Zinc finger protein composition for regulating huntingtin protein (HTT)

Abstract

The present disclosure relates to the field of regulating Htt gene expression and HTT protein levels.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 659,552, filed April 18, 2018, and U.S. Provisional Application No. 62 / 780,605, filed December 17, 2018, the disclosures of which are incorporated herein by reference in their entireties.

[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 April 15, 2019, named 8325-0173_SL.txt, and is 34,379 bytes in size. Technical Field

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

[0006] background

[0007] 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.

[0008] Huntington's disease is an example of a trinucleotide repeat expansion disorder that was first characterized in the early nineties (see Di Prospero and Fischbeck (2005) Nature Reviews Genetics 6: 756-765). These disorders involve local expansion of unstable repeat sequences of three nucleotide groups and may lead to loss of function, acquisition of toxic function, or both of genes in which the expanded repeat sequences are retained. Trinucleotide repeat sequences can 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 lead to abnormal expression of genes, while amplified repeat sequences in coding regions (also known as codon reiteration disorders) may lead to misfolding and protein aggregation. The exact cause of the pathophysiology associated with abnormal proteins is often unknown. Typically, in wild-type genes that undergo trinucleotide expansion, these regions contain a variable number of repeat sequences in normal populations, but in diseased populations, the number of repeat sequences can 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 large cytoplasmic protein huntingtin (Htt). Normal Htt alleles contain 15-20 CAG repeats (SEQ ID NO:71), while alleles containing 35 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 (SEQ ID NO:89) are considered 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 fully penetrant. In fact, asymptomatic individuals with HD alleles having 40 or more repeats have been reported. 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 this region will encode a polyserine polypeptide (in the case of the +1 frameshift, encoded by the AGC repeat sequence) trace rather than polyglutamine (Davies and Rubinsztein (2006) Journal of Medical Genetics 43:893-896).

[0009] In HD, the mutant Htt 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, the HD allele may also display a prognostic phenomenon where an increase in severity or a decrease in age of onset is observed over several generations due to the unstable nature of the repeat region during spermatogenesis.

[0010] In addition, trinucleotide expansion in Htt leads to neuronal loss in the striatal medium spiny gamma-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).

[0011] The role of normal Htt protein is poorly understood, but may be related to neurogenesis, programmed cell death and / or 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. It has been shown that the progression of HD is associated with reduced 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 adeno-associated virus (AAV) vector-mediated gene delivery in a HD mouse model can protect striatal neurons (Kells et al. (2004) Molecular Therapy 9(5):682-688).

[0012] Multiple mechanisms of HD pathogenesis have been investigated, including toxicity of mutant HTT (mHTT) proteins and loss of normal HTT proteins (Ross and Tabrizi (2011) The Lancet. Neurology 10:83-98; Zuccato et al. (2010) Physiological Reviews 90:905-981). Results from multiple HD mouse models expressing mHtt suggest that it exerts its pathogenic activity primarily through gain-of-function toxicity (Brooks and Dunnett (2015) Curr Top Behav Neurosci 22:101-133). The discovery that inactivation of an inducible mHTT transgene results in disease reversal (Yamamoto et al. (2000) Cell 101:57-66) has led to the development of HTT lowering agents as potential therapeutic agents. RNA interference (RNAi) and antisense oligonucleotides (ASOs) targeting HTT mRNA have been shown to be effective in multiple preclinical models of HD (Boudreau et al. (2009) Mol Ther 17:1053-1063; Harper et al. (2005) Proc Natl Acad Sci 102:5820-5825; Kordasiewicz et al. (2012) Neuron 74:1031-1044; Stanek et al. (2014) Human Gene Ther. 25(5):461), and a recent phase 1 / 2a trial showed that multiple intrathecal administrations of ASOs reduced normal and mutant HTT levels in the cerebrospinal fluid of participants.

[0013] 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.

[0014] For treatment, some potential approaches designed to prevent protein aggregation-related toxicity have been shown to reduce these toxicities in in vitro models, where protein aggregation occurs through extended polyglutamine tracts, such as overexpression of chaperones 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, HD Htt, which is mutated by caspase cleavage, 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, supra).

[0015] Recombinant transcription factors containing DNA binding domains from zinc finger proteins ("ZFPs") have the ability to regulate gene expression of endogenous genes (including Htt genes). See, for example, U.S. Patent Nos. 9,943,565; 9,499,597; 9,234,016; and 8,841,260 and U.S. Patent Publication Nos. 2015 / 0335708; 2017 / 0096460; and 20150255877. 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, for example, Yu et al. (2006) FASEB J. 20: 479-481).

[0016] Furthermore, artificial nucleases comprising ZFPs have the ability to modify gene expression of endogenous genes through nuclease-mediated gene modification, including homology-directed repair (HDR), by end capture after non-homologous end joining (NHEJ) and / or in a process driven by non-homologous end joining (NHEJ). See, e.g., U.S. Pat. Nos. 9,873,894; 9,394,545; 9,150,847; 9,206,404; 9,222,105; 9,045,763; 9,005,973; 8,956,828; 8,936,936; 8,945,868; 8,871,905; 8,586,526; 8,563,314; 8,329,986; 8,399,218; 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; 8,771,985; 8,895,264; U.S. Patent Publication Nos. 2003 / 0232410; 2005 / 0208489; 2005 / 0026157; 2005 / 0064474; 2006 / 0063231; 2011 / 0265198; and 2013 / 0177960, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Therefore, these methods generally involve using engineered cutting systems to induce double-strand breaks (DSBs) or nicks in target DNA sequences, therefore repairing breaks by error-prone processes such as non-homologous end joining (NHEJ) or using repair templates (homologous directed repair or DHA) to repair can cause knockout genes or insert sequences of interest (targeted integration). Introducing double-strand breaks in the absence of an externally provided repair template (e.g., "donor" or "transgenic") is generally used to inactivate target genes via sudden changes (insertion and / or deletion referred to as "indels") introduced by cell NHEJ approaches.

[0017] However, there remains a need for methods for diagnosing, studying, treating and / or preventing Huntington's disease, including detection of mHTT for monitoring disease progression, for increasing understanding of HD neuropathology, and for evaluating disease-modifying HD therapies. Summary of the invention

[0018] Disclosed herein are methods and compositions for diagnosing and / or treating Huntington's disease. Specifically, provided herein are zinc finger protein compositions that regulate Htt expression, including selective repression of mutant Htt genes (mHtt) expressing toxic mutant HTT (mHTT) proteins. The compositions and methods selectively repress >99% of alleles that cause HD within an 80-fold dose range, while retaining >86% of the expression of wild-type alleles present in the HD population. The expression of other CAG-containing genes is minimally affected, and the artificial transcription factor repressors described herein are active and well tolerated in HD neurons over 100 days of culture and in mouse brains for at least 9 months or longer.

[0019] Thus, in one aspect, a ZFP genetic modulator of the Htt gene is described. In certain embodiments, the genetic modulator comprises an artificial ZFP transcription factor or zinc finger nuclease (ZFN) and a functional domain (transcription activator, transcription repressor, nuclease domain, etc.) that modulates the expression of an HD allele (e.g., Htt). In certain embodiments, the zinc finger protein has a recognition helix as shown in any of the attached tables and figures. In certain embodiments, the ZFP-TF is formulated into a pharmaceutical composition, e.g., for administration to a subject.

[0020] In one aspect, the genetic modulators described herein are ZFP repressors (artificial transcription factors or nucleases that inhibit Htt expression). These ZFP repressors can specifically bind to wild-type and / or mutant Htt alleles. Certain repressors bind to wild-type and mutant Htt genes; others only bind to wild-type Htt genes, while others only bind to mutant Htt genes. In some embodiments, the artificial transcription factor forms a multimeric stable complex of a given size and is therefore able to preferentially interact with CAG tracts exceeding a certain minimum size, wherein the minimum size is greater than the length of the wild-type CAG tract.

[0021] In certain embodiments, the ZFP genetic regulators described herein preferentially regulate the expression of mutant Htt alleles. In some embodiments, the ZFP genetic regulator (e.g., a repressor) specifically binds to mutant Htt alleles, wherein the amplified tract encodes polyglutamine, and in other embodiments, the genetic regulator specifically binds to mutant Htt alleles, wherein the amplified tract encodes polyserine. Therefore, in some embodiments, the ZFP genetic regulator regulates wild-type and mutant Htt alleles. In certain embodiments, the ZFP genetic regulator only regulates wild-type Htt alleles. In certain embodiments, the ZFP genetic regulator only regulates mutant Htt.

[0022] In other embodiments, a repressive regulator comprising a ZFP as described herein is provided that preferentially binds to a known SNP associated with an amplified HD Htt allele. In this way, the genetic repressor is specific for a mutant Htt allele comprising a SNP, thereby allowing specific repression of the mutant Htt allele. In another aspect, a genetic regulator is provided that specifically activates a wild-type Htt allele by interacting with a SNP associated with a wild-type allele. In this way, only the wild-type Htt allele is activated.

[0023] In some embodiments, the ZFP genetic regulator comprises at least one regulatory domain (or functional domain). For example, the functional domain may be a transcriptional activation domain, a transcriptional repression domain, and / or a nuclease (cleavage) domain. By selecting an activation domain or a repression domain for fusion with a ZFP DNA binding domain, these artificial transcription factors can be used to activate or repress gene expression. In some embodiments, such a fusion molecule is provided, which comprises a ZFP DNA binding domain targeting mutant Htt as described herein, and the ZFP DNA binding domain is fused with a transcriptional repression domain that can be used to downregulate mutant Htt expression. In some embodiments, such an artificial transcription factor is provided, which comprises a ZFP DNA binding domain targeting a wild-type Htt allele, and the ZFP DNA binding domain is fused with 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 ligand, so that in the absence of an exogenous ligand, the interaction with the cell transcription machinery structure does not occur. Such external ligands control the degree of interaction between artificial transcription factors and transcription machinery structures. One or more regulatory (functional) domains may be operably linked to or associated in any manner with any portion of one or more ZFP DNA binding domains, including between one or more zinc finger domains, outside of one or more ZFPs, and any combination thereof.Any fusion protein described herein may be formulated into a pharmaceutical composition.

[0024] In some embodiments, the genetic modulators described herein comprise artificial nucleases comprising an engineered ZFP protein described herein and a cleavage (nuclease domain). These artificial zinc finger nucleases (ZFNs) can be used to target mutant Htt alleles in stem cells such as induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESC cells), mesenchymal stem cells (MSCs), or neuronal stem cells, wherein the activity of the nuclease fusion will produce an Htt allele comprising a wild-type number of CAG repeats. In certain embodiments, a pharmaceutical composition comprising modified stem cells is provided for ex vivo administration to a subject.

[0025] In another aspect, polynucleotides encoding any of the ZFP DNA binding genetic modulators described herein (or components thereof) are provided. Such polynucleotides can be administered to a subject in whom treatment of Huntington's disease is desired.

[0026] In another aspect, the present invention provides methods and compositions for generating a specific model system for studying Huntington's disease. In certain embodiments, models are provided herein, wherein mutant Htt alleles are produced using embryonic stem cells to generate cell lines or animal strains, wherein specific trinucleotide amplification bundles (e.g., 50 (SEQ ID NO: 72), 80 (SEQ ID NO: 73), 109 (SEQ ID NO: 74) and 180 CAG repeats (SEQ ID NO: 75)) are inserted into wild-type Htt alleles using artificial nucleases targeting Htt as described herein to drive targeted integration. In certain embodiments, the model system comprises an in vitro cell line, while in other embodiments, the model system comprises a transgenic animal. In any animal model described herein, for example, the animal can be a rodent (e.g., rat, mouse), a primate (e.g., non-human primate) or a rabbit.

[0027] In another aspect, a gene delivery vector comprising any polynucleotide encoding a ZFP regulator described herein is provided. In certain embodiments, the vector is an adenoviral vector (e.g., an Ad5 / F35 vector), a lentiviral vector (LV), including an integration-competent or integration-defective lentiviral vector, or an adenovirus-associated viral vector (AAV). Therefore, an adenovirus (AD) vector, LV, or adenovirus-associated viral vector (AAV) comprising such a sequence is also provided herein, wherein the sequence encodes at least one genetic regulator described herein, and optionally, further encodes one or more donor sequences that are targeted for integration into a target gene after nuclease-mediated cleavage. In certain embodiments, the Ad vector is a chimeric Ad vector, for example, an Ad5 / F35 vector. In certain embodiments, the lentiviral vector is an integrase-defective lentiviral vector (IDLV) or an integration-competent lentiviral vector. In certain embodiments, the vector is a pseudotyped vector with a VSV-G envelope or with other envelopes.

[0028] In some embodiments, a model system for Huntington's disease is provided, wherein the target allele (e.g., mutant Htt) is labeled with an expression marker tag. In certain embodiments, the mutant allele (e.g., mutant Htt) is labeled with a tag. In some embodiments, the wild-type allele (e.g., wild-type Htt) is labeled with a tag, and in other embodiments, wild-type and mutant alleles are labeled with different expression marker tags. In certain embodiments, the model system comprises an in vitro cell line, and in other embodiments, the model system comprises a transgenic animal.

[0029] In addition, pharmaceutical compositions comprising one or more genetic modulators described herein are also provided. For example, certain compositions include a combination of a nucleic acid containing a sequence encoding a ZFP-TF described herein and a pharmaceutically acceptable carrier or diluent, such that upon administration to a subject, the ZFP-TF is expressed in the subject and modulates Htt expression. In certain embodiments, the encoded genetic modulator is specific for the HD Htt allele. In these pharmaceutical compositions, the protein ZFP-TF is also used in conjunction with or as a substitute for the nucleic acid encoding the genetic modulator described herein.

[0030] In yet another aspect, isolated cells are provided that contain any of the ZFP genetic regulators and / or compositions described herein.

[0031] In other aspects, methods of treating and / or preventing Huntington's disease using the methods and compositions described herein are also provided herein. In some embodiments, the method relates to a composition wherein the polynucleotide and / or protein can be delivered using a viral vector, a non-viral vector (e.g., a plasmid), and / or a combination thereof. In some embodiments, the method relates to a composition comprising a stem cell population.

[0032] These and other aspects will be apparent to those skilled in the art based on the disclosure as a whole.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A-1K Design and testing of an allele-selective ZFP targeting mHtt is shown. Figure 1A is the structure and expected behavior of the HTT allele-specific repressor. Designed ZFP-KRAB proteins that are engineered to bind within poly-CAG tracts with appropriate affinity and target spacing should exhibit highly selective repression of amplified disease alleles. ZFP-TFs were screened for differential repression of wild-type (<22) and disease (>39) alleles in patient cell lines carrying downstream SNPs, enabling independent quantification of allele levels using SNP-specific qRT-PCR targeting rs363099, rs362331, and rs362307. Figure 1B 1- and 2-base spanning linkers and 1- and 2-finger units used to assemble candidate ZFPs are shown. Alternative shading indicates alternative designs with different sets of DNA contact residues and binding properties. See Table 1 for sequence details. Figure 1CShown are ZFP-TF sense (left) and antisense (right) targeting designs screened for selective inhibition of mHTT. 1- and 2-finger modules targeting either poly-CAG strand are linked together using alternative linkers to generate 3, 4, 5, or 6-finger ZFPs, enabling the skipping of 0, 1, or 2 bases between adjacent fingers. Target sequences are indicated by left, middle, and right DNA sequences. Figure 1D Shown are transcript levels of normal (top panel) and disease (bottom panel) alleles of HTT, assessed 24 hours after delivery of 100 ng of ZFP-TF mRNA by nucleofection to patient fibroblasts carrying poly-CAG tracts of varying lengths (GM02151 or GM04723). Allele-selective repressors (ZFP A, B, and D) and non-selective repressor (ZFP-C) were selected for further study. n = 3 biological replicates; mean ± SD. Figure 1D SEQ ID Nos: 81, 82, 79 and 80 are disclosed respectively in order of appearance. Figure 1E Shows Figure 1D Allele-specific repression of the three ZFPs shown were further investigated and analyzed. Studies were performed in patient GM04723 fibroblasts. Total HTT levels are also shown. n = 3 biological replicates; mean ± SD. Figure 1E SEQ ID Nos: 81 and 82 are disclosed respectively in order of appearance. Figure 1F Shown is the selective downregulation of mHTT (upper band at the top of the blot) protein by ZFP-A and ZFP-B detected by Western blot 72 hours after transfection of GM04723 fibroblasts with 100ng or 30ng ZFP or control mRNA. Wild-type HTT (lower band at the top of the blot) was not attenuated. Calnexin loading control (lower blot). Figure 1G ( Figure 1G SEQ ID No: 81 and 82), 1I ( Fig. 1I SEQ ID Nos: 79 and 80) and 1J ( Figure 1J SEQ ID Nos: 83 and 84 are disclosed in order of appearance, respectively) show the dose response of allele-specific repression in fibroblasts from patients GM04723 (Panel G), GM02151 (Panel I), or GM30259 (Panel J). qRT-PCR for total, WT, and mutant HTT was performed on RNA isolated from patient fibroblasts transfected with ZFP mRNA at a dose range of 100,000-fold (half-log dose steps, 1,000-0.01 ng). n=3 biological replicates; mean ± SD. Figure 1His a graph showing the frequency of various CAG repeat lengths in the HD cohort. HD patient allele frequencies were calculated based on the third enrollment-HD periodicity data set. All entries with CAG high > 35 (n=6602) were used to analyze the mean and median mHTT allele lengths. The CAG17 (SEQ ID NO: 76) and CAG43 (SEQ ID NO: 77) boxes show the median alleles. The circles under the CAG numbers indicate the allele lengths tested in this study. Figure 1K Activity of ZFP 45249 in GM02151 and GM30259 fibroblasts is shown, where the data are shown as doublets and the bars to the right of each doublet represent Htt expression on the amplified allele, while the bars to the left of each doublet represent Htt expression on the wild-type Htt allele. Figure 1K SEQ ID Nos: 79, 80, 83 and 84 are disclosed respectively in order of appearance.

[0035] Figure 2A-2N Allele-selective suppression, specificity, and phenotypic correction in HD neurons are demonstrated. Figure 2A Shown is a timeline overview of studies performed using NSCs derived from GENEA020 hESCs (CAG17 / 48, EQ ID NO 76 / 78). Figure 2B Shown is the dose response of allele-specific repression in HD NSCs. Total HTT or SNP-specific (rs362307) qRT-PCR for WT (CAG17) and mutant HTT (CAG48) was performed on RNA isolated from NSCs transfected with ZFP mRNA at doses ranging from 3,000-fold (half-log dose steps, 3,000-1 ng). n=3 biological replicates; mean±SD. Figure 2B SEQ ID Nos: 76 and 78 are disclosed respectively in order of appearance. Figure 2C The cells were expressed with AAV6-ZFP or AAV6-GFP (1x10 5 vg / cell) for 21 days. The figure shows qRT-PCR for WT and mHTT. Figure 2B ; n = 2 biological replicates; mean ± SD. Figure 2C SEQ ID Nos: 76 and 78 are disclosed respectively in order of appearance. Figure 2D HD NSCs infected with lentivirus encoding ZFP-2A-GFP or GFP were shown, and cell sorting for GFP expression was performed 27 days after infection to enrich for transduced cells. qRT-PCR data at 74 days are shown, as Figure 2B ; n = 3 biological replicates; mean ± SD. Figure 2DSEQ ID Nos: 76 and 78 are disclosed respectively in order of appearance. Figure 2E Differentiation of GFP sorted NSCs into neurons from panel D is shown. qRT-PCR data 25 days after the initiation of neuronal differentiation (103 days after lentiviral infection) are shown, as Figure 2B ; n = 3 biological replicates; mean ± SD. Figure 2E SEQ ID Nos: 76 and 78 are disclosed respectively in order of appearance. Figure 2F Shown is a genome-wide specific assessment of HD neurons (CAG17 / 48 (SEQ ID NOS 76 / 78)) using microarrays (Affymetrix GeneChip Primeview, n=4-6 biological replicates / treatment) 24 hours after transfection with 1500ng of ZFP mRNA. Each point represents the fold change in transcript level (x-axis) and p-value (y-axis) of a single gene in cells treated with the indicated ZFP compared to control-treated cells. Genes with >2-fold regulation and p-value <0.01 are shown. Figure 2G Shows Figure 2F Venn diagram of genes regulated in . Figure 2H Shown is an increase in intracellular ATP levels in neurons treated with ZFP-B. Intracellular ATP levels in HD and normal neurons were measured 21 days after ZFP-2A-GFP or CMV-GFP lentiviral infection. Fig.2I Shown is a decrease in apoptosis in neurons treated with ZFP-2A. Apoptosis was assessed by TUNEL staining of neurons infected with lentivirus encoding ZFP-2A-GFP or CMV-GFP for 5 days followed by growth factor withdrawal for 48 hours. Figure 2J is an overview of experiments with HdhQ50 mice. Figure 2K Shown are striatal WT (Q7) and KI (Q50) Htt mRNA levels in heterozygous Q50 mice measured by allele-specific qRT-PCR 7 weeks after injection of AAV2 / 6 encoding ZFP-B or GFP. n=6-8 hemispheres / group, mean±SD. Figure 2L is a regression analysis of Q50 or Q7 and ZFP-B mRNA levels assessed by qRT-PCR. All striatal subsections are included. 95% confidence bands are shown. Figure 2MShown is a genome-wide specificity assessment in HD neurons (CAG17 / 48 (SEQ ID NOS 76 / 78)) and HD fibroblasts (CAG18 / 45 (SEQ ID NOS 79 / 80)) using microarrays (Affymetrix GeneChip Primeview, n=4-6 biological replicates / treatment) 24 hours after transfection with 1500ng of ZFP mRNA. Figure 2M In the figure, two ZFP-TFs were compared: ZFP-B and ZFP-45249. Dots represent genes that were >2-fold regulated compared to the no ZFP-TF treatment. Figure 2N Shown is repression of mHtt alleles and wild-type alleles in GeneA020 neurons using ZFP-45249. Also shown is repression of CPEB1 and MBD5 by these same ZFPs (see Example 5). Figure 2N SEQ ID Nos: 76 and 78 are disclosed respectively in order of appearance.

[0036] Figures 3A-3G Improvement of behavioral deficits and neuroprotection in R6 / 2 mice was shown. Figure 3A is a timeline overview of the behavioral and molecular endpoints assessed in wild-type and AAV-treated R6 / 2 mice. Figure 3B Shown are the percentages of mice exhibiting clasping behavior assessed weekly following treatment with AAV2 / 6 encoding ZFP-B (left bar) or GFP (right bar); n=14 / group; P=0.024 for treatment effect, log-rank test. Figure 3C and 3D Shown are the results of the open field test of rearing frequency (3C) or total distance traveled (3D) performed on wild-type (left), ZFP-B-treated R6 / 2 (center), or GFP-treated (right) R6 / 2 mice at 4 (baseline), 6, 8, 10, and 12 weeks of age. Repeated measures ANOVA, (3C, 3D) genotype main effect P < 0.0001, ( Figure 3C ) Treatment effect P<0.009, (3D) Treatment effect P<0.038. Figure 3E Shown are WT and mutant Htt(R6 / 2) mRNA measured by qRT-PCR 7 weeks after delivery. n=14-20 hemispheres; mean±SD. Figure 3F is qRT-PCR assessment of mRNA levels of striatal neuronal markers DARPP32, PDE10A, DRD1A, and DRD2 as in (3E); n = 6 (wild type), 14 (GFP), and 20 (ZFP-B) hemispheres per group; means ± SD are shown. Figure 3Gis the correlation between mHtt, DARPP32, PDE10A, DRD1A or DRD2 levels and ZFP-B mRNA levels. All striatal subregions are included. 95% confidence bands are shown.

[0037] Figures 4A-4T ZFP-driven repression of mHTT in a knock-in HD mouse model is shown. Figure 4A and 4B Mouse or mutant HTT mRNA ( Figure 4A ) or soluble protein ( Figure 4B ) levels. mRNA was assessed by qRT-PCR. Protein was assessed on the Singulex (mouse HTT) or MSD (mHTT) platform. Figure 4C is an overview of the timeline showing endpoints in the zQ175 heterozygous mouse study. Figure 4D Representative images of mEM48 immunostaining in heterozygous zQ175 striatum injected with AAV2 / 1+2 encoding ZFP-B.T2A.GFP at 2 months of age and analyzed by staining for mEM48, GFP, DARPP-32, and DAPI at 4 months of age are shown (colors not shown but as follows: HTT inclusions (mEM48): yellow, GFP: green, DARPP-32: red, DAPI: blue). Amplified GFP+ and GFP- areas are shown on the right. Figure 4E and 4F The perinuclear ( Figure 4E ) and core( Figure 4F ) Quantification of mHTT inclusion bodies; n=5; mean±SD. Statistical analysis used in 4E-4O: Two-tailed t-test with Welch correction. Figure 4G Quantification of mEM48 intensity in GFP+ cells is shown as in (4E and 4F); n=5; mean±SD. Figure 4H Data as in 4D are shown, except that heterozygous mice injected with AAV2 / 1+2 encoding ZFP-B.T2A.GFP at 6 months of age and analyzed at 10 months of age were used. Fig. 4I and 4J Shown are data as in 4E and 4F , except that the 6-10 month old groups were treated with AAV2 / 1+2ZFP-B.T2A.GFP, ΔDBD.T2A.GFP, or GFP; n=5; mean±SD. Figure 4KAs in 4G , except that the 6-10 month old groups were treated with AAV2 / 1+2ZFP-B.T2A.GFP, ΔDBD.T2A.GFP, or GFP; n=5; mean±SD. Figure 4L Shown are DARPP32 in the striatum of 10 month old WT and heterozygous zQ175 mice measured by IHC; n=5; mean±SD. Figure 4M Shown are DARPP32 levels in the striatum of mice injected with AAV2 / 1+2ZFP-B.T2A.GFP, ΔDBD.T2A.GFP, or GFP at 6 months of age and analyzed at 10 months of age; n=5; mean±SD. Figure 4N Representative images of GFAP and IBA1 immunostaining in the striatum of heterozygous zQ175 injected with AAV2 / 1+2 encoding ZFP-B.T2A.GFP at 6 months of age and analyzed for GFP, DAPI, GFAP, and IBA1 at 10 months of age are shown (colors not shown but as follows: GFP: green, DAPI: blue, GFAP: red, IBA1: yellow). Fig.4O and 4P Shown is the quantification of GFAP+ (o) and IBA+ (p) cells compared to all cells in the striatum of mice treated with AAV2 / 1+2ZFP-B.T2A.GFP, ΔDBD.T2A.GFP or GFP at 6 months of age and analyzed at 10 months of age; n=5; mean±SD. Figure 4Q Shown are line scans from which bAP-evoked Ca2+ images were taken at proximal and distal locations on the same dendrite and used to calculate the dendritic index. Figure 4R Shown are Ca2+ transients in mice injected at 4 months with genes encoding ZFP-D.T2A.tdTomato (zQ175, left), ΔDBD.T2A.tdTomato zQ175 het (zQ175, center), or ZFP-D.T2A.tdTomato (WT littermates, right) and tested at 6 months. Figure 4S and 4T Shown is the rescue of dendritic index of zQ175 HET treated with ZFP-D.T2A.tdTomato (n=10-17) in 2-6 month old (s) or 4-6 month old (t) groups compared to ΔDBD.T2A.tdTomato zQ175 (n=6-11) and ZFP-D.T2A.tdTomato WT mice (n=10-15). Mann-Whitney U, single tail.

[0038] Figures 5A-5F Restoration of imaging markers expressed by ZFPs in the striatum of Q175 het mice is shown. Figures 5A-5FShown are autoradiographic analyses of mice (n=10 / group) injected unilaterally with AAV2 / 1+2 encoding ZFP-D (right) or GFP (left). Mice were injected at 2 months of age and re-injected at 6 months of age ( Figure 5A ) or 4 months of age, and at 10 months of age ( Figure 5A ) analysis, using the Figure 5B )、D2( Figure 5C ) and PDE10( Figure 5D In both studies, specific binding to D1, D2, and PDE10 was significantly increased in mice treated with AAV2 / 1+2ZFP-D (paired t-test). Figure 5E The % difference in BPND of [18F]MNI-659 in the striatum of zQ175 mice injected and uninjected at 6.5 / 7M and 10M of age treated with AAV2 / 1+2ZFP encoding ZFP-D or GFP is shown (n=33-41 mice / group / time point). The percentage difference between the left and right striatum between the two treatment groups was compared by unpaired t-test. Fig. 5F ) Average [18F]MNI-659%SUV images of 10M-old zQ175 het mice treated with AAV2 / 1+2ZFP-D, averaged from 15 to 63 minutes. Template MRI (upper row) and PET (middle row) are co-registered in the bottom row. Both left (untreated) and right (treated) striatum are shown in the sagittal plane (n=36). Asterisks mark the increase in PDE10 binding obtained in the ZFP-D-treated striatal hemisphere.

[0039] Fig. 6A and 6B Validation of the allele-specific qRT-PCR assay is shown. Fig. 6AResults obtained using plasmid templates corresponding to rs63099T (099T) and rs363099C (099C) SNPs of HTT are shown. The 099T SNP is in phase with WT HTT in GM02151 CAG18 / 45 (SEQ ID NOS 79 / 80) and GM04723 CAG15 / 67HD (SEQ ID NOS 81 / 82) fibroblasts, while 099C is in phase with mutant HTT in the same cells. The plasmids were then used to prepare serial dilutions of each other, as shown. To this end, the 099C template was serially diluted two-fold from 5fg to 0.156fg (099C:099T from 0.125:1 to 4:1) in the presence of a fixed amount (1.25fg) of the 099T template; and the 099T template was serially diluted two-fold from 5fg to 0.156fg (099T:099C from 0.125:1 to 4:1) in the presence of a fixed amount (1.25fg) of the 099C template. The serial dilutions were assembled in the presence of a fixed amount of total cDNA, which was prepared from a third patient fibroblast cell line in which HTT and mHTT expression had been depleted (see the method in the examples). In this line, HTT and mHTT are also in phase with 099T and 099C (respectively). The plasmid levels were selected so that the qRT-PCR signal from the 1:1 plasmid ratio was approximately the same as the HTT and mHTT cDNA from the non-depleted cells. 099C-specific (right column) and 099T-specific (left column) qRT-PCR analysis was performed on each diluted sample. Relative amounts are plotted as a fraction of the values ​​obtained for samples with a 1:1 ratio of 099T and 099C templates. Figure 6BShown are results from plasmid templates from rs362331T (331T, in phase with WT HTT in ND30259 CAG21 / 38 (SEQ ID NO 83 / 84) HD fibroblasts) and rs362331C (331C, in phase with mHTT in ND30259 CAG21 / 38 (SEQ ID NO 83 / 84) HD fibroblasts) serially diluted into cDNA made from GM21756 fibroblasts (CAG15 / 70 (SEQ ID NO 81 / 85)) that had been transfected with a biallelic ZFP repressor that repressed WT and mutant HTT by >90%. The 331C template was serially diluted two-fold from 2fg to 0.0625fg in the presence of a fixed amount (0.5fg) of 331T template; the 331T template was serially diluted two-fold from 2fg to 0.0625fg in the presence of a fixed amount (0.5fg) of 331C template. 331C-specific (red columns) and 331T-specific (blue columns) qRT-PCR analysis was performed on each diluted sample. Relative amounts are plotted as a fraction of the value obtained for samples with a 1:1 ratio of 331T and 331C templates.

[0040] Fig. 7A and 7B Dose-dependent ZFP protein expression with 100-fold more transfected ZFP mRNA is shown. Fig. 7A Displayed in 2×10 5 HD fibroblasts (GM04723, CAG15 / 67 (SEQ ID NOS 81 / 82)) were transfected with ZFP mRNA at doses of 1000, 300, 100, 30 and 10 ng per cell. Five hours after transfection, cells were harvested for ZFP Western blotting using anti-FLAG antibody (Sigma F1804). Figure 7B ZFP and GAPDH protein levels quantified using the Odyssey CLx Imager are shown, with the ZFP / GAPDH ratio for each sample scaled to that of the 1000 ng ZFP-C sample (set to 1).

[0041] Fig. 8A and 8B Testing of candidate ZFP designs in Hdh Q7 / Q111 striatal cells is shown. Fig. 8A and 8B Candidate ZFP-TF designs in the screening system used in this study (patient fibroblasts; CAG18 / 45 (SEQ ID NOS79 / 80)) are shown ( Figure 8B) compared to cells used in previous work (cells from the STHdhQ111 / HdhQ7 mouse model; CAG 4 / 111 (SEQ ID NOS 86 / 87)) ( Fig. 8A ) in mouse cells. Only 3 designs tested (ZFP-A, ZFP-B, ZFP-D) exhibited highly allele-specific repression in the patient fibroblast system (>75% repression of the mutant allele and <10% repression of the wild-type allele). In contrast, 25 designs exhibited this behavior in less stringent mouse cells. For mouse cell studies, WT (CAG4 (SEQ ID NO: 86)) and KI (CAG111 (SEQ ID NO: 87)) Htt alleles were measured using allele-specific qRT-PCR 24 hours after transfection of STHdhQ111 / HdhQ7 mouse striatal cells with 100 ng of ZFP or GFP mRNA. Htt signals were normalized to the mean of EIF4a2, ATP5b, and GAPDH, and then scaled to GFP (set to 1); n=3 biological replicates; mean±SD.

[0042] Fig. 9 Shown is testing of a CAG-targeted ZFP from Garriga-Canut et al. (2012) Proc Natl Acad Sci USA 109(45):e3136-45 in HD fibroblasts and comparison of allele-selective inhibition in patient fibroblasts from a previous study (CAG 18 / 45 (SEQ ID NO 79 / 80)) (ZF6 and ZF11, see Garriga-Canut et al. (2012) Proc Natl Acad Sci USA 109(45):e3136-45)). Allele-specific qRT-PCR of total, WT (CAG18 (SEQ ID NO:86)) and mutant (CAG45 (SEQ ID NO:87)) HTT mRNA 24 hours after transfection of GM02151 fibroblasts with 1000, 300 or 100 ng of ZFP mRNA. HTT expression was normalized as ( Figure 1D ). The published ZF6-Kox and ZF11-Kox sequences were cloned into the same expression cassette used in the current study and prepared in parallel. Figure 1D In vitro transcribed mRNA of all ZFPs tested in ; n = 3 technical replicates; mean ± SD.

[0043] Figures 10A-10D The expression of NSC and neuronal markers in differentiated CAG17 / 48 (SEQ ID NO 76 / 78) hESCs is shown. Fig. 10A Shown are NSC-specific PAX6 (light shading) and Nestin (dark shading) expression in NSCs differentiated from CAG17 / 48 (SEQ ID NO 76 / 78) ESCs (GENEA020) confirmed by IHC. Fig. 10B Shown are expression levels of a panel of pluripotency markers (OCT4, NANOG, and REX1) or NSC markers (PAX6, SOX1, and NES) in ESCs or differentiated NSCs measured by qRT-PCR. Gene expression was normalized to GAPDH and scaled to normalized expression levels (set to 1) in ESCs (upper panels) or NSCs (lower panels); n=2 biological replicates; mean ± SD. Fig. 10C Shown is neuron-specific βIII-tubulin expression (dark shading) in differentiated CAG17 / 48 (SEQ ID NO 76 / 78) neurons (DAPI, light shading) confirmed by IHC. Fig. 10D Shown are expression levels of a panel of neuronal markers (MAP2, GAD1, and FOXG1) or NSC markers (PAX6, SOX1, and NES) from CAG17 / 48 (SEQ ID NO 76 / 78) NSCs or differentiated neurons measured by qRT-PCR. Gene expression was normalized to GAPDH and scaled to normalized expression levels (set to 1) in neurons (top) or NSCs (bottom); n=2 biological replicates; mean ± SD.

[0044] Figures 11A-11D Shown is the assessment of ZFP specificity in HD fibroblasts. Fig.11A Fold changes in gene expression with the indicated ZFP-TFs are shown. Each point represents the fold change in transcript level (x-axis) and p-value (y-axis) of a single gene in cells treated with the indicated ZFP compared to control treated cells. Genes with >2-fold regulation and p-value <0.01 are shown. Fig. 11B Fold repression of indicated genes is shown. Fig. 11B SEQ ID Nos: 79 and 80 are disclosed respectively in order of appearance. Fig. 11B Normalized expression percentages of indicated genes are shown. Fig.11D Shows Fig. 11C Venn diagram of genes regulated in . See further description in the Examples text.

[0045] Fig. 12A and 12Bis a graph showing confirmation of allele-selective HTT repression in samples used for microarray studies. Allele-specific qRT-PCR analysis was performed to confirm mHTT regulation in RNA samples (6 biological replicates per treatment) that were subjected to PCR for GENEA020 neurons (shown in Fig. 12A (in order of appearance, SEQ ID Nos: 76 and 78 respectively); corresponding to Figure 2F and 2G ) and GMO2151 fibroblasts (shown in Fig. 12B (SEQ ID NOs 79 and 80, respectively, in order of appearance); corresponding to Fig.11A ) microarray analysis. Figure 2B (GENEA020 Neuron) and Fig. 1I (GMO2151 fibroblasts) Allele-specific qPCR analysis as shown.

[0046] Figure 13A-13C Body weight and other behavioral data from the R6 / 2 mouse study are shown for wild-type ("R62_WT") subjects and subjects treated with the GFP transgene ("R62_Tg GFP") or the ZFP-TF 33074 ("R62_Tg 33074"). Fig.13A is a graph depicting body weight for a given week. Separate graphs for male and female subjects for gender combinations are shown. Fig. 13B is a graph depicting the fall time for a given condition. Separate graphs are shown for male and female subjects for the gender combination. Fig. 13C is a graph depicting the normalized ratio of forelimb grip strength: body weight for the indicated week. See also the Examples text for further description.

[0047] Figures 14A-14C Injection of AAV1 / 2-hSYN1-GFP and

[0048] IHC analysis of zQ175 mice injected with AAV1 / 2-hSYN1-GFP. Representative images of mEM48 immunostaining in the striatum of heterozygous zQ175 mice injected with control construct. Fig.14A Results are shown for subjects injected with AAV2 / 1+1-hSYN1-GFP at 2 months of age and analyzed at 4 months of age; Fig. 14B Results are shown for subjects injected with AAV2 / 1+1-hSYN1-GFP at 6 months of age and analyzed at 10 months of age; Fig. 14CResults are shown for subjects injected with AAV2 / 1+1-hSYN1-ΔDBD.T2A.GFP at 6 months of age and analyzed at 10 months of age. Colors not shown are as follows: HTT inclusion bodies (mEM48): yellow, GFP: green, DARPP-32: red, DAPI: blue.

[0049] Figures 15A-15G Neuroinflammation assessment in treated wild-type and zQ175 mice is shown. Fig.15A Results of immunostaining against the indicated molecules (GFP, DAP, GFAP, BA1) are shown. Fig. 15B are graphs showing the ratio of ba1 cells to all cells at 2-6 months in subjects treated as indicated. Fig. 15C are graphs showing the ratio of GFAP cells to all cells at 2-6 months in subjects treated as indicated. Fig.15D are graphs showing GFAP F intensity in GFAP cells at 2-6 months in subjects treated as indicated. Fig.15E are graphs showing the ratio of ba1 cells to all cells at 6-12 months in subjects treated as indicated. Fig.15F are graphs showing the ratio of GFAP cells to all cells at 6-12 months in subjects treated as indicated. Figure 15G is a graph showing GFAP F intensity in GFAP cells at 6-12 months in subjects treated as indicated. See also the Examples text for further description.

[0050] Figures 16A-16E Molecular and histopathological bridging data for ZFP-D in zQ175 mice are shown. Fig.16A Normalized mRNA expression of wild-type and mutant Htt in neurons of subjects treated with ZFP-D or mock is shown. Fig. 16B HTT protein expression of wild-type and mutant Htt under indicated conditions. Fig. 16C Shown are the densities of perinuclear inclusion bodies in subjects treated as indicated at 2-6 months (left) or 6-12 months (right) post-treatment. Fig.16D Shown are nuclear inclusions in ZFP+ and NueN+ neurons from subjects treated as indicated at 2-6 months (left) or 6-12 months (right) post-treatment. Fig.16E Shown are EM48 nuclear intensities in ZFP+ and NueN+ neurons of subjects treated as indicated at 2-6 months (left) or 6-12 months (right) post-treatment. See also the Examples text for further description.

[0051] Figures 17A-17FAnalysis of CAG repeat content in the human or mouse genome for genes with greater than or equal to 6 CAG repeats ("≥CAGx6") is shown. Fig.17A Results from screening for 0 mismatches / 6-mers are shown. Fig. 17B Results are shown for screening for 0 mismatches within 1 kb of the transcription site site (TSS). Fig. 17C Results for screening of ≤3 mismatches / 6-mer are shown. Fig.17D Results are shown for screening for ≤3 mismatches within 1 kb of the TSS. Fig.17E Comparison of mouse and human is shown for the top 100 genes with 0 mismatches (left) and the top 100 genes with ≤ 3 mismatches (right). Fig.17F Comparison of mouse and niyse for the top 100 genes with 0 mismatches (left) and the top 100 genes with ≤ 3 mismatches (right) is shown. See also the Examples text for further description. DETAILED DESCRIPTION

[0052] Disclosed herein are compositions and methods for reducing toxic mHTT protein levels in subjects using mutant Htt allele ZFP genetic regulators. The genetic repressors described herein selectively repress >99% of alleles that cause HD over an 80-fold dose range while retaining expression of >86% of wild-type alleles present in the HD population. At the same time, the expression of other CAG-containing genes is minimally affected, and the repressors delivered by the virus are active and well tolerated in HD neuron cultures for more than 100 days and in mouse brains for at least 4 months. In addition, the genetic repressors described herein result in HD molecular, histopathological, electrophysiological, and phenotypic corrections in a variety of accepted animal models.

[0053] The methods and compositions described herein provide allele-selective transcriptional repression at the native HTT locus. Results from extensive testing in patient-derived cells indicate that expression of mHTT alleles with ≥38 CAG repeats can be repressed by 79-93%, while expression of normal alleles with ≤21 CAG repeats (the longest normal repeat length tested) is only inhibited by 0-31%. Thus, allele-selective ZFP-TFs exhibit a significant ability to distinguish 100% of fully penetrant mutant alleles from at least 86% of normal HTT alleles in the HD population. This is in contrast to SNP-based allele-selective mHTT reduction methods, each of which is limited to a subset of HD patients (Pfister et al. (2009) CurBiol 19(9):774-778; Southwell et al. (2014) Mol Ther

[0054] 22(12):2093-106)), the CAG-targeted ZFP repressor described in this study has the potential to selectively downregulate the expression of the pathogenic allele in the majority of HD patients.

[0055] Although the non-human primate striatum can tolerate a partial reduction (about 45%) in normal HTT expression (Grondin et al. (2012) Brain 135(4):1197-1209; McBride et al. (2011) Mol Ther 19:2152-2162; Stiles et al. (2012) Exp Neurol 233:463-471), the long-term effects of further reduction are unclear. HTT plays a role in a variety of biological functions (Zuccato, cited above), including intracellular trafficking, energy, transcriptional regulation, and autophagy. In mice, Htt gene knockout is embryonic lethal (Duyao et al. (1995) Science 269:407-410; Zeitlin et al. (1995) Nat Genet 11:155-163; Nasir et al. (1995) Cell 81:811-823), and perinatal loss leads to motor dysfunction and other neuropathologies (Dragatsis et al. (2000) Nat Genet 26:300-306; Arteaga-Bracho et al. (2016) Neurobiol Dis 96:144-155), suggesting an important role in the development of the nervous system. Although conditional knockout in adult mouse brain appears to be tolerated (Wang et al. (2016) Proc Natl Acad Sci USA 113:3359-3364), heterozygous hypomorphic HTT variants have been linked to Rett Syndrome-like disorders (Lopes, F., et al. (2016) J Med Genet 53:190-199; Rodan et al. (2016) Eur J Hum Genet 24:1833), suggesting a critical role in humans. Therefore, lowering normal HTT, especially below 50% of baseline levels, that coexists with mHTT will carry unknown risks. Therefore, the development of allele-selective mHTT-lowering methods suitable for clinical use has been a long-term goal.

[0056] A variety of targeting RNA patterns have been investigated to selectively downregulate mHTT transcripts by degradation or translational inhibition. For example, ASO- and RNAi-based strategies targeting single nucleotide polymorphisms (SNPs) associated with mHTT alleles have been shown to selectively reduce mHTT in HD mouse models (Pfister et al. (2009) Cur Biol 19(9):774-778; Lombardi et al. (2009) Exp Neurol 217:312-319; Carroll et al. (2011) Mol Ther. 19(12):2178-85). A significant obstacle to the clinical development of these approaches is that various SNP-specific therapies can only treat a subset of patients carrying that particular SNP (Kay et al. (2015) Mol Ther 23:1759-1771). Alternatively, direct targeting of the CAG repeat sequence would in principle constitute a single therapy for all HD patients. However, ASO- and RNAi-based agents targeting CAG repeats have so far yielded only modest levels of allelic discrimination (Evers et al. (2011) PLoS One 6(9):e24308; Gagnon et al. (2010) Biochemistry 49:10166-10178; Hu et al. (2009) Nat Biotech 27:478-484; Yu et al. (2012) Cell 150:895-908; Fiszer et al. (2011) Nuc Acids Res 39:5578-5585), likely due to a general reliance on a large number of effectors to achieve selectivity.

[0057] As described herein, repressing transcription by directly binding to an expanded CAG tract near the promoter effectively reduces mHtt levels. Without being bound by any theory, this method sometimes provides a greater degree of allele discrimination in transcriptional regulation by taking advantage of functional synergy (Lutz et al. (2000) BiochemSoc Trans 28: 386-389) and cooperativity (Reiter et al. (2017) Curr Opin Genet Dev 43: 73-81), and can use a variety of local regulatory elements to set the level of transcript synthesis. Our method provides the additional benefit of reducing the level of mutant RNA, which itself is a potential pathogenic factor (Marti (2016) Brain Pathol 26: 779-786). Transcriptional inhibition can also provide a more efficient method to achieve complete removal of mHTT, because compared with other methods that bind thousands of mutant RNA or protein substances to each cell, this method only requires the identification of a single copy of the therapeutic target-the endogenous mHTT gene. In previous studies, engineered transcription factors targeting poly-CAG were attempted for allele-selective regulation, albeit via a simpler and less functional bulk mechanism of action (Garriga-Canut et al. (2012) Proc Nat Acad Sci USA 109(45):e3136-45). Differential repression was observed only in mouse cells carrying a non-pathogenic repeat array that was too small to encompass a single target of the tested agent (CAG4 (SEQ ID NO:86)). In addition, allele skew was dramatically exaggerated (4 (SEQ ID NO:86) vs. 111 CAGs (SEQ ID NO:87); length ratio of 28) and more than tenfold that present in typical patients (17 (SEQ ID NO:76) vs. 43 CAGs (SEQ ID NO:77); length ratio of 2.5).

[0058] Furthermore, using the allele-selective repressor described herein (ZFP-B), which is also highly specific genome-wide, we demonstrated robust in vivo repression of mHtt expression in 3 different HD mouse models at time points between 7 weeks and 4 months, and thereby corrected molecular, histological, electrophysiological, and phenotypic pathologies. We also established sustained ZFP expression and efficacy in HD neurons for >100 days and rescued MSN marker gene expression in R6 / 2 and zQ175 mice, indicating that long-term expression of highly potent mHTT-selective ZFPs is well tolerated in target brain regions and human cell types.

[0059] Here, we present a targeted transcriptional approach that differs from previous approaches in terms of scale, design complexity of the assay, and use of screens that directly interrogate desired behaviors: allele-selective repression of endogenous human genes over a wide range of doses. We identified a novel class of functionally synergistic ZFPs whose repression is highly dependent on poly-CAG tract length. These ZFP repressors highly discriminate between >86% of combinations of normal and disease alleles in HD patients (Landwehrmeyer et al. (2017) Movement Disorders Clinical Practice 4:212-224) and exhibit high specificity for mHTT compared to other CAG repeat genes. Using 3 HD mouse models, we demonstrated improvements in molecular, histopathological, electrophysiological, and functional endpoints. Finally, we demonstrated that allele-selective ZFPs were well tolerated for at least 9 months after administration to the mouse striatum and were effective in multiple HD pathology models.

[0060] Overview

[0061] 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; 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.

[0062] definition

[0063] 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.

[0064] 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.

[0065] "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.

[0066] Zinc finger "DNA binding proteins" (or binding domains) are proteins or domains within larger proteins that can bind to 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 zinc ion coordination. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Artificial nucleases and transcription factors can include ZFP DNA binding domains and functional domains and functional domains (nuclease domains of ZFNs or transcriptional regulatory domains of ZFP-TFs). The term "zinc finger nuclease" includes a ZFN and a pair of ZFNs (including a first and a second ZFN, referred to as a left and right ZFN), which dimerize to cut a target gene.

[0067] Zinc finger binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example, by engineering (changing one or more amino acids) the recognition helix region of a naturally occurring zinc finger. Thus, engineered DNA binding proteins (zinc fingers) are non-naturally occurring proteins. Non-limiting examples of methods for engineering DNA binding proteins are design and selection. Designed DNA binding proteins are non-naturally occurring proteins whose design / composition is primarily derived from rational criteria. Rational criteria for design include applying substitution rules and computer algorithms to process information in database storage information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261; and 8,585,526; see also International Patent Publication Nos. WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496.

[0068] "Selected" zinc finger proteins are proteins that do not exist in nature and are generated primarily as a result of empirical processes such as phage display, interaction traps or hybrid selection. See, for example, U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; 8,586,526; and 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; and WO 02 / 099084.

[0069] "TtAgo" is a prokaryotic Argonaute protein that is believed to be involved in gene silencing. TtAgo is derived from thermophilic bacteria (Thermus thermophilus). See, e.g., Swarts et al. (2014) Nature 507(7491):258-261; G. Sheng et al. (2013) Proc. Natl. Acad. Sci. USA 111:652). The "TtAgo system" is all the components required, including, e.g., a guide DNA for cleavage by the TtAgo enzyme.

[0070] "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 purpose 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 a homology-directed repair mechanism. This process requires nucleotide sequence homology, and the "target" molecule (i.e., the molecule undergoing double-strand breaks) is repaired using a "donor" molecule template, which is also called "non-crossover gene conversion" or "short-track gene conversion" because it causes genetic information to be transferred from the donor to the target. Without being limited to any particular theory, this transfer can involve mismatch correction of heteroduplex DNA formed between the broken target and the donor, and / or "synthesis-dependent chain annealing" using the donor to resynthesize the genetic information that will become part of the target, and / or related processes. This special HR usually results in a change in the sequence of the target molecule, so that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

[0071] In the methods disclosed herein, one or more targeted nucleases described herein generate double-strand breaks (DSBs) at predetermined sites (e.g., cellular chromatin) in a target sequence (e.g., cellular chromatin). DSBs can result in deletions and / or insertions by homology-directed repair or by non-homologous directed repair mechanisms. Deletions can include any number of base pairs. Similarly, insertions can include any number of base pairs, including, for example, integration of a "donor" polynucleotide, optionally with homology to the nucleotide sequence in the break region. The donor sequence can be physically integrated, or the donor polynucleotide is used as a template for break repair by homologous recombination, resulting in the introduction of all or part of the nucleotide sequence in the donor into the cellular chromatin. Therefore, the first sequence in the cellular chromatin can be changed, and in some embodiments, can be converted into a sequence present in the donor polynucleotide. Therefore, the use of the term "replacement" or "substitution" can be understood to mean that a nucleotide sequence is replaced by another nucleotide sequence (i.e., replacement of a sequence in an information sense), without necessarily requiring that a polynucleotide is physically or chemically replaced by another polynucleotide.

[0072] Any of the methods described herein can be used to insert any size donor and / or partially or completely inactivate one or more target sequences in a cell by targeted integration of a donor sequence that disrupts expression of a gene of interest. Also provided are cell lines with partially or completely inactivated genes.

[0073] In any of the methods described herein, the exogenous nucleotide sequence ("donor sequence" or "transgene") may comprise a sequence that is homologous but not identical to the genomic sequence in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the sequence in the region of interest exhibits a sequence identity of about 80-99% (or any integer therebetween) with the genomic sequence to be replaced. In other embodiments, the homology between the donor and genomic sequences exceeds 99%, for example, the genomic sequence of more than 100 consecutive base pairs differs from the donor by only 1 nucleotide. In certain cases, the non-homologous portion of the donor sequence may comprise a sequence that is not present in the region of interest, thereby introducing a new sequence into the region of interest. In these cases, the non-homologous sequence is generally flanked by 50-1,000 base pairs (or any integer value therebetween) or a sequence of any number of base pairs greater than 1,000, which is homologous or identical to the sequence in the region of interest. In other embodiments, the donor sequence is non-homologous to the first sequence and is inserted into the genome by a non-homologous recombination mechanism.

[0074] "Cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand cleavage and double-strand cleavage can be used, and double-strand cleavage can be caused by different single-strand cleavage events. DNA cleavage can result in blunt ends or staggered ends. In certain embodiments, a fusion polypeptide is used for targeted double-stranded DNA cleavage.

[0075] A "cleavage half-domain" is a polypeptide sequence that can form a complex with a second polypeptide (the two are the same or different) that has cleavage activity (preferably double-stranded cleavage activity). The terms "first and second cleavage half-domains," "+ and - cleavage half-domains," and "right and left cleavage half-domains" are used interchangeably to refer to a pair of dimerized cleavage half-domains.

[0076] An "engineered cleavage half-domain" is a cleavage half-domain that has been modified to form an obligate heterodimer with another cleavage half-domain (e.g., another engineered cleavage half-domain). See also U.S. Patent Nos. 8,623,618; 7,888,121; 7,914,796; and 8,034,598, which are incorporated herein by reference in their entireties.

[0077] 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, 2-100,000,000 nucleotides in length (or any integer value therebetween or thereon), preferably about 100-100,000 nucleotides in length (or any integer therebetween), more preferably about 2000-20,000 nucleotides in length (or any value therebetween), and even more preferably about 5-15 kb in length (or any value therebetween).

[0078] "Chromatin" is a nuclear protein structure containing the cell genome. Cellular chromatin contains nucleic acids and proteins, nucleic acids are mainly DNA, and proteins include histones and non-histone chromosomal proteins. Eukaryotic cell chromatin mainly exists in the form of nucleosomes, wherein the nucleosome core contains about 150 base pairs of DNA associated with octamers, and the octamers contain two copies of histones H2A, H2B, H3 and H4; and linker DNA extending between the nucleosome cores (the length varies depending on the organism). Histone H1 molecules are usually associated with linker DNA. For the purposes of this disclosure, the term "chromatin" is intended to cover all types of cellular nuclear proteins, including prokaryotic and eukaryotic ones. Cellular chromatin includes chromosomes and episomal chromatin.

[0079] A "chromosome" is a chromatin complex that contains all or part of the genome of a cell. A cell genome is typically characterized by its karyotype, which is the collection of all chromosomes that comprise the cell genome. A cell genome may contain one or more chromosomes.

[0080] An "episome" is a replicating nucleic acid, nucleoprotein complex, or other structure containing nucleic acid that is not part of the chromosomal nucleotype of a cell. Examples of episomes include plasmids and certain viral genomes.

[0081] An "accessible region" is a site in cellular chromatin where a target site present in a nucleic acid can be bound by an exogenous molecule that recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is a region that is not packaged into a nucleosome structure. The different structures of accessible regions can generally be detected by their sensitivity to chemical and enzymatic probes, such as nucleases.

[0082] 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. A target site can be of any length, e.g., 9-20 or more nucleotides and length, and the bound nucleotides can be contiguous or non-contiguous.

[0083] 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.

[0084] A "fusion" molecule is a molecule in which two or more subunit molecules are linked (preferably covalently linked). 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.

[0085] 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.

[0086] 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.

[0087] "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.

[0088] "Regulation" of gene expression refers to a change 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 mutation) can be used to regulate expression. A gene regulator refers to any change in gene expression compared to a cell that does not contain a ZFP gene regulator as described herein. Thus, gene inactivation can be partial or complete.

[0089] 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.

[0090] "Eukaryotic" cells include, but are not limited to, fungal cells (eg, yeast), plant cells, animal cells, mammalian cells, and human cells (eg, B cells), including stem cells (pluripotent and multipotent).

[0091] 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.

[0092] For fusion polypeptides, the term "operably linked" may refer to the same function that each component performs in connection with other components as when not linked. For example, for a fusion molecule in which a ZFP DNA binding domain is fused or otherwise associated with a transcriptional repression domain, if in the fusion molecule, the ZFP DNA binding domain portion is capable of binding to its target site and / or its binding site, and the activation domain is capable of up-regulating gene expression, then the DNA binding domain and the repression domain are operably linked. In the case of a fusion polypeptide in which a ZFP DNA binding domain is fused to a cleavage domain, if in the fusion polypeptide, the ZFP DNA binding domain portion is capable of binding to its target site and / or its binding site, and the cleavage domain is capable of cleaving DNA near the target site, then the ZFP DNA binding domain and the cleavage domain are operably linked.

[0093] 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.

[0094] "Vector" is capable of transferring a gene sequence to a target cell. "Vector construct", "expression vector" and "gene transfer vector" generally refer to a nucleic acid construct that is capable of directing the expression of a gene of interest and of transferring a gene sequence to a target cell. Thus, the term includes cloning and expression vectors, as well as integrating vectors.

[0095] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as laboratory animals such as rabbits, dogs, cats, rats, mice and other animals. Thus, the terms "subject" or "patient" as used herein refer to any mammalian patient or subject to which the nucleases, donors and / or genetically modified cells of the invention can be administered. Subjects of the invention include those suffering from a disease.

[0096] Zinc finger DNA binding domain

[0097] Described herein are DNA binding domains comprising zinc finger proteins that bind to the Htt gene. See, e.g., U.S. Pat. No. 8,841,260.

[0098] Compared to naturally occurring zinc finger proteins, engineered zinc finger binding domains may have new binding specificities. Engineering methods include, but are not limited to, rational design and different selection types. For example, rational design includes the use of a database comprising trisomic (or tetrasomic) nucleotide sequences and individual zinc finger amino acid sequences, wherein each trisomic or tetrasomic nucleotide sequence is associated with one or more zinc finger amino acid sequences that bind to the specific trisomic or tetrasomic sequence. See, for example, co-owned U.S. Patent Nos. 6,453,242 and 6,534,261, which are incorporated herein by reference in their entirety.

[0099] Exemplary selection methods include phage display and two-hybrid systems, disclosed in U.S. Pat. Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568, and International Patent Publication Nos. WO 98 / 37186, WO 98 / 53057, WO 00 / 27878, and WO 01 / 88197, and GB 2,338,237. In addition, enhancement of the binding specificity of zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02 / 077227.

[0100] In addition, as disclosed in these and other references, zinc finger domains and / or multi-finger zinc finger proteins can be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are also described, for example, in U.S. Pat. Nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein can include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of the binding specificity of zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02 / 077227.

[0101] 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. 6,140,0815; 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 WO 03 / 016496.

[0102] In addition, as disclosed in these and other references, zinc finger domains and / or multi-finger zinc finger proteins can be linked together using any suitable linker sequence, including, for example, linkers of 5 or more amino acids in length. Exemplary linker sequences of 6 or more amino acids in length are also described, for example, in U.S. Pat. Nos. 6,479,626, 6,903,185, and 7,153,949. The proteins described herein can include any combination of suitable linkers between the individual zinc fingers of the protein.

[0103] In certain embodiments, the DNA binding domain is an engineered zinc finger protein that binds to a target site in the Htt gene (in a sequence-specific manner) and regulates the expression of Htt. ZFPs can selectively bind to mutant Htt alleles or wild-type Htt sequences. 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). ZFPs typically include at least three fingers. Some ZFPs include 4, 5 or 6 fingers, while some ZFPs include 8, 9, 10, 11 or 12 fingers. ZFPs including three fingers typically recognize target sites including 9 or 10 nucleotides; ZFPs including four fingers typically recognize target sites including 12-14 nucleotides; and ZFPs with six fingers can recognize target sites including 18-21 nucleotides. ZFPs can also be fusion proteins including one or more regulatory domains, which may be transcriptional activation or repression domains. In some embodiments, the fusion protein comprises ZFP DN binding domains linked together. Thus, these zinc fingers can comprise 8, 9, 10, 11, 12 or more fingers. In some embodiments, 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 4, 5 or 5 zinc fingers. In some embodiments, the linker is a standard inter-finger linker, so the finger array comprises a DNA binding domain containing 8, 9, 10, 11 or 12 or more fingers. In other embodiments, the linker is an atypical linker, such as a flexible linker. The DNA binding domain is fused to at least one regulatory domain and can be considered as a "ZFP-ZFP-TF" architecture. Specific examples of these embodiments can be referred to as "ZFP-ZFP-KOX" and "ZFP-KOX-ZFP-KOX", wherein the "ZFP-ZFP-KOX" comprises two DNA binding domains connected to a flexible linker and fused to a KOX inhibitor, and the two ZFP-KOX fusion proteins in the "ZFP-KOX-ZFP-KOX" are fused together by a linker.

[0104] 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.

[0105] "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, while 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 interval between two target sequences can encompass many nucleotides. A two-handed ZFP can include a functional domain, for example, fused to one or two ZFPs. Thus, it will be apparent that a functional domain can be attached to the outside of one or two ZFPs, or can be located between the ZFPs (connecting two ZFPs).

[0106] Fusion Protein

[0107] Also provided is such a fusion protein, which comprises a DNA binding protein (e.g., ZFP) as described herein and a heterologous regulatory (functional) domain (or a functional fragment thereof). Common domains include, for example, transcription factor domains (activators, repressors, coactivators, co-repressors), silencers, oncogenes (e.g., 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 (e.g., kinases, acetylases and deacetylases); and DNA modification enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. For more information on the fusion of a DNA binding domain and a nuclease cleavage domain, see U.S. Patent Application Publication Nos. 2005 / 0064474; 2006 / 0188987; and 2007 / 0218528, which are incorporated herein by reference in their entireties.

[0108] 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 κB (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 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.

[0109] It will be appreciated by those skilled in the art that in the formation of a fusion protein (or nucleic acid encoding the same) between a DNA binding domain and a functional domain, an activation domain or a molecule that interacts with an activation domain is suitable as a functional domain. Basically, any molecule that can recruit an activation complex and / or activation activity (e.g., histone acetylation) to a target gene can be used as an activation domain of a fusion protein. Insulator domains, localization domains, and chromatin remodeling proteins (e.g., proteins containing domains of ISWI and / or methyl binding domains) suitable for use as functional domains in fusion molecules are described, for example, in shared U.S. Patent Publication Nos. 2002 / 0115215 and 2003 / 0082552, and shared International Patent Publication No. WO 02 / 44376.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] In certain embodiments, the target site bound by the DNA binding domain is present in an accessible region of cell chromatin. The accessible region can be determined as described in, for example, the shared International Patent Publication No. WO 01 / 83732. If the target site is not present in an accessible region of cell chromatin, one or more accessible regions can be generated as described in the shared International Patent Publication No. WO 01 / 83793. In other embodiments, the DNA binding domain of the fusion molecule is capable of binding to cell chromatin, regardless of whether its target site is located in an accessible region. For example, the DNA binding domain is capable of binding to linker DNA and / or nucleosomal DNA. Examples of such "pioneer" DNA binding domains are present in certain steroid receptors and in hepatocyte nuclear factor 3 (HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990) Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

[0114] 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.

[0115] 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.

[0116] Other exemplary functional domains are disclosed, for example, in commonly owned US Pat. No. 534,261 and US Patent Publication No. 2002 / 0160940.

[0117] Functional domains that can be regulated by exogenous small molecules or ligands can also be selected. technology, where the functional domain is only external to RheoChem TM Its active conformation is assumed in the presence of a ligand (see, e.g., U.S. Patent Publication No. 2009 / 0136465). Thus, a ZFP can be operably linked to a regulatable functional domain, wherein the activity of the resulting ZFP-TF is controlled by an external ligand.

[0118] deliver

[0119] Nucleases, polynucleotides encoding these nucleases, donor polynucleotides and compositions comprising proteins and / or polynucleotides described herein can be delivered by any suitable means. In certain embodiments, nucleases and / or donors are delivered in vivo. In other embodiments, nucleases and / or donors are delivered to isolated cells (e.g., autologous or allogeneic stem cells) to provide modified cells that can be delivered to patients ex vivo.

[0120] Methods for delivering nucleic acids containing the nucleases 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.

[0121] Any nucleic acid delivery mechanism can also be used to deliver nucleases and / or donor constructs described herein, including naked DNA and / or RNA (e.g., mRNA) and vectors, which contain sequences encoding one or more of the components. Any vector system can be used, including but not limited to plasmid vectors, DNA minicircles, retroviral vectors, lentiviral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, and adeno-associated virus vectors, and combinations thereof. See also, for example, U.S. Patent Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824; and U.S. Patent Publication No. 2014 / 0335063, which is incorporated herein by reference in its entirety. In addition, it is apparent that any of these systems may contain one or more sequences required for treatment. Thus, when one or more nucleases and donor constructs are introduced into a cell, the nucleases and / or donor polynucleotides can be carried by the same delivery system or by different delivery mechanisms. When multiple systems are used, each delivery mechanism can comprise a sequence encoding one or more nucleases and / or donor constructs (e.g., mRNA encoding one or more nucleases and / or AAV or mRNA carrying one or more donor constructs).

[0122] Conventional viral and non-viral transgenic methods can be used to introduce nucleic acids encoding nucleases or donor constructs into cells (e.g., mammalian cells) and target tissues. Non-viral vector delivery systems include DNA plasmids, DNA minicircles, naked nucleic acids, and nucleic acids compounded with delivery vehicles (such as liposomes or poloxamer). Viral vector delivery systems include DNA and RNA viruses that 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.

[0123] Non-viral delivery methods for nucleic acids include electroporation, lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, lipid nanoparticles (LNPs), naked DNA, naked RNA, capped RNA, artificial virions, and agent-enhanced DNA uptake. Sonoporation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

[0124] 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 and Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor-recognition lipid transfection of polynucleotides include those of Felgner, International Patent Publication Nos. WO 91 / 17424, WO 91 / 16024. In some aspects, the nuclease is delivered as mRNA, while the transgene is delivered by other means, such as viral vectors, minicircle DNA, plasmid DNA, single-stranded DNA, linear DNA, liposomes, nanoparticles, etc.

[0125] 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).

[0126] 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).

[0127] The use of RNA or DNA virus-based systems to deliver nucleic acids encoding engineered CRISPR / Cas systems utilizes highly evolved processes to target viruses to specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be directly administered to a subject (in vivo), or they can be used to treat cells in vitro and administer modified cells to a subject (ex vivo). Conventional virus-based systems for CRISPR / Cas systems include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated, vaccinia, and herpes simplex virus vectors for gene delivery. Integration in the host genome can be performed using retroviral, lentiviral, and adeno-associated viral 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.

[0128] 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 1994 / 026877).

[0129] 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. Any AAV serotype may be used, including AAV1, AAV3, AAV4, AAV5, AAV6 and AAV8, AAV 8.2, AAV9 and AAV rh10 and pseudotyped AAVs such as AAV9.45, AAV2 / 8, AAV2 / 5 and AAV2 / 6.

[0130] 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.

[0131] 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).

[0132] Recombinant adeno-associated virus vector (rAAV) is a promising alternative gene delivery system based on the defective and non-pathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from such plasmids that retain only the AAV 145 base pair (bp) inverted terminal repeats flanking the transgene 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 AAAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV9.45 and AAVrh10, and all variants thereof, may also be used according to the present invention.

[0133] 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: 1083-1089). 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; and Topf et al. (1998) Gene Ther. 5:507-513.

[0134] 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 production cell lines that package nucleic acid vectors into viral particles. The vector usually contains the minimum viral sequences 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, 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. Viral DNA is packaged into a cell line, which contains a helper plasmid encoding other AAV genes, i.e., rep and cap, but lacks ITR sequences. The cell line is also infected with adenovirus (as an auxiliary). The helper virus promotes the replication of the AAV vector and the expression of 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).

[0135] In many gene therapy applications, it is desirable to deliver gene therapy vectors to specific tissue types with a high degree of specificity. Therefore, viral vectors can be modified to have specificity 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.

[0136] Gene therapy vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, sublingual, or intracranial infusion), local administration, as described below, or by pulmonary inhalation. 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.

[0137] The vector (e.g., retrovirus, adenovirus, liposome, etc.) containing the nuclease and / or donor construct can also be directly administered to the organism for in vivo cell transduction. Alternatively, naked DNA can be administered. Administration is carried out by any conventional route commonly used for introducing molecules and ultimately contacting blood or tissue cells, including but not limited to injection, infusion, topical application, inhalation, and electroporation. Suitable methods for administering the nucleic acid are available and well known to those skilled in the art, and, although more than one approach can be used to administer a particular composition, a particular approach can generally provide a more direct and more effective response than another approach.

[0138] Suitable vectors for introducing the polynucleotides described herein include non-integrating lentiviral vectors (IDLV). See, for example, Ory, et al. (1996) Proc. Natl. Acad. Sci. USA

[0139] 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; U.S. Patent No. 8,936,936.

[0140] 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).

[0141] It is apparent that the same or different systems may be used to deliver the sequence encoding the nuclease and the donor construct. For example, the donor polynucleotide may be carried by AAV, while one or more nucleases may be carried by mRNA. In addition, different systems may be administered by the same or different routes (intramuscular injection, tail vein injection, other intravenous injection, intraperitoneal administration, and / or intramuscular injection. Multiple vectors may be delivered simultaneously or in any order.

[0142] Formulations for in vitro and in vivo administration include suspensions in liquids or emulsified liquids. The active ingredient is usually mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the composition may contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, or other agents that enhance the efficacy of the pharmaceutical composition.

[0143] Example

[0144] Example 1: Materials and Methods

[0145] mRNA production for transient transfection

[0146] Templates for in vitro transcription were generated from pVAX-ZFP or pVAX-GFP plasmids using PCR (forward primer GCAGAGCTCTCTGGCTAACTAGAG (SEQ ID NO: 1); reverse primer (T(180)): CTGGCAACTAGAAGGCACAG (SEQ ID NO: 2)). mRNA was synthesized using the T7 ULTRA transcription kit (ThermoFisher Scientific) according to the manufacturer's instructions and purified using RNeasy96 columns (Qiagen).

[0147] Production of lentiviral vectors

[0148] The coding sequence of ZFP-2A-GFP was cloned into the lentiviral transfer vector pRRL downstream of the CMV promoter. HEK293T cells cultured in 15-cm tissue culture dishes were transfected with the following plasmids: pRRL-CMV-ZFP-2A-GFP (37.5 μg), VSVG envelope plasmid (18.75 μg) and packaging plasmids (pMDL and pREV, 18.75 μg each (Dull, T., et al. (1998) J Virol 72(11):8463-8471) using Lipofectamine 2000 (Thermo Fisher Scientific). Viral supernatants (30 ml) were harvested 48 and 72 hours after transfection and filtered through a 0.45-μm filter and then centrifuged at 50,000×g at 4°C (Optima L-80K preparative ultracentrifuge, Beckman Coulter). Coulter) for 90 minutes to concentrate 300 times. The pellet was then suspended in an appropriate volume of Hank's buffered saline (Lonza). To determine the infection titer, 2×10 cells were transduced with 100 μl of serially diluted vector preparations. 4 HEK293T cells were incubated overnight (in triplicate) and then incubated for an additional 48 hours at 37°C with 5% CO 2. The infectivity titer was determined using the dilution that exhibited a linear dose response for GFP expression.

[0149] Production of AAV vectors

[0150] AAV6-CMV-GFP and AAV6-CMV-ZFP (A, B and C) were generated by baculovirus-based AAV production according to standard methods in the art. All AAV6 vectors were purified by double CsCl ultracentrifugation and buffer exchanged into PBS with 0.001% Pluronic F-68. AAV titer was determined using qPCR.

[0151] Chimeric AAV serotype 1 / 2 (Hauck, B., et al. (2003) Mol Ther 7:419-425) vectors were generated by Evotec AG (Hamburg, Germany) to express ZFP-2A-GFP and GFP control under the control of the human synapsin 1 (hSYN1) promoter. AAV1 / 2 vectors were produced by HEK293 transfection and purified by iodixanol gradients as described (Heikkinen et al. (2012) PLoS ONE 7:e50717, doi:10.1371 / journal.pone.0050717).

[0152] Cell culture and mRNA transfection

[0153] The immortalized mouse striatal cell line STHdhQ111 / HdhQ7 (Q111 / Q7, Trettel et al. (2000) Human Molecular Genetics 9:2799-2809, doi:10.1093 / hmg / 9.19.2799) was a gift from the CHDI Foundation (CHDI). Q111 / Q7 cells were grown in DMEM containing 10% FBS, penicillin / streptomycin and G418 (0.4 mg / ml) at 33°C. mRNA transfection was performed using a 96-well Shuttle Nucleofector (Lonsard). 1×10 5 Each cell was transfected with 100 ng of ZFP mRNA using P3 solution and program EN- 132. 24 hours after transfection, cells were harvested for gene expression analysis by qRT-PCR.

[0154] HD fibroblast cell lines GM04723 (CAG15 / 67 (SEQ ID NO: 81 / 82)), GM02151 (CAG18 / 45 (SEQ ID NO: 79 / 80)) and ND30259 (CAG21 / 38 (SEQ ID NO: 83 / 84)) were obtained from the Coriell Cell Repository and maintained in complete MEM containing 20% ​​FBS. The CAG repeat length was confirmed by sequencing. mRNA transfection was performed using a 96-well Shuttle Nucleofector (Lonsard). Each 1.5×10 5 Each cell was transfected with 0.01-1000 ng ZFP mRNA using P2 solution and program CA-137; for doses less than 100 ng ZFP mRNA, GFP mRNA was added to bring the total amount of transfected mRNA to 100 ng. 24 hours after transfection, cells were harvested for gene expression analysis by qRT-PCR.

[0155] HD ESC line GENEA018 (CAG17 / 48 (SEQ ID NO:76 / 78), Bradley et al. (2011) Stem Cells Dev 20:495-502, doi:10.1089 / scd.2010.0120) was a gift from CHDI. The CAG repeat length was confirmed by sequencing. HD-ESCs were passaged with accutase and cultured in E8 medium on Matrigel-coated plates (Thermo Fisher Scientific). Neural stem cells were derived using StemPro Neural Induction Medium (Thermo Fisher Scientific). Briefly, ESCs were cultured at 2×10 5 Cells / well were seeded into 6-well geltrex-coated dishes and the medium was changed to StemPro Neural Induction Medium (Thermo Fisher Scientific) at 10-20% confluence. The medium was changed every two days and NSCs were harvested and expanded on day 7. HD-NSCs and non-HD NSCs (HIP TM Neural stem cells, Globalstem. NSCs were passaged on geltrex-coated plates using accutase. mRNA transfection was performed using a 96-well Shuttle Nucleofector (Lonsard). NSCs were prepared according to the NSC subculture protocol (StemPro NSC, Life Technologies) and 2x10 cells were transfected using 20 μL SF solution and program CM. 5 Immediately thereafter, 80 μL of culture medium was added to the wells, which were then transferred to a 96-well plate containing 50 μL of warm culture medium.

[0156] Induce neuronal differentiation from NSCs by changing to neural differentiation medium (NDIFF), which consists of Neurobasal medium with B-27 serum-free supplement and GlutaMAX TM (Thermo Fisher Scientific). The medium was changed every 3-4 days. After 7 days in NDIFF, the cells showed neuronal morphology.

[0157] To confirm NSC and neuronal differentiation, HD NSC or neuronal cultures were cultured on chamber slides (Lab-Tek, Thermo Fisher Scientific) for immunohistochemical analysis of neuronal marker genes. TMKit (BD Biosciences) to fix and permeabilize cells. Slides were blocked with 4% NGS and stained overnight at 4C with primary antibodies against PAX6 (AB2237, Millipore), anti-nestin-488 (MAB5326A4, Millipore) or anti-β-III tubulin (MAB119, R&D Systems). Appropriate Alexa 488 or 555-conjugated secondary antibodies (Molecular Probes) were diluted 1:500 for secondary staining.

[0158] Western blot analysis of HD protein knockdown

[0159] GMO4723 (CAG15 / 67 (SEQ ID NO: 81 / 82)) human fibroblasts were transfected with ZMO (300 ng) at 150,000 cells / transfection (quadruplicates) using Lonza program CA-137 in P2 solution. After transfection, cells were pooled in complete medium and then split into 4 wells of a 24-well plate and incubated at 37°, 5% CO2 for 72 hours. Cells were trypsinized, washed, pelleted and lysed in hot 95° Laemmli sample buffer and incubated at 95° for 5 minutes. Samples were loaded onto a Bio-Rad 4-15% TGX gel at 5 μL / lane and run at 150 V for 3.5 hours at room temperature in Tris / Glycine / SDS running buffer. Wet transfer to PVDF membrane was performed at 90° V at 4° for 2.5 hours in transfer buffer containing MeOH (10%). The membrane was blocked in Odyssey blocking buffer at 4° and then incubated with primary antibodies for 3 hours at room temperature in 0.2% Tween-20 Odyssey blocking buffer: mouse anti-Htt (1:500; Millipore MAB2166) and rabbit anti-calnexin (1:5000; Sigma C4731). The blot was washed 3 times × 10 minutes in PBS-T (PBS + 0.1% Tween-20). The blot was then incubated with secondary antibodies for 1 hour at room temperature in the dark in Odyssey blocking buffer containing 0.2% Tween-20, 0.01% SDS: goat anti-mouse IgG1 (1:5000; Li-Cor IRDye800CW 926-32350) and goat anti-rabbit (1:10,000; LiCor IRDye 680RD#926-68071). Blots were washed 3 times x 10 min in PBS-T under light protection, dried between Whatman papers, and scanned on a Li-Cor Odyssey near-infrared fluorescence imaging system.

[0160] Generation of stable NSC lines expressing ZFPs

[0161] HD-NSCs expressing ZFPs were established using a lentiviral vector with a CMV promoter driving the expression of ZFPs linked to GFP with a 2A peptide. 1 × 10 6 The cells were inoculated with the concentrated vector. After several passages, the resulting cells were sorted to enrich for GFP-positive cells. The sorted cells were further expanded and differentiated into neurons as described above.

[0162] Gene expression analysis using qRT-PCR

[0163] Total RNA was extracted from cultured cells and mouse striatum using the HighPure RNA Isolation Kit (Roche) and the Purelink RNA Mini Kit (Ambion), respectively. cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and quantitative PCR was performed using the SsoAdvanced Universal Probe Supermix (Bio-Rad) and a CFX Real-Time PCR Instrument (Bio-Rad). qRT-PCR primer / probe sets for gene expression analysis were ordered from IDT: mouse total Htt (Mm.PT.58.6953479), DRD1A (Mm.PT.56a.43576955.g), DRD2 (Mm.PT.56a.7811767), PDE10A (Mm.PT.56a.16919824), DARPP-32 (Mm.PT.53a.9253526.gs), ATP5B (Mm.PT.53a.17279462), EIF4A2 (Mm.PT.53a.9498195.g), RPL38 (Mm.PT.58.42993403.g) AIF1 (Mm.PT.58.42993403.g).

[0164] (Mm.PT.58.7014816); human total HTT (Hs.PT.49a.14676852.g), STC1

[0165] (Hs.PT.51.14992722), NAP1L3 (Hs.PT.56a.24655549.g), ATXN2 (Hs.PT.58.40126607), ORC4 (Hs.PT.56a.24527823.g), THAP11

[0166] (Hs.PT.49a.15404553.g), GLS (Hs.PT.53a.20624732), FBXO11

[0167] (Hs.PT.58.2665601), DNM1 (Hs.PT.58.25262501), TBP (Hs.PT.56a.20792004), FOXP2 (Hs.PT.58.153 57735), ENO2 (Hs.PT.53a.25227282), B2M (Hs.PT.20234084), TOP1 (Hs.PT.53a.19541381), NES (Hs.PT .53a.20758620), SOX1 (Hs.PT.53a.28041414.g), PAX6 (Hs.PT.53a.814314), OCT4 (Hs.PT.58.144941 69.g), REX1(Hs.PT.53a.23001209), NANOG(Hs.PT.53a.21480849), GAD1(Hs.PT.56a.21283000), MAP2

[0168] (Hs.PT.53a.40791337.g) and FOXG1 (Hs.PT.56a.26906112.g).

[0169] Detection of ZFP and GFP mRNA expressed from AAV6 vectors by universal primers / probes targeting the 5'UTR:

[0170] Forward primer: GGAACGGTGCATTGGAACG (SEQ ID NO: 3)

[0171] Reverse primer: GTTCGAATCCCAATTCTTTGCC (SEQ ID NO: 4)

[0172] Probe: AGCACGTTGCCCAGGAGGTCAC (SEQ ID NO: 5).

[0173] The following primer / probe sets were used to detect mutant Htt mRNA in R6 / 2 mice:

[0174] Forward primer: CGCAGGCTGCAGGGTTAC (SEQ ID NO: 6)

[0175] Reverse primer: GCTGCACCGACCGTGAGT (SEQ ID NO: 7)

[0176] Probe: CAGCTCCCTGTCCCGGCGG (SEQ ID NO: 8).

[0177] Allele-specific detection of human HTT expression in HD fibroblasts GM04723 (CAG15 / 67 (SEQ ID NOS: 81 / 82)) and GM02151 (CAG18 / 45 (SEQ ID NOS: 79 / 80)) was performed using primers customized based on SNP rs363099C / T (exon 29):

[0178] 363099C-F (099-C forward primer): AGTTTGGAGGGTTTCTC (SEQ ID NO: 9)

[0179] 363099T-F (099-T forward primer): AGTTTGGAGGGTTTCTT (SEQ ID NO: 10)

[0180] 363099T-BL (099-T Blocker):

[0181] AGGGTTTCTCCGCTCAGC / phos / (SEQ ID NO:11)

[0182] 363099-R (reverse primer, used with 099-C and 099T forward primers): TCGACTAAAGCAGGATTTCAGG (SEQ ID NO: 12).

[0183] The 099-T blocker oligonucleotide was designed to anneal to the "C / G" allele and was phosphorylated at the 3' terminal base to inhibit amplification of the mutant allele (Morlan et al. (2009) PLOS ONE 4:e4584), which was added at a 2:1 molar ratio relative to the 099T forward primer. For each sample, a 10 μL qPCR reaction for 099T and 099C was set up as follows: 5 μL SsoFast Evagreen supermix (Bio-Rad), 0.5 μL of 20x 099C or 099T master mix (final concentrations of 0.5 μM and 1 μM blocker oligonucleotides for each primer in the 099T assay), 2.5 μL H2O, and 2 μL of cDNA sample. Thermal cycling conditions were as follows: 1) 98°C for 2 minutes, 2) 98°C for 5 seconds, 3) 58.3°C for 9 seconds for 099C assay, obtained from 55.6°C for 9 seconds for 099T assay, 4) read plate, 5) repeat steps 2-4 45 times, 6) end.

[0184] Allele-specific detection of human HTT expression in HD fibroblasts ND30259 (CAG21 / 38 (SEQ ID NOS:83 / 84)) was performed using primers customized based on SNP rs362331C / T (exon 50): 362331-F (331 forward primer): TCTCCTCCAGAGTTTGTGA (SEQ ID NO: 13), 362331-R (331 reverse primer):

[0185] CCTTCTTTCTGGACTAAGAAGCTG (SEQ ID NO: 14), 362331C probe: TCC CTC ATC+C+ACTGT GT (SEQ ID NO: 15), and 362331T probe: CTC+A+T+C+T+A+C TGT GT (SEQ ID NO: 16).

[0186] For each assay, allele-specific Taqman probes were added to detect 331C or 331T, which contained locked nucleic acid (LNA) bases (indicated by "+N"). The use of LNA probes improves allele discrimination compared to unmodified DNA probes (Latorra et al. (2003) Hum Mutat 22:79-85, doi:10.1002 / humu.10228 and You et al. (2006) Nucleic Acids Research 34:e60, doi:10.1093 / nar / gkl175). For each sample, a 10 μL qPCR reaction for 331T and 331C was set up as follows: 5 μL SsoFast probe supermix (Bio-Rad), 1 μL 10x 331-C master mix (final concentrations in the assay: 0.5 μM for each of the F and R primers and 0.25 μM for the 331C probe) or 331T master mix (final concentrations in the assay: 0.5 μM for each of the F and R primers and 0.25 μM for the 331-T probe), 2 μL H2O, and 2 μL of cDNA sample. Thermocycling conditions were as follows: 1) 95° for 45 seconds, 2) 95° for 5 seconds, 3) 62° for 1 minute for 331C assays, or 62.7° for 1 minute for 331T assays, 4) plate read, 5) repeat steps 2-4 45 times, 6) end.

[0187] Microarray

[0188] In vitro transcribed mRNA encoding ZFP-TF (100 ng) was transfected into 150,000 HD fibroblasts (GM02151) in biological six replicates (Amaxa shuttle, set CA-137, solution P2, Lonza). After 24 hours, cells were washed once with PBS and then processed to extract total RNA (High Pure, Roche). Each replicate sample (50 ng total RNA) was processed according to the manufacturer's protocol for sample preparation, hybridization, flow control and scanning (Human Primeview GeneChip array, Affymetrix). The robust multi-array average (RMA) was used to normalize the raw signal from each probe set. Fold change analysis was performed using the Transcriptome Analysis Console 3.0 (Affymetrix) with the "Gene Level Differential Expression Analysis" option. Samples transfected with ZFP-A, ZFP-B, and ZFP-C were compared to samples treated with a ZFP-TF that does not target CAG, which has two known targets in fibroblasts (PAPPA and LMCD1, both repressed with <2FC). Changes in transcripts (probe sets) are reported for which the mean signal differed more than 2-fold relative to control and P value <0.05 (unpaired T test, one-tailed ANOVA analysis for each probe set). For any gene with more than one probe set, the probe set with the highest fold change was selected for downstream analysis.

[0189] Bioinformatics analysis

[0190] The hexameric CAG motif (ZFP-A and ZFP-C) or CAGCAGnnGCAGCAnCAGCAG motif (SEQ ID NO: 17) (ZFP-B) of human chromosomes 1-22, X and Y (GRCh38 version) was scanned using the 'moods_dna.py' Python script from the MOODS software package (version 1.9.2) and a threshold set to detect zero to a maximum of 3 mismatches in the motif. The regions found using this method were converted to BED format using a custom python script. BED regions were sorted and adjacent or overlapping motif regions were fused into a single region using the BEDtools (v2.26.0) "merge" command, which is independent of strandedness.

[0191] ENSEMBL people's transcript and its transcription start site coordinates (Ensembl version 87) are obtained from ENSEMBLBiomart, and only those samples with GENCODE (v25) BASIC annotation and " protein_coding " biotype are filtered. The distance (vice versa) of CAG motif and given transcription start site is determined by bedtools command " bedtools closest-D ". Affymetrix Primeview probe group ID is used to map to ENSEMBL transcript. Affymetrix Primeview probe group that is not overlapped with ENSEMBL transcript will be mapped to multiple genes or removed from subsequent studies according to ENSEMBL biomart annotation (GRCh38.7, version 87). If multiple transcripts are mapped to Primeview probe group, the transcript with the minimum distance between CAG motif and TSS is selected.

[0192] Expression RPKM values ​​for human tissue expression were obtained from the GTEx project data accessible through the UCSC genome browser (Mele et al. (2015) Science 348:660-665, doi: 10.1126 / science.aaa0355) for the following categories: brain_caudate, brain_cortex, brain_putamen, and transformed_fibroblasts.

[0193] Measuring ATP levels in cultured neurons

[0194] In vitro differentiated CAG17 / 48 (SEQ ID NO: 76 / 78) neurons and normal neurons (about 1.5 x 105 cells) were infected with Lenti-CMV-ZFP-2A-GFP or Lenti-CMV-GFP at an MOI of 500 in triplicate. Luminescent assay (Promega) was used to measure intracellular ATP levels. The number of cells in each sample was determined using the ATP assay (Promega). ATP levels in each cell from different cells / treatments were then normalized to those in mock-infected HD neurons.

[0195] Measuring growth factor withdrawal-induced apoptosis in cultured neurons

[0196] In vitro differentiated CAG17 / 48 (SEQ ID NO: 76 / 78) neurons and normal neurons (about 1.5x105 cells) were infected in triplicate with Lenti-CMV-ZFP-2A-GFP or Lenti-CMV-GFP at an MOI of 500. Five days after infection, the culture medium was replaced with fresh neurobasal medium without growth factors. After 48 hours of growth factor withdrawal, the level of cell death was measured using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (ApoBrdU Red DNA cleavage kit, BioVision). The percentage of apoptotic cells (anti-BrdU staining positive) in lentiviral transduced (GFP positive) cells was measured using flow cytometry.

[0197] Mouse studies

[0198] Studies with HdhQ50 / Hdh+(Q50) and R6 / 2 mice were performed at PsychoGenics, Inc. (Tarrytown, NY) in accordance with the U.S. Public Health Service policy on the humane care and use of laboratory animals, and procedures were approved by the Institutional Animal and Use Committee of PsychoGenics, Inc. Studies with zQ175 mice were performed at Evotec AG (Hamburg, Germany) in accordance with the German Animal Welfare Act and EU regulations (EU Directive 2010 / 63 / EU). Animals were grouped into treatment groups based on age, sex, CAG number, and body weight.

[0199] Q50 Mouse Studies

[0200] HdhQ50 / Hdh+(Q50)C57BL / 6J mice were provided by CHDI. At 11 weeks of age, AAV6-CMV-ZFP or AAV6-CMV-GFP (1x10 13vg / ml) was delivered bilaterally to the striatum of Q50 mice (n=4 / group, 2 males and 2 females, group size was determined initially). To increase vector coverage of the striatum, two injection sites were used in each striatum: 5 μL was delivered to the anterior site (coordinates: A / P+1.4mm, M / L+ / -1.7mm, D / V-3.5mm) and 4 μL was delivered to the posterior site (coordinates: A / P+0.2mm, M / L+ / -2.3mm, D / V–3.2mm) using a stepped cannula design at a rate of 0.5 μL / min. Body weight was monitored twice a week throughout the study. Seven weeks after injection, the mice were decapitated and the brains were quickly removed from the skull and rinsed in ice-cold saline to remove any surface blood. The striatum was dissected on an ice-cold surface and subdivided into 3 equal-sized fragments representing the rostral, middle, and tail parts of the striatum; the striatal tissue was treated in RNALater (Qiagen) at 4°C overnight and then stored at -80°C until RNA extraction. Because AAV injection did not result in complete and uniform coverage of the striatum (data not shown), each striatum was divided into 3 parts to reduce the possibility that the baseline Htt level in the poorly transduced area interfered with the detection of Htt regulation in the well-transduced area. For each striatal section, Htt and ZFP expression were quantified by qRT-PCR.

[0201] zQ175 Mouse Studies

[0202] zQ175 C57B / L6J knock-in mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). At 2 or 6 months of age, heterozygous zQ175 mice (n=5 / group, mixed sex) were injected with AAV1 / 2-hSYN1-ZFP-2A-GFP (1x10 13 vg / mL) was injected intrastriatically, while the left hemisphere received AAV1 / 2-hSYN1-GFP (1x10 13 A total of 2 μL of AAV vector was delivered to each striatum at a constant flow rate of 200 0.5 μL / min using a Hamilton gas-tight syringe (, model 1801RN) and a custom-made 26-gauge needle, with injection coordinates of A / P + 0.8 mm, M / L + / - 1.8 mm, D / V - 3.8 mm.

[0203] zQ175 mice were euthanized 2 or 4 months after transcranial infusion of 30 mL of ice-cold PBS followed by 50 mL of 4% paraformaldehyde using a peristaltic pump. Brain samples were removed from the skull and sectioned; immunohistochemistry, image acquisition, and automated image analysis were performed as previously described (Carty et al. (2015) PLoS One 10:e0123527, doi:10.1371 / journal.pone.0123527).

[0204] R6 / 2 Mouse Studies

[0205] R6 / 2 transgenic mice were bred in the PsychoGenics colony by crossing ovarian transplanted females (Jackson Laboratory) of CBAxC57BL / 6 background with C57BL / 6 wild-type males. The CAG sequence repeat length in transgenic mice was confirmed to be 123 ± 0.6. All experiments were blinded to the treatment of each group. At 5 weeks of age, AAV6-CMV-ZFP or AAV6-CMV-GFP (1x10 13 vg / mL) was delivered bilaterally to the striatum of R6 / 2 mice (n=14 / group, 7 males and 7 females, group size was selected by studies testing other agents in the same model); the same injection volume and coordinates were used in the Q50 mouse study. The survival of the mice was monitored twice a day. Body weight (BW) was measured once a week until the mice were euthanized at 12 weeks of age. There was no statistically significant difference in BW between the two groups.

[0206] Clasping behavior was recorded during each weight test. Briefly, each mouse was removed from its home cage, the cage lid was inverted, and the mouse was placed on the surface. The observer then gently pulled the animal backward and upward in a steady motion until the animal was suspended approximately 12 inches above the surface. The animals were observed for 30 seconds. Complete clasping, defined as simultaneous clasping of the hind and forelimb claws to the core, was recorded and used for analysis.

[0207] Mice were also tested in an open field (OF) at 4 (baseline), 6, 8, 10, and 12 weeks of age. Animals were placed in a plexiglass cube (27.3 x 27.3 x 20.3 cm; Med Associates Inc., St Albans, VT) surrounded by an infrared light source for 30 minutes. Total horizontal activity (distance traveled) and vertical activity (rearing) were measured by continuous beam breaks.

[0208] During the study, three mice in the AAV-CMV-ZFP group and five mice in the AAV-CMV-GFP group died. After euthanasia of mice at 12 weeks of age, the striatum was dissected (n=7 and 10 mice for the GFP and ZFP groups, respectively) for gene expression analysis as described for the Q50 mouse study.

[0209] Analysis of HTT aggregates in ZFP-treated zQ175 mice (Animal Research)

[0210] Male and female zQ175 C57B / L6J knock-in mice were obtained from Jackson Laboratory, Inc. (Bar Harbor, ME, USA). The zQ175 line was derived from a spontaneous expansion of CAG copy number in CAG 140 knock-in mice and was generated at Psychogenics, Inc. (Tarrytown, NY, USA). Transgenic mice were backcrossed to C57BL / 6J to generate heterozygous zQ175 mice and wild-type littermates. Animals were housed in European standard type II long cages and allowed ad libitum access to food and water. The following environmental conditions were maintained: 21 ± 1 °C ambient temperature, 55 ± 10% humidity and a 12:12 light:dark cycle with lights on from 7 am to 7 pm. The health status of the animals was checked daily. All animal handling was performed in accordance with the German Animal Welfare Act and EU regulations (EU Directive 2010 / 63 / EU). The study protocol has been approved by the Regional Ethics Committee of the City and State Office of Health and Consumer Protection of Hamburg (“Association for Consumer Protection ( und Verbraucherschutz)” BGV) document number # V11307 / 591 00.33.

[0211] AAV vector construction and production

[0212] To express ZFPs, plasmids were modified from the adeno-associated virus (AAV) vector pAAV-6P-SWB (Minderer et al. (2012) J Physiol 590(1):99-107). ZFP-C (FLAG-tagged) was cloned behind the human synapsin 1 promoter (phSyn1) to generate pAAV-SWB-ZFP-C. In addition, an inactive ZFP control construct was generated by deleting the ZFP DNA binding domain (ZFP-ΔDBD) from ZFP-B. Pseudotyped rAAV2 / 1+2 particles were generated and purified as previously described (Zolotukhin, S., et al. (1999) Gene Ther. 6(6):973-85. PMID:10455399); Carty, N., et al. (2015) PLOS ONE 10:e0123527).

[0213] In brief, HEK293 cells were co-transfected with an AAV vector carrying the transcription unit of interest and a plasmid containing the rep and cap genes (pDP1rs and pDP2rs, Plasmid Factory) in an equimolar ratio by polyethylenimine-mediated plasmid transfection. 48 hours after transfection, the cells were lysed by 3 freeze-thaw cycles, and cell debris was removed by centrifugation. The supernatant containing the viral particles was treated with nuclease and subjected to iodixanol density centrifugation at 60,000 rpm (S6, S7). Iodixanol was removed, and the viral particles were concentrated in PBS 300MK (300 mM NaCl, 1 mM MgCl2, 2.5 mM KCl) by filter centrifugation. The remaining rAAV solution was filtered through a Millex GV 0.22 μm pore size. Sterile rAAV particles were stored at 4°C and diluted 1:1 with sterile PBS buffer to obtain PBS MK (150 mM NaCl, 0.5 mM MgCl2, 1.25 mM KCl) for in vivo application. AAV titer was determined using qPCR. Before in vivo application, rAAV particles expressing ZFPs were tested in vitro for downregulation of WT or mutant Htt in primary striatal neurons of zQ175 het mice. Briefly, striatal cultures were prepared at a cell density of 2E5 cells / well in 24-well plates. rAAV particles (containing 3E8 copies of genome, GS) were added at 3DIV, and cells were harvested at 14DIV. Wt and mutant Htt knockdown were assessed by qPCR using the following primers: forward CAG GTC CGGCAG AGG AAC C (SEQ ID NO: 18) and reverse TTC ACA CGG TCT TTC TTG GTG G (SEQ ID NO: 19) for WT, and forward GCC CGG CTG TGG CTG A (SEQ ID NO: 20) and reverse TTC ACA CGG TCTTTC TTG GTG G (SEQ ID NO: 21) for Htt mutant, respectively.

[0214] In vivo application of AAV-ZFP

[0215] Two groups of 16 zQ175 het mice (8 males and 8 females) received bilateral intrastriatal injections of rAAV constructs encoding HTT allele-specific ZFP30640 or ZFP-ΔDBD control at 2 months of age. Each mouse was anesthetized with 3% isoflurane at a flow rate of 1 L / min and placed in a stereotaxic apparatus (Kopf, model 940). Anesthesia was maintained throughout the surgical procedure by delivery of 2% isoflurane through a nose cone at a flow rate of 0.5 L / min. After sterilization with 70% ethanol and iodine solution and application of lidocaine, a longitudinal midsagittal incision of 1 cm in length was made on the scalp. After incision of the skin, a small hole corresponding to the striatal injection site was made in the skull using an electric drill (Foredom; model H.30). The coordinates measured from the mouse bregma were 0.8 mm anterior, 1.8 mm right, 3.8 mm deep from the bregma, and a flat skull nose setting. A total volume of 4 μL (4E10 GC) of ZFP viral vector was administered at a constant flow rate of 200 nL / min using a Hamilton gastight syringe (model 1801RN, custom-made 26-gauge needle) connected to an automated microinjection pump. After injection, the surgical wound was sealed and the animal was placed on a heating pad until full recovery.

[0216] Histology and immunohistochemistry

[0217] Mice were euthanized by transcardial infusion at 6 and 10 weeks of age. For the infusion, mice were deeply anesthetized by intraperitoneal injection of a ketamine / xylazine mixture (120 mg / 15 mg / kg (15 μl / g body weight)) using a small diameter 27G needle. Before starting the infusion, the animals were assessed for loss of toe pinch reflex and corneal reflex to ensure that the correct level of anesthesia was achieved. Using a peristaltic pump, mice were transcardially infused with 30 mL of ice-cold PBS and 50 mL of 4% paraformaldehyde. Brain samples were removed from the skull and fixed overnight in the same fixative at 4°C and then cryoprotected by incubation in a 30% sucrose solution until saturated. The whole brain was embedded in TissueTek and stored at -80°C. 25 μm coronal sections were cut using a cryostat, collected in free-floating form in 24-well plates, and used directly for staining or stored in -20°C cryoprotectant solution (25 mM sodium phosphate buffer pH 7.4, 30% ethylene glycol, 20% glycerol) until use. The following primary antibodies were used for immunostaining: monoclonal mouse anti-mutant huntingtin (1:100; EM48, Millipore, MAB5374, lot 2135055), monoclonal rabbit anti-DARPP-32 (1:250; clone 19A3, Cell Signaling #2306, lot 2), polyclonal rabbit anti-NeuN (1:1000; Millipore, ABN78, lot 2140086), monoclonal mouse anti-GFAP (1:1500, Millipore, MAB3402, lot 1990686), polyclonal rabbit anti-Iba1 (1:1000, Wako, #019-19741, lot SAE6921). All staining was performed using the floating section method. The slices were permeabilized in 0.3% Triton X-100 / PBS, blocked in 10% normal goat serum / PBS, and incubated overnight at 4°C with a primary antibody diluted in 1% normal goat serum, 0.1% Triton X-100PBS. The slices were washed 3 times in PBS for 15 minutes and incubated in secondary antibodies for 2 hours at room temperature. The slices were washed in PBS as described above, and fixed in 24-well glass bottom plates (Sensoplate, Greiner, #662892) using an aqueous mounting medium (Fluoroshield, Sigma, F6057) containing DAPI, which was suitable for imaging using Opera High Content Screening system (PerkinElmer Inc.).

[0218] Image acquisition and automated image analysis

[0219] Image acquisition and analysis were performed as previously described (Carty, N., supra). The High Content Screening System and Opera software 2.0.1 (PerkinElmer Inc.) were used for automated image acquisition using a 40x water immersion objective (Olympus, NA 1.15, pixel size: 0.32 μm) for imaging mHTT inclusion bodies or a 20x water immersion objective (Olympus, NA 0.7, pixel size: 0.64 μm) for analyzing glial cells in tissues. Studio 3.1 (ParkinElmer Inc.) and integrated Batch analysis (as The algorithm was used to develop an image analysis script for characterizing and quantifying mHTT inclusions (part of the GFAP and Iba1 systems). To identify astrocytes and microglia, the algorithm searched for neurite-like cell extensions in the GFAP and Iba1 channels. Extensions connected to the previously determined nuclear boundaries in the 4-pixel wide edge around the nucleus were considered valid. Only when extensions could be detected were cells considered to be GFAP or Iba1 positive. Subsequently, the "local" background signal intensity was determined in the outer nuclear edge region separated by 3px width and 2px distance from the previously determined nuclear boundary. Finally, only cells with nuclear GFAP or Iba1 intensity higher than the average "local" background intensity were considered to be astrocytes or microglia, respectively. The image data of 6 slices of each animal were averaged, and 5 animals in each treatment group were used for statistical evaluation.

[0220] Tissue Homogenization for Meso Scale Discovery (MSD) Analysis

[0221] Whole striatum dissected from cerebral hemispheres was lysed in 80 μL tissue lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM PMSF, phosphatase inhibitor cocktail II (Sigma), phosphatase inhibitor cocktail III (Sigma), protease inhibitors (Roche Diagnostics)) using a FastPrep24 homogenizer (MP Biomedicals). The crude lysate was centrifuged three times at 16,000 rcf and 4°C for 10 minutes, and the supernatant was collected after each centrifugation step. The total protein concentration was determined using the bicinchoninic acid assay (BCA; Thermo Fisher Scientific) and adjusted to 1 mg / mL using lysis buffer. The homogenate was aliquoted, snap frozen, and stored at -80°C until analysis.

[0222] Mesoscale Discovery Analysis

[0223] MSD plates (384 wells) were coated overnight at 4°C with 10 μL of coated antibody carbonate-bicarbonate coating buffer (15mM Na2CO3 / 35mM NaHCO3, pH 9.6) per well. Then, each well was washed 3 times with 35 μL of washing buffer (PBS with 0.2% Tween-20), and each well was blocked with 35 μL of blocking buffer (PBS with 2% probumin / 0.2% Tween-20) for 1 hour at RT and shaken. Striatal extracts were diluted to 0.5 mg / mL in a mixture of 50% tissue lysis buffer and 50% blocking buffer. After an additional washing step, 10 μL of each sample was transferred to each well of the antibody-coated MSD plate and incubated for 1 hour at RT with shaking. After processing the sample and performing 4 wash cycles with 35 μL of washing buffer, 10 μL of detection antibody was added to each well and incubated for 1 hour at RT with shaking. After washing 3 times with wash buffer, 35 μL of read buffer T and surfactant (Mesoscale Discovery) were added to each well and the plate was imaged on a Sector Imager 6000 (Mesoscale Discovery) according to the manufacturer's instructions. The following antibody combinations were used: 4 μg / mL 2B7 / 0.1 μg / mL 4C9-ST; 4 μg / mL MW8 / 1 mg / mL 4C9-ST; 4 μg / mL MW8 / 5 μg / mL MW8-ST (ST: SULFO-tag). Samples were quantified according to standard curves of human HTT-Q73, aa 1-573 (for testing 2B7 / 4C9-ST) or aggregated exon 1-Q46 (for MW8 / 4C9-ST and MW8 / MW8-ST determinations).

[0224] Data Analysis and Statistics

[0225] Using GraphPad Statistical analysis was performed using 6.0 software. For all analyses, p values ​​less than 0.05 were considered statistically significant. Quantitative analysis (n = 5 animals, 6 sections per animal in each treatment group) was performed using a t-test with Welch correction; p < 0.05*; p < 0.01**; p < 0.001***.

[0226] Animals used in receptor autoradiography (ARG) and microPET studies

[0227] Heterozygous zQ175 mice (mixed sexes) were injected unilaterally with AAV1 / 2-hSYN1-ZFP-D or AAV2 / 1-hSYN1-ΔDBD-ZFP at 2 or 4 months of age, respectively, by Evotec, Hamburg, Germany.

[0228] The brains of two groups of zQ175 het mice were probed with ARG using three striatal markers [3H]raclopride (D2 ligand), [3H]MNI-659 (PDE10 ligand), and [3H]NNC112 (D1 ligand). One group of animals injected at 2 months of age (n=10 mice / group) were sacrificed at 6 months, transported to Karolinska Institutet, and the brains were probed with ARG in vitro using radioligands.

[0229] The second group (n=33-41 mice / group) was injected at 4 months of age. The animals were transported to the Karolinska Institute (Stockholm, Sweden) and microPET imaging was performed at 6.5-7 and 10 months of age with the dopamine D2 / D3 receptor radioligand [11C] raclopride and [18F] MNI-659 (radioligand for the PDE10A enzyme). At the end of the 10-month imaging study, a subset of this group (n=10 animals / group) was sacrificed and used for ARG with [3H] raclopride (D2 ligand), [3H] MNI-659 (PDE10 ligand), and [3H] NNC112 (D1 ligand).

[0230] While at Karolinska Institutet, animals were housed in the animal unit of Karolinska University Hospital in a temperature (±21°C) and humidity (±40%) controlled environment with a 12 h light / dark cycle (light on at 7:00 AM) and were allowed ad libitum access to food and water. Animals were allowed at least one week to acclimate to the animal unit before starting the imaging procedure. All experiments were performed during the light phase of the cycle. All experiments were performed under a protocol approved by the Animal Ethics Review Committee of North Stockholm, Sweden (N558 / 11) in accordance with the guidelines of the Swedish National Committee for Experimental Animals.

[0231] Brain resection and sectioning

[0232] The brain was quickly removed from the skull and frozen in isopentane (2-methylbutane 99% solution) at about -40°C for 15-20 seconds. The frozen brain was wrapped in tin foil and stored at -80°C until use. The central part or striatum (approximately 1 mm) was cut at a thickness of 14 μm (coronal) on a cryostat (Leica CM1860). Three sections per slide (75 sections = 1 mm tissue = 25 slides). The sections were thawed and mounted on microscope slides ( Plus, German Menzel- ), air-dried and refrozen directly in the cryostat. The slides were kept at -20 °C until use. The sections were made sequentially, meaning that there were three horizontal sections on the same slide.

[0233] Fluorescent immunohistochemistry (IHC) of HTT aggregates

[0234] The sections were placed in phosphate buffered saline (PBS) and then incubated for 72 hours with mouse anti-HTT primary antibody (MAB5374 from Chemicon, anti-huntingtin antibody, clone mEM48, mouse origin) diluted 1 / 300 in 0.3% TX-100, 0.1% NaN3 in PBS. The sections were washed in Tris-HCl buffered saline (pH 7.4) containing 0.5% Tween, blocked (Perkin Elmer), and incubated in blocking buffer containing 488-conjugated anti-mouse secondary antibody (1 / 200, Jackson), and then washed several times in Tris-HCl buffered saline containing 0.5% Tween. All sections were stained with the nuclear marker Hoechst (1 / 5.000), and tissue autofluorescence was blocked using 1% Sudan Black (in 70% ethanol) and mounted with polyvinyl alcohol / glycerol (Sigma) containing 2.5% DABCO. All IHC slides were analyzed using MetaViewer imaging software from MetaSystems.

[0235] Radioligand

[0236] [3H]NNC112 (77 Ci / mmol) was synthesized by the Department of Clinical Neuroscience, Karolinska Institutet. [3H]Raclopride (81 Ci / mmol) was purchased from Novandi Chemistry AB (Södertälje, Sweden). MNI659 (57 Ci / mmol) was provided by the CHDI Foundation. The radiochemical purity of all radioligands was measured before ARG experiments (>95%).

[0237] In vitro autoradiography (ARG)

[0238] The slides were thawed at room temperature and pre-incubated in binding buffer (Tris HCl 50 mM, pH 7.4, including 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2) for about 20 minutes. The slides were then incubated with 1 nM radioligand in binding buffer for 60 minutes at room temperature.

[0239] In a duplicate set of containers, 10 μM butaclamol ([3H] NNC112 and [3H] raclopride) or MP-10 ([3H] MNI659) was added to establish an irremovable binding. After incubation, the slides were washed 3 times for 10 minutes each in Tris HCl 50mM, pH 7.4, and then briefly washed in distilled water. The slides were dried overnight at room temperature or on a heated (37°C) plate for about 30 minutes. The slides were placed with autoradiographic micro-standards (American Radiolabeled Chemicals Inc.) and exposed for 90 hours on tritium-sensitive phosphor imaging plates (Fuji plates BAS-TR2025, Fujifilm, Tokyo, Japan).

[0240] Before exposure to phosphor imaging plates, tissue sections for [3H]MNI659 were fixed in 4% paraformaldehyde (PFA). This results in reduced signal intensity, which is critical for analyzing signals because radioligands are very powerful. This technique with post fixation of tissues was previously used to make multiple uses of phosphor imaging plates with low-energy isotopes such as tritium (3H) possible. In this case, we used post fixation to reduce the signal intensity of [3H]MNI659 in tissues to utilize binding at higher concentrations (e.g., 1 nM), which is critical for achieving equilibrium between bound and free fractions. Without a post fixation step, we can only bind at concentrations below 0.15 nM, and this is not enough to reach equilibrium, and therefore not enough to show the difference between wild type and Q175 [3H]MNI659 binding in vitro.

[0241] The disadvantage of post-fixation with [3H]MNI659 is that the removed signal affects the image analysis with lower specific binding quantification values ​​(fmol / mg tissue). Nevertheless, the ratios between the analyzed brain regions were still the same, which means that the differences in specific binding between the groups were equal.

[0242] Image analysis

[0243] Phosphor imaging plate is scanned, and the obtained image is processed in Fuji BAS-5000 phosphor imager (Fuji Corporation, Tokyo, Japan).Region of interest (ROI) analysis is applied by manually delimiting the injection site, and the manually delimiting injection site is based on the EM48 immunoreactivity shown by fluorescence IHC.Then, the mean pixel value of the ROI of 6 sections is converted to radioactivity value and binding density (fmol / mg tissue, tissue wet weight) using micro-standard.Multi Gauge 3.2 phosphor imager software (Fuji Corporation, Tokyo, Japan) is used to carry out quantitative analysis.Specific binding is calculated by deducting nonspecific binding level from all combinations of each section.

[0244] Radiosynthesis

[0245] Radiosynthesis of [11C] Raclopride: [11C] Raclopride was synthesized at the Karolinska Institutet by methylation of the demethylated precursor analog using [11C] trifluoromethanesulfonic acid methyl ester as previously described (Langer et al. (1999) Nucl Med Biol 26(5):509-18). Incorporation was >50% and radiochemical purity was >99%.

[0246] Radiosynthesis of [18F]MNI659: Radiosynthesis was performed as described in PMID 27856625.

[0247] In vivo imaging using [11C]raclopride and [18F]MNI-659

[0248] use PET-MRI and PET measurements were performed using the PET-CT Preclinical Small Animal Imaging System. Both systems have identical PET performance (Nagy et al. (2013) J. Nucl Med 54(10):1825-32) and are calibrated to provide consistent results. The first PET measurement was performed when the animals were 6.5-7 months old. On the day of the experiment, the animals were anesthetized by inhalation of isoflurane (4-5% isoflurane in 100% oxygen). After induction of anesthesia, the isoflurane concentration was reduced to 1.5-2% (50 / 50 air / oxygen) and the animals were placed in a designated mouse bed in the scanner. A cannula was inserted into the tail vein through which the radioligand was administered. A 63-minute dynamic PET scan was started immediately after intravenous injection of the radioligand. After completion of the imaging procedure, the animals were returned to their cages. The animals were housed in the animal department of the Karolinska Institute until 10 months of age, where imaging was repeated using the same radioligand.

[0249] Image analysis and statistical analysis

[0250] The acquired list-mode data were reconstructed into 25 time frames (63 min scan = 4x10 s, 4x20 s, 4x60 s, 7x180 s, 6x360 s). Image reconstruction was performed using a full three-dimensional maximum-likelihood expectation maximization algorithm (MLEM) with 20 iterations without scatter and attenuation correction. The reconstructed dynamic PET images were co-registered with the built-in mouse MRI template in PMOD, which also included the volume of interest (VOI) settings (PMOD Technologies Ltd., Zurich, Switzerland). With the help of these VOI sets, attenuation-corrected time-activity curves (TACs) were generated. Using the cerebellum as a reference region, the binding potentia (BPND) was calculated in PMOD with a simplified reference tissue model (SRTM). The % difference between the left and right striatum was calculated according to the following formula:

[0251] Difference % = ((right striatum - left striatum)) / (left striatum) x 100

[0252] The differences between the two sides of the striatum were analyzed using the within-subject paired t-test using GraphPad Prism version 5.00 for Windows (GraphPad Software Inc, San Diego, CA, USA). In addition, the % difference between the left and right striatum between the control and inhibitor-treated groups was analyzed using an unpaired t-test between subjects.

[0253] Electrophysiological recordings

[0254] For electrophysiological recordings, slices were transferred to an immersion recording chamber fixed on an Olympus BX51 upright microscope (60X / 0.9NA objective lens) equipped with infrared differential interference contrast. Whole-cell patch clamp electrophysiological recordings were performed with a Multiclamp 700B amplifier. Signals were filtered at 1 KHz and converted to digital format with a Digidata 1400. Stimulation and display of electrophysiological recordings were obtained with custom-written WinFluor software (John Dempster, University of Strathclyde, Glasgow, UK), which synchronizes two-photon imaging and electrophysiology. Targeted electrophysiological recordings were obtained from iSPNs or dSPNs. Patch-pipettes (4-6 MΩ) were made with a Sutter Instruments horizontal puller using borosilicate glass with a filament and filled with the following (mM): 135KMeSO4, 5KCl, 5HEPES, 0.05EGTA, 2ATP-Mg2, 0.5GTP-Na, 10phosphocreatine di(tris); pH was adjusted to 7.25 with KOH and osmolarity was adjusted to 270–280mosM. To record Ca2+ transients in dendritic spines and rods, cells were filled with 100μM fluorine-4 pentapotassium salt and 25μM Alexa Fluor 568 hydrazide sodium salt (Invitrogen). All recordings were performed 30 minutes after the whole cell configuration was established. Electrophysiological characterization of neurons was performed in the current clamp configuration. The amplifier bridge circuit was adjusted to compensate for the electrode resistance. The access resistance was continuously monitored and the experiment was abandoned if a change of >20% was observed. Only neurons with stable recordings were used for analysis. We discarded 30% of the recordings because of changes in access resistance. The membrane potential was held at -80 mV. The digitized data were imported for analysis using commercial software (IGOR Pro 6.0, WaveMetrics, OR).

[0255] Two-photon Ca 2+ Imaging

[0256] Ca2+ transients were stimulated by back-propagating action potentials (bAPs) (5 bAP triplets each; 50 Hz intra-training; 5 Hz inter-training; in ZFP experiments, bAPs were generated with single 50 Hz triplets). Ca2+ transients were recorded at the proximal (50-60 μm) and distal dendritic spines (>100 μm) of the same dendrite in-plane sections using an Ultima laser scanning microscope system (Bruker Technologies, formerly Prairie) connected to a tunable laser (Chameleon, Coherent Laser Group, Santa Clara, California) with an excitation wavelength of 820 nm (pulse frequency of 80 MHz; pulse duration of 250 fs). The red signal (580-630 nm) from Alexa Fluor 568 was used to visualize dendrites, while the green signal (490-560 nm) from Fluor-4 was used to record Ca2+ transients. The red channel was used to normalize the signals obtained from the proximal and distal sections. Therefore, Ca2+ transients are expressed as the ratio (G / R) between green and red fluorescence. In order to avoid dye diffusion artifacts, only proximal and distal dendritic lysates with red fluorescence differences <10% in the same focal plane were considered for further analysis. Line scan signals were acquired with a resolution of 512 pixels per line and a dwell time of 10 μs / pixel. For ZFP experiments using triplet bAPs, IGOR Pro (WaveMetrics, Lake Oswego, Oregon) was used for data smoothing and statistics. The mean fluorescence function (F(t)) is the spatial average of 5 adjacent pixels, while the basic fluorescence Fo is the average of the first 30 time points in the line scan. The standardized difference (ΔF / Fo) caused by current injection in the Ca2+ signal is defined as the maximum fluorescence change standardized by the basic fluorescence.

[0257] Two-photon laser uncage

[0258] Two-photon Ca2+ imaging and two-photon laser uncaging were performed simultaneously using two different femtosecond pulses connected to a microscope (Ultima, Bruker Group). MNI-glutamate (5mM) was fused into the recorded area and excited at 720nm by a photostimulation laser (Chameleon, Dry Laser Group, Santa Clara, California). The two laser beams on the sample were controlled by two independent galvanometer scanning mirrors. A 1ms pulse (about 10mW) was delivered to a single spine located in the same focal plane (5-10 columns). Single-thorn stimulation was calibrated so that each stimulated spine produced a somatic excitatory postsynaptic potential of 1-2mV. Custom writing software (WinFluor) was used to synchronize Ca2+ transients, electrophysiological recordings, and two-photon laser stimulation.

[0259] ZFP constructs

[0260] Plasmids containing ZFP-D cDNA and unbound ZFP cDNA were subcloned and packaged into AAV9 by Virovek (Hayward, CA). Expression of human mutant Htt-repressor ZFP and tdTomato tagged with N-terminal NLS and C-terminal FLAG tags were bridged by viral 2A cleavage peptide. The striatal derived cell line ST HDH Q7 / 111 (Coriell, CHDI-90000072) from knock-in transgenic mice containing a hybrid huntingtin (HTT) locus was used to test ZFP-mediated repression of mHTT expression, wherein the locus has humanized exon 1 with 111 polyglutamine repeats and the rest has wild-type mouse HTT with 7 polyglutamine repeats. After incubation overnight, cells were plated in 12-well plates and then infected with AAV carrying ZFP-30645 and harvested 72 hours after infection for RNA isolation.

[0261] ZFP-mediated repression in A2a-Q175 HETs

[0262] Stereotaxic injections of AAV carrying ZFP and tdTomato genes were performed in the striatum of 4-month-old A2a-EGFP and A2a-EGFP / Q175 het mice under isoflurane anesthesia (ML=-1.7, AP=-0.98, DV=-3.6). Mice were allowed to recover for at least 2 months after injection.

[0263] Real-time quantitative PCR analysis of Htt mRNA expression

[0264] RNA was isolated from ST HDH Q7 / 111 cells and striatal tissue of mice injected with ZFP AAV using RNAeasy kit (Qiagen). RNA was reverse transcribed using Superscript III RT enzyme (Life Technologies). Real-time quantitative PCR was performed using ABI StepOnePlus rtPCR system with SYBR-Green PCR master mix (Applied Biosystems, Foster City, California). The relative abundance of different transcripts was assessed by SYBR quantitative PCR using the 2-[Δ][Δ]Ct method (ABS, User Bulletin 2). The following primers (IDT) were used for PCR amplification: wild-type mouse Htt_fw: CAG GTC CGG CAG AGG AAC C (SEQ ID NO: 22), coated mouse Htt_Q175_fw: GCC CGG CTG TGG CTG A (SEQ ID NO: 23), mutant and wild-type Htt_rv*: TTC ACA CGG TCTTTC TTG GTG G (SEQ ID NO: 24), ZFP_fw: CTG GCT GGT GGA GAG AGA AAT T (SEQ ID NO: 25) and ZFP_rv: TCG TCG TCC TTG TAG TCA ACT GA (SEQ ID NO: 26), (*wild-type and mutant HTT share the same reverse primer sequence). Briefly, the experimental Ct was normalized to GAPDH values ​​using the following formula: ΔCt = Ct (Htt) - Ct (GAPDH). The expression level relative to the control was calculated using the following formula: ΔΔCt=Ct(treated)-ΔCt(control). The final expression level was obtained using the formula 2-ΔΔCt.

[0265] Example 2: Identification of ZFP-TFs that selectively repress HTT alleles carrying expanded poly-CAG tracts

[0266] At the time these studies were conducted, it could not have been predicted that the binding properties of ZFP-TFs targeting poly-CAG would display a high degree of synergy and a strong dependence on poly-CAG tract length in their ability to repress Htt ( Figure 1A ). Furthermore, to address the fact that environmental factors that can induce allele-specific responses are difficult to mimic in model systems, the strategy we pursued emphasized screening different panels of ZFP-TFs directed against allele-specific repression in situ in the context of the endogenous HTT promoter in patient cells.

[0267] Therefore, different panels of ZFP-TFs were screened for direct allele-specific repression in situ in the context of the endogenous Htt promoter in patient cells. Figure 1B and 1C As shown in Tables 1 and 2 below, a set of 41 different ZFPs was assembled (essentially as described in U.S. Pat. No. 8,841,260) with a variety of structures, target lengths, and binding periodicities within the poly-CAG tract. Each ZFP was linked to the KRAB transcriptional repression domain from the KOX1 protein as previously described. By convention, ZFPs are assembled from one-finger modules or two-finger modules, and the proteins use different finger linkers between the ZFP modules (referred to as "Mod links" in Table 1 below). The linkers used are as follows: "0a" is TGEKPFQ (SEQ ID NO: 27); "0c" is TGSQKPFQ (SEQ ID NO: 28); "1c" is

[0268] THPRAPIPKPFQ (SEQ ID NO: 88); "2f" is TPNPHRRTDPSHKPFQ (SEQ ID NO: 29). The 0a and 0c linkers do not skip any nucleotide bases between modules, while 1c and 2f skip 1 bp and 2 bp, respectively.

[0269] Table 1: Zinc finger design

[0270]

[0271]

[0272]

[0273] Table 2: Zinc finger target sites

[0274]

[0275]

[0276] *Uppercase letters indicate nucleotides that contact the DNA binding domain; lowercase letters indicate nucleotides that do not contact

[0277] These proteins were then screened for allele-selective repression when delivered via RNA nucleofection to fibroblasts from two different HD patients, each carrying a different combination of mutant and wild-type poly-CAG tract lengths ( Figure 1D Independent monitoring of wild-type and mutant transcript levels was achieved via SNP-based allele-specific qRT-PCR ( Figure 1A and Figure 6 ).

[0278] This screen yielded a diverse range of Htt repressive behaviors, including clear evidence of allele specificity for some designs (see, e.g., Figure 1D ).

[0279] Example 3: Allele-selective repression of mHTT across a wide ZFP dosage range

[0280] We next expanded our evaluation of the dose range compatible with allele-specific repression, as well as quantification of Htt protein product, using two ZFP-TFs that selectively repressed the mutant alleles in the initial screen (ZFP-A and ZFP-B, Figure 1C ). The following Table 3 provides the complete sequences of ZFP-A to ZFP-D.

[0281] Table 3: Amino acid sequences of ZFP-A, ZFP-B, ZFP-C and ZFP-D

[0282]

[0283] like Figure 1E As shown, in studies of CAG15 / 67 (SEQ ID NOS: 81 / 82) fibroblasts, ZFP-A and ZFP-B ZFP-TFs showed exclusive repression of the amplified allele. Figure 1F As shown, both repressors were shown to selectively reduce only mHtt protein, as determined by Western blotting ( Figure 1F , compare the upper band (mHtt) and lower band (wtHtt) measurements.

[0284] Next, we evaluated these ZFP-TFs in titration studies as described above.

[0285] like Figure 1G As shown, allele-specific repression was observed over a wide dose range. In particular, ZFP-B exhibited >92% repression of the disease allele over a 100-fold delivered RNA dose range (10 ng–1000 ng delivered RNA) and its EC 50 2.58ng(R 2 0.96), and no repression of wild-type HTT. Notably, ZFP expression reflected the delivered RNA levels within this dose range. In summary, these studies determined that the designed ZFP-TF can exclusively downregulate mHTT transcripts within a 100-fold ZFP expression range, resulting in a selective reduction in mHTT protein product.

[0286] Example 4: Allele-selective repression among multiple HD genotypes

[0287] We next performed titration studies in fibroblasts carrying poly-CAG tracts that represent the HD population ( Figure 1H To achieve this, the repressors ZFP-A and ZFP-B, which exhibit poly-CAG tracts with 18 (SEQ ID NO: 79) and 45 (SEQ ID NO: 80) repeats, were first evaluated in GM02151 fibroblasts.

[0288] like Figure 1G As shown, ZFP-A and ZFP-B selectively repressed mHTT over an approximately 100-fold dosage range, and repression of the CAG18 (SEQ ID NO: 79) allele was observed to be <15% at any dose.

[0289] In addition, the effects of the repressor were also examined in patient fibroblasts that had an atypical narrow separation between wild-type and mutant poly-CAG tract lengths, 21 (SEQ ID NO: 83) and 38 repeats (SEQ ID NO: 84).

[0290] like Figure 1H As shown, ZFP-A and ZFP-B drove selective and potent repression (up to 93% reduction) of the CAG38 allele (SEQ ID NO: 84) at 100-fold doses (ZFP-A EC50 = 9.8 ng, R2 = 0.94; ZFP-B EC50 = 3.9 ng, R2 = 0.94), although some repression (usually < 25%) of the CAG21 allele (SEQ ID NO: 83) was observed at higher doses (Figure IJ).

[0291] These results demonstrate that the designed genetic repressors described herein can drive endogenous allele-selective repression over a 100-fold range of ZFP levels, which is substantially greater than other approaches previously demonstrated to target poly-CAG (e.g., ASOs or RNAi, see, Gagnon et al., supra; Hu et al., supra; and Yu et al., supra). Furthermore, by bracketing allele lengths that are either susceptible (CAG ≥ 38) or resistant (CAG ≤ 21) to repression, these studies highlight the ability of ZFP-A and ZFP-B to downregulate 100% of fully penetrant mutant alleles (CAG>39) while distinguishing at least 87% of normal alleles present in HD patients ( Figure 1H ).

[0292] The high degree of allele selectivity exhibited by ZFP-A and ZFP-B contrasts with HTT repressors reported in previous studies (Garriga-Canut et al., supra), which showed much lower sensitivity to poly-CAG tract length. We suspect that this distinction is due to the fact that we have screened for allele selectivity in a more stringent and disease-relevant setting (patient fibroblasts), which allows us to distinguish rare repressors that exhibit true functional synergy, as opposed to more common designs that discriminate alleles with lower effectiveness by simple mass action. Consistent with this, evaluation of our candidate designs in Garriga-Canut's mouse cell system (HdhQ7 / Q111, including 4 (SEQ ID NO: 86) and 111 (SEQ ID NO: 87) consecutive CAG repeat alleles, respectively) yielded a higher frequency of clearly allele-selective hits, with 25 ZFP-TFSs exhibiting >75% reduction in mHTT and <10% reduction in wild-type HTT ( FIG. 8 ). In contrast, only 3 ZFP-TFs produced quantitatively comparable behavior in our more stringent patient fibroblast screening model (CAG 18 / 45 (SEQ ID NOS: 79 / 80)). Consistent with this observation, a control ZFP (ZFP-C) (SEQ ID NOS 86 / 87) that was allele selective only in CAG 4 / 111 cells was not allele selective in the same follow-up study used to characterize ZFP-A and ZFP-B (see Figures 1E-1J Finally, direct evaluation of the two lead ZFPs from Garriga-Canut et al., supra, showed that when tested in fibroblasts (GMO2151) with a disease-associated poly-CAG tract (CAG 18 / 45 (SEQ ID NOS: 79 / 80)) ( Fig. 9 ) do not show allele selectivity.

[0293] Example 5: Long-term allele-selective repression of mHTT in human neurons and mouse striatum and amelioration of HD-related phenotypes

[0294] Because HD pathology is characterized by neuronal dysfunction, we performed a series of studies to evaluate ZFP performance in neuronal stem cells (NSCs) and neurons differentiated from the well-characterized CAG17 / 48 (SEQ ID NOS:76 / 78) embryonic stem cell (ESC; Genea020) line. Figure 2A ; Figure 10). Initial studies interrogated allele-specific repression as described above.

[0295] like Figure 2BAs shown, delivery of ZFPs via transient mRNA transfection revealed allele-selective repression over a wide dose range, similar to the results described in Example 1. In particular, ZFP-B exhibited >90% repression of the disease allele over a 100-fold delivered RNA dose range (30 ng–3,000 ng) and no repression of wild-type HTT. In differentiated neurons, delivery of ZFPs via AAV transduction also produced highly allele-specific repression ( Figure 2C ; see Table 4 below for a summary of all AAV vectors used in this study), although modestly reduced in CAG17 (SEQ ID NO: 76) (approximately 17% and 8% for ZFP-A and ZFP-B, respectively), which may reflect higher ZFP expression levels in this system. Similar to its behavior in HD fibroblasts, ZFP-C strongly repressed both normal and disease alleles in NSCs and neurons.

[0296] Table 4: AAV serotypes and transgenes used in this study

[0297]

[0298] We next tested ZFPs to demonstrate cell tolerance and lasting efficacy. To this end, lentiviral delivery was used to drive long-term ZFP expression in NSCs and neurons for a 103-day study. After 27 days, infected NSCs were expanded and FACS-enriched for ZFP expression, expanded for 51 days, and finally differentiated into neurons and cultured for a further 25 days; allele expression was assessed at days 74 and 103 ( Figure 2A ).

[0299] like Figure 2D and 2E As shown, regulation was highly selective (>88% reduction for CAG48 (SEQ ID NO: 78), <12% repression for CAG17 (SEQ ID NO: 76)) and did not decrease on the second sampling day, suggesting that ZFP expression was well tolerated during all phases studied. Consistent with this result, transcriptome-wide specificity analysis by microarray (n=18,149 queried genes) revealed robust on-target allele-specific regulation of CAG17 / 48 (SEQ ID NO: 76 / 78) neurons ((n=5, ZFP-A; n=16, ZFP-B), Figure 2F ; Table 5 below) and CAG18 / 45 (SEQ ID NO: 79 / 80) fibroblasts ((n=10, ZFP-A; N=5, ZFP-B), Fig.11A; Table 6 below) (Figure 11). A significantly higher number of genes were repressed by non-allelic selective ZFP-C in neurons (n=53) and fibroblasts (n=45), most of which contained CAG arrays within 1 kb of TSS ( Figure 2G , Fig.11D ). Microarray performance was extensively confirmed by quantitative RT-PCR of a control transcript panel ( Fig. 11B and 11C Importantly, membership analysis of the microarray data revealed no overlap between genes regulated by ZFP-A and ZFP-B ( Figure 2G , Fig.11D ), and examination of corresponding promoters showed no strict correspondence between whether a gene was regulated and the presence, length, or position of CAG repeats (see, Tables 5 and 6). Taken together, these results rule out the possibility that allele-specific mHTT repression by the CAG-targeted approach might inevitably repress some other gene with a longer and / or more readily bound repeat array, and instead suggest that further optimization of our design will yield ZFP-TFs that uniquely repress mHtt.

[0300] Table 5: Microarray analysis results of ZFP A, B and C in neurons

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] We next evaluated the amelioration of HD-associated phenotypes in ZFP-B-treated CAG17 / 48 (SEQ ID NO: 76 / 78) neurons ( Figure 2A ).

[0308] like Figure 2H As shown, compared with non-HD neurons ( Figure 2H and 2I), intracellular ATP levels in CAG17 / 48 (SEQ ID NO: 76 / 78) neurons were significantly reduced and susceptibility to apoptosis was increased. After 21 days of ZFP-B expression, ATP levels increased by approximately 70% compared to control-treated neurons, indicating that ZFP-mediated mHTT repression can improve HD-associated metabolic defects. Cultured HD neurons also exhibited increased susceptibility to apoptosis. Seven days after growth factor withdrawal, the percentage of CAG17 / 48 (SEQ ID NO: 76 / 78) neurons undergoing apoptosis was 3.3 times higher than that of non-HD neurons, and ZFP expression restored apoptosis to baseline levels. Fig.2I As shown, expression of ZFP-B reduced the level of apoptosis to that of normal neurons.

[0309] Thus, selective repression of the mHTT allele by ZFP-TFs resulted in reversal of cellular phenotypes associated with key pathological features of HD.

[0310] Given the need for artificially long CAG tracts to induce HD-like disease in rodents, we evaluated ZFP-B performance in heterozygous Q50 (HdhQ50 / HDH+) mice, which have a knock-in allele encoding human exon 1 of 48 CAGs (SEQ ID NO: 78) at the endogenous mouse Htt gene, before conducting phenotypic studies. Although phenotypically normal, the Q50 model enables the assessment of mHtt repression in the context of a typical HD patient CAG array and is the same length used in our human neuronal studies. AAV encoding ZFP-B or GFP was injected bilaterally intrastriatically at 2 doses (3×10 10 or 9x10 10 VG / Hemisphere; Figure 2J ) was administered to 11-week-old Q50 heterozygous animals. Seven weeks after delivery, transgene expression and Htt regulation were assessed by qRT-PCR.

[0311] like Figure 2K As shown, the low and high doses repressed the Q50 allele by 55% (P<0.001) or 67% (P<0.0001), respectively. The endogenous mouse Q7 allele (CAG4 (SEQ ID NO: 86)) was not repressed by any dose of ZFP-B, and total Htt levels were consistent with the observed allele-selective Q50 repression. In addition, as Figure 2L As shown, regression analysis of all striatal sections revealed an inverse correlation between ZFP-B and Q50 expression levels (P < 0.001, R 2=0.36), but not Q7. The variability in ZFP expression and Q50 repression levels in striatal slices is consistent with the incomplete AAV striatal coverage we observed throughout the stereotaxic delivery studies (usually 30%-70% striatal coverage). In addition, ZFP-TF 45794 was also tested in GENEA020 neurons (see Figure 2N ).

[0312] Together, these results demonstrate that ZFPs can achieve durable and selective repression of the disease-associated CAG allele in human HD neurons and mouse striatum.

[0313] A key consideration for any therapeutic strategy is to assess specificity and off-target effects. Of particular interest to this approach is the sum of the prevalence of the other 1,053 endogenous CAG arrays (length ≥ 6 to account for the approximate DNA footprint of 6-finger ZFPs) in the human genome adjacent to the promoters of 176 genes (see Methods). The abundance of partially discontinuous arrays (up to 3 mismatches per hexamer) was even higher, totaling nearly 21,456 sites (full CAG arrays) in 1,872 human genes. Although no other human gene has a TSS-adjacent CAG tract of the same size as the smallest fully penetrant mHTT allele (CAG40 (SEQ ID NO: 91); see Figure 1H ), but the potential regulation of some CAG-adjacent off-target genes may pose a risk to the clinical application of this strategy.

[0314] To investigate this, we employed global transcriptional profiling in patient neurons and fibroblasts. In contrast to previous strategies targeting CAGs, we chose an unbiased approach to interrogate specificity due to the uncertain influence of promoter context, epigenetic modifications, and ZFP-TFs acting in concert with other factors, any of which could unpredictably influence off-target behavior. In our first design attempt (the focus of the current report), we identified ZFPs that exhibited a high degree of genome-wide specificity without the need for any optimization for this property. Particularly useful information was the effect of structure on allele-selective ZFP specificity. Although ZFP-A and ZFP-B had similar on-target behavior for all queried mHTT alleles, they had mutually exclusive off-target profiles in the two cellular contexts ( Figure 2G , Figure 11). For example, MBD5 is a CAG-adjacent gene with a discontinuous CAG tract (CAG19 (SEQ ID NO: 92), 0 bp from TSS), which is repressed by ZFP-B but not by ZFP-A ( Fig. 11B and 11C). ZFP-A and ZFP-B target different frameworks of the CAG tract, are composed of different ZF modules, and use different linker structures, suggesting that their off-target properties may be design-dependent and can therefore be optimized. We also note that recent advances in engineering ZF modules (e.g., increased preference for targetable frameworks in CAG / CTG arrays) and elimination of nonspecific interactions between ZFs and the DNA backbone provide potentially complementary avenues for optimizing ZFP specificity and developing ZFPs suitable for clinical use.

[0315] While our initial design work generated allele-selective ZFPs with high genome-wide specificity in human fibroblasts and neurons, we anticipated that our proteins would also exhibit some off-target activity in mouse, despite being located at different loci given the substantial lack of CAG repeat conservation between orthologs. Surprisingly, 85 of the top 100 mouse genes with the largest TSS-adjacent CAG arrays have no corresponding repeats in the human genome. However, we performed biased off-target analysis in the mouse genome using the largest TSS-adjacent CAG array (+ / - 1 kb of TSS) following striatal ZFP treatment in wild-type and zQ175 het mice. Mouse genes that were significantly regulated in vivo by ZFP-B and / or ZFP-D all had significantly more CAG repeats in the mouse ortholog than in humans. For example, DNAJC12, a gene with 19 CAG repeats (SEQ ID NO: 92) present in the mouse promoter but absent in the human ortholog, was regulated in mouse striatum but not in human fibroblasts or neurons, highlighting the need to assess human cell specificity. Of the orthologs, only NAP1L3 was regulated in human neurons and fibroblasts, and only by ZFP-A (3.1-5.8-fold repression), but not ZFP-B (no detectable changes), further confirming our observation that CAG off-target regulation is greatly affected by ZFP design.

[0316] Crucially, we performed extensive long-term tolerance and safety studies to understand whether the ZFPs cause detectable toxicity at the molecular, cellular, or behavioral level (Figures 4, 16, and 17). We note that we did not observe neurodegeneration, neuroinflammation, or toxicity at least 15 months of age and 9 months of ZFP exposure under conditions of long-term expression in human neurons and mouse striatum using a variety of promoters and AAV serotypes, suggesting that the ZFPs are well tolerated under these delivery and expression conditions. Given the on-target and allele selectivity windows of ZFP-A and ZFP-B, and the fact that we performed off-target analysis at ZFP doses 1-2.5 orders of magnitude above the on-target EC50 dose, it may be useful to use weaker cell-specific promoters (e.g., other serotypes or routes of administration) when using these reagents to study aspects of HTT biology outside of our experimental systems.

[0317] Example 6: ZFP-driven neuroprotection and improvement of behavioral deficits in the R6 / 2 model

[0318] Having established robust mHTT repression in multiple in vitro settings and in the brain, we proceeded to evaluate whether allele-selective ZFPs could ameliorate neuropathological hallmarks and behavioral phenotypes in HD mice. We first investigated ZFP performance in the R6 / 2 mouse model, in which a human HTT exon 1 fragment with an expanded CAG array (approximately 120 CAGs) is overexpressed. R6 / 2 mice display early and progressive changes in neurophysiology, body weight (BW) loss, motor and cognitive alterations including paw clasping and hypolocomotor states, and significant reductions in medium spiny neuron (MSN) markers including DARPP32, phosphodiesterase 10a (PDE10a), dopamine receptors D1 (DRD1) and D2 (DRD2) (see Example 1).

[0319] In these studies, 5-week-old R6 / 2 mice were injected bilaterally intrastriatically with AAV encoding ZFP-B or GFP and compared to non-transgenic age-matched controls for all endpoints ( Figure 3A ).

[0320] like Figure 3B As shown, ZFP-B resulted in a significant reduction in paw clasping over the 7-week study (P = 0.024, log-rank test), and fewer ZFP-treated mice clasped their paws compared to the weekly GFP-treated group. The ZFP-treated group was 2.4 times less likely to clasp its paw than the GFP group over the 7-week study ( Figure 3B ).

[0321] An open-field test was also used every two weeks to monitor motor deficits. Figure 3CAs shown, in a two-week open-field assessment of spontaneous locomotion, ZFP treatment was significantly higher than GFP treatment in terms of rearing frequency (P = 0.009) and total distance traveled ( Figure 3D , P = 0.038) conferred significant longitudinal improvement. In addition, ZFP-treated mice performed significantly better at multiple time points after ZFP administration, and the greatest benefit relative to control-treated mice was observed at the final time point (12 weeks). Notably, there were no significant BW differences in ZFP-treated mice compared to controls, and we did not detect an effect of ZFP treatment on grip strength or rotarod performance (Figure 13).

[0322] The improvements we observed in some behavioral endpoints raised the prospect that neurons expressing ZFPs in AAV-transduced striatal regions were protected. To investigate this possibility, mice were sacrificed after 12 weeks of behavioral assessments, and striatal tissue was subjected to qRT-PCR analysis for expression of ZFPs, HTT, and MSN markers (see Methods above).

[0323] like Figure 3E This is similar to our results from bulk tissue analysis in Q50 mice ( Figure 2K and 2L ), ZFP treatment resulted in a 62% reduction in mHtt (P < 0.0001), while native mouse Htt expression was unchanged. We examined GFP fluorescence in a subset of control animals and found that AAV coverage ranged from approximately 50-70% of the striatum, indicating near-maximal repression of mHTT in the delivered region (data not shown). Transcript levels of DARPP32, PDE10A, DRD1A, and DRD2 were 1.6-2.0-fold higher in ZFP-treated mice than in GFP-treated mice (all P < 0.0001; Figure 3F ), although 1.5- to 2.3-fold lower than age-matched non-transgenic controls (all P < 0.0001), consistent with incomplete vector coverage of the analyzed striatal regions and the presence of neurodegeneration prior to the intervention age. Importantly, regression analysis of individual striatal samples revealed a significant negative correlation between ZFP and mHTT transcript levels (R 2 =0.48, P<0.0001; Figure 3G ), and the corresponding positive correlations between ZFP expression and levels of all MSN markers (R 2 =0.38-0.62, all P<0.0001). In summary, our findings in R6 / 2 mice provide evidence that ZFPs drive improvements in behavioral and molecular neuropathological endpoints and motivate further investigation of potential neuroprotective effects of allele-selective ZFPs at the cellular level in less aggressive disease models.

[0324] Example 7: Suppression of mHtt and correction of histopathological and electrophysiological defects in zQ175 heterozygous mice

[0325] Due to the limitations imposed by intraparenchymal AAV delivery and rapid R6 / 2 disease progression in mice, we focused on whether ZFP-TFs could affect key neuropathological deficits in the slowly progressive zQ175 model. These mice carry a knock-in mHtt exon 1-like allele with approximately 188 CAG repeats and display HD-associated molecular, histological, electrophysiological, and behavioral phenotypes. See Example 1. Heterozygous zQ175 mice develop hallmark mHTT-containing inclusions at 3-4 months of age that continue to accumulate by 12 months of age, develop molecular and electrophysiological disease features at 4-6 months of age, and develop a relatively mild delayed onset at 10-12 months of age (Menalled et al. (2012) PLoS ONE 7:e49838; Heikkinen et al. (2012) PLoS ONE 7:e50717; Carty et al. (2015) PLoS One 10:e0123527; Beaumont et al. (2016) Neuron 92:1220-1237), thus allowing the evaluation of ZFP efficacy at different stages of disease progression. We first confirmed that AAV delivery of ZFP-B resulted in selective repression of mutant HTT mRNA (99%, P < 0.0001) and protein (99%; P < 0.0001) in cultured primary heterozygous zQ175 striatal neurons ( Figure 4A and 4B As expected, no reduction in wild-type mouse HTT was observed, and a control virus lacking the ZFP DNA-binding domain (ΔDBD) was unable to significantly reduce mutant HTT.

[0326] To evaluate the effects of ZFPs on mHTT inclusion, we monitored EM48 immunoreactivity in heterozygous zQ175 mice after intrastriatal treatment either before (early treatment) or after (late treatment) the onset of neuropathology (Fig. 4c). For the early treatment group, AAV encoding ZFP-B or GFP was injected into the dorsal striatum of 2-month-old mice, and HTT aggregation was assessed at 4 months of age. A self-cleaving T2A-GFP tag was used to label cells expressing ZFPs. In transduced MSNs (DARPP32+GFP+), ZFP-B almost completely prevented nuclear mHTT aggregation (99.6% reduction, P<0.0001) ( Figure 4D and 4E ; Figure 14). Significant reductions in the density of perinuclear HTT inclusions (88% reduction; P < 0.0001) and total EM48 immunofluorescence (48% reduction; P < 0.01) were also observed. Figure 4F and 4G For the late treatment groups, ZFP-B.T2A.GFP, ΔDBD.T2A.GFP, or GFP vector was administered to the striatum of 6-week-old heterozygous zQ175 mice, which have a large preload of HTT aggregates (Mangiarini et al. (1996) Cell 87:493-506). Four months after injection, ZFP-B treatment reduced the number of nuclear EM48+ inclusions by 19% relative to GFP controls (P < 0.01, Figure 4H and 4I ) and 87% of the number of perinuclear inclusions ( P < 0.0001; Figure 4H and 4J ). EM48 intensity in cells with nuclear inclusions was reduced by 24% (P<0.05, Figure 4k). In these studies, there were no significant differences between ΔDBD- and GFP-treated mice for any endpoint.

[0327] Together, these results suggest that allele-selective ZFPs can reduce and potentially block mHTT aggregation both before and after disease onset.

[0328] We also assessed the impact of ZFP expression on the health of transduced MSNs in late treatment groups by monitoring DARRP32 levels. Ten-month-old zQ175 heterozygotes exhibited a 17% (P < 0.01) reduction in striatal DARPP-32 immunoreactivity compared to age-matched WT mice. Figure 4L ). Consistent with our findings in R6 / 2 mice, administration of ZFP-B to 6-month-old zQ175 mice resulted in a 20% increase in DARPP32 immunoreactivity at 10 months of age (P < 0.01) ( Figure 4M Importantly, this effect was unique to ZFP-expressing MSNs, suggesting a protective effect conferred by ZFP expression. To assess whether long-term ZFP expression induced a neuroinflammatory response in vivo, we monitored markers of astrogliosis (GFAP) and microgliosis (Iba1). With the exception of the transient effect of the injection itself (located at the needle track), we observed no changes in marker intensity or GFAP+( Figure 4N and 4O ) or Iba+( Figure 4N and 4P Importantly, GFP expression had no effect on these markers (Figure 15).

[0329] The amelioration of mHTT aggregation pathology and DARPP32 levels prompted us to investigate whether ZFPs could also alleviate important electrophysiological defects affecting zQ175 indirect pathway projection neurons (iSPNs) that are evident at 4 months of age (PMID: 24991961, reviewed in PMID: 25700146). In these studies, 2-month-old or 4-month-old mice received intrastriatal injections of AAV encoding ZFP-D.T2A.tdTomato or ΔDBD.T2A.tdTomato, and iSPN dendritic excitability was assessed at 6 months of age ( Figure 4C and 4N ; Figure 15).

[0330] We first confirmed that the extent of mHTT-specific repression by ZFP-D was consistent with our previous stereotaxic delivery studies and was not altered in nontargeted cortical areas (data not shown). We then tested dendritic excitability in tdTomato+ iSPNs using a simplified backpropagating action potential (bAP) burst protocol consisting of 3 APs at 50 Hz ( Fig.4O Compared with control-treated zQ175 iSPNs, which exhibited an average of 35% inhibition of dendritic excitability (P<0.01), the dendritic index of ZFP-D-treated zQ175 iSPNs was significantly increased in the 2-month-old group ( Figure 4S , P<0.05) and 4-month-old group ( Figure 4T , P<0.05) to near wild-type levels. Therefore, iSPN electrophysiological defects in zQ175 mice can be prevented and reversed by ZFPs targeting mHTT. In addition, because mHTT repression is limited to the striatum, all our findings are consistent with a regional autonomous model of dendritic hypoexcitability rather than a secondary consequence of dysfunctional innervating neurons (Cummings et al. (2009) J. Neurosci 29(33): 10371-86).

[0331] Example 8: Restoration of translational biomarkers in ZFP-treated zQ175 mice

[0332] Our results in R6 / 2 mice demonstrate that early ZFP treatment can partially restore the expression of genes that are downregulated in HD, including PDE10A and D1 and D2 receptors. Monitoring these genes is important because PET imaging studies have demonstrated an early, progressive, and significant decrease in the levels of PDE10A enzyme and D2-like receptors in HD patients, which begins many years before clinical diagnosis (unpublished results from CHDI / Karolinska Institutet, see review by Niccolini et al. (2018) J Neurol Neurosurg Psychiatry PMID: 28889093). In similar studies in heterozygous zQ175 mice (PMID: 27856625), clinical imaging ligands identified early and progressive changes in orthologous mouse proteins. Therefore, we investigated whether these markers are sensitive to mHTT reduction after ZFP administration using two methods: in vitro autoradiography (ARG) of striatal brain slices, and longitudinal microPET imaging in live animals.

[0333] In the first study, 2- or 4-month-old heterozygous zQ175 mice received unilateral intrastriatal injections of AAV encoding ZFP-d or GFP ( Figure 5A ). Using drugs targeting D1-like receptors ([ 3 H]NNC112), D2-like receptors ([ 3 H] raclopride) or PDE10A ([ 3 Expression was assessed with tritiated ligands of 5-H]MNI-659. Binding was measured by ARG in striatal regions of interest (ROIs) delineated by areas showing reduced mHTT accumulation (assessed by EM48 staining of adjacent sections). In mice treated with ZFP-D, injected striatal RO1 [ 3 H]NNC112 binding was significantly higher in the contralateral striatum ROI in both the early 2-6 months (34%, P < 0.0001) and late 4-10 months (24%, P < 0.0001) phases. Figure 5B ). For both the early (26%, P < 0.0001) and late (7%, P < 0.001) ZFP-treated groups, the injected striatal ROI [ 3 H] raclopride binding was also significantly higher than that in the uninjected striatal ROI ( Figure 5C ). Similarly, in the striatal ROI injected with ZFP, [ 3 H]MNI-659 binding was significantly higher in the early (31%, P < 0.0001) and late (11%, P < 0.001) groups ( Figure 5D ).

[0334] Together, these data demonstrate that AAV-delivered ZFP repressors can prevent and restore specific binding of the PDE10A enzyme, D1-like receptors, and D2-like receptors in mice with established disease, with the greatest benefit in animals expressing the ZFPs observed prior to symptom onset and reduction in biomarker expression.

[0335] Then, to investigate whether in vitro alterations in the expression of D2-like and PDE10 ARGs could be monitored in living animals, we assessed the effects of ZFP treatment on [ 11 C] raclopride and [ 18 F] Effect of MNI-659 binding. We previously demonstrated that raclopride and MNI-659 binding progressively decreased in zQ175 striatum from 6 to 9 months of age, from 60% to 56% and 52 to 41% during the period (PMID 27856625). Therefore, we injected 4-month-old mice unilaterally with AAV ZFP-D or ΔDBD and monitored binding longitudinally from 6.5-10 months of age. Consistent with the greater magnitude of the effect obtained with ARG in these mice, ZFP-injected striatum showed a significant decrease in [ 18 F]MNI-659BPND increased significantly at 6.5 (12.8%, P < 0.0001) and 10 (16.5%, P < 0.001) months of age, whereas there was no statistically significant increase in the ΔDBD group at each time point (1.9% at 7 months and -1.5% at 10 months; Figure 5E and 5F Moreover, when the percentage difference in BPND between hemispheres was compared between treatments, the ZFP-D group showed a significant increase in BPND compared to GPF mice at 6.5-7 (11.0%, P < 0.001) and 10 (18.1%, P < 0.0001) months of age ( Figure 5E Interestingly, despite the 7% increase in raclopride binding seen in the 4-10 month ARG group, we could not detect ZFP-D in our microPET study [ 11 C] A significant increase in BPND with raclopride (Tables 7 and 8).

[0336] Table 7: Mean injected radioactivity levels (RA injected), injected mass and weight of zQ175 mice imaged with [11C] Raclopride and [18F] MNI-659

[0337]

[0338] Table 8: Mean %SUV and BPND values ​​for [11C]raclopride or [18F]MNI-659 in the striatum of Q175 animals treated with ZFP-D relative to control virus

[0339]

[0340]

[0341] Differences between left and right striatum were calculated using within-subject paired t-test. Values ​​are expressed as mean ± SD. n = 33-41 mice / group.

[0342] In summary, our molecular findings in R6 / 2 and Q175 mice demonstrate that allele-selective ZFP-TFs can prevent and reverse the loss of key markers of disease progression and that these changes can be monitored in living subjects using clinically translatable markers.

[0343] Example 9: Assessment of tolerance and specificity following long-term ZFP-TF expression in vivo

[0344] Although we did not observe evidence of allele-selective ZFP toxicity in our in vitro and in vivo studies, we sought to more closely examine markers of tolerance and specificity following both short-term and long-term in vivo ZFP treatment. To assess whether long-term ZFP expression leads to neuroinflammatory responses or neurodegeneration, we monitored markers of astrogliosis (GFAP), microglia (Iba1), and general neuronal viability (NeuN) in zQ175 and wild-type mice treated with ZFPs in both early (2-6 months of age) and late (6-12 months of age) paradigms. In addition to the transient effects of the injection itself (located at the needle track) ( Fig.16A ), we did not observe a significant increase in marker intensity ( Fig. 15B and 15G ), or the number of GFAP+ (Figure 4r, s and Fig. 15C , 15D , 15F, 15G) or Iba+ (Figures 4r, t and Fig. 15B and 15E Importantly, GFP expression had no effect on these markers ( Fig.15A ). Consistent with the absence of elevated neuroinflammatory markers, we did not detect any reduction in NeuN+ cells or intensity or number of neurons in WT or zG175 het striatum treated with either ZFP-B or ZFP-D ( Fig. 16C , 16D, 17). These results indicate that long-term striatal expression is generally well tolerated and does not induce overt neuroinflammatory responses in WT and zQ175 mice before and after disease onset. We further evaluated ZFP-B and ZFP-D in early (2-6 months of age) and late (6-12 months of age) treatment groups of WT and zG175 het mice following unilateral striatal AAV injection. Our observations were that there was no loss in BW during the study, and there were no changes in general activity, spontaneous behavior, grooming, nesting, food and fluid intake, or body temperature. Histological analysis showed no evidence of brain volume changes, no loss of NeuN expression, and no elevation of astrocyte markers compared to the contralateral hemisphere ( Figures 16C-16E ).

[0345] To assess the potential for off-target regulation in our in vivo studies, we first mapped the closest CAG array position (length ≥ 6 CAG repeats to minimally consider one ZFP binding site; see Methods) to each annotated transcription start site (TSS) in the mouse and human genomes. We considered perfect CAG arrays, as well as those with more than 3 mismatches per CAG hexamer, to allow for longer and discontinuous repeat bundles. These searches showed that the mouse genome has significantly more CAG content than the human locus (3,472 vs. 1,053 for perfect CAG repeats; 32,328 vs. 21,456 for subperfect repeats). However, imposing a requirement of 1 kb distance from the TSS showed that the two genomes were similar in terms of the total number of genes with TSS-adjacent CAG arrays (150 vs. 176 for perfect CAG repeats; 1720 vs. 1872 for subperfect repeats). We then performed a bidirectional ortholog analysis, whereby each annotated protein-coding mouse gene was mapped to the corresponding human ortholog and vice versa (see Methods). Importantly, we found poor CAG conservation between orthologs, with only 15 of the top 100 CAG mouse genes having orthologs in the human ortholog with TSS-adjacent CAG arrays of any length, and for the reverse comparison, only 12 of the top 100 human genes.

[0346] These informatics results were used to guide in vivo off-target analysis of ZFPs used in our functional mouse studies. We injected the right hemisphere of 6-month-old wild-type (WT) and zQ175 mice intrastriatically with AAV encoding ZFP-B, ZFP-D, or ΔDBD, and injected the left hemisphere with PBS. Striatal tissues were collected 1 month later for qRT-PCR analysis.

[0347] Consistent with the allelic selectivity of these ZFPs, we observed a >70% reduction in mutant HTT mRNA levels, but did not significantly repress mouse HTT in any genotype. We then examined the 12 genes identified in our off-target search, focusing on those with the largest TSS-adjacent CAG repeats in the mouse genome. This group included genes with and without TSS-adjacent CAG arrays in the human ortholog.

[0348] In summary, we observed no repression for genes with CAG arrays ≤19 CAGs (MTUS2, RSLD241, AKT3, KCNA6, ITGA7); the exception was DNAJC12, which was regulated 70% by ZFP-B and 62% by ZFP-D, and lacks CAG repeats in the human ortholog. Examination of the mouse Dnajc12 promoter revealed that 12 CAA repeats are directly adjacent to the CAG array, which may explain why it is regulated while other CAG candidates of similar length are not. For the examined mouse genes with arrays containing ≥24 CAGs, we observed repression ranging from 38% for ZFP-D for RUNX2 (CAG28 (SEQ ID NO: 93), TSS 100 bp) to 79% reduction for ZFP-B for NAP1L3 (CAG28 (SEQ ID NO: 93), TSS 511 bp). In most cases, and regardless of statistically equivalent zQ175 repression, ZFP-D resulted in significantly less off-target repression than ZFP-B, except in the case of RUNX2 and NAP1L3. Genotype did not appear to substantially alter the pattern or extent of regulation. We also examined whether ZFP-B or ZFP-D treatment resulted in loss of neuronal biomarker transcripts DRD1a, DRD2, DARPP32, PDE10a, and RBFOX3 / NEUN. Consistent with our previous long-term analysis, we found no significant reduction in the levels of these neuronal markers 1 month after the treatment course. Finally, to investigate the general tolerance and potential for any behavioral deficits conferred by long-term ZFP expression, we conducted two additional studies with large groups of mice using extended treatment times. In the first study, we evaluated the safety of ZFP-B and ZFP-D after unilateral intrastriatal injections at different ages. We injected early (2-6 months of age) and late (6-12 months of age) groups of WT or zG175 het mice with ZFP-B and ZFP-D. Uninjected age-matched littermates were included. Our observations were that there was no weight loss over the course of the study (early group, 4 months; late group, 6 months), and no changes in general activity, spontaneous behavior, grooming, nesting, food and fluid intake, or body temperature. Consistent with our previous studies, histological analysis showed no evidence of brain volume changes, no loss of NeuN expression, and no increase in Iba1 and GFAP compared to the contralateral side (data not shown), further supporting long-term ZFP tolerance in healthy and diseased striatum.

[0349] In a second study, a total of 164 6-month-old female and male zQ175 heterozygous mice were injected bilaterally with AAV encoding ZFP-D or eGFP or vehicle and monitored for 9 months in empty field, rotarod, paw clasping, neurological index measurements, brain MRI, and body weight (n=22 animals / group for all groups, except for the zQ175 het uninjected group, n=15 animals). This study also included uninjected age-matched WT control littermates (n=44). In summary, we did not observe any degree of deleterious effects of ZFP-D in zQ175 heterozygous mice after disease manifestation. zQ175 het mice did not show obvious paw clasping phenotype or spontaneous movement changes in empty field or neurological index deficits. Striatal ZFP treatment did not significantly alter the minor zQ175 het rotarod performance deficits observed at 15 months, weight changes during disease progression, or affect neurological index scores. Furthermore, intrastriatal ZFP-D administration had no effect on whole brain, striatal, and cortical volumes of zQ175 as assessed by MRI.

[0350] Taken together, the results of these extensive in vivo studies support the conclusion that allele-selective ZFPs are well tolerated at both the cellular and behavioral levels in the striatum of wild-type and diseased mice.

[0351] Together, our data demonstrate that ZFP gene regulators targeting mutant Htt sequences can be used to treat HD.

[0352] in conclusion

[0353] Our study is the first to directly demonstrate selective transcriptional repression of alleles at the native HTT locus in mice. Results from extensive testing in patient-derived cells showed that expression of mHTT alleles with ≥38 CAG repeats could be repressed by 79-93%, whereas expression of normal alleles with ≤21 CAG repeats (the longest normal repeat length tested) was only repressed by 0-31%. Thus, allele-selective ZFP-TFs exhibited a remarkable ability to discriminate between 100% of fully penetrant mutant alleles and at least 86% of normal HTT alleles in the HD population ( Figure 1H Compared to SNP-based allele-selective mHTT lowering approaches, each of which is limited to a subset of HD patients, the CAG-targeted ZFP repressor described in this study has the potential to selectively downregulate the expression of the pathogenic allele in the majority of HD subjects.

[0354] Furthermore, in 3 different HD mouse models, we demonstrated that allele-selective ZFPs robustly repressed mHtt expression in vivo over sustained exposures ranging from 6 weeks to 9 months or longer and ameliorated molecular, histological, electrophysiological, and some behavioral deficits. We also established sustained ZFP expression and efficacy for >100 days in human HD neurons and rescued electrophysiological deficits and MSN marker gene expression in striatal projection neurons in vivo, indicating that long-term expression of mHTT-selective ZFPs in target brain regions and relevant human cell types is well tolerated and effectively alleviates core disease phenotypes.

[0355] The ZFP we developed exhibits a high combination of allele selectivity, genome-wide specificity, and long-term sustained repression, and to our knowledge, this is unprecedented for synthetic transcription factors. These studies have established new benchmarks for the performance of these factors and their potential applications. The results also raise interesting questions about our R&D strategy that can contribute to the successful identification of ZFPs with desired properties, especially compared to previous attempts to repress different alleles (Agustin-Pavon et al. (2016) Mol Neurodegener 11:64). In this regard, it seems that a key aspect of our work is to recognize that by looking for this behavior in a sufficiently diverse candidate design group, it is possible to identify such a poly-CAG-targeted ZFP repressor, which has a very strong functional dependence on the length of the repeat bundle, which is stronger than by simple mass action. It has long been recognized that (Pavletich and Pabo (1991) Science 252:809-817) that macromolecular systems can provide the ability to perform highly synergistic behaviors, in which the initial binding event promotes subsequent binding events, resulting in a consistent all-or-nothing response. The most important requirement for this type of behavior is a method of communication between the substrate-bound ligand. Given the interaction of the KRAB repressor domain with large co-repressor complexes whose components form multimers and, at higher levels, oligomers, ZFPs bound to chromosomal repeat arrays offer a myriad of possibilities for such communication, including noncovalent contacts between adjacent DNA-binding fingers (Nekludova and Pabo (1994) Proc Natl Acad Sci USA 91:6948-6952), binding-dependent DNA distortion (Mirny (2010) Proc Natl Acad Sci USA 107:22534-22539), nucleosome ejection (Iyengar and Farnham (2011) J Biol Chem 286:26267-26276), and the potential for avidity effects (Lupo et al. (2013) Curr Genomics 14:268-278; Peng et al. (2000) J Biol Chem 275:18000-18010; Hinde et al. (2015) Sci Rep 5:12001; Brasher et al. (2000) EMBO J19:1587-1597; Sathasivam et al. (2013) Proc Natl Acad Sci USA 110:2366-2370).

[0356] These considerations motivated our choice of ZFP architecture (5F and 6F proteins, containing multiple fingers, linkers, and targeting poly-CAG frameworks), design scale (large enough to identify relatively rare ZFP designs that best discriminate between alleles) and methodology (independent monitoring of endogenous normal and mutant allele expression in patient cells). With these features in place, a relatively small screen effort (41 designs) yielded proteins with the desired properties. In contrast, previous studies that employed only a single repeat finger and screened fewer allele-discriminating candidates (4) using unrealistic environments such as chimeric episomal reporters and mouse cells (Agustin-Pavon, supra) demonstrated allele-selective mHTT repression, but only in the non-disease-relevant context of mouse cells carrying the non-pathogenic allele (CAG 4 (SEQ ID NO: 86)), which even lacked a single full-length target. Furthermore, the initially identified ZFPs were not well tolerated and required further modifications to the ZFP backbone, KRAB repression domain, and promoter to achieve in vivo repression for more than 2 weeks (Gersbach and Perez-Pinera (2014) Expert Opin Ther Targets 18:835-839). We note that in our own testing of these agents in HD patient cells, we observed an inability to discriminate between the disease-associated human HTT alleles (18 (SEQ ID NO: 79) vs. 45 (SEQ ID NO: 80) CAG). The differences in the strategy we developed compared to previous studies appear to have resulted in significant differences in ZFP performance.

[0357] Our findings also provide some insights into possible mechanisms of allele-selective repression. Specifically, we observed that this phenomenon is design-dependent ( Figure 1D ), which seems to be detrimental to the possibility of indirect communication between bound ZFP-TFs, such as by propagating DNA structural distortions or nucleosome ejection (Mirny, supra, Iyengar and Farnham, supra). In addition, the lack of binding synergy observed in gel shift studies using purified components is inconsistent with the occurrence of direct contacts between adjacent bound ZFPs (data not shown). Finally, we note that the observed properties require the KRAB functional domain. This raises the possibility that the allele-selective behavior originates from an avidity effect, which may be mediated by the interaction of the KRAB repressor domain and the large co-repressor complex complex.

[0358] A key consideration for any therapeutic strategy is to assess specificity and off-target effects. Of particular interest to this approach is the sum of the prevalence of the other 1,053 endogenous CAG arrays (length ≥ 6, to account for the approximate DNA footprint of 6-finger ZFPs) in the human genome adjacent to the promoters of 176 genes. Partial discontinuous arrays (up to 3 mismatches per hexamer) are even more abundant, with a total of 21,456 sites (all CAG arrays) approaching 1,872 human genes. Although no other human gene has a TSS-adjacent CAG tract of the same size as the smallest fully penetrant mHTT allele (CAG40 (SEQ ID NO: 91); see Figure 1H ), but the potential regulation of some CAG-adjacent off-target genes may pose a risk to the clinical application of this strategy.

[0359] To investigate this, we employed global transcriptional profiling in patient neurons and fibroblasts. In contrast to previous strategies targeting CAGs (Gagnon, supra; Hu, supra; Yu, supra; Fiszer, supra; Lutz, supra), we chose an unbiased approach to interrogate specificity due to the uncertain influence of promoter context, epigenetic modifications, and the synergy of our ZFP-TFs with other factors, any of which could unpredictably affect off-target behavior. We identify, for example, ZFPs that exhibit high genome-wide specificity without any optimization for this property. Particularly useful information is the effect of structure on allele-selective ZFP specificity. Although ZFP-A and ZFP-B have similar on-target behavior for all queried mHTT alleles, they have mutually exclusive off-target profiles in two cellular contexts ( Figure 2G , Figure 11 , Figure 17 ). For example, MBD5, a CAG-proximal gene with a discontinuous CAG tract (CAG19 (SEQ ID NO: 92), 0 bp from TSS), was repressed by ZFP-B but not by ZFP-A ( Fig. 11B and 11C). ZFP-A and ZFP-B target different frameworks of the CAG tract, consist of different ZF modules, and use different linker structures, suggesting that their off-target properties may be design-dependent and can therefore be optimized. Specificity is also important for effectively targeting one or more pathogenic agents. Although it has long been recognized that CAG expansions in the HTT gene are the cause of HD, there is still a need to elucidate the molecular species that may cause neurodegeneration. For example, Htt isoforms containing abnormal splicing of exon 1 and intron 1 are present in multiple HD rodent models and postmortem HD brains, and may contribute to the formation of N-terminal protein species containing pathogenic polyQ (Banez-Coronel et al. (2015) Neuron 88:667-677). In addition, it has been demonstrated that an increasing number of microsatellite expansions implicated in the disease (including CAG repeats of mHTT) undergo antisense transcription as well as repeat-associated non-ATG (RAN) translation, producing neurotoxic dipeptides that accumulate in patient brains (Arber (2017) EMBO Mol Med 9:281-284). These HTT subtypes are expected to be resistant to ASO and RNAi-based strategies targeting sequences downstream of exon 1. In contrast, it is expected that allele-selective ZFP-TFs will reduce the production of all sense and antisense mHtt subtypes containing expanded CAGs, which may provide important therapeutic advantages. We note that although other approaches targeting CAG expansion in exon 1 have been developed, allele-selective behavior has been reported to be applicable only to a narrow dose range (2-5 times) (Pfister et al. (2009) Current Biology 19: 774-778; Gagnon, et al.; Hu. et al.; Yu, et al., Fiszer, et al.; Lutz, et al.). Importantly, we demonstrated that ZFP-TFs can achieve allele-selective repression in a much wider dose window (at least 100 times), which is a key factor in clinical translatability, considering the current technical difficulties in delivering a uniform dose to all cells in the target tissue.

[0360] While R6 / 2 mice treated with ZFPs showed significant improvements in some behavioral endpoints (claw and open field tests), we did not observe significant treatment effects on other standard HD model endpoints (e.g., speeding up the rotarod or grip strength) in either R6 / 2 or zQ175 het mice. An important consideration in this regard is the limited AAV coverage that can be achieved by stereotaxic delivery to the mouse brain. In our studies, intraparenchymal injections achieved coverage of 30-70% of the mouse striatum, limiting the ability to assess the effects of ZFPs on some measures of disease progression, most importantly at the behavioral level. In addition, R6 / 2 is an invasive model, and the introduction of ZFPs at 5 weeks of age (the earliest time point at which stereotaxic injections can be reliably performed) may be too late to affect some behavioral endpoints. Although striatal inclusion is critical for motor control, it is unclear whether striatal-selective suppression of mHTT is sufficient to alleviate these symptoms given the role of the broader cortico-striatal-thalamocortical circuit in motor pattern learning and execution. In addition, it is unclear whether the changes in motor behavior in HD mice are driven solely by striatal mHTT expression. Clearly, other brain regions contribute to spontaneous motor control in mice, including motor-related cortical areas, the cerebellum, and brainstem nuclei, which can also be affected in these models (Capelli et al. (2017) Nature 551:373-377; Datson et al. (2017) PLoS One 12:e0171127). Therefore, widespread inhibition of mHTT may be necessary to rescue these disease phenotypes in HD mouse models. In support of this notion, improvements in spontaneous movement and rotarod performance have been reported with alternative approaches (e.g., ASOs or siRNAs) that have been shown to be more widely distributed in the mouse brain, although their effects are generally mild or partial restoration (Stanek et al. (2014) Human Gene Therapy 25(5):461-474; Russell et al. (2014) JAMA Neurol 71:1520-1528). Given these limitations and the understanding that mHTT is an underlying etiology of HD, we focused our analyses primarily on readouts of target engagement, correction of histopathological and electrophysiological deficits, and biomarker improvements, which may represent more translatable and directly relevant efficacy readouts of interest for delaying and / or reversing neurodegeneration in HD patients.

[0361] Results using autoradiography for markers of dopamine D1-like and D2-like receptors and the PDE10A enzyme showed that lowering mHTT in Q175 heterozygous mice was associated with increased specific binding of [3H]NNC112, [3H]raclopride, and [3H]MNI-659 in both early and late treatment studies (after symptom onset and decline in expression). The greatest differences were observed for D1-like receptors, followed by PDE10A and D2-like receptors, suggesting that the effects of lowering mHTT after symptom onset in mice were more pronounced in striatal neurons of the direct pathway because PDE10 is expressed in both populations of SPNs. The effects of reducing mHTT were also more pronounced in the early treatment studies, suggesting that lowering mHTT may have a greater effect if intervention is initiated early. However, treatment was initiated at 4 months after downregulation of these targets in Q175 heterozygous mice (expected ZFP expression within 1-2 weeks of in vivo viral transduction; data not shown) (Monod et al. (1965) J Mol Biol 12:88-118), suggesting that ZFP administration can partially restore pre-existing losses of these proteins. The PDE10A ARG results were confirmed using microPET with [18F]MNI-659, where we demonstrated a significant increase in binding potential even after symptom onset with clinically relevant translational endpoints. In contrast, raclopride binding was not altered in the microPET study, which may reflect methodological differences between the D2-like in vitro ARG and in vivo microPET, as well as limited tissue distribution of the viral vector used to detect small effects on this marker. In summary, our findings provide encouraging support for the use of imaging ligands as an early readout of striatal mHTT reduction in human trials (Wilson et al. (2016) Journal of the Neurological Sciences 368:243-248; Marks et al. (2016) Hum Gene Ther 27:522-527). Additional work is needed to investigate markers for other brain regions involved in HD (such as the cortex) where mHTT reduction is clinically required.

[0362] All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

[0363] 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. CHDI FOUNDATION, INC. <120> Zinc finger protein composition for regulating huntingtin protein (HTT) <130> 8327-0173.40 <140> <141> <150> 62 / 659,552 <151> 2018-04-18 <150> 62 / 780,605 <151> 2018-12-17 <160> 93 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 1 gcagagctct ctggctaact agag 24 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 2 ctggcaacta gaaggcacag 20 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 3 ggaacggtgc attggaacg 19 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 4 gttcgaatcccaattctttg cc 22 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <400> 5 agcacgttgc ccaggaggtc ac 22 <210> 6 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 6 cgcaggctgc agggttac 18 <210> 7 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 7 gctgcaccga ccgtgagt 18 <210> 8 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <400> 8 cagctccctg tcccggcgg 19 <210> 9 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 9 agtttggagg gtttctc 17 <210> 10 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 10 agtttggagg gtttctt 17 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 11 agggtttctc cgctcagc 18 <210> 12 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 12 tcgactaaag caggatttca gg 22 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 13 tctcctccac agagtttgtg a 21 <210> 14 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 14 ccttctttct ggactaagaa gctg 24 <210> 15 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <400> 15 tccctcatcc actgtgt 17 <210> 16 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic probes <400> 16 ctcatctact gtgt 14 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <220> <221> Modified_base <222> (7)..(8) <223> a, c, t, g, unknown or other <220> <221> Modified_base <222> (15) <223> a, c, t, g, unknown or other <400> 17 cagcagnngc agcancagca g 21 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 18 caggtccggc agaggaacc 19 <210> 19 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 19 ttcacacggt ctttcttggt gg 22 <210> 20 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 20 gcccggctgt ggctga 16 <210> twenty one <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty one ttcacacggt ctttcttggt gg 22 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty two caggtccggc agaggaacc 19 <210> twenty three <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty three gcccggctgt ggctga 16 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> twenty four ttcacacggt ctttcttggt gg 22 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 25 ctggctggtg gagagagaaa tt 22 <210> 26 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic primers <400> 26 tcgtcgtcct tgtagtcaac tga 23 <210> 27 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 27 Thr Gly Glu Lys Pro Phe Gln 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 28 Thr Gly Ser Gln Lys Pro Phe Gln 1 5 <210> 29 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 29 Thr Pro Asn Pro His Arg Arg Thr Asp Pro Ser His Lys Pro Phe Gln 1 5 10 15 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 30 Gln Ser Ser Asp Leu Ser Arg 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 31 Gln Trp Ser Thr Arg Lys Arg 1 5 <210> 32 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 32 Gln Ser Gly Asp Leu Thr Arg 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 33 Arg Ser Asp Asn Leu Ser Glu 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 34 Lys Arg Cys Asn Leu Arg Cys 1 5 <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 35 Met Ala Cys Cys Arg Tyr Ala 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 36 Arg Leu Trp Asn Arg Lys Gln 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 37 His Arg Ser Thr Arg Asn Arg 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 38 Arg Ser Asp Thr Leu Ser Glu 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 39 Arg Arg Trp Thr Leu Val Gly 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 40 Gln Ser Ser Asp Leu Arg Arg 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 41 Arg Ser Ala Val Leu Ser Glu 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 42 Arg Ser Asp Val Leu Ser Glu 1 5 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 43 gcagcagcag cagcagca 18 <210> 44 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 44 gcagcagcag cagca 15 <210> 45 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 45 gcagcagcag cagcagcag 19 <210> 46 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 46 gcagcagcag cagcag 16 <210> 47 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 47 gcagcagcag cagcagcagc a 21 <210> 48 <211> 13 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 48 gcagcagcag cag 13 <210> 49 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 49 cagcagcagc agcagcag 18 <210> 50 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 50 cagcagcagc agcagcagca 20 <210> 51 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 51 cagcagcagc agcagcagca g 21 <210> 52 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 52 cagcagcagc agcagca 17 <210> 53 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 53 cagcagcagc agca 14 <210> 54 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 54 gctgctgctg ctgctgct 18 <210> 55 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 55 gctgctgctg ctgctgctg 19 <210> 56 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 56 gctgctgctg ctgct 15 <210> 57 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 57 gctgctgctg ctgctg 16 <210> 58 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 58 ctgctgctgc tgctgctg 18 <210> 59 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 59 ctgctgctgc tgctgctgct 20 <210> 60 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 60 ctgctgctgc tgctgctgct g 21 <210> 61 <211> 9 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 61 gctgctgct 9 <210> 62 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 62 ctgctgctgc tgct 14 <210> 63 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 63 ctgctgctgc tgctg 15 <210> 64 <211> 266 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 64 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 Gln Ser Ser Asp 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 Gly Ser Gln Lys Pro Phe 65 70 75 80 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Ser Asp Leu Ser 85 90 95 Arg His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 100 105 110 Cys Gly Arg Lys Phe Ala Gln Trp Ser Thr Arg Lys Arg His Thr Lys 115 120 125 Ile His Thr Gly Glu 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> 65 <211> 307 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 65 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> 66 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 66 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 Gly Ser Gln Lys Pro Phe 65 70 75 80 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp Asn Leu Ser 85 90 95 Glu His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 100 105 110 Cys Gly Arg Lys Phe Ala Lys Arg Cys Asn Leu Arg Cys His Thr Lys 115 120 125 Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Arg Ile Cys Met Arg 130 135 140 Asn Phe Ser Arg Ser Asp Asn Leu Ser Glu His Ile Arg Thr His Thr 145 150 155 160 Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Lys 165 170 175 Arg Cys Asn Leu Arg Cys His Thr Lys Ile His Leu Arg Gln Lys Asp 180 185 190 Ala Ala Arg Gly Ser Gly Met Asp Ala Lys Ser Leu Thr Ala Trp Ser 195 200 205 Arg Thr Leu Val Thr Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu 210 215 220 Glu Trp Lys Leu Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val 225 230 235 240 Met Leu Glu Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr 245 250 255 Lys Pro Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu 260 265 270 Val Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala 275 280 285 Phe Glu Ile Lys Ser Ser Val 290 295 <210> 67 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 67 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 Ser Asp Leu Ser 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 Trp Ser Thr Arg Lys 115 120 125 Arg His Thr Lys Ile His Thr Gly Glu Lys Pro Phe Gln Cys Arg Ile 130 135 140 Cys Met Arg Lys Phe Ala Gln Ser Gly Asp Leu Thr Arg His Thr Lys 145 150 155 160 Ile His Leu Arg Gln Lys Asp Ala Ala Arg Gly Ser Gly Met Asp Ala 165 170 175 Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val Thr Phe Lys Asp Val 180 185 190 Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu Leu Asp Thr Ala Gln 195 200 205 Gln Ile Val Tyr Arg Asn Val Met Leu Glu Asn Tyr Lys Asn Leu Val 210 215 220 Ser Leu Gly Tyr Gln Leu Thr Lys Pro Asp Val Ile Leu Arg Leu Glu 225 230 235 240 Lys Gly Glu Glu Pro Trp Leu Val Glu Arg Glu Ile His Gln Glu Thr 245 250 255 His Pro Asp Ser Glu Thr Ala Phe Glu Ile Lys Ser Ser Val 260 265 270 <210> 68 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 68 cagcagcagc agcagcagca 20 <210> 69 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 69 Asp Thr Gly Leu Leu Asn Arg 1 5 <210> 70 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 70 Ser Ser Tyr Asn Leu Lys Thr 1 5 <210> 71 <211> 60 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (1)..(60) <223> This sequence may include 15-20 "CAG" repeats <400> 71 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 <210> 72 <211> 150 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 72 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag 150 <210> 73 <211> 240 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 73 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 240 <210> 74 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 74 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 240 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 300 cagcagcagc agcagcagca gcagcag 327 <210> 75 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 75 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 240 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 300 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 360 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 420 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 480 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 540 <210> 76 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic oligonucleotide <400> 76 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca g 51 <210> 77 <211> 129 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polynucleotide <400> 77 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcag 129 <210> 78 <211> 144 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 78 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcag 144 <210> 79 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 79 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcag 54 <210> 80 <211> 135 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 80 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcag 135 <210> 81 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 81 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcag 45 <210> 82 <211> 201 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 82 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 <210> 83 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 83 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cag 63 <210> 84 <211> 114 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 84 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcag 114 <210> 85 <211> 210 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 85 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca gcagcagcag 210 <210> 86 <211> 12 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 86 cagcagcagc ag 12 <210> 87 <211> 333 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 87 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 180 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 240 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 300 cagcagcagc agcagcagca gcagcagcag cag 333 <210> 88 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 88 Thr His Pro Arg Ala Pro Ile Pro Lys Pro Phe Gln 1 5 10 <210> 89 <211> 117 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <220> <221> misc_feature <222> (1)..(117) <223> The sequence may include 36-39 "CAG" repeats <400> 89 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag 117 <210> 90 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 90 gcagcagcag cagc 14 <210> 91 <211> 120 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 91 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 120 <210> 92 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic oligonucleotides <400> 92 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcag 57 <210> 93 <211> 84 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic polynucleotides <400> 93 cagcagcagc agcagcagca gcagcagcag cagcagcagc agcagcagca gcagcagcag 60 cagcagcagc agcagcagca gcag 84

Claims

1. A zinc finger protein transcription factor repressor (ZFP-TF repressor), comprising a repressor domain and a zinc finger protein (ZFP) that binds to Huntingtin protein (HTT), wherein the amino acid sequence of the ZFP-TF repressor is as follows: (a) MAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSQSSDLSRHIRTHTGEKPFACDICGRKFAQWSTRKRHTKIHTGSQKPFQCRICMRNFSQSSDLSRHIRTHTGEKPFACDICGRKFAQWSTRKRHTKIHTGEKP FQCRICMRKFAQSGDLTRHTKIHLRQKDAARGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO:64); (b) MAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSRSDNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHTHPRAPIPKPFQCRICMRNFSQSGDLTRHIRTHTGEKPFACDICGRKFAQSGDLTRHTKIHTPNPHRRTDPSHKPFQCRICM RNFSRSDNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHLRQKDAARGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO:65); (c) MAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSRSDNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHTGSQKPFQCRICMRNFSRSDNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHTGSQKPFQCRICMRNFSRS DNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHLRQKDAARGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO:66); or (d) MAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSRSDNLSEHIRTHTGEKPFACDICGRKFAKRCNLRCHTKIHTHPRAPIPKPFQCRICMRNFSQSSDLSRHIRTHTGEKPFACDICGRKFAQWSTRKRHTKIHTGE KPFQCRICMRKFAQSGDLTRHTKIHLRQKDAARGSGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO:67).

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

3. An mRNA comprising one or more polynucleotides according to claim 2.

4. An adeno-associated virus serotype 2 (AAV2) vector comprising one or more polynucleotides of claim 2.

5. A pharmaceutical composition comprising one or more ZFP-TF repressors according to claim 1, one or more polynucleotides according to claim 2, one or more mRNAs according to claim 3 and / or one or more AAV2 vectors according to claim 4.

6. A cell comprising one or more ZFP-TF repressors according to claim 1, one or more polynucleotides according to claim 2, one or more mRNAs according to claim 3, and / or one or more AAV2 vectors according to claim 4 and / or the pharmaceutical composition according to claim 5.

7. The cell according to claim 6, wherein The cells are neurons or fibroblasts.

8. Use of one or more ZFP-TF repressors according to claim 1, one or more polynucleotides according to claim 2, one or more mRNAs according to claim 3, one or more AAV2 vectors according to claim 4 and / or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating and / or preventing Huntington's disease (HD) in a subject in need thereof.

9. The use according to claim 8, by administering one or more of the ZFP-TF repressors, one or more of the polynucleotides, one or more of the mRNAs, one or more of the AAV2 vectors and / or the pharmaceutical composition to the striatum of the subject.

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