Therapeutic agents for treating polyQ disorders

Abstract

The present invention relates to therapeutic proteins (therapeutic proteins) for use in the treatment of polyQ diseases, mRNA encoding said therapeutic proteins, gene therapy vectors or viral particles, and compositions comprising them.

Description

[Technical field]

[0001] The present invention relates to therapeutic proteins (therapeutic proteins) for use in the treatment of polyQ diseases, mRNA encoding said therapeutic proteins, gene therapy vectors or viral particles, and compositions comprising them.

[0002] Polyglutamine diseases (polyQ diseases) are a group of neurodegenerative disorders caused by the expansion of cytosine-adenine-guanine (CAG) repeats that code for long polyQ tracts in the respective proteins. At least nine polyQ disorders have been described so far: six types of spinocerebellar ataxia (SCA) types 1, 2, 6, 7, and 17; Machado-Joseph disease (MJD / SCA3); Huntington's disease (HD); dentatorubral-pallidoluysian atrophy (DRPLA); and X-linked spinobulbar muscular atrophy type 1 (SMAX1 / SBMA).

[0003] Currently, there is no cure or prevention for these diseases, only symptomatic treatment for polyQ disorders. Long-term drug therapy has so far been disappointing, probably due to undesirable complications and reduced efficacy.

[0004] Among polyQ disorders, Huntington's disease (HD) is the most prevalent monogenic neurodegenerative disorder among Caucasians (prevalence approximately 7–11 per 100,000). It is inherited in an autosomal dominant manner, given that a single copy of the mutated HTT gene is sufficient to confer a pathological phenotype in both patients and experimental models.

[0005] The disease is caused by an abnormal expansion (>36) of CAG triplets in the huntingtin (HTT) gene, which results in the formation of mutant HTT proteins containing polyQ repeats. Wild-type HTT proteins contain 9-35 Q residues at the NH2-terminus and are involved in neural tube formation, resistance to apoptotic stimuli, and transcriptional regulation of BDNF and related genes. In fact, the expansion of the polyglutamine-encoding CAG trinucleotide repeat sequence confers toxic gain-of-function activity to mutant HTT, leading to abnormal accumulation of aggregation-prone proteins, increased sensitivity to glutamate toxicity, mitochondrial damage, and misregulation of the transcriptional program. However, it is still difficult to know which processes are the initial causative events and which are the consequence. Moreover, although the HTT protein is ubiquitously expressed, HD is characterized by specific damage of cell populations, loss of efferent medium spiny neurons in the striatum of the basal ganglia, and extensive degeneration of cortical structures.

[0006] Despite all the knowledge gained about the pathogenesis of HD, effective therapeutic interventions are still unavailable. Although animal models have shown promising results in improving cellular processes impaired in HD, not all clinical trials to date have demonstrated efficacy. Therefore, the art needs to find different therapeutic strategies to treat polyQ disorders. Summary of the Invention

[0007] The inventors focused on identifying factors, i.e. therapeutic proteins, that can reduce the toxicity of polyQ mutant proteins that cause polyQ diseases, i.e. counteract the cell death, oxidative stress and / or transcriptional changes caused by said mutant polyQ-expressing proteins.

[0008] The inventors have identified at least nine genes that, when expressed in vitro and / or in vivo (in animal models), via suitable gene therapy means such as suitable viral vectors (e.g. AAV vectors) or in the form of mRNA, significantly ameliorate the deleterious effects of mutant HTT genes, thereby providing new therapeutic strategies against polyQ-diseases. The genes code for polypeptide molecules, i.e. proteins, defined herein as "therapeutic proteins", that share the functional feature of reducing the cytotoxicity caused by one or more polyQ-mutated proteins that cause polyQ-diseases, when expressed in cells or animals expressing said polyQ-mutated proteins, thereby reducing the pathological effects of the mutant proteins that cause polyQ-diseases (see Table 1).

[0009] The present invention relates to one or more therapeutic proteins capable of reducing the toxicity of mutant proteins causing polyQ diseases, or nucleotide sequences encoding said therapeutic proteins, for use in the treatment of said diseases, preferably by gene therapy (herein intended gene delivery of said therapeutic proteins).

[0010] Thus, the present invention relates to one or more therapeutic proteins capable of reducing the toxicity of mutant proteins expressed by one or more of the ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP genes, wherein said proteins contain a pathological number of polyQ residues (as described in Table 1) for use in treating polyQ diseases, e.g. by gene therapy.

[0011] According to the present invention, polyQ diseases are, in the state of the art, DRPLA (Dentatorubral-Pallidoluysian Atrophy), HD (Huntington's Disease), SBMA (Spinobulbar Muscular Atrophy), SCA1 (Spinocerebellar Ataxia Type 1), SCA2 (Spinocerebellar Ataxia Type 2), SCA3 (Spinocerebellar Ataxia Type 3 or Machado-Joseph Disease), SCA6 (Spinocerebellar Ataxia Type 6), SCA7 (Spinocerebellar Ataxia Type 7), SCA12 (Spinocerebellar Ataxia Type 12) and SCA17 (Spinocerebellar Ataxia Type 17).

[0012] According to the present invention, gene therapy is a therapeutic strategy that involves the delivery of nucleic acids that encode factors capable of treating a disease by preventing or modifying the progression of said disease.

[0013] The objectives of the present invention are as follows: one or more therapeutic proteins or nucleotide sequences encoding said therapeutic proteins capable of reducing the toxicity of mutant proteins causing polyQ diseases, for use in the treatment of said diseases, preferably by gene therapy; a delivery system comprising a nucleotide sequence encoding said therapeutic protein, isoform or homologue as defined or claimed herein; an expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP composition comprising one or more cDNA or RNA sequences encoding a therapeutic protein capable of reducing the toxicity of a mutant protein causing polyQ disease; cDNA or mRNA molecules (free or complexed with one or more suitable carrier molecules) encoding therapeutic proteins capable of reducing the toxicity of mutant proteins causing polyQ disease; said delivery system, or expression vector, or cDNA molecule, or mRNA molecule (free or complexed with one or more suitable carrier molecules), for use in the treatment of polyQ disease; A method for treating a polyQ disorder comprising administering to a subject in need of treatment a therapeutically effective amount of: A delivery system, an expression vector comprising a cDNA or RNA sequence encoding a therapeutic protein capable of reducing the toxicity of a mutant protein causing polyQ disease as defined in any embodiment or claim herein, or A cDNA or mRNA molecule (free or in a suitable delivery system) encoding a therapeutic protein capable of reducing the toxicity of a mutant protein causing polyQ disease as defined in any embodiment or claim herein, or A therapeutic protein capable of reducing the toxicity of mutant proteins causing polyQ disease as defined in any of the embodiments and claims herein.

[0014] For all purposes of this invention, The mutant protein is expressed by one or more of the ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP genes and contains a pathological number of polyQ residues (as described in Table 1); The one or more Therapeutic proteins, isoforms or homologs thereof can reduce the toxicity of a mutant protein expressed by one or more of the ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP genes that contains a pathological number of polyQ residues (as described in Table 1); the one or more therapeutic proteins are selected from metal regulated transcription factor 1 (gene: MTF1-mtf1), lysine specific demethylase 5B (gene: KDM5B-Kdm5b), lysine specific demethylase 2B (gene: KDM2B-Kdm2b), F-box only protein 34 (gene: FBX034-Fbx034), essential MCU regulator, mitochondria (gene: SMDT1-Smdt1), ephrin type A receptor 4 (gene: EPHA4-Ephha4), transducin-like transcriptional enhancer protein 4 (gene: TLE4-Tle4), arfaptin 1 (gene: ARFIP1-Arfip1), and AT-rich interaction domain containing protein 5B (gene: ARID5B-Arid5B); Said isoforms or homologs retain the ability of the therapeutic protein of which it is an isoform or homolog as defined herein to reduce the toxicity of a given polyQ disease-causing mutant protein.

[0015] Gene therapy as described herein has its commonly recognized meaning in the art and thus refers to treatment in a subject in need of treatment by introduction of genetic material (e.g., replacing a mutant gene with a healthy copy, or inactivating an improperly functioning mutant gene, or introducing a new gene into the body, such as a gene encoding a therapeutic protein according to the present invention), i.e., therapeutic delivery of a nucleic acid to a patient's cells as a drug to treat a disease. Gene therapy according to the technology and the present invention can be accomplished by using mRNA molecules or non-viral or viral methods to introduce the genetic material of interest into a subject in need of treatment. Viral expression vectors commonly used in human gene therapy include retroviruses, adenoviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, and adeno-associated viruses. The viral vector genome either integrates into the host's genome or remains episomal.

[0016] The adeno-associated virus (AAV) vector described herein has the meaning generally recognized in the art. AAV belongs to the parvoviruses. It is a single-stranded non-enveloped DNA virus of 4.7 kb size that causes latent infection in human cells. Parvoviruses are an alternative to retroviruses associated with malignant tumors. Many naturally occurring AAV serotypes and variants have been isolated from various animal species, including mammals, birds and reptiles. Among these, common AAV serotypes include AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. In addition to these serotypes, there are many AAV variants and mutants that have been used for gene delivery via AAV vectors, including AAV-DJ, AAV-LK03, AAV-PHP.B, AAV-PHP.eB, AAV-Retro, and AAV2.7m8.

[0017] A typical AAV vector contains two ITR (inverted terminal repeat) regions that contain at least the following elements: a promoter, a gene of interest, and a terminator / polyadenylation signal.

[0018] The optimal serotype for brain gene therapy as described herein refers to an AAV serotype that is well known in the art to be effective and suitable for brain gene therapy, i.e., a serotype that exhibits strong neurotropism. Thus, according to the present specification, the optimal serotype is the serotype that exhibits the strongest tropism and infection efficiency depending on the brain cells to be treated.

[0019] A viral particle according to the present specification refers to a viral capsid that contains a nucleotide construct that leads to the expression of a molecule of interest when introduced into an appropriate cell. A viral particle according to the present invention includes a viral capsid that contains a gene construct, also known in the art as a "gene transfer vector", that is introduced into an infected cell.

[0020] An mRNA molecule according to the present invention is a therapeutically suitable mRNA molecule, ie, a molecule that comprises 3'UTR and 5'UTR elements flanking the coding sequence, a 5' cap and a polyA tail.

[0021] According to the present specification, the term polyglutamine disease, or polyQ disease, refers to a genetic disease caused by trinucleotide repeat expansion, which is a type of mutation in which the copy number of three nucleotide repeats (trinucleotide repeats) increases beyond a threshold that causes instability. PolyQ diseases are caused by CAG repeat expansions in the protein-coding portion of certain genes. In all cases, the expanded CAG repeats are translated into a continuous sequence of glutamine residues, forming polyQ strands, and the accumulation of polyQ proteins impairs important cellular functions.

[0022] Known polyQ diseases herein are shown in Table 1 and include DRPLA (dentatorubral-pallidoluysian atrophy), HD (Huntington's disease), SBMA (spinal-bulbar muscular atrophy), SCA1 (spinocerebellar ataxia type 1), SCA2 (spinocerebellar ataxia type 2), SCA3 (spinocerebellar ataxia type 3 or Machado-Joseph disease), SCA6 (spinocerebellar ataxia type 6), SCA7 (spinocerebellar ataxia type 7), SCA12 (spinocerebellar ataxia type 12), and SCA17 (spinocerebellar ataxia type 17).

[0023] A polyQ mutant protein according to the present specification refers to a mutant protein that contains an abnormally increased number of Q residues compared to a wild-type protein due to the expansion of the triplet coding for said amino acid in the gene encoding said protein, where said mutant protein is a pathogen and causes polyQ disease. The additional Q residues in a polyQ mutant protein result in a chain of Q residues that are clusters of additional Q residues, and their number is correspondingly increased compared to the wild-type protein. The average number of additional Q residues in a polyQ mutant protein is known in the art, and an example is provided in Table 1.

[0024] PolyQ mutant proteins according to the present invention are listed in Table 1.

[0025] As used herein, the phrase "capable of reducing toxicity" with respect to said Therapeutic protein or its isoform or homolog means that expression of a Therapeutic protein of the invention reduces cell death in a model animal of polyQ disease or a model cell of polyQ disease (i.e., a model animal or cell expressing a polyQ mutant protein that causes polyQ disease) compared to cell death observed in said model in the absence of expression of a Therapeutic protein of the invention, and / or reduces oxidative stress in said model compared to oxidative stress observed in said model in the absence of expression of a Therapeutic protein of the invention, and / or reduces transcriptional changes in said model compared to transcriptional changes observed in said model in the absence of expression of a Therapeutic protein of the invention.

[0026] According to the present specification, a therapeutic protein capable of reducing the toxicity of a mutant protein causing a given polyQ disease is a protein (i.e., a therapeutic protein) that exerts a therapeutic effect after being administered to a subject in need thereof or expressed in said subject through gene therapy.

[0027] According to this specification, expression that "is capable of reducing the toxicity of a mutant protein that causes a polyQ disease" means that expression of the therapeutic protein or nucleotide sequence "is capable of reducing the toxicity of the mutant protein that causes a polyQ disease when introduced into a mammalian cell that expresses the mutant protein that causes a polyQ disease or when administered to a patient suffering from a polyQ disease."

[0028] According to the present specification, the expression "toxicity of polyQ mutant proteins" includes cell death, oxidative stress and transcriptional changes caused by said mutant polyQ expressed proteins.

[0029] Pathogenic mutant proteins that cause polyQ diseases are proteins that have an increased number of Q residues in the form of polyQ sequences, as shown in Table 1.

[0030] Any reference herein to "use" in therapy also encompasses the use of one or more therapeutic proteins (including isoforms or homologues), constructs, vectors, mRNAs as defined herein for the preparation of a medicament for said therapy, wherein said one or more therapeutic proteins (including isoforms or homologues), constructs, vectors, mRNAs are formulated with one or more suitable excipients and / or carriers, as exemplified herein, into a medicament that can be administered to treat polyQ disease. [Brief description of the drawings]

[0031] Figure 1 – Establishment and characterization of ES cells expressing mutant HTT a. Experimental strategy to generate and characterize wild-type ES cells (Rex1GFP-d2) expressing the N-terminal fragment of mutant (128 CAG repeats) or wild-type (15 CAG repeats) HTT by DNA transfection and puromycin selection, named Q15 and Q128 cells, respectively. b. Western blot of HTT confirmed accurate production of 80 kDa and 65 kDa HTT proteins in Q128 and Q15 cells, respectively. The expression of Q128 HTT protein was lower compared to Q15 HTT. GAPDH was used as a loading control. c. Proliferation assays of Q128 (orange) and Q15 (blue) ES cells showed a marked impairment of cell proliferation by mutant HTT expression. Bars show the mean ± SEM of six independent experiments, as indicated by dots. p values ​​were calculated by two-way repeated measures ANOVA. d. Measurement of cell death by propidium iodide uptake and flow cytometry. Q128 cells (orange) had more cell death compared to Q15 cells (blue). The percentage of PI positive cells (dashed line) was calculated for each sample and the fold change was calculated relative to the Q15 sample. Bars indicate the mean ± SEM of four independent experiments indicated by dots. A representative flow cytometry plot is shown on the right. p values ​​were calculated by one-tailed one-sample Mann-Whitney U test. e. Measurement of the median fluorescence of 2',7'-dichlorofluorescein diacetate (DCFDA) as an assessment of reactive oxygen species generation in Q15 (blue) versus Q128 (orange) cells. Bars indicate the mean ± SEM of four independent experiments indicated by dots. Fold changes were calculated relative to Q15 samples. Representative flow cytometry profiles of ROS detection are shown in the right panel. p values ​​were calculated by one-tailed one-sample Mann-Whitney U test. f. Transcriptome analysis of Q128 and Q15 cells. Decreased (Log2 fold change <0.5, p-value <0.05) and increased (Log2 fold change >0.5, p-value <0.05) genes are shown in blue and red, respectively. Known genes previously associated with Huntington's disease are highlighted. The threshold used is indicated by a dashed line. p-values ​​were calculated by Wald test without adjustment. g. Gene list enrichment analysis of decreased (left panel, blue bars) and increased (right panel, red bars) genes revealed statistically significant enrichment (p-value = 0.05, indicated by black dashed line) of genes involved in metabolism (phosphorylation, glycolysis), proteasome (apoptosis, ubiquitin conjugation), and transcription in general. p-values ​​were calculated by Fisher's exact test using the DAVID database.

[0032] Figure 2 - Gain-of-function screen for suppressors of mutant HTT toxicity a. Top panel: Diagram of piggyBac vector pGG134 containing the MSCV 5'LTR followed by a splice donor site from exon 1 of the mouse Foxf2 gene, which activates genes adjacent to the integration site. The ITRs allow random integration at TTAA sites, and a DsRed-IRES-hygromycin cassette was used to identify cells in which the vector had stably integrated. Bottom panel: Schematic of the screening strategy used to identify novel proteins involved in HTT-dependent toxicity. Electroporation of pGG134 in Q128 ES cells generated thousands of independent mutants, each with a different hyperactivated gene. Resistant mutants were expanded and further characterized, allowing the identification of genes as novel suppressors of mutant HTT toxicity. b. Number of viable cells recorded by quantification of crystal violet staining positive colonies upon treatment with selected compounds for 48 h. Bafilomycin A and rotenone had no effect on Q128 cells at doses that affected Q15 cell viability, whereas MG132 and tamoxifen further decreased Q128 cell viability. Bars indicate the mean ± SEM of at least two independent experiments. Data were normalized to the Q15 vehicle sample. c. Left panel: Representative images of parental ES cells electroporated with PB vector encoding GFP (parental ES_GFP, top), Q128 line electroporated with PB_GFP (Q128_GFP, middle), and Q128 cells electroporated with pGG134 vector treated with MG132 for 5 days and stained with crystal violet (Q128_pGG134, bottom). Rare Q128_pGG134 cells survived even after MG132 treatment. Right panel: After mutagenesis and selection in the presence of MG132 (left) or tamoxifen (right), the intensity of the CV signal of surviving Q128_pGG134 colonies was significantly increased compared to Q128_GFP. Bars show the mean ± SEM of four independent experiments indicated by dots. Each experiment was normalized to the PB_GFP vector. p values ​​were calculated by two-tailed one-sample Mann-Whitney U test. d. Identification of the integration site of resistant clones. The gel electrophoresis in the left panel shows the Sp-PCR products from mutant clones MG15, MG16 and MG17. A single band corresponding to the amplification of the 5' end of the PB vector in MG15 (red dashed box) was excised from the gel, purified and sequenced. The resulting sequence (upper right panel) contains a portion of genomic DNA followed by a BstYI restriction site (GATC sequence) and an adapter sequence. The genomic sequence was then aligned to the mouse genome, which allowed identification of the precise site of integration in each mutant cell line. Here, the PB vector was found to be inserted upstream of the Kdm5b gene (lower panel). e. Expression analysis by qPCR of Synj2, Kdm5b, Mtf1, Fbxo34, and Arid1b genes confirmed increased expression of such candidate targets in the corresponding clones compared to both parental and Q128 cell lines. Bars indicate the mean ± SEM of three technical repeats indicated by dots. Expression was normalized to the highest value. f. Representative crystal violet staining images of five clones selected from the Q128 cell and Q128_pGG134 mutant populations (left panel). All clones were resistant to exogenous stress factors, but most of the Q128 cells died, while the parental ES cells survived. g. Intensity of crystal violet signal in all five clones analyzed after 48 h treatment with MG132 or tamoxifen (MG132 is shown, whereas tamoxifen is shown in Fig. 8c). Data were normalized to Q128_GFP1. Bars indicate the mean ± SEM of two independent experiments indicated by dots.

[0033] Figure 3 – Secondary validation of mutant HTT inhibitors a, Schematic diagram of the secondary validation experiment performed by stably expressing vectors containing the cDNA of the candidate genes under the control of the constitutive CAG promoter in Q128 cells. Empty vector (EV) and vector containing mCherry cDNA served as negative controls. b. Gene expression analysis by qPCR of Mtf1, Kdm2b, Kdm5b, and Fbxo34 confirmed increased gene levels in the corresponding overexpressed cell lines. Bars indicate mean ± SEM of three independent experiments (Mtf1, Fbxo34, Kdm2b) and points indicate mean ± SEM of two independent experiments (Kdm5b). Expression was normalized to the highest value. c. Western blot analysis showed that mutant HTT protein was present at similar levels in Q128 cells transfected with the different constructs. GAPDH was used as a loading control. An unharvested gel is provided in the source data file. d. Proliferation assay of the indicated cell lines. Bars show the mean ± SEM of three independent experiments, indicated by dots. p-values ​​were calculated by two-way repeated measures ANOVA comparing each candidate with the Q128_EV sample. e. Quantification of crystal violet showing the number of viable colonies in 128_Mtf1, Q128_Kdm5b and Q128_Fbxo34 cells after 48 h treatment with MG132 (left panel) or tamoxifen (right panel) compared to parental and Q128 cell lines. Mtf1 significantly improved cellular resistance to mutant HTT, whereas milder effects were observed with overexpression of the other candidates. Bars show the average of two technical repeats of a single experiment reported in the source data file (the same trend was observed in two additional experiments).

[0034] Figure 4 - Mtf1 regulates HD-related genes. a. Differentially expressed genes recovered by Mtf1. Among the 552 DEGs between Q15 and Q128 (Fig. 1f), 181 genes (pink) were recovered by Mtf1 overexpression (p-value >= 0.05 between Q128_Mtf1 and Q15_EV, log2FC >|0.5| between Q128_Mtf1 and Q128_EV). b. Heatmap showing the restorative effect of Mtf1 overexpression on 552 DEGs between Q128 and Q15 cells. Z-scores calculated based on row-scale expression values ​​(counts per million) are displayed for each cell line (Q15, Q128, and Q128_Mtf1). Red and blue colors indicate high and low expression, respectively. HD-associated genes Dpf1, Prnp, and Ube2cbp are highlighted. c. Transcriptome analysis of Q128_Mtf1 compared to Q128 cells. Decreased (Log2 fold change < 0.5, p-value < 0.05) and increased (Log2 fold change > 0.5, p-value < 0.05) genes are shown in blue and red, respectively. Mt1 and Mt2 are highlighted as the most significantly increased genes, along with Mtf1 itself. The threshold used is indicated by the dashed line. p-values ​​were calculated by Wald test without adjustment. d. Gene list enrichment analysis of the transcriptional response induced by Mtf1 overexpression in Q128 cells. Black dashed line corresponds to p-value = 0.05. p-values ​​were calculated by Fisher's exact test using the DAVID database. e. Proliferation assay of Q15 cells transfected with an empty vector (Q15_EV) or Mtf1 (Q15_Mtf1)-encoding plasmid. No significant changes in cell proliferation rate were observed upon Mtf1 overexpression. Bars indicate the mean ± SEM of three independent experiments, as indicated by dots. p values ​​were calculated by two-way repeated measures ANOVA. f. Measurement of cell death by propidium iodide uptake and flow cytometry. The staining revealed that Q128_Mtf1 (green) overexpression reduced cell death compared to Q128 cells (orange). The percentage of PI positive cells (dashed lines) was calculated for each sample and the fold change was calculated relative to the Q15 sample. Bars indicate the mean ± SEM of two independent experiments indicated by dots. Representative flow cytometry plots are shown on the right. g. Measurement of the median fluorescence value of 2',7'-dichlorofluorescein diacetate (DCFDA) as an assessment of reactive oxygen species generation. Overexpression of 128_Mtf1 (green) was able to reduce ROS generation in cells expressing mutant HTT. Bars show the mean ± SEM of two independent experiments, indicated by dots. Fold changes were calculated relative to the Q15 sample. Representative flow cytometry profiles of ROS detection are shown in the right panel. Source data are provided as source data files. h. Gene expression analysis by qPCR confirmed a strong increase in Mt1 and Mt2 genes in Q128_Mtf1 cells (green). Bars show the average of three independent experiments indicated by dots. Expression was normalized to the highest value.

[0035] Figure 5 – Mtf1 antagonizes mutant HTT effects in zebrafish. a. Exon 1 huntingtin (HTT) coding sequences containing 16 or 74 CAG repeats were cloned into the pCS2 plasmid and fused in-frame with the eGFP coding sequence. After in vitro transcription, Q16eGFP and Q74eGFP mRNA were injected into the yolk of single-cell stage embryos. At 24 hours post fertilization (hpf), embryos were collected for phenotypic and molecular analysis. b. Representative images of 24-h embryos injected with either Q16eGFP+mCherry, Q74eGFP+mCherry, or Q74eGFP+Mtf1 (250 pg / embryo + 250 pg / embryo) and stained with acridine orange. Top: Brightfield images show that embryo morphology was affected by Q74eGFP+mCherry mRNA injection. Bottom: Fluorescence microscopy shows that Q74eGFP+mCherry injection increased acridine orange-positive foci (white arrows). Images are anterior-up lateral views. c. Percentage of dead, malformed (severe or mild), and healthy embryos counted at 24 hpf in eight independent injection experiments (shown as dots). A total of 407, 511, and 621 embryos were analyzed for Q16+mCherry, Q74+mCherry, and Q74+Mtf1 samples, respectively. Box plots show the first, second, and third quartiles, with whiskers indicating minimum and maximum values. p values ​​were calculated by unpaired two-tailed Mann-Whitney U test. d. eGFP gene expression analysis by qPCR of zebrafish embryos microinjected with eGFP and Q74+Mtf1 mRNA. Bars indicate the mean ± SEM of four independent experiments indicated by dots. Expression was normalized to the highest value. e. Representative images of Tunel assays for 30-hour stage embryos injected with either Q16+mCherry, Q74+mCherry or Q74+Mtf1 from two independent experiments. For each embryo, multiple focal planes spanning the entire depth of the anterior structures were scanned and Z-projections were acquired in either brightfield and fluorescent channels. Embryos injected with Q16+mCherry showed some basal Tunel positivity due to the physiological apoptosis-dependent remodeling that occurs at this developmental stage. f. Quantification of the percentage of Tunel positive area relative to the total area (excluding yolk region). Each point represents an embryo (Q16+mCherry=15, Q74+mCherry=14, Q74+Mtf1=17). Box plots show the first, second and third quartiles, with whiskers showing the minimum and maximum values. The percentage of malformed and healthy embryos is shown in red and green, respectively. Healthy embryos are characterized by a reduced Tunel signal.

[0036] Figure 6 – AAV vector delivery of Mtf1 reduces motor deficits in R6 / 2 mice. a, Schematic diagram of the experimental strategy for motor behavior testing in HD mouse models injected with AAV loaded with Mtf1 or GFP. Tail vein injections were performed at 4 weeks of age. Motor behavior was assessed in the horizontal ladder task and rotarod test. b. Western blot of GFP confirmed viral expression in brain lysates of 4-week-old R6 / 2 mice injected with AAV-containing GFP via the tail vein, thus confirming that AAV can cross the blood-brain barrier. Mice injected with AAV-Mtf1 were used as negative control. Act B was used as loading control. c. Injection of AAV-Mtf1 improves motor function in R6 / 2 mice (red line). Motor performance assessed by rotarod test takes into account the latency to fall. R6 / 2 mice injected with AAV-GFP vector fall more rapidly than wild-type littermates or R6 / 2 mice injected with AAV-Mtf1. Arrows indicate the start time of treatment. Top: Line graphs show the mean + / - standard deviation of each experimental group at each time point. Bottom: Box plots of the indicated experimental groups at 11 weeks of age. Box plots show the first, second and third quartiles, whiskers show minimum and maximum values. For each group, the number of mice injected is indicated by the dots (WT AAV-GFP=8; WT AAV-Mtf1=9; R6 / 2 AAV-GFP=9; R6 / 2 AAV-Mtf1=10). p values ​​were calculated using unpaired two-tailed Mann-Whitney U tests with Bonferroni correction. d. Analysis of motor coordination in the horizontal ladder task. R6 / 2 mice injected with AAV-Mtf1 (red line) had lower sum error scores, especially in the later weeks when symptoms appear, compared with R6 / 2 mice injected with AAV-GFP vector (dashed black line). Arrows indicate the start time of treatment. Box plots show the first, second and third quartiles, with whiskers showing minimum and maximum values. For each group, the number of mice injected is indicated by dots (WT AAV-GFP=8; WT AAV-Mtf1=8; R6 / 2 AAV-GFP=9; R6 / 2 AAV-Mtf1=10). p-values ​​were calculated by unpaired two-tailed Mann-Whitney U test with Bonferroni correction. e. Mean body weights of R6 / 2 and WT mice after viral injection with either AAV-GFP or AAV-Mtf1. Box plots show the first, second and third quartiles, with whiskers showing minimum and maximum values. For each group, the number of mice injected is indicated by dots (WT AAV-GFP=8; WT AAV-Mtf1=9; R6 / 2 AAV-GFP=9; R6 / 2 AAV-Mtf1=10). p values ​​were calculated by unpaired two-tailed Mann-Whitney U test with Bonferroni correction. f. Efficient in vivo brain delivery of Mtf1 was confirmed in both brain cortex and striatal tissues by PCR of total DNA. Act B was used as a positive control for the PCR reaction.

[0037] Figure 7 – Establishment and characterization of ES cells expressing mutant HTT a. Gene expression analysis by qPCR of the HTT gene (left) and pluripotency markers Oct4, Sox2, Nanog (right panel) in Q15 (blue) and Q128 cells (orange) compared to the parental ES cell line (Rex1GFP-d2). Bars represent the mean ± SEM (standard error of the mean) of two (for Nanog), three (for HTT), or four (for Oct4 and Sox2) independent experiments indicated by dots. Results showed that expression levels of HTT mRNA were similarly elevated in both HD lines, contextually retaining pluripotency properties. Expression was normalized to the highest value. b. Gene expression analysis by qPCR of the HTT gene in wild-type mouse ES cells (E14TG2a) expressing either Q15 (blue) or Q128 (orange) constructs compared to the parental ES cell line. Bars indicate the mean ± SEM of three independent experiments indicated by dots. Results showed similarly elevated expression levels of HTT mRNA in both HD lines. Expression was normalized to the highest value. c. Western blot of HTT confirmed accurate production of 80 kDa and 65 kDa HTT proteins in E14_Q128 and E14_Q15 cells, respectively. GAPDH was used as a loading control. d. Proliferation assays of E14_Q128 (orange) and E14_Q15 (blue) ES cells showed a marked impairment of cell proliferation by mutant HTT expression. Bars show the mean ± SEM of three independent experiments indicated by dots. p values ​​were calculated by two-way repeated measures ANOVA. e. Measurement of cell death by propidium iodide uptake and flow cytometry. E14_Q128 cells (orange) have increased cell death compared to E14_Q15 (blue) cells. Bars represent the mean ± SEM of four independent experiments indicated by dots. Fold changes were calculated relative to the Q15 sample. p-values ​​were calculated with a one-tailed one-sample Mann-Whitney U test. Source data are provided as source data files. f. Measurement of 2',7'-dichlorofluorescein diacetate (DCFDA) fluorescence as an assessment of reactive oxygen species generation in E14_Q15 (blue) and E14_Q128 (orange) cells. Bars indicate the mean ± SEM of four independent experiments indicated by dots. Fold changes were calculated relative to the Q15 sample. p values ​​were calculated by one-tailed one-sample Mann-Whitney U test.

[0038] Figure 8 - Gain-of-function screen for suppressors of mutant HTT toxicity a, Number of viable parental ES cells after 48 h treatment with the indicated compounds as quantified by crystal violet staining. Bars represent the mean ± SEM of two independent experiments. Data were normalized to vehicle-treated cells. b. List of target genes identified by Sp-PCR and PCR band analysis of all collected MG or tamoxifen clones. Due to technical limitations such as multiple integration sites or failure to detect signals, it was not possible to identify the integration site for nine clones (out of 44 collected). c. Quantification of crystal violet showing the number of viable colonies in all five clones analyzed after 48 h of tamoxifen treatment. Data were normalized to the Q128 control. Bars represent the mean ± SEM of two independent experiments with dots indicated. d. qPCR analysis showed similar high expression levels of HTT mRNA in all clones and Q128 Htt ​​ES cells, confirming that Q128 mRNA was not silenced in the screening procedure. Bars show the mean ± SEM of three technical repeats indicated by dots. Expression was normalized to the highest value. e. Western blot confirmed that HTT protein was still present in all clones. GAPDH was used as a loading control. Values ​​shown under each clone are the average (three technical replicates) of HTT intensity normalized to GAPDH intensity.

[0039] Figure 9 – Network analysis of candidate suppressors of mutant HTT toxicity Gene list enrichment analysis of genes identified from the NGS screen revealed statistically significant enrichment for categories related to proteasomal degradation (e.g., ubiquitin conjugation, p-value=9.77E-04) or vesicular trafficking (e.g., acetylation, p-value=0.005) and transcriptional regulators (e.g., methylation, p-value=0.001). p-values ​​were calculated with Fisher's exact test using the DAVID database.

[0040] Figure 10 – Secondary validation of mutant HTT inhibitors a, qPCR analysis of HTT mRNA showed that HTT expression was not affected by co-expression of the candidate genes. Bars represent the mean ± SEM of three independent experiments indicated by dots. Expression was normalized to the highest value.

[0041] Figure 11 - Mtf1 regulates HD-related genes. a. Proliferation assay of E14 cells transfected with empty vector (E14_EV) or Mtf1-encoding plasmid (E14_Mtf1). No significant changes in cell proliferation rate were observed upon Mtf1 overexpression. Bars indicate the mean ± SEM of three independent experiments, as indicated by dots. p values ​​were calculated by two-way repeated measures ANOVA.

[0042] FIG. 12 – Mtf1 antagonizes mutant HTT effects in zebrafish. Representative images of 24-h embryos injected with Q74eGFP mRNA (250 pg / embryo, right panel) and observed under a fluorescent microscope compared to uninjected controls (left panel). The dashed lines indicate the region of interest, and the yolk (Y) shows autofluorescence. Embryos injected with Q74eGFP mRNA showed fluorescent signals throughout the entire embryo. b. Percentage of dead, malformed and healthy embryos after increasing doses of Q74eGFP mRNA ranging from 150 to 1000 pg / embryo at 24 hpf. Doses above 500 pg / embryo were highly toxic to embryos, resulting in the deaths of many fish, whereas doses below 500 pg / embryo were relatively well tolerated. The dose of 250 pg / embryo resulted in the lowest mortality and highest incidence of malformations, and was therefore selected for the following experiments. Each point represents an independent experiment.

[0043] Figure 13. Proliferation assay of Q128 cells transfected with empty vector (Q128_EV) or plasmids encoding the candidate therapeutic genes: Arid5b (Q128_Arid5b), Smdt1 (Q128_Smdt1), Epha4 (Q128_Epha4), Tle4 (Q128_Tle4), and Arfip1 (Q128_Arfip1). Cell proliferation was assessed by seeding 30,000 ES cells in 12-well plates in the presence of 6 μg / ml puromycin, and cells were counted every 24 hours for 4 days. Significant restoration of cell proliferation rate was observed upon overexpression of each candidate gene. Bars represent mean ± SEM of three independent experiments for Arid5b, Tle4 and Arfip1 and five independent experiments for Smdt1 and Epha4, indicated by dots. p-values ​​were calculated by two-way repeated measures ANOVA comparing each candidate with the Q128_EV sample. Detailed Description of the Invention

[0044] The inventors have been able to identify a number of therapeutic proteins that, when expressed recombinantly or introduced into cells that are model cells for polyQ disease or administered to model animals for polyQ disease (i.e. cells or animals expressing the mutant proteins that cause polyQ disease, as shown in Table 1), are able to reduce the toxicity of said mutant proteins that cause polyQ disease, thereby providing new tools for treating said disease.

[0045] The present invention therefore relates to therapeutic proteins capable of reducing the toxicity of mutant proteins causing polyQ diseases, for use in the treatment of said diseases, for example by protein therapy or gene therapy.

[0046] In other words, the present invention relates to therapeutic proteins, i.e. proteins whose expression or delivery in a cell / tissue / organism of a subject reduces the toxicity caused by polyQ mutant proteins that cause polyQ diseases, i.e. pathogenic polyQ mutant proteins that result from the expression of mutant genes with an abnormal increase in CAG triplets compared to the wild-type gene.

[0047] In one embodiment of the invention, the pathogenic mutant protein causing polyQ disease is expressed by one or more of the following mutant genes: ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, wherein the mutation is an abnormal expansion of a CAG triplet within a CAG repeat, and the mutant protein contains a pathological number of polyQ residues.

[0048] PolyQ disease is well known in the art. Table 1 below shows a list of genes that express mutant proteins that contain abnormal repeats of Q residues (also defined here as polyQ repeats) due to mutations consisting of CAG triplet expansions in CAG repeat regions. Said proteins cause so-called polyQ disease.

[0049] Table 1 also lists the range of typical numbers of polyQ residues within the Q-rich region in normal versus mutated pathogenic proteins. Table 1 JPEG2025508694000001.jpg197154JPEG2025508694000002.jpg81154

[0050] Therefore, according to the present invention, the polyQ disease is any of dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

[0051] In a preferred embodiment, the polyQ disorder causing protein is expressed by the HTT gene and the associated polyQ disorder is Huntington's disease, also known as Huntington's Korea.

[0052] According to the present invention, a therapeutic protein for use in the treatment of polyQ disease, capable of reducing the toxicity of a mutant protein causing polyQ disease, is either metal regulating transcription factor 1, lysine specific demethylase 5B, lysine specific demethylase 2B, F-box only protein 34, essential MCU regulator, mitochondrion, ephrin type A receptor 4, transducin-like transcriptional enhancer protein 4, alphaptin 1, AT-rich interaction domain containing protein 5B or an isoform thereof, or a homologue of a therapeutic protein having sequence homology defined as at least 55% coverage and at least 56% identity with one of said therapeutic proteins. Said isoforms and homologue proteins retain the ability of a therapeutic protein, being an isoform or homologue protein as defined herein, to reduce the toxicity of a given mutant protein causing polyQ disease.

[0053] Table 2 below summarizes the names of the human and mouse genes that code for the above-mentioned Therapeutic proteins and the names of the Therapeutic proteins encoded by each gene.

[0054] Each gene listed below is well characterized in the art, and both the gene and cDNA sequences are associated with NBCI gene IDs, and the encoded protein sequences are available in public protein databases. Thus, reference to the gene name, gene NCBI ID, and protein name provides the skilled artisan with all the necessary information regarding the nucleotide sequence encoding the therapeutic protein of interest of the present invention, as well as the respective amino acid sequence of said therapeutic protein. Table 2 JPEG2025508694000003.jpg212137JPEG2025508694000004.jpg54137

[0055] The therapeutic proteins identified by the inventors are suitable for use in the treatment of polyQ diseases.

[0056] Also encompassed by the present invention are isoforms of said therapeutic proteins, and the nucleotide sequences encoding same, for use in the treatment of polyQ diseases, which retain the ability to reduce the toxicity of the mutant proteins causing said diseases.

[0057] Examples of suitable isoforms according to the invention, designated with the annotation numbers NM, XM, or NR NCBI, are shown in Table 3 below. Table 3 JPEG2025508694000005.jpg207136

[0058] Also encompassed by the present invention are therapeutic protein homologues and their encoding nucleotide sequences that have sequence homology defined as at least 55% coverage and at least 49% identity with one of the above therapeutic proteins for use in treating polyQ diseases and that retain the ability to reduce the toxicity of the mutant protein that causes the disease.

[0059] In a preferred embodiment, a sequence homology defined as at least 88% coverage and at least 49% identity with one of said therapeutic proteins, or a sequence homology defined as at least 55% coverage and at least 58% identity with one of said therapeutic proteins, or a sequence homology defined as at least 95% coverage and at least 55% identity with one of said therapeutic proteins, or a sequence homology defined as at least 95% coverage and at least 64% identity with one of said therapeutic proteins, or a sequence homology defined as at least 97% coverage and at least 64% identity with one of said therapeutic proteins, for use in the treatment of polyQ diseases. or sequence homology defined as at least 99% coverage and at least 64% identity with one of said Therapeutic proteins, or sequence homology defined as at least 99% coverage and at least 70% identity with one of said Therapeutic proteins, or sequence homology defined as at least 99% coverage and at least 77% identity with one of said Therapeutic proteins, or sequence homology defined as at least 99% coverage and at least 79% identity with one of said Therapeutic proteins, and which retain the ability to reduce the toxicity of the disease-causing mutant protein, are also encompassed by the invention.

[0060] Examples of suitable homologues according to the invention are shown in Table 4 below: Table 4 JPEG2025508694000006.jpg89163TIFF2025508694000007.tif70165

[0061] In one embodiment, a therapeutic protein listed in Tables 2, 3 and 4, or an isoform or homologue thereof (or a combination thereof, preferably in the form of a recombinant protein) is administered via a suitable delivery means to a subject in need of treatment for a polyQ disease, where the polyQ disease is one of the diseases listed in Table 1.

[0062] In a preferred embodiment, a therapeutic protein listed in Table 2, or its isoforms or homologs listed in Tables 3 and 4 (or a combination thereof) is provided to a subject in need of treatment of a polyQ disease via protein delivery or via a delivery system of a nucleotide sequence that is expressed in the host, for example by gene therapy. In a preferred embodiment, said therapeutic protein (and / or its isoforms and / or homologs) is expressed by the host after administration of a suitable expression vector or delivery system or vector suitable for gene therapy, or an adeno-associated viral vector AAV or lentiviral vector, or a nanoparticle, or a LNP or a nucleotide construct. Such an embodiment is also generally defined herein as "gene therapy".

[0063] Gene therapy is well known in the art. Preferably, according to the present invention, gene therapy is performed by administering to a subject in need of treatment for polyQ disease a suitable expression vector or delivery system or vector suitable for gene therapy, or an adeno-associated viral vector AAV or lentiviral vector, or a nanoparticle, or an LNP, or a nucleotide sequence (e.g., an mRNA molecule) encoding a therapeutic protein of Table 2, and / or its isoforms as defined herein (e.g., Table 3), and / or its homologs as defined herein (e.g., Table 4), or a mixture thereof, to induce expression of the therapeutic protein of interest in the administered cell or host.

[0064] The present invention therefore also relates to an expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence encoding a therapeutic protein, its isoforms or homologues as defined above, preferably selected from the therapeutic proteins, isoforms or homologues of tables 2 to 4, to said vector or delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence for use in the treatment of a polyQ disease, such as the diseases of table 1, to a pharmaceutical composition comprising said vector or delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence and at least a pharma- ceutical acceptable carrier, and to a method for the treatment of a polyQ disease comprising administering a therapeutically effective amount of said vector or composition to a patient in need of treatment.

[0065] Expression vectors, particularly gene therapy vectors, are known in the art. Preferred vectors are those that, when administered, are capable of inducing the synthesis of said therapeutic protein, its isoforms or homologues in a subject affected by a polyQ disease, preferably in tissues affected by said disease.

[0066] In one embodiment of the invention, the construct used for the treatment of polyQ diseases according to the invention comprises a nucleotide sequence, e.g. a cDNA or RNA, encoding said therapeutic protein of interest, or an isoform or homologue thereof as defined above, preferably any of Tables 2 to 4, under the control of (i.e. operably linked to) a promoter (e.g. a constitutive promoter, an inducible promoter, a ubiquitous promoter, a tissue-specific promoter, preferably a constitutive promoter), thereby providing expression of the desired therapeutic protein.

[0067] Preferably, the construct comprising the nucleotide sequence, e.g., cDNA or RNA, will be designed to provide recombinant expression of a therapeutic protein of the same species to be treated. Thus, in a preferred embodiment, if the subject is a human, a cDNA of a human gene will be expressed, resulting in expression of a recombinant human therapeutic protein of interest, its isoform or homologue. In a preferred embodiment, the nucleotide sequence of the present invention may be designed to contain optimized codons.

[0068] In one embodiment of the invention, the therapeutic protein, its isoforms or homologs are recombinantly expressed by an AAV vector.

[0069] Therefore, the present invention also relates to a gene therapy expression adeno-associated virus vector, AAV, comprising a DNA sequence (cDNA) encoding a therapeutic protein, its isoform or homologue, capable of reducing the toxicity of mutant proteins causing polyQ diseases as defined above. Suitable examples are shown in Tables 2 to 4.

[0070] In one embodiment of the invention, the vector is an AAV vector.

[0071] For polyQ diseases that affect the CNS, AAV vectors can be selected that have the most suitable capsid serotype for CNS gene therapy.As known to those skilled in the art, for example, the most suitable capsid serotypes for CNS gene therapy are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP.B, AAV-PHP.eB, AAV-BR1, AAV-Retro, scAAV, AAVrh.8, AAVrh.10, AAV.miRNA AAV-DJ, AAV-LK03, and AAV2.7m8.

[0072] Protocols for producing AAV vectors are known in the art, and any of the known protocols can be used to practice the present invention.

[0073] In order to carry out gene therapy, the vector of the present invention must be delivered to target cells.

[0074] Therefore, the present invention also relates to a viral particle comprising a viral capsid and a nucleotide sequence encoding a therapeutic protein or an isoform or homologue thereof according to any of the above embodiments and claims.

[0075] In a preferred embodiment, said viral particle is an AAV particle, thus consisting of an AAV capsid and an adeno-associated viral vector (AAV) according to any of the above defined embodiments.

[0076] In one embodiment of the invention, the vector has a serotype optimal for CNS gene therapy, as described above.

[0077] For AAV vectors, several AAV capsid proteins are known in the art, and several AAV capsids can be designed depending on the target cells of therapy.

[0078] AAV is known to enter cells through interactions with carbohydrates present on the surface of target cells, typically sialic acid, galactose, and heparin sulfate, and it is known that differences in the sugar-binding preferences encoded in the capsid sequence can affect the cell type transduction preferences of various AAVs.

[0079] The preferential galactose binding of AAV9 is thought to confer the virus the ability to cross the blood-brain barrier (BBB) ​​and infect CNS cells, including primary neuronal cells.

[0080] According to the present invention, suitable AAV capsids are any of AAV-PHP, AAV9, AAV-BR1, AAV-Retro capsid, scAAV, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP.B, AAV-PHP.eB, AAV-BR1, AAV-Retro, scAAV, AAVrh.8, AAVrh.10, AAV.miRNA AAV-DJ, AAV-LK03, AAV2.7m8.

[0081] In one embodiment of the invention, the AAV-PHP capsid is AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, or AAV-PHP.A. The AAV-PHP capsid protein (and related capsids) are well-known engineered AAV capsid proteins made based on the AAV serotype 9 capsid.

[0082] According to the invention, the nucleotide sequence packaged in said viral particles encodes a therapeutic protein, its isoforms or homologues as defined above and in the claims, for the treatment of polyQ diseases as already defined in Table 1. Non-limiting examples of said therapeutic proteins, their isoforms or homologues are given in Tables 2-4.

[0083] According to the invention, the therapeutic protein, its isoform or homologue, when expressed by gene therapy, is capable of reducing the toxicity of a mutant pathogenic protein expressed by one or more of the ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP genes, wherein the pathogenic protein contains a pathological number of polyQ residues.

[0084] According to the invention, the therapeutic protein, its isoform or homologue is in any of Tables 2, 3 or 4.

[0085] The object of the present invention is an expression vector, a delivery system or a vector suitable for said gene therapy or an adeno-associated viral vector AAV or lentiviral vector, a nanoparticle, LNP, a nucleotide sequence for use in the gene therapy treatment of polyQ diseases, preferably a gene therapy expression vector, more preferably a gene therapy expression adeno-associated viral vector, AAV or viral particle as defined herein and in the claims.

[0086] Therefore, according to the present invention, the polyQ disease is selected from dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

[0087] In a preferred embodiment, the disease is Huntington's disease and the therapeutic protein, its isoform or homologue reduces the toxicity of a pathogenic protein produced by a mutant HHT gene as shown in Table 1.

[0088] The present invention also encompasses a method for treating said polyQ disease, preferably Huntington's disease, comprising administering to a subject in need thereof a therapeutically effective amount of a vector or viral particle according to any of the embodiments and claims described herein.

[0089] All definitions and examples of gene therapy methods / use provided for the gene therapy vectors of the invention apply mutatis mutandis to the viral particles of the invention.

[0090] The present invention also relates to nucleotide sequences, such as cDNA or mRNA molecules, encoding therapeutic proteins, their isoforms or homologues, as defined herein, which are capable of reducing the toxicity of mutant proteins causing polyQ diseases, and which are optionally complexed to one or more carrier molecules (Table 1), preferably therapeutic proteins, their isoforms or homologues as disclosed in Tables 2-4.

[0091] Preferably, the mRNA comprises 3'UTR and 5'UTR elements flanking the coding sequence, a 5' cap and a polyA tail. 5' and 3' mRNA untranslated regions (UTRs) are well known in molecular genetics. 5'UTRs are sequences that contain the Kozak consensus sequence that is recognized by ribosomes and allows the ribosomes to bind to the mRNA molecule and initiate its translation. 3'UTR regions are found after the stop codon and play a role in translation termination and post-transcriptional modification. UTRs are well known to those skilled in the art as well as their role in enhancing protein production in non-viral gene therapy. Those skilled in the art can easily select suitable 5' and 3' sequences for the mRNA construct of the present invention from among the UTRs commonly used in non-viral gene therapy.

[0092] The mRNA construct may also include a 5' cap and a polyA tail, both of which stabilize the mRNA molecule and enhance protein translation.

[0093] 5' caps, of various versions known in the art, can be added during or after the transcription reaction, using vaccinia virus capping enzyme, or by incorporating a synthetic cap or an anti-reverse cap analog.

[0094] The mRNA molecules of the present invention can also include optimized codons, which are selected to match the more abundant tRNAs of the organism to which the mRNA is administered. In a preferred embodiment, the codons can be optimized for humans.

[0095] Further, according to the present invention, the mRNA molecule, in any of the embodiments disclosed herein, can include one or more modified nucleosides to enhance its stability, safety, and / or its translation. Suitable examples of modified nucleosides include, but are not limited to, pseudouridine and 1-methylpseudouridine.

[0096] In yet another embodiment, which applies to all embodiments relating to the mRNA molecules of the invention, said molecules may be naked or may be complexed with one or more carrier molecules, e.g. in the form of cationic nanoemulsions, nanoparticles, liposomes, cationic polymer liposomes, polysaccharide particles, cationic lipid nanoparticles, cationic lipid cholesterol nanoparticles, or cationic lipid cholesterol PEG nanoparticles, according to common knowledge in the art.

[0097] Common delivery methods and carrier molecules for therapeutic mRNA molecules include naked mRNA (part a); naked mRNA by in vivo electroporation; protamine (cationic peptide) complexed mRNA; mRNA conjugated to positively charged cationic oil-in-water nanoemulsions; mRNA conjugated to chemically modified dendrimers and complexed with polyethylene glycol (PEG) lipids; protamine complexed mRNA in PEG lipid nanoparticles; mRNA conjugated to cationic polymers such as polyethyleneimine (PEI); mRNA complexed with cationic polymers such as PEI and lipid components; mRNA complexed with polysaccharide (such as chitosan) particles or gels; mRNA in cationic lipid nanoparticles (e.g. 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA complexed with cationic lipids and cholesterol (part k); mRNA complexed with cationic lipids, cholesterol and PEG lipids.

[0098] The cationic peptide protamine has been shown to protect mRNA from degradation by serum RNases.

[0099] According to a preferred embodiment of the invention, the mRNA molecule codes for one or more therapeutic proteins selected from metal regulated transcription factor 1, lysine specific demethylase 5B, lysine specific demethylase 2B, F box only protein 34, essential MCU regulator, mitochondria, ephrin type A receptor 4, transducin-like transcriptional coactivator protein 4, alphaptin 1, AT-rich interaction domain containing protein 5B (Table 2), or isoforms or homologs thereof as previously defined (e.g. Tables 3 and 4).

[0100] Further, according to one embodiment of the present invention, the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17 (see Table 1).

[0101] In a preferred embodiment of the invention, the mRNA molecule according to any of the above embodiments is advantageously used for gene therapy of a polyQ disease as defined in Table 1. In a preferred embodiment, said polyQ disease is Huntington's disease.

[0102] The present invention also relates to a method for the treatment of said diseases, said method comprising administering to a subject in need of treatment a therapeutically effective amount of an mRNA molecule as defined above.

[0103] Another object of the invention is a pharmaceutical composition comprising or consisting of a therapeutic protein, its isoforms or homologues, or an expression vector or delivery system or a vector suitable for gene therapy as defined above or in the claims, or an adeno-associated viral vector AAV or lentiviral vector as defined above or in the claims, or a nanoparticle, or an LNP or a nucleotide sequence, or a viral particle as defined above or in the claims, or a cDNA or mRNA as defined above or in the claims, and a pharma- ceutically acceptable carrier. When using mRNA, said molecule can be formulated with a gene transfer reagent. Suitable gene transfer reagents are commercially available. Said composition is suitable for systemic injection (including intravenous), central nervous system delivery, or aerosol / nasal delivery. By way of example, said injection can be intravenous injection, intraparenchymal administration into specific areas of the brain such as the ventricles, cisterns, lumbar spine, etc., or intrathecal administration, or intra-arterial injection (carotid artery injection may be used for administration), or direct administration into the cerebrospinal fluid.

[0104] Pharmaceutical carriers suitable for the above administration are well known to those skilled in the art, and for example, physiological saline can be used.

[0105] For medical use, all products described herein are rendered sterile.

[0106] Thus, a further embodiment of the invention is a pharmaceutical composition and a pharma- ceutically acceptable carrier as defined herein for use in the treatment of polyQ disease.

[0107] According to the present invention, the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

[0108] In a preferred embodiment, the polyQ disease is Huntington's disease.

[0109] Therefore, a further object of the present invention is a medical treatment comprising the administration of a therapeutic protein as defined above or in the claims, or an isoform or homologue thereof, or an expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector as defined above or in the claims, or a nanoparticle, or a LNP or a nucleotide sequence, or a viral particle as defined above or in the claims, or a cDNA or mRNA as defined above or in the claims, or a pharmaceutical composition of the invention, to a patient in need thereof, in a therapeutically effective amount.

[0110] The invention also relates to the use of a therapeutic protein as defined above or in the claims, or an isoform or homologue thereof, or an expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle or a LNP as defined above or in the claims, or a nucleotide sequence, or a viral particle as defined above or in the claims, or a cDNA or mRNA as defined above or in the claims, for the manufacture of a medicament for the treatment of a polyQ-disease of table 1, in particular Huntington's disease.

[0111] In said embodiment, the therapeutic protein, its isoforms or homologues as defined above or in the claims, or the expression vector or delivery system or vector suitable for gene therapy, or the adeno-associated viral vector AAV or lentiviral vector as defined above or in the claims, or the nanoparticle, or the LNP or the nucleotide sequence, or the viral particle as defined above or in the claims, or the cDNA or mRNA as defined above or in the claims, is formulated with one or more suitable pharmaceutical carriers and / or excipients selected from those commonly used by the skilled artisan for the desired formulation for the treatment of polyQ diseases of table 1 (see pharmaceutical compositions above).

[0112] Therefore, the present invention can be summarized in the following items. 1. A Therapeutic protein, or an isoform or homolog thereof, said homolog having sequence homology defined as at least 55% coverage and at least 49% identity with said Therapeutic protein, said Therapeutic protein, its isoform or homolog thereof, capable of reducing the toxicity of mutant proteins causing polyQ disease and used in the treatment of said disease. 2. The therapeutic protein, its isoform or homologue used according to item 1, wherein said mutant protein is expressed by one or more of the following genes: ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, and said mutant protein comprises a pathological number of polyQ residues. 3. The therapeutic protein used according to item 1 or 2, wherein said therapeutic protein is selected from metal regulated transcription factor 1, lysine specific demethylase 5B, lysine specific demethylase 2B, F-box only protein 34, essential MCU regulator, mitochondria, ephrin type A receptor 4, transducin-like activator protein 4, alphaptin 1, AT-rich interaction domain containing protein 5B. 4. The therapeutic protein isoform used according to item 1 or 2, wherein said isoform is selected from Table 3 below. JPEG2025508694000008.jpg1791325. A therapeutic protein homolog for use according to item 1 or 2, wherein said homolog is selected from table 4 below. JPEG2025508694000009.jpg1981326. The therapeutic protein, its isoform or homologue for use according to any one of items 1 to 5, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17. 7. The therapeutic protein, its isoform or homologue used according to item 6, wherein the disease is Huntington's disease. 8. The therapeutic protein, its isoform or homologue used according to any one of items 1 to 5, wherein said therapeutic protein, its isoform or homologue is expressed by an expression vector or delivery system, or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP, or a nucleotide sequence. 9. An expression vector or delivery system or vector suitable for gene therapy, or adeno-associated viral vector AAV or lentiviral vector, or nanoparticle, or LNP, comprising a nucleotide sequence encoding a therapeutic protein, its isoform or homologue, wherein said isoform or homologue has sequence homology defined as at least 55% coverage and at least 49% identity with said therapeutic protein, wherein said therapeutic protein, its isoform or homologue is capable of reducing the toxicity of a mutant protein causing polyQ disease. 10. The expression vector or delivery system or vector suitable for gene therapy, or an adeno-associated viral vector AAV or lentiviral vector, or a nanoparticle, or a LNP, according to item 9, wherein the mutant protein is expressed by one or more of the following genes: ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, and the protein comprises a pathological number of polyQ residues. 11. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP, according to item 9 or 10, wherein the nucleotide sequence encodes a therapeutic protein as defined in items 3 to 5, or an isoform or a homologue thereof. 12. The vector according to any one of items 9 to 11, wherein the vector is an AAV viral vector having a capsid serotype optimal for CNS gene therapy. 13. The viral vector AAV according to item 12, wherein the capsid serotype is any one of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP.B, AAV-PHP.eB, AAV-BR1, AAV-Retro, scAAV, AAVrh.8, AAVrh.10, AAV.miRNA AAV-DJ, AAV-LK03, and AAV2.7m8. 14. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence, as defined in any one of items 9 to 13, for use in the treatment of a polyQ disease. 15. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence for use according to item 14, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17. 16. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence for use according to item 15, wherein the polyQ disease is Huntington's disease. 17. A viral particle comprising a viral capsid and a nucleotide sequence encoding a therapeutic protein or an isoform or homologue thereof as defined in any one of items 1 to 5. 18. The viral particle according to item 17, wherein the capsid is an AAV capsid having a serotype optimal for CNS gene therapy. 19. The viral particle according to item 18, wherein the AAV capsid is any one of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-PHP.B, AAV-PHP.eB, AAV-BR1, AAV-Retro, scAAV, AAVrh.8, AAVrh.10, AAV.miRNA AAV-DJ, AAV-LK03, and AAV2.7m8. 20. The viral particle described in item 19, wherein the AAV-PHP capsid is AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, or AAV-PHP.A. 21. The viral particle according to any one of items 17 to 20, for use in gene therapy for a polyQ disease. 22. Viral particles for use according to item 21, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17. 23. The viral particle for use according to item 22, wherein the polyQ disease is Huntington's disease. 24. An mRNA encoding a therapeutic protein, its isoform or homologue, said isoform or homologue having sequence homology defined as at least 55% coverage and at least 49% identity with said therapeutic protein, said therapeutic protein, its isoform or homologue being capable of reducing the toxicity of a mutant protein causing a polyQ disease. 25. The mRNA of item 24, wherein the mutant protein is expressed by one or more of the following genes: ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, and the protein contains a pathological number of polyQ residues. 26. The mRNA according to item 24 or 25, which encodes a therapeutic protein as defined in items 3 to 5, or an isoform or homolog thereof. 27. The mRNA according to any one of items 24 to 26, wherein the mRNA comprises 3'UTR and 5'UTR elements flanking the coding sequence, a 5' cap and a polyA tail. 28. The mRNA according to any one of items 24 to 27, wherein the mRNA comprises one or more modified nucleosides. 29. The mRNA according to any one of items 24 to 28, wherein the mRNA is complexed to one or more carrier molecules. 30. The mRNA of any one of claims 24 to 29, wherein the mRNA is complexed in the form of a cationic nanoemulsion, nanoparticle, liposome, cationic polymer liposome, polysaccharide particle, cationic lipid nanoparticle, cationic lipid cholesterol nanoparticle, or cationic lipid cholesterol PEG nanoparticle. 31. The mRNA according to any one of items 24 to 30, for use in treating a polyQ disease. 32. The mRNA for use according to item 31, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12 or spinocerebellar ataxia type 17. 33. The mRNA for use according to item 32, wherein the polyQ disease is Huntington's disease. 34. A pharmaceutical composition comprising a therapeutic protein as defined in items 1 to 5, or an isoform or a homologue thereof, or an expression vector or a delivery system or a vector suitable for gene therapy according to any one of items 8 to 13, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or an LNP or a nucleotide sequence, or a viral particle according to any one of items 17 to 20, or an mRNA according to any one of items 24 to 30, and a pharma- ceutically acceptable carrier. 35. The pharmaceutical composition according to item 34, for use in the treatment of polyQ diseases. 36. The pharmaceutical composition for use according to item 35, wherein said polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17. 37. The pharmaceutical composition for use according to item 36, wherein the polyQ disease is Huntington's disease. 39. The pharmaceutical composition according to item 35, for administration by systemic injection, central nervous system delivery, or aerosol / nasal delivery. 40. The pharmaceutical composition according to item 39, for intravenous injection, for intraparenchymal administration to specific areas of the brain such as the ventricles, cisterns, lumbar vertebrae, etc., or for intrathecal administration, or for intra-arterial injection, or for direct administration into the cerebrospinal fluid. Detailed description of the sequence

[0113] Table 5 JPEG2025508694000010.jpg86160TIFF2025508694000011.tif58160

[0114] The following examples demonstrate the therapeutic effects in cell and / or animal models of the therapeutic proteins identified by the inventors and listed in Table 2. These examples are provided to fully illustrate one method for practicing the invention and the results observed in vivo.

[0115] All animal experiments were performed in accordance with Italian and European Community legislation on the use of laboratory animals and were approved by the Bioethical Committee of the University of Bicocca.

[0116] Embryonic stem (ES) cells, designated herein as “Rex1GFP-d2” and “E14TG2a,” were obtained from mouse blastocyst embryos and were a gift from the Austin Smith laboratory at the University of Exeter, UK. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0117] Establishment and characterization of ES cells expressing mutant HTT Embryonic stem (ES) cells, designated herein as “Rex1GFP-d2” and “E14TG2a,” were obtained from mouse blastocyst embryos and were a gift from the Austin Smith laboratory at the University of Exeter, UK.

[0118] ES cells (Figures 1a and 7a) stably expressing either mutant HTT (containing 128 CAG repeats) or wild-type HTT (containing 15 CAG repeats), named Q15 and Q128 cells, respectively, were generated. Expression of mutant HTT did not change the expression of pluripotency markers (Figure 7a, right). Translation of mutant and wild-type Q128 and Q15 HTT proteins in cells was confirmed by Western blot (Figure 1b). Cytotoxicity induced by expression of Q128 HTT was verified. The cell numbers obtained in 4 days of culture were measured, and it was observed that Q128 cells were significantly reduced compared to Q15 cells (Figure 1c). Conversely, Q15 cells proliferated robustly. Similar results were obtained with independent parental ES cell lines (Figures 7b-d). Furthermore, Q128 cells showed increased cell death (Fig. 1d and Fig. 7e) and increased production of reactive oxygen species (ROS, Fig. 1e and Fig. 7f), as previously reported in the art. Collectively, these data demonstrate that expression of Q128 HTT, but not Q15 HTT, reduced ES cell viability.

[0119] To further validate the model of Q128 HTT-expressing ES cells, we performed transcriptome analysis and identified differentially expressed genes (DEGs) in Q128 and Q15 cells (Fig. 1f). Gene list enrichment analysis (Fig. 1g) identified misregulation of genes related to processes involved in HD pathogenesis, such as metabolism, transcriptional regulation, and post-translational modifications, such as ubiquitination, methylation, acetylation, and metal homeostasis. Furthermore, among the DEGs, we found several genes that have been previously associated with Huntington's disease as biomarkers or because of their altered expression in HD models, such as Dpf1, Med29, Stub1, Polr3b, Psmc1, Setdb1, Ube2cbp, Pqbp1, and Prnp (Fig. 1f). Thus, Q128 cells recapitulate molecular features associated with HD.

[0120] Gain-of-function screening for suppressors of mutant HTT toxicity With the aim of performing a genetic screen, we explored experimental conditions that would exacerbate the toxic effects of mutant HTT to facilitate the isolation of fully resistant mutants. We envisioned that treating Q128 cells with various cellular stressors would induce cell death and identify genetic mutants that are resistant to the toxic effects of mutant HTT (Figure 2a). We explored small molecules and inhibitors that were previously shown to exacerbate the effects of mutant HTT acting on biological processes implicated in HD, such as autophagy, the proteasomal degradation system, and mitochondrial metabolism, and selected a panel of inhibitors and titrated them to find a dose that was non-lethal to parental ES cells (Figure 8a). We then treated Q128 cells with the inhibitors and observed that MG132 and tamoxifen further reduced the viability of Q128 cells (Figure 2b). Thus, we selected these as stressors that selectively induce cell death in Q128 cells to be used for the genetic screen (Figure 2a). To better identify candidate genes that act specifically on mutant HTT, rather than the stressors themselves, we used two independent stressors that affect unrelated biological processes.

[0121] A gain-of-function approach was chosen for the screen. This has the advantage that candidates are selected from all genes present in the complete genome of ES cells, and not only from genes expressed in ES cells or in response to mutant HTT, as in the case of loss-of-function approaches, thus broadening the discovery possibilities. To generate the gene mutants, we used PB vectors based on transposons, DNA elements stably integrated into the genome. Electroporation of the PB vector in the presence of the transposase leads to random integration into TTAA sites abundant in the genome. For the gain-of-function screen in ES cells, the PB vector used (pGG134, Figure 2a) was optimized and consists of a murine stem cell virus (MSCV) transcription enhancer / transcription start sequence followed by a splice donor site, which allows hyperactivation of endogenous genes adjacent to the integration site. Q128 cells were electroporated with pGG134 vector or a PB vector encoding GFP (PB_GFP) as a control. After 5 days of treatment with exogenous stressors, surviving colonies were counted. As a control, parental ES cells expressing GFP proliferated robustly in the presence of stress factors, whereas Q128 cells expressing GFP barely survived MG132 or tamoxifen treatment (Fig. 2c). In contrast, Q128 cells electroporated with pGG134 significantly formed resistant colonies in the presence of both stress factors (Fig. 2c), indicating successful generation of mutants resistant to Q128 HTT. A total of 44 mutant clonal lines that emerged from the four mutagenesis experiments were individually picked and grown in the presence of the stress factors MG132 or tamoxifen, respectively, according to their induction, and were named MG♯ or T♯. To identify which genes were activated in individual clones, we performed splinterlet-PCR (Sp-PCR), which allows the amplification of genomic regions flanking the integration site. For 35 clones, one or two major bands (see Fig. 2d, MG15) corresponding to the genomic regions at the 5' and 3' ends of the PB vector were obtained. The Sp-PCR bands were then excised and sequenced.Sequence alignment against the reference genome allowed us to identify the precise integration site and orientation in each mutant cell line. In the example shown in Figure 2d, it was confirmed that the pGG134 was inserted upstream of the Kdm5b gene in the correct orientation to allow its activation (Figure 2d, lower panel). 23 genes flanking pGG134 (a complete list of integration sites is shown in Figure 8b) were identified that represent genes, or "suppressors," that may confer resistance to mutant HTT.

[0122] When the PB vector is integrated near a specific gene, overexpression of that gene occurs.

[0123] qPCR confirmed that endogenous Kdm5b transcripts appeared to be increased in MG15 clones compared to Q128 cells electroporated with PB_GFP or parental ES cells (Fig. 2e). Integration sites were clearly identified and characterized for four additional mutant clones corresponding to Fbxo34 genes (MG21 clone), Mtf1 (MG18), Synj2 (MG9), and Arid1b (T4). In each clonal line, an increase in the cognate gene was confirmed compared to the control (Fig. 2e). To validate the ability of the screening method to select mutants resistant to Q128-induced toxicity, five selected clones were exposed individually to both exogenous stressors. All clones survived comparably to parental ES cells, whereas Q128 cells electroporated with PB_GFP vector showed a very reduced viability (Fig. 2f and Fig. 8c). The clonal lines were resistant to both stressors. This suggests that the mutagenesis method activated genes that conferred resistance to mutant HTT, but not to MG132 or tamoxifen per se. Because it is possible that the clones acquired resistance to mutant HTT simply by silencing the Q128 HTT transgene, we monitored both HTT mRNA (Fig. 8d) and protein (Fig. 8e), which were robustly detected in all clones. Thus, the screening procedure identified five genes as bona fide suppressors of mutant HTT-induced toxicity.

[0124] Secondary validation of mutant HTT suppressors Next, to confirm that the identified candidates (therapeutic proteins) were able to confer resistance to mutant HTT, vectors containing the cDNAs of the candidate genes under the control of constitutive promoters were introduced into Q128 cells (Figure 3a). For such validation experiments, we selected Mtf1, Kdm5b and Fbxo34, which were identified in mutant clones, and Kdm2b, which was identified multiple times in genome-wide screening (Figure 3b). First, we confirmed whether the expression of Mtf1, Kdm2b, Kdm5b and Fbxo34 was indeed increased in cells expressing each candidate (named Q128_Mtf1, Q128_Kdm2b, Q128_Kdm5b and Q128_Fbxo cells, respectively). We observed that in all the generated cell lines, the expression levels of the candidate genes were higher compared to the relevant controls (Figure 3b). Meanwhile, the HTT Q128 levels were unchanged (Figure 3c and Figure 10a). Finally, we checked whether the proliferation of cells expressing Q128 was affected by the co-expression of selected therapeutic proteins. While expression of Fbxo34 or Kdm5b had only a modest effect, increasing the levels of Mtf1 and Kdm2b increased the proliferation of Q128 cells (Fig. 3d). The cells were then exposed to the stressors MG132 or tamoxifen, and among all the candidates, Mtf1 stood out for its ability to confer resistance to mutant HTT in the presence of both stressors (Fig. 3e). We concluded that expression of Mtf1 consistently conferred resistance to mutant HTT, corroborating the phenotype observed in clones in which this gene was increased. For this reason, we selected it for further molecular characterization and in vivo testing.

[0125] Mtf1 regulates HD-related genes. MTF1 is a transcription factor that functions as a sensor for various stress conditions in cells. Upon accumulation of metals (e.g., cadmium, zinc, or iron) or upon hypoxia or oxidative stress, MTF1 translocates into the nucleus and activates the transcription of a series of genes, including transporters of metals and endogenous metal chelators called metallothioneins (MTs).

[0126] Given that MTF1 is a transcription factor, RNaseq was performed to identify the transcriptional programs controlled by Mtf1 that confer protection against mutant HTT. First, we asked whether DEGs controlled by mutant HTT (Fig. 1f) were affected by Mtf1, and observed that 32.78% (181 out of 552) were significantly restored (Fig. 4a). Of the 258 genes increased by Q128, 66 were restored, including interestingly the HD-associated genes DPF1 and Ube2cbp (Fig. 4b). Similarly, of the 294 genes decreased by Q128, 115 were restored by Mtf1, including the HD-associated gene Prnp.

[0127] We next analyzed the global transcriptional response induced by Mtf1 in Q128 cells and found that Mt1 and Mt2 were the most significantly increased genes along with Mtf1 itself (Fig. 4c). Gene enrichment analysis revealed the regulation of genes related to metal homeostasis, such as zinc transporters Slc30a1 and Slc30a2 and regulation of ROS (Fig. 4d). Importantly, Mtf1 did not induce any oncogenic signatures, and known regulators of cell proliferation or apoptosis were not affected. Indeed, no significant effect on cell proliferation rate was observed upon Mtf1 overexpression in parental ES cells or Q15 cells (Fig. 11a and Fig. 4e). We concluded that Mtf1 controls the expression and ROS regulation of genes related to HD and genes involved in metal homeostasis in Q128 cells. Given the increased cell death and ROS production in Q128 cells (Fig. 1d and Fig. 1e), we assessed whether Mtf1 overexpression could prevent these processes. Compared with Q128 cells, reduced cell death (Fig. 4f) and lower ROS production (Fig. 4g) were detected in Q128_Mtf1.

[0128] We next assessed whether mutant HTT affected Mtf1 expression or activity. No changes in Mtf1 were observed in Q128 cells (Fig. 3b). As a surrogate for Mtf1 activity, the expression levels of Mt1 and Mt2 were measured by qPCR and RNAseq (Fig. 4h), and no significant differences were observed in Q128 cells. This indicates that endogenous Mtf1 is not activated in response to the cytotoxic effects of mutant HTT. In contrast, Q128_Mtf1 cells showed a very strong induction of Mt1 and Mt2, which was associated with reduced ROS generation and reduced cell death (Fig. 4f-g). Collectively, these results indicated that the presence of mutant HTT causes increased ROS generation and cell death.

[0129] Although the endogenous Mtf1 pathway appears unable to counter such effects, expression of exogenous Mtf1 leads to a strong increase in the endogenous metal chelators Mt1 / 2, reduced ROS generation, and decreased cell death in Q128 cells.

[0130] Mtf1 antagonizes mutant HTT effects in zebrafish. Zebrafish is a powerful vertebrate model system that is widely used for human disease modeling, including HD. Therefore, we decided to use the zebrafish HD model to test whether Mtf1 exerts a protective effect in vivo. We expressed Q16 and Q74 HTT fused in-frame with eGFP in single-cell stage fertilized eggs in vivo by mRNA microinjection and characterized the phenotype (identified by GFP expression, Figure 11a) of injected embryos at 24 hours post fertilization (hdp) (Figure 5a).

[0131] Injection of titrated doses of Q74eGFP (Fig. 11b) resulted in malformations such as shortened body length and loss of head structure. Injected larvae were divided into three main groups based on the severity of their phenotype (representative images of each group are shown in the bright-field panel of Fig. 5b): healthy embryos (H), mildly malformed embryos (M) (if embryos showed reduced head volume or ocelli, shortened axial length, and curled tail), and severely malformed embryos (S) (if embryos appeared as a disorganized mass with indistinguishable anterior and posterior parts). Finally, some embryos rapidly degenerated and died extensively, and were classified as dead. Quantification of eight independent experiments showed that injection of Q74eGFP resulted in mild and severe malformations and larval death. In contrast, injection of Q16eGFP showed that more than 90% of injected embryos were healthy (Fig. 5c), even though the same levels of the two mRNAs were detected in the embryos (Fig. 5d). We conclude that expression of mutant HTT alone in zebrafish embryos inhibits embryonic development of anterior structures.

[0132] We next assessed whether embryonic degeneration was involved in cell death. To this end, we performed acridine orange staining in 24 hpf microinjected embryos and detected areas of particularly strong signal in Q74eGFP embryos exhibiting malformations (Fig. 5b, lower panel). Increased cell death in Q74eGFP-injected embryos was also confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (Tunel) assays in tissues and subsequent confocal microscopy analysis and quantification. Control embryos injected with Q16eGFP mRNA showed Tunel positivity in somatic regions where apoptosis occurs physiologically at that developmental stage (e.g., optic vesicle, diencephalon, telencephalon) (Fig. 5e). In contrast, Q74eGFP-injected embryos showed widespread, strong Tunel-positive signals (64.2% of injected embryos, approximately 5-fold increase in Tunel-positive areas), especially in anterior regions with severe malformations (Fig. 5e-f). We conclude that microinjection of mutant HTT into zebrafish embryos leads to widespread cellular apoptosis.

[0133] We next assessed whether Mtf1 could suppress the deleterious effects of mutant HTT. We observed that co-injection of Q74eGFP and Mtf1 mRNA reduced the percentage of severely malformed (28% to 17%, p = 0.002) and dead embryos (16.5% to 8.5%, p = 0.036, Fig. 5c), and finally doubled the percentage of healthy embryos (20% to 46.5%, p = 0.005). Importantly, Mtf1 expression also significantly reduced the acridine orange signal intensity (Fig. 5b, lower panel) and Tunel-positive area (Fig. 5e-f), indicating reduced cell death.

[0134] AAV vector delivery of Mtf1 attenuates motor deficits in R6 / 2 mice. Observations of the protective effects of Mtf1 in vertebrate models prompted us to test its function in more established HD models, such as the commonly used R6 / 2 mouse. R6 / 2 mice display an early HD-related phenotype (at approximately 3 weeks of age) characterized by motor hyperactivity and learning deficits, followed by progressive neurodegeneration and full onset of the disease with severe motor coordination deficits at around 8–15 weeks of age. These changes are characterized by cell loss in the striatum and global brain atrophy.

[0135] To express Mtf1 in mouse brain, we used AAV-PHP.eB, a capsid engineered to efficiently cross the blood-brain barrier upon intravenous injection. The viral vector spread to broad neural regions, including the basal ganglia, resulting in transduction of over 90% of neurons in the striatum after a single injection in multiple mouse models. First, we assessed whether the selected AAV-PHP.eB vector could efficiently cross the blood-brain barrier in R6 / 2 mice. To this end, we injected AAV-containing GFP into the tail vein of 4-week-old R6 / 2 mice and confirmed viral expression by immunoblotting (Figure 6b).

[0136] Next, R6 / 2 mice and WT littermates were given a single tail vein injection of AAV-PHP.eB packages of either GFP (AAV-GFP) or Mtf1 (AAV-Mtf1), used as a control, and motor performance was assessed weekly by the rotarod and horizontal ladder task (HLT) as a functional readout of striatal neuronal loss (Figure 6a). In the rotarod test, R6 / 2 mice injected with AAV-GFP fell faster than wild-type littermates from 7 weeks of age, as previously reported. R6 / 2 mice injected with AAV-Mtf1 maintained similar performance to wild-type littermates throughout the entire period of analysis (Figure 6c). The HLT revealed that the number of errors in R6 / 2 mice was increased compared to wild-type mice at 7 weeks of age, as previously reported. Such effects were restored by injection of AAV-Mtf1 (Figure 6d). Finally, weight loss was detected in R6 / 2 mice when injected with AAV-GFP, compared to WT littermates. Mtf1 did not significantly affect the weight of R6 / 2 mice (Fig. 6e). To confirm the expression of exogenous Mtf1, mice were sacrificed at the endpoint and striatal and cortical tissues were harvested. PCR of total DNA reliably detected copies of Mtf1 virus in both brain regions (Fig. 6f), thus confirming accurate in vivo delivery of Mtf1. Collectively, these results indicate that the motor deficits observed in R6 / 2 mice were greatly and specifically ameliorated by injection of AAV-Mtf1 vectors. Methods

[0137] ES cell culture ES cell lines (Rex1GFP-d2 and E14TG2a) were cultured in feeder-free conditions (plastic coated with 0.2% gelatin [Sigma, catalog no. G1890]) and replated at a split ratio of 1:10 every 3–4 days after dissociation with cell detachment enzyme (GE Healthcare, catalog no. L11-007) or 0.25% trypsin (Life Technologies). The cells were cultured in serum-free N2B27-based medium (DMEM / F12 and Neurobasal mixed in a 1:1 ratio, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1:200 N2, and 1:100 B27 [all reagents from Life Technologies]) or serum-containing KSR medium (10% KSR [Life Technologies], 2% FBS [Sigma, Cat. No. F7524], 100 mM EDTA, 0.1 mM EDTA, 0.1 mM L-glutamine, 1:200 N2, and 1:100 B27 [all reagents from Life Technologies]) supplemented with two small molecule inhibitors (2i) PD (PD (1 μM, PD0325901) and CH (3 μM, CHIR99021) purchased from Axon (Cat. Nos. 1386 and 1408) and LIF (100 units / ml, Qkine - Cambridge, UK)). Cells were cultured in GMEM [Sigma, catalog no. G5154]) supplemented with 2-mercaptoethanol [Sigma, catalog no. M7522], 1× MEM non-essential amino acids [Invitrogen, catalog no. 1140-036], 2 mM L-glutamine, and 1 mM sodium pyruvate [both from Invitrogen].

[0138] Generation of ES cell lines expressing HTT Q15 and Q128 cells were generated by DNA transfection of vectors containing the N-terminus of the human huntingtin gene with 128 or 15 CAG repeats, respectively (provided by Prof. Elena Cattaneo). Plasmid DNA was linearized overnight with the restriction enzyme PvuI. For DNA transfection, reverse transfection was performed using Lipofectamine 2000 (Life Technologies, Cat. No. 11668-019). For one well of a 6-well plate, 6 μl of transfection reagent, 2 μg of plasmid DNA and 300,000 cells in 2 ml of medium were used. After overnight incubation, the medium was changed. Antibiotic selection (puromycin 1 μg / ml) was started 24 hours after transfection.

[0139] Generation of ES cells stably expressing a gene of interest Stable transgenic ESCs expressing candidate genes were generated by introducing PB transposon plasmids (1 μg of CAG-Mtf1, CAG-Kdm2b, CAG-Kdm5b, CAG-Fbxo34) into cells with the PB transposase expression vector pBase (1 μg). Reverse transfection was performed using Lipofectamine 2000 as described for generation of HD lines. Antibiotic selection (hygromycin B; 150 μg / ml, Invitrogen 10687010) was initiated 24 h after transfection.

[0140] Proliferation assay Cell proliferation was performed by inoculating 15,000 ES cells (7,500 cells / cm) into 24-well plates in the presence of 6 μg / ml puromycin. 2 ) were seeded in 24-well plates in the presence of inhibitors (and 6 μg / ml puromycin) and counted every 24 hours for 4 days. 2) were seeded for 48 h to assess the response to stress factors and scored by quantification of viable cell numbers with crystal violet (CV) staining (CV solution: 0.05% w / v crystal violet [Sigma], 1% formaldehyde solution 37% [Sigma], 1% methanol, 10% PBS). For PB mutagenesis after stress treatment, cells were plated at 2,500 cells / cm in 6 μg / ml puromycin. 2 Cells were seeded at a density of 1000× and selected in the presence of MG132 or tamoxifen for 5 days.

[0141] Propidium iodide (PI) staining PI staining was performed on live single ES cells according to the manufacturer's instructions (Ebioscience, Cat. No. 88-8007-72). After washing with PBS, cells were resuspended in 100 μl of 1X binding buffer (Cat. No. 00-0055) and incubated with 2.5 μl of fluorescent dye-conjugated Annexin V (Cat. No. 17-8007) for 10 min at room temperature. Then, cells were washed once with PBS and resuspended in 100 μl of 1X binding buffer. Finally, 5 μl of propidium iodide staining solution (Cat. No. 00-6990) was added to the cell suspension and flow cytometry analysis was performed within 1 h while samples were stored at 2–8 °C in the dark.

[0142] ROS measurement assay Reactive oxygen species (ROS) generation was detected by staining live single ES cells with 20,70-dichlorodihydrofluorescein diacetate (H2DCFDA) (Life Technologies, Cat. No. D399) as follows: a) Just before performing the experiment, the ROS indicator was reconstituted to create a concentrated stock solution (10 mM); b) cells were harvested, c) washed once with PBS, and d) resuspended in PBS containing the probe to provide a final working concentration of 0.5 μM dye; e) cells were incubated for 10 min in the dark at 37 °C; f) after removing the staining solution, g) samples were washed twice with PBS, and h) analyzed by flow cytometry using a BD FACSCanto II cytometer.

[0143] Electroporation of the PB system in ES cells For genome-wide screening, PB-mediated mutagenesis by electroporation was performed. The PB vector is stably integrated in the genome after random insertion at the TTAA site. The PB pGG134 vector used (shown in Figure 2a) was optimized for gain-of-function screening. It consists of a murine stem cell virus (MSCV) transcriptional enhancer / transcriptional start sequence followed by a splice donor (SD) site, which allows hyperactivation of nearby genes. The PB 5'ITR also has weak directional promoter activity, i.e., the construct can activate genes in either orientation. The vector also contains a second cassette containing a constitutive promoter followed by DsRed and hygromycin resistance genes.

[0144] Conditions were optimized to obtain a low number of integration events by adjusting the ratio of PB vector to transposase pBase. For the screening procedure, mutagenesis was performed using optimal amounts of pGG134 (0.5 μg) and pBase (20 μg). For one electroporation, 10^7 cells and 20.5 μg of DNA were mixed and placed in an electroporation cuvette (Biorad Gene Pulser Cuvette, Cat. No. 165-2088). The cuvette was placed in the electroporation holder of a Biorad GenePulser (Cat. No. 165-2076) and the cells were electroporated. Settings used: 250V, 500 μF, time constant 5.6-7.5. Electroporated cells were gently harvested from the cuvette and plated. Antibiotic selection was started 24 hours after electroporation.

[0145] Genomic DNA extraction and Sprinkle-RT-PCR Cells were harvested and incubated overnight at 56°C in lysis buffer (10 mM Tris, pH 7.5; 10 mM EDTA; 10 mM NaCl; 0.5% w / v Sarkosyl supplemented with proteinase K [Sigma, Cat. No. P2308] to a final concentration of 1 mg / ml). The next day, 2 ml of a mixture of NaCl and ethanol (30 μl of 5 M NaCl mixed with 20 ml of cold absolute ethanol) was added to obtain DNA precipitation. The cell extract was centrifuged for 45 min at 4°C and the soluble fraction was removed. The precipitated gDNA was washed three times by dropwise addition of 2 ml of 70% ethanol and finally resuspended in Milli-Q water at 70°C.

[0146] The procedure for Sprinklerette (Sp)-PCR for PB-integration mapping was adapted from the method of Potter and Luo (2010). The procedure consisted of the following: a) 2 μg of genomic DNA was digested with 10 U BstYI (10,000 U / ml) in a volume of 30 μl. The reaction was incubated overnight at 60°C and the enzyme was inactivated the next day at 80°C for 20 min. Sp adapters were generated by annealing 150 pmol of adapter A and B primers in a final volume of 100 μl (10X NEB buffer 2). Oligos were denatured at 65°C for 5 min and then cooled. b) Ligation was performed in a total of 6 μl containing 2X Ligation Mix (Takara), 2.5 μl of digested gDNA, and 0.5 μl of annealed Sp adapters. Ligation reactions were incubated overnight at 16°C and the next day at 65°C for 10 min for enzyme inactivation. A purification step was included prior to step C using a QIaquick PCR purification kit according to the manufacturer's instructions. PCR amplification used Phusion HF DNA Pol (NEB) in 5x Phusion GC buffer, which is recommended for GC-rich templates or templates with secondary structure. The PCR mixture contained 5X GC buffer, 10 mM dNTPs, DMSO, and Phusion Pol. c) The first PCR was amplified with 15 μl of ligated DNA (or ligation product of 50% of each reaction of PB5' and PB3' transposon / host junctions), 0.5 μM of each primer (adapter PCR1 and PB5' or PB3'-ITR PCR1), and 6.5 μl of PCR mixture (final volume 25 μl). Sp-PCR1 program: 95°C for 2 min; 2 cycles of 95°C for 20 s, 65°C for 30 s, 68°C for 2 min; then 30 cycles of 95°C for 30 s, 60°C for 30 s, 68°C for 2 min; then 68°C for 10 min. d) For the second PCR, 5 μl of a 1:500 dilution of PCR1 product, 0.5 μM of each primer (Adapter PCR2 and PB5' or PB3'-ITR PCR2) and 6.5 μl of PCR mix (final volume 25 μl) were used. Sp-PCR2 program: 95°C for 2 min; 2 cycles of 95°C for 20 sec, 65°C for 30 sec, 68°C for 2 min; then 5 cycles of 95°C for 30 sec, 60°C for 30 sec, 68°C for 2 min; then 25 cycles of 95°C for 30 sec, 58°C for 30 sec, 68°C for 2 min; then 68°C for 10 min. e) PCR2 products were treated with Antarctic phosphatase and Exonuclease I (both from NEB) and sequenced using PB5' or PB3'-ITR PCR2 primers. Primer and adapter sequences are listed in Table 1 of Potter and Luo (2010).

[0147] Next-generation sequencing analysis of genomic target sites Genomic DNA was extracted from the entire mutant population using the Gentra Puregene cell kit. Library production and sequencing were performed as described by Lackner et al. (2020, bioRxiv). A custom-built bioinformatic pipeline allowed mapping each read to a genomic locus and relating each site of integration to genes located within a distance of 20 kb. The data were then organized into a network of HD-interacting genes using Cytoscape software.

[0148] RNA isolation, reverse transcription and quantitative PCR For cell lysates, RNA was isolated using a total RNA purification kit (Norgen Biotek 37500) and complementary DNA (cDNA) was generated from 1 μg using M-MLV reverse transcriptase (Invitrogen 28025-013) and oligo dT18 (500 μg / ml) primers. For zebrafish larvae, total RNA was isolated using phenol-chloroform extraction. Total RNA was isolated from a pool of 10 animals using Trizol reagent (Life Technologies, Cat. No. 15596026) following the manufacturer's instructions for a standard Trizol-chloroform-ethanol extraction procedure. To increase the yield of the RNA precipitation step, RNase-free glycogen was used as recommended in the protocol. Two micrograms of total RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen, catalog no. 18080044) and a mixture of oligo dT18 primer (500 μg / ml), dNTP mix (10 mM), DTT (0.1 M), 5X first strand buffer, and RNaseOUT (40 units / μl).

[0149] For real-time PCR, SYBR green master mix (Bioline BIO-94020) was used. Primers used are shown in Table 5 above. Technical replicates were performed for all quantitative PCR. For ESCs, Gapdh was used as an endogenous control to normalize expression. The Ct averages of zebrafish gapdh, eef1a, tuba1b, and b2m were used for normalization as endogenous housekeeping controls due to the variability in the expression levels of these genes. QuantStudio TM qPCR data were acquired with 6 & 7 Flex software 1.0.

[0150] Western blotting Cells were washed with cold PBS and harvested in lysis buffer (50 mM Hepes pH 7.8, 200 mM NaCl, 5 mM EDTA, 1% NP40, 5% glycerol) freshly supplemented with 1 mM DTT, protease inhibitor (Roche 39802300), and phosphatase inhibitor (Sigma-Aldrich P5726). Samples were exposed to ultrasound in a sonicator (Diagenode Bioruptor) and supernatants were prepared by centrifugation at 13000 rpm for 10 min. Protein concentrations were measured by the Bradford assay. Total protein (10 μg) was fractionated on a 4–12% Nupage MOPS acrylamide gel (Life Technologies; BG04125BOX / BG00105BOX) and electrophoretically transferred to a PVDF membrane (Millipore; IPFL00010) in transfer solution (50 mM Tris, 40 mM glycine, 20% methanol, 0.04% SDS).

[0151] Membranes were then saturated with 5% nonfat dry milk (BioRad, 170-6405-MSDS) in TBSt (8 g NaCl, 2.4 g Tris, 0.1% Tween 20 / liter, pH 7.5) for 1 h at room temperature and incubated overnight at 4°C with HTT or GAPDH primary antibodies (Millipore Cat. No. MAB2166 and Millipore Cat. No. MAB374). Mice were sacrificed by cervical dislocation and tissues were homogenized and sonicated in lysis buffer containing 20 mM Tris, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, and a 1:1000 mixture of protease inhibitors (Sigma-Aldrich). 40 μg of total protein lysates were immunoblotted with anti-GFP and anti-actin antibodies (Cell Signaling Technologies, Cat. No. 3700). The membrane was then incubated with secondary antibodies conjugated with peroxidase diluted in 1% milk in TBSt. The membrane was incubated with Pico SuperSignal West chemiluminescence reagent (Thermo Scientific, 34078) and digital images were acquired with an ImageQuant LAS 4000 (GE Healthcare).

[0152] RNA Sequencing: Library Construction Total RNA was quantified using the Qubit 2.0 fluorescent assay (Thermo Fisher Scientific). Libraries were prepared by aliquoting (125 * -250 ** - 250 *** ) ng of total RNA using a 3′DGE mRNA-seq (research* / clinical** / diagnostic***) grade sequencing service (Next Generation Diagnostics srl) including library production, quality assessment, and sequencing on a NovaSeq 6000 sequencing system using the single-end, 100 cycle method (Illumina Inc.).

[0153] Bioinformatics workflow Raw data were analyzed by Next Generation Diagnostics srl's proprietary 3'DGE mRNA-seq pipeline (v2.0), including cleaning steps by quality filtering and trimming, alignment to the reference genome, and counting by gene. All genes with less than 1 cpm and more than 20% Perc MM reads in samples below n_min were simultaneously excluded. Differential expression analysis was performed in the R environment (v. 3.5.3) using Bioconductor (v.3.7) utilizing the DESeq2 R package (v.1.24.0) and edgeR. DESeq2 estimates the size of therapeutic proteins and the variance of each gene, fitting a negative binomial generalized linear model using Wald statistics. Genes with p-values ​​≥ 0.05 between Q128_Mtf1 and Q15_EV and log2FC > |0.5| between Q128_Mtf1 and Q128_EV were considered significant and defined as differentially expressed (DEGs).

[0154] Gene set enrichment analysis of DEGs was performed using Database for Annotation, Visualization, and Integration utilizing the UniProt Keywords Database (DAVID) ( https: / / david.ncifcrf.gov ).

[0155] Volcano plots were generated with Log2 fold changes and -Log10 p-values ​​using the ggscatter function from the ggpubr R package (v.0.2.5). Heatmaps were generated using CPM values ​​with the pheatmap function from the pheatmap R package (v.1.0.12).

[0156] Creating a HD model of zebrafish HD zebrafish were generated by microinjecting single-cell stage embryos with mRNA encoding the first exon of human HTT containing 74Q or 16Q, fused in-frame to the eGFP coding sequence. Q74eGFP and Q16eGFP were cloned into the pCS2+ plasmid to allow in vitro transcription. pCS2_Mtf1 and pCS2_mCherry plasmids were also generated to obtain Mtf1 and mCherry mRNAs used for injection into HD zebrafish embryos.

[0157] For in vitro transcription of RNA, 2.5 μg of pCS2_Q74eGFP, pCS2_Q16eGFP, pCS2_Mtf1, and pCS2_mCherry were linearized by digestion with HF-Not I (New England Biolabs, Cat. No. R3189S) overnight at 37 °C. The digestion solution was then concentrated with a DNA Cleanup & Concentration Kit (Zymo Research, Cat. No. D4003) and used for a capped transcription reaction with SP6 RNA polymerase (mMESSAGE mMACHINETM SP6 Transcription Kit, Thermo Fisher Scientific, Cat. No. AM1340). After removing the DNA template by DNase treatment (Thermo Fisher Scientific, Cat. No. AM2238) for 15 min at 37 °C, RNA was purified by phenol-chloroform extraction (as described in the paragraph "RNA isolation, reverse transcription, and quantitative PCR"). RNA was quantified with a Nanodrop ND-1000 and then diluted as necessary with a mixture of 10% Danieau buffer (8 mM NaCl, 0.7 Mm KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 2.5 mM HEPES, Ph 7.6), 10% phenol red (Merck, Cat. No. 1072410025), and RNase-free water.

[0158] To select the injection dose that would result in the lowest mortality and highest rate of malformations, we injected increasing doses of Q74eGFP mRNA, ranging from 150 to 1000 pg / embryo, and assessed the phenotype of 24 hpf embryos.

[0159] After establishing the dosage at 250 pg / embryo, the transcribed mRNA was injected into the embryos under a light microscope. The microinjected embryos were then transferred to fish water and incubated at 28°C. Unfertilized eggs were identified and discarded 4 hours after microinjection. The 24 hpf tadpoles were dechorionated using a specialized needle under a light microscope.

[0160] Whole specimen staining Injected embryos were anesthetized with tricaine, fixed in 1.5% methylcellulose or 2% low melting point agarose, and analyzed using a Leica M165FC fluorescence microscope. Confocal images of zebrafish were acquired with a Nikon C2 H600L confocal microscope.

[0161] For acridine orange hemi(zinc chloride) in vivo staining (Merck, Cat. No. A6014), membranes of 24 hpf embryos were peeled, transferred to 6-well plates, and incubated for 15 min at 28 °C with approximately 2 ml of acridine orange (20 μg / ml) per well in fish water. The acridine solution was then removed and the embryos were washed three times with 1 ml of fish water. Tadpoles were anesthetized with tricaine before observation by fluorescence microscopy on glass slides.

[0162] ApopTag Fluorescein In-Tissue Apoptosis Detection Kit (Merck, Cat. No. S7110) and collagenase (Merck, Cat. No. C9891) were used for the Tunel assay. Thirty hours after microinjection, seven embryos per condition were left in an Eppendorf, anesthetized with tricaine, and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. The PFA was then removed and samples were washed three times with phosphate-buffered saline. Each wash was performed for 10 min with shaking. Embryos were dehydrated in a series of methanol solutions (10%-100%) and frozen overnight at -20°C. Embryos were then rehydrated in a series of methanol solutions (70-50-30%) and washed in PBS containing Tween-20 (PBST) for 10 min each time with shaking. Collagenase was then applied for 8 min with shaking, followed by three 5 min washing steps with PBST to wash off excess collagenase. Samples were incubated for 1 h with shaking in equilibration buffer, then incubated for 2 h with working concentrations of TdT at 37°C. The reaction was stopped by washing samples twice with working concentrations of stop / wash buffer. A 1 h blocking step with PBST with shaking was then performed, after which embryos were incubated overnight with working concentrations of anti-digoxigenin conjugate in the dark at 4°C. The following morning, the antibody solution was removed and samples were washed with PBS (4 times, 10 min each) and analyzed by confocal microscopy.

[0163] Image analysis For quantitative analysis, all images were acquired with similar exposure parameters and processed using FIJI software. Statistical analysis was performed using Past4 software.

[0164] For the Tunnell assay, the anterior structure of zebrafish larvae was scanned with 70 stacks of 3.475 μM each across the entire depth. The fraction of fluorescent positive area relative to the total area (excluding the yolk region) was quantified.

[0165] AAV-PHP.eB vector injections, mouse phenotyping and tissue harvesting AAV-PHP.eB viral particles were produced and titrated in the Broccoli laboratory as described by Morabito et al. (2017). The viral vector was modified to express either the candidate gene Mtf1 or eGFP as a control under the control of the Ef-1α promoter. Intravascular injections were performed in a restrainer with the tail placed in a heated groove. The tail was wiped with alcohol and then injected intravenously. WT and R6 / 2 mice were randomly grouped and injected into the tail vein at 4.2 weeks of age. After injection, all mice were weighed twice weekly. Phenotyping was performed twice weekly in mice blinded to genotype and treatment. Balance and motor coordination were assessed by the rotarod test and horizontal ladder task. Total DNA was isolated from animal tissues (cortex and striatum) using the Qiagen DNeasy Blood and Tissue Kit (QIAGEN).

[0166] Animal husbandry All zebrafish experiments were performed in the Fish Facility of the Department of Biology, University of Padua. Zebrafish larvae were maintained for up to 3 days in Petri dishes with fish water (60 mg Instant Ocean per liter of distilled water, Cat. No. SS15-10) at neutral pH and 28°C, according to standard procedures (http: / / ZFIN.org). Mice were maintained in sterile microisolators and supplied with autoclaved food and water at the Mouse Facility of the IRCCS Neuromed Institute (Pozzili, Italy). Breeding pairs of transgenic female mice of the R6 / 2 strain with an approximately 160±10 (CAG) repeat expansion [strain name: B6CBA-tgN (HDexon1) 62Gpb / 1J] were purchased from the Jackson Laboratory. Male R6 / 2 mice were mated with female B6CBA WT mice for colony maintenance. All procedures were performed according to protocols approved by the in-house Institutional Animal Care and Use Committee (IACUC) and reported to the Italian Ministry of Health, in accordance with the European Commission Council Directive 2010 / 63 / EU and the Italian law on animal experiments (Decreto Legislativo D.Lgs 26 / 2014).

Claims

1. A therapeutic protein, or an isoform or homolog thereof, said homolog having sequence homology defined as at least 55% coverage and at least 49% identity with said therapeutic protein, said therapeutic protein, isoform or homolog thereof being capable of reducing the toxicity of mutant proteins causing polyQ disease, and for use in the treatment of said disease.

2. 2. The therapeutic protein, its isoform or homologue of claim 1, wherein the mutant protein is expressed by one or more of the following genes: ATN1, HTT, AR, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, TBP, and the mutant protein comprises a pathological number of polyQ residues.

3. The therapeutic protein of claim 1 or 2, wherein the therapeutic protein is selected from metal regulating transcription factor 1, lysine-specific demethylase 5B, lysine-specific demethylase 2B, F-box only protein 34, essential MCU regulator, mitochondria, ephrin type A receptor 4, transducin-like transcription factor protein 4, alphaptin 1, AT-rich interaction domain-containing protein 5B.

4. 3. The therapeutic protein isoform of claim 1 or 2, wherein the isoform is selected from Table 3 below.

5. 3. The therapeutic protein homolog of claim 1 or 2, wherein the homolog is selected from Table 4 below.

6. The therapeutic protein, its isoform or homologue according to any one of claims 1 to 5, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

7. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP, comprising a nucleotide sequence encoding a therapeutic protein, an isoform or a homologue thereof, according to claims 1 to 5.

8. The vector according to any one of claims 9 to 11, wherein the vector is an AAV viral vector having a capsid serotype optimal for CNS gene therapy.

9. 9. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence as defined in any one of claims 7 or 8 for use in the treatment of a polyQ disease.

10. 10. An expression vector or a delivery system or a vector suitable for gene therapy, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or a LNP or a nucleotide sequence for use according to claim 9, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

11. A viral particle comprising a viral capsid and a nucleotide sequence encoding a therapeutic protein, an isoform or a homologue thereof, according to any one of claims 1 to 5.

12. A viral particle as described in claim 11 for use in gene therapy for polyQ disease.

13. The viral particle of claim 12, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

14. An mRNA encoding a therapeutic protein, an isoform or a homologue thereof according to claims 1 to 5.

15. The mRNA of claim 14, wherein the mRNA is complexed to one or more carrier molecules.

16. The mRNA of any one of claims 14 or 15, wherein the mRNA is complexed in the form of a cationic nanoemulsion, nanoparticle, liposome, cationic polymer liposome, polysaccharide particle, cationic lipid nanoparticle, cationic lipid cholesterol nanoparticle, cationic lipid cholesterol PEG nanoparticle.

17. The mRNA according to any one of claims 14 to 16 for use in the treatment of polyQ disease.

18. The mRNA of claim 17, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinal-bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12 or spinocerebellar ataxia type 17.

19. A pharmaceutical composition comprising a therapeutic protein, an isoform or a homologue thereof, according to claims 1 to 5, or an expression vector or a delivery system or a vector suitable for gene therapy according to any one of claims 7 to 9, or an adeno-associated viral vector AAV or a lentiviral vector, or a nanoparticle, or an LNP or a nucleotide sequence, or a viral particle according to claim 11, or an mRNA according to any one of claims 14 to 16, and a pharma- ceutical acceptable carrier.

20. 20. The pharmaceutical composition of claim 19 for use in the treatment of polyQ disease.

21. 21. The pharmaceutical composition of claim 20, wherein the polyQ disease is dentatorubral-pallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 12, or spinocerebellar ataxia type 17.

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