery vehicles (e.g., as mRNAs, plasmids, AAV vectors, lentiviral vectors, Ad vectors) encoding the genetic modulators described herein (or one or more of their components on the same or different polynucleotides). In some embodiments, the gene delivery vehicle includes an AAV vector, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, AAV rh10, pseudotypes of these vectors (e.g., as AAV2/8 , AAV2/5, AAV2/6, AAV2/9, etc.), including AAV vector variants known in the art (e.g., from US Pat. . Also provided are pharmaceutical compositions and isolated cells containing one or more genetic modulators, one or more polynucleotides, and/or one or more gene delivery vehicles. The present invention also provides methods and uses for modulating MAPT expression in a subject in need thereof, including by providing the subject with one or more polynucleotides, one or more gene delivery vehicles, and/or a pharmaceutical composition as described herein. In some embodiments, the compositions described herein are used to suppress the expression of MAPT in a subject, including the treatment and/or prevention of tauopathy (eg, by reducing the amount of tau in a subject). The compositions described herein reduce tau levels over extended periods of time (6 months to a year or more) in the brain (including, but not limited to, frontal cortex (lobe), anterior cortex, posterior cortex, hippocampus, brainstem, striatum , thalamus, midbrain, cerebellum) and spinal cord (including but not limited to lumbar, thoracic and cervical regions). The compositions described herein may be provided to a subject by any route of administration, including, but not limited to, intracerebroventricular, intrathecal, intracranial, intravenous, orbital (retroorbital (RO)) and/or intracisternal administration. Also provided are kits comprising one or more of the compositions (eg, genetic modulators, polynucleotides, pharmaceutical compositions, and/or cells) described herein, as well as instructions for use of these compositions.

Thus, in one aspect, engineered (non-naturally occurring) genetic modulators (eg, repressors) of one or more tau genes are provided. Tau genetic modulators may include systems (eg, zinc finger proteins, TAL effector proteins (TALE) or CRISPR/dCas-TF) that modulate (eg, repress) the expression of the tau allele. Engineered zinc finger or TALE proteins are non-naturally occurring zinc finger or TALE proteins whose DNA-binding domains (e.g., helix recognition or RVD) have been altered (e.g., by selection and/or design) to bind to preformed selected target site. Any of the zinc finger proteins described herein can include 1, 2, 3, 4, 5, 6 or more zinc fingers, with each zinc finger having a recognition helix that binds to a target subsite in the selected sequence(s) ( e.g. gene(s)). Likewise, any of the TALE proteins described herein may include any number of RVD TALEs. In some embodiments, at least one RVD has non-specific DNA binding characteristics. In some embodiments, at least one recognition helix (or RVD) does not occur naturally. CRISPR/Cas-TF includes a chimeric guide RNA that binds to a target sequence. In some embodiments, the engineered transcription factor binds (e.g., via a ZFP, TALE, or sgRNA DNA binding domain) to a target site of at least 12 bp in a gene encoding tau, e.g., a target site of at least 12 base pairs (e.g., 12, 13, 14, 15, 16, 17, 18 or more) from Tables 1-3 (SEQ ID NOs: 1-6, 33 and 44-46), including contiguous or non-contiguous sequences in these target sites. In some embodiments, the DNA binding domains of zinc finger proteins have recognition helices in the proteins shown in any of Tables 1-3, including ZFPs designated 52288, 52322, 52366, 57890, 57880, 65888, 52364, 52389, 65894, 57930 , 65918, 65920, 65887, 57947, 65968, 65976, or 65860 of Tables 1, 2, and 3. In some embodiments, the genetic modulator is a genetic repressor that includes the DNA binding domain (ZFP, TALE, chimeric guide RNA) described here, operably linked to a transcriptional repressive domain. In other embodiments, the genetic modulator is a genetic repressor comprising a DNA binding domain (ZFP, TALE, chimeric guide RNA) described herein, operably linked to at least one nuclease domain (e.g., one, two or more nuclease domains). The resulting nuclease is capable of genetic modification (by insertions and/or deletions) of the target gene, for example, in the target sequence(s) of the DNA binding domain; at the cleavage site(s); close (1-50 or more base pairs) to the target sequence(s) and/or cleavage site(s); and/or between paired target sites when a pair of nucleases is used for cleavage.

In some embodiments, zinc finger proteins (ZFPs), the CRISPR/Cas system Cas protein, or the TALE proteins described herein can be placed in operable association with a regulatory domain (or functional domain) as part of a fusion protein. A functional domain may be, for example, a transcription activating domain, a transcription repressive domain, and/or a nuclease (cleaving) domain. By choosing either an activating domain or a repressive domain for use with a DNA binding domain, such molecules can be used to either activate or repress tau expression. In some embodiments, functional or regulatory domains may play a role in post-translational histone modifications. In some cases, the domain is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methylase, or an enzyme that sumolizes or biotinylates histone, or another enzymatic domain that allows post-translational modifications of histones to regulate gene repression (Kousarides (2007 ) Cell 128:693-705). In some embodiments, a molecule comprising a ZFP, dCas, or TALE targeted to a tau gene (eg, MAPT) as described herein, fused to a transcription repressive domain, is provided that can be used to suppress tau expression. In some embodiments, the methods and compositions of the present invention are useful in the treatment of eukaryotes. In some embodiments, the activity of the regulatory domain is regulated by an exogenous small molecule or ligand such that interaction with the transcriptional machinery of the cell will not occur in the absence of the exogenous ligand. Such external ligands control the degree of interaction of ZFP-TF, CRISPR/Cas-TF, or TALE-TF with the transcriptional machinery. The regulatory domain(s) may be operably linked to any portion(s) of one or more of the ZFPs, dCas, or TALEs, including between one or more ZFPs, dCas, or TALEs, outside of one or more of the ZFPs, dCas, or TALEs, and any their combination. In preferred embodiments, the regulatory domain leads to the suppression of gene expression of the target tau gene. Any of the fusion proteins described herein can be formulated into a pharmaceutical composition.

In some embodiments, the methods and compositions of the present invention include the use of two or more fusion proteins described herein, such as two or more tau modulators (eg, tau repressors). Two or more fusion proteins may bind to different target sites and include the same or different functional domains. Alternatively, two or more fusion proteins described herein may bind to the same target site but include different functional domains. In some cases three or more fusion proteins are used, in others four or more fusion proteins are used, while in others 5 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 the cell as nucleic acids. In preferred embodiments, the fusion proteins cause downregulation of the expression of the target gene. In some embodiments, the two fusion proteins are administered at doses where each protein is active on its own, but in combination the inhibitory activity is additive. In preferred embodiments, the two fusion proteins are administered at doses where neither is active on its own, but in combination the inhibitory activity is synergistic.

In some embodiments, the engineered DNA binding domains described herein can be placed in operable association with nuclease (cleavage) domains as part of a fusion protein. In some embodiments, the implementation of the nuclease includes the nuclease Ttago. In other embodiments, nuclease systems, such as the CRISPR/Cas system, can be used with a specific chimeric guide RNA to target a nuclease to a target site in DNA. In some embodiments, such nucleases and fusions with nuclease(s) can be used to target the tau allele in stem cells such as induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), or neural stem cells in which nuclease fusion activity will result in decreased expression of the tau allele. In some embodiments, pharmaceutical compositions containing modified stem cells are provided.

In yet another aspect, a polynucleotide is provided that encodes any of the DNA-binding proteins, nucleases, and/or transcription factors described herein. In some embodiments, the polynucleotide comprises at least one AAV vector (or a pseudotype or variant thereof), including, but not limited to, one or more AAV2, AAV2/9, AAV6, or AAV9 vectors, including, but not limited to, one or more AAV vectors described in US Patent No. 9,585,971 or US Patent No. 7,198,951) and/or one or more AAV vectors described in US Provisional Application No. 62/503,121.

In other aspects, the present invention includes the delivery of a donor nucleic acid into a target cell. The donor may be delivered before, after, or together with the nucleic acid encoding the nuclease(s). The donor nucleic acid may include an exogenous sequence (transgene) for integration into the cell's genome, such as an endogenous locus. In some embodiments, the donor may include a full length gene or a fragment thereof flanked by regions of homology to the target cleavage site. In some embodiments, the donor lacks homologous regions and integrates into the target locus via a homology-independent mechanism (ie, NHEJ). The donor may include any nucleic acid sequence, such as a nucleic acid that, when used as a substrate for homology-directed nuclease-induced double-strand break repair, results in a donor-specific deletion to be created at an endogenous chromosomal locus, or alternatively (or additionally), a new allelic forms (eg, point mutations that remove the transcription factor binding site) of the endogenous locus to be created. In some aspects, the donor nucleic acid is an oligonucleotide, wherein the integration results in a gene editing event or targeted deletion. In some embodiments, the donor encodes a transcription factor capable of repressing Tau expression. In other embodiments, the donor encodes an RNA molecule that inhibits the expression of a tau protein.

In some embodiments, the polynucleotide encoding the DNA binding protein is mRNA. In some aspects, the mRNA may be chemically modified (see, for example, Kormann et al., (2011) Nature Biotechnology 29(2):154-157). In other aspects, the mRNA may include an ARCA cap (see US Pat. Nos. 7,074,596 and 8,153,773). In other embodiments, the mRNA may include a mixture of unmodified and modified nucleotides (see US Patent Application Publication No. 2012/0195936).

In yet another aspect, a gene delivery vector is provided, comprising any of the polynucleotides (eg, encoding genetic modulators (repressors)) described herein. In some embodiments, the vector is an adenoviral vector (eg, an Ad5/F35 vector), a lentiviral vector (LV) comprising integration-competent or integration-defective lentiviral vectors, or an adeno-associated viral vector (AAV). In some embodiments, the AAV vector is an AAV2, AAV6, or AAV9 vector. In some embodiments, the AAV vector is an AAV variant capable of crossing the blood-brain barrier (eg, US Patent No. 9,585,971 and US Provisional Application No. 62/503,121). Also provided herein are adenoviral (Ad) vectors, LV vectors, or adeno-associated virus (AAV) vectors comprising a sequence encoding at least one nuclease (ZFN or TALEN) and/or a donor sequence for targeted integration into a target gene. In some embodiments, the Ad vector is a chimeric Ad vector, such as an Ad5/F35 vector. In some embodiments, the lentiviral vector is an integrase-defective lentiviral vector (IDLV) or an integration-competent lentiviral vector. In some embodiments, the vector is pseudotyped with the VSV-G coat protein or other coat proteins.

In addition, pharmaceutical compositions are also provided containing nucleic acids (for example, vectors (for example, AAV) for delivery, including sequences encoding artificial transcription factors (tau repressors) described herein, and/or proteins (for example, ZFP, Cas or TALE or fusion proteins comprising ZFP, Cas, or TALE.) For example, some compositions include a nucleic acid comprising a sequence that encodes one of the ZFP, Cas, or TALE described herein, operably linked to a regulatory sequence, in association with a pharmaceutically acceptable carrier or diluent, wherein the regulatory sequence provides expression of the nucleic acid in the cell.In some embodiments, the encoded ZFP, CRISPR/Cas, or TALE are specific for the mutant tau allele.In some embodiments, the pharmaceutical compositions contain ZFP, CRISPR/Cas, or TALE that modulate the mutant tau allele, and ZFP, CRISPR/Cas or TA LE that modulate neurotrophic factor. Protein-based compositions include one of several ZFP, CRISPR/Cas, or TALE described herein and a pharmaceutically acceptable carrier or diluent.

In yet another aspect, an isolated cell is also provided, comprising any of the proteins, polynucleotides, and/or compositions described herein.

In another aspect, provided herein are methods for treating and/or preventing tauopathy, such as Alzheimer's disease or an epileptic seizure, using the methods and compositions described herein. In some embodiments, the methods include compositions in which the polynucleotides and/or proteins can be delivered using a viral vector, a non-viral vector (eg, a plasmid), and/or combinations thereof. In some embodiments, the methods include compositions containing stem cell populations containing ZFP or TALE or modified with ZFN, TALEN, Ttago, or the CRISPR/Cas nuclease system of the present invention. The introduction of the compositions described here (proteins, polynucleotides, cells and/or pharmaceutical compositions containing these proteins, polynucleotides and/or cells) leads to a therapeutic (clinical) effect, including, but not limited to, reducing the intensity or eliminating any of clinical symptoms associated with AD, taupathies or epileptic seizures, as well as an increase in the functioning and/or number of CNS cells (eg, neurons, astrocytes, myelin, etc.). In some embodiments, the compositions and methods described herein reduce tau expression (compared to controls not receiving the artificial repressors described here) by at least 30% or 40%, preferably by at least 50%, even more preferably by at least 70%. In some embodiments, a reduction of at least 50% is achieved.

In yet another aspect, a method for delivering a tau repressor to the brain of a subject (e.g., a mammalian subject such as a mouse, human, or non-human primate) (NHP) using a viral or non-viral vector is described. In some embodiments, the viral vector is an AAV vector, such as an AAV9 vector, or an AAV vector variant as described in US Pat. No. 9,585,971 or US Provisional Application No. 62/503,121. Delivery can be to any region of the brain, such as the hippocampus or entorhinal cortex, by any suitable means, including the use of a cannula or any other delivery technology. Any AAV vector that provides substantially ubiquitous delivery of a repressor to a subject's brain, including via antergrade and retrograde axonal transport in a region of the brain, without direct introduction of the vector (e.g. delivery to the putamen results in delivery to other structures such as the cerebral cortex brain, substantia nigra, thalamus, etc.). In some embodiments, the subject is a human, and in other embodiments, the subject is a non-human primate. The introduction can be in a single dose or in several doses (at any time between injections).

Thus, in other aspects, a method is described herein for preventing and/or treating tauopathy (eg, AD) in a subject, the method comprising administering a tau allele repressor to the subject using one or more AAV vectors. In some 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, or intracisternal delivery. In other embodiments, an AAV encoding a repressor is administered directly to the parenchyma (eg, hippocampus and/or entorhinal cortex) of a subject. In other embodiments, the AAV encoding the repressor is administered intravenously (IV). In any of the methods described herein, the administration may be performed once (single administration) or may be performed multiple times (with any time between administrations). Multiple administrations may use the same or different doses and/or delivery vehicles for administration routes (eg, different AAV vectors, IV and/or ICV administration). The methods include methods of reducing tau aggregation in a subject (eg, reducing NFTs indicative of tau aggregation), eg, in neurons of a subject with AD; methods for reducing apoptosis in a neuron or population of neurons (eg, an AD neuron or an AD population of neurons); ways to reduce the excessive excitability of neurons; methods of reducing amyloid-induced beta toxicity (eg, synapse loss and/or axonal degeneration); and/or methods of reducing the loss of one or more cognitive functions in subjects with AD, all compared to a subject not subjected to the method, or compared to the subject himself prior to exposure to the methods. Thus, the methods described herein lead to a reduction in biomarkers and/or symptoms of taupathies, including one or more of the following: neurotoxicity, gliosis, dystrophic axons, loss of processes, excitotoxicity, thinning of the cerebral cortex and hippocampus, accumulation of tau in axons, cognitive impairment (eg, radial arm maze and Morris water maze in rodent models, fear formation, etc.) and/or motor impairment.

In some aspects, the methods and compositions of the present invention are provided for reducing the number of pathogenic tau species in a cell. In some embodiments, the methods result in a decrease in hyperphosphorylated tau. In some cases, a decrease in hyperphosphorylated tau results in a decrease in soluble or granular tau. In other embodiments, the reduction of pathogenic tau species reduces tau aggregation and causes a decrease in neurofibrillary tangles (NFTs) compared to a cell or subject that has not been treated in accordance with the methods and/or compositions of the present invention. In further embodiments, methods are provided for reverting the amount of NFTs observed in a cell. In yet other embodiments, the methods and compositions of the present invention cause a slowdown in the spread of pathogenic tau species (NFTs, hyperphosphorylated tau) in the brain of a subject. In some embodiments, the spread of pathogenic tau in the brain is stopped, and in other embodiments, the spread of pathogenic tau in the brain is stopped. In other embodiments, the implementation reduces the number of dystrophic axons associated with amyloid β-plaques in the brain. In some embodiments, the number of dystrophic axons is reduced to levels found in an age-matched wild-type brain. In further embodiments, provided herein are methods and compositions for reducing hyperphosphorylated tau associated with amyloid β-plaques in the brain of a subject.

In any of the methods described herein, the tau allele repressor may be a ZFP-TF, eg, a fusion protein comprising ZFP that specifically binds to the tau allele and a transcriptional inhibitory domain (eg, KOX, KRAB, etc.). In other embodiments, the tau allele repressor may be TALE-TF, eg, a fusion protein comprising a TALE polypeptide that specifically binds to the tau allele and a transcriptional repressor domain (eg, KOX, KRAB, etc.). In some embodiments, the tau allele repressor is CRISPR/Cas-TF and the nuclease domains in the Cas protein have been inactivated such that the protein no longer cleaves DNA. The resulting RNA-guided Cas DNA-binding domain is fused to a transcriptional repressor (eg, KOX, KRAB, etc.) to repress the tau allele.

In some embodiments, the sequence encoding the genetic modulator (genetic repressor) described herein (e.g., ZFP-TF, TALE-TF, or CRISPR/Cas-TF) is inserted (integrated) into the genome, while in other embodiments, the sequence encoding the repressor , is supported episomally. In some instances, the TF fusion nucleic acid is inserted (eg, via nuclease(s) mediated integration) into a secure harbor site comprising a promoter such that the endogenous promoter drives expression. In other embodiments, a repressor (TF) donor sequence is inserted (via nuclease(s) mediated integration) into a safe harbor site and the donor sequence includes a promoter that directs expression of the repressor. In some embodiments, the genetic modulator coding sequence is conserved extrachromosomally (episomally) after delivery and may include a heterologous promoter. The promoter may be a constitutive or inducible promoter. In some embodiments, the promoter sequence is widely expressed, while in other embodiments, the promoter is tissue or cell/type specific. In preferred embodiments, the promoter sequence is specific for nerve cells. In particularly preferred embodiments, the selected promoter is characterized in that it has low expression. Non-limiting examples of preferred promoters include the nerve cell-specific NSE, Synapsin, CAMKiia and MECP promoters. Non-limiting examples of ubiquitous promoters include CAS and Ubc. Further embodiments include the use of the self-regulating promoters described in US Patent Application Publication No. 2015/0267205.

In still other embodiments, the repressor may include a nuclease (eg, ZFN, TALEN, and/or the CRISPR/Cas system) that represses the tau allele by cleaving and thereby inactivating the tau allele. In some embodiments, the nuclease introduces an insertion and/or deletion ("Indel") via non-homologous end joining (NHEJ) after cleavage with the nuclease. In other embodiments, the nuclease introduces a donor sequence (via homology-directed and non-homology-directed methods), wherein integration of the donor inactivates the tau allele.

In any of the methods described herein, the repressor may be delivered to the subject (eg, the brain) as a protein, polynucleotide, or any combination of protein and polynucleotide. In some embodiments, the repressor(s) are delivered using an AAV vector. In other embodiments, at least one repressor component (eg, CRISPR/Cas sgRNA) is delivered in the form of RNA. In other embodiments, the repressor(s) are delivered using a combination of any of the expression constructs described herein, such as one repressor (or part thereof) on one expression construct (AAV9) and one repressor (or part thereof) on a single expression construct (AAV or another viral or non-viral construct).

In addition, in any of the methods described here, the repressors can be delivered at any concentration (dose) that provides the desired effect. In preferred embodiments, the repressor is delivered using an adeno-associated virus (AAV) vector at an MOI (multiplicity of infection) of 10,000-50,000 vector genomes/cell (or any value in between). In some embodiments, the repressor is delivered using a lentiviral vector at an MOI of 250 to 1000 (or any value in between). In other embodiments, the repressor is delivered using a plasmid vector in an amount of 0.01-1000 ng/100,000 cells (or any value in between). In other embodiments, the repressor is delivered as mRNA at 150-1500 ng/100,000 cells (or any value in between).

In any of the methods described herein, the method may result in repression of tau alleles in one or more neurons of a subject with AD by about 50% or more, 55% or more, 60% or more, 65% or more, about 70% or more, about 75% or more, about 85% or more, about 90% or more, about 92% or more, or about 95% or more.

In further aspects, the tau-modulating transcription factors described herein, such as tau-modulating transcription factors comprising one or more of zinc finger protein (ZFP-TF), TALE (TALE-TF), and CRISPR/Cas-TF, e.g., ZFP- TF, TALE-TF or CRISPR/Cas-TF, are used to suppress the expression of a mutant tau allele or a wild-type tau allele in the brain (eg, neuron) of a subject. Suppression may be repression of about 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, about 75% or more, about 85% or more, about 90% or more, about 92% or more, or about 95% or more, of tau alleles in one or more neurons of the subject compared to unexposed (wild-type) neurons of the subject. In some embodiments, a tau modulating transcription factor may be used to achieve one or more of the methods described herein.

Also provided is a kit comprising one or more AAV tau modulators (eg, repressors) and/or polynucleotides comprising components of tau modulators and/or encoding tau modulators (or components thereof) described herein. Kits may further include cells (eg, neurons), reagents (eg, to detect and/or quantify tau protein, eg in CSF), and/or instructions for use, including the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1A-1D depict MAPT expression following administration of the engineered MAPT genetic modulators described herein. Fig. 1A is a diagram showing the target sites in the MAPT gene for a genetic modulator DNA-binding molecule. The repressor was the modulator for these experiments. In FIG. 1B is a graph showing MAPT expression 24 hours after administration of the indicated amounts of the MAPT repressor (52322, 52364, 52366, 52389, 57880, 57890, and 52288) or GFP or mRNA false controls (at the indicated doses of 1000 ng, 300 ng, 100 ng, 30 ng, 10 ng, or 3 ng, from left to right, respectively) into Neuro 2A cells. In FIG. 1C is a graph showing MAPT expression 7 days after administration of the MAPT repressor (52322, 52364, 52366, 52389, 57880, 57890 and 52288) or GFP or mock controls using the AAV9 vector (with CMV promoter) at indicated doses of 3×10 ⁵ vector genes/cell, 1×10 ⁵ vector genes/cell, 3×10 ⁴ vector genes/cell, 1×10 ⁴ vector genes/cell in primary mouse cerebral cortex neurons (MCN). In FIG. 1D are diagrams showing tau repression (MAPT) and off-target repression by 57880 and two typical derivatives (65887 and 65888) containing mutations in contact with phosphate residues in the ZFP backbone.

In FIG. 2A-2C show the levels of proteins (tau, GAPDH and ZFP) in MCNs treated with the indicated repressor (52389) carried by the AAV9 vector. Fig. 2A is a Western blot showing the levels of the indicated proteins at the indicated doses of AAV9. Fig. 2B and FIG. 2C are graphs showing the ratio of tau/GAPDH (upper panels) and ZFP/GAPDH (lower panels) proteins at indicated doses of AAV9-52389 or mock control (M).

In FIG. 3 shows the results of microarray analysis showing the specificity of the indicated repressors (52322, 52364, 52366, 52389, 57880, 57890 or 52288) for the MART gene. The analysis was performed 24 hours after the introduction of repressors in the form of mRNA into Neuro2A cells in the amount of 300 ng. The results are discussed in Example 3. The numbers above each graph represent the number of genes whose expression was increased (up arrow) or decreased (repression) (down arrow).

In FIG. 4 shows the results of the analysis using microarrays of the indicated repressors (52322, 52364, 52366, 52389, 57880, 57890, 52288) 24 hours after the introduction of repressors in the form of mRNA into primary human fibroblasts in an amount of 300 ng. The results are discussed in Example 3. The numbers above each plot represent the number of genes whose expression increased (up arrow) or decreased (repression) (down arrow).

In FIG. 5A and 5B show the results of the analysis using microarrays of the indicated repressors (52322, 52364, 52366, 52389, 57880, 57890). In FIG. 5A shows the results 7 days after injection of AAV vector-borne (AAV) repressors into mouse cortex neurons at a dose of 1×10 ⁵ viral genomes/cell. The results are discussed in Example 3. The numbers above each graph represent the number of genes whose expression increased (up arrow) or decreased (repression) (down arrow). In FIG. 5B shows the results of microarray analysis comparing two repressors 57880 and 65888 with identical helices. The data for the 57880 protein is shown in the top row, while the data for the 65888 protein is shown in the bottom row. In 65888, some potential contacts with phosphate residues were removed from the zinc finger backbone (see Table 2), and the data demonstrate a significant increase in the specificity of the 65888 protein (57880 activated 75 genes and downregulated 110 genes under these experimental conditions, while 65888 activated 3 genes and suppressed 4 genes, including tau) while maintaining similar tau inhibitory activity in neurons of the mouse cerebral cortex.

Fig. 6A and 6B are graphs depicting mRNA expression in cells using genetic repressors carried by the AAV vector (AAV9), the expression of the repressor being driven by the indicated promoter (CMV, Synapsin (SYN1), alpha-calcium/calmodulin-dependent protein kinase (CamKII) promoter and a 229 bp fragment of the methyl CpG binding protein 2 (MeCP2) promoter Figure 6A shows expression in MCN Figure 6B shows expression in hippocampal primary neurons.

In FIG. 7A-7D show mRNA levels (for human tau and ZFP) in human iPSC-derived neurons. Fig. 7A is a diagram showing a partial sequence of target sites in human and mouse MAPT (SEQ ID NOS: 31, 32 and 48-51, respectively, from left to right). In FIG. 7B is a graph showing mRNA expression in iPSC 18 days after administration of the indicated AAV vector-borne repressors at the indicated doses. In FIG. 7C is a graph showing mRNA expression in iPSC 18 days after administration of the indicated repressor (52366) carried by an AAV vector with the indicated promoters. Fig. 7D is a series of microarray plots using typical ZFP-TFs in human iPSC-derived neurons and shows that ZFP-TFs are highly specific. Cells were exposed to 1E5 AAV6 containing donor ZFP-TF for 19 days prior to analysis.

In FIG. 8A-8H show efficient neuronal ZFP-TF transduction and in vivo effects on mRNA expression (for tau and ZFP) in murine subjects using various AAV vectors to deliver tau repressors. See example 6. FIG. 8A shows efficient transduction of the motor cortex (upper panels), hippocampus (middle panels), and cerebellum (lower panels) after administration of either AAV (right panels) or ICV (left panels). In FIG. 8B shows a strong decrease in tau (~50-70% decrease) with both ICV and IV administration in major areas of tauopathy, including the motor cortex (left panel, top row), the brainstem (right panel, top row), and hippocampus (lower left panel)). In FIG. 8C shows the decrease in mRNA for tau (~40-70% decrease) throughout the brain and spinal cord after IV or ICV administration of AAV-ZFP-TF vectors. In FIG. 8D shows mRNA levels for tau (MAPT) and FIG. 8E shows mRNA levels for the repressor (ZFP) for each section of the hippocampus. Fig. 8F are graphs showing sustained tau repression in the entire CNS and spinal cord following administration of the tau repressors described here (left panel shows the 52389 repressor and right panel shows the 57890 repressor) using AAV vectors. In FIG. 8G are graphs showing rapid and sustained tau repression over several months in the cerebral cortex (left panels) and hippocampus (right panels) of animals treated with the compositions ("GFP" refers to GFP as a control; "172" refers to irrelevant ZFP-TF as a control that does not bind to MAPT; "52389" refers to the 52389 repressor transferred to the AAV constructs, "57890" refers to the 57890 repressor described here transferred to the AAV constructs, and "PBS" refers to the control animals receiving only PBS). The top row in Fig. 8G shows mRNA expression for tau, and the bottom row shows the levels of tau protein. Fig. 8H are graphs showing levels of mouse total tau in the CSF (top row, left panel), cortex (top row, right panel), and hippocampus (top row, rightmost graph) of animals receiving the indicated compositions ("VEH 'refers to vehicle-only control animals: '172V' refers to animals receiving irrelevant ZFP-TF as a control that does not bind to MAPT carried by AAV vector, '389V' refers to repressor 52389 transferred to AAV constructs, and "890V" refers to the 57890 repressor described here transferred to AAV constructs). Also presented (bottom row) is a statistical analysis showing the correlation between CSF and levels in the cerebral cortex (right panel) and hippocampus (left panel).

9A-9D show further analysis of MAPT modulation (repression) in vivo using statistical analysis as described in Example 6. FIG. 9A shows analysis of variance (ANOVA) followed by Sidak's post hoc test for all sections (top panel shows tau, bottom panel shows ZFP). In FIG. 9B shows the mean maximum tau reduction (top panel shows tau, bottom panel shows ZFP). In FIG. 9C shows a high correlation between MAPT and ZFP-TF levels in animals treated with the MAPT 52322 repressor. FIG. 9D shows a high correlation between MAPT and ZFP-TF levels in animals treated with the MAPT 52389 repressor.

Fig. 10A-10D are graphs depicting mRNA repression for MAPT (tau) following in vivo treatment with tau-specific ZFP-TF following injection into the hippocampus. In this experiment, four different promoters (CMV, SNY1, CAMKII or MeCP2) were used to drive the expression of the ZFP-TF-venus construct. In FIG. 10A shows tau expression 6 weeks post-injection and shows a decrease in all animals that received ZFP-TF, regardless of the promoter chosen. Asterisks indicate signal significance compared to PBS control (p<0.0001 for all promoters). In FIG. 10B shows ZFP-TF expression from various promoters, with the highest ZFP-TF expression found in animals treated with the CAMKII-driven vector. The presence of activated astrocytes was determined for each treatment group (FIG. 10C) by detection of GFAP. In this experiment, the MeCP2 promoter did not result in an increase in GFAP compared to animals injected with PBS, SYN1 resulted in a 4.0-fold increase in GFAP levels (P<0.0001), CMV resulted in a 3.2-fold increase in levels (P <0.01), and CAMKII resulted in a 2.4-fold increase in levels (P<0.05). Similarly, the presence of microglia was also determined (Fig. 10D) in the treatment groups, with the MeCP2 promoter not leading to significant changes in IBA1 levels compared to animals injected with PBS, SYN1 leading to a 4.7-fold increase in IBA1 levels (P< 0.0001), CMV resulted in a 3.0-fold increase in levels (P<0.05), and CAMKII resulted in a 3.2-fold increase in levels (P<0.001).

Fig. 11 is a graph showing the reduction in tau expression in the groups described in FIG. 10. Compared to control levels after PBS injection (269 ng/mL), the rAAV9-SYN1-52389V construct produced 23% of control levels (61 ng/mL, P<0.0001), the rAAV9-CAMKII-52389V construct produced 18 % of control levels (49 ng/mL, P<0.0001), the rAAV9-SYN1-52389V construct produced 12% of control levels (32 ng/mL, P<0.0001). Thus, nerve cell promoters are able to induce >80% reduction in mRNA for tau and tau protein throughout the mouse hippocampus.

In FIG. 12A-12F present histological evidence for a decrease in tau in the hippocampus and associated brain regions. AAVs comprising tau-specific ZFP-TF driven by the CMV promoter were delivered to the hippocampus to evaluate endogenous tau reduction by immunofluorescence staining. ZFP-TF was coupled to Venus fluorescent protein for detection and animals were sacrificed 6 weeks after injection. Fig. 12A is a cross section of the hippocampus and shows 4 areas in boxes for closer examination. Fig. 12B is a close-up of frame 1 in FIG. 12A and shows GFP/DAPI staining (left panel); GFP/tau/DAPI (middle panel) and tau (right panel). Fig. 12C is a plot of fluorescence intensity in box 1 showing the decrease in tau on the ipsilateral side of the section (right panel). Fig. 12D shows a close-up of frame 2 while FIG. 12E is a close-up of frame 3 showing the reduction in tau staining on the ipsilateral side (right panel). In FIG. 12F shows frame 4, with the top panel showing the GFP signal (indicating ZFP-TF) on the ipsilateral side of the injection with concomitant reduction in tau staining (bottom panel). The lower middle panel also shows a graphical representation of the reduction in tau staining. Lower left and right panels show staining in ipsilateral (left) and contralateral (right) sections. The data are discussed in more detail in the examples.

Fig. 13A-13J demonstrate the safety and sustained expression of ZFP-TF over six months in the hippocampus in vivo. In FIG. 13A shows the signal for transduced cells in the hippocampus as a whole at the 6-week time point, indicating that the expression of vectors encoding ZFP-TF and GFP alone is almost the same. Fig. 13B shows similar results specifically for astrocytes, while FIG. 13C shows results for microglia. In FIG. 13D shows transduction data similar to FIG. 13A except for the 6 month time point. Similarly, in FIG. 13E shows astrocyte data for a 6 month time point, and FIG. 13F shows microglial data for a 6 month time point. Fig. 13G demonstrates that the mean thickness of the hippocampal region is maintained across different treatment groups, demonstrating no apparent neurotoxicity from long-term expression of ZFP-TF. In FIG. 13H shows the injection site in the anterior (left panel) and posterior (right panel) hippocampus. The data are discussed in more detail in the examples. Fig. 13I are graphs showing plotted expression levels in subjects treated with the indicated ZFs (tau in upper left, ZF in upper middle, VG/cell in upper right, GFPA (glial fibrillary acidic protein, a marker for astrocytes) in lower left). diagram, IBA1 (ionized calcium-binding adapter molecule 1, a microglial marker) in the lower middle diagram, and NeuN (a well-recognized marker of postmitotic neurons that is highly conserved across species, (Wang et al., (2015) Sci Reports 5: 17383, doi 10.1038/spre17383)) in the lower right chart). In FIG. 13J are graphs showing the levels of expression of the indicated proteins in subjects treated with the indicated compositions ("PBS" means no tau modulator; "89V 6 weeks" refers to mice treated with the 52389 Venus construct, with selection at 6 weeks, and "89V 11m" refers to construct 52389 Venus, with sampling at 11 months (tau, ZFP, GFAP, IBA1 from left to right in the upper panels; NeuN, MAP1, MAP1A, MAP2 from left to right in the lower panels).

Fig. 14A-14F are micrographs of brain sections from APP/PS1 mice treated with ZFP-TF. In these images, GFP is green, RFP is red, and Aβ-specific antibodies are labeled with a second antibody (blue). Fig. 14A and 14C are images of wild-type brain cortex (CTX), with the left CTX exposed to irrelevant ZFP-TF (Fig. 14A) and the right CTX exposed to AAV encoding the tRFP fluorescent protein (Fig. 14C). In FIG. 14B and 14D are cortical images of APP/PS1 mice with left CTX exposed to AAV encoding tau-specific ZFP-TF ("389dV", Fig. 14B) or AAV encoding tRFP (Fig. 14D). The images illustrate the reduction of dystrophic axons (identified as mottled staining around Aβ plaques and indicated by arrows) in CTX treated with tau-specific ZFP-TF. Fig. 14E and 14F are high magnification images of two examples of CEX slices from APP/PS1 treated with either tau-specific ZFP-TF (FIG. 14E) or tRFP (FIG. 14F), with more dystrophic axons (indicated by arrows) present. in a tRFP-exposed section than in a ZFP-TF389-exposed section.

Fig. 15 is a graph showing a quantitative representation of the number of dystrophic axons per Aβ plaque in APP/PS1 mice. Each dot represents the average number of dystrophic axons per plaque for all plaques on a single cortical section; 3-5 sections were analyzed per hemisphere per mouse. As can be seen, there was a statistically significant reduction in dystrophic axons in CTX exposed to 389dV ZFP-TF compared to CTX exposed to tRFP (leftmost (grey) bar with 389dV treatment, second left (white) bar with tRFP treatment). ). Also shown is a comparison of CTX in mice treated with irrelevant ZFP-TF ("172dV", gray bar with stripes, 2nd from right bar) or tRFP (white bar with bars, rightmost bar). As can be seen, there were no differences between these samples in the number of dystrophic axons/plaques.

Fig. 16 is a chart showing a quantitative comparison of the data shown in FIG. 15, accounting for variation among animals in dystrophic axons per plaque at baseline. The number of dystrophies per Aβ plaque was averaged for each ZFP-TF-exposed hemisphere or for the tRFP-exposed contralateral hemisphere and compared using a paired two-tailed T-test. Values for each group were adjusted to the mean of the tRFP-exposed side (set at 100%). Using this pairwise analysis, it was found that the number of dystrophic axons was significantly reduced (mean reduction of 34%) in the case of the 52389V treated group (leftmost (grey) bar with 389dV treatment, second left (white) bar with 52389V treatment). tRFP), but not for the irrelevant ZFP-TF treated group (172dV, second from the right (grey striped) bar; tRFP, rightmost (banded, white) bar).

In FIG. 17 shows exemplary results of microarray analysis on hippocampal tissue obtained from mice treated with ZFP-TF. ZFP-TF was delivered to animals using the AAVs described here (2.5e12 viral genomes/animal) via retroorbital delivery. Mice were treated and sacrificed ten weeks later and the results displayed are data compared to irrelevant ZFP-TF as a control.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Disclosed here are compositions and methods for preventing and/or treating taupathies. In particular, the compositions and methods described herein are used to suppress MAPT (tau) protein expression to prevent or treat taupathies such as Alzheimer's disease (AD), frontotemporal dementia, progressive supranuclear paresis, epilepsy, and/or corticobasal degeneration. MAPT repressors (eg, MAPT-modulating transcription factors, such as MAPT-modulating transcription factors, including zinc finger proteins (ZFP-TF), TALE (TALE-TF), and/or CRISPR/Cas-TF) modify the CNS such that the effects and/or symptoms of tauopathy are reduced or eliminated, for example, by reducing tau aggregation in the brain of a subject with tauopathy (e.g., AD) and reducing the occurrence of neurofibrillary tangles.In preferred embodiments, MAPT-modulating transcription factors are delivered to the brain via a viral a vector such as AAV AAV has been shown to be well suited for delivery to the brain, so the use of these viral vectors to deliver MAPT-modulating transcription factors is particularly useful in the treatment of diseases such as Alzheimer's disease associated with inappropriate expression and therefore tau protein aggregation.

General information

In the practice of the methods, as well as the preparation and use of the compositions disclosed herein, unless otherwise indicated, methods generally accepted in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields are used, which are within the limits of competence in a particular field of technology. These methods 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 updates from time to time; METHODS IN ENZYMOLOGY series, 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" (P.B. Becker, ed.) Humana Press, Totowa, 1999.

Definitions

The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymer of deoxyribonucleotides or ribonucleotides in a linear or circular structure and in single or double stranded form. For the purposes of this disclosure, these terms should not be construed as limiting the length of the polymer. The terms may include known analogs of naturally occurring nucleotides, as well as nucleotides that are modified in base, sugar and/or phosphate moieties (eg, phosphorothioate backbones). Typically, an analog of a particular nucleotide will have the same specificity for base pairing; those. the analog of A would be a base pair with T.

The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acid.

The term "binding" refers to a sequence-specific non-covalent interaction between macromolecules (eg, between a protein and a nucleic acid). Not all binding interaction components need to be sequence-specific (eg, in the case of contacts with phosphate residues in the DNA backbone) if the interaction as a whole is sequence-specific. Such interactions are typically characterized by a dissociation constant (K _d ) of 10 ^-6 M -1 or less. "Affinity" refers to the strength of binding: increased affinity correlates with lower K _d .

A "binding protein" is a protein that is capable of non-covalently binding to another molecule. The 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 may bind to itself (to form homodimers, homotrimers, etc.) and/or it may bind to one or more molecules of another protein or proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activities.

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

The term "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repetitive domains are involved in the binding of TALE to its corresponding target DNA sequence. A single "repeating unit" (also called a "repeat") is typically 33-35 amino acids long and shows at least some degree of sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, for example, U.S. Patent No. 8586,526.

"TtAgo" is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo comes from Thermus thermophilus bacteria. See, for example, Swarts et al., (2014) Nature 507(7491):258-261, G. Sheng et al., (2013) Proc. Natl. Acad. sci. U.S.A. 111, 652). The "TtAgo system" is all the necessary components, including, for example, guide DNAs for cleavage by the TtAgo enzyme.

"Recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, donor capture via non-homologous end joining (NHEJ) and homologous recombination. For the purposes of this disclosure, the term "homologous recombination (HR)" refers to a specialized form of such exchange that occurs, for example, during the repair of double-strand breaks in cells by homology-directed repair mechanisms. This process requires homology of nucleotide sequences, uses a "donor" molecule to repair the template of the "target" molecule (i.e., the one that has undergone a double-strand break), and is known by various names as "non-crossover gene conversion" or "gene conversion on a short stretch" because it results in the transfer of genetic information from the donor to the target. While not wishing to be bound by any particular theory, such transfer may include editing a mismatch in the heteroduplex DNA that forms between the damaged 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 a change in the sequence of the target molecule such that part or all of the donor polynucleotide sequence is incorporated into the target polynucleotide.

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 constructing an sgRNA that binds to a selected target site, or by constructing (changing one or more amino acids) the recognition helical region of a naturally occurring zinc finger protein or by constructing the RVD protein TALE. Therefore, engineered zinc finger or TALE proteins are proteins that do not occur naturally. Non-limiting examples of methods for creating DNA binding domains are design and selection. An "engineered" zinc finger protein or TALE is a non-naturally occurring protein whose design/construction is predominantly derived from rational criteria. Rational design criteria include the use of substitution rules and computer algorithms to process information in a database that stores information about existing ZFP constructs and binding data. A "selected" zinc finger protein, or TALE, is a non-naturally occurring protein that is derived primarily from an empirical process such as phage display, interaction trap, or hybrid selection. See, for example, US Pat. Nos. 8,586,526; 6140081; 6453242; 6746838; 7241573; 6866997; 7241574; and 6534261; see also International Patent Application Publication No. WO 03/016496.

The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA; may be linear, circular or branched and may be either single or double stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into the genome. The donor sequence may be of any length, for example, from 2 to 10,000 nucleotides in length (or any integer in between or greater), preferably from about 100 to 1000 nucleotides in length (or any integer in between), more preferably from approximately 200 to 500 nucleotides in length.

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

An "exogenous" molecule is one that is not normally present in a cell, but can be introduced into the cell by one or more genetic, biochemical, or other means. "Normal presence in the cell" is defined taking into account the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is only present during embryonic muscle development is an exogenous molecule with respect to the adult muscle cell. Similarly, a heat shock induced molecule is an exogenous molecule with respect to a cell not subjected to heat shock. The exogenous molecule may include, for example, a functioning variant of a misfunctioning endogenous molecule or a misfunctioning variant of a normally functioning endogenous molecule.

An exogenous molecule may be, but is not limited to, a small molecule such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex , including one or more of the above molecules. Nucleic acids include DNA and RNA, may be single or double stranded; may be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes as well as triplex-forming nucleic acids. See, for example, US Pat. phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.

The exogenous molecule may be the same type of molecule as the endogenous molecule, such as an exogenous protein or nucleic acid. For example, the exogenous nucleic acid may include the genome of the infecting virus, a plasmid or episome introduced into the 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 transfer (i.e., liposomes including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, coprecipitation calcium phosphate, DEAE-dextran mediated transfer and viral vector mediated transfer. The exogenous molecule can also be the same type of molecule as the endogenous molecule, but derived from a species different from the species from which the cell is derived. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under certain environmental conditions. For example, an endogenous nucleic acid may include a chromosome, a mitochondrial genome, a chloroplast or other organelle, or a naturally occurring episomal nucleic acid. Additional endogenous molecules may include proteins such as transcription factors and enzymes.

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

Expression of a fusion protein in a cell may result from the delivery of the fusion protein into the cell, or from the delivery of a polynucleotide encoding the fusion protein into the cell, where the polynucleotide is transcribed and the transcript is translated to form the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation may also be involved in protein expression in a cell. Methods for delivering polynucleotides and polypeptides to cells are presented elsewhere in this specification.

A "multimerization domain" (also referred to as a "dimerization domain" or "protein interaction domain") is a domain included in the N-, C- or N- and C-terminal regions of ZFP-TF or TALE-TF. These domains allow multimerization of multiple ZFP-TF or TALE-TF units such that larger stretches of trinucleotide repeat domains become preferentially bound by multimerized ZFP-TF or TALE-TF compared to shorter stretches with wild-type length numbers. Examples of multimerization domains include leucine zippers. Multimerization domains can also be regulated by small molecules, with the multimerization domain adopting the proper conformation to allow interaction with another multimerization domain only in the presence of a small molecule or an external ligand. Thus, exogenous ligands can be used to regulate the activity of these domains.

For the purposes of the present invention, "gene" includes the DNA region encoding the gene product (see below) as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational control 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 areas.

"Gene expression" refers to the transformation of the information contained in a gene into a gene product. A gene product may be a direct transcription product of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein resulting from translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

"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 (eg, cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any decrease in gene expression compared to a cell that does not include the ZFP or TALE protein, as described here. Thus, gene inactivation can be partial or complete.

The term "genetic modulator" refers to any molecule that changes the expression and/or sequence of one or more genes. Non-limiting examples of genetic modulators include transcription factors (such as the artificial transcription factors described herein) that bind to a target gene and alter its expression, and nucleases that modify the sequence of the target gene, which in turn alters its expression (e.g. , target inactivation by insertions and/or deletions). Thus, a genetic modulator can be a genetic repressor (which represses and/or inactivates gene expression) or a genetic activator.

A "region of interest" is any region of cellular chromatin, such as, for example, a gene or non-coding sequence within or near a gene, to which it is desired to associate an exogenous molecule. The binding may be for the purpose of targeted DNA cleavage and/or targeted recombination. The region of interest may be present, for example, in a chromosome, episome, genome of an organelle (eg, mitochondria, chloroplast), or in the genome of an infecting virus. The region of interest may be in the coding region of a gene, in transcribed non-coding regions such as, for example, leader sequences, trailer sequences, or introns, or in non-transcribed regions, either upstream (5') or downstream (3') of the coding region. The region of interest can be as small as one nucleotide pair, or up to 2000 nucleotide pairs in length, or equal to any integer number of nucleotide pairs.

"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).

The terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably to refer to a juxtaposition of two or more components (such as elements of a sequence) in which the components are arranged such that both components function normally and allow for the possibility that at least one of the components may mediate a function that affects at least one of the other components. By way of illustration, a transcription control sequence, such as a promoter, is operably linked to a coding sequence if the transcription control sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcription control factors. The transcription control sequence is typically cis-operably linked to the 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 if they are not contiguous.

With regard to fused polypeptides, the term "operably linked" may refer to the fact that each of the components in connection with the other component performs the same function as if it were not connected in this way. For example, with respect to a fusion molecule in which a ZFP or TALE DNA-binding domain is fused to an activating domain, the ZFP or TALE DNA-binding domain and the activating domain are operably linked if, in the fusion polypeptide, a portion of the ZFP or TALE DNA-binding domain is capable of binding with its target site and/or binding site, while the activating domain is able to enhance gene expression. ZFPs fused to domains capable of regulating gene expression are collectively referred to as "ZFP-TF" or "zinc finger transcription factors", while TALEs fused to domains capable of regulating gene expression are collectively referred to as "TALE-TF" or "TALE transcription factors". When 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 part of the DNA-binding domain is in the fusion polypeptide The ZFP is capable of binding to its target site and/or binding site, while the cleavage domain is able to cleave DNA in close proximity to the target site. When a fusion polypeptide in which a TALE DNA-binding domain is fused to a cleavage domain ("TALEN" or "nuclease to TALE"), the TALE DNA-binding domain and the cleavage domain are operably linked if part of the TALE DNA-binding domain is in the fusion polypeptide is capable of binding to its target site and/or binding site, while the cleavage domain is able to cleave DNA in close proximity to the target site. With respect to a fusion molecule in which a Cas DNA-binding domain (e.g., a chimeric guide RNA) is fused to an activating domain, the Cas DNA-binding domain and the activating domain are operably linked if, in the fusion polypeptide, a portion of the Cas DNA-binding domain is capable of binding to its target site and/or binding site, while the activating domain is able to enhance gene expression. When 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, a portion of the Cas DNA-binding domain is capable of binding to its target site and/or site binding, while the cleavage domain is able to cleave DNA in close proximity to the target site.

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 retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment may contain 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 (eg, coding function, ability to hybridize with another nucleic acid) are well known in the art. Similarly, methods for determining the function of a protein are well known. For example, the DNA binding function of a polypeptide can be determined, for example, using a filter binding assay, electrophoretic mobility shift, or immunoprecipitation. DNA cleavage can be analyzed using 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 complementation, either genetic or biochemical. See, for example, Fields et al., (1989) Nature 340: 245-246; US Patent No. 5585245 and International Patent Application Publication No. WO 98/44350.

The "vector" is capable of transferring gene sequences to target cells. In general, "vector construct", "expression vector" and "gene transfer vector" means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression agents as well as integrating vectors.

"Reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is easily measured, preferably, though not necessarily, in a conventional 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 stained or fluorescent or luminescent proteins (eg, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase) and proteins that mediate enhanced cell growth and/or gene amplification (eg, dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. "Expression tags" include sequences that encode reporters that can be operably linked to a desired gene sequence to control the expression of a gene of interest.

Tau and Alzheimer's disease

The tau protein is encoded by the MAPT gene, which includes 16 exons. Interestingly, exons 1, 4, 5, 7, 9, 11, and 12 are constitutively expressed, while exons 2, 3, and 10 may be present in tau protein species derived from alternatively spliced variants, resulting in the presence of six distinct tau isoforms. protein in the adult brain. Tau binds to microtubules via 3 or 4 repetitive tubulin-binding motifs in the C-terminal half of the protein and is thought to stabilize tubules, with tau4R (4 tubulin-binding motifs) thought to interact more strongly with microtubules than tau3R. The ratio of 3R to 4R is usually unchanged, but may be disturbed in pathological conditions. The form of tau that interacts with microtubules is phosphorylated, and hyperphosphorylation appears to cause tau to detach from microtubules. Hyperphosphorylated tau can be sequestered in the cell, which then leads to protein conformational changes and aggregation. These aggregates may represent the initial step in the formation of pathogenic neurofibrillary tangles (NFTs), however, hyperphosphorylated tau can be pathogenic both in soluble form and when present in tangles (Bodea et al., (2016) J of Neurochem 138 (Suppl 1): 71-94). In the early stages of AD, NFTs are limited to the presence in the entorhinal cortex and mid-temporal lobe, and by the time severe clinical symptoms of the disease appear, NFTs are widespread throughout the brain. Due to the presence of a large number of NFTs, amyloid plaques are also widespread. In fact, it appears that the deposition of amyloid in the cerebral cortex leads to an increase in the rate of tau propagation and the spread of NFT to the 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).

Tau contains 95 amino acid residues that are capable of being phosphorylated, and several kinases have been identified that may be responsible for tau phosphorylation, which may be potential candidate targets for novel therapeutics, including 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.).

The amyloid β (Aβ) protein is a major component of senile plaques, which, together with NFTs, are hallmarks of neuropathological confirmation of Alzheimer's disease. Aβ is a peptide that contains chains of 39-42 amino acids; The 42 amino acid form aggregates more actively and is believed to be involved in the pathogenesis of the disease and is the basis of the amyloid hypothesis (suggestions that the accumulation of Aβ in the brain is the main cause of AD, see review Hardy and Selkoe (2002) Science 297:353). Aβ are proteolytic cleavage products of the amyloid precursor protein (APP), a ubiquitous, glycosylated, sulfated and phosphorylated integral membrane protein (Sorrentino et al., (2014) FEBS Lett 588:641-652). However, it is becoming clear that the pathogenesis leading to AD is extremely complex, and that the pathogenesis of Aβ accumulation may play a role in the abnormal behavior of tau (Ando et al., (2016) PLoS Genet 12(3):e1005917.

Decreasing tau in the brain has been shown to attenuate the pathology of AD. Controlled downregulation of tau transgene expression in a mouse model of AD demonstrated a reduction in transgene-associated tau aggregates and a reduction in hyperphosphorylated tau and NFT. In fact, this scientific study also demonstrated a complete loss of NFT, indicating that NFT accumulation may be reversible (Polydoro et al., (2013) J of Neurosci 33(33):13300-13311). In addition, studies performed with intracellular anti-tau antibody delivered by AAV directly via injection into the hippocampus demonstrated a reduction in insoluble tau species, NFTs, and rescue from hippocampal atrophy that occurs in an untreated mouse model (Liu et al., (2016) J Neurosci 36 (49): 12425-12435).

DNA binding domains

The methods described herein use compositions, such as tau-modulating transcription factors, comprising a DNA-binding domain that specifically binds to a target sequence in the tau gene (MATP). Any polynucleotide or polypeptide DNA binding domain may be used in the compositions and methods disclosed herein, for example DNA binding proteins (eg ZFP or TALE) or DNA binding polynucleotides (eg chimeric guide RNAs). Thus, genetic modulators (repressors) of tau genes have been described.

In some embodiments, the tau repressor, or the DNA binding domain therein, comprises a zinc finger protein. Selection of target sites; ZFPs and methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art and are detailed in US Pat. Nos. 6,140,081; 5789538; 6453242; 6534261; 5925523; 6007988; 6013453; 6200759; and publication of international patent application No. WO 95/19431; W096/06166; W098/53057; W098/54311; WO00/27878; WO 01/60970; WO 01/88197; WO 02/099084; W098/53058; W098/53059; W098/53060; WO 02/016536 and WO 03/016496.

Target sites in tau typically involve at least one zinc finger, but may involve multiple zinc fingers (eg, 2, 3, 4, 5, 6 or more fingers). Typically, ZFPs include at least three fingers. Some of the ZFPs include four, five, or six fingers, while some ZFPs include 8, 9, 10, 11, or 12 fingers. ZFPs that include three fingers usually recognize a target site that is 9 or 10 nucleotides long; ZFPs that include four fingers typically recognize a target site that is 12 to 14 nucleotides long; while six-finger ZFPs can recognize target sites that include 18 to 21 nucleotides. ZFPs can also be fusion proteins that include one or more regulatory domains, which domains can be transcription activating or repressive domains. In some embodiments, the fusion protein includes two ZFP DNA-binding domains linked together. These zinc finger proteins may thus include 8, 9, 10, 11, 12 or more fingers. In some embodiments, two DNA-binding domains are connected via an extendable flexible linker such that one DNA-binding domain includes 4, 5, or 6 zinc fingers and the second DNA-binding domain includes an additional 4, 5, or 5 zinc fingers. In some embodiments, the linker is a standard finger-to-toe linker such that the finger array includes a single DNA binding domain including 8, 9, 10, 11, or 12 or more fingers. In other embodiments, the implementation of the linker is an atypical linker, such as a flexible linker. The DNA binding domains are fused to at least one regulatory domain and can be considered as a "ZFP-ZFP-TF" structure. Specific examples of these embodiments may be referred to as "ZFP-ZFP-KOX", which includes 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 are fused together with a linker.

The engineered zinc finger binding domain may have novel binding specificity compared to a naturally occurring zinc finger protein. Design methods include, but are not limited to, design analysis and various types of selection. Design calculation includes, for example, the use of databases including triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more zinc finger amino acid sequences that link a particular triplet or quadruplet sequence. . See, for example, co-owned US Pat. Nos. 6,452,442 and 6,534,261, which are incorporated herein by reference in their entirety.

In addition, as disclosed in these and other references, zinc finger domains and/or zinc finger proteins can be linked together using any suitable linker sequences, including, for example, linkers of 5 or more amino acids in length. See also US Pat. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein.

The ZFP may be operably linked (associated) with one or more transcription-regulating domains (eg, suppressor domains) to form a ZF-TF (eg, a repressor). The methods and compositions can also be used to increase the specificity of ZFP for its intended target over other unintended cleavage sites known as off-target sites, for example, by mutations in the ZFP backbone as described in US Patent Application No. 15/685580. Thus, the tau repressors described herein may contain mutations in one or more of the backbone regions of the DNA binding domains and/or one or more mutations in their transcriptional regulatory domains. These ZFPs may include mutations in amino acids within the ZFP DNA-binding domain (in the "ZFP backbone") that can non-specifically interact with phosphates in the DNA backbone, but do not contain changes in DNA recognition strands. 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 mutation of a cationic amino acid residue to a neutral or anionic amino acid residue. In some embodiments, these mutations in the ZFP backbone include the mutation of a polar amino acid residue to a neutral or non-polar amino acid residue. In preferred embodiments, mutations occur at position (-5), (-9) and/or position (-14) relative to the DNA-binding helix. In some embodiments, the implementation of the zinc finger may contain one or more mutations in position (-5), (-9) and/or (-14). In further embodiments, one or more zinc fingers in a zinc finger protein may contain mutations at position (-5), (-9) and/or (-14). In some embodiments, the amino acids at (-5), (-9), and/or (-14) (e.g., arginine (R) or lysine (K)) are mutated to alanine (A), leucine (L), Ser (S ), Asp (N), Glu (E), Tyr (Y) and/or glutamine (Q).

Alternatively, the DNA binding domain may be derived from a nuclease. For example, recognition sequences of 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 US Pat. No. 5420032; US Pat. No. 6833252; 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 New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be designed to bind 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.

Two-handed zinc finger proteins are those in which two clusters of zinc finger DNA binding domains are separated by intermediate amino acids such that the two zinc finger domains bind to two discrete target sites. An example of a two-handed type of zinc finger binding protein is SIP1, in which a cluster of four zinc fingers is located at the N-terminus of the protein and a cluster of three fingers is located at the C-terminus (see Remacle et al., (1999) EMBO Journal 18 ( 18):5073-5084). Each zinc finger cluster in these proteins is capable of binding to a unique target sequence, and the distance between two target sequences can span multiple nucleotides. Two-handed ZFPs may include a functional domain, eg fused to one or both of the ZFPs. Thus, it will be apparent that the functional domain may be attached to the outside of one or both ZFPs, or may be located between the ZFPs (attached to both ZFPs).

In some embodiments, the DNA binding domain includes a naturally occurring or engineered (non-naturally occurring) TAL effector (TALE) DNA binding domain. See, for example, US Pat. No. 8,586,526, incorporated herein by reference in its entirety. In some embodiments, the TALE DNA binding protein binds to 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides of the target site in tau as shown in Table 1 or 2 (SEQ ID NO:1-6 or 33). The RVDs of the TALE DNA binding protein that binds to the target site in tau may be naturally occurring or non-naturally occurring RVDs. See US Patent Nos. 8586526 and 9458205.

Plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important agricultural crops. The pathogenicity of Xanthomonas depends on a conserved type III secretion system (T3S), which adds more than 25 different effector proteins to the plant cell. Among these added proteins are transcription activator-like effectors (TALE), 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-activating 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 WO2010079430). TALE contain a centered domain of tandem repeats, each containing approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activating domain (for a review, see Schornack S, et al., (2006) J Plant Physiol 163(3):256-272). In addition, two genes, designated brg11 and hpx17, that are homologous to the AvrBs3 Xanthomonas family in R. solanacearum biovar 1 strain GMI1000 and biovar 4 strain RS1000 were found in the phytopathogenic bacteria Ralstonia solanacearum (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, but differ in a 1575 bp deletion. in the hpx17 repeat domain. However, both gene products are less than 40% identical in sequence to Xanthomonas AvrBs3 family proteins.

The specificity of these TALEs depends on the sequences found in the tandem repeats. The repeat sequence is approximately 102 bp, and the repeats are typically 91-100% homologous to each other (Bonas et al., ibid.). The repeat polymorphism is typically found at positions 12 and 13, and there appears to be a one-to-one correspondence between the identity of the two hypervariable residues (HD) at positions 12 and 13 and the identity of consecutive nucleotides in the TALE target sequence (see Moscou and Bogdanove (2009) Science 326:1501 and Boch et al. (2009) Science 326:1509-1512). Experimentally, the code for DNA recognition by these TALEs was determined such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI 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 repeat numbers to create artificial transcription factors capable of interacting with the new sequences. In addition, U.S. Patent No. 8,586,526 and U.S. Patent Application Publication No. 2013/0196373, incorporated herein by reference in their entirety, describe TALEs with N-capped polypeptides, C-capped polypeptides (e.g., +63, +231, or +278) and/or new (atypical) RVDs.

Examples of TALE are described in US Pat. Nos. 8,586,526 and 9,458,205, which are incorporated by reference in their entirety.

In some embodiments, DNA binding domains include a dimerization and/or multimerization domain, such as a coiled helix (CC) and a dimerizing zinc finger (DZ). See U.S. Patent Application Publication No. 2013/0253040.

In still other embodiments, the DNA binding domain comprises a chimeric CRISPR/Cas guide RNA, such as sgRNA, as described in 20150056705.

Recently, there has been strong evidence for an RNA-mediated genome defense pathway in archaea and many bacteria, presumably parallel to the RNA interference pathway in eukaryotes (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). This pathway, known as the CRISPR-Cas system or prokaryotic RNA interference (pRNAi), is thought to arise from two evolutionarily and often physically related gene loci: the CRISPR (short palindromic repeats, regularly arranged in clusters) locus, which encodes the RNA components of the system , and the cas locus (CRISPR-associated) that codes for proteins (Jansen et al., 2002. Mol. Microbiol. 43:1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30:482-496; Makarova et al. 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 as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage. Individual Cas proteins do not have significant sequence similarity to the protein components of the eukaryotic RNA interference mechanism, 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. Over forty different families of Cas proteins have been described. Of these protein families, Cas1 appears to be ubiquitous among the various CRISPR/Cas systems. Specific combinations of cas genes and repeat structures were used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated cryptic proteins (RAMPs) . More than one CRISPR subtype can occur in the same genome. The random distribution of CRISPR/Cas subtypes suggests that the system undergoes horizontal gene transfer during microbial evolution.

Type II CRISPR, originally described in S. pyogenes, is one of the best-characterized systems and performs targeted DNA double-strand breakage in four successive steps. First, two non-coding RNAs, an array of pre-crRNA (crRNA precursors) and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to repetitive regions of pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing separate spacer sequences, the processing being mediated by double-stranded RNA-specific RNase III in the presence of the Cas9 protein. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base pairing between a spacer in crRNA and a protospacer in the target DNA next to the protospacer adjacent motif (PAM), which is an additional requirement for target recognition. In addition, tracrRNA must also be present as it undergoes base pairing with crRNA at its 3' end and this association triggers Cas9 activity. Finally, Cas9 mediates target DNA cleavage to create a double-strand break within the protospacer. The activity of the CRISPR/Cas system consists of three steps: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks, in a process called "adaptation", (ii) expression of the appropriate proteins, and expression and processing of the array, followed by (iii ) RNA-mediated interference using a foreign nucleic acid. Thus, in the bacterial cell, some of the so-called "Cas" proteins are involved in the natural function of the CRISPR/Cas system.

Type II CRISPR systems have been found in many different bacteria. BLAST searches on publicly available genomes by Fonfara et al. ((2013) Nuc Acid Res 42(4):2377-2590) revealed Cas9 orthologs in 347 bacterial species. In addition, this group demonstrated in vitro CRISPR/Cas cleavage of target DNA using Cas9 orthologs from S. pyogenes, S. mutans, S. therophilus, C. jejuni, N. meningitides, P. multocida, and F. novicida. Thus, the term "Cas9" refers to an RNA-guided DNA nuclease comprising a DNA binding domain and two nuclease domains, wherein the gene encoding Cas9 may be derived from any suitable bacteria.

The Cas9 protein has at least two nuclease domains: one nuclease domain is similar to the HNH endonuclease and the other resembles the Ruv endonuclease domain. The HNH-type domain appears to be responsible for cleaving the DNA strand that is complementary to crRNA, while the Ruv-type domain cleaves the non-complementary strand. It is possible to design the Cas 9 nuclease so that only one of the nuclease domains is functional, creating a Cas nickase (see Jinek et al., (2012) Science 337:816). Nicases can be created by specific mutation of amino acids in the catalytic domain of an enzyme, or by truncation of part or all of the domain so that it no longer functions. Since Cas9 contains two nuclease domains, this approach can be used for either of the two domains. A double-strand break in the target DNA can be achieved using two such Cas9 nicases. Each of the nicases will cleave one strand of DNA, and the use of two will create a double-strand break.

The need for a crRNA-tracrRNA complex can be avoided by using an engineered "chimeric guide RNA" (sgRNA) that includes a hairpin, usually formed by crRNA and tracrRNA annealing (see Jinek et al., ibid., and Cong et al., (2013 ) Sciencexpress/10.1126/science.1231143). In S. pyrogenes, an engineered tracrRNA:crRNA, or sgRNA fusion directs Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer is formed between the Cas-associated RNA and the target DNA. This system, including the Cas9 protein and engineered sgRNA containing the PAM sequence, has been used for RNA-guided genome editing (see Ramalingam et al., Stem Cells and Development 22(4):595-610 (2013)) and has been applicable to embryonic genome editing. zebrafish in vivo (see Hwang et al., (2013) Nature Biotechnology 31(3):227) with similar editing efficiency to ZFN and TALEN.

The primary products of CRISPR loci appear to be short RNAs that contain intruding targeting sequences and are referred to as guide RNAs or prokaryotic silencing RNAs (psiRNAs), given their putative role in the pathway (Makarova et al., 2006. Biol. Direct 1:7; Hale et al., 2008. RNA, 14:2572-2579). RNA analysis shows that transcripts from the CRISPR locus cleave within repeat sequences to release ~60-70 nt RNA intermediates that contain distinct intruding targeting 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 archaea of Pyrococcus furiosus, these intermediate RNAs are further processed into abundant, stable mature psiRNAs of ~35-45 nucleotides in size (Hale et al., 2008. RNA, 14:2572-2579).

The need for a crRNA-tracrRNA complex can be avoided by using an engineered "chimeric guide RNA" (sgRNA) that includes a hairpin usually formed by crRNA and tracrRNA annealing (see Jinek et al., (2012) Science 337:816 and Cong et al ., (2013) Sciencexpress/10.1126/science.1231143). In S. pyrogenes, an engineered tracrRNA:crRNA, or sgRNA fusion directs Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer is formed between the Cas-associated RNA and the target DNA. This system, including the Cas9 protein and an engineered sgRNA containing the PAM sequence, has been used for RNA-guided genome editing (see Ramalingam ibid.) and has been applicable to in vivo zebrafish embryo genome editing (see Hwang et al., (2013) Nature Biotechnology 31 (3):227) with editing efficiency similar to ZFN and TALEN.

Chimeric or sgRNA can be designed to include a sequence that is complementary to any desired target. In some embodiments, the length of the guide sequence is about or greater than 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 or more nucleotides. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. In some embodiments, the sgRNA includes a sequence that binds to 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides of a target site in tau as shown in Table 1 or 2 (SEQ ID NO:1-6 or 33). In some embodiments, the RNAs comprise 22 bases of target complementarity and in 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, sgRNAs can be constructed by using a known ZFN target in a gene of interest by (i) aligning a ZFN heterodimer recognition sequence with a reference sequence of the appropriate genome (human, mouse, or specific plant species); (ii) defining a spacer region between half sites for ZFN; (iii) locating the G[N20]GG motif that is closest to the spacer region (when more than one such motif overlaps the spacer, the motif that is centered on the spacer is selected); (iv) using this motif as the sgRNA core. This method is predominantly based on proven nuclease targets. Alternatively, sgRNAs can be designed to target any region of interest by simply identifying the appropriate target sequence corresponding to the formula G[n20]GG. Along with the complementarity region, the sgRNA can include additional nucleotides to extend to the tail region of the tracrRNA portion of the sgRNA (see Hsu et al., (2013) Nature Biotech doi: 10.1038/nbt.2647). The length of the tail regions can be from +67 to +85 nucleotides, or any number in between, with the preferred length being +85 nucleotides. Truncated sgRNAs, "tru-gRNAs" can also be used (see Fu et al., (2014) Nature Biotech 32(3):279). In tru-gRNA, the length of the complementarity region is reduced to 17 or 18 nucleotides.

In addition, alternative PAM sequences can also be used, where the PAM sequence can be NAG as an alternative to NGG (Hsu 2013, ibid.) using S. pyogenes Cas9. Additional PAM sequences may also include sequences lacking the initial G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the S. pyogenes encoded PAM sequences for Cas9, other PAM sequences that are specific for Cas9 proteins from other bacterial sources can be used. For example, the PAM sequences below (adapted from Sander and Joung, ibid., and Esvelt et al., (2013) Nat Meth 10(11):1116) are specific for these Cas9 proteins:

View 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. meningitis NNNNGATT P. multocida GNNNCNNA F. novicida NG

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

It is most preferred to select a target sequence with the highest probability of specificity that avoids potential off-target sequences. These undesirable off-target sequences can be identified by considering the following attributes: i) target sequence similarity followed by a PAM sequence known to function with the Cas9 protein used; ii) a similar target sequence with less than three mismatches compared to the desired target sequence; iii) a target sequence similar to that in ii) in which all mismatches are located in the distal region of the PAM, and not in the proximal region of the PAM (there is evidence that nucleotides 1-5 immediately adjacent or proximal to the PAM, sometimes called the region " primers (Wu et al., (2014) Nature Biotech doi: 10.1038/nbt2889) are the most important for recognition, so putative off-target sites with mismatches located in the primer region may be the least likely to be recognized by sgRNA); and iv) a similar target sequence in which the mismatches are not sequentially spaced or more than four nucleotides apart from each other (Hsu 2014, ibid.). Thus, by performing an analysis of the number of potential off-target sites in the genome for any CRIPSR/Cas system used, using the above criteria, a suitable sgRNA target sequence can be determined.

In some 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 has qualitative biological properties in common with the native sequence polypeptide. "Functional derivatives" include, but are not limited to, native sequence fragments and derivatives of a native sequence polypeptide and fragments thereof, provided that they have a biological activity in common with the corresponding native sequence polypeptide. The biological activity considered here is the ability of a functional derivative to be hydrolyzed into fragments by a DNA substrate. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent modifications, and fusions thereof. In some aspects, a functional derivative may comprise the only 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 fragment thereof include, but are not limited to, mutants, fusions, covalent modifications of the Cas protein or fragment thereof. The Cas protein, which includes the Cas protein or fragment thereof, as well as derivatives of the Cas protein or fragment thereof, can be obtained from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces a Cas protein, or a cell that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein at a higher expression level, or to produce a Cas protein from an exogenously introduced nucleic acid that encodes Cas, which is the same as or different from the endogenous Cas. In some cases, the cell does not naturally produce the Cas protein and is genetically engineered to produce the Cas protein.

Exemplary CRISPR/Cas nuclease systems targeting specific genes (including safe harbor genes) are disclosed, for example, in US Publication No. 2015/0056705.

Thus, a nuclease includes a DNA binding domain that specifically binds to a target site in any gene into which a donor (transgene) is desired to be inserted, in combination with a nuclease domain that cleaves the DNA.

Tau gene modulators

Tau DNA binding domains can be fused or otherwise linked to any additional molecules (eg, polypeptides) for use in the methods described herein. In some embodiments, methods use fusion molecules comprising at least one DNA-binding molecule (eg, ZFP, TALE, or chimeric guide RNA) and a heterologous regulatory (functional) domain (or functional fragment thereof).

In some embodiments, the tau modulator functional domain includes a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activators, repressors, coactivators, corepressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, members of the mos family, etc. .d.); DNA repair enzymes and related factors and modifiers; DNA rearrangement enzymes and related factors and modifiers; chromatin-associated proteins and their modifiers (eg, kinases, acetylases, and deacetylases); and DNA modifying enzymes (eg, methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See, for example, US Patent Application Publication No. 2013/0253040, incorporated herein by reference in its entirety.

Suitable domains to achieve activation include the HSV VP16 activating domain (see e.g. Hagmann et al., J. Virol. 71, 5952-5962 (1997)), nuclear hormone receptors (see e.g. Torchia et al., Curr. Opin Cell Biol 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron (Molinari et al. (1999) EMBO J. 18, 6439-6447). Additional exemplary activating domains include Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipel et al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2 See, 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. 46:77-89; 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. by way of example, activating 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. See, 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. al. al., (1999) Plant Mol Biol 40:419-429 Ulmason et al., (1999) Pro c. 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 96:15348-15353.

Exemplary inhibitory domains that can be used to generate tau repressors include, but are not limited to, KRAB A/B, KOX, TGF-beta inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (eg, 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 inhibitory domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al., (1996) Plant Cell 8:305-321; and Wu et al., (2000) Plant J. 22:19-27.

In some cases, the domain is involved in the epigenetic regulation of the chromosome. In some embodiments, the domain is a histone acetyltransferase (HAT), e.g., type A, nuclear localized, such as MYST family members MOZ, Ybf2/Sas3, MOF and Tip60, GNAT family members Gcn5 or pCAF, p300 CBP family members, p300 or Rtt109 (Berndsen and Denu (2008) Curr Opin Struct Biol 18 (6): 682-689). In other cases, 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 (HDAC 6 and 10)), class IV (HDAC-11), class III (also known as sirtuins (SIRT); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898-3941). Another domain that is used in some embodiments is histone phosphorylase or β-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 methylating domain is used and may be selected from groups such as Ezh2, PRMT1/6, PRMT5/7, PRMT 2/6, CARM1, set7/9, MLL, ALL-1, Suv 39h, G9a, SETDB1 , Ezh2, Set2, Dot1, PRMT 1/6, PRMT 5/7, PR-Set7 and Suv4-20h. Domains involved in sumoylation and biotinylation (Lys9, 13, 4, 18 and 12) can also be used in some embodiments (for a review, see Kousarides (2007) Cell 128:693-705).

Fusion molecules are constructed by cloning and biochemical conjugation methods that are well known to those skilled in the art. Fusion molecules include a DNA-binding domain and a functional domain (eg, a transcription-activating or repressing domain). The fusion molecules also optionally include nuclear localization signals (such as, for example, the signal from the SV40 middle T antigen) and epitope marks (such as, for example, FLAG and hemagglutinin). Fusion proteins (and the nucleic acids encoding them) are designed so that the translational reading frame is conserved among the fusion components.

Fusions between a functional domain polypeptide component (or a functional fragment thereof) on the one hand and a non-protein DNA binding domain (e.g. antibiotic, minor groove intercalator, nucleic acid) on the other hand are constructed by biochemical conjugation methods known to those skilled in the art. in this field of technology. See, for example, the catalog of Pierce Chemical Company (Rockford, IL). Methods and compositions have been described for fusions of a minor groove binder and a polypeptide. Mapp et al., (2000) Proc. Natl. Acad. sci. USA 97:3930-3935. Similarly, CRISPR/Cas-TF and nucleases comprising a sgRNA nucleic acid component in association with a polypeptide component as a functional domain are also known to those skilled in the art and are described in detail herein.

The fusion molecule may be formulated with a pharmaceutically acceptable carrier as known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985, and co-owned International Patent Application No. WO 00/42219.

The functional component/domain of the fusion molecule can be selected from any of a variety of different components capable of influencing gene transcription when the fusion molecule binds to the target sequence via its DNA binding domain. Therefore, a functional component may include, but is not limited to, various transcription factor domains such as activators, repressors, coactivators, corepressors, and silencers.

In some embodiments, the fusion molecule comprises a DNA binding domain and a nuclease domain to generate functional molecules that are capable of recognizing the intended target nucleic acid through their engineered (ZFP or TALE or sgRNA) DNA binding domains and to generate nucleases (e.g., nuclease with zinc fingers or TALE nucleases or CRISPR/Cas nucleases), which cause DNA cleavage near the DNA binding site through nuclease activity. This cleavage leads to inactivation (repression) of the tau gene. Thus, tau repressors also include tau nucleases.

Thus, the methods and compositions described herein are broadly applicable and may include any nuclease of interest. Non-limiting examples of nucleases include meganucleases, TALEN, and zinc finger nucleases. The nuclease may include heterologous DNA-binding and cleaving domains (e.g., zinc finger nucleases; TALEN; meganuclease DNA-binding domains with heterologous cleavage domains, sgRNA in association with nuclease domains), or alternatively, the DNA-binding domain of a naturally occurring nuclease can be modified to bind to a selected target site (eg, a meganuclease that has been engineered to bind to a site other than its corresponding binding site).

The nuclease domain can be derived from any nuclease, such as any endonuclease or exonuclease. Non-limiting examples of suitable nuclease (cleavage) domains that can be fused to the tau DNA binding domains described herein include domains from any restriction enzyme, eg a type IIS restriction enzyme (eg FokI). In some embodiments, cleavage domains are cleavage half domains (semi-domains) that require dimerization for cleavage activity. See, for example, US Pat. Typically, two fusion proteins are required for cleavage if the fusion proteins include a half domain for cleavage. Alternatively, a single protein can be used that includes two half domains for cleavage. 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 different endonucleases (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably positioned relative to each other such that binding of the two fusion proteins to their respective target sites places the half domains in such a spatial orientation to each other that allows the half domains to be cleaved to form a functional cleavage site. domain, for example, as a result of dimerization.

The nuclease domain can also be derived from any meganuclease (homing endonuclease), and a cleavage domain from the nucleases described herein can also be used, 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.

In some embodiments, the implementation of the nuclease includes compact TALE-nuclease (cTALEN). These are single-stranded fusion proteins that link the DNA-binding domain of TALE to the nuclease domain of TevI. The fusion protein can either act as a nickase restricted to the TALE region or create a double-strand break, depending on where the TALE DNA-binding domain is located relative to the nuclease domain of the meganuclease (e.g., TevI) (see Beurdeley et al., (2013) Nat Comm : 1-8 DOI: 10.1038/ncomms2782).

In other embodiments, the TALE nuclease is mega-TAL. These mega-TAL nucleases are fusion proteins comprising the TALE DNA-binding domain and the meganuclease cleavage domain. The cleaving domain of the meganuclease is active as a monomer and does not require dimerization to be active. (See Boissel et al., (2013) Nucl Acid Res: 1-13, doi: 10.1093/nar/gkt1224).

In addition, the nuclease domain of a meganuclease can also exhibit DNA-binding functionality. Any TALEN may be used in conjunction with additional TALENs (for example, one or more TALENs (cTALEN or FokI-TALEN) with one or more mega-TALs and/or ZFNs.

In addition, the cleavage domains may include one or more changes from the wild type, for example, to form obligate heterodimers, which reduce or eliminate the effects of off-target cleavage. See, for example, US patent No. 7914796, 8034598 and 8623618, fully incorporated here by reference.

The nucleases described herein can create double-stranded or single-stranded breaks in a double-stranded target (eg, a gene). The creation of single-strand breaks ("nicks") is described, for example, in US patents with No. 8703489 and 9200266, included here by reference, which describe how a mutation of the catalytic domain of one of the nuclease domains leads to the appearance of nikase.

Thus, a nuclease (cleavage) domain or cleavage half domain can be any portion of a protein that retains cleaving activity or that retains the ability to multimerize (eg, dimerize) to form a functional cleavage domain.

Alternatively, nucleases can be assembled in vivo at the target site of a nucleic acid using so-called "split enzyme" technology (see, for example, US Patent Application Publication No. 2009/0068164). The components of such split enzymes may be expressed either on separate expression constructs or may be linked into a single open reading frame in which the individual components are separated by, for example, a 2A self-cleaving peptide or an IRES sequence. The components may be individual zinc finger binding domains or nucleic acid binding domain domains of a meganuclease.

Nucleases can be screened for activity prior to use, for example in a yeast-based chromosome system, as described in US Patent Application Publication No. 2009/0111119. Nuclease-expressing constructs can be readily generated using methods known in the art.

Expression of the fusion proteins (or components thereof) can be controlled by a constitutive promoter or an inducible promoter, such as the galactokinase promoter, which is activated (derepressed) in the presence of raffinose and/or galactose and downregulated in the presence of glucose. Non-limiting examples of preferred promoters include the nerve cell-specific NSE, Synapsin, CAMKiia and MECP promoters. Non-limiting examples of ubiquitous promoters include CAS and Ubc. Further embodiments include the use of self-regulating promoters (by including high affinity binding sites for the DNA binding for the tau domain) as described in US Patent Application Publication No. 2015/0267205).

Delivery

Proteins and/or polynucleotides (e.g., tau modulators) and compositions containing the proteins and/or polynucleotides described herein can be delivered to a target cell by any suitable method, including, for example, injection of proteins, using mRNA and/or an expression construct (e.g. , plasmid, lentiviral vector, AAV vector, Ad vector, etc.). In preferred embodiments, the repressor is delivered using an AAV vector, including but not limited to AAV9 (see US Pat. No. 7,198,951), an AAV vector as described in US Pat. No. 9,585,971, and/or an AAV vector as described in Provisional Application No. US Patent No. 62/503121.

Methods for delivering proteins including zinc finger proteins described herein are described, for example, in US Pat. Nos. 6,452,442; 6503717; 6534261; 6599692; 6607882; 6689558; 6824978; 6933113; 6979539; 7013219; and 7163824, the disclosure of all of which is incorporated here by reference in their entirety.

Any vector system can be used, including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; vectors based on herpes virus and adeno-associated virus, etc. See also US Pat. No. 8,586,526; 6534261; 6607882; 6824978; 6933113; 6979539; 7013219 and 7163824, incorporated herein by reference in their entirety. In addition, it will be obvious that any of these vectors may include one or more sequences encoding a DNA binding protein. Thus, when one or more tau modulators (eg, repressors) are introduced into a cell, sequences encoding protein components and/or polynucleotide components may be carried in the same vector or in different vectors. When multiple vectors are used, each vector may include a sequence encoding one or more tau modulators (eg, repressors), or components thereof. In preferred embodiments, the vector system is an AAV vector, such as AAV9 or an AAV variant as described in US Patent No. 9,585,971 and/or US Provisional Application No. 62/503,121.

Conventional viral and non-viral-based gene transfer techniques can be used to introduce nucleic acids encoding engineered tau modulators into cells (eg, mammalian cells) and target tissues. Such methods can also be used to introduce nucleic acids encoding such repressors (or components thereof) into cells in vitro. In some embodiments, nucleic acids encoding repressors are administered for use in in vivo or ex vivo gene therapy. Non-viral vector-based delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector-based delivery systems include DNA- and RNA-containing viruses that have either an episomal or an integrated genome upon delivery into a cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Böhm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, ballistic transformation, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and agent-enhanced DNA uptake. Sonoporation using, for example, 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. Also preferred is the use of capped mRNAs to improve translation efficiency and/or mRNA stability. Particularly preferred are ARCA (anti-reverse cap analog) caps or variants thereof. See US Pat. Nos. 7,074,596 and 8,153,773, incorporated herein by reference.

Additional 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, US Pat. No. 6,008,336). Lipofection is described, for example, in US patents No. 5049386; 4946787; and 4897355), and lipofection reagents are commercially available (eg Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides via receptor recognition include Felgner lipids, International Patent Publication No. WO 91/17424 and WO 91/16024. Delivery can be to cells (ex vivo administration) or to target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled in the art (see, for example, Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2 :291-297 (1995) Behr et al Bioconjugate Chem 5:382-389 (1994) Remy et al Bioconjugate Chem 5:647-654 (1994) Gao et al Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992);

Additional delivery methods include the use of packaging of delivered nucleic acids in EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies, with one arm of the antibody specific for the target tissue and the other specific for the EDV. The antibody carries the EDV to the surface of the target cell, and then the EDV is introduced into the cell by endocytosis. Upon entering the cell, the contents are released (see MacDiarmid et al., (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA virus-based systems to deliver nucleic acids encoding engineered ZFPs, TALEs, or CRISPR/Cas systems takes advantage of highly evolved processes to target the virus to specific cells in the body and transfer the 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 are administered to patients (ex vivo). Common virus-based delivery systems for ZFP, TALE, or CRISPR/Cas systems include, but are not limited to, retroviral, lentiviral, adenovirus, adeno-associated, vaccinia, and herpes simplex vectors for gene transfer. Retroviral, lentivirus, and adeno-associated virus gene transfer techniques can integrate into the host genome, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Retrovirus tropism can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and typically produce high viral titers. The choice of retroviral system for gene transfer depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb. foreign sequence. Minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the therapeutic gene into the target cell to ensure continued expression of the transgene. Commonly used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J Virol 66:2731-2739 (1992), Johann et al., J. Virol 66:1635-1640 (1992), Sommerfelt et al., Virol 176:58-59 (1990), Wilson et al. , J. Virol 63:2374-2378 (1989); Miller et al., J. Virol 65:2220-2224 (1991); International Patent Application Publication No. WO 1994/026877).

In applications where transient expression is preferred, adenovirus-based systems may be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. When using such vectors, a high titer and a high level of expression were obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides and in in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; International Patent Publication No. WO 93/24641; Kotin Human Human Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94 :1351 (1994) The construction of recombinant AAV vectors has been described in a number of publications, including US Patent No. 5,173,414, Tratschin et al., Mol. Cell Biol. Mol Cell Biol 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al, J. Virol 63:03822-3828 (1989).

At least six viral vector-based approaches for gene transfer in clinical trials are currently available that use approaches that involve complementing defective vectors with genes inserted in helper cell lines to generate a transduction agent.

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

Recombinant adeno-associated virus (rAAV) vectors are promising alternative gene delivery systems based on the defective and non-pathogenic type 2 adeno-associated virus of the Parvoviridae family. All vectors are derived from a plasmid that retains only the 145 bp inverted terminal repeats. AAV flanking the transgene expression cassette. Efficient gene transfer and stable delivery of transgenes due to integration into the genomes of the transduced cell are the key features of this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)). Other AAV serotypes including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9 and AAV rh10 and pseudotyped AAV such as AAV2/8, AAV2/5, AAV2/9 and AAV2/6 may also be used in in accordance with the present invention. New AAV serotypes capable of crossing the blood-brain barrier can also be used in accordance with the present invention (see, for example, US patent No. 9585971). In preferred embodiments, an AAV9-based vector (including AAV9 variants and pseudotypes) is used.

Recombinant replication-defective adenoviral vectors (Ad) can be produced at high titer and easily infect a number of different cell types. Most adenoviral vectors are designed such that the transgene replaces the E1a, E1b and/or E3 Ad genes; subsequently, the replication-defective vector is propagated in human 293 cells, which provide the function of the deleted gene transiently. Adenovirus vectors can transduce many tissue types in vivo, including non-dividing, differentiated cells such as those found in liver, kidney, and muscle. Conventional adenoviral vectors have a large capacity. An example of the use of an adenoviral vector in clinical trials included polynucleotide therapy for antitumor immunization by intramuscular injection (Sterman et al., Hum. Gene Ther. 7: 1083-9 (1998)). Additional examples of the use of adenoviral vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

The packaging cells are used to form viral particles capable of infecting the host cell. Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually produced by a producer cell line that packages the NK vector into a viral particle. Vectors typically contain 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. Missing viral functions are supplied transiently via a packaging cell line. For example, AAV vectors used in gene therapy typically contain only inverted terminal repeat (ITR) sequences from the AAV genome that are required for packaging and integration into the host genome. The viral DNA is packaged into a cell line that contains a helper plasmid encoding other AAV genes, namely rep and cap, but without the ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the vector used in gene therapy be delivered with a high degree of specificity to a particular tissue type. Accordingly, the viral vector can be modified to be cell type specific by expressing the ligand as a fusion protein to the viral envelope protein on the outer surface of the virus. The selected ligand has an affinity for the receptor known to be present in the cell type of interest. For example, Han et al., Proc. Natl. Acad. sci. USA 92:9747-9751 (1995), reported that Moloney mouse leukemia virus can be modified to express human heregulin fused to gp70 and the recombinant virus infects some human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus-cell-target pairs in which the target cell expresses the receptor and the virus expresses a fusion protein comprising a ligand for the receptor on the cell surface. For example, filamentous phage can be engineered to display antibody fragments (eg, FAB or Fv) that have specific affinity for virtually any chosen 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 designed to contain specific uptake sequences that promote uptake by specific target cells.

Vectors useful in gene therapy can be delivered in vivo by administration to an individual patient, usually by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, intracisternal, intracerebroventricular, or intracranial infusion, including direct injection into the brain, including into any region of the brain such as the hippocampus, cerebral cortex, striatum, etc.), or topical 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 aspirates, tissue biopsy) or donor universal hematopoietic stem cells, followed by reimplantation of the cells into the patient, usually after selection of the cells that included vector.

In some embodiments, compositions (eg, polynucleotides and/or proteins) described herein are delivered directly in vivo. 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. The target may be one or more regions of the brain, including, but not limited to, the hippocampus, the substantia nigra, the nucleus basalis of Meinert (NBM), the striatum, and/or the cerebral cortex. Alternatively, or in addition to CNS delivery, the compositions may be administered systemically (eg, by intravenous, intraperitoneal, intracardiac, intramuscular, subcutaneous, intrathecal, intracisternal, intracerebroventricular, and/or intracranial infusion). Methods and compositions for delivering the compositions described herein directly to a subject (including directly to the CNS) include, but are not limited to, direct injection (eg, stereotaxic injection) using collection needles. Such methods are described, for example, in US Pat.

The effective amount to be administered will vary from patient to patient and according to the route of administration and site of administration. Accordingly, effective amounts are best determined by the physician administering the compositions, and appropriate doses can be readily determined by one of ordinary skill in the art. Once sufficient time has been allowed for integration and expression (typically, for example, 4-15 days), analysis of serum or other tissue levels of the therapeutic polypeptide and comparison with pre-administration baseline will determine if the amount administered is too low, in the correct range, or too high. Suitable regimens for initial and subsequent administrations are also variable, but are characterized by an initial administration followed by subsequent administrations, if necessary. Subsequent administrations can be made at various intervals ranging from daily to annual and every few years.

To deliver ZFP using adeno-associated virus (AAV) vectors directly to the human brain, a dose range of 1×10 ¹⁰ -5×10 ¹⁵ (or any number in between) vector genomes per striatum can be used. As already noted, doses may vary for other brain structures and for different delivery protocols. Methods for delivering AAV vectors directly to the brain are known in the art. See, for example, US Pat. Nos. 9,089,667; 9050299; 8337458; 8309355; 7182944; 6953575 and 6309344.

Transfection of cells ex vivo for diagnosis, research, or gene therapy (eg, by reinfusion of the transfected cells into the host) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from the subject, transfected with at least one tau modulator (eg, repressor) or component thereof, and reintroduced back into the subject (eg, patient). In a preferred embodiment, one or more nucleic acids encoding a tau modulator (eg, repressor) are delivered using AAV9. In other embodiments, one or more nucleic acids encoding a tau modulator (eg, repressor) are delivered as mRNA. Also preferred is the use of capped mRNAs to improve translation efficiency and/or mRNA stability. Particularly preferred are ARCA (anti-reverse cap analog) caps or variants thereof. See US Pat. Nos. 7,074,596 and 8,153,773 incorporated herein by reference in their entirety. Various cell types suitable for ex vivo transfection are well known to those skilled in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein. , to explore how to isolate and culture cells from patients).

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 differentiate into another type of cell in vitro or can be injected into a mammal (eg a cell donor) where they will take root in the bone marrow. Methods are known to differentiate CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ and TNF-α (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992 )).

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 using antibodies that bind unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and Iad ( differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified can also be used in some embodiments. For example, neural stem cells that have been made resistant to apoptosis can be used as therapeutic compositions, the stem cells also containing the TF-ZFP of the present invention. Apoptosis resistance can arise, for example, by knocking out BAX and/or BAK using BAX- or BAK-specific TALEN or ZFN (see US Pat. No. 8,599,912), in stem cells or in cells that have abnormalities in caspase, again using, for example, caspase-6-specific ZFNs. These cells can be transfected with ZFP-TF or TALE-TF, which are known to regulate the tau gene.

Vectors (eg, retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids encoding ZFP can also be administered directly to the body for in vivo cell transduction. Alternatively, naked DNA can be introduced. Administration is by any route commonly used to bring the molecule into maximum contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route can often provide a more direct and more efficient reaction than another route.

Methods for introducing DNA into hematopoietic stem cells are disclosed in, for example, US Pat. No. 5,928,638. Vectors useful for introducing transgenes into hematopoietic stem cells, such as CD34+ cells, include adenovirus type 35.

Suitable vectors for introducing transgenes into immune cells (eg, 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.

Pharmaceutically acceptable carriers are determined in part by the particular composition administered, as well as by the particular route used to administer the composition. Accordingly, there is a wide range of suitable pharmaceutical formulations available as described below (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1989).

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 skilled 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 cells fungi such as Saccharomyces, Pischia and Schizosaccharomyces. Progeny, variants and derivatives of these cell lines may also be used. In a preferred embodiment, the methods and compositions are delivered directly to a brain cell, such as the striatum.

Models of CNS disorders

Studies of CNS disorders can be conducted in animal model systems such as non-human primates (e.g. for 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); epileptic seizure (Hsiao et al., (2016) E Bio Med 9:257-77), canine (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); epileptic seizure (Varatharajah et al., (2017) Int J Neural Syst 27(1):1650046) and mice (e.g. for epileptic seizure (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 by Webster et al., (2014) Front Genet 5 ar t 88, doi:10.3389f/gene.2014.00088). These models can be used even in the absence of an animal model that completely replicates the CNS disease, as they can be used to study the constellations of certain symptoms of the disease. Models may be useful in determining the efficacy and safety profiles of the therapeutic methods and compositions (genetic repressors) described here.

Applications

The tau modulators (e.g., tau repressors) described herein, including the MAPT-binding molecules (e.g., ZFP, TALE, CRISPR/Cas systems, Ttago, etc.) described herein, and the nucleic acids encoding them, can be used for various applications. These applications include therapeutic methods in which a MAPT binding molecule (including a nucleic acid encoding a DNA binding protein) is administered to a subject using a viral (eg, AAV) or non-viral vector and is used to modulate target gene expression within the subject. The modulation may be in the form of repression, for example downregulation of tau expression, which contributes to the course of AD disease. Alternatively, the modulation may be in the form of activation, where upregulation of expression or increased expression of an endogenous cellular gene can reduce the disease state. In still other embodiments, the modulation may be repression via cleavage (eg, with one or more nucleases), eg, to inactivate the MAPT gene. As noted above, for such applications, the MAPT-binding molecules, or more typically the nucleic acids encoding them, are formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition.

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 "sprayed") for administration by inhalation. Aerosol formulations may be placed in suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatics, and solutes that render the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. The compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, retro-orbitally (RO), intracranially (for example, to any region of the brain, including, but not limited to, the hippocampus and/or cerebral cortex), or intrathecally. Compositions of the compounds may be presented in sealed single-dose or multi-dose containers such as ampoules and vials. Injectable solutions and suspensions may be prepared from sterile powders, granules and tablets of the type previously described.

The dose administered to the patient should be sufficient to provide a positive therapeutic response in the patient over time. The dose is determined by the potency and K _d of the particular MAPT binding molecule used, the target cell and the condition of the patient, as well as the body weight or surface area of the patient being treated. The size of the dose is also determined by the presence, nature and extent of any adverse side effects that accompany the administration of a particular compound or vector to a particular patient.

The following examples relate to exemplary embodiments of the present invention in which the MART modulator comprises a zinc finger protein. It is understood that this is for illustrative purposes only, and that other MAPT modulators (e.g., repressors) may be used, including, but not limited to, TALE-TF, CRISPR/Cas system, additional ZFPs, ZFN, TALEN, additional systems CRISPR/Cas, homing endonucleases (meganucleases) with engineered DNA-binding domains. It will be apparent that these modulators can be easily obtained using methods known to the person skilled in the art for binding to target sites, as illustrated below. Likewise, the following examples refer to exemplary embodiments in which the delivery vehicle is any AAV vector, but it will be apparent that any viral (Ad, LV, etc.) or non-viral (plasmid , mRNA, etc.) vector.

EXAMPLES

Example 1: MAPT repressors

Screening of approximately 185 zinc finger proteins constructed essentially as described in US Pat. No. 6,534,261; U.S. Patent Application Publication No. 2015/0056705; 2011/0082093; 2013/0253040; and 2015/0335708 has been executed and ZFPs are linked to their target sites in MAPT. The zinc finger proteins 52288, 52322, 52364, 52366, 52389, 57880, 57890 and 65888 (see Tables 1-3 below) targeted to mouse MAPT were selected for further study. Phosphate contact mutants listed under 65888 are as previously described (see, for example, US Patent Application No. 15/685580). Table 1 shows the helix recognition DNA-binding domain of these ZFPs and the target sequences of these ZFPs. A set of ZFPs have also been created that target MAPT target sequences common to mouse and human genes. They are shown in Table 2. Table 3 lists parental and derivative ZFP-TFs in which the ZFP backbone has been mutated at the indicated locations to remove potential non-specific contacts with phosphate residues. The ZFPs were evaluated using the standard SELEX assay and demonstrated binding to their target sites.

Table 1: Mouse MAPT-specific repressor constructs

SBS#, Target Design F1 F2 F3 F4 F5 F6 SBS#52364 cgACAGAAGGCGAG gacagaagaggaca
(SEQ ID NO:1) RSDNLAR (SEQ ID NO:7) DRSHLAR
(SEQ ID NO:8) QSGNLAR (SEQ ID NO:9) QSNTRIM
(SEQ ID NO:10) No data No data SBS#52389
ccGTTGCGCCTGAT
tGATGCCcagctcc
(SEQ ID NO:2) ERGTLAR (SEQ ID NO:11) TSANLSR
(SEQ ID NO:12) TSGNLTR
(SEQ ID NO:13) HRTSLTD
(SEQ ID NO:14) RSHSLLR
(SEQ ID NO:15) HPSARKR (SEQ ID NO:16) SBS#52322
gtGGCGGAGACTGA GAGcgcgcgcggcc (SEQ ID NO:3) RSANLTR (SEQ ID NO:17) DSSHLEL (SEQ ID NO:18) DRSNLTR
(SEQ ID NO:19) DRSHLTR
(SEQ ID NO:20) DRSHLAR
(SEQ ID NO:8) No data SBS#57930 cgGCAGAAGGTGG GcGGTGGCggcggcg (SEQ ID NO:44) DRSHLTR
(SEQ ID NO:20) LKQHLTR
(SEQ ID NO:37) RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR (SEQ ID NO:35) SBS#57947 gcGGCGGCgGCAG AAGGTGGGcggtggc (SEQ ID NO:45) RSAHLSR (SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) DRSHLSR (SEQ ID NO:38) DRSHLAR (SEQ ID NO:8)

Table 2: Human/Mouse MAPT-Specific Repressor Constructs

SBS#, Target Design F1 F2 F3 F4 F5 F6 SBS#52366 ctTCTGTCGATTAT CAGgtaagcgccgc (SEQ ID NO:4) RSDNLSE (SEQ ID NO:21) TSSNRKT
(SEQ ID NO:22) TSGNLTR
(SEQ ID NO:13) DRSALAR
(SEQ ID NO:23) RNSDRTK (SEQ ID NO:24) No data SBS#57880 ctGGTGGGtGGCGG AGACTGAgagcgcg (SEQ ID NO:5) DSSHLEL
(SEQ ID NO:18) DRSNLTR
(SEQ ID NO:19) DRSHLTR (SEQ ID NO:20) DRSHLAR
(SEQ ID NO:8) RSAHLSR
(SEQ ID NO:25) TSGHLSR
(SEQ ID NO:26) SBS#57890 tgGTGCTGGAGCT GGTGGGTggcggaga (SEQ ID NO:6) LRHHLTR
(SEQ ID NO:27) RRFTLSK
(SEQ ID NO:28) RSDVLSE
(SEQ ID NO:29) KHSTRRV
(SEQ ID NO:30) RSDVLSE
(SEQ ID NO:29) RLYTLHK
(SEQ ID NO:47) SBS#52288 agGGGCGGGCAGCG aggcctgggcgggc (SEQ ID NO:33) RSADLTR (SEQ ID NO:34) QSGDLTR
(SEQ ID NO:35) RSDHLSE
(SEQ ID NO:36) RSAHLSR
(SEQ ID NO:25) No data No data SBS#65976 ctCCAGAAGGGGAT CATGACctcctcac (SEQ ID NO:46) DRSNLSR (SEQ ID NO:39) LRQNLIM
(SEQ ID NO:40) TSANLTV
(SEQ ID NO:41) RSDHLSR
(SEQ ID NO:42) QSGNLAR
(SEQ ID NO:9) QRNDRKS
(SEQ ID NO:43) 65976 Phosphate contact mutants Qm5 No Qm5 No Qm5 No

ZFPs comprising one or more mutations in the framework regions as described in US Patent Application No. 15/685580 (e.g., mutations at the (-5), (-9) and/or (-14) position relative to the DNA helices, e.g. R or K in A, L, S, N, E, Y or Q) have also been obtained (see e.g. tables 1-3). All of the ZFPs described here were operably linked to the KRAB inhibitory domain to form ZFP-TF, and all repressed MAPT expression.

Exemplary ZFP-TFs were transfected into mouse Neuro2a cells or primary mouse cortical neurons. After 24 hours, total RNA was extracted and expression of MAPT and two control genes (ATP5b, RPL38) was monitored using real-time RT-qPCR. As shown in FIG. 1, ZFP-TFs were found to be effective in downregulating MAPT expression with dose-response diversity and MAPT inhibitory activity (see FIG. 1B).

ZFP-TF tau repressors were constructed that included mutations in the framework region. Table 3 lists typical ZFP-TF repressors (and their parent compounds). Results with these optimized ZFP-TFs (eg, the exemplary results comparing 65888 to 57880) showed dramatically increased specificity (greater than 10-fold) without affecting activity in primary neurons (tau repression, see FIG. 1B). In addition, two derivatives of the parent 57880, 65887 and 65888, were tested in Neuro2A cells for inhibitory activity for off-target regions of BOD1 and MOSPD1 (see Figure 1D), with off-target repression by proteins involving mutations in contact with phosphate residues in the backbone ZFP, has been reduced. See also US Patent Application No. 15/685580.

Table 3: Optimized ZFP-TF designs

SBS#, Target Design F1 F2 F3 F4 F5 F6 SBS#57930 cgGCAGAAGGTGG GcGGTGGCggcggcg (SEQ ID NO:44)
[Original] DRSHLTR (SEQ ID NO:20) LKQHLTR
(SEQ ID NO:37) RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) SBS#65916
cgGCAGAAGGTGG
GcGGTGGCggcggcg
(SEQ ID NO:44) DRSHLTR (SEQ ID NO:20) LKQHLTR
(SEQ ID NO:37) RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) 65918 Phosphate contact mutants Qm5 No No No Qm5 No SBS#65920
cgGCAGAAGGTGG GcGGTGGCggcggcg
(SEQ ID NO:44) DRSHLTR
(SEQ ID NO:20) LKQHLTR
(SEQ ID NO:37) RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) 65920 Phosphate contact mutants Qm5 No Qm5 No Qm5 No SBS#57890
tgGTGCTGGAGCT
GGTGGGTggcggaga
(SEQ ID NO:6)
[Original] LRHHLTR
(SEQ ID NO:27) RRFTLSK
(SEQ ID NO:28) RSDVLSE
(SEQ ID NO:29) KHSTRRV
(SEQ ID NO:30) RSDVLSE
(SEQ ID NO:29) RLYTLHK
(SEQ ID NO:30) SBS#65894
tgGTGCTGGAGCT
GGTGGGTggcggaga
(SEQ ID NO:6) LRHHLTR
(SEQ ID NO:27) RRFTLSK
(SEQ ID NO:28) RSDVLSE
(SEQ ID NO:29) KHSTRRV
(SEQ ID NO:30) RSDVLSE
(SEQ ID NO:29) RLYTLHK
(SEQ ID NO:47) 65894 Phosphate contact mutants Qm5 No No No Qm5 No SBS#57947
gcGGCGGCgGCAG
AAGGTGGGcggtggc (SEQ ID NO:45)
[Original] RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) DRSHLSR
(SEQ ID NO:38) DRSHLAR
(SEQ ID NO:8) SBS#65968
gcGGCGGCgGCAG
AAGGTGGGcggtggc (SEQ ID NO:45) RSAHLSR
(SEQ ID NO:25) TSGHLST
(SEQ ID NO:26) QSGNLAR
(SEQ ID NO:9) QSGDLTR
(SEQ ID NO:35) DRSHLSR
(SEQ ID NO:38) DRSHLAR
(SEQ ID NO:8) 65968 Phosphate contact mutants Qm5 No Qm5 No Qm5 No SBS#57880
ctGGTGGGtGGCGG AGACTGAgagcgcg (SEQ ID NO:5)
[Original] DSSHLEL
(SEQ ID NO:18) DRSNLTR
(SEQ ID NO:19) DRSHLTR
(SEQ ID NO:20) DRSHLAR
(SEQ ID NO:8) RSAHLSR
(SEQ ID NO:25) TSGHLSR
(SEQ ID NO:26) SBS#65888
ctGGTGGGtGGCGG
AGACTGAgagcgcg
(SEQ ID NO:5) DSSHLEL
(SEQ ID NO:18) DRSNLTR
(SEQ ID NO:19) DRSHLTR
(SEQ ID NO:20) DRSHLAR
(SEQ ID NO:8) RSAHLSR
(SEQ ID NO:25) TSGHLSR
(SEQ ID NO:26) 65888 Phosphate contact mutants Qm5 No Qm5 No Qm5 No SBS#65887 ctGGTGGGtGGCGG AGACTGAgagcgcg (SEQ ID NO:5) DSSHLEL
(SEQ ID NO:18) DRSNLTR
(SEQ ID NO:19) DRSHLTR
(SEQ ID NO:20) DRSHLAR
(SEQ ID NO:8) RSAHLSR
(SEQ ID NO:25) TSGHLSR
(SEQ ID NO:26) 65887 Phosphate contact mutants No No Qm5 No Qm5 No SBS#52389
ccGTTGCGCCTGAT tGATGCCcagctcc
(SEQ ID NO:2)
[Original] ERGTLAR
(SEQ ID NO:11) TSANLSR (SEQ ID NO:12) TSGNLTR
(SEQ ID NO:13) HRTSLTD
(SEQ ID NO:14) RSHSLLR
(SEQ ID NO:15) HPSARKR
(SEQ ID NO:16) SBS#65860 ccGTTGCGCCTGAT tGATGCCcagctcc (SEQ ID NO:2) ERGTLAR
(SEQ ID NO:11) TSANLSR
(SEQ ID NO:12) TSGNLTR
(SEQ ID NO:13) HRTSLTD
(SEQ ID NO:14) RSHSLLR
(SEQ ID NO:15) HPSARKR
(SEQ ID NO:16) 65860 Phosphate contact mutants No No No No Qm5 No

Example 2 Tau Repression in Mouse Neurons

All ZFP repressors were cloned into rAAV2/9 vectors using the CMV promoter to drive expression. Virus was produced in HEK293T cells, purified using a CsCl density gradient, and titrated by real-time qPCR according to methods known in the art. The purified virus was used to infect cultured mouse primary cortex neurons at a dose of 3E5, 1E5, 3E4 and 1E4 viral genes (VG)/cell. After 7 days, total RNA was extracted and expression of MAPT and two control genes (ATP5b, EIF4a2) was monitored using real-time RT-qPCR.

It was set as shown in Fig. 1C that all AAV vectors encoding ZFP-TF effectively suppress mouse MAPT over a wide range of infective doses, with some ZFPs reducing target expression by more than 95% at multiple doses. In contrast, MAPT repression was not observed for rAAV2/9 virus encoding CMA-GFP tested at equivalent doses or for sham-treated neurons (see FIG. 1).

rAAV2/9 encoding CMV-52389-KRAB was used to infect mouse cortical neurons at 3E5, 1E5 and 3E4 VG/cell. After 10 days, the total protein was isolated from one set of repeats; a parallel group of infections was used for total RNA extraction. Equal amounts of total cell lysate were analyzed by Western blotting for total tau protein (anti-tau-5), 52389-KRAB (anti-ZNF10) and GAPDH (anti-GAPDH) (see Fig. 2A). Quantitative fluorescence was determined using the Odyssey LiCOR system, revealing a 98% reduction in tau protein at 3E5 VG/cell compared to sham-treated neurons (see FIG. 2). Expression of MAPT, 52389-KRAB and two control genes (ATP5b, EIF4a2) was monitored using real-time RT-qPCR. MAPT transcript levels were reduced by 98% in the 3E5 VG/cell group compared to sham-treated neurons (see FIG. 2).

Thus, the genetic modulators (e.g., repressors) described herein, including those that bind to target sites as shown in Tables 1 and 2, were functional repressors when designed as plasmids, in mRNA form, in Ad vectors and/or AAV vectors.

Example 3: Specificity of MAPT Repression

The overall specificity of ZFP-TF, shown in Tables 1 and 2, was assessed by microarray analysis in mouse Neuro2A cells. Briefly, 300 ng of mRNA encoding ZFP-TF was transfected into 150,000 Neuro2A cells in biological quadruple. After 24 hours, total RNA was extracted and processed according to the manufacturer's protocol (Affymetrix Genechip MTA1.0). A robust multi-chip average (RMA) was used to bring the raw signals from each set of probes. The analysis was performed using the Transcriptome Analysis Console 3.0 (Affymetrix) with the "Gene Level Differential Expression Analysis" option. Samples transfected with ZFP were compared with samples that were treated with irrelevant ZFP-TF (which does not bind to the target site in MAPT). Transitions to change were reported for transcripts (probesets) with >2-fold difference in mean signal relative to control and P value<0.05 (one-way analysis of variance (ANOVA), unpaired T-test for each probeset).

As shown in FIG. 3, SBS#52322 downregulated 5 genes in addition to MAPT and caused an increase in 3 other genes. SBS#52364 and #52366 suppressed 4 genes in addition to MAPT, while SBS#52389 induced repression of only the MAPT gene and increased expression of 1 gene. SBS#57880 reduced the expression of 2 genes in addition to MAPT and increased the expression of 1 gene. SBS#57890 downregulated 4 genes in addition to MAPT and increased expression of 1 gene. SBS#52288 downregulated 124 genes in addition to MAPT and increased the expression of 25 genes.

The specificity of ZFP-TF was also evaluated in primary human fibroblasts, which are more sensitive to transfection and allow specificity to be assessed even at higher effective levels of ZFP-TF. Four biological replicates of each treatment were used, consisting of 300 ng of mRNA encoding ZFP-TF transfected into 150,000 human fibroblasts. After 24 hours, total RNA was extracted and processed according to the manufacturer's protocol (Affymetrix Human Primeview). Processing and analysis were the same as described for Neuro2A cells. Importantly, human fibroblasts do not express MAPT, so no changes in MAPT levels were found. Based on the previously described fold change criterion, SBS#52322 repressed 58 genes and caused an increase in 53 other genes. SBS#52364 suppressed 43 genes and increased the expression of 25 genes. SBS#52366 suppressed 63 genes and caused an increase in the expression of 63 other genes. SBS#52389 did not repress any genes and increased the expression of 13 other genes. SBS#57880 decreased the expression of 7 genes and increased the expression of 2 genes. SBS#57890 reduced the expression of 1 gene and increased the expression of 2 genes, while SBS#52288 downregulated 594 genes, increasing the expression of 302 genes (see Fig. 4).

The specificity of ZFP-TF was further evaluated in primary mouse cortical neurons following AAV delivery of ZFP-TF. Six biological replicates of each treatment were used, consisting of rAAV2/9 encoding CMV-ZFP-TF infected at 1E5 VG/cell, in 160,000 primary neurons in the mouse cerebral cortex. After 7 days, total RNA was extracted and processed according to the manufacturer's protocol (Affymetrix Human Primeview). Processing and analysis were the same as described for Neuro2A cells and fibroblasts.

Based on the previously described fold change criterion, SBS#52322 downregulated 80 genes in addition to MAPT and caused an increase in the expression of 19 other genes. SBS#52364 downregulated 29 genes in addition to MAPT and increased the expression of 2 genes. SBS#52389 suppressed 1 gene in addition to MAPT. SBS#57880 reduced the expression of 60 genes in addition to MAPT and increased the expression of 3 genes. SBS#57890 only downregulated MAPT and increased expression of 1 gene (see Fig. 5).

Experiments were also performed to examine the effect on specificity of the modification(s) (described here in US Patent Application No. 15/685,580) of potential contacts of the zinc finger backbone with phosphate residues. Two typical ZFP tau suppressing proteins (57880 and 65888) were compared, which have identical helical domains (see Table 3), but 65888 has zinc finger backbone mutations to remove potential phosphate-contacting amino acid residues.

As shown in FIG. 5B, the number of genes that were either up- or down-expressed by 57800 (75 and 110 respectively) was markedly down-regulated (3 up- and 4 down-expressed, including tau) by the 65888 protein, demonstrating an increase in specificity through modifications in core area.

Example 4 Nerve Cell Promoters for CNS Restricted ZFP-TF Expression

Three neural cell promoters were evaluated for their ability to express ZFP-TF in primary mouse neurons: a 469 bp fragment. human Synapsin1 promoter (SYN1; Kügler et al., (2001) Methods Mol Med 53:139-50), 375 bp fragment. alpha-calcium/calmodulin-dependent protein kinase promoter (CamKII; Dittgen et al., (2004) Proc Natl Acad Sci USA 101(52):18206-11), and a 229 bp fragment. methyl CpG binding protein 2 promoter (MeCP2; Adachi et al., (2005) Hum Mol Genet 14 (23): 3709-22). Each fragment was cloned in place of the CMV promoter used in Example 2, and rAAV2/9 was generated for each promoter construct driving 52389-KRAB linked by the 2A peptide sequence to allow co-expression of both proteins (Nagai et al., (2002) Nat Biotechnol 20 (1):87-90 and MD Ryan et al., (1991) J. Gen. Virol. 72 p. 2727).

The purified virus was used to infect cultured primary neurons in the mouse cerebral cortex at a dose of 3E5, 1E5, 3E4 and 1E4 viral genomes/cell. After 7 days, total RNA was extracted and expression of MAPT and two control genes (ATP5b, EIF4a2) was monitored using real-time RT-qPCR.

All promoters resulted in ZFP expression, with both SYN1 and CamKII achieving transcription levels similar to CMV. In contrast, expression with MeCP2 was at levels that were about 10 times lower. MAPT was also downregulated over a wide range of infective doses, with SYN1 showing the strongest response, followed by CAMKII, then CMV, and finally MeCP2 with the lowest levels of MAPT repression. At the maximum dose of 3E5 viral genomes/cell, all promoters were able to downregulate MAPT by >90%, with SYN1 showing the strongest MAPT reduction of 99.4% (~175-fold lower) compared to sham-treated neurons ( see Fig. 6A).

The purified virus was also used to infect cultured primary hippocampal neurons and samples were analyzed and processed as described for cortical neurons. All neuronal promoters resulted in ZFP expression and MAPT repression with the same rank order of activity, similar degree of repression and dose-response profiles observed in primary cortical neurons (see Fig. 6B).

Taken together, these results suggest that any of the neuronal promoters would be sufficient to achieve ZFP expression and MAPT repression in the main regions of interest for a MAPT-related indication, namely the hippocampus and cerebral cortex.

In addition, the use of a strong neuronal promoter (such as SYN1 or CAMKII) can be used to reduce the level of a highly expressed target transcript when the corresponding delivery of ZFP-TF is a limitation (eg, via intravenous or intrathecal delivery) and/or when ZFP -TF with low affinity can be chosen to minimize the regulation of the gene outside the target. Conversely, a weaker nerve cell promoter (such as MeCP2) may be selected when the target gene of interest is expressed at moderate or low levels, when sufficient delivery to target areas is not limited (eg, direct intracranial delivery), and/or for minimizing activity outside the high affinity ZFP-TF target.

Example 5: Human MAPT repression in iPS-derived neurons

Based on a standard SELEX specificity assay for all ZFPs described here, it was predicted that the ZFPs listed in Tables 2 and 3 would be tolerant of one (in the case of SBS#57890 and SBS#52366) or two (in the case of SBS) mismatches. #57880) non-conservative positions in the human MAPT sequence (see FIG. 7A, SEQ ID NOS: 31, 32, and 48-51, respectively, from left to right).

Representative ZFP-TFs in Tables 2 and 3 were cloned into an AAV vector (AAV6, AAV9 AAV2/9 or variants thereof) with a 469 bp fragment. SYN1 promoter described in Example 4. rAAV virus was produced in HEK293T cells, purified using a CsCl density gradient, and titrated by qPCR. The purified virus was used to infect human iPS-derived neurons at a dose of 3E5, 1E5, 3E4 and 1E4 viral genomes/cell (iCell Neurons, Cellular Dynamics International Inc.). After 18 or 19 days, total RNA was extracted and the expression of human MAPT, ZFP-KRAB, and three control genes (ATP5b, EIF4a2, GAPDH) was assessed using real-time RT-qPCR. ZFP expressions at similar levels were established for each virus over the dose range tested.

Illustrative results are shown in Fig. 7. At the maximum dose of 3E5 viral genomes/cell, 57880-KRAB suppressed human MAPT by 95%, 52366-KRAB reduced MAPT expression by 87%, and 57890-KRAB reduced MAPT expression by 45% compared to false infected cells (see Fig. 7B). Similar results were obtained for other transcription factors described here (eg, ZFP-TF, as shown in tables 1-3). One explanation for the reduced human MAPT inhibitory activity of SBS#57890 compared to the behavior observed for mouse MAPT is its exceptional degree of genome-wide specificity, consistent with poor tolerance for mismatches at its target site (see example 3).

CMV, SYN1, and CAMKII promoters were evaluated for human ZFP-TF expression and human MAPT repression using ZFP-TF 52366. For each promoter construct driving 52366-KRAB, rAAV2/9 was generated and used to infect human iPS-derived neurons as described. The SYN1 promoter resulted in the highest levels of ZFP expression and a decrease in MAPT (5% of sham-treated neurons) compared to CAMKII (40% of sham control) and CMV (71% of sham control) (see Fig. 7C).

Human iPS-derived neurons were also subjected to microarray analysis as described above to analyze the degree of off-target repression in a population of 19959 coding transcripts. In each case, ZFP-TF was compared to 65976 protein based on the previously described fold test, and the graphs show the profile change for the ZFP-TF under investigation (see FIG. 7D). The results show that some of the ZFP-TFs as human tau repressors are highly specific in human neurons.

Example 6: In vivo MAPT repression driven by AAV-delivered ZFP-TF

Various ZFP-TFs described here were delivered using various AAV vectors to the mouse hippocampus to assess MAPT repression in vivo. AAVs encoding tau repressors were administered either intravenously (IV) or intracerebroventricularly (ICV) with 57890.T2A.Venus or intracranially (IC) via stereotaxic injection.

Briefly, in the case of AAV 2/9, a total dose of 8E9 rAAV2/9-CMV-ZFP-TF vector genomes per hemisphere was administered by stereotaxic injection using double bilateral 2 μl injections. In the case of the AAV vectors described in US Pat. No. 9,585,971, AAV was administered intracerebroventricularly (ICV) or intravenously (IV) at total doses of 1E12. The AAV vectors described in US Provisional Application No. 62/503121 were administered with ICV at a dose of 1E12 vector genomes/animal, or at a dose of 2E12 vector genomes/animal in the case of IV.

Animals were sacrificed four (IV and ICV) or five weeks (IC) after injection. In IV and ICV treated animals, the right hemisphere was dissected into different regions of the brain for RT-qPCR analysis and the left hemisphere was used for histology. For IC-treated animals, each hippocampus was divided into three sections for RT-qPCR analysis (A, anterior; B, middle; C, posterior hippocampus). Expression of MAPT and ZFP-TF was also analyzed by real-time RT-qPCR and resulted in the geometric mean of the three housekeeping genes (ATP5b, EIF4a2 and GAPDH).

The data showed that AAV vectors efficiently transduced target nerve cells when administered intracerebroventricularly or intravenously, and that ZFP-TF resulted in a strong and sustained decrease in tau throughout the CNS (brain and spinal cord, including the frontal cortex, anterior cortex, posterior cortex). cortex, hippocampus, brainstem, striatum, thalamus, midbrain, cerebellum, lumbar spinal cord, thoracic spinal cord, and cervical spinal cord). In FIG. 8A-8C show exemplary results using ZFP-TF SBS#52322, SBS#52389, and SBS#57890 after administration of the AAV vectors described in 62/503121 ("SGMO" in Fig. 8B) compared to AAV9 ("9" in Fig. 8B). The micrograph shown in Fig. 8A was done 4 weeks after delivery of the SGMO vector, using doses of 1e11 vector genomes/mouse for ICV delivery and 2e12 vector genomes/mouse for intravenous delivery. In FIG. 8C shows changes in mRNA levels for tau after a single ICV or IV dosing from the same experiment. The same results were found for all additional ZFP-TFs tested.

Fig. 8D and 8E present exemplary results using the AAV2/9 vector and demonstrated that, when delivered intracranially to the hippocampus, both 52389-KRAB and 52322-KRAB were able to effectively suppress MAPT (see FIG. 8D). The area of action was assessed by the relative levels of ZFP expression on three sections for each hemisphere. This analysis showed that while many sections had excellent scoping, some had little or no ZFP-TF expression, suggesting heterogeneous scoping throughout the hippocampus in several animals after intracranial stereotaxic delivery (see Fig. 8E). Similar results were obtained with other ZFP-TFs as described here.

In addition, the tau repressors described here, delivered using the AAV vector described in US Pat. No. 9,585,971, also significantly suppressed tau in the CNS (including the spinal cord). Illustrative results are shown in Fig. 8F-8H. For all ZFP-TFs described here, repression in the CNS was rapid and maintained over time at both the protein and mRNA levels. In addition, tau levels in CSF correlate with levels in the brain and thus can be used as an indicator of tau repressor function (see exemplary Fig. 8H).

In general, any tau repressor described herein, delivered by any AAV vector by any route, effectively modulates (suppresses) tau over long periods.

Three methods of analysis were used to further understand the extent of ZFP-TF-induced MAPT repression in the hippocampus. First, ZFP-TF and MAPT expression values from all six sections from each animal in the treatment group were averaged and compared to the PBS group by analysis of variance (ANOVA) followed by Sidak's post hoc test. Very significant (P<0.0001) MAPT repression was observed for both 52322 (66% mean MAPT decrease) and 52389 (55% mean MAPT decrease) (see Fig. 9A).

Second, a slice from each animal with the highest level of ZFP-TF expression was identified and used to calculate the mean maximum tau reduction under the ideal coverage scenario. Again, very significant (P<0.0001) MAPT repression was observed for all ZFP-TFs tested. The exemplary results shown in FIG. 9B show a decrease of more than 70% using 52322 (73% average MAPT decrease) and 52389 (89% average MAPT decrease).

Third, the correlation between MAPT and ZFP-TF levels (presented here as the absolute value of ZFP copies/ng RNA introduced into the RT-qPCR reaction) was assessed. A very significant relationship was observed for both 52322 (P<0.0001, R ² =0.53) and 52389 (P<0.0001, R ² =0.82) (see FIGS. 9C and 9D).

The reduction of tau in a mouse model of tauopathy transgenic for the (P301L) human tau mutant (rTg4510, Jackson Labs) is also assessed after administration of the genetic repressors described here. In addition, the clinical and therapeutic efficacy of repressors is being evaluated in this and other mouse models of AD (e.g., APPswe/PS1d9, Jackson Labs) to determine if there is a reduction in biomarkers and symptoms of taupathies, including one or more of the following: neurotoxicity, gliosis, dystrophic axons, loss of processes, excitotoxicity, thinning of the cerebral cortex and hippocampus, accumulation of tau in axons, cognitive (eg, radial arm maze and Morris water maze, fear formation, etc.) and/or motor impairment. See, for example, 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. In addition, models of chemically induced epileptic seizures, e.g., in wild-type mice treated with 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, also evaluated 4-8 weeks after the administration of the genetic repressors described here, to determine whether a decrease in tau has a protective effect against seizure, including death, associated with an attack, a prolonged delay in an attack and/or a decrease in the severity of an attack.

The tau modulators (e.g., repressors) described herein have been delivered using non-viral or viral (e.g., AAV) vectors described herein directly to the brain, for example, by intracranial injection into the hippocampus or cerebral cortex, intracerebroventricular or intravenous delivery. After a certain period of time after the administration of the repressors (1-11 months after the administration of the repressor), the brain was collected, dissected and subjected to immunohistochemical and/or image analysis (e.g., 2-photon imaging) to assess the viability of nerve cells, gliosis, dystrophic axons, density vertebrae, cortical and hippocampal thickness, mRNA expression for tau and tau protein levels.

At 8 weeks after administration of the ZFP repressors, mice treated with the repressors showed 80-90% or more tau (mRNA and protein) repression in ZFP-expressing neurons. After prolonged (6 months) tau knockdown, no significant neuronal loss or increased gliosis was observed. Tau repression in mice also protects against axonal dystrophies. Thus, the tau modulators (eg, repressors) described herein provide in vivo clinical and therapeutic benefits for subjects with taupathies.

Example 7 In vivo MAPT repression and protein reduction induced by AAV-delivered ZFP-TFs expressed from neuronal promoters.

Recombinant AAV vectors encoding the ZFP-TF 2A-Venus constructs described herein driven by the CMV, SYN1, CAMKII or MeCP2 promoters described in Example 5 were delivered to the mouse hippocampus for mRNA evaluation for MAPT and protein reduction in vivo. Briefly, a total dose of 2.4E10 vector genomes per hemisphere was administered by stereotaxic injection to wild-type adult mice using double bilateral injections of 1.5 μl. Animals were sacrificed six weeks after injection and each hippocampus was well homogenized with a razor blade and the resulting tissue was divided into two equal amounts. One half was taken for mRNA analysis, with MAPT, ZFP-TF, GFAP and IBA1 expression analyzed by real-time RT-qPCR and normalized to the geometric mean of the three housekeeping genes (ATP5b, EIF4a2 and GAPDH). The other half was taken for tau quantification by ELISA and representative results are shown in FIG. 10. Compared to control animals, mRNA for tau was reduced by at least 85% after administration of AAV encoding ZFP-TF. Indicative results after administration of rAAV9-CMV-52389V were as follows: 63% reduction in tau expression with rAAV9-SYN1-52389V; 74% reduction in tau expression with rAAV9-CAMKII-52389V; and an 83% reduction with the rAAV9-MeCP2-52389V construct (P<0.0001 for all promoters). The mean ZFP expression was found to be highest for the vector encoding the CAMKII promoter (set at 1), lowest for the MeCP2 promoter (15% of CAMKII levels), and intermediate for the CMV promoters (59% of CAMKII levels) and SYN1 ( 90% of CAMKII levels).

The presence of microglia and activated astrocytes was also assessed using RT-qPCR reagents for IBA1 and GFAP, respectively. The MeCP2 promoter did not result in significant changes in IBA1 levels compared to PBS-treated animals, SYN1 resulted in a 4.7-fold increase in IBA1 levels (P<0.0001), CMV resulted in a 3.0-fold increase in levels (P< 0.05) and CAMKII resulted in a 3.2-fold increase in levels (P<0.001). Also, the MeCP2 promoter did not increase GFAP compared to PBS-treated animals, SYN1 resulted in a 4.0-fold increase in GFAP levels (P<0.0001), CMV resulted in a 3.2-fold increase in levels (P< 0.01) and CAMKII resulted in a 2.4-fold increase in levels (p<0.05).

A sustained decrease in total tau was also observed for all three neural promoter constructs (illustrative results using SBS 52389 are shown in Figure 11). Compared to control levels after PBS injection (269 ng/ml), the rAAV9-SYN1-52389V construct gave 23% of control levels (61 ng/ml, P<0.0001), the rAAV9-CAMKII-52389V construct gave 18% of control levels (49 ng/mL, P<0.0001), the rAAV9-SYN1-52389V construct resulted in 12% of control levels (32 ng/mL, P<0.0001). Similar or high levels of reduction are achieved with other ZFP-TFs described here (e.g., 57890, 65894, 57930, 65918, 65920, 57880, 65887, 65888, 57947, 65968, 52322, 52364, 52366, 52288, and/or 523898 ).

Thus, nerve cell promoters are able to induce >80% reduction in mRNA for tau and tau protein throughout the mouse hippocampus.

Example 8: Histological confirmation of a decrease in tau throughout the hippocampus and associated brain regions

The artificial transcription factors described here were delivered to the mouse hippocampus for in vivo assessment of tau repression by immunofluorescence staining. A total dose of 2.4E10 of rAAV-CMV-ZFP-TF vector genomes (rAAV2/9, rAAV6, etc.) was administered by stereotaxic injection using double unilateral 1.5 μl injections into wild-type adult mice. ZFP-TFs were linked to Venus fluorescent protein to determine the site of TF expression. Animals were sacrificed six weeks after injection and whole brains were harvested and sectioned for histological staining of nuclei (DAPI), tau and GFP using standard methods. A fluorescence-activated cell sorting (FACS) assay was also performed. Briefly, to evaluate the efficiency of tau knockdown in ZFP-dVenus AAV-transduced cells, the present inventors dissociated hippocampus injected with ZFP-dVenus AAV CMV using a papain-assisted cell dissociation kit (Worthington Biochem. Corp.) according to with the manufacturer's manual, and then analyzed the cell suspension using flow cytometry (Miltenyi MACSQuant VYB flow cytometer). Cells expressing dVenus or GFP in the same samples were then separated by FACS using a 488 nm green laser (BioRadS3 cell sorter; 30-50 thousand GFP-positive cells in each sample). After sorting, the cells were pelleted by centrifugation for 10 minutes at 1000×g and resuspended in 100 µl of reagent (ThermoFisher) for RNA analysis.

Illustrative results are shown in Fig. 12 (animals were injected with rAAV2/9-CMV-ZFP-TF SBS#52389). In FIG. 12A is a cross-sectional view of the mouse brain through the anterior hippocampus and identifies four regions, shown in boxes and numbered 1-4. The injection was carried out in the left hemisphere (ipsilateral side), which leads to an increase in fluorescence in the left side of the cross section. The following panels are close-ups of each of these frames. Frames are described below:

Box 1 (FIG. 12B) shows a quantitative analysis of fluorescence intensity in the retrosplenial cortex, with ZFP-TF expression clearly demarcated between the ipsilateral and contralateral hemispheres. In the left panel of Fig. The 12B fluorescent signal was higher on the ipsilateral side of the midline ("ipsi") than on the contralateral side ("contra"). ZFP expression here probably originates from the axonal processes of the hippocampus. Most of the signal on the right side of the panel was due to staining of the DAPI nuclei. The middle image shows total fluorescence using labeled anti-tau antibodies, with the signal on the ipsilateral (left) side of the panel being almost entirely due to the GFP signal, while the signal on the opposite (right) side of the panel was mainly due to the presence of tau and basic staining. DAPI. In the right panel of Fig. 12B shows a cross-sectional frame that was analyzed to quantify the tau signal (depicted in FIG. 12C). The figure shows reduced tau-specific fluorescence from the injected (treated) side ("ipsi" versus "contra").

Frame 2, fig. 12D (ipsilateral) and box 3, FIG. 12E (contralateral) are images of the hippocampus showing a decrease in tau on the ipsilateral (treated) side compared to the contralateral side, tau knockdown was particularly pronounced in the dentate gyrus on the contralateral side compared to the ipsilateral side (marked with an arrow).

Box 4 is an area of the hypothalamus that had focal ZFP expression on the ipsilateral side (upper panel shows GFP fluorescence, left oval) and total tau fluorescence (lower panel) shows a decrease in total tau signal by ~50% (see graph showing comparison between the ipsilateral side and the contralateral side). Similar results are obtained using AAV based constructs including ZFP-TF as described in Tables 1, 2 and 3.

Thus, the ZFP-TFs described here suppress tau expression in vivo.

Example 9 In vivo safety, tau reduction and stable expression of ZFP-TF in the mouse hippocampus.

To evaluate the safety of both short-term and long-term expression in the hippocampus of the tau-targeting artificial transcription factors described herein, the ZFP-TFs described herein were delivered in vivo to the mouse hippocampus to monitor ZFP expression as well as astrocyte and microglia response and gross hippocampal anatomy. In one experiment, a total dose of 2.4E10 vector genomes rAAV2/9-CMV-52389-2A-Venus ("389dV"), rAAV2/9-CMV-ZFP-TF ("389"), rAAV2/9-CMV GFP ("GFP") or PBS ("PBS") were administered by stereotaxic injection using double unilateral injections of 1.5 μl to wild-type adult mice. Two groups were sacrificed either six weeks or six months after injection and the entire brain was harvested and sectioned for histological nuclear staining (DAPI), GFP, GFAP (to detect activated astrocytes) and IBA1 (to detect microglia) using standard methods, and the results are shown in FIG. 13. In FIG. 13H are images showing the fluorescence of two regions of the hippocampus, demonstrating that after injections, tau was suppressed in both the anterior and posterior regions of the hippocampus.

At the time point of 6 weeks (FIG. 13A), representing the signal for transduced cells in the hippocampus, the signal for ZFP-TF is almost the same as for the vector encoding GFP. In FIG. 13B shows the results for astrocytes and also shows almost the same results for the three treatments also observed in hippocampal microglia (FIG. 13C).

For a time point of 6 weeks, the data in FIG. 13D show very similar fluorescence levels for the vectors encoding GFP and ZFP-TF as seen at the 6 week time point, suggesting that there was not much change in the number of transduced cells in the hippocampus over time. Fig. 13E also shows that the signal between different treatments was very similar and did not change significantly between the 6-week and 6-month time points (Figure 13B versus 13E - note the difference in y-axis between the two figures). Microglia at the 6 month time point after treatment with the AAV vectors are essentially the same, with a slight decrease in PBS-treated samples.

Measurement of the mean thickness of CA1 (one of the regions of the hippocampus) showed no significant change after 6 months of ZFP expression (Fig. 13G), demonstrating no apparent neurotoxicity from long-term ZFP expression.

An additional analysis was performed in which ZFP-TF was administered as above except that only PBS and constructs 52288, 52364 and 52389 associated with 2a-Venus were compared. Dosing was the same as described in Example 6. After dosing, animals were sacrificed 4 weeks later and the data are shown in FIG. 13I. The data presented show that treatment with construct 52288 resulted in significantly attenuated vector genomes and attenuated ZFP expression compared to other constructs and did not suppress tau expression in any significant manner. However, this led to an increase in the level of GFAP and IBA1. ZFP 52364 produced higher levels of vector genome and ZFP expression than construct 52288, but resulted in equally high levels of GFAP and IBA1, and did not significantly downregulate tau expression. Conversely, construct 52389 was able to cause a significant reduction in tau expression without affecting GFAP or IBA1 expression.

The lack of in vivo tau reduction and elevation of neuroinflammatory biomarkers for ZFP 52288 and 52364 correlate with ZFP specificity profiles assessed in Neuro2A cells, human fibroblasts, and primary neurons (FIGS. 3, 4, 5), showing that highly specific ZFP significantly reduced tau expression and were valid in vivo.

The study of a portion of the dosing groups receiving PBS or 52389-2a-Venus was extended by 11 months. In addition to analyzing the degree of tau repression, the samples were also analyzed for mRNA expression levels against the amount of ZFP expressed and any changes in the amount of transcripts encoding GFAP, IBA1, NeuN, MAP1A, MAP1B and MAP2. Briefly, each hippocampus was well homogenized with a razor blade and the resulting tissue was divided into two equal amounts. One half was taken for mRNA analysis, and expression of MAPT, ZFP-TF, GFAP and IBA1 was analyzed by real-time RT-qPCR and resulted in the geometric mean of the three housekeeping genes (ATP5b, EIF4a2 and GAPDH). Illustrative results are shown in Fig. 13J, which also includes data acquired at a 6-week time point for comparison. The data demonstrates that overall there was not much change in most of the analyzed datasets between the 6 week and 11 month time frames. However, a decrease in the amount of detectable ZFP expression was found. No significant compensatory changes were observed for the other three microtubule-associated proteins (MAP1A, MAP1B, and MAP2), and no decrease in NeuN levels was observed, consistent with the preservation of hippocampal volume in the ZFP-treated hemispheres assessed six months after delivery. ZFP. Additional experiments were performed using ZFP-TF as described here and gave similar results.

Thus, the results demonstrate that after a single administration, ZFP expression is maintained over time in vivo and does not lead to CNS toxicity in the subject, and that the ZFP-mediated reduction of tau in the hippocampus of adult mice is robust and well tolerated for at least 11 months.

Using wild-type C57BL/6 mice (8-week-old females), another study was conducted in which ZFP-TF was delivered using the AAVs described here via retroorbital delivery. 2.5x12 viral genomes (vg) per animal were used and the animals were sacrificed ten weeks later. Microarray analysis was performed on hippocampal tissue from treated mice, the data presented being compared to irrelevant ZFP-TF delivered in the same manner. In FIG. 17 shows that tau was specifically inhibited by 57890V and 52389V proteins compared to treatment with GFP-encoding AAV. These data demonstrate that the specificity of these proteins observed in vitro is also detected in vivo.

Example 10 Tau Reduction in Mouse AD Model.

Studies were performed in a mouse model of AD in addition to those performed above in wild-type mice. APP/PS1 mice (reviewed in Li et al., ibid.) were treated with ZFP-TF as described here to analyze efficacy in this model.

Briefly, in one experiment, 4 APP/PS1 mice and 2 wild-type controls were treated at 4.5 months of age. Single injections were performed by injecting 3 μl of the AAV composition into opposite cortical layers (CTX) of the brain. The left CTX of each mouse received 3 µl of AAV9 containing GFP-bound ZFP-TF driven by the Synapsin1 promoter (or an irrelevant ZFP-TF control ("172"; AAV9 syn1-172v)) or 52389 ZFP-TF (" 389"; AAV9 syn1-389v). The right CTX of each mouse received 3 μl of AAV9 containing an RFP expression construct driven by the Synapsin1 promoter ("AAV9 syn1-tRFP").

Eleven (11) weeks post-treatment, 7-month-old mice were perfused with 4% PFA/PBS. Then the entire brain was removed and then fixed for 2 days using 4% PFA/PBS at 4°C. The tissue was then cryoprotected in 30% sucrose/PBS. 50 μm thick coronal sections were analyzed for immunofluorescent labeling for tRFP, GFP, and beta-amyloid ("Aβ"). 3 brain slices per mouse were analyzed and 3-5 images of each slice were obtained. Dystrophic axons were counted in Aβ-rich plaques such that 121 to 256 plaques were analyzed per animal. 708 plaques were counted for CTX exposed to AAV9 syn1-389v and 287 plaques were counted for CTX exposed to AAV9 syn1-tRFP. Illustrative data is shown in Fig. 14A-D and they show a statistically significant difference in the number of dystrophic axons per number of Aβ plaques in ZFP-TF52389 treated cortices compared to tRFP treated cortices (FIGS. 15 and 16). In contrast, there was no difference in plaque dystrophies in animals treated with irrelevant ZFP-TF. In addition, the number and volume of plaques did not change in both treated and untreated animals. Further experiments performed using other AAV vectors, routes of administration and/or ZFP-TF described here gave similar results.

Thus, the tau genetic repressors described here provide clinical and therapeutic benefits for taupathies such as AD, as shown in studies in known mouse models of AD.

Example 11 Tau Reduction in Vivo in the Primate Brain.

Primate tau-specific ZFP-TFs were tested in cynomolgus monkeys (M. ascicularis) to observe repression of tau expression in primates (non-human primate (NHP) models). The cynomolgus monkeys are housed in stainless steel cages equipped with a stainless steel mesh floor and an automatic drinker. The study complies with all applicable sections of the provisions of the Final Rules of the Animal Welfare Act (CFR Title 9).

Initial experiments are being conducted to determine whether the monkey hippocampus can be targeted accordingly. Control product (prescription buffer, PBS and 0.001% Pluronic F 68, pH 7.1) and test product are thawed and dispensed on day 1 of studies, with test and control products administered by MRI-guided delivery of AAV9-GFP or AAV9-ZFP -TF in the hippocampus.

In the first experiment, AAV9 containing the hSYN1-driven GFP gene was delivered at a dose of 3.84e11 vector genomes/hemisphere to the left hemisphere and 7.68e11 vector genomes/hemisphere to the right hemisphere. One animal receives a single dose of study product in a volume = 40 µl to the left hemisphere and a single dose = 80 µl to the right hemisphere. The second animal receives two doses of 20 μl in the left hemisphere and two doses of 40 μl in the right hemisphere. For both test products, the dose concentration is 9.6x12 vector genomes/ml. After 14 days, the animals are sacrificed and the brain is bisected and divided into approximately 17 sections (3 mm each). Sections including regions of the hippocampus/entorhinal cortex are used for GFP intrinsic fluorescence analysis and GFP immunohistochemistry. Other sections are used to collect tissue stamps for mRNA analysis by RT-qPCR, where tau and housekeeping gene expression levels are analyzed. In addition, some stamps are collected and stored for preliminary tau protein analysis. The results show that the hSYN1 promoter is able to drive GFP expression, which is found in the hippocampal region after delivery.

A second experiment was performed to evaluate the effect of the route of administration on GFP expression and location in the brain. In this study, the GFP transgene is carried by the SB3 AAV particle (see US Provisional Application No. 62/503121) and the dose concentration of the test product is 2.5x13 vector genomes/ml. The dose is administered by the intracerebroventricular, intrathecal, or intravenous route, with the dose volume (ml/animal) being approximately 1, 1.5, and 10-16, respectively. 14 days after dosing, the animals are sacrificed and the brain is divided in half, as described above. In addition to the analysis of tau and housekeeping gene expression levels by RT-qPCR, genes associated with inflammation (GFAP and Iba1) are also analyzed. Trial studies are also being conducted to analyze tau protein levels in brain tissue samples and in cerebrospinal fluid. These studies show that all dosing routes are well tolerated and able to deliver the GFP transgene to the hippocampus.

A study is then carried out to analyze the suppression of tau expression by the artificial transcription factors described herein in the brain of cynomolgus monkeys. Three or more of the ZFP-TFs described here (Tables 1-3) are being investigated, with three ZFPs being characterized as proteins that have high specificity (0 off-target) and high (≥90% repression) in vitro potency (ZFP-TF1 ); moderate specificity (5-10 out of target) and high in vitro efficiency (ZFP-TF2); and high specificity and moderate (50-60%) in vitro efficacy (ZFP-TF3). Animals are sacrificed at 1, 3 and 6 months and analyzed as described above. Significant repression of tau throughout the brain and CSF is observed without significant loss of neurons.

Studies show that tau-specific ZFP-TF reagents suppress tau expression (including at a therapeutic level) in the primate brain.

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

Although the invention has been provided in some detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications may be practiced without departing from the spirit or scope of the present invention. Accordingly, the foregoing descriptions and examples should not be construed as limiting.

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SEQUENCE LIST

<110> SANGAMO THERAPEUTICS, INC.

<120> TAU MODULATORS AND METHODS AND COMPOSITIONS FOR THEIR DELIVERY

<130> 8325-0140.40

<140> PCT/US2017/064181

<141> 2017-12-01

<150> 62/584.342

<151> 2017-11-10

<150> 62/500.807

<151> 2017-05-03

<150> 62/466.198

<151> 2017-03-02

<150> 62/450.895

<151> 2017-01-26

<150> 62/428.871

<151> 2016-12-01

<160> 51

<170> Patent in version 3.5

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<213> Mouse sp.

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gtggcggaga ctgagagcgc gcgcggcc 28

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<211> 28

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

human or mouse oligonucleotide

<400> 4

cttctgtcga ttatcaggta agcgccgc 28

<210> 5

<211> 28

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

human or mouse oligonucleotide

<400> 5

ctggtgggtg gcggagactg agagcgcg 28

<210> 6

<211> 28

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

human or mouse oligonucleotide

<400> 6

tggtgctgga gctggtggggt ggcggaga 28

<210> 7

<211> 7

<212> Protein

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Arg Ser Asp Asn Leu Ala Arg

15

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Asp Arg Ser His Leu Ala Arg

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Gln Ser Gly Asn Leu Ala Arg

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<212> Protein

<213> Artificial sequence

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Gln Ser Asn Thr Arg Ile Met

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<212> Protein

<213> Artificial sequence

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<212> Protein

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Thr Ser Ala Asn Leu Ser Arg

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<212> Protein

<213> Artificial sequence

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Thr Ser Gly Asn Leu Thr Arg

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<210> 14

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<212> Protein

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His Arg Thr Ser Leu Thr Asp

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<210> 15

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<212> Protein

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15

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<212> Protein

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His Pro Ser Ala Arg Lys Arg

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<212> Protein

<213> Artificial sequence

<220>

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Arg Ser Ala Asn Leu Thr Arg

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<210> 18

<211> 7

<212> Protein

<213> Artificial sequence

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Asp Ser Ser His Leu Glu Leu

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Asp Arg Ser Asn Leu Thr Arg

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Asp Arg Ser His Leu Thr Arg

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Thr Ser Ser Asn Arg Lys Thr

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<211> 7

<212> Protein

<213> Artificial sequence

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Asp Arg Ser Ala Leu Ala Arg

15

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<212> Protein

<213> Artificial sequence

<220>

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15

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<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

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Arg Ser Ala His Leu Ser Arg

15

<210> 26

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

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Thr Ser Gly His Leu Ser Arg

15

<210> 27

<211> 7

<212> Protein

<213> Artificial sequence

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<212> Protein

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<212> Protein

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Lys His Ser Thr Arg Arg Val

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<210> 31

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<213> Homo sapiens

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<211> 19

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<213> Mus sp.

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<210> 33

<211> 28

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

human or mouse oligonucleotide

<400> 33

aggggcgggc agcgaggcct gggcgggc 28

<210> 34

<211> 7

<212> Protein

<213> Artificial sequence

<220>

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Arg Ser Ala Asp Leu Thr Arg

15

<210> 35

<211> 7

<212> Protein

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<220>

<223> Artificial sequence description: synthetic peptide

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Gln Ser Gly Asp Leu Thr Arg

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<210> 36

<211> 7

<212> Protein

<213> Artificial sequence

<220>

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Arg Ser Asp His Leu Ser Glu

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<210> 37

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

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<210> 38

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

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Asp Arg Ser His Leu Ser Arg

15

<210> 39

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 39

Asp Arg Ser Asn Leu Ser Arg

15

<210> 40

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 40

Leu Arg Gln Asn Leu Ile Met

15

<210> 41

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 41

Thr Ser Ala Asn Leu Thr Val

15

<210> 42

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 42

Arg Ser Asp His Leu Ser Arg

15

<210> 43

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 43

Gln Arg Asn Asp Arg Lys Ser

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<210> 44

<211> 28

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<212> DNA

<213> Mouse sp.

<400> 45

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<211> 28

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

human or mouse oligonucleotide

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ctccagaagg ggatcatgac ctcctcac 28

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<212> Protein

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<---

Claims (42)

1. A microtubule-associated tau (MAPT) gene transcription repressor, wherein said repressor comprises A zinc finger protein (ZFP) DNA binding domain that binds to a target site of at least 12 nucleotides in the MAPT gene, wherein said ZFP DNA binding domain contains four, five, or six zinc finger domains where each zinc finger domain contains a recognition helix region, and where the ZFP-based DNA-binding domain contains recognition helix regions, in the following order: (i) RSDNLAR (SEQ ID NO:7), DRSHLAR (SEQ ID NO:8), QSGNLAR (SEQ ID NO:9), QSNTRIM (SEQ ID NO:10); (ii) ERGTLAR (SEQ ID NO:11), TSANLSR (SEQ ID NO:12), TSGNLTR (SEQ ID NO:13), HRTSLTD (SEQ ID NO:14), RSHSLLR (SEQ ID NO:15), HPSARKR ( SEQ ID NO:16); (iii) RSANLTR (SEQ ID NO:17), DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:20), DRSHLAR (SEQ ID NO:8); (iv) DRSHLTR (SEQ ID NO:20), LKQHLTR (SEQ ID NO:37), RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:26), QSGNLAR (SEQ ID NO:9), QSGDLTR ( SEQ ID NO:35); (v) RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:26), QSGNLAR (SEQ ID NO:9), QSGDLTR (SEQ ID NO:35), DRSHLSR (SEQ ID NO:38), DRSHLAR ( SEQ ID NO: 8); (vi) RSDNLSE (SEQ ID NO:21), TSSNRKT (SEQ ID NO:22), TSGNLTR (SEQ ID NO:13), DRSALAR (SEQ ID NO:23), RNSDRTK (SEQ ID NO:24); (vii) DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:20), DRSHLAR (SEQ ID NO:8), RSAHLSR (SEQ ID NO:25), TSGHLSR ( SEQ ID NO:26); (viii) LRHHLTR (SEQ ID NO:27), RRFTLSK (SEQ ID NO:28), RSDVLSE (SEQ ID NO:29), KHSTRRV (SEQ ID NO:30), RSDVLSE (SEQ ID NO:29), RLYTLHK ( SEQ ID NO:47); or (ix) RSADLTR (SEQ ID NO:34), QSGDLTR (SEQ ID NO:35), RSDHLSE (SEQ ID NO:36), RSAHLSR (SEQ ID NO:25); And transcription-regulating domain or nuclease domain, where the specified transcriptional repressor suppresses the expression of the MAPT gene. 2. The transcription repressor of claim 1, wherein said ZFP-based DNA-binding domain comprises RSADLTR (SEQ ID NO:34), QSGDLTR (SEQ ID NO:35), RSDHLSE (SEQ ID NO:36), and RSAHLSR (SEQ ID NO:36). NO:25) for F1-F4 respectively. 3. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises RSANLTR (SEQ ID NO:17), DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:19). NO:20) and DRSHLAR (SEQ ID NO:8) for F1-F5, respectively. 4. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises RSDNLSE (SEQ ID NO:21), TSSNRKT (SEQ ID NO:22), TSGNLTR (SEQ ID NO:13), DRSALAR (SEQ ID NO:13). NO:23) and RNSDRTK (SEQ ID NO:24) for F1-F5, respectively. 5. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises LRHHLTR (SEQ ID NO:27), RRFTLSK (SEQ ID NO:28), RSDVLSE (SEQ ID NO:29), KHSTRRV (SEQ ID NO:29). NO:30), RSDVLSE (SEQ ID NO:29) and RLYTLHK (SEQ ID NO:47) for F1-F6, respectively. 6. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:20), DRSHLAR (SEQ ID NO:20). NO:8), RSAHLSR (SEQ ID NO:25) and TSGHLSR (SEQ ID NO:26) for F1-F6, respectively. 7. The transcription repressor of claim 1, wherein said ZFP-based DNA-binding domain comprises DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:20), DRSHLAR (SEQ ID NO:20). NO:8), RSAHLSR (SEQ ID NO:25) and TSGHLSR (SEQ ID NO:26) for F1-F6, respectively, and a Qm5 mutation in the F1, F3 and F5 backbone region. 8. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises RSDNLAR (SEQ ID NO:7), DRSHLAR (SEQ ID NO:8), QSGNLAR (SEQ ID NO:9) and QSNTRIM (SEQ ID NO:10) for F1-F4 respectively. 9. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises ERGTLAR (SEQ ID NO:11), TSANLSR (SEQ ID NO:12), TSGNLTR (SEQ ID NO:13), HRTSLTD (SEQ ID NO:13). NO:14), RSHSLLR (SEQ ID NO:15) and HPSARKR (SEQ ID NO:16) for F1-F6, respectively. 10. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises LRHHLTR (SEQ ID NO:27), RRFTLSK (SEQ ID NO:28), RSDVLSE (SEQ ID NO:29), KHSTRRV (SEQ ID NO:29). NO:30), RSDVLSE (SEQ ID NO:29) and RLYTLHK (SEQ ID NO:47) for F1-F6, respectively, and a Qm5 mutation in the F1 and F5 backbone. 11. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises DRSHLTR (SEQ ID NO:20), LKQHLTR (SEQ ID NO:37), RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:25). NO:26), QSGNLAR (SEQ ID NO:9) and QSGDLTR (SEQ ID NO:35) for F1-F6, respectively. 12. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises DRSHLTR (SEQ ID NO:20), LKQHLTR (SEQ ID NO:37), RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:25). NO:26), QSGNLAR (SEQ ID NO:9) and QSGDLTR (SEQ ID NO:35) for F1-F6, respectively, and a Qm5 mutation in the F1 and F5 backbone region. 13. The transcription repressor of claim 1, wherein said ZFP-based DNA-binding domain comprises DRSHLTR (SEQ ID NO:20), LKQHLTR (SEQ ID NO:37), RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:25). NO:26), QSGNLAR (SEQ ID NO:9) and QSGDLTR (SEQ ID NO:35) for F1-F6, respectively, and a Qm5 mutation in the F1, F3 and F5 backbone region. 14. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises DSSHLEL (SEQ ID NO:18), DRSNLTR (SEQ ID NO:19), DRSHLTR (SEQ ID NO:20), DRSHLAR (SEQ ID NO:20). NO:8), RSAHLSR (SEQ ID NO:25) and TSGHLSR (SEQ ID NO:26) for F1-F6, respectively, and a Qm5 mutation in the F3 and F5 backbone region. 15. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:26), QSGNLAR (SEQ ID NO:9), QSGDLTR (SEQ ID NO:9). NO:35), DRSHLSR (SEQ ID NO:38) and DRSHLAR (SEQ ID NO:8) for F1-F6, respectively. 16. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises RSAHLSR (SEQ ID NO:25), TSGHLSR (SEQ ID NO:26), QSGNLAR (SEQ ID NO:9), QSGDLTR (SEQ ID NO:9). NO:35), DRSHLSR (SEQ ID NO:38) and DRSHLAR (SEQ ID NO:8) for F1-F6, respectively, and a Qm5 mutation in the F1, F3 and F5 backbone region. 17. The transcription repressor of claim 1, wherein said ZFP-based DNA binding domain comprises ERGTLAR (SEQ ID NO:11), TSANLSR (SEQ ID NO:12), TSGNLTR (SEQ ID NO:13), HRTSLTD (SEQ ID NO:13). NO:14), RSHSLLR (SEQ ID NO:15) and HPSARKR (SEQ ID NO:16) for F1-F6, respectively, and a Qm5 mutation in the F5 backbone region. 18. The transcription repressor according to any one of paragraphs. 1-17, wherein said transcriptional regulatory domain comprises a KRAB domain. 19. The transcription repressor according to any one of paragraphs. 1-18, wherein said ZFP-based DNA binding domain binds to a target site in the human MAPT gene. 20. A polynucleotide encoding a transcriptional repressor according to any one of paragraphs. 1-19. 21. A gene delivery vehicle comprising an adeno-associated viral (AAV) vector, wherein said AAV vector contains a polynucleotide according to claim 20 between the nucleotide sequences of inverted terminal repeats of said AAV vector, where said gene delivery means allows expression of said polynucleotide in a cell transduced with said vector . 22. The gene delivery vehicle of claim 21, wherein said AAV vector is an AAV6, AAV9, or AAV2/9 vector. 23. Pharmaceutical composition for suppressing the expression of MAPT, containing one or more different transcription repressors according to any one of paragraphs. 1-19, one or more different polynucleotides according to claim 20 and/or one or more different gene delivery vehicles according to claim 21 or 22. 24. The pharmaceutical composition of claim 23, wherein said transcriptional repressor comprises a nuclease domain that cleaves the MAPT gene. 25. A pharmaceutical composition according to claim 24, further comprising a donor nucleic acid molecule for integration into the cleaved MAPT gene. 26. The use of one or more different transcription repressors according to any one of paragraphs. 1-19, one or more different polynucleotides according to claim 20, one or more different gene delivery vehicles according to claim 21 or 22 and/or a pharmaceutical composition according to any one of claims. 23-25 to suppress MAPT expression in a subject. 27. Use according to claim 26, wherein the administration to the subject is intracerebroventricular, intrathecal, intracranial, intravenous, retroorbital, or intracisternal. 28. Use according to claim 26 or 27, wherein suppression of MAPT expression in a subject treats and/or prevents tauopathy. 29. Application according to any one of paragraphs. 26-28, where the amount of tau in the subject is reduced. 30. Kit to suppress the expression of MAPT, including one or more different transcription repressors according to any one of paragraphs. 1-19, one or more different polynucleotides according to claim 20, one or more different gene delivery vehicles according to claim 21 or 22, a pharmaceutical composition according to any one of paragraphs. 23-25 and instructions for use.

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