HTT repressor and its application

Abstract

Disclosed herein are HTT repressors and methods and compositions using the same. Disclosed herein are methods and compositions for diagnosing, preventing and / or treating Huntington's disease. In particular, provided herein are methods and compositions for modifying (e.g., regulating expression) HD HTT alleles to prevent or treat Huntington's disease, comprising an mHTT repressor (which represses mHTT transcripts and thus also mHTT protein expression).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 792,701, filed on January 15, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 3, 2020, is named 8327-018740_SL.txt, and is 22,157 bytes in size. Technical Field

[0005] The present disclosure relates to the field of diagnosis and treatment of Huntington's disease. Background Art

[0006] Huntington's disease (HD), also known as Huntington's Chorea, is a progressive disorder of movement, cognition and psychiatric impairment. The average age of disease onset is 35-44 years, but in about 10% of cases, onset occurs before the age of 21, and the average life expectancy after disease diagnosis is 15-18 years. The prevalence is about 3-7 per 100,000 people of Western European descent.

[0007] Huntington's disease is an example of a trinucleotide repeat expansion disorder first characterized in the early nineties (see Di Prospero and Fischbeck (2005) Nature Reviews Genetics 6: 756-765). These disorders involve local amplification of unstable repeat sequences of three nucleotide groups and may result in loss of function, acquisition of toxic function, or both of genes in which the amplified repeat sequences are retained. Trinucleotide repeat sequences may be located in any part of a gene, including non-coding and coding gene regions. Repeat sequences located in coding regions generally involve repeated glutamine coding triplets (CAG) or alanine coding triplets (CGA). Amplified repeat sequence regions in non-coding sequences may result in abnormal expression of genes, while amplified repeat sequences in coding regions (also known as codon reiteration disorders) may result in misfolding and protein aggregation. The exact cause of the pathophysiology associated with abnormal proteins is often unknown. Typically, in wild-type genes undergoing trinucleotide amplification, these regions contain a variable number of repeat sequences in normal populations, but in diseased populations, the number of repeat sequences may increase from doubling to a logarithmic increase in the number of repeat sequences. In HD, the repeat sequence is inserted into the N-terminal coding region of the gene encoding the large cytoplasmic protein huntingtin (HTT). Normal HTT alleles contain 15-24 CAG repeats ("CAG" repeats disclosed in SEQ ID NO:23), while alleles containing 36 or more repeats are likely to be considered potential HD pathogenic alleles and increase the risk of developing the disease. Alleles containing 36-39 repeats are considered to be incompletely penetrant, and individuals with these alleles may or may not develop the disease (or may develop symptoms later in life), while alleles containing 40 repeats or more are considered to be fully penetrant. In fact, no person with an HD allele with such a large number of repeats has ever been reported as an asymptomatic person. Those individuals with juvenile onset HD (age <21 years) are often found to have 60 or more CAG repeats. In addition to the increase in CAG repeats, it has been demonstrated that HD can involve +1 and +2 frameshifts within the repeat sequence, so that the region will encode a polyserine polypeptide (in the case of a +1 frameshift, encoded by the AGC repeat sequence) trace instead of polyglutamine (Davies and Rubinsztein (2006) Journal of Medical Genetics 43:893-896). In HD, the mutant HTT (mHTT) allele is usually inherited from one parent as a dominant trait.Any child of an HD patient has a 50% chance of developing the disease if the other parent does not have the disease. In some cases, a parent may have an intermediate HD allele and may be asymptomatic, but due to the repeat expansion, the child displays the disease. Additionally, HD alleles may also display a prognostic phenomenon where increased severity or decreased age of onset is observed over generations due to the unstable nature of the repeat region during spermatogenesis.

[0008] In addition, trinucleotide expansion in HTT leads to neuronal loss in the striatal medium spiny γ-aminobutyric acid (GABA) projection neurons, and neuronal loss also occurs in the neocortex. Compared with neurons containing substance P and projecting to the internal globus pallidum, the correlation of medium spiny neurons containing enkephalin and projecting to the external globus pallidum is higher. Other brain areas that are greatly affected in Huntington's disease patients include: substantia nigra, cortex 3, 5 and 6, CA1 area of ​​the hippocampus, angular gyrus of the parietal lobe, Purkinje cells of the cerebellum, lateral tubular nucleus of the hypothalamus and centromedial parafascicular complex of the thalamus (Walker (2007) Lancet 369: 218-228).

[0009] The role of normal HTT protein is poorly understood, but may be involved in neurogenesis, programmed cell death, and vesicle trafficking. In addition, there is evidence that wild-type HTT stimulates the production of brain-derived neurotrophic factor (BDNF), a pro-survival factor for striatal neurons. The progression of HD has been shown to be associated with decreased BDNF expression in a mouse model of HD (Zuccato et al. (2005) Pharmacological Research 52(2):133-139), and delivery of BDNF or glial cell line-derived neurotrophic factor (GDNF) by recombinant adeno-associated virus (rAAV) vector-mediated gene delivery can protect striatal neurons in HD mouse models (Kells et al. (2004) Molecular Therapy 9(5):682-688).

[0010] Currently, the diagnostic and therapeutic options for HD are very limited. In terms of diagnosis, altered (mutated) HTT (mHTT) levels are significantly correlated with disease burden scores, and the concentration of soluble mHTT species increases with disease progression. However, it is difficult to quantify low-abundance mHTT in the patient's CNS, which limits the study of HD neuropathological effects in vivo and excludes the demonstration of target engagement of drugs that reduce HTT. See, for example, Wild et al. (2014) J Neurol Neurosurg Psychiatry 85:e4.

[0011] For treatment, some potential approaches designed to prevent protein aggregation-related toxicities have been shown to reduce these toxicities in in vitro models, where protein aggregation occurs through extended polyglutamine tracts, such as overexpression of chaperone proteins or heat shock responses induced by the compound geldanamycin. Other treatments target the role of apoptosis in the clinical manifestations of the disease. For example, in the offspring of a pair of mice in which one parent contains an HD allele and the other parent has a dominant negative allele of caspase 1, it has been demonstrated in animal models that blockade of caspase activity alleviates disease symptoms. In addition, cleavage of mHTT by caspases may play a role in the pathogenicity of the disease. It was found that transgenic mice carrying caspase-6 resistant mutant HTT maintained normal neuronal function and did not develop striatal neurodegeneration compared to mice carrying non-caspase resistant mutant HTT alleles (see Graham et al. (2006) Cell 125: 1179-1191). Molecules targeting members of the apoptosis pathway have also been shown to alleviate symptoms. For example, compounds zVAD-fmk and minocycline, which inhibit caspase activity, have been shown to slow disease manifestations in mice. The drug remacemide has also been used in small HD human trials because the compound is thought to prevent mutant HTT from binding to NDMA receptors, thereby preventing toxic effects on nerve cells. However, in these trials, no statistically significant improvement in neuronal function was observed. In addition, the Huntington Research Group conducted a randomized double-blind study using coenzyme Q. Although a trend toward slower disease progression was observed in patients treated with coenzyme Q10, there was no significant change in the rate of decline in total functional capacity. (Di Prospero and Fischbeck (2005) Nature Reviews Genetics 6:756-765).

[0012] Recombinant transcription factors and nucleases comprising a DNA binding domain from a zinc finger protein ("ZFP"), a TAL effector domain ("TALEs"), and a CRISPR / Cas transcription factor system (including Cas and / or Cfp1 systems) have the ability to regulate gene expression of endogenous genes. See, e.g., U.S. Patent Nos. 9,045,763; 9,005,973; 8,956,828; 8,945,868; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S. Patent Publication Nos. 2003 / 0232410; 2005 / 0208489; 2005 / 0026157; 2005 / 0064474; 2006 / 0063231; 2008 / 0159996; 2010 / 00218264; 2012 / 0017290; 2011 / 0265198; 2013 / 0137104; 2013 / 0122591; 2013 / 0177983, 2013 / 0177960, 2015 / 0335708; and 2015 / 0056705; Perez-Pinera et al. (2013) Nature Methods 10:973-976; Piatek et al. (2015) Plant Biotechnology J. 13(4):578-89, doi:10.1111 / pbi.12284), the disclosures of which are incorporated herein by reference in their entirety for all purposes. For example, U.S. Patent Nos. 9,234,016; 9,943,565; 8,841,260; 9,499,597; and U.S. Patent Publication Nos. 2018 / 0200332; 2017 / 0096460; 2017 / 0035839; 2016 / 0296605 and 2019 / 0322711 relate to DNA binding proteins that regulate the expression of HD alleles such as HTT. U.S. Patent Publication No. 2015 / 0335708 relates to methods for modifying medium spiny neurons.

[0013] In addition, targeted nucleases are based on the Argonaute system (e.g., from T. thermophilus, called 'TtAgo', see Swarts et al. (2014) Nature 507(7491):258-261), which may also have the potential for genome editing and gene therapy. Clinical trials using these engineered transcription factors containing zinc finger proteins have shown that these novel transcription factors can treat a variety of diseases. (See, e.g., Yu et al. (2006) FASEB J. 20:479-481). Nuclease-mediated cleavage involves the use of engineered nucleases to induce double-strand breaks (DSBs) or nicks in the target DNA sequence, so that repair of the break by an error-born process such as non-homologous end joining (NHEJ) or repair using a repair template (homologous directed repair or DHA) can lead to knockout of genes or insertion of sequences of interest (targeted integration). Introduction of double-strand breaks in the absence of an externally provided repair template (e.g., a "donor" or "transgene") is often used to inactivate the target gene via mutations (insertions and / or deletions referred to as "indels") introduced by the cellular NHEJ pathway.

[0014] However, there remains a need for methods for diagnosing, studying, treating and / or preventing Huntington's disease, including approaches that exhibit broad delivery to the brain. Summary of the invention

[0015] Disclosed herein are methods and compositions for diagnosing, preventing and / or treating Huntington's disease. In particular, provided herein are methods and compositions for modifying (e.g., regulating expression) HD HTT alleles to prevent or treat Huntington's disease, comprising an mHTT repressor (which represses mHTT transcripts and thus also represses mHTT protein expression). The compositions described herein (mHTT repressors) provide therapeutic benefits in subjects, for example, by reducing cell death, reducing apoptosis, increasing cell function (metabolism) and / or reducing motor defects in subjects. Therefore, described herein are non-naturally occurring zinc finger proteins (ZFPs) that bind to the CAG repeat sequence domain of the mHTT gene, the zinc finger proteins comprising 4, 5 or 6 zinc finger domains, in the order of F1 to F4, F1 to F5 or F1 to F6 as described herein, including ZFPs comprising recognition helical regions of ZFPs designated as 45643, 46025, 45294, 45723 or 33074. In certain embodiments, provided herein are ZFPs designated as 45294 or 45723, comprising a recognition helical region in the order shown in a single row of Table 1. Artificial transcription factors (ZFP-TFs) are also described, comprising these ZFPs operably linked to a transcriptional repression domain (e.g., KRAB, KOX, etc.), and optionally comprising other elements, such as a nuclear localization signal (NLS) and / or a promoter (e.g., a constitutive promoter, such as a CMV promoter) that drives expression of a ZFP-TF coding sequence (e.g., a ZFP-TF comprises a ZFP designated as 45294 or 45723, further comprising a sequence encoding a transcriptional repression domain, and optionally comprising a sequence encoding an NLS and / or a promoter that drives expression of a ZFP-TF). In certain embodiments, provided herein are one or more ZFP-TFs (in the form of proteins and / or polynucleotides) having an amino acid sequence or nucleotide sequence (specific repressor) as shown in a single row of Table 3.

[0016] Disclosed herein is a zinc finger protein transcription factor (ZFP-TF) comprising a zinc finger protein (ZFP) designated as 45294 or 45723 or comprising the amino acid sequence of a ZFP-TF as shown in Table 3. Also described herein is one or more polynucleotides encoding one or more ZFP-TFs described herein, wherein the one or more polynucleotides may encode one or more identical and / or different ZFP-TFs, optionally wherein the one or more polynucleotides comprise one or more rAAV vectors (e.g., rAAV comprising a sequence encoding one or more ZFP-TFs, the ZFP-TF comprising a ZFP designated as 45294 or 45723), or wherein the rAAV vector comprises a polynucleotide having a sequence as shown in Table 3, optionally wherein the one or more rAAV vectors further comprise other elements, such as a sequence encoding a nuclear localization signal (NLS) and optionally a promoter driving the expression of the ZFP-TF, such as a constitutive promoter (e.g., CMV). Also described herein is a pharmaceutical composition comprising one or more ZFP-TFs described herein, one or more polynucleotides and / or one or more rAAV vectors. Also provided are methods of modifying the expression of an HTT gene (e.g., a mutant HTT (mHTT) gene) in a cell (e.g., a neuronal cell in the brain, optionally in the striatum) or a subject, the method comprising administering to the subject a cell one or more ZFP-TFs, one or more polynucleotides, one or more rAAV vectors, and / or pharmaceutical compositions described herein. Also provided are methods of treating and / or preventing Huntington's disease (HD) in a subject in need thereof, the method comprising administering to a subject in need thereof one or more ZFP-TFs, one or more polynucleotides, one or more rAAV vectors, and / or pharmaceutical compositions described herein, optionally wherein the one or more ZFP-TFs, polynucleotides, rAAV vectors, and / or pharmaceutical compositions are administered bilaterally to the striatum of the subject. Also provided are uses of one or more ZFP-TFs, one or more polynucleotides, one or more rAAV vectors, and / or pharmaceutical compositions described herein in suppressing the expression of mutant HTT (mHTT) in a subject in need thereof. Treatment and / or prevention of HD may involve reducing mHTT aggregates and / or motor deficits in a subject. Furthermore, in any of the methods or uses described herein, one or more ZFP-TFs, one or more polynucleotides, one or more rAAV vectors, and / or pharmaceutical compositions can be delivered to the brain of a subject, optionally bilaterally to the striatum of a subject, at any dose, including but not limited to 1×10 7 Up to 1×10 15 (or any value therebetween).

[0017] Therefore, in one aspect, an engineered (non-naturally occurring) mHTT repressor is provided. The repressor may comprise a system (e.g., a zinc finger protein, a TAL effector (TALE) protein, or a CRISPR / dCas-TF) that regulates the expression of an HD allele (e.g., mHTT). An engineered zinc finger protein or TALE is a non-naturally occurring zinc finger protein or TALE protein, whose DNA binding domain (e.g., a recognition helix or RVD) has been modified (e.g., by selection and / or rational design) to bind to a pre-selected target site. Any zinc finger protein described herein may include 1, 2, 3, 4, 5, 6, or more zinc fingers, each of which has a recognition helix that binds to a sub-target site (target subsite) in a selected sequence (e.g., a gene). Similarly, any TALE protein described herein may include any number of TALE RVDs. In some embodiments, at least one RVD has non-specific DNA binding. In some embodiments, at least one recognition helix (or RVD) is non-naturally occurring. In certain embodiments, the repressor comprises a DNA binding domain (ZFP, TALE, single guide RNA) operably linked to a transcriptional repression domain to create an artificial transcription factor (repressor). Optionally, the artificial repressor comprises other components, including but not limited to a nuclear localization signal (NLS). In some embodiments, these artificial TFs (e.g., ZFP-TF, CRISPR / dCas-TF or TALE-TF) include a protein interaction domain (or "dimerization domain") that allows multimerization when bound to DNA.

[0018] In certain embodiments, the position of the zinc finger protein (ZFP) described herein, the Cas protein of the CRISPR / Cas system, or the TALE protein can be operably connected to the regulatory domain (or functional domain) as part of a fusion protein. For example, the functional domain can be a transcriptional activation domain, a transcriptional repression domain, and / or a nuclease (cleavage) domain. By selecting an activation domain or a repression domain and using it with a DNA binding domain, such a molecule can be used to activate or repress gene expression. In some embodiments, such a molecule is provided, which comprises a ZFP, dCas or TALE targeting mHTT as described herein, which is fused to a transcriptional repression domain that can be used to downregulate mutant HTT expression. In some embodiments, such a fusion protein is provided, which comprises a ZFP, CRISPR / Cas or TALE targeting a wild-type HTT allele, which is fused to a transcriptional activation domain that can upregulate a wild-type HTT allele. In some embodiments, the activity of the regulatory domain is regulated by an exogenous small molecule or a ligand, so in the absence of an exogenous ligand, the interaction with the cell transcription machinery structure does not occur, and in other embodiments, an exogenous small molecule or a ligand prevents interaction. Such external ligands control the degree of interaction between ZFP-TF, CRISPR / Cas-TF or TALE-TF and the transcription machinery structure. One or more regulatory domains can be operably connected to any part of one or more ZFPs, dCas or TALEs, including between one or more ZFPs, dCas or TALEs, outside one or more ZFPs, dCas or TALEs, and any combination thereof. Any fusion protein described herein can be formulated into a pharmaceutical composition.

[0019] In some embodiments, the position of the engineered DNA binding domain described herein can be operably connected to the nuclease (cutting) domain as part of the fusion protein. In some embodiments, the nuclease includes Ttago nuclease. In other embodiments, a nuclease system such as a CRISPR / Cas system can utilize a specific single guide RNA to target the nuclease in the DNA. In certain embodiments, such nucleases and nuclease fusions can be used to target stem cells such as induced pluripotent stem cells (iPSC), human embryonic stem cells (hESC cells), mesenchymal stem cells (MSC) or neuronal stem cells. Mutation HTT alleles, wherein the activity of the nuclease fusion will produce HTT alleles containing wild-type number of CAG repeats. Therefore, any HTT (e.g., mHTT) repressor described herein can also include a dimerization domain and / or a functional domain (e.g., a transcriptional activation domain, a transcriptional repression domain, or a nuclease domain). In certain embodiments, a pharmaceutical composition comprising a modified cell (e.g., a stem cell) is provided. In certain embodiments, the repressor comprises a ZFP, which comprises a recognition spiral region in the order shown in the single row of Table 1. Also provided are ZFP-TFs (repressors) as shown in Table 3 (one repressor per row as indicated in Table 3). Also provided are compositions comprising one or more fusion molecules (e.g., a ZFP-TF comprising a ZFP of Table 1 and / or a ZFP-TF as shown in Table 3).

[0020] In yet another aspect, a polynucleotide is provided, which encodes one or more DNA binding proteins and / or fusion molecules (e.g., artificial transcription factors) described herein. In certain embodiments, the polynucleotide is carried on a viral (e.g., AAV or Ad) vector and / or a non-viral (e.g., a plasmid or mRNA vector or an aptamer) vector. A pharmaceutical composition is also provided, which comprises a host cell of these polynucleotides (e.g., an rAAV vector) and / or comprises a polynucleotide, protein, and / or host cell described herein. In certain embodiments, the polynucleotide comprises at least one sequence shown in Table 3 (column 2). A composition comprising one or more of these polynucleotides is also provided.

[0021] In other aspects, the present invention includes the delivery of donor nucleic acid to target cells.Donors can be delivered before, after or simultaneously to the nucleic acid encoding nuclease.Donor nucleic acid may include exogenous sequences (transgenes) integrated in the cell genome, such as endogenous loci.In some embodiments, the donor may include a full-length gene or a fragment thereof, which is flanked by a region homologous to the target cleavage site.In some embodiments, the donor lacks a homology region and is integrated in the target locus by a mechanism (i.e., NHEJ) unrelated to homology.Donors may include any nucleic acid sequence, such as such nucleic acid, when used as a substrate for homology-directed repair of a double-strand break induced by a nuclease, it may cause donor-specific deletions to occur at endogenous chromosomal loci, or (or in addition) may produce new allele forms of endogenous loci.In some aspects, donor nucleic acid is an oligonucleotide, wherein integration results in gene correction events or targeted deletions.

[0022] In some embodiments, the polynucleotide encoding the DNA binding protein and / or artificial transcription factor (e.g., ZFP-TF) is mRNA. In some aspects, the mRNA can be chemically modified (see, e.g., Kormann, et al. (2011) Nature Biotechnology 29(2): 154-157). In other aspects, the mRNA can include an ARCA cap (see U.S. Patent Nos. 7,074,596 and 8,153,773). In other embodiments, the mRNA can include a mixture of unmodified and modified nucleotides (see U.S. Patent Publication No. 2012 / 0195936).

[0023] In yet another aspect, a gene delivery vector comprising one or more polynucleotides described herein is provided. In certain embodiments, the vector is an adenovirus vector (e.g., Ad5 / F35 vector), or a lentiviral vector (LV), including an integration-capable or integration-defective lentiviral vector, or an AAV vector (AAV), also referred to as a recombinant adeno-associated virus vector (rAAV). In certain embodiments, the AAV vector is an AAV6 or AAV9 vector. The AAV vector may include one or more polynucleotides as shown in the single row of Table 3 (any one or more of SEQ ID NO:13-17). In certain embodiments, the AAV vector may carry a naturally occurring capsid sequence or an artificially engineered capsid sequence. Therefore, an adenovirus (Ad) vector, LV or a recombinant adeno-associated virus vector (rAAV) is also provided herein, comprising a sequence encoding at least one nuclease (ZFN or TALEN) and / or a donor sequence for targeted integration into a target gene. In certain embodiments, the Ad vector is a chimeric Ad vector, such as an Ad5 / F35 vector. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector (IDLV) or an integration-competent lentiviral vector. In some embodiments, the vector is a pseudotyped vector with a VSV-G envelope or with other envelopes.

[0024] In addition, pharmaceutical compositions comprising nucleic acids and / or proteins (e.g., ZFPs, Cas or TALEs) and / or fusion molecules (e.g., artificial transcription factors comprising ZFPs, Cas or TALEs) are also provided. For example, certain compositions include a nucleic acid comprising a sequence encoding one of the ZFPs, Cas or TALEs described herein, operably linked to a regulatory sequence, and combined with a pharmaceutically acceptable carrier or diluent, wherein the regulatory sequence allows the nucleic acid to be expressed in a cell. In certain embodiments, the encoded ZFP, CRISPR / Cas or TALE is specific to the HD HTT allele. In some embodiments, the pharmaceutical composition comprises a ZFP, CRISPR / Cas or TALE that regulates the HD mHTT allele and a ZFP, CRISPR / Cas or TALE that regulates a neurotrophic factor. The protein-based composition comprises one or more ZFPs, CRISPR / Cas or TALEs described herein and a pharmaceutically acceptable carrier or diluent. In certain embodiments, the pharmaceutical composition comprises one or more proteins and / or polynucleotides of Table 3 for suppressing HTT. In certain embodiments, the pharmaceutical composition comprising an AAV vector described herein comprises 1×10 7 Up to 5×10 15 vg (or any value therebetween), more preferably 1×10 7 Up to 1×10 11 vg (or any value therebetween), more preferably 1×108 and 1×10 10 vg (or any value therebetween). In certain embodiments, the AAV vector is expressed at 1×10 8 Up to 1×10 10 (or any value therebetween) vg dose administration, including but not limited to 3e8, 3e9 or 3e10, 9.2e9, 3.1e10 or 9.2e10 vg per striatum. Intrastriatal administration can be administered to a single hemisphere, or preferably (at the same or different doses) bilaterally. In yet another aspect, an isolated cell is also provided, comprising any protein, polynucleotide and / or composition described herein.

[0025] In another aspect, described herein are methods of modifying HTT gene expression in a cell (e.g., a neuronal cell in vitro or in vivo in the brain of a subject, such as in the striatum), comprising administering to the cell one or more proteins, polynucleotides, pharmaceutical compositions, and / or cells described herein. Administration (e.g., of a pharmaceutical composition comprising an AAV ZFP-TF described herein) can be performed before and / or after the onset of disease symptoms at any dose (e.g., 1×10 7 Up to 5×10 15 AAV vg (or any value therebetween)). Administration can be performed once or repeatedly at any interval, and repeated administration can be the same or different doses. The HTT gene may comprise at least one wild-type and / or mutant HTT allele. In certain embodiments, HTT expression is repressed, for example, wherein expression of mutant HTT (mHTT) is preferentially repressed compared to wild-type expression. After administration of one or more ZFP-TFs described herein, repression of HTT (including selective repression of mHTT) can last for days, weeks, months or years. In certain embodiments, after a single administration, selective repression of mHTT (compared to wild-type HTT) lasts for 6 months or more.

[0026] In another aspect, provided herein are methods for treating and / or preventing Huntington's disease using the methods and compositions (proteins, polynucleotides and / or cells) described herein. In some embodiments, the method relates to compositions in which polynucleotides and / or proteins can be delivered using viral vectors, non-viral vectors (e.g., plasmids), and / or combinations thereof. Pharmaceutical compositions can also be delivered to a subject using standard techniques. In some embodiments, the method relates to compositions comprising a stem cell population containing a ZFP or TALE, or altered with a ZFN, TALEN, Ttago, or CRISPR / Cas nuclease system of the present invention. The subject may comprise at least one mutant and / or wild-type HTT allele.

[0027] In yet another aspect, described herein is a method of delivering one or more inhibitors of HTT (e.g., mHTT) to the brain of a subject using an rAAV (e.g., capsid AAV9 or AAV6) vector. It can be delivered to any brain region, such as the striatum (e.g., dura mater; intrastriatal injection includes stereotactic intrastriatal injection) by any suitable means including the use of a cannula. Administration into the brain (e.g., striatum) can be administered to a single hemisphere, or can be bilateral administration (e.g., when bilaterally administered, with the same or different doses). In some embodiments, delivery is performed by direct injection into the intrathecal space. In other embodiments, delivery is performed by intravenous injection. The rAAV vector provides a wide range of delivery of inhibitors to the brain of the subject, including delivery to brain regions to which the vector is not directly administered (e.g., delivery to the striatum) through anterograde and retrograde axonal transport, thereby delivering to other structures such as the forebrain, posterior cerebral cortex, substantia nigra, thalamus, etc. In certain embodiments, the subject is a human, and in other embodiments, the subject is a non-human mammal. In certain embodiments, one or more proteins and / or polynucleotides of Table 3 (or pharmaceutical compositions comprising these proteins and / or polynucleotides) are delivered to a subject. Any one or combination of repressors (e.g., 1, 2, 3, 4, or 5 repressors in any combination) as shown in Table 3 can be used.

[0028] Thus, in other aspects, described herein are methods for preventing and / or treating HD in a subject, the methods comprising administering to the subject a repressor of at least one mutant HTT (mHTT) allele. The repressor may be administered in polynucleotide form (e.g., using a viral (e.g., AAV) and / or non-viral vector (e.g., plasmid and / or mRNA)), in protein form, and / or via a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising one or more polynucleotides, one or more AAV vectors, one or more fusion molecules, and / or one or more cells described herein). In certain embodiments, the repressor is administered to the CNS (e.g., striatum) of the subject. The repressor may provide therapeutic benefits, including, but not limited to: reducing the formation of mHTT aggregates in HD neurons of subjects with HD (including reducing mHTT aggregation without affecting nuclear aggregation); reducing cell death in neurons or neuronal populations (e.g., HD neurons or HD neuronal populations); and / or reducing motor deficits (e.g., clasping, chorea, balance problems, etc.) in HD subjects. In certain embodiments, mutant HTT expression is suppressed by delivering one or more proteins and / or polynucleotides of Table 3 (or pharmaceutical compositions comprising these proteins and / or polynucleotides) to a subject.

[0029] In any of the methods described herein, the repressor of the mutant HTT allele can be a ZFP-TF, such as a fusion protein comprising a ZFP that specifically binds to a mutant HTT allele and a transcriptional repression domain (e.g., KOX, KRAB, etc.). In certain embodiments, the ZFP-TF comprises a ZFP having a recognition helical region of a ZFP as shown in a single row of Table 1, including a ZFP-TF repressor having an amino acid sequence as shown in Table 3 or encoded by a polynucleotide as shown in Table 3. In other embodiments, the repressor of the mutant HTT allele can be a TALE-TF or a CRISPR / Cas-TF, in which the nuclease domain in the Cas protein has been inactivated so that the protein no longer cuts DNA. In yet other embodiments, the repressor may comprise one or more nucleases (e.g., ZFNs, TALENs, and / or CRISPR / Cas systems) that repress the mutant HTT allele by cutting the mutant HTT allele and thereby inactivating it. In certain embodiments, the nuclease introduces insertions and / or deletions ("indels") by non-homologous end joining (NHEJ) and subsequent nuclease cleavage. In some embodiments, two nucleases cleave the CAG expansion region to generate a large deletion in the region.

[0030] In other embodiments, a nuclease is introduced into the donor sequence (by homology or non-homology directed approaches), wherein integration of the donor inactivates the mutant HTT allele.

[0031] In any of the methods described herein, the repressor can be delivered to a subject (e.g., brain) as a protein, a polynucleotide, or any combination of proteins and polynucleotides. In certain embodiments, the repressor is delivered with an AAV (e.g., AAV9 or AAV6) vector. In other embodiments, at least one component of the repressor (e.g., sgRNA of a CRISPR / Cas system) is delivered in RNA form. In other embodiments, the repressor is delivered with a combination of any expression constructs described herein, such as one repressor (or a portion thereof) on an expression construct (e.g., AAV, such as AAV9 or AAV6), and another repressor (or a portion thereof) on another expression construct (rAAV or other viral or non-viral construct).

[0032] In addition, in any of the methods described herein, the repressor can be delivered at any concentration (dose) that can provide the desired effect. As shown herein, HTT repression can be achieved in vivo by exposure to as little as 1VG / cell in a subject. In a preferred embodiment, the repressor is delivered using a recombinant adeno-associated viral vector of 10,000 to 500,000 vector genomes / cell (or any value therebetween). In certain embodiments, the repressor is delivered using a lentiviral vector with an MOI of 250 to 10,000 (or any value therebetween). In other embodiments, the repressor is delivered using a plasmid construct of 150 to 1,500 ng / 100,000 cells (or any value therebetween). In other embodiments, the repressor is delivered using 0.003 to 1,500 ng / 100,000 cells (or any value therebetween) of mRNA. In some embodiments, the AAV dose is calculated for each animal (subject). For example, the AAV vector described herein may contain 1×10 7 Up to 5×10 15 vg (or any value therebetween), more preferably 1×10 7 Up to 1×10 13 vg (or any value therebetween), more preferably 1×10 8 and 1×10 13 vg (or any value therebetween). Intrastriatal administration can be administered to a single hemisphere, or preferably bilaterally (at the same or different doses). For example, in some embodiments, the repressor is delivered at about 9e9 VG / mouse, or about 9e9 VG / mouse to 3e10 VG / mouse, or about 3e10 VG / mouse to 9e10 VG / mouse. In some embodiments, the AAV dose is less than 9e9 VG / mouse (e.g., 6e8 VG / mouse or less), and in other embodiments, the AAV dose is greater than 9e10 VG / mouse.

[0033] In any of the methods described herein, the compositions and methods described herein can achieve 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 suppression of mutant HTT allele expression in one or more HD neurons of a subject. In addition, the compositions and methods described herein can achieve at least 50%, preferably 50%-90% (or any value therebetween), more preferably greater than 90% selectivity in HTT (e.g., mHTT) suppression (compared to suppression of off-target sites) compared to a control.

[0034] In other aspects, the invention described herein includes one or more HTT regulatory transcription factors, such as HTT regulatory transcription factors comprising one or more zinc finger proteins (ZFP TFs), TALEs (TALE-TFs), and CRISPR / Cas-TFs, such as ZFP-TFs, TALE-TFs, or CRISPR / Cas-TFs. In certain embodiments, the HTT regulatory transcription factor can repress the expression of a mutant HTT allele in one or more HD neurons of a subject. The repression can be 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 of the mutant HTT allele in one or more HD neurons of a subject compared to an untreated (e.g., wild-type) neuron of the subject. In certain embodiments, the HTT regulatory transcription factor can be used to implement one or more methods described herein. In certain embodiments, the ZFP-TF comprises the amino acid sequence of the mHTT repressor as shown in Table 3.

[0035] In some embodiments, the efficacy of the treatment is determined using the Unified Huntington's Disease Rating Scale (UHDRS) (Huntington's Disease Research Group (1996) Mov Disord 11(2): 136-142) for analyzing overt clinical symptoms. In other embodiments, the efficacy in patients is determined using PET and MRI imaging. In some embodiments, treatment with mutant HTT regulatory transcription factors prevents any further development of overt clinical symptoms and prevents any further loss of neuronal function. In other embodiments, treatment with mutant HTT regulatory transcription factors improves clinical symptoms (e.g., motor function determined using known measurements, such as grasping behavior, rotating rod analysis, etc.) and improves neuronal function.

[0036] Also provided is a kit comprising one or more HTT regulators (e.g., repressors) as described herein, and / or components comprising HTT regulators and / or polynucleotides encoding HTT regulators (or components thereof). The kit may also comprise cells (e.g., neurons), reagents (e.g., for detecting and / or quantifying mHTT proteins in, e.g., CSF), and / or instructions for use including the methods described herein.

[0037] These and other aspects will be apparent to those skilled in the art based on the disclosure as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 An alignment of the protein (amino acid) sequences (SEQ ID NOs: 18-22) of the indicated ZFP-TFs is shown. See also Table 3.

[0039] Figure 2A and Figure 2B is a graph showing relative HTT expression (wtHTT and mHTT) after introduction of the indicated ZFP-TFs. Figure 2A The expression of HTT containing 17 CAG repeats (CAG 17 is shown in the left column of each pair of columns) (the "CAG" repeat sequence disclosed in SEQ ID NO: 23) or 48 CAG repeats (CAG 48 is shown in the right column of each pair of columns) (the "CAG" repeat sequence disclosed in SEQ ID NO: 23) in human neural stem cells (NSCs) after the introduction of the specified ZFP-TF or control (GFP or mock) in the form of mRNA. For each ZFP, the leftmost pair of columns shows the results when 1500 ng of mRNA is transfected into the cells; the second pair of columns from the left shows the results when 300 ng of mRNA is transfected into the cells; the second pair of columns from the right shows the results when 150 ng of mRNA is transfected into the cells, and the rightmost pair of columns shows the results when 15 ng of mRNA is transfected into the cells. Figure 2B The upper panel shows the expression of ZFP-TFs or controls (eg Figure 2A HD neurons (with Figure 2A The relative HTT expression in each pair of columns (CAG17 (17 "CAG" repeats disclosed in SEQ ID NO: 23) and CAG48 (48 "CAG" repeats disclosed in SEQ ID NO: 23)) is shown in the lower part of the figure (the columns from left to right represent 500K, 300K, 100K, 10K and double repeats). Figure 2B The lower panel shows the ZFP-TF copy number under the indicated conditions. The results using ZFP-TFs (45643 and 46025) are boxed.

[0040] Figure 3 Relative expression levels of mutant or wild-type HTT in subjects treated at low (3E10) or high (9E10) doses with rAAV carrying the indicated ZFP-TFs and GFP control (GFP), as well as vehicle and no injection controls are shown. Suppression of mutant HTT (KI allele Q50) expression in Q50 mice carrying a knock-in CAG repeat sequence (KI CAG48) (48 "CAG" repeat sequences disclosed in SEQ ID NO:23) is shown.

[0041] Figure 4The results of the microarray analysis of off-target regulation by the 4 designated ZFPs in HD neurons are shown. Unshaded areas indicate off-target expression is 50-90% of the control. Shaded areas indicate off-target expression levels are greater than 90% of the control. The cross-hatched boxes (column 1 of 45294, 45643, and 45723) indicate off-target expression is less than 50% of the control. As shown, the specificity of the ZFP-TFs is ranked from most specific to least specific as follows: 46025>45723>45643>45294.

[0042] The off-target sites are referred to by the abbreviated gene names (e.g., SRPX refers to Sushi-Repeat Containing Protein, X-linked gene, etc.).

[0043] Figure 5A and Figure 5B is a graph showing the relative expression of the indicated mRNA encoding wild-type HTT (CAG18 (18 "CAG" repeats disclosed in SEQ ID NO: 23)) or mutant HTT (mHTT (CAG45 (45 "CAG" repeats disclosed in SEQ ID NO: 23))) after administration of the indicated doses of ZFP 45643 mRNA (ng). The left (white) column under each condition (dose) shows HTT mRNA expression, and the right (black) column shows mHTT expression. Figure 5A Results in fibroblasts at the indicated ZFP mRNA doses are shown. Figure 5B Results in neurons are shown (mRNA expression, expressed as percentage of GFP expression).

[0044] Figure 6 Off-target analysis of ZFP-TF 45643 in neurons (upper panel) and fibroblasts (lower panel) is shown. Unshaded areas indicate off-target repression less than or equal to 2-fold repression of control. Shaded areas indicate no change in regulation compared to control. Cross-hatched grids (column 1 showing sprx targets in neurons and columns 1-5 (SPRX, TTC12, MAB211.1, STC1, and CNK5R2 target sites in fibroblasts)) indicate off-target repression greater than 2-fold.

[0045] Figure 7Shown are graphs depicting wild-type and mHTT expression in the striatum of control (untreated) and treated Q175 mice, a rodent model of HD that carries a knock-in allele of the human mutant Huntington allele, 11 weeks after treatment with rAAV vectors encoding GFP or ZFP-TF 45643 at the indicated doses. The graph on the left shows the relative expression of the mutant (mHTT) and the graph on the right shows the relative expression of wild-type HTT (wtHTT). As shown, significant and preferential repression of mHTT was seen in animals treated with all doses of ZFP-TFs.

[0046] Figure 8 Shown are graphs depicting viral genome copies / cell and mHTT mRNA levels in the striatum of control (untreated) and treated Q175 mice 11 weeks after treatment with AAV vectors encoding GFP or ZFP-TF 45643 at the indicated doses. The graph on the left shows the viral genome copies / cell under the indicated conditions, and the graph on the right shows the mHTT mRNA levels under the indicated conditions, expressed as a percentage of GFP levels. Significant suppression of mHTT mRNA levels was seen in animals treated with all doses of the ZFP-TF, including exposures as low as 1 VG / cell.

[0047] Fig. 9 Figures depicting soluble mHTT protein (% GFP treatment) in the indicated brain regions (striatum, cortical forebrain, and cortical hindbrain) of Q175 mice 11 weeks (left) and 33 weeks (right) after treatment with rAAV vectors carrying GFP or ZFP-TF 45643 at the indicated doses are shown. The bars for each brain region represent, from left to right: GFP 5.5e10 VG / mouse; rAAV vector encoding ZFP-TF 45643 9.2e9 VG / mouse; rAAV vector encoding ZFP-TF 45643 3.1e10 VG / mouse; and rAAV vector encoding ZFP-TF 45643 9.2e10 VG / mouse. After a single administration of rAAV vector encoding ZFP-TF 45643, a significant dose-dependent reduction in soluble mHTT persisted for 33 weeks.

[0048] Fig.10Figures depicting soluble mHTT protein (% GFP treatment) in the indicated brain regions (striatum, cortical forebrain, and cortical hindbrain) of R6 / 2HD mice treated with AAV vectors carrying GFP or ZFP-TF 45643 at the indicated doses are shown. The bars for each brain region represent, from left to right: GFP 5.5e10 VG / mouse; rAAV vector encoding ZFP-TF 45643 9.2e9 VG / mouse; rAAV vector encoding ZFP-TF 45643 3.1e10 VG / mouse; and rAAV vector encoding ZFP-TF 45643 9.2e10 VG / mouse. ZFP-TF significantly reduced the production of soluble mHTT protein in the severe R6 / 2HD mouse model.

[0049] Fig.11 Figures showing mHTT nuclear aggregation in Q175 and R6 / 2 mice following administration of rAAV vectors encoding ZFP-TF 45643 at the indicated doses. The left graph shows mHTT nuclear aggregates in transgene-positive neurons (number of dots / number of transgene-positive neurons) in Q175 subjects. The right graph shows mHTT nuclear aggregates in all neurons under the indicated conditions (number of dots / number of neurons). ZFP-TF administration reduced mHTT nuclear aggregates in Q175 and R6 / 2 subjects.

[0050] Fig.12 is a graph depicting the relative expression of wild-type (wtHTT) and mutant (mHTT) mRNA in striatal neurons of 12-month-old Q175 mice dosed single-time under the indicated conditions and analyzed 8 weeks after dosing.

[0051] Fig.13 is a graph depicting perinuclear aggregates (expressed as a percentage of vehicle) in 12 month old Q175 mice at 8 and 16 weeks post-dosing under the indicated treatment conditions. ZFP-TF 45643 produced perinuclear mHTT aggregates upon treatment in 12 month old Q175 mice.

[0052] Fig.14 Shown are graphs depicting motor function of R6 / 2 mice under the indicated treatment conditions. The left graph shows the results of the rotarod performance test, which measures how long a subject can stay on a rotating rod. The right graph shows the percentage of mice that exhibited clasping behavior under the indicated conditions at the indicated age (in weeks). DETAILED DESCRIPTION

[0053] Disclosed herein are compositions and methods for broad CNS delivery of compositions for detecting, monitoring disease progression, treating and / or preventing Huntington's disease (HD). In particular, the compositions and methods described herein use AAV9 vectors to deliver mHTT repressors, which provide diffusion of functional mHTT repressors beyond the delivery site. The mHTT repressor (e.g., mHTT regulatory transcription factors, such as ZFP-TFs, TALEs (TALE-TFs) or CRISPR / Cas-TFs, such as mHTT regulatory transcription factors that suppress expression of mutant HTT alleles) modifies the CNS to reduce or eliminate the effects and / or symptoms of HD, for example, by reducing the aggregation of HTT in HD neurons, by increasing HD neuron energy (e.g., increasing ATP levels), by reducing apoptosis in HD neurons, and / or by reducing motor deficits in HD subjects.

[0054] Overview

[0055] Unless otherwise indicated, the practice of the present methods and the preparation and use of the compositions described herein employ conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields within the skill of the art. These techniques are fully described in the literature. See, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory Press, 1989 and 3rd ed., 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and regularly updated; METHODS IN ENZYMOLOGY series, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, 3rd ed., Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, 3rd ed., Academic Press, San Diego, 1998; IN ENZYMOLOGY, Vol. 304, “Chromatin” (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (PB Becker, ed.), Humana Press, Totowa, 1999.

[0056] definition

[0057] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, which can be in a linear or cyclic configuration, in single-stranded or double-stranded form. For the purposes of this disclosure, these terms are not intended to limit the length of the polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides modified at the base, sugar and / or phosphate moieties (e.g., phosphorothioate backbones). In general, analogs of a particular nucleotide have the same base pairing specificity; that is, an analog of A will base pair with T.

[0058] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein 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 acids.

[0059] "Binding" refers to sequence-specific, non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids). It is not required that all components of a binding interaction be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are often characterized by a dissociation constant (K d ) is 10 -6 M -1 or lower. “Affinity” refers to the strength of binding: increased binding affinity is associated with a lower K d association.

[0060] A "binding protein" is a protein that can non-covalently bind to another molecule. A binding protein can bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein) and / or a protein molecule (protein binding protein). In the case of a protein binding protein, it can bind to itself (forming homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of one or more different proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding and protein binding activities.

[0061] A zinc finger "DNA binding protein" (or binding domain) is a protein or domain within a larger protein that can bind DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

[0062] A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains / units. The repeat domain is involved in the binding of a TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids long and shows at least some sequence homology to other TALE repeat sequences in naturally occurring TALE proteins. See, e.g., U.S. Patent No. 8,586,526.

[0063] "TtAgo" is a prokaryotic Argonaute protein that is believed to be involved in gene silencing. TtAgo is derived from thermophilic bacteria (Thermus thermophilus). See, for example, Swarts et al. (2014) Nature 507(7491):258-261; G. Sheng et al. (2013) Proc. Natl. Acad. Sci. USA 111:652). "TtAgo system" is all the components required, including, for example, guide DNA for cleavage by the TtAgo enzyme. "Recombination" refers to the process of exchanging genetic information between two polynucleotides, including but not limited to donor capture by non-homologous end joining (NHEJ) and homology recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to a specific form of this exchange, such as during the repair of double-strand breaks in cells by homology-directed repair mechanisms. This process requires nucleotide sequence homology, and the use of a "donor" molecule template to repair a "target" molecule (i.e., a molecule that has undergone a double-strand break) is also referred to as "non-crossover gene conversion" or "short-segment gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, this transfer may involve mismatch correction of heteroduplex DNA formed between the broken target and donor, and / or "synthesis-dependent strand annealing" using the donor to resynthesize genetic information that will become part of the target, and / or related processes. This specialized HR typically results in a change in the sequence of the target molecule, such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0064] Zinc finger binding domains and TALE DNA binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering the recognition helix region of a naturally occurring zinc finger protein (changing one or more amino acids) or by engineering the RVD of a TALE protein. Therefore, an engineered zinc finger protein or TALE is a non-naturally occurring protein. Non-limiting examples of methods for engineering zinc finger proteins or TALEs are design and selection. A "designed" zinc finger protein or TALE is a non-naturally occurring protein whose design / composition is primarily derived from reasonable criteria. Reasonable criteria for design include applying substitution rules and computer algorithms to process information in a database storing information of existing ZFP designs and binding data. A "selected" zinc finger protein or TALE is a protein that does not exist in nature, and the results of its generation are primarily derived from empirical processes, such as phage display, interaction traps, or hybrid selection. See, e.g., U.S. Patent Nos. 5,605,806; 6,140,081; 6,453,242; 6,746,838; 7,241,573; 6,866,997; 7,241,574 and 6,534,261; see also International Patent Publication No. WO 03 / 016496.

[0065] 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 single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. The donor sequence may be of any length, for example, 2-10,000 nucleotides in length (or any integer value therebetween or thereon), preferably 100-1,000 nucleotides in length (or any integer therebetween), and more preferably 200-500 nucleotides in length.

[0066] A "target site" or "target sequence" is a nucleic acid sequence that defines the portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.

[0067] An "exogenous" molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normal presence in a cell" is determined relative to the specific developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule relative to a non-heat shocked cell. For example, an exogenous molecule can include a functional form of a malfunctioning endogenous molecule or a malfunctioning form of a normally functioning endogenous molecule.

[0068] Exogenous molecules can be small molecules or macromolecules, such as those produced by combinatorial chemistry methods, macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes comprising one or more of the above molecules. Nucleic acids include DNA and RNA, which can be single-stranded or double-stranded; can be linear, branched or circular; and can have any length. Nucleic acids include those that can form duplexes, as well as nucleic acids that form triplexes. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins can include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetyltransferases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrase and helicase.

[0069] Exogenous molecules can be molecules of the same type as endogenous molecules, such as exogenous proteins or nucleic acids. For example, exogenous nucleic acids can include infectious viral genomes, plasmids or episomes introduced into cells, or include chromosomes that are not usually present in cells. Methods for introducing exogenous molecules into cells are known to those skilled in the art, including but not limited to lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electrotransduction, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. Exogenous molecules can also be molecules of the same type as endogenous molecules but derived from species different from the cell source. For example, human nucleic acid sequences can be introduced into cell lines originally derived from mice or hamsters.

[0070] Differently, "endogenous" molecules are molecules that are usually present in specific cells of a specific developmental stage under specific environmental conditions. For example, endogenous nucleic acids can include chromosomes, mitochondrial genomes, chloroplasts or other organelles, or naturally occurring additional nucleic acids. Other endogenous molecules can include proteins, for example, transcription factors and enzymes.

[0071] A "fusion" molecule is a molecule in which two or more subunit molecules are connected (preferably covalently connected). The subunit molecules can be molecules of the same chemical type or molecules of different chemical types. Examples of the first type of fusion molecules may include, but are not limited to, fusion proteins (e.g., fusions between ZFP or TALE DNA binding domains and one or more activation domains) and fusion nucleic acids (e.g., nucleic acids encoding the fusion proteins described above). Examples of the second type of fusion molecules include, but are not limited to, fusions between triplex-forming nucleic acids and polypeptides, and fusions between minor groove binders and nucleic acids. The term also includes molecules in which a polynucleotide component is associated with a polypeptide component to form a functional molecule (e.g., a CRISPR / Cas system in which a single guide RNA is associated with a functional domain to regulate gene expression).

[0072] The expression of the fusion protein in the cell can be caused by the delivery of the fusion protein into the cell or by delivering a polynucleotide encoding the fusion protein to the cell, wherein the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. The expression of the protein in the cell can also involve trans-splicing, polypeptide cleavage and polypeptide connection. Methods for delivering polynucleotides and polypeptides to cells are presented elsewhere in this disclosure.

[0073] "Multimerization domains" (also called "dimerization domains" or "protein interaction domains") are domains incorporated into the amino, carboxyl, or amino and carboxyl terminal regions of a ZFPTF or TALE TF. These domains allow for the multimerization of multiple ZFPTF or TALE TF units so that larger segments of trinucleotide repeat domains are preferentially bound by multimerized ZFP TFs or TALE TFs relative to shorter segments of wild-type length. Examples of multimerization domains include leucine zippers. Multimerization domains can also be regulated by small molecules, wherein the multimerization domain assumes an appropriate conformation, thereby allowing interaction with another multimerization domain only in the presence of a small molecule or external ligand. In this way, exogenous ligands can be used to regulate the activity of these domains.

[0074] For purposes of this disclosure, a "gene" includes DNA regions that encode a gene product (see above), as well as DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0075] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include modified RNAs modified by processing such as capping, polyadenylation, methylation, and editing, and proteins modified by processing such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0076] "Regulation" of gene expression refers to changes in gene activity. Regulation of expression may include, but is not limited to, gene activation and gene repression. Genome editing (e.g., cutting, altering, inactivating, random mutations) can be used to regulate expression. Gene inactivation refers to any reduction in gene expression compared to cells that do not contain the ZFP or TALE described herein. Therefore, gene inactivation can be partial or complete.

[0077] A "region of interest" is any region of cellular chromatin to which an exogenous molecule is to be bound, for example, a gene or a non-coding sequence within or adjacent to a gene. Binding may be for the purpose of targeted DNA cutting and / or targeted recombination. The region of interest may be present in, for example, a chromosome, an episome, an organelle genome (e.g., mitochondria, chloroplasts), or an infectious viral genome. The region of interest may be in the coding region of a gene, in a transcribed non-coding region such as a leader sequence, a trailer sequence, or an intron, or upstream or downstream of a coding region in a non-transcribed region. The region of interest may be as small as a single nucleotide pair, or as long as 2,000 nucleotide pairs, or any integer value of nucleotide pairs.

[0078] "Eukaryotic" cells can include, but are not limited to, fungal cells (eg, yeast), plant cells, animal cells, mammalian cells, and human cells (eg, T cells).

[0079] The terms "operably linked" and "operably connected" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (e.g., sequence elements) that are arranged so that the components can function properly and allow at least one of the components to mediate an effect exerted on at least one other component. For example, a transcription regulatory sequence such as a promoter is operably linked to a coding sequence if the transcription regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcription regulatory factors. Transcription regulatory sequences are generally operably linked to a coding sequence in cis, but need not be immediately adjacent to the sequence. For example, enhancers are transcription regulatory sequences that are operably linked to a coding sequence, although they are discontinuous.

[0080] For fusion polypeptides, the term "operably linked" may refer to the fact that the functions performed by each component in connection with other components are the same as their functions when not linked. For example, for a fusion polypeptide in which a ZFP or TALE DNA binding domain is fused to an activation domain, if in the fusion polypeptide, the ZFP or TALE DNA binding domain portion is able to bind to its target site and / or its binding site, and the activation domain is able to upregulate gene expression, then the ZFP or TALE DNA binding domain and the activation domain are operably linked. ZFPs fused to a domain capable of regulating gene expression are collectively referred to as "ZFP-TFs" or "zinc finger transcription factors", while TALEs fused to a domain capable of regulating gene expression are collectively referred to as "TALE-TFs" or "TALE transcription factors". In the case of a fusion polypeptide in which a ZFP DNA binding domain is fused to a cleavage domain ("ZFN" or "zinc finger nuclease"), if in the fusion polypeptide, the ZFP DNA binding domain portion can bind to its target site and / or its binding site, while the cleavage domain can cleave DNA near the target site, then the ZFP DNA binding domain and the cleavage domain are operably linked. In the case of a fusion polypeptide in which a TALE DNA binding domain is fused to a cleavage domain ("TALEN" or "TALE nuclease"), if in the fusion polypeptide, the TALE DNA binding domain portion can bind to its target site and / or its binding site, while the cleavage domain can cleave DNA near the target site, then the TALE DNA binding domain and the cleavage domain are operably linked. For a fusion polypeptide in which a Cas DNA binding domain is fused to an activation domain, if in the fusion polypeptide, the Cas DNA binding domain portion can bind to its target site and / or its binding site, while the activation domain can upregulate gene expression, then the Cas DNA binding domain and the activation domain are operably linked. In the case of a fusion polypeptide in which the Cas DNA binding domain is fused to the cleavage domain, the Cas DNA binding domain and the cleavage domain are operably linked if, in the fusion polypeptide, the Cas DNA binding domain portion can bind to its target site and / or its binding site, while the cleavage domain can cleave DNA near the target site.

[0081] A "functional fragment (or functional fragment)" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid that is not identical in sequence to the full-length protein, polypeptide or nucleic acid but retains the same function of the full-length protein, polypeptide or nucleic acid. A functional fragment may have more, less 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 nucleic acid function (e.g., encoding function, ability to hybridize with another nucleic acid) are well known in the art. Similarly, methods for determining protein function are also well known in the art. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift or immunoprecipitation assays. DNA cleavage can be analyzed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation analysis, which can be either genetic or biochemical. See, e.g., Fields, et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and International Patent Publication No. WO 98 / 44350.

[0082] "Vector" can transfer a gene sequence to a target cell. "Vector construct", "expression vector" and "gene transfer vector" generally refer to a nucleic acid construct that can direct the expression of a gene of interest and can transfer a gene sequence to a target cell. Thus, the term includes cloning and expression vectors, as well as integration vectors. The term includes viral and non-viral vectors, including but not limited to plasmids, mRNA, AAV (also referred to herein as "recombinant AAV" or "rAAV"), adenoviral vectors (Ad), lentiviral vectors (e.g., IDLV), and the like.

[0083] "Reporter gene" or "reporter sequence" refers to any sequence that produces such a protein product, which is easily detected, which is not necessary but preferred in routine tests. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins (e.g., dihydrofolate reductase) that mediate enhanced cell growth and / or gene amplification. Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence. "Expression tag" includes a sequence encoding a reporter that is operably connected to a desired gene sequence to monitor the expression of a gene of interest.

[0084] DNA binding domain

[0085] The methods disclosed herein utilize compositions, such as HTT regulatory transcription factors, which include a DNA binding domain that specifically binds to a target sequence in the HTT gene, particularly a DNA binding domain that binds to a mutant HTT allele (mHTT) comprising multiple trinucleotide repeats. Any polynucleotide or polypeptide DNA binding domain, such as a DNA binding protein (e.g., ZFP or TALE) or a DNA binding polynucleotide (e.g., a single guide RNA) can be used in the compositions and methods disclosed herein. In certain embodiments, the DNA binding domain binds to a target site comprising 9 to 28 (or any value therebetween, including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) consecutive copies of the nucleotides of SEQ ID NO: 6.

[0086] In certain embodiments, the mHTT regulatory transcription factor or the DNA binding domain therein comprises a zinc finger protein. Target site selection; ZFPs and methods for designing and constructing fusion proteins (and their encoding polynucleotides) are known to those of skill in the art and are described in detail in U.S. Patent Nos. 6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; 6,200,759; International Patent Publication Nos. WO 95 / 19431; WO 96 / 06166; WO 98 / 53057; WO 98 / 54311; WO 00 / 27878; WO 01 / 60970; WO 01 / 88197; WO 02 / 099084; WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO03 / 016496.

[0087] In certain embodiments, the ZFP can selectively bind to a mutant HTT allele or a wild-type HTT sequence. The HTT target site typically includes at least one zinc finger, but may include multiple zinc fingers (e.g., 2, 3, 4, 5, 6, or more fingers). See, for example, U.S. Patent Nos. 9,234,016; 9,943,565; 8,841,260; 9,499,597 and U.S. Patent Publication Nos. 2015 / 0335708; 2018 / 0200332; 2017 / 0096460; 2017 / 0035839; 2016 / 0296605; 2019 / 0322711. ZFPs typically include at least three fingers. Some ZFPs include 4, 5, or 6 fingers, while some ZFPs include 7, 8, 9, 10, 11, or 12 fingers. ZFPs comprising three fingers generally recognize target sites comprising 9 or 10 nucleotides; ZFPs comprising four fingers generally recognize target sites comprising 12-14 nucleotides; and ZFPs having six fingers can recognize target sites comprising 18-21 nucleotides. ZFPs may also be fusion proteins comprising one or more regulatory domains, which may be transcriptional activation or repression domains. In some embodiments, the fusion protein comprises two ZFP DNA binding domains linked together. Thus, these zinc finger proteins may comprise 8, 9, 10, 11, 12 or more fingers. In some embodiments, the two DNA binding domains are connected by an extendable flexible linker, so that one DNA binding domain comprises 4, 5 or 6 zinc fingers, and the second DNA binding domain comprises an additional 4, 5 or 5 zinc fingers. In some embodiments, the linker is a standard inter-finger linker so that the finger array comprises a DNA binding domain comprising 8, 9, 10, 11, 12 or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. The DNA binding domain is fused to at least one regulatory domain and can be considered a "ZFP-ZFP-TF" structure. Specific examples of these embodiments can be referred to as "ZFP-ZFP-KOX" and "ZFP-KOX-ZFP-KOX", wherein "ZFP-ZFP-KOX" comprises two DNA binding domains connected by a flexible linker and fused to a KOX repressor, and "ZFP-KOX-ZFP-KOX" wherein the two ZFP-KOX fusion proteins are fused together by a linker.

[0088] Alternatively, the DNA binding domain may be derived from a nuclease. For example, recognition sequences for targeting endonucleases and meganucleases such as I-Scel, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-Crel, I-TevI, I-TevII, and I-TevIII are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs product catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., 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 Publication No. 2007 / 0117128.

[0089] "Two handed" zinc finger proteins are proteins in which two clusters of zinc finger DNA binding domains are separated by an intervening amino acid, thereby allowing the two zinc finger domains to 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 amino terminus of the protein and a cluster of three zinc fingers is located at the carboxyl terminus (see, Remacle et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins is capable of binding to a unique target sequence, and the spacing between two target sequences can encompass many nucleotides. A two-handed ZFP can comprise, for example, a functional domain fused to one or two ZFPs. Thus, it will be apparent that the functional domain can be attached to the exterior of one or two ZFPs (see Figure 1C), or can be located between ZFPs (connecting two ZFPs) (see Figure 4 ).

[0090] ZFPs targeting HTT are disclosed in Table 1 and U.S. Patent Nos. 9,234,016; 8,841,260; 6,534,261; U.S. Patent Publication Nos. 2017 / 0096460; 2015 / 0056705; 2015 / 0335708; 2019 / 0322711, which are incorporated herein by reference in their entirety for all purposes. The first column in the table is the internal reference name (number) of the ZFP, corresponding to the same name in column 1 of Table 2. "F" refers to the zinc finger, and the number after "F" refers to which zinc finger (e.g., "F1" refers to finger 1).

[0091] Table 1: Zinc finger proteins targeting HTT

[0092]

[0093] The sequences and positions of the target sites for these proteins are disclosed in Table 2. Nucleotides in the target site that contact the ZFP recognition helix are indicated in uppercase letters; nucleotides that are not contacted are indicated in lowercase letters.

[0094] Table 2: Target sites on human and mouse HTT

[0095] SBS Number Target site 45643 agCAGCAGcaGCAGCAGCAgcagcagca(SEQ ID NO:6) 46025 agCAGCAGCAGcaGCAGCAgcagcagca (SEQ ID NO:6) 45294 caGCAGCAGCAGCAGCAgcagcagcagc (SEQ ID NO:11) 45723 agCAGCAGcaGCAGCAgcagcagcagca (SEQ ID NO:6) 33074 CAGCAGcaGCAGCAgCAGCAG (SEQ ID NO:12)

[0096] The ZFP-TFs described herein may also comprise one or more mutated external recognition helical regions (e.g., to the backbone region), including mutations described in U.S. Patent Publication No. 2018 / 0087072.

[0097] In certain embodiments, the DNA binding domain comprises a naturally occurring or engineered (non-naturally occurring) TAL effector (TALE) DNA binding domain. See, e.g., U.S. Pat. No. 8,586,526, which is incorporated herein by reference in its entirety.

[0098] Plant pathogens of the genus Xanthomona are known to cause many diseases in important crops. The pathogenicity of Xanthomonas depends on a conservative type III secretion (T3S) system that injects more than 25 different effector proteins into plant cells. These injected proteins include transcription activator-like effectors (TALEs) that mimic plant transcription activators and manipulate plant transcriptomes (see Kay et al., (2007) Science 318: 648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most fully characterized TALEs is AvrBs3 from Xanthomonas campestris pv.vesicatoria (see Bonas et al. (1989) MolGen Genet 218: 127-136 and International Patent Publication No. WO2010 / 079430). TALE contains a centralized domain of tandem repeats, each containing about 34 amino acids, which are the key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for review, see S. Schornack et al. (2006) J Plant Physiol 163 (3): 256-272). In addition, in the plant pathogenic bacteria Ralstonia solanacearum, two genes, called brg11 and hpx17, have been found in Ralstonia solanacearum biovar 1 strain GMI1000 and biovar 4 strain RS1000, which have homology with the AvrBs3 family of Xanthomonas (see Heuer et al. (2007) Appland Envir Micro 73 (13): 4379-4384). These genes have 98.9% identity to each other in nucleotide sequence, but are distinguished by a 1,575bp deletion in the hpx17 repeat domain. However, both gene products have less than 40% sequence identity with AvrBs3 family proteins of Xanthomonas.

[0099] The specificity of these TALEs depends on the sequences present in the tandem repeats. The repeat sequences contain about 102 base pairs, and the repeat sequences are usually 91-100% homologous to each other (Bonas et al., (1989) Mol Gen Genet 218:127-136). The polymorphism of the repeats is usually located at positions 12 and 13, and there seems to be a one-to-one correspondence between the types of hypervariable diresidues at positions 12 and 13 and the types 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 coding for DNA recognition of these TALEs has been determined, so the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG binds to T, NI binds to A, C, G or T, NN binds to A or G, and NG binds to T. These DNA binding repeat sequences have been assembled into proteins with new combinations and numbers of repeat sequences to make artificial transcription factors that can interact with new sequences. In addition, U.S. Patent No. 8,586,526 and U.S. Patent Publication No. 2013 / 0196373 describe TALEs with N-cap polypeptides, C-cap polypeptides (e.g., +63, +231 or +278) and / or novel (atypical) RVDs, which are incorporated herein by reference in their entirety.

[0100] Exemplary TALEs are described in U.S. Patent Publication No. 2013 / 0253040, which is incorporated herein by reference in its entirety.

[0101] In certain embodiments, the DNA binding domain comprises a dimerization and / or multimerization domain, such as a coiled coil (CC) and a dimerization zinc finger (DZ). See U.S. Patent Publication No. 2013 / 0253040.

[0102] In yet other embodiments, the DNA binding domain comprises a single guide RNA of the CRISPR / Cas system, such as the sgRNA described in U.S. Patent Publication No. 2015 / 0056705.

[0103] Convincing evidence has recently emerged suggesting the existence of an RNA-mediated genome defense pathway in archaea and many bacteria, which has been hypothesized to parallel the eukaryotic RNAi pathway (for reviews, see Godde and Bickerton (2006) J. Mol. Evol. 62:718-729; Lillestol et al. (2006) Archaea 2:59-72; Makarova et al. (2006) Biol. Direct 1:7; Sorek et al. (2008) Nat. Rev. Microbiol. 6:181-186). It has been proposed that the pathway known as the CRISPR-Cas system or prokaryotic RNAi (pRNAi) arises from two evolutionarily and often physically linked genetic loci: the CRISPR (clustered regularly interspaced short palindromic repeats) locus encoding the RNA component of the system, and the Cas (CRISPR-associated) locus encoding the protein (Jansen et al. (2002) Mol. Microbiol. 43:1565-1575; Makarova et al. (2002) Nucleic Acids Res. 30:482-496; Makarova et al. (2006) Biol. Direct 1:7; Haft et al. (2005) PLoS Comput. Biol. 1:e60). The CRISPR locus in a microbial host contains a combination of CRISPR-associated (Cas) genes and noncoding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. Individual Cas proteins have no significant sequence similarity to protein components of the eukaryotic RNAi machinery, but have similar predicted functions (e.g., RNA binding, nuclease, helicase, etc.) (Makarova et al. (2006) Biol. Direct 1:7). CRISPR-associated (Cas) genes are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Among these protein families, Cas1 seems to be ubiquitous in different CRISPR / Cas systems. Specific combinations of cas genes and repeat sequence structures have been used to define eight CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with other gene modules encoding repeat-associated unknown proteins (RAMPs). More than one CRISPR subtype may appear in a genome. The sporadic distribution of CRISPR / Cas subtypes suggests that the system has been affected by horizontal gene transfer during microbial evolution.

[0104] Type II CRISPR, originally described in Streptococcus pyogenes (S.pyogenes), is one of the most well-characterized systems and performs targeted DNA double-strand breaks in 4 consecutive steps. First, two non-coding RNAs are transcribed from the CRISPR locus: the pre-crRNA array and the tracrRNA. Second, tracrRNA hybridizes with the repeat sequence region of the pre-crRNA and mediates the processing of the pre-crRNA into a mature crRNA containing a single spacer sequence, which is processed by double-stranded specific RNase III in the presence of the Cas9 protein. Third, the mature crRNA:tracrRNA complex guides Cas to the target DNA through Watson-Crick base pairing, which is located between the spacer on the crRNA and the prototype spacer on the target DNA next to the prototype spacer adjacent motif (PAM), and the PAM is an additional requirement for target identification. In addition, tracrRNA must also be present because it base pairs with the 3' end of the crRNA, and this association triggers Cas9 activity. Finally, Cas9 mediates the cutting of the target DNA to produce a double-strand break in the prototype spacer. The activity of the CRISPR / Cas system involves three steps: (i) insertion of foreign DNA sequences into the CRISPR array through a process called "adaptation" to prevent future attacks, (ii) expression of associated proteins, and expression and processing of the array, and then (iii) RNA-mediated interference with foreign nucleic acids. Thus, in bacterial cells, there are multiple so-called "Cas" proteins involved in the natural function of the CRISPR / Cas system.

[0105] Type II CRISPR systems have been found in many different bacteria. Fonfara et al. ((2013) Nuc Acid Res 42(4):2377-2590) performed a BLAST search of publicly available genomes and found Cas9 orthologs in 347 bacteria. In addition, the group demonstrated in vitro CRISPR / Cas cleavage of DNA targets using Cas9 orthologs from Streptococcus pyogenes (S. pyogenes), Streptococcus mutans (S. mutans), Streptococcus thermophilus (S. thermophilus), Campylobacter jejuni (C. jejuni), Neisseria meningitidis (N. meningitides), Pasteurella multocida (P. multocida), and Francisella novicida (F. novicida). Therefore, the term "Cas9" refers to an RNA-guided DNA nuclease comprising a DNA binding domain and two nuclease domains, wherein the gene encoding Cas9 can be derived from any suitable bacterium.

[0106] The Cas9 protein has at least two nuclease domains: one nuclease domain is similar to the HNH endonuclease, and the other is similar to the Ruv endonuclease domain. The HNH-type domain appears to be responsible for cutting the DNA strand complementary to the crRNA, while the Ruv domain cuts the non-complementary strand. The Cas 9 nuclease can be engineered so that only one nuclease domain is functional, thereby creating a Cas nickase (see Jinek et al. (2012) Science 337:816). The nickase can be produced by specific mutations of amino acids in the catalytic domain of the enzyme, or by truncating some or all of the domains so that they are no longer functional. Since Cas 9 contains two nuclease domains, this method can be used on either domain. By using two such Cas 9 nickases, double-strand breaks can be achieved in the target DNA. The nickase will cut one strand of the DNA separately, and the use of two nickases will produce double-strand breaks.

[0107] The need for a crRNA-tracrRNA complex can be avoided by using an engineered "single guide RNA" (sgRNA) containing a hairpin that is typically formed by annealing crRNA and tracrRNA (see Jinek et al. (2012) Science 337:816 and Cong et al. (2013) Science 339(6121):819-823, Sciencexpress / 10.1126 / science.1231143). In S. pyrogenes, an engineered tracrRNA:crRNA fusion or sgRNA guides 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, which consists of the Cas9 protein and an engineered sgRNA containing a PAM sequence, has been used for RNA-guided genome editing (see Ramalingam (2013) Genome Biol. 14(2):107) and has been used in vivo for zebrafish embryo genome editing (see Hwang et al. (2013) Nature Biotechnology 31(3):227), with editing efficiencies similar to those of ZFNs and TALENs.

[0108] The major products of CRISPR loci appear to be short RNAs containing invader-targeting sequences, termed guide RNAs or prokaryotic silencing RNAs (psiRNAs) based on their postulated role in this pathway (Makarova et al. (2006) Biol. Direct 1:7; Hale et al. (2008) RNA 14:2572-2579). RNA analysis indicates that CRISPR locus transcripts are cleaved within the repetitive sequence to release an RNA intermediate of approximately 60 to 70 nt, which contains a single invader targeting sequence and flanking repetitive sequence fragments (Tang et al. (2002) Proc. Natl. Acad. Sci. 99:7536-7541; Tang et al. (2005) Mol. Microbiol. 55:469-481; Lillestol et al. (2006) Archaea 2:59-72; Brouns et al. (2008) Science 321:960-964; Hale et al. (2008) RNA 14:2572-2579). In the archaeon Pyrococcus furiosus, these intermediate RNAs are further processed into abundant, stable mature psiRNAs of approximately 35 to 45 nt (Hale et al. (2008) RNA 14:2572-2579).

[0109] The need for a crRNA-tracrRNA complex can be avoided by using an engineered "single guide RNA" (sgRNA) containing a hairpin that is typically formed by annealing crRNA and tracrRNA (see Jinek et al. (2012) Science 337:816 and Cong et al. (2013) Science 339(6121):819-823, Sciencexpress / 10.1126 / science.1231143). In S. pyrogenes, an engineered tracrRNA:crRNA fusion or sgRNA guides 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, which consists of the Cas9 protein and an engineered sgRNA containing a PAM sequence, has been used for RNA-guided genome editing (see Ramalingam (2013) Genome Biol. 14(2):107) and has been used in vivo for zebrafish embryo genome editing (see Hwang et al. (2013) Nature Biotechnology 31(3):227), with editing efficiencies similar to those of ZFNs and TALENs.

[0110] Chimeric or sgRNA can be engineered to include a sequence complementary to any desired target. In some embodiments, the length of the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides or longer. In some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. In some embodiments, RNA comprises 22 bases complementary to the target in the form of G[n19], followed by a protospacer adjacent motif (PAM) in the form of NGG or NAG, for use in the S.pyogenes CRISPR / Cas system. Thus, in one approach, sgRNAs can be designed by exploiting known ZFN targets in a gene of interest by: (i) aligning the recognition sequences of the ZFN heterodimers with reference sequences of the relevant genome (human, mouse, or a particular plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) determining the position of the motif G[N20]GG closest to the spacer region (when more than one such motif overlaps the spacer, the one centered relative to the spacer is selected); (iv) using this motif as the core of the sgRNA. This approach advantageously relies on a validated nuclease target. Alternatively, sgRNAs can be designed to target any region of interest by simply identifying a suitable target sequence that conforms to the formula G[n20]GG. Along with the complementary 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 tail can be +67 to +85 nucleotides, or any number therebetween, preferably +85 nucleotides in length. Truncated sgRNAs, "tru-gRNAs" (see Fu et al. (2014) Nature Biotech 32(3):279) can also be used. In tru-gRNAs, the length of the complementary region is reduced to 17 or 18 nucleotides.

[0111] In addition, alternative PAM sequences may be used, where S. pyogenes Cas9 is used, the PAM sequence may be NAG instead of NGG (Hsu (2013) Nature Biotech 31:827-832, doi:10.1038 / nbt.2647). Other PAM sequences may also include those lacking an initial G (Sander and Joung (2014) Nature Biotech 32(4):347). In addition to the Cas9 PAM sequence encoded by S. pyogenes, other PAM sequences may be used that are specific for Cas9 proteins from other bacterial sources. For example, the PAM sequences shown below (adapted from Sander and Joung, supra, and Esvelt et al. (2013) Nat Meth 10(11):1116) are specific for these Cas9 proteins:

[0112]

[0113] Therefore, suitable target sequences for the S. pyogenes CRISPR / Cas system can be selected according to the following guidelines: [n17, n18, n19, or n20] (G / A) G. Alternatively, the PAM sequence can follow the guideline G [n17, n18, n19, n20] (G / A) G. For Cas9 derived from bacteria other than S. pyogenes, the same guide can be used, with the alternative PAM replaced with the S. pyogenes PAM sequence.

[0114] It is most preferred to select a target sequence with the highest probability of specificity to avoid potential off-target sequences. These unwanted off-target sequences can be identified by considering the following attributes: i) similarity of the target sequence followed by a PAM sequence known to work with the employed Cas9 protein; ii) similar target sequences with fewer than three mismatches to the desired target sequence; iii) target sequences similar to those in ii), where the mismatches are all located in the PAM distal region rather than the PAM proximal region (there is evidence that the 1-5 nucleotides directly adjacent to or close to the PAM, sometimes referred to as the "seed" region (Wu et al. (2014) Nature Biotech 32:670-676, doi:10.1038 / nbt2889) are most critical for recognition, so putative off-target sites with mismatches in the seed region may be least likely to be recognized by sg RNA); and iv) similar target sequences where the mismatches are discontinuously spaced or spaced greater than four nucleotides (Hsu et al. (2014) Cell 157(6):1262-78). Therefore, by analyzing the number of potential off-target sites in the genome using any CRIPSR / Cas system, appropriate target sequences for sgRNAs can be identified using these criteria.

[0115] In some embodiments, a CRISPR-Cpf1 system is used. The CRISPR-Cpf1 system identified in Francisella spp is a type 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Despite functional conservation, Cpf1 and Cas9 differ in many ways, including in their guide RNA and substrate specificity (see Fagerlund et al., (2015) Genom Bio 16:251). The main difference between Cas9 and Cpf1 proteins is that Cpf1 does not utilize tracrRNA, and therefore only crRNA is required. FnCpf1 crRNA is 42–44 nucleotides in length (19 nucleotide repeats and 23–25 nucleotide spacers), and contains a single stem loop that tolerates sequence changes that maintain secondary structure. In addition, Cpf1 crRNA is significantly shorter than the approximately 100 nucleotides of engineered sgRNA required for Cas9, and the PAM requirement of FnCpf1 is 5'-TTN-3' and 5'-CTA-3' on the displacement strand. Although both Cas9 and Cpf1 produce double-strand breaks in the target DNA, Cas9 uses its RuvC- and HNH-like domains to produce blunt-end cuts within the seed sequence of the guide RNA, while Cpf1 uses the RuvC-like domain to produce misplaced cuts outside the seed. Because the misplaced cuts produced by Cpf1 are away from the critical seed region, NHEJ will not destroy the target site, thus ensuring that Cpf1 can continue to cut the same site until the desired HDR recombination event occurs. Therefore, in the methods and compositions described herein, it should be understood that the term "Cas" includes Cas9 and Cpf1 proteins. Therefore, the "CRISPR / Cas system" used in this article refers to the CRISPR / Cas and / or CRISPR / Cpf1 system, and also includes nuclease, nickase and / or transcription factor systems.

[0116] In some embodiments, other Cas proteins can be used. Some exemplary Cas proteins include Cas9, Cpf1 (also known as Cas12a), C2c1, C2c2 (also known as Cas13a), C2c3, Cas1, Cas2, Cas4, CasX and CasY; and include their engineered and natural variants (Burstein et al. (2017) Nature 542: 237-241) such as HF1 / spCas9 (Kleinstiver et al. (2016) Nature 529: 490-495; Cebrian-Serrano and Davies (2017) MammGenome28(7): 247-261); split Cas9 system (Zetsche et al. (2015) Nat Biotechnol33(2): 139-142), trans-splicing Cas9 based on the intron-extron system (Troung et al. (2015) Nucl Acid Res 43(13):6450-8); mini-SaCas9 (Ma et al. (2018) ACS Synth Biol 7(4):978-985). Therefore, in the methods and compositions described herein, it should be understood that the term "Cas" includes all Cas variant proteins, whether natural or engineered.

[0117] In certain embodiments, the 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 a common qualitative biological property with a native sequence polypeptide. "Functional derivatives" include, but are not limited to, fragments of native sequences or derivatives of native sequence polypeptides and fragments thereof, provided that they have a common biological activity with the corresponding native sequence polypeptide. The biological activity contemplated herein is the ability of a functional derivative to hydrolyze a DNA substrate into fragments. The term "derivative" encompasses amino acid sequence variants, covalently modified forms, and fusions thereof of a polypeptide. In some aspects, a functional derivative may comprise a single biological property of a naturally occurring Cas protein. In other aspects, a functional derivative may comprise a subset of the biological properties of a naturally occurring Cas protein. Suitable derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, covalently modified forms, or fragments thereof of Cas proteins. Cas proteins including Cas proteins or fragments thereof and derivatives of Cas proteins or fragments thereof may be obtained from cells or by chemical synthesis or by a combination of these two methods. 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, wherein the genetic engineering enables the cell to produce an endogenous Cas protein at a higher expression level or produce a Cas protein from an exogenously introduced nucleic acid, wherein the nucleic acid encodes a Cas that is the same as or different from the endogenous Cas. In some cases, the cell does not naturally produce a Cas protein, but is genetically engineered to produce a Cas protein.

[0118] Exemplary CRISPR / Cas nuclease systems that target specific genes are disclosed, for example, in U.S. Patent Publication No. 2015 / 0056705.

[0119] Thus, the nuclease comprises a DNA binding domain that, in conjunction with a nuclease domain that cleaves DNA, binds specifically to a target site in any gene into which it is desired to insert the donor (transgene).

[0120] Fusion molecules

[0121] The DNA binding domain can be fused to any other molecule (e.g., polypeptide) for use in the methods described herein. In certain embodiments, the method employs a fusion molecule comprising at least one DNA binding molecule (e.g., ZFP, TALE or single guide RNA) and a heterologous regulatory (functional) domain (or its functional fragment).

[0122] In some embodiments, the functional domain includes a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activators, repressors, coactivators, co-repressors), silencers, oncogenes (such as myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; Chromatin-related proteins and their modifiers (such as kinases, acetylases and deacetylases); and DNA modification enzymes (such as methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See, for example, U.S. Patent Publication No. 2013 / 0253040, which is incorporated herein by reference in its entirety.

[0123] Suitable domains for achieving activation include HSV VP16 activation domain (see, e.g., Hagmann et al. (1997) J. Virol. 71:5952-5962), nuclear hormone receptors (see, e.g., Torchia et al. (1998) Curr. Opin. Cell. Biol. 10:373-383); the p65 subunit of nuclear factor kappa B (Bitko and Barik (1998) J. Virol. 72:5610-5618 and Doyle and Hunt (1997) Neuroreport 8:2937-2942); Liu et al. (1998) Cancer Gene Ther. 5: 3-28), or artificial chimeric functional domains, such as VP64 (Beerli et al. (1998) Proc. Natl. Acad. Sci. USA 95: 14623-33), and degradation determinants (degron) (Molinari et al. (1999) EMBO J. 18: 6439-6447). Other exemplary activation domains include Oct 1, Oct-2A, Sp1, AP-2 and CTF1 (Seipel et al. (1992) EMBO J. 11: 4961-4968) and p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, e.g., 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. Other exemplary activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7 and -8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1.See, e.g., Ogawa et al. (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

[0124] Exemplary repressor domains include, but are not limited to, KRAB A / B, KOX, TGF-β-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, DNMT family members (e.g., DNMT1, DNMT3A, DNMT3B), Rb and MeCP2. See, e.g., 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. Other exemplary repressor domains include, but are not limited to, ROM2 and AtHD2A. See, e.g., Chem et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

[0125] Fusion molecules are constructed by cloning and biochemical coupling methods well known to those skilled in the art. Fusion molecules include a DNA binding domain and a functional domain (e.g., a transcriptional activation or repression domain). Fusion molecules also optionally include a nuclear localization signal (e.g., from SV40 medium T-antigen) and an epitope tag (e.g., FLAG and hemagglutinin). Fusion proteins (and their encoding nucleic acids) are designed so that the translation reading frame is retained between the components of the fusion.

[0126] Fusions between the functional domain (or functional fragment thereof) polypeptide component on the one hand and the non-protein DNA binding domain (e.g., antibiotic, intercalator, minor groove binder, nucleic acid) on the other hand are constructed by biochemical coupling methods known to those skilled in the art. See, e.g., Pierce Chemical Company (Rockford, IL) product catalog. Methods and compositions for generating fusions between minor groove binders and polypeptides have been described. Mapp et al. (2000) Proc. Natl. Acad. Sci. USA 97: 3930-3935.

[0127] The fusion molecules can be formulated with pharmaceutically acceptable carriers / vehicles, as known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 17th edition, 1985; and co-owned International Patent Publication No. WO 00 / 42219.

[0128] The functional components / domains of the fusion molecule can be selected from any of a variety of different components that can affect gene transcription once the fusion molecule is bound to the target sequence via its DNA binding domain. Thus, functional components can include, but are not limited to, various transcription factor domains, such as activators, repressors, co-activators, co-repressors, and silencers.

[0129] In certain embodiments, the fusion molecule comprises one or more ZFP-TF (repressor), wherein the ZFP is operably linked to a transcriptional repression domain. Non-limiting examples of repression domains include KOX (KRAB) domains, etc. Other elements may also be included, such as NLS, and any linker may be used between zinc finger domains and / or between ZFP and repression domains (and / or any other elements). The polynucleotides encoding these ZFP-TF repressors may further include other elements, such as promoters, enhancers, insulators, etc., that drive ZFP-TF expression.

[0130] Table 3 shows the polynucleotide and amino acid sequences of exemplary ZFP-TFs comprising the ZFPs described herein (identified by name in the first column). The recognition helix region sequences (Table 1) are underlined in Table 3; the NLS peptide is in bold and the repression domain is in italics.

[0131] Table 3: Nucleotide and amino acid sequences of ZFP-TFs

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] The polynucleotides encoding the repressors described herein can be delivered using any suitable expression vector, including but not limited to viruses (e.g., AAV, Ad, etc.) and non-viral vectors (e.g., mRNA, plasmids, minicircles, etc.). The expression vector may include other elements, such as a nuclear localization signal (NLS) and / or a promoter (e.g., a constitutive promoter, such as a CMV promoter) that drives the expression of the repressor. One or more polynucleotides (e.g., expression vectors) of the same or different forms (e.g., viruses and / or non-viral vectors) can be delivered to a subject and can be formulated in one or more pharmaceutical compositions. The polynucleotides described herein can be maintained additionally (extrachromosomally) and / or can be stably integrated into cells after delivery.

[0139] In certain embodiments, the fusion protein comprises a DNA binding domain and a nuclease domain to create a functional entity that is capable of recognizing its intended nucleic acid target through its engineered (ZFP or TALE) DNA binding domain and creating a nuclease (e.g., zinc finger nuclease or TALE nuclease) that causes DNA to be cleaved near the DNA binding site through nuclease activity.

[0140] Thus, the methods and compositions described herein are broadly applicable and may involve any nuclease of interest. Non-limiting examples of nucleases include meganucleases, TALENs, and zinc finger nucleases. Nucleases may comprise a heterologous DNA binding domain and a cleavage domain (e.g., zinc finger nucleases; TALENs; meganuclease DNA binding domains with heterologous cleavage domains), or alternatively, the DNA binding domains of naturally occurring nucleases may be altered to bind to a selected target site (e.g., with a meganuclease engineered to bind to a site different from a cognate binding site).

[0141] 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 HTT DNA binding domain described herein include domains from any restriction enzyme, such as a type IIS restriction enzyme (e.g., FokI). In certain embodiments, the cleavage domain is a cleavage half-domain that requires dimerization for cleavage activity. See, e.g., U.S. Pat. Nos. 8,586,526; 8,409,861 and 7,888,121, which are incorporated herein by reference in their entirety. In general, if the fusion protein comprises a cleavage half-domain, two fusion proteins are required for cleavage. Alternatively, a single protein comprising two cleavage half-domains can be used. The two cleavage half-domains can be derived from the same endonuclease (or a functional fragment thereof), or each cleavage half-domain can be derived from a different endonuclease (or a functional fragment thereof). Furthermore, the target sites of the two fusion proteins are preferably arranged relative to each other such that binding of the two fusion proteins to their respective target sites places the cleavage half-domains in a spatial orientation relative to each other that allows the cleavage half-domains to form a functional cleavage domain (e.g., by dimerization).

[0142] The nuclease domain can also be derived from any meganuclease (homing endonuclease) domain with cleavage activity and can also be used with the nucleases described herein, including but not limited to I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.

[0143] In certain embodiments, the nuclease comprises a compact TALEN (cTALEN). There is a single-stranded fusion protein that connects the TALE DNA binding domain to the TevI nuclease domain. The fusion protein can act as a nickase positioned by the TALE region, or can produce a double-strand break, depending on the position of the TALE DNA binding domain relative to the nuclease domain of the meganuclease (e.g., TevI) (see Beurdeley et al. (2013) Nat Comm: 41762 DOI: 10.1038 / ncomms2782). In other embodiments, the TALE-nuclease is a megaTAL. These megaTAL nucleases comprise a fusion protein of a TALE DNA binding domain and a meganuclease cleavage domain. The meganuclease cleavage domain is active as a monomer and does not require dimerization to obtain activity. (See Boissel et al. (2013) Nucl Acid Res 42 (4): 2591-601, doi: 10.1093 / nar / gkt1224).

[0144] In addition, the nuclease domain of a mega-nuclease may also have DNA binding functionality.Any TALEN may be used in combination with other TALENs (eg, one or more TALENs (cTALENs or FokI-TALENs) with one or more mega-TALEs) and / or ZFNs.

[0145] In addition, the cleavage domain may include one or more changes compared to the wild type, such as forming an obligate heterodimer that reduces or eliminates off-target cleavage effects. See, e.g., U.S. Patent Nos. 7,914,796; 8,034,598 and 8,623,618, which are incorporated herein by reference in their entireties.

[0146] Nucleases as described herein can produce double-stranded or single-stranded breaks in double-stranded targets (e.g., genes). The generation of single-stranded breaks ("nicks") is described in, for example, U.S. Patent No. 8,703,489, which is incorporated herein by reference, which describes how a mutation in the catalytic domain of one of the nuclease domains results in a nickase.

[0147] A nuclease (cleavage) domain or cleavage half-domain can be any portion of a protein that retains cleavage activity, or retains the ability to multimerize (e.g., dimerize) to form a functional cleavage domain.

[0148] Alternatively, the nuclease can be assembled in vivo at the nucleic acid target site using a technique known as "split-enzyme" (see, e.g., U.S. Patent Publication No. 2009 / 0068164). The components of such split enzymes can be expressed on separate expression constructs, or can be linked to an open reading frame where the individual components are separated from one another, e.g., by a self-cleaving 2A peptide or IRES sequence. The components can be separate zinc finger binding domains or domains of a meganuclease nucleic acid binding domain.

[0149] Nuclease activity can be screened prior to use, for example, in a yeast-based chromosomal system as described in US Patent Publication No. 2009 / 0111119. Nuclease expression constructs can be readily designed using methods known in the art.

[0150] The expression of the fusion protein can be under the control of a constitutive promoter or an inducible promoter, such as a galactokinase promoter, which is activated (de-repressed) in the presence of raffinose and / or galactose and repressed in the presence of glucose. In certain embodiments, the promoter self-regulates the expression of the fusion protein, for example by including a high affinity binding site. See, for example, U.S. Patent No. 9,624,498.

[0151] deliver

[0152] Proteins and / or polynucleotides (e.g., HTT repressors) and compositions comprising proteins and / or polynucleotides described herein can be delivered to target cells by any suitable means, including, for example, by injection of protein, via mRNA, and / or using expression constructs (e.g., plasmids, lentiviral vectors, AAV bodies, Ad vectors, etc.). In a preferred embodiment, the repressor is delivered using AAV9 or AAV6.

[0153] Methods for delivering proteins containing the zinc finger proteins described herein are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219 and 7,163,824, the entire disclosures of which are incorporated herein by reference in their entirety.

[0154] Any vector system may be used, including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors, etc. See also, for example, U.S. Patent Nos. 8,586,526; 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219 and 7,163,824, which are incorporated herein by reference in their entirety. In addition, it is apparent that any of these vectors may contain one or more DNA binding protein encoding sequences. Thus, when one or more HTT repressors are introduced into cells, sequences encoding protein components and / or polynucleotide components may be carried on the same vector or on different vectors. When multiple vectors are used, each vector may contain sequences encoding one or more HTT repressors or components thereof.

[0155] Conventional viral and non-viral transgenic methods can be used to introduce nucleic acids encoding engineered HTT repressors into cells (e.g., mammalian cells) and target tissues. Such methods can also be used to give cells nucleic acids encoding such repressors (or components thereof) in vitro. In certain embodiments, the nucleic acids encoding repressors given are used for in vivo or ex vivo gene therapy applications. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles (e.g., liposomes or poloxamer). Viral vector delivery systems include DNA and RNA viruses, which have an additional or integrated genome after delivery to cells. Gene therapy procedures are reviewed in Anderson (1992) Science 256:808-813; Nabel and Felgner (1993) TIBTECH 11:211-217; Mitani and Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al. (1995) Current Topics in Microbiology and Immunology Doerfler and (eds); and Yu et al. (1994) Gene Therapy 1:13-26.

[0156] Non-viral delivery methods of nucleic acids include electroporation, lipofection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and reagent-enhanced DNA uptake. The sonoporous effect of, 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 in the form of mRNA. It is also preferred to use capped mRNA to increase translation efficiency and / or mRNA stability. Especially preferred ARCA (anti-inversion cap analog) caps or their variations. See U.S. Patent Nos. 7,074,596 and 8,153,773, which are incorporated herein by reference.

[0157] Other exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockwell, Maryland), BTX Molecular Delivery Systems (Holliston, Massachusetts), and Copernicus Therapeutics Inc. (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described in, e.g., U.S. Pat. Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam TM 、Lipofectin TM and Lipofectamine TM RNAiMAX). Cationic and neutral lipids suitable for efficient receptor-recognition lipid transfection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91 / 17424 and WO 91 / 16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

[0158] The preparation of lipid:nucleic acid complexes (including targeted liposomes, such as immunolipid complexes) is well known to those skilled in the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992 ... Res. 52:4817-4820; U.S. Patent Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

[0159] Other methods of delivery include packaging the nucleic acid to be delivered into EnGeneIC delivery vectors (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies, wherein one arm of the antibody is specific to the target tissue, and the other arm is specific to the EDV. The antibody brings the EDV to the surface of the target cell, and then the EDV enters the cell by endocytosis. Once inside the cell, the contents are released (see MacDiarmid et al. (2009) Nature Biotechnology 27 (7): 643).

[0160] The use of RNA or DNA virus-based systems to deliver nucleic acids encoding engineered ZFP, TALE or CRISPR / Cas systems utilizes highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. The viral vector can be directly administered to the patient (in vivo) or it can be used to treat cells in vitro, and the modified cells are administered to the patient (ex vivo). Conventional virus-based systems for delivering ZFP, TALE or CRISPR / Cas systems include, but are not limited to, retrovirus, lentivirus, adenovirus, adeno-associated, vaccinia and herpes simplex virus vectors for gene delivery. Integration in the host genome can be performed using retrovirus, lentivirus and adeno-associated virus gene delivery methods, typically resulting in long-term expression of the inserted transgene. In addition, high transduction efficacy has been observed in many different cell types and target tissues.

[0161] The tropism of retroviruses can be changed by introducing foreign envelope proteins and expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually produce high viral titers. The choice of retroviral transgenic system depends on the target tissue. Retroviral vectors contain cis-acting long terminal repeats, which have the ability to package foreign sequences up to 6-10kb. The minimal cis-acting LTR is sufficient for replication and packaging of the vector, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgenic expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; International Patent Publication No. WO 94 / 026877).

[0162] In applications where transient expression is preferred, adenovirus-based systems may be used. Adenovirus-based vectors are able to achieve extremely high transduction efficiencies in many cell types and do not require cell division. High titers and high levels of expression have been achieved using such vectors. The vectors 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, e.g., for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy (see, e.g., West et al., Virology 160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No. WO 93 / 24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260; Tratschin et al. (1984) Mol. Cell. Biol. 4:2072-2081; Hermonat and Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol. 63:03822-3828.

[0163] There are currently at least six viral vector approaches available for gene delivery in clinical trials that employ methods involving complementation of defective vectors by inserting the gene into helper cell lines to produce the transducing agent.

[0164] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94(22):12133-12138). PA317 / pLASN was the first therapeutic vector used in gene therapy trials. (Blaese et al. (1995) Science 270:475-480). Transduction efficiencies of 50% or more have been observed in MFG-S packaged vectors. (Ellem et al. (1997) Immunol Immunother. 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2).

[0165] Recombinant adeno-associated virus vector (rAAV) is a promising alternative gene delivery system based on defective and non-pathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from plasmids that retain only the AAV145bp inverted terminal repeat sequence flanking the transgenic expression cassette. Efficient gene transfer and stable transgene delivery are key features of this vector system, which is attributed to integration into the genome of the transduced cells. (Wagner et al. (1998) Lancet 351 (9117): 1702-3; Kearns et al. (1996) Gene Ther. 9: 748-55). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9 and AAV rh10 and pseudotyped AAV, such as AAV2 / 8, AAV2 / 5 and AAV2 / 6, can also be used according to the present invention. In a preferred embodiment, AAV9 or AAV6 capsids are used.

[0166] Replication-deficient recombinant adenoviral vectors (Ad) can be produced in high titers and easily infect a variety of different cell types. Most adenoviral vectors are engineered so that the transgene replaces the Ad E1a, E1b and / or E3 genes; the replication-deficient vector is then propagated in human 293 cells that provide the missing transgene function. Ad vectors can transduce a variety of tissue types in vivo, including non-dividing, differentiated cells, such as those found in the liver, kidney, and muscle. Traditional Ad vectors have a large carrying capacity. An example of the use of Ad vectors in clinical trials involves polynucleotide therapy for anti-tumor immunity using intramuscular injection (Sterman et al. (1998) Hum. Gene Ther. 7: 1083-9). Other examples of the use of adenoviral vectors for transgenesis in clinical trials include Rosenecker et al. (1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther. 9(7):1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez et al. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther. 5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

[0167] Packaging cells are used to form viral particles capable of infecting host cells. The cells include 293 cells, which package adenoviruses, and ψ2 cells or PA317 cells, which package retroviruses. Viral vectors for gene therapy are usually produced by a production cell line that packages nucleic acid vectors into viral particles. The vector usually contains the minimum viral sequence required for packaging and subsequent integration into the host (if feasible), and other viral sequences are replaced by expression cassettes encoding proteins to be expressed. The lost viral function is provided in trans by the packaging cell line. For example, if there is an AAV replication protein, the AAV vector used for gene therapy usually only processes the reverse terminal repeat (ITR) sequence from the AAV genome, which is required for packaging and integration into the host genome. The viral genome is trans-supplemented to the cell line, which includes a helper plasmid encoding other AAV genes (i.e., rep and cap) but lacking ITR sequences. The cell line is also infected with adenovirus (as an auxiliary). The helper virus promotes the replication of the AAV genome and expresses the AAV gene from the helper plasmid. Because of the lack of ITR sequences, the helper plasmid is not packaged in a significant amount. Adenovirus contamination can be reduced by, for example, heat treatment (adenovirus is more sensitive to heat treatment than AAV).

[0168] In many gene therapy applications, it is desirable to deliver gene therapy vectors with high tropism to specific tissue types. Therefore, viral vectors can be modified to have tropism for a given cell type by expressing ligands as proteins on the outer surface of the virus that are fused to viral coat proteins. Select ligands with affinity for receptors known to be present on the cell type of interest. For example, Han et al. (1995), Proc. Natl. Acad. Sci. USA 92: 9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, where the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell surface receptor. For example, filamentous phages can be engineered to display antibody fragments (e.g., FAB or Fv) with specific binding affinity for almost any selected cell receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. The vectors can be engineered to contain specific uptake sequences that facilitate uptake by specific target cells.

[0169] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion, including direct injection into the brain) or local administration, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as explanted cells (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells from an individual patient, which are then reimplanted into the patient, typically after selection of cells into which the vector has been introduced.

[0170] In certain embodiments, the compositions described herein (e.g., polynucleotides and / or proteins) are delivered directly in vivo. The compositions (cells, polynucleotides and / or proteins) can be administered directly to the central nervous system (CNS), including but not limited to direct injection into the brain or spinal cord. One or more regions of the brain can be targeted, including but not limited to the hippocampus, substantia nigra, nucleus basalis of Menert (NBM), striatum, and / or cortex. Instead of CNS delivery or in addition to CNS delivery, the composition can be systemically administered (e.g., intravenous, intraperitoneal, intracardiac, intramuscular, intrathecal, subcutaneous, and / or intracranial infusion). Methods and compositions for delivering compositions described herein directly to an object (including direct delivery to the CNS) include but are not limited to direct injection (e.g., stereotactic injection) via a needle assembly. Such methods are described in, for example, U.S. Patent Nos. 7,837,668; 8,092,429, which relate to delivering compositions (including expression vectors) to the brain, and are also described in U.S. Patent Publication No. 2006 / 0239966, which is incorporated herein by reference in its entirety.

[0171] The effective amount administered may vary from patient to patient and may vary depending on the mode of administration and site of administration. Thus, the effective amount may be determined by one of ordinary skill in the art. After sufficient time has been given to express the repressor (e.g., typically 4-15 days), the serum or other tissue levels of the therapeutic polypeptide are analyzed and compared to the initial levels before administration, which will determine whether the dose is too low, within the correct range, or too high. In certain embodiments, when a viral vector such as AAV is used, the dose administered is 1×10 7 Up to 5×10 15 vg / ml (or any value therebetween), more preferably 1×10 11 Up to 1×10 14 vg / ml (or any value therebetween), more preferably 1×10 12 Up to 1×10 13 vg / ml (or any value therebetween). AAV doses may also be administered per kg or per striatum of the subject, including 1×10 7 Up to 5×10 15 vg / kg or vg / striatum (or any value therebetween), more preferably 1×10 7Up to 1×10 13 vg / kg or vg / striatum (or any value therebetween), more preferably 1×10 8 Up to 1×10 12 Any dose of vg / kg or vg / striatum (or any value therebetween).

[0172] To deliver ZFPs directly to the human brain using recombinant adeno-associated virus (rAAV) vectors, 1 × 10 7 -5×10 15 vg / mL (or any value therebetween, including, for example, 1×10 11 Up to 1×10 14 Any value between vg / ml or 1×10 12 Up to 1×10 13 vg / mL) vector genome dose range. The dose range is 1×10 7 Up to 5×10 15 vg / kg or vg / striatum (or any value therebetween), more preferably 1×10 7 Up to 1×10 13 vg / kg or vg / striatum (or any value therebetween), more preferably 1×10 8 Up to 1×10 12 vg / kg or vg / striatum (or any value therebetween). As described, doses for other brain structures and different delivery regimens may vary. Methods for delivering rAAV vectors directly to the brain are known in the art. For example, see U.S. Patent Nos. 9,089,667; 9,050,299; 8,337,458; 8,309,355; 7,182,944; 6,953,575; and 6,309,634.

[0173] Ex vivo cell transfection for diagnosis, research or for gene therapy (e.g., by re-infusing transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are separated from the subject's body, transfected with at least one HTT repressor or a component thereof, and re-infused back into the subject's body (e.g., a patient). In a preferred embodiment, AAV9 is used to deliver one or more nucleic acids of HTT repressors. In other embodiments, one or more nucleic acids of HTT repressors are delivered in the form of mRNA. It is also preferred to use capped mRNA to increase translation efficiency and / or mRNA stability. ARCA (anti-inversion cap analog) caps or variations thereof are particularly preferred. See U.S. Patent Nos. 7,074,596 and 8,153,773, which are incorporated herein by reference in their entirety. A variety of cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)). The references cited herein discuss how to isolate and culture cells from a patient).

[0174] In one embodiment, stem cells are used in ex vivo protocols for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (e.g., a donor of the cells), where they will be engrafted into the bone marrow. Methods for using cytokines such as GM-CSF, IFN-γ, and TNF-α to differentiate CD34+ cells into clinically important immune cell types in vitro are known (see Inaba et al. (1992) J. Exp. Med. 176: 1693-1702).

[0175] Stem cells can be isolated for transduction and differentiation using known methods. For example, stem cells can also be isolated from bone marrow cells by screening the bone marrow cells with antibodies that bind to 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. (1992) J. Exp. Med. 176: 1693-1702).

[0176] In some embodiments, modified stem cells can be used. For example, neuronal stem cells made resistant to apoptosis can be used as therapeutic compositions, wherein the stem cells also contain the ZFP TF of the present invention. Resistance to apoptosis can come from, for example, knocking out BAX and / or BAK in stem cells using BAX- or BAK-specific TALEN or ZFN (see, U.S. Patent Application No. 8,597,912), or those that interfere with caspases, such as caspase 6-specific ZFNs. These cells can be transfected with ZFP TFs or TALE TFs known to regulate mutant or wild-type HTT.

[0177] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic ZFP nucleic acids can also be administered directly to an organism for in vivo cell transduction. Alternatively, naked DNA can be administered. Administration is by any conventional route commonly used to introduce molecules and ultimately contact them with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable methods for administering the nucleic acids are available and are well known to those skilled in the art, and, although more than one route may be used to administer a particular composition, a particular route will generally provide a more direct and more effective response than another route.

[0178] Methods for introducing DNA into hematopoietic stem cells are disclosed in, for example, U.S. Pat. No. 5,928,638. + Cells) include adenovirus type 35.

[0179] Vectors suitable for introducing transgenes into immune cells (e.g., T cells) include non-integrating lentiviral vectors. See, for example, Naldini 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.

[0180] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, a variety of suitable pharmaceutical composition formulations are available, as described below (see, e.g., Remington's Pharmaceutical Sciences; 17th edition, 1989).

[0181] As described above, disclosed methods and compositions can be used for 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 may include but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, insect cells such as Spodoptera fugiperda (Sf), and fungal cells such as yeast (Saccharomyces), Pichia (Pichia) and fission yeast (Schizosaccharomyces). Progeny, variants, and derivatives of these cell lines may also be employed.In a preferred embodiment, the methods and compositions are delivered directly to brain cells, such as the striatum.

[0182] application

[0183] The HTT binding molecules described herein (e.g., ZFPs, TALEs, CRISPR / Cas systems, Ttago, etc.) and nucleic acids encoding them can be used in a variety of applications. These applications include therapeutic methods in which HTT binding molecules (including nucleic acids encoding DNA binding proteins) are administered to a subject (e.g., AAV, such as AAV9) and used to regulate the expression of a target gene (and thus a protein) in the subject. Regulation can be in the form of suppression, such as suppression of mHTT that causes the HD disease state. Alternatively, when activation or increased expression of an endogenous cellular gene can improve the disease state, regulation can be in the form of activation. In yet other embodiments, regulation can be cutting (e.g., by one or more nucleases), for example, for inactivating a mutant HTT gene. As described above, for such applications, the HTT binding molecules or more typically the nucleic acids encoding them are formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier.

[0184] HTT binding molecules or vectors encoding them, alone or in combination with other suitable ingredients (e.g., liposomes, nanoparticles, or other ingredients known in the art), can be formulated into aerosol formulations (i.e., they can be "nebulized") for administration by inhalation. Aerosol formulations can be placed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, etc. Formulations suitable for parenteral administration, such as by intravenous, intramuscular, intradermal, and subcutaneous routes, include: aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The composition can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, intracranially, or intrathecally. Formulations of the compound may be present in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injections and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

[0185] The dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient for a prolonged period of time. The dose depends on the potency and K of the particular HTT binding molecule employed. d , target cells, the condition of the patient, and the body weight or surface area of ​​the patient to be treated. The size of the dose may also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular compound or vector to a particular patient.

[0186] Beneficial treatment responses can be measured in a variety of ways. For example, improvements in Huntington's-related movement disorders, such as involuntary twitches or twists, muscle problems, such as stiffness or muscle contractures (dystonia), slow or abnormal eye movements, impaired gait, posture, and balance, difficulty expressing speech, or impairment of swallowing and voluntary movements can be measured. Other impairments, such as cognitive and psychiatric disorders, can also be monitored to look for signs of improvement associated with treatment. The UHDRS scale can be used to quantify the clinical features of the disease. Other biomarker measurements can also be used to determine outcomes, including measuring mHTT in CSF.

[0187] Treatment is particularly important for presymptomatic patients because it provides an opportunity to treat the disease before the extensive neurodegeneration that occurs in HD. This damage begins before the onset of overt symptoms described above. HD pathology primarily involves the toxic effects of mutant HTT in striatal medium spiny neurons. These medium spiny neurons express high levels of phosphodiesterase 10A (PDE10A), which regulates cAMP and cGMP signaling cascades involving gene transcription factors, neurotransmitter receptors, and voltage-gated channels (Niccolini et al. (2015) Brain 138:3016-3029), and PDE10A expression is known to be reduced in HD mice, as has been found in post-mortem studies in humans. Recently, positron emission tomography (PET) ligands (e.g., ligands for the PDE10A enzyme) have been developed. 11 C-IMA107, see, e.g., Niccolini et al., supra; 18 FMNI-659, see, e.g., Russell et al. (2014) JAMA Neurol 71(12):1520-1528), and these molecules have been used to evaluate pre-symptomatic HD patients. Studies have shown that PDE10A levels in HD patients are altered even before symptoms appear. Therefore, PDE10A levels can be assessed by PET before, during, and after treatment to measure the therapeutic efficacy of the compositions of the invention. "Therapeutic efficacy" can refer to improvements in clinical and molecular measures, and can also refer to protection of patients from any further decline in medium spiny neuron function or increase in spiny neuron loss, or further development of overt clinical manifestations associated with HD.

[0188] The following examples relate to exemplary embodiments of the present disclosure, wherein the HTT regulator comprises a zinc finger protein. It will be apparent that this is for illustrative purposes only, and other HTT regulators (e.g., repressors) may be used, including but not limited to TALE-TFs, CRISPR / Cas systems, other ZFPs, ZFNs, TALENs, other CRISPR / Cas systems (e.g., Cfp systems), homing endonucleases (mega-nucleases) and engineered DNA binding domains.

[0189] Example

[0190] Example 1: mHTT repressor

[0191] Zinc finger proteins were engineered to target mHTT essentially as described in U.S. Pat. Nos. 9,234,016; 8,841,260; 6,534,261; U.S. Patent Publication Nos. 2019 / 0322711; 2017 / 0096460; 2015 / 0056705; 2015 / 0335708 and 2019 / 0322711. Table 1 shows the recognition helices of the DNA binding domains of these ZFPs, and Table 2 shows the target sequences of these ZFPs. The ZFPs were evaluated and shown to bind to their target sites.

[0192] The ZFP was operably linked to the KOX repressor domain to form a ZFP-TF that represses HTT. Table 3 shows the amino acid and nucleotide sequences of the designated ZFP-TF repressors.

[0193] ZFP TF transcripts were transfected into human cells (e.g., cells from HD patients) and the expression of HTT and mHTT transcripts was monitored using real-time qRT-PCR. All ZFP-TFs were found to be effective in selectively repressing mutant HTT expression. ZFP-TFs were functional repressors when formulated as plasmids in recombinant viral vectors, including Ad vectors, lentiviral vectors, and / or AAV vectors (e.g., AAV9 or AAV6) in mRNA form.

[0194] Example 2: In vitro studies

[0195] The ability of the ZFP-TFs shown in Tables 1 and 3 to selectively repress mHTT transcription relative to the wtHTT allele was evaluated in HD patient fibroblasts and stem cell-derived neurons.

[0196] Briefly, human neuronal stem cells (CAG17 or CAG48 (17 and 48 "CAG" repeats disclosed in SEQ ID NO: 23)) were transfected with mRNA encoding ZFP-TF or GFP in Table 3 at 0-1500 ng (1500, 300, 150 or 15 ng), and wtHTT and mHTT levels were measured by qPCR after 24 hours. In addition, HD neurons were transduced with AAV6 carrying the ZFP-TF coding sequence or GFP in Table 3 at an MOI of 10K to 500K (500K, 300K, 100K or 10K).

[0197] As shown in Figure 2, ZFP-TF significantly repressed mHTT expression while minimally repressing wild-type HTT.

[0198] ZFP-TF 45643 was selected for further study and the transgenic mRNA was transiently transfected into HD patient fibroblasts to evaluate the ability of the transgenic protein to repress transcription of the mHTT and wtHTT genes. GM02151 fibroblasts (CAG18 or CAG 45 (18 or 45 "CAG" repeats disclosed in SEQ ID NO: 23)) were transfected with 0-1000 ng mRNA encoding the rAAV9-ZFP-TF 45643 transgene or GFP and the levels of wtHTT and mHTT mRNA, and the levels of wtHTT and mHTT mRNA were measured by qPCR after 24 hours.

[0199] Meta-analysis of data from 6 independent experiments (one-way ANOVA and Dunnett's multiple comparison test) showed that mHTT mRNA was significantly reduced (p<0.001 compared to GFP-transfected cells) and wtHTT mRNA was not significantly reduced in cells transfected with ≥0.3 ng of transgenic mRNA. See Figure 5. These results were confirmed in two other HD fibroblast cell lines, GM04723 (CAG15 or CAG 67 (15 or 67 "CAG" repeats disclosed in SEQ ID NO: 23)) and ND30259 (CAG21 or CAG38 (21 or 38 "CAG" repeats disclosed in SEQ ID NO: 23).

[0200] To assess the repression of mHTT in neurons, HD patient ES cells (GENEA020; CAG17 or CAG 48 (17 or 48 "CAG" repeats disclosed in SEQ ID NO: 23)) were differentiated into neurons, transiently transfected with 0, 15, 150 or 300 ng of mRNA encoding the ZFP-TF 45643 transgene or GFP, and the levels of wtHTT and mHTT mRNA were measured by qPCR 48 hours later. Neurons transfected with mRNA encoding the ZFP-TF 45643 transgene had approximately 90% less mHTT mRNA than cells transfected with the same amount of mRNA expressing GFP. The level of wtHTT mRNA did not change in all treatment groups. See Figure 5.

[0201] In addition, the repression of mHTT and wtHTT transcription was examined 17 days after rAAV9-ZFP-TF 45643 was transduced into neurons derived from HD patient ES cells (GENEA020; CAG 17 / 48 (the "CAG" repeat sequence disclosed in SEQ ID NO:23)). Cells were transduced with rAAV9-ZFP-TF 45643 at 5e4, 1e5, 5e5, 5e6, or 1e7 vg / cell, and the levels of transgenic mRNA, wtHTT, and mHTT mRNA were measured by qPCR 17 days later. The level of transgenic mRNA / cell was estimated based on the number of transduced cells and increased from 2 to 123 copies / cell in a dose-dependent manner. Despite the dose-dependent increase in transgenic mRNA, the levels of mHTT and wtHTT mRNA were equal at all doses. The level of mHTT was approximately 75% lower than that of mock- or AAV9-GFP-transduced cells, while the level of wtHTT mRNA was unaffected.

[0202] Taken together, these results demonstrate that the ZFP-TF proteins described herein selectively target the expanded CAG repeat sequence in the mHTT allele. Furthermore, the results demonstrate that low levels of transgene expression are sufficient to achieve maximal repression of mHTT.

[0203] Example 3: Specificity

[0204] For both targeted and off-target sites, genome-wide selectivity was assessed in vitro in HD patient cells using microarray analysis. A subset of off-target genes was also analyzed by qPCR analysis.

[0205] like Figure 4 and 6 As shown, for the listed off-target sites, the ZF-TFs exhibited high specificity for their target sites as determined by qPCR analysis.

[0206] Example 4: In vivo studies

[0207] The in vivo pharmacology of rAAV9-ZFP-TF 45643 was evaluated in two HD mouse models, the severe R6 / 2 model and the more progressive Q175 model. See, e.g., Crook & Housman (2011) Neuron 69:423-435. In some studies, rAAV9-ZFP-TF 45643 was administered before the onset of disease symptoms, while in other studies, it was administered after disease onset. In all of these studies, the primary endpoint was suppression of mHTT expression. In addition, the effects of rAAV9-ZFP-TF45643 on motor and cognitive function were also evaluated in some studies.

[0208] The effects of rAAV9-ZFP-TF 45643 on mHTT mRNA and protein aggregates were evaluated in the aged Q175 mouse model. Q175 mice aged 52 / 53 weeks were given vehicle, rAAV9-ZFP-TF 45643 (3e8, 3e9, or 3e10 VG / striatum), or rAAV9-GFP (3e8, 3e9, or 3e10 VG / striatum) by stereotaxic intrastriatal injection. Fig.13 Tissues were collected 8 weeks after surgery for qPCR analysis and 8 or 16 weeks after surgery for IHC analysis.

[0209] Furthermore, treatment with rAAV9-ZFP-TF 45643 resulted in a dose-dependent increase in transgene mRNA and a dose-dependent decrease in mHTT mRNA. Figure 7 and 8 . The effects on HTT in wild-type mice were similar to those observed in the rAAV9-GFP treated groups. Cytoplasmic mHTT protein aggregates were reduced in rAAV9-ZFP-TF 45643 treated striatum compared with vehicle or rAAV9-GFP treated striatum. No effects on nuclear aggregates were observed in any treatment group. Therefore, rAAV9-ZFP-TF 45643 can repress mHTT transcription and reduce the levels of cytoplasmic mHTT aggregates when administered after disease onset in the Q175 mouse model.

[0210] The effects of rAAV9-ZFP-TF 45643 on mHTT mRNA, soluble mHTT protein, and mHTT aggregates were evaluated in young Q175 mice. Five-week-old Q175 mice were given rAAV9-ZFP-TF45643 (9.2e9, 3.1e10, 9.2e10 VG / mouse) or rAAV9-GFP (5.5e10 VG / mouse) by stereotaxic bilateral intrastriatal injection. Tissues were collected 11 weeks after surgery for qPCR, ELISA, and IHC analysis.

[0211] The effects of rAAV9-ZFP-TF 45643 on mHTT mRNA, soluble mHTT protein, and mHTT aggregates were evaluated in Q175 mice 33 weeks after treatment. Five-week-old Q175 mice were given rAAV9-ZFP-TF 45643 (9.2e9, 3.1e10, 9.2e10 VG / mouse) or rAAV9-GFP (5.5e10 VG / mouse) by stereotaxic bilateral intrastriatal injection. Motor function (open field, rotamex, tapered balance beam as described in Carter (1999) J Neurosci. 19(8):3248-3257) was assessed at 16 / 17, 26 / 27, and 36 / 37 weeks of age, and cognitive function (FR5 / PR) was assessed at 28-35 weeks of age. Tissues were collected 33 weeks post-surgery (38 weeks of age) for qPCR, ELISA, and IHC analysis. At the end of the study, brains of spare mice (4-6 per group) were processed for histopathology.

[0212] The effects of rAAV9-ZFP-TF 45643 on mHTT mRNA, soluble mHTT protein, and mHTT aggregates were evaluated in R6 / 2 mice. Four-week-old R6 / 2 mice were given rAAV9-ZFP-TF45643 (9.2e9, 3.1e10, 9.2e10 VG / mouse) or rAAV9-GFP (5.5e10 VG / mouse) by stereotaxic bilateral intrastriatal injection. Motor function was assessed at 5, 7, 9, and 11 weeks of age. Tissues were collected 7 weeks post-surgery (11 weeks of age) for qPCR, ELISA, and IHC analysis.

[0213] like Figure 7-13 As shown, the results show that intrastriatal injection of rAAV9-ZFP-TF 45643 suppressed the synthesis of mHTT in cells expressing the transgenic protein. Figure 7 and Figure 8 Statistically significant selective repression of mHTT was shown at all ZFP-TF doses (no reduction in wild-type HTT expression levels). Fig. 9 It was shown that soluble mHTT protein was significantly reduced in subjects treated with ZFP-TF (all doses) in the striatum and cortical forebrain at 11 weeks post-treatment, and in the striatum, cortical forebrain, and cortical hindbrain at 33 weeks post-treatment. Fig.10 It was shown that at high doses (9.2×10 12 vg) After 8 weeks of treatment, soluble mHTT was significantly reduced in the striatum. Fig.11 Shown is a significant reduction in mHTT protein aggregates (compared to GFP control) in neurons expressing GFP control or ZFP-TF (all doses). Fig.12 and 13Significant repression of mHTT expression in subjects treated with the AAV ZFP-TFs described herein was demonstrated.

[0214] also, Fig.14 It showed that the motor function of the treated subjects was significantly improved compared with the control.

[0215] Similar in vivo results were obtained using any of the ZFP-TFs described herein (including ZFPs designated as 46025, 45294, 45723, or 33074).

[0216] In vitro, in HD patient cells, the ZFP-TFs described herein repressed transcription of the mHTT allele without significant effects on the wtHTT allele or other CAG repeat-containing genes. In vivo studies in 2 different HD mouse models demonstrated that a single intrastriatal (intrastriate) administration of rAAV9-ZFP-TF 45643, given either before or after the onset of disease symptoms, effectively repressed the synthesis of mHTT mRNA and protein for up to 33 weeks.

[0217] All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety for all purposes.

[0218] Although for the purpose of clear understanding, the present invention provides some details by way of explanation and example, it will be appreciated by those skilled in the art that various changes and modifications can be implemented without departing from the spirit or scope of the present invention. Therefore, the above description and embodiments should not be understood as limiting. Sequence Listing <110> SANGAMO THERAPEUTICS, INC. SHIRE HUMAN GENETIC THERAPIES, INC. <120> HTT repressor and its application <130> 8327-018740 <140> <141> <150> 62 / 792,701 <151> 2019-01-15 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 1 Gln Ser Gly Asp Leu Thr Arg 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Lys His Gly Asn Leu Ser Glu 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Lys Arg Cys Asn Leu Arg Cys 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 4 Cys Pro Ser His Leu Thr Arg 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Arg Gln Phe Asn Arg His Gln 1 5 <210> 6 <211> 28 <212> DNA <213> unknown <220> <223> Unknown description: Human or mouse HTT sequence <400> 6 agcagcagca gcagcagcag cagcagca 28 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Ser Pro Glu Gln Leu Ser Arg 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Gln Trp Ser Thr Arg Lys Arg 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Lys Gln Gly Asn Leu Val Glu 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Arg Ser Asp Asn Leu Ser Glu 1 5 <210> 11 <211> 28 <212> DNA <213> unknown <220> <223> Unknown description: Human or mouse HTT sequence <400> 11 cagcagcagc agcagcagca gcagcagc 28 <210> 12 <211> twenty one <212> DNA <213> unknown <220> <223> Unknown description: Human or mouse HTT sequence <400> 12 cagcagcagc agcagcagca g 21 <210> 13 <211> 825 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 13 atggccccca agaaaaagcg gaaagtgggc atccacgggg tacccgccgc tatggctgag 60 aggcccttcc agtgtcgaat ctgcatgcgt aagtttgccc agtccggcga cctgacccgc 120 cataccaaga tacacacggg cgagaagccc ttccagtgtc gaatctgcat gcgtaacttc 180 agtcagtccg gcgacctgac ccgccacatc cgcacccaca ccggcgagaa gccttttgcc 240 tgtgacattt gtgggaggaa atttgcccag tccggcgacc tgacccgcca taccaagata 300 cacacgccga acccgcaccg ccgcaccgac ccgtcccaca agcccttcca gtgtcgaatc 360 tgcatgcgta acttcagtaa gcacggcaac ctgtccgagc acatccgcac ccacaccggc 420 gagaagcctt ttgcctgtga catttgtggg aggaaatttg ccaagcgctg taacctgcgc 480 tgtcatacca agatacacct gcgccaaaaa gatgcggccc ggggatccgg catggatgct 540 aagtcactaa ctgcctggtc ccggacactg gtgaccttca aggatgtatt tgtggacttc 600 accagggagg agtggaagct gctggacact gctcagcaga tcgtgtacag aaatgtgatg 660 ctggagaact ataagaacct ggtttccttg ggttatcagc ttactaagcc agatgtgatc 720 ctccggttgg agaagggaga agagccctgg ctggtggaga gagaaattca ccaagagacc 780 catcctgatt cagagactgc atttgaaatc aaatcatcag tttaa 825 <210> 14 <211> 827 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 14 atggccccca agaaaaagcg gaagtgggc atccacgggg tacccccgc tatggctgag 60 aggcccttcc agtgtcgaat ctgcatgcgt aacttcagtt gtccgtcccca cctgacccgc 120 cacatccgca cccacaccgg cgagaagcct ttgctgtg acatttgtgg gaggaattt 180 gcccagtccg gcgacctgac ccgccatacc aagatacaca cgcctaatcc tcatcgccgc 240 actgatccca gccataagcc cttccagtgt cgaatctgca tgcgtactt cagtaagcac 300 ggcaacctgt ccgagcacat ccgcaccac accggcgaga agccttttgc ctgtgacatt 360 tgtgggagga aatttgccaa gcgctgtaac ctgcgctgtc ataccaagat acacacgggc 420 tcccaatccc ccttccagtg tcgaatctgc atgcgtaagt tgcccgcca gttcaaccgc 480 caccagcata ccaagataca cctgcgccaa aagatgcgg cccggggatc cggcatggat 540 gctaagtcac taactgctg gtcccggaca ctggtgacct tcaggtgt atttgtggac 600 ttcaccagggg aggagtggaa gctgctggac actgctcagc agatcgtta cagaaatgtg 660 atgctggaga actataagaa cctggtttcc ttggtttc agcttactaa gccagatgtg 720 atcctccggt tggagaaggg agaagagccc tggctggtgg agagagaaat tcaccaagag 780 acccatcctg attcagagac tgcatttgaa atcaaatcat cagttta 827 <210> 15 <211> 800 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 15 atggccccca agaaaaagcg gaaagtgggc atccacgggg tacccgccgc tatggctgag 60 aggcccttcc agtgtcgaat ctgcatgcgt aacttcagtt gtccgtccca cctgacccgc 120 cacatccgca cccacaccgg cgagaagcct tttgcctgtg acatttgtgg gaggaaattt 180 gcccagtccg gcgacctgac ccgccatacc aagatacaca cgggcgagaa gcccttccag 240 tgtcgaatct gcatgcgtaa cttcagttgt ccgtcccacc tgacccgcca catccgcacc 300 cacaccggcg agaagccttt tgcctgtgac atttgtggga ggaaatttgc ccagtccggc 360 gacctgaccc gccataccaa gatacacacg ggatctcaga agcccttcca gtgtcgaatc 420 tgcatgcgta agtttgccca gtccggcgac ctgacccgcc ataccaagat acacctgcgc 480 caaaaagatg cggcccgggg atccggcatg gatgctaagt cactaactgc ctggtcccgg 540 acactggtga ccttcaagga tgtatttgtg gacttcacca gggaggagtg gaagctgctg 600 gacactgctc agcagatcgt gtacagaaat gtgatgctgg agaactataa gaacctggtt 660 tccttgggtt atcagcttac taagccagat gtgatcctcc ggttggagaa gggagaagag 720 ccctggctgg tggagagaga aattcaccaa gagacccatc ctgattcaga gactgcattt 780 gaaatcaaat catcagttta 800 <210> 16 <211> 740 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 16 atggccccca agaaaaagcg gaaagtgggc atccacgggg tacccgccgc tatggctgag 60 aggcccttcc agtgtcgaat ctgcatgcgt aacttcagtt ccccggagca gctgtcccgc 120 cacatccgca cccacaccgg cgagaagcct tttgcctgtg acatttgtgg gaggaaattt 180 gcccagtggt ccacccgcaa gcgccatacc aagatacaca cgccgaaccc gcaccgccgc 240 accgacccgt cccacaagcc cttccagtgt cgaatctgca tgcgtaactt cagtaagcag 300 ggcaacctgg tggagcacat ccgcacccac accggcgaga agccttttgc ctgtgacatt 360 tgtgggagga aatttgccaa gcgctgtaac ctgcgctgtc ataccaagat acacctgcgc 420 caaaaagatg cggcccgggg atccggcatg gatgctaagt cactaactgc ctggtcccgg 480 acactggtga ccttcaagga tgtatttgtg gacttcacca gggaggagtg gaagctgctg 540 gacactgctc agcagatcgt gtacagaaat gtgatgctgg agaactataa gaacctggtt 600 tccttgggtt atcagcttac taagccagat gtgatcctcc ggttggagaa gggagaagag 660 ccctggctgg tggagagaga aattcaccaa gagacccatc ctgattcaga gactgcattt 720 gaaatcaaat catcagttta 740 <210> 17 <211> 922 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 17 atgcccccaa gaaaaagcgg aaagtgggca tccacggggt acccgccgct atggctgaga 60 ggcccttcca gtgtcgaatc tgcatgcgta acttcagtcg ctccgacaac ctgtccgagc 120 acatccgcac ccacaccggc gagaagcctt ttgcctgtga catttgtggg aggaaatttg 180 ccaagcgctg taacctgcgc tgtcatacca agatacacac gcatcccagg gcacctattc 240 ccaagccctt ccagtgtcga atctgcatgc gtaacttcag tcagtccggc gacctgaccc 300 gccacatccg cacccacacc ggcgagaagc cttttgcctg tgacatttgt gggaggaaat 360 ttgcccagtc cggcgacctg acccgccata ccaagataca cacgccgaac ccgcaccgcc 420 gcaccgaccc gtcccacaag cccttccagt gtcgaatctg catgcgtaac ttcagtcgct 480 ccgacaacct gtccgagcac atccgcaccc acaccggcga gaagcctttt gcctgtgaca 540 tttgtgggag gaaatttgcc aagcgctgta acctgcgctg tcataccaag atacacctgc 600 gccaaaaaga tgcggcccgg ggatccggca tggatgctaa gtcactaact gcctggtccc 660 ggacactggt gaccttcaag gatgtatttg tggacttcac cagggaggag tggaagctgc 720 tggacactgc tcagcagatc gtgtacagaa atgtgatgct ggagaactat aagaacctgg 780 tttccttggg ttatcagctt actaagccag atgtgatcct ccggttggag aagggagaag 840 agccctggct ggtggagaga gaaattcacc aagagaccca tcctgattca gagactgcat 900 ttgaaatcaa atcatcagtt ta 922 <210> 18 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 18 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Met Ala Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Lys Phe 20 25 30 Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Thr Gly Glu 35 40 45 Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Gly 50 55 60 Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala 65 70 75 80 Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg 85 90 95 His Thr Lys Ile His Thr Pro Asn Pro His Arg Arg Thr Asp Pro Ser 100 105 110 His Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Lys His 115 120 125 Gly Asn Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe 130 135 140 Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys Arg Cys Asn Leu Arg 145 150 155 160 Cys His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg Gly Ser 165 170 175 Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val Thr 180 185 190 Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu Leu 195 200 205 Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn Tyr 210 215 220 Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val Ile 225 230 235 240 Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu Ile 245 250 255 His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys Ser 260 265 270 Ser Val <210> 19 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 19 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Met Ala Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe 20 25 30 Ser Cys Pro Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu 35 40 45 Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly 50 55 60 Asp Leu Thr Arg His Thr Lys Ile His Thr Pro Asn Pro His Arg Arg 65 70 75 80 Thr Asp Pro Ser His Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn 85 90 95 Phe Ser Lys His Gly Asn Leu Ser Glu His Ile Arg Thr His Thr Gly 100 105 110 Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys Arg 115 120 125 Cys Asn Leu Arg Cys His Thr Lys Ile His Thr Gly Ser Gln Ser Pro 130 135 140 Phe Gln Cys Arg Ile Cys Met Arg Lys Phe Ala Arg Gln Phe Asn Arg 145 150 155 160 His Gln His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg Gly 165 170 175 Ser Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val 180 185 190 Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu 195 200 205 Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn 210 215 220 Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val 225 230 235 240 Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu 245 250 255 Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys 260 265 270 Ser Ser Val 275 <210> 20 <211> 266 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 20 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Met Ala Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe 20 25 30 Ser Cys Pro Ser His Leu Thr Arg His Ile Arg Thr His Thr Gly Glu 35 40 45 Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly 50 55 60 Asp Leu Thr Arg His Thr Lys Ile His Thr Gly Glu Lys Pro Phe Gln 65 70 75 80 Cys Arg Ile Cys Met Arg Asn Phe Ser Cys Pro Ser His Leu Thr Arg 85 90 95 His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys 100 105 110 Gly Arg Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile 115 120 125 His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Lys 130 135 140 Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys Ile His Leu Arg 145 150 155 160 Gln Lys Asp Ala Ala Arg Gly Ser Gly Met Asp Ala Lys Ser Leu Thr 165 170 175 Ala Trp Ser Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe 180 185 190 Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr 195 200 205 Arg Asn Val Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr 210 215 220 Gln Leu Thr Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu 225 230 235 240 Pro Trp Leu Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser 245 250 255 Glu Thr Ala Phe Glu Ile Lys Ser Ser Val 260 265 <210> 21 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 21 Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Met Ala Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe 20 25 30 Ser Ser Pro Glu Gln Leu Ser Arg His Ile Arg Thr His Thr Gly Glu 35 40 45 Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Trp Ser 50 55 60 Thr Arg Lys Arg His Thr Lys Ile His Thr Pro Asn Pro His Arg Arg 65 70 75 80 Thr Asp Pro Ser His Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn 85 90 95 Phe Ser Lys Gln Gly Asn Leu Val Glu His Ile Arg Thr His Thr Gly 100 105 110 Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys Arg 115 120 125 Cys Asn Leu Arg Cys His Thr Lys Ile His Leu Arg Gln Lys Asp Ala 130 135 140 Ala Arg Gly Ser Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg 145 150 155 160 Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu 165 170 175 Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met 180 185 190 Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys 195 200 205 Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val 210 215 220 Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe 225 230 235 240 Glu Ile Lys Ser Ser Val 245 <210> twenty two <211> 307 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty two Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 1 5 10 15 Ala Met Ala Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe 20 25 30 Ser Arg Ser Asp Asn Leu Ser Glu His Ile Arg Thr His Thr Gly Glu 35 40 45 Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys Arg Cys 50 55 60 Asn Leu Arg Cys His Thr Lys Ile His Thr His Pro Arg Ala Pro Ile 65 70 75 80 Pro Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser 85 90 95 Gly Asp Leu Thr Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe 100 105 110 Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Gln Ser Gly Asp Leu Thr 115 120 125 Arg His Thr Lys Ile His Thr Pro Asn Pro His Arg Arg Thr Asp Pro 130 135 140 Ser His Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg 145 150 155 160 Ser Asp Asn Leu Ser Glu His Ile Arg Thr His Thr Gly Glu Lys Pro 165 170 175 Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys Arg Cys Asn Leu 180 185 190 Arg Cys His Thr Lys Ile His Leu Arg Gln Lys Asp Ala Ala Arg Gly 195 200 205 Ser Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val 210 215 220 Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu 225 230 235 240 Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn 245 250 255 Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val 260 265 270 Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu 275 280 285 Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys 290 295 300 Ser Ser Val 305 <210> twenty three <211> 201 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (1)..(201) <223> The sequence can contain 15-67 "cag" repeat units <220> <223> For a detailed description of alternative and preferred embodiments, see the filed specification. <400> twenty three cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca g 201

Claims

1. A zinc finger protein transcription factor ZFP-TF, comprising (i) a zinc finger protein ZFP denoted as 45294 or 45723, comprising an amino acid sequence in the order shown in the following table: as well as (ii) Transcription repression domain. 2 . The zinc finger protein transcription factor ZFP-TF according to claim 1 , comprising the amino acid sequence of SEQ ID NO: 21 or 22.

3. A polynucleotide encoding the ZFP-TF according to claim 1 or 2.

4. An rAAV vector comprising one or more polynucleotides according to claim 3.

5. The rAAV vector of claim 4, further comprising a sequence encoding a nuclear localization signal NLS.

6. A pharmaceutical composition comprising one or more ZFP-TFs as described in claim 1 or 2, one or more polynucleotides as described in claim 3 and / or one or more rAAV vectors as described in claim 4 or 5.

7. The pharmaceutical composition of claim 6 for use in the treatment of Huntington's disease.

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