Crispr-CAS9 based tools: potential novel therapeutic approaches for machado-joseph disease

Abstract

The present disclosure relates to Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), an autosomal dominantly inherited neurodegenerative disorder that is caused by an over-repetition of the polyglutamine-codifying region in the ataxin-3 (ATX7V3) gene. Expanded ATXN3 is prone to aggregate and disturbs diverse cellular systems, ultimately leading to cell dysfunction and death in specific neuronal populations. To date, no treatment able to reverse or stall MJD / SCA3 progression has been developed. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease, producing an incomplete and / or transient therapeutic effect in target cells or tissues. Recently, gene-based therapeutics, including the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) systems for gene edition, have been successfully used to permanently inactivate and correct disease-related genes, holding promise for the development of a definitive cure for inherited diseases.

Description

D E S C R I P T I O NCRISPR-CAS9 BASED TOOLS: POTENTIAL NOVEL THERAPEUTIC APPROACHES FOR MACHADO-JOSEPH DISEASETechnical Field

[0001] The present disclosure relates to Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), an autosomal dominantly inherited neurodegenerative disorder that is caused by an overrepetition of the polyglutamine-codifying region in the ataxin-3 (ATXN3) gene. Expanded ATXN3 is prone to aggregate and disturbs diverse cellular systems, ultimately leading to cell dysfunction and death in specific neuronal populations. To date, no treatment able to reverse or stall MJD / SCA3 progression has been developed. Strategies based on the suppression of the deleterious gene products have demonstrated promising results in pre-clinical studies. Nonetheless, these strategies do not target the root cause of the disease, producing an incomplete and / or transient therapeutic effect in target cells or tissues. Recently, gene-based therapeutics, including the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) systems for gene edition, have been successfully used to permanently inactivate and correct disease-related genes, holding promise for the development of a definitive cure for inherited diseases.Background

[0002] Machado-Joseph disease (MJD) / SCA3 is one of the nine known polyglutamine (polyQ) expansion diseases. Despite rare, MJD / SCA3 is the most common autosomal dominantly inherited ataxia worldwide.

[0003] The disease mutation has been mapped to chromosome 14 (14q32.12) and it consists on the abnormal repetition of the trinucleotide CAG in the coding region of the ATXN3 gene, which encodes a pathogenic glutamine repeat expansion at the C-terminus of the ATXN3 protein12. The expanded protein (stretch over 61 repeats) undergoes proteolytic cleavage, generating polyQ-containing fragments that cannot be properly eliminated due to an inefficient activation of autophagy. As a consequence, this mutant ATXN3 is prone to form insoluble aggregates in neurons, eliciting several cellular events that trigger neuronal dysfunction and degeneration in specific brain regions, such as the cerebellum, brainstem and striatum.

[0004] Symptoms usually start in adult age and are highly variable between individuals. Although some patients benefit from symptomatic therapy, no effective causative treatment able to prevent, cure or delay the progression of this disorder has been developed.

[0005] Considering the toxic gain-of-function acquired by expanded ATXN3 species, strategies aiming at silencing the expression of MJD / SCA3's causative gene in affected brain regions have been explored as potential therapies. However, therapeutic strategies based on gene silencing only provide partial and transient suppression of ATXN3, demanding successive administrations or the permanent expression of gene silencing sequences using viral vectors.

[0006] Gene editing tools relying on programmable nucleases allow the interference with the diseasecausing genes at the most upstream level, assuring a complete and permanent gene disruption3. Among the currently available genome editing technologies, the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) system is the most rapidly developing class of endonucleases3.

[0007] One of the best characterized and most widely used CRISPR systems for gene editing purposes derives from Streptococcus pyogenes. Briefly, the bacterial CRISPR-associated (Cas) nuclease 9 (SpCas9) is recruited to the region of interest by a short single guide RNA (sgRNA) molecule that binds to a complementary 20 bp DNA sequence, via Watson-Crick pairing. A critical feature of this system is the existence of an obligatory protospacer adjacent motif (PAM) sequence immediately downstream of the DNA target site. SpCas9 targets DNA sites flanked by 5'-NGG PAM sequences, catalysing a double-strand break (DSB) at approximately 3 bp upstream of the PAM, through the activation of the HNH and RuvC nuclease domains4-7. Apart from its high efficiency, the mode of recognition used by this system significantly facilitates the re-targeting of Cas9 nuclease to new DNA sequences, by simply changing the 20 bp guide sequence of the gRNA.

[0008] Upon the introduction of double-strand breaks (DSBs), gene editing is achieved through the activation of endogenous cellular repair machinery. In eukaryotic cells there are two major repair mechanisms to restore DSBs, although the error-prone NHEJ constitutes the most active repair mechanism throughout the cell cycle. NHEJ repairs the lesion by re-joining the two cleaved ends in a process that does not require a repair template. Consequently, this repair process results in nucleotide insertions or deletions (indels) at the lesion site. When introduced into a genomic coding sequence, these indels will often result in frameshift mutations, creating premature stop codons. Thus, similarly to RNA silencing methods, NHEJ can be used to supress gene function, with the major advantage of being a permanent approach.

[0009] Several gene editing applications of SpCas9 have been reported in a variety of species and cell types, dramatically expanding the range of treatable genetic diseases and accelerating the generation of cellular and animal models of disease. Nonetheless, the issue of target specificity is still a big concern, since Cas9-derived gene editing causes permanent gene modifications. Although SpCas9 binding specificity is determined by the Watson-Crick base-pairing interactions of a 20-nucleotide guide sequencewith its target DNA, SpCas9 tolerates mismatches throughout the guide sequence, especially at PAM distant sites. Moreover, off-target sites followed by a 5'-NAG PAM sequence, have also been reported8

[0010] To minimize these off-targets, several efforts to increase targeted specificity of CRISPR-Cas9 systems are in progress. One of these strategies consists on the use of paired nickases (Cas9n) to target a specific locus9. The family of Cas9 proteins is characterized by two nuclease domains, RuvC and HNH, each of which generates site-specific cleavage on opposite DNA strands, consequently generating a DSB7Cas9 variants, developed by the mutation of each of the two catalytic domains (D10A or H840A point mutations into the RuvC or HNH, respectively) retained DNA-binding specificity, cutting either the complementary (D10A nickase) or non-complementary (H840A nickase) DNA strands4-10-X1. This approach brings a considerable advantage, since individual single-stranded breaks in the genome are repaired with high-fidelity, while a DSB will only be generated if a pair of opposite oriented Cas9 nickases are in close proximity12.

[0011] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.General description

[0012] In the present disclosure, it was demonstrated the successful application of the CRISPR-Cas9 system in the context of MJD / SCA3, the most common dominantly inherited ataxia worldwide. While the pathogenic events underlying neurodegeneration remain unclear, it is well established that the disease is caused by an abnormal expansion of the trinucleotide CAG in the ATXN3 gene. Therefore, in order to prevent the downstream toxic pathways, the present disclosure relates to the development of CRISPR- Cas9-based strategies that could efficiently promote the ATXN3 gene silencing or the removal of the deleterious mutation associated with MJD / SCA3.

[0013] A knock-out approach was initially developed by directing the CRISPR-Cas9 system to exon 2 of the ATXN3 gene. After a first validation in a human cellular line, the developed system was tested in a lentiviral-based mouse model of MJD / SCA3. This system demonstrated its efficiency on gene disruption and the consequent decrease of ATXN3 protein levels was observed in a human cellular line. Importantly, the inactivation of the mutant ATXN3 gene in a lentiviral-based mouse model of MJD / SCA3 led to a drastic reduction of ATXN3 aggregates, along with an improvement in key neuropathological markers of the disease. These data provide the first in vivo evidence for the potential of CRISPR-based gene inactivation as a new therapeutic option for MJD / SCA3.

[0014] In parallel, a second CRISPR-Cas9-based approach was developed in the present disclosure to excise the causative mutation of MJD / SCA3. Upon the simultaneous introduction of two double-strandbreaks flanking the CAG tract, this genomic region was efficiently removed by the activation of non- homologous end-joining repair pathway. As a consequence of this excision, truncated variants of the protein were generated, and although characterization of their function is still lacking, this approach can be particularly useful for the correction of patient-derived stem cells, providing a basis for molecular mechanism research and autologous stem cell transplantation.

[0015] Despite the demonstration of the effectiveness of the previous approach, the issue of target specificity is still a big concern, since Cas9-derived gene editing causes permanent gene modifications. Therefore, in the third approach of the present disclosure, a Cas9 nickase-based strategy has been adapted to excise the CAG tract with increased targeted specificity, which may enhance the therapeutic benefit of this strategy. Upon the simultaneous introduction of two double-strand breaks flanking the CAG tract, it was observed in the present disclosure the appearance of PCR amplicons with lower sizes and the formation of versions of ATXN3 protein with decreased molecular weight, as already observed in the second approach.

[0016] Despite achieving different outcomes with different efficiencies, all of the developed gene editing systems may constitute promising candidates for the treatment of this devastating neurodegenerative disorder.

[0017] An aspect of the present disclosure relates to a method for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 (MJD / SCA3 targeting the ATXN3 gene, comprising the administration of a Cas protein, or a nucleic acid molecule encoding a Cas protein, along with guide RNAs, or a nucleic acid molecule encoding guide RNAs, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to a protospacer-adjacent motif (PAM) in the target gene, specifically using a single guide RNA targeting exon 2 of the gene, which includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a combination of two guides to excise the deleterious ATXN3 mutation, with one guide targeting intron 9 (SEQ ID NO: 5) and one guide targeting exon 10 (SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8), or a combination of four guides to excise the deleterious ATXN3 mutation, wherein a pair of guides target intron 9 (SEQ ID NO: 5 and SEQ ID NO: 9) and a pair of guides target exon 10 (SEQ ID NO: 8 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 12 and SEQ ID NO: 13).

[0018] Another aspect of the present disclosure relates to a method for the treatment of Machado- Joseph disease or Spinocerebellar ataxia type 3 (MJD / SCA3 targeting the ATXN3 gene, comprising the administration of a Cas protein, or a nucleic acid molecule encoding a Cas protein, along with guide RNAs, or a nucleic acid molecule encoding guide RNAs, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to a protospacer-adjacent motif (PAM) in the target gene, specifically using a single guide RNA targeting exon 2 of the gene, which includes SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, or SEQID NO: 4. In an embodiment, the method as described in the present disclosure, wherein the guide RNA comprises SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.

[0019] Another aspect of the present disclosure relates to a method for the treatment of Machado- Joseph disease or Spinocerebellar ataxia type 3 (MJD / SCA3 targeting the ATXN3 gene, comprising the administration of a Cas protein, or a nucleic acid molecule encoding a Cas protein, along with guide RNAs, or a nucleic acid molecule encoding guide RNAs, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to a protospacer-adjacent motif (PAM) in the target gene, specifically using a combination of two guides to excise the deleterious ATXN3 mutation, with one guide targeting intron 9 (SEQ ID NO: 5) and one guide targeting exon 10 (SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8). In an embodiment, the method previously described, wherein the guide RNAs comprise a combination of a guide targeting intron 9 (SEQ ID NO: 5) and a guide targeting exon 10 (SEQ ID NO: 7, or SEQ ID NO: 8).

[0020] An aspect of the present disclosure relates to a method for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 (MJD / SCA3 targeting the ATXN3 gene, comprising the administration of a Cas protein, or a nucleic acid molecule encoding a Cas protein, along with guide RNAs, or a nucleic acid molecule encoding guide RNAs, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to a protospacer-adjacent motif (PAM) in the target gene, specifically using a combination of four guides to excise the deleterious ATXN3 mutation, wherein a pair of guides target intron 9 (SEQ ID NO: 5 and SEQ ID NO: 9) and a pair of guides target exon 10 (SEQ ID NO: 8 and SEQ ID NO: 10, or SEQ ID NO: 11 and SEQ ID NO: 12, or SEQ ID NO: 12 and SEQ ID NO: 13).

[0021] In an embodiment, the method described in the present disclosure, wherein the guide RNAs comprise a combination of four guides, wherein a pair of guides targets intron 9 (SEQ ID NO: 5 and SEQ ID NO: 9) and a pair of guides targets exon 10 (SEQ ID NO: 8 and SEQ ID NO: 10).

[0022] An aspect of the present disclosure relates to the use of the method as described in the present disclosure for the treatment of MJD / SCA3, wherein the gene delivery system used to deliver Cas protein and guide RNAs is suitable for ex vivo or in vivo ATXN3 gene targeting.

[0023] In an embodiment, the use of the method as described in the present disclosure for the treatment of MJD / SCA3, wherein the gene delivery system used to deliver Cas protein and guide RNAs is suitable for the usage in neurons and / or oligodendrocytes and / or glial cells.

[0024] An aspect of the present disclosure relates to a combination of guide RNAs for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3, wherein said combination comprises: a first guide RNA for targeting intron 9 of ataxin-3 (ATXN3) gene, wherein said guide RNA comprises a sequence identical to the following list: SEQ ID NO: 5, and a second guide RNA for targeting exon10 of ATXN3 gene, wherein said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 8, or combinations thereof; or a first and a second guide RNA for targeting intron 9 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 5 and SEQ ID NO: 9, or combinations thereof, and a first and a second guide RNA for targeting exon 10 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or combinations thereof.

[0025] In an embodiment, the first and a second guide RNA for targeting intron 9 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to the combination of sequences of the following list: SEQ ID NO: 5 and SEQ ID NO: 9.

[0026] In an embodiment, the first and a second guide RNA for targeting exon 10 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 8 and SEQ ID NO: 10; or SEQ ID NO: 11 and SEQ ID NO: 12; or SEQ ID NO: 12 and SEQ ID NO: 13.

[0027] In an embodiment, said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 13, or combinations thereof or a guide RNA consisting in a sequence identical SEQ ID NO: 2.

[0028] In an embodiment, said guide RNA comprises a single sequence identical to a sequence of the following list: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or combinations thereof.

[0029] In an embodiment, said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 13, or combinations thereof.

[0030] An aspect of the present disclosure relates to a CRISPR-Cas system for the treatment of Machado- Joseph disease or Spinocerebellar ataxia type 3 comprising a human codon-optimized Cas protein, a guide RNA or a combination of guide RNAs, and a protospacer adjacent motif, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to the protospacer-adjacent motif in the ATXN3 gene.

[0031] In an embodiment, the said human codon-optimized Cas protein, comprises a Cas protein; preferably the Cas9 protein may comprise one or more mutations to improve the efficiency and safety.

[0032] In an embodiment, the mutated Cas is a Cas9 nickase. Preferably, the Cas9 nickase is Streptococcus pyogenes Cas9 (SpCas9) D10A nickase.

[0033] In an embodiment, the Cas9 protein further includes a functional domain.

[0034] In an embodiment, the Cas protein is selected from a list comprising: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus Cas9 (StlCas9), Neisseria meningitidis Cas9 (NmCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), Streptococcus canis Cas9 (ScCas9), or Staphylococcus simulans Cas9 (SsCas9), Geobacillus stearothermophilus Cas9 (GeoCas9), Acidaminococcus sp. Casl2a (AsCasl2a), Lachnospiraceae bacterium Casl2a (LbCasl2a), Bacillus hisashii, Casl2b (BhCasl2b), Delta proteobacteria Casl2e (DpbCasl2e), Planctomycetes Casl2e (PlmCasl2e), and Unultured archeon Casl2f.

[0035] In an embodiment, the composition as described in the present disclosure, wherein Cas protein comprises one or more mutations to improve the efficiency and safety.

[0036] In an embodiment, the composition as described in the present disclosure, wherein said mutated Cas protein is a nickase.

[0037] In an embodiment, the composition as described in the present disclosure, wherein Cas protein further includes a functional domain.

[0038] In an embodiment, the composition as described in the present disclosure, wherein Cas protein is a Cas9.

[0039] An aspect of the present disclosure relates to a vector or a vector combination for the use in the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 comprising the CRISPR-Cas system of the present disclosure.

[0040] In an embodiment, the said vector may be selected from the group consisting of lipid nanoparticles, polymeric nanoparticle, liposome, extracellular vesicle, plasmid or viral vector; preferably a plasmid or a viral vector; more preferably adenovirus, lentivirus, retrovirus, or adeno-associated virus vector.

[0041] An aspect of the present disclosure relates to a composition for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 comprising a guide RNA or a combination of guide RNAs as described in the present disclosure, or a CRISPR-Cas system as described in the present disclosur, or a vector or a vector combinations as described in the present disclosur.

[0042] In an embodiment, the gene delivery vector consists of a single vector or several vectors.

[0043] In an embodiment, the gene delivery vector is a viral vector, e.g. adeno-associated vectors (AAV) or lentiviral vectors, and / or a non-viral vector, e.g. a liposome, a nanoparticle, an exosome or a microvesicle.

[0044] An aspect of the present disclosure relates to an in vitro or ex vivo method for editing the ATXN3 gene in a cell by a CRISPR-Cas genome editing system, the method may comprise:a step of introducing the vector or the combinations of vectors of the present disclosure into the cell to achieve either: the excision of the CAG tract, by targeting by intron 9 and exon 10 of the ATXN3 gene; or the gene knock-out, by targeting exon 2 of the ATXN3 gene..

[0045] In an embodiment, the cell is an induced pluripotent stem cell, or a cell of the central nervous system; preferably neurons, or oligodendrocytes, or glial cells.

[0046] An aspect of the present disclosure relates to a guide RNA or a combination of guide RNAs of the present disclosure, or a CRISPR-Cas system of the present disclosure, or vector or combinations of vectors of the present disclosure, or a composition of the present disclosure for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3, wherein the said guide RNA or combination of guide RNAs, or the said CRISPR-Cas system, or the said vector, or the said composition may be administrated systemically, intravenously, intratumorally, orally, intranasally, intraperitoneally, intramuscularly, intravertebrally, intracerebrally, intracerebroventriculally, intracisternally, intrathecally, intraocularly, intracardiacally, intradermally, or subcutaneously, preferably intravenously, intracisternally, intrathecally or, in situ, by intracerebral administration.

[0047] An aspect of the present disclosure relates to the use of guide RNA or a combination of guide RNAs of the present disclosure, or a CRISPR-Cas of the present disclosure, or vector or combinations of vector of the present disclosure, or a composition of the present disclosure for the manufacture of a medicament for the treatment of a neurodegenerative disease, wherein the neurodegenerative disease is an ATXN3 gene-related condition or disorder; preferably the ATXN3 gene-related condition or disorder is Machado-Joseph disease or Spinocerebellar ataxia type 3.

[0048] An aspect of the present disclosure relates to a method for treating or preventing ATXN3 gene- related condition or disorder in a subject, the method comprising administering guide RNA or a combination of guide RNAs of the present disclosure, or a CRISPR-Cas system of the present disclosure, or vector or combinations of vectors of the present disclosure, or a composition of the present disclosure to the subject.Brief description of the drawings

[0049] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

[0050] Figure 1 - CRISPR-Cas9 system designed to permanently inactivate the human ATXN3 gene decreases ATXN3 protein expression in HEK293T cells. (A) Four guide sequences (sgKO.l-SEQ. ID NO1, sgKO.2-SEQ ID NO2, sgKO.3-SEQ ID NO3, sgKO.4-SEQ ID NO4) were designed to recognize different regions of exon 2 of the human ATXN3 gene, recruiting SpCas9 to the locus of interest. sgRNA target sequencesare displayed. SpCas9 mediates the insertion of a DSB at approximately 3 base pairs upstream of a PAM sequence (arrowheads) and stimulates genome editing via NHEJ repair pathway for the permanent blocking of ATXN3 gene expression. (B) A sequence targeting the bacterial lacZ gene (sgCTRL-SEQ ID NO14) and four A7XA / 3-targetting guide sequences (SEQ ID NO1, SEQ ID NO2, SEQ ID NO3 and SEQ ID NO4) were cloned into a sgRNA scaffold-codifying region of a lentiviral expression vector (lentiCRISPRv2, plasmid #52961). In addition to the sgRNA, this vector also codifies for FLAG-tagged SpCas9 and a puromycin resistance cassette. For the validation of the guide sequences, HEK293T cells were transfected with each of the plasmids and maintained in culture for 72 hours (selection medium with puromycin 10 pg / mL for 48 hours). (C-D) Locus modification efficiencies were analysed using Surveyor nuclease assay. Lane 1: DNA ladder (GeneRuler 100 bp, Thermo Scientific); Lane 2: Cells transfected with the sgCTRL construct (SEQ ID NO14); Lanes 3-6: Cells transfected with the sgRNA knock-out guide sequences (sgKO.l- SEQ ID NO1, sgKO.2-SEQ ID NO2, sgKO.3-SEQ ID NO3, sgKO.4-SEQ ID NO4, respectively). Arrowheads indicate the expected DNA fragments cleaved by Surveyor nuclease. The estimated indel occurrence within human ATXN3 locus is represented as a percentage (n=4). (E-F) Western blot analysis revealed a significant decrease (approximately 0.5-fold change) in ATXN3 protein levels after SpCas9 targeting of ATXN3 locus in comparison with the control sequence (n=3). Results are presented as fold change relative to cells transfected with sgCTRL (SEQ ID NO14) expressing plasmid. Optical densitometry analysis of ATXN3 fractions were normalized with -actin and FLAG signals. Statistical significance was evaluated with one-way ANOVA with Dunnett's post hoc test (*p<0.05). Data are expressed as mean ± SEM.

[0051] Figure 2 - Bioinformatic analysis of potential off-targets in the human genome for the knockout sequences. (A) Benchling bioinformatic tool allowed the prediction of the number of potential off- target sites for each guide sequence, according to the attributed scores. (B) The amount of off-targets separated according to the number of mismatches (1 to 4 mm), existing between each predicted off -target site and the respective sgKO sequence, are displayed. (C) Number of off-targets located in different genomic regions (location mapped through UCSC genome browser). The large majority of putative off- targets are located in intergenic or intronic regions.

[0052] Figure 3 -ATXN3 gene editing in vivo reduces mutant ATXN3 expression in the striatum of adult mice. (A) Schematic representation of the stereotaxic co-injection of viral vectors in the striatum of C57BL / 6 mice. Lentivirus encoding for the human mutant ATXN3 protein with 72 glutamines (Myc- tagged), AAVs encoding for SpCas9 (HA-tagged) and AAVs encoding for the CTRL guide sequence (SEQ ID NO14, EGFP-KASH co-expression) were injected in the left hemisphere, serving as experimental control. In the contralateral hemisphere AAVs encoding for the ATXN3 guide sequence (sgKO.2-SEQ ID NO2) were injected, along with LV-PGK-A TXN3 72Q and the AAV-SpCas9. Four weeks after surgery animals were sacrificed. (B) Editing efficiency was evaluated in striatal homogenates. Surveyor gel showed the modification of ATXN3 locus in treated hemispheres, when compared with the contralateral controlhemispheres. CRISPR-induced indels, in percentage, were determined for each treated hemisphere (n=5) and are represented in (C). (D-E) Western blot analysis demonstrated that CRISPR-A TX / V3 (SEQ ID NO2) promoted a significant reduction of ATXN3 aggregates in the striatum of mice, together with a 0.5-fold change in the mutant ATXN3 soluble form. No changes were observed in the mouse endogenous Atxn3 levels (n=5). Results are presented as fold change relative to control hemispheres (CRISPR-CTRL; SEQ ID NO14). Optical densitometry analysis of ATXN3 fractions was normalized with p-tubulin signals. Statistical significance was evaluated with paired Student's t-test (*p<0.05, ns=non-significant). Data are expressed as mean ± SEM.

[0053] Figure 4 - ATXN3 gene editing in vivo alleviates MJD / SCA3 neuropathology in a lentiviral-based mouse model of the disease. (A-B) Immunohistochemical peroxidase staining upon labelling of striatal sections with anti-ubiquitin antibody, 4 weeks after stereotaxic surgery. (C) CRISPR-ATX / V3 injected hemispheres (SEQ ID NO2) display a drastic reduction in the number of ubiquitin-positive inclusions in comparison with the contralateral control hemisphere, injected with CRISPR-CTRL (SEQ ID NO14). Scale bar, 50 pm (D-E) Immunohistochemical analysis using anti-DARPP-32 antibody for expanded ATXN3- derived lesion identification. Treated hemispheres, injected with CRISPR-A TXN3 (SEQ ID NO2) showed a statistically-significant reduction of DARPP-32 depleted volume, as quantified in (F). Scale bar, 200 pm. Statistical significance was evaluated with paired Student's t-test (**p<0.01, n=5). Data are expressed as mean ± SEM.

[0054] Figure 5 - CRISPR-A TX / V3 induced a diminishment of neuroinflammatory markers in a lentiviral- based mouse model of MJD / SCA3. Fluorescent immunohistochemical analysis was performed 4 weeks after stereotaxic delivery of viral vectors. (A-C) lba-1 immunoreactivity in the mouse striatum indicated that microglial recruitment triggered by the mutant ATXN3 expression in control hemisphere (A) is reduced in the CRISPR-ATX / V3-edited hemisphere (B), as quantified in (C). (D-F) GFAP immunoreactivity in mouse striata. Non-edited striatum (D) displays an increased GFAP immunoreactivity, although not statistically significant, in comparison with the ATX / V3-edited striatum (E), as quantified in (F). Scale bar, 200 pm in general view images and 50 pm in detail magnifications. Statistical significance was evaluated with paired Student's t-test (*p<0.05, ns=non-significant, n=4). Data are expressed as mean ± SEM.

[0055] Figure 6 - CRISPR-Cas9-mediated deletion of the CAG repeat tract of the human ATXN3 gene. (A) Graphical illustration of the DNA target sites for the designed sgRNAs (target sequences are displayed). One sgRNA (sgl9Tl-SEQ ID NO5) targets an intronic region, while the three remaining sequences (sgElOTl- SEQ ID NO6; sgE10T2-SEQ ID NO7; sgE10T3-SEQ ID NO8) target the exon 10 of the gene, immediately downstream of the CAG motif. Importantly, since sgRNA guide sequence (SEQ ID NO6) is designed to target a DNA sequence that includes a known single-nucleotide polymorphism (G nucleotide is underlined), the change of this specific nucleotide (G to C) might be of relevance for the development of an allele specific editing approach. (B) HEK293T cells were transfected with plasmids encoding the SpCas9and each of the designed sgRNAs targeting the vicinity of the CAG tract. As a negative control, cells were transfected with a sgRNA sequence targeting the bacterial lacZ gene (sgCTRL-SEQ ID NO14). Cells were maintained in culture for 72h, in the presence of puromycin (10 pg / mL). (C) Surveyor assay revealed the presence of fragments (arrowheads) resulting from SpCas9-induced DSBs. Fragment sizes correspond to the predicted DSB position for each target sequence. SpCas9 cleavage efficiency (indel percentage) for each sgRNA is indicated below (n=l). (D) The simultaneous introduction of two DSBs flanking the repeat tract at either end can be achieved by the combination of guide sequences targeting the upstream (sgl9Tl- SEQ ID NO5) and downstream region (sgElOTl-SEQ ID NO6, sgE10T2-SEQ ID NO7 or sgE10T3-SEQ ID NO8) of the repeat tract. The two broken ends may be re-joined by NHEJ repair pathway, while the repeatcontaining fragment is eliminated. (E) Electrophoretic resolution of PCR products comprising the expected DNA DSB sites. PCR amplicons with lower molecular weight were visible in multiplexed targeting conditions (lanes 6 to 8). (F) Western blot analysis of cell extracts showed the presence of ATXN3 species with reduced molecular weight (arrowheads) in several conditions. Targeting the nuclease activity of SpCas9 to both CAG ends led to the prominent detection of a band of approximately 35 kDa mainly in conditions sgl9Tl-SEQ ID NO5 + sgE10T2-SEQ ID NO7 and sgl9Tl-SEQ ID NO5 + sgE10T3-SEQ ID NO8 (lanes 7 and 8, n=l), that may possibly result from mutation-induced truncation of ATXN3.

[0056] Figure 7 - CRISPR-Cas9 targeted nickases induced the excision of the CAG repeat motif of the ATXN3 gene. (A) Graphical illustration of the DNA target sites for the designed sgRNAs (target sequences are displayed). One pair of sgRNAs (sgNickl9TlA-SEQ ID NO9 and sgNickl9TlB-SEQ ID NO5) target opposite DNA strands of intron 9, while the three remaining pairs of sequences (sgNickElOTlA-SEQ ID NO8 and sgNickElOTIB-SEQ ID NO10; sgNickE10T2A-SEQ ID NO11 and sgNickE10T2B-SEQ ID NO12; sgNickE10T3A- SEQ ID NO13 and sgNickE10T2B-SEQ ID NO12) target the exon 10 of the gene, immediately downstream of the CAG motif. Since these sgRNA sequences recruit a SpCas9 D10A nickase variant to the locus of interest, single strand breaks (arrowheads) are inserted in the DNA strand that is complementary to the sgRNA. Since each pair of sgRNAs was designed to simultaneously target the gene in close proximity, SpCas9 D10A nickase generates a DSB (5'-overhang) with an improved specificity in both sides of the CAG tract. (B) HEK293T cells were transfected with plasmids encoding the SpCas9 D10A nickase and each pair of the designed sgRNAs targeting the vicinity of the CAG tract (SEQ ID NO9 and SEQ ID NO5, SEQ ID NO8 and SEQ ID NO10, SEQ ID NO11 and SEQ ID NO12 or SEQ ID NO13 and SEQ ID NO12) . As a negative control, cells were transfected with a sgRNA sequence targeting the bacterial lacZ gene (sgCTRL-SEQ ID NO14). Cells were maintained in culture for 48h after transfection. (C) The simultaneous introduction of two DSBs flanking the repeat tract at either end can be achieved by the combination of guide sequences targeting the upstream (SEQ ID NO9 combined with SEQ ID NO5) and downstream region (SEQ ID NO8 combined with SEQ ID NO 10, SEQ ID NO11 combined with SEQ ID NO12 or SEQ ID NO13 combined with SEQ ID NO12) of the repeat tract. The two broken ends may be re-joined by NHEJ repair pathway, while the repeat-containing fragment is eliminated. (E) Electrophoretic resolution of PCR products comprising theexpected DNA DSB sites. PCR amplicons with lower molecular weight were visible in multiplexed targeting conditions (lanes 7 to 9). (F) Western blot analysis of cell extracts showed the presence of ATXN3 species with reduced molecular weight (arrowheads) in conditions where SpCas9 D10A nickase was recruited to both CAG ends (lanes 6 to 8, n=l), that may possibly result from mutation-induced truncation of ATXN3.Detailed Description

[0057] The specificity of the CRISPR-Cas9 system is defined by a 20-nucleotide guide sequence within the sgRNA, and therefore the selection of the appropriate target DNA sequence is critical to maximize the efficiency of cleavage. Moreover, considering that the RNA-guided SpCas9 nuclease targets DNA sites in immediate vicinity of a 5'-NGG PAM sequence (where N= A, T, C or G), this constraint had to be considered while screening potential SpCas9 cleavage sites in the human ATXN3 gene. The selection of suitable target sites in the ATXN3 gene to achieve either i) gene knock-out or ii) the excision of the CAG tract was performed using bioinformatic tools, in which the selection criteria tend to maximize on-target activity, while minimizing the number of putative off-targets.

[0058] In the present disclosure, four sgRNAs to target exon 2 of the human ATXN3 gene (sgKO.l-SEQ ID NO1, sgKO.2-SEQ ID NO2, sgKO.3-SEQ ID NO3, sgKO.4-SEQ ID NO4) were designed and used these sequences for ATXN3 gene knock-out experiments.

[0059] In the present disclosure, sgRNA sequences were designed to remove the region comprising the CAG tract, located at the exon 10 of the human ATXN3 gene.

[0060] For that, in a first approach four sgRNA sequences (sgl9Tl-SEQ ID NO5, sgElOTl-SEQ ID NO6, sgE10T2-SEQ ID NO7, sgE10T3-SEQ ID NO8) have been designed to target the immediate vicinity of the CAG tract: sgRNA guide sequence SEQ ID NO5 targets intron 9 (upstream region of the CAG tract), while the remaining guide sequences (SEQ ID NO6, SEQ ID NO7 and SEQ ID NO8) target exon 10 downstream of the CAG tract. The simultaneous introduction of two DSB flanking the repeat tract at either end will be achieved by the recruitment of a catalytically active SpCas9. Importantly, in an embodiment, since sgRNA guide SEQ ID NO6 is designed to target a DNA sequence that includes a known exonic single-nucleotide polymorphism (C987GG / G987GG: rsl2895357), this might be relevant for the development of an allelespecific CRISPR-Cas9-based approach: the G nucleotide might be substituted by a C nucleotide in the sgRNA sequence.

[0061] In a second approach to remove the region comprising the CAG tract, a panel of sgRNA sequences able to recruit SpCas9 nickases (sgNickl9TlA-SEQ ID NO9; sgNickl9TlB-SEQ ID NO5; sgNickElOTlA-SEQ ID NO8; sgNickElOTIB-SEQ ID NQ10; sgNickE10T2A-SEQ ID NO11; sgNickE10T2B-SEQ ID NO12;sgNickE10T3A-SEQ ID N013) was also designed in the present disclosure. This approach brings a considerable advantage, since a DSB will only be generated if a pair of opposite oriented SpCas9 nickases is in close proximity12, while individual single-stranded breaks inserted in potential off-target sites are repaired with high-fidelity. During the design process of the double-nicking strategy by a SpCas9 variant, in the present disclosure, the following aspects were took into consideration: a) PAM sites were located outside of the target region ("PAM-out" configuration); b) a D10A nickase variant (D10A point mutation into the RuvC) was used in the scope of the present disclosure; c) combined sgRNAs were designed to target opposite DNA strands in close proximity, i.e., nick sites were separated by 37-68 base pairs (optimal spacing for D10A nickases). In this approach the pair of sequences SEQ ID NO9 and SEQ ID NO5 were designed to target the upstream region of the repeat tract (nick sites separated by 48 base pairs), while the remaining sequences were designed to target the downstream region of CAGs, being paired as follows: SEQ ID NO8 and SEQ ID NQ10 (nick sites separated by 43 base pairs); SEQ ID NO11 and SEQ ID NO12 (nick sites separated by 49 base pairs); SEQ ID NO13 and SEQ ID NO12 (nick sites separated by 60 base pairs).

[0062] The non-targeting control sgRNA sequence used in the framework of the present disclosure, targets the lacZ gene from Escherichia coli (sgCTRL-SEQ ID NO14) and has previously been described elsewhere.

[0063] In the present disclosure, the design of the guide sequences was based on the annotated reference sequence for the human ATXN3 gene (SEQ ID NO15) available at the National Center for Biotechnology Information (NCBI) database (accession code NG_008198.2 - https: / / www.ncbi.nlm.nih.gov / gene / 4287, date of last access 19thApril 2023).

[0064] In the present disclosure, the co-expression of SpCas9 and sgRNAs (SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8 or SEQ ID NO14) was achieved through the use of the lentiCRISPRv2 expression plasmid, obtained from Addgene (plasmid #52961, Watertown, USA). This third-generation lentiviral backbone includes a humanized SpCas9-codifying sequence (FLAG-tagged), a puromycin resistance gene and a chimeric sgRNA-codifying region with a cloning site for the specific 20-nucleotide guide sequences immediately upstream of an invariant scaffold sequence. The cloning site includes a BsmBI (Esp3l) restriction site, so that upon digestion, a pair of annealed oligonucleotides codifying for the 20-nucleotide guide sequences can be cloned into the sgRNA backbone.

[0065] In the present disclosure, the lentiCRISPRv2 vector was initially digested and dephosphorylated with BsmBI (Esp3l, Thermo Fisher Scientific, Waltham, USA) and the resulting product was purified upon electrophoretic separation, using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany).

[0066] In an embodiment, pairs of partially complementary oligonucleotides (top and bottom) encoding the guide sequences (SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO6, SEQ ID NO7, SEQ ID NO8 or SEQ ID NO14) and containing overhangs matching the BsmBI digested plasmid were synthetized by Invitrogen (Waltham, USA). Importantly, since the U6 RNA polymerase III promoter used to express the sgRNA requires a guanine nucleotide at the 5' terminus of the codifying strand, an extra guanine was included in all cases. Each pair of oligonucleotides was phosphorylated and annealed by mixing 1 pL of top and bottom oligonucleotides (100 pM, Invitrogen, Waltham, USA), 2 pL lOx Reaction buffer (New England Biolabs, Ipswich, USA), 1 pL ATP 25mM (New England Biolabs, Ipswich, USA) and 1 pL T4 Polynucleotide Kinase (New England Biolabs, Ipswich, USA), in a total reaction volume of 20 pL. The mixture was incubated in a Veriti 96-Well thermal cycler (Applied Biosystems, Waltham, USA), initially at 379C for 30 minutes, followed by an incubation at 959C for 5 minutes and a ramp down to 49C by 0.19C / second.

[0067] In an embodiment, the annealed oligonucleotide pairs (1 pL of the previous reaction at a 1:50 dilution) were finally ligated to the open vector (25 ng) using 0.75 pL of T7 DNA ligase (New England Biolabs, Ipswich, USA) and 5 pL 2x T7 DNA ligase reaction buffer (New England Biolabs, Ipswich, USA) in a reaction of 10 pL total volume, incubated at room temperature for 30 minutes.

[0068] In order to avoid the potential recombination of the long-terminal repeats (LTRs), in the present disclosure, the ligation product was transformed into One Shot Stbl3 chemically competent E. coli (Invitrogen, Waltham, USA). Briefly, 1 pl of each ligation product was added to 25 pL of Stbl3 cells and mixed gently. After the incubation for 20 minutes on ice, competent cells underwent a heat-shock step for 45 seconds at 429C and were immediately transferred to ice (2 minutes). Aseptic S.O.C. medium (Invitrogen, Waltham, USA) was added and competent bacteria were incubated at 379C for 1 hour at 225 rpm. Subsequently, each transformation was spread on a pre-warmed selective plate (100 pg / mL ampicillin, Enzo Life Sciences, Farmingdale, USA) and incubated overnight at 379C.

[0069] Surviving colonies were then selected and inoculated into 4 mL of lysogeny broth (LB, Fisher Scientific, Pittsburgh, USA) medium supplemented with 100 pg / mL ampicillin (Enzo Life Sciences, Farmingdale, USA) for 8 hours at 379C with constant shaking (200 rpm). Plasmid DNA was isolated from each culture using a NZYMiniprep kit (NZYTech, Lisbon, Portugal), following the manufacturer's instructions.

[0070] In the present disclosure, the correct insertion of each sgRNA-codifying oligonucleotide pair was confirmed by Sanger sequencing (GATC Biotech, Konstanz, Germany), using a primer designed to the U6 promoter and available at Addgene repository (primer LKO.l 5'-SEQ ID NO16, Watertown, USA). Furthermore, the evaluation of the LTRs' integrity was performed by restriction analysis using Hindlllrestriction enzyme (Thermo Fisher Scientific, Waltham, USA), followed by electrophoresis in a 1.5% agarose gel prepared with lx TAE buffer (Sigma-Aldrich, St. Louis, USA).

[0071] In the present disclosure, the preparation of sgRNAs (SEQ ID NO2, SEQ ID NO9, SEQ ID NO5, SEQ ID NO8, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13 or SEQ ID NO14) for adeno-associated viral (AAV) delivery was performed in a two-vector system due to constraints related with AAV packaging capacity.

[0072] In the present disclosure, sequences used for knock-out experiments in the adult brain (SEQ ID NO2 or SEQ ID NO14) were cloned into the AAV-SpGuide vector (plasmid #60958, pX552, Addgene, Watertown, USA). This plasmid codifies the sgRNA, downstream of a U6 promoter, and the enhanced green fluorescent protein (EGFP) fused to the KASH nuclear transmembrane domain under the control of the human synapsin 1 (hSynl) promoter, which allows the identification of positively transduced neurons. The AAV-SpGuide backbone includes a Sapl (Lgul) restriction site upstream of the sgRNA scaffold and was used to clone sequences SEQ ID NO2 and SEQ ID NO14. In each oligonucleotide the overhangs for the ligation were properly adapted to this vector, while maintaining the 20-nucleotide target sequences and the appended 5'-guanine, required by the U6 promoter.

[0073] After the digestion of the AAV-SpGuide vector with the Sapl (Lgul, Thermo Fisher Scientific, Waltham, USA) restriction enzyme, the resulting product was purified upon electrophoretic separation, using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany). Each pair of oligonucleotides was phosphorylated, annealed and ligated as described above. The ligation product was transformed into recombination-deficient bacteria (One Shot Stbl3, Invitrogen, Waltham, USA). Surviving colonies were then selected and inoculated into LB medium (Fisher Scientific, Pittsburgh, USA) supplemented with ampicillin (100 pg / mL, Enzo Life Sciences, Farmingdale, USA).

[0074] Plasmid DNA was isolated from each culture using the NZYMiniprep kit (NZYTech, Lisbon, Portugal) and sequenced (GATC Biotech, Konstanz, Germany) with the U6-forward primer (SEQ ID NO16) to confirm the correct insertion of the SEQ ID NO2 or SEQ ID NO14-codifying oligonucleotide pairs. The evaluation of the invert terminal repeats' (ITRs) integrity was performed by restriction analysis using Smal (Thermo Fisher Scientific, Waltham, USA), followed by electrophoresis in a 1.5% agarose gel in lx TAE buffer (Sigma-Aldrich, St. Louis, USA).

[0075] In the knock-out experiments, in combination with the cloned AAV-SpGuide vector, AAV-SpCas9 construct (plasmid #60957, pX551, Addgene, Watertown, USA) was also used to mediate the expression of the catalytically active SpCas9 in the mammalian brain. This plasmid drives the neuronal expression of HA-tagged SpCas9, under the control of the mouse methyl CpG binding protein 2 (pMecp2) promoter.

[0076] In the present disclosure, sequences designed to excise the CAG tract of ATXN3 gene using a nicking strategy (SEQ ID NO9, SEQ ID NO5, SEQ ID NO8, SEQ ID NO10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13), were cloned into a AAV-SpGuide vector adapted to accomplish the simultaneous delivery of two sgRNA molecules. In an embodiment, the original AAV-SpGuide vector (plasmid #60958, pX552, Watertown, USA), which already includes a Sapl (Lgul) restriction site to clone a guide sequence upstream of the sgRNA scaffold, was adapted to include an additional cloning site, which includes an Ajul restriction site. The generated plasmid, henceforward called AAV-SpGuide_Plus vector, was produced at GenScript Biotech (New Jersey, USA).

[0077] In an embodiment, the cloning procedure was performed as follows. After the digestion of the AAV-SpGuide_Plus vector with the Sapl (Lgul, Thermo Fisher Scientific, Waltham, USA) restriction enzyme, the resulting product was purified upon electrophoretic separation, using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany). Each pair of the designed oligonucleotides (Invitrogen, Waltham, USA) encoding the guide sequences SEQ ID NO9, SEQ ID NO8, SEQ ID NO11 or SEQ ID NO13, the appended 5' -guanine required by the U6 promoter, and containing overhangs matching the Sapl (Lgul) digested plasmid, was phosphorylated, annealed and ligated as described above. The ligation product was transformed into recombination-deficient bacteria (One Shot Stbl3, Invitrogen, Waltham, USA). Surviving colonies were then selected and inoculated into LB medium (Fisher Scientific, Pittsburgh, USA) supplemented with ampicillin (100 pg / mL, Enzo Life Sciences, Farmingdale, USA).

[0078] Plasmid DNA was isolated from each culture using the NZYMiniprep kit (NZYTech, Lisbon, Portugal) and sequenced (GATC Biotech, Konstanz, Germany) with the 5'-Sequencing primer gene insertl (SEQ ID NO17) from Addgene repository (Watertown, USA) to confirm the correct insertion of sequences SEQ ID NO9, SEQ ID NO8, SEQ ID NO11 or SEQ ID NO13-codifying oligonucleotide pairs. The evaluation of the invert terminal repeats' (ITRs) integrity was performed by restriction analysis using Smal (Thermo Fisher Scientific, Waltham, USA), followed by electrophoresis in a 1.5% agarose gel in lx TAE buffer (Sigma-Aldrich, St. Louis, USA). In the present disclosure, a clone of each of the intended sequences exhibiting a low percentage of ITR recombination was selected for further cloning.

[0079] In the present disclosure, each of the generated plasmids, already containing a sgRNA guide sequence (SEQ ID NO9, SEQ ID NO8, SEQ ID NO11 or SEQ ID NO13), was digested with Ajul (Thermo Fisher Scientific, Waltham, USA) restriction enzyme. The resulting product was purified upon electrophoretic separation, using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany). At this point, a second pair of annealed oligonucleotides codifying for the 20-nucleotide guide sequences (SEQ ID NO5, SEQ ID NQ10 or SEQ ID NO12), the appended 5' -guanine required by the U6 promoter, and the overhangs matching the Ajul digested plasmids was ligated as follows: i) AAV-SpGuide_Plus vector containing SEQ ID NO9 guide sequence was ligated with the annealed oligonucleotides encoding SEQ ID NO5; i) AAV-SpGuide_Plus vector containing SEQ ID NO8 guide sequence was ligated with the annealedoligonucleotides encoding SEQ ID NO10; iii) i) AAV-SpGuide_Plus vector containing SEQ ID NO11 guide sequence was ligated with the annealed oligonucleotides encoding SEQ ID NO12; iv) i) AAV-SpGuide_Plus vector containing SEQ ID NO13 guide sequence was ligated with the annealed oligonucleotides encoding SEQ ID NO12. The ligation product was transformed into recombination-deficient bacteria (One Shot Stbl3, Invitrogen, Waltham, USA). Surviving colonies were then selected and inoculated into LB medium (Fisher Scientific, Pittsburgh, USA) supplemented with ampicillin (100 pg / mL, Enzo Life Sciences, Farmingdale, USA).

[0080] Plasmid DNA was isolated from each culture using the NZYMiniprep kit (NZYTech, Lisbon, Portugal) and sequenced (GATC Biotech, Konstanz, Germany) with the 5'-Sequencing primer gene insertl (SEQ ID NO17) from Addgene (Watertown, USA) to confirm the correct insertion of the intended SEQ ID NO5, SEQ ID NQ10 or SEQ ID NO12-codifying oligonucleotide pairs. The evaluation of the invert terminal repeats' (ITRs) integrity was performed by restriction analysis using Smal (Thermo Fisher Scientific, Waltham, USA), followed by electrophoresis in a 1,5% agarose gel in lx TAE buffer (Sigma-Aldrich, St. Louis, USA).

[0081] In the present disclosure, for the precise excision of the repeat tract using a paired Cas9 nickase strategy, the previously generated plasmids were employed in combination with a AAV-SpCas9 nickase codifying plasmid obtained from Addgene (plasmid #112719, Watertown, USA). This plasmid drives the neuronal expression of HA-tagged SpCas9 D10A nickase variant (D10A point mutation into the RuvC nuclease domain), under the control of the mouse methyl CpG binding protein 2 (pMecp2) promoter.

[0082] After sgRNA design and construction, in the present disclosure, functional characterization of the designed systems was performed in HEK293T cells. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, USA) high glucose, supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen, Waltham, USA) and 1% penicillin / streptomycin (Gibco, Invitrogen, Waltham, USA) at 379C, under a humidified atmosphere containing 5% CO2.

[0083] For transfection experiments, 2.75 x 105cells were plated per well in 12-well plates 24 hours before transfection and plasmid DNA was transfected using Lipofectamine 3000 reagent (Invitrogen, Waltham, USA). Complex formation was performed by combining a mixture of Lipofectamine-Opti-MEM reduced serum media (Gibco, Invitrogen, Waltham, USA) and DNA-Qpti-MEM-P3000 Reagent (Invitrogen, Waltham, USA) in a 1:1 ratio, in accordance with the manufacturer's instructions. Initially, Lipofectamine 3000 was diluted in Opti-MEM and added to a second solution containing the plasmid DNA diluted in Opti- MEM and P3000 reagent. The mixture was incubated at room temperature for 5 minutes and added dropwise to cell cultures. Medium was completely replaced 4 hours later.

[0084] In knock-out experiments, the assessment of sgRNAs' functionality was performed upon transfection of HEK293T cells with 1.6 pg of the generated lentiviral plasmids in technical triplicates: LV-SpCas9-sgKO.l (SEQ ID NO1), LV-SpCas9-sgK0.2 (SEQ ID NO2), LV-SpCas9-sgK0.3 (SEQ ID NO3), LV- SpCas9-sgK0.4 (SEQ ID NO4). Cells transfected with LV-SpCas9-sgCTRL (SEQ ID NO14) were used as controls.

[0085] In an embodiment, sequences designed to target the vicinity of exon 10 in the human ATXN3 gene, were initially validated individually to assess each sgRNA's efficiency to recruit the catalytically active SpCas9 to the locus of interest, and subsequently in combination to evaluate the ability of the generated constructs to excise the CAG tract. Individual transfections were performed in technical triplicates as previously described using 1.6 pg of the following sequences: LV-SpCas9-sgl9Tl (SEQ ID NO5), LV-SpCas9-sgE10Tl (SEQ ID NO6), LV-SpCas9-sgE10T2 (SEQ ID NO7), LV-SpCas9-sgE10T3 (SEQ ID NO8) or the non-targeting sequence LV-SpCas9-sgCTRL (SEQ ID NO14), serving as the experimental control. Combined transfections were performed using 800 ng of each plasmid DNA in technical triplicates as follows: i) LV-SpCas9-sgCTRL (SEQ ID NO14) & LV-SpCas9-sgl9Tl (SEQ ID NO5); ii) LV-SpCas9-sgCTRL (SEQ ID NO14) & LV-SpCas9-sgE10Tl (SEQ ID NO6); iii) LV-SpCas9-sgCTRL (SEQ ID NO14) & SpCas9-sgE10T2 (SEQ ID NO7); iv) LV-SpCas9-sgCTRL (SEQ ID NO14) & LV-SpCas9-sgE10T3 (SEQ ID NO8); v) LV-SpCas9- sgl9Tl (SEQ ID NO5) & LV-SpCas9-sgE10Tl (SEQ ID NO6); vi) LV-SpCas9-sgl9Tl (SEQ ID NO5) & LV-SpCas9- sgE10T2 (SEQ ID NO7) and vii) LV-SpCas9-sgl9Tl (SEQ ID NO5) & LV-SpCas9-sgE10T3 (SEQ ID NO8). Control condition consisted on the transfection of 1.6 ug of the LV-SpCas9-sgCTRL construct (SEQ ID NO14).

[0086] Twenty-four hours after transfection cell medium was completely substituted by fresh medium containing 10 pg / mL puromycin (Gibco, Invitrogen, Waltham, USA) and cultures were maintained for 48 hours in antibiotic selection conditions. Cell collection was performed 72 hours post-transfection, for either genomic DNA purification or protein extract preparation.

[0087] In an embodiment, sequences designed to excise the CAG tract of the ATXN3 gene using a paired Cas9 nickase strategy (SEQ ID NO9, SEQ ID NO5, SEQ ID NO8, SEQ ID NQ10, SEQ ID NO11, SEQ ID NO12, SEQ ID NO13), were validated in combination of a total of two or four sequences. Combined transfections were performed as previously described, using Lipofectamine 3000 complexes, and 700 ng of each plasmid DNA (in a total of 2.1 ug DNA per condition) in technical triplicates as follows: i) AAV-SpCas9 (D10A) & AAV-sgCTRL (SEQ ID NO14) & AAV-sgRNA expressing sequences sgNickl9TlA (SEQ ID NO9) and sgNickl9TlB (SEQ ID NO5); ii) AAV-SpCas9 (D10A) & AAV-sgCTRL (SEQ ID NO14) & AAV-sgRNA expressing sequences SgNickElOTlA (SEQ ID NO8) and SgNickElOTIB (SEQ ID NQ10); iii) AAV-SpCas9 (D10A) & AAV- sgCTRL (SEQ ID NO14) & AAV-sgRNA expressing sequences sgNickE10T2A (SEQ ID NO11) and sgNickE10T2B (SEQ ID NO12); iv) AAV-SpCas9 (D10A) & AAV-sgCTRL (SEQ ID NO14) & AAV-sgRNA expressing sequences sgNickE10T3A (SEQ ID NO13) and sgNickE10T2B (SEQ ID NO12); v) AAV-SpCas9 (D10A) & AAV-sgRNA expressing sequences sgNickl9TlA (SEQ ID NO9) and sgNickl9TlB (SEQ ID NO5) & AAV-sgRNA expressing sequences SgNickElOTlA (SEQ ID NO8) and SgNickElOTIB (SEQ ID NQ10); vi) AAV- SpCas9 (D10A) & AAV-sgRNA expressing sequences sgNickl9TlA (SEQ ID NO9) and sgNickl9TlB (SEQ IDN05) & AAV-sgRNA expressing sequences sgNickE10T2A (SEQ ID N011) and sgNickE10T2B (SEQ ID NO12); vii) AAV-SpCas9 (D10A) & AAV-sgRNA expressing sequences sgNickl9TlA (SEQ ID NO9) and sgNickl9TlB (SEQ ID NO5) & AAV-sgRNA expressing sequences sgNickE10T3A (SEQ ID NO13) and sgNickE10T2B (SEQ ID NO12). Control condition consisted on the transfection of 700 ng of AAV-SpCas9 (D10A) and 1.4 ug of the AAV-sgCTRL (SEQ ID NO14).

[0088] Given the absence of a selection gene in the AAV constructs used to drive the expression of the sgRNA and SpCas9 nickase in vitro, cell collection was performed 48 hours post-transfection, for either genomic DNA purification or protein extract preparation.

[0089] In an embodiment, potential off-target loci in the human genome were computationally predicted for the designed knock-out sequences (SEQ ID NO1, SEQ ID NO2, SEQ ID NO3, SEQ ID NO4) using the Benchling web tool (https: / / benchling.com / ). This tool is based on a previously designed algorithm and takes into consideration that SpCas9 can mediate the cleavage of genomic off-targets in the presence of either 5'-NGG or 5'-NAG PAM sequences. The screen identifies 50 putative off-targets per guide sequence, scored in accordance with the likelihood of off-target binding. Genomic locations of the predicted off- targets in the human genome were determined using the UCSC genome browser (https: / / genome.ucsc.edu / ).

[0090] In the present disclosure, lentiviral vectors were produced in HEK293T cells with a four-plasmid system, as previously described. Human immunodeficiency virus type 1 (HIV-1) vectors were pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) envelope and encoded for the human mutant ATXN3 with 72 glutamines (LV-PGK-ATXN3 72Q).

[0091] Lentiviral particles were concentrated by ultracentrifugation of the culture medium containing virus and resuspended in sterile 0.5% bovine serum albumin (BSA, Millipore, Burlington, USA) in phosphate-buffered saline (PBS). The viral particle content of each batch was determined using an ELISA kit to detect HIV-1 p24 antigen levels (Retro Tek, Gentaur, Brussels, Belgium), following the instructions provided by the manufacturer.

[0092] In the present disclosure, adeno-associated virus serotype 1 / 2 (AAV1 / 2), encoding for AAV- SpCas9, AAV-sgCTRL (SEQ ID NO14) and AAV-sgKO.2 (SEQ ID NO2) were produced in HEK293T cells as described elsewhere. Apart from the transgene of interest, cells were transfected with plasmids encoding for the AAV1 and AAV2 serotypes and with the adenoviral helper genes (pHelper). AAV vectors were harvested from transfected cells and purified by fast protein liquid chromatography (FPLC), using an AKTA pure 25 system (GE Healthcare, Life Sciences). AAV titer was determined by quantitative real-time PCR using the AAVpro Titration kit (for Real Time PCR) Ver.2 (Takara Bio Inc, Kusatsu, Japan), following the manufacturer's instructions.

[0093] Six-week-old C57BL / 6 mice were obtained from Charles River Laboratories (Barcelona, Spain). Animals were housed in a temperature-controlled room and maintained on a 12 hour light / dark cycle. Food and water were provided ad libitum. Experiments involving animals were carried out in accordance with the European Community directive (2010 / 63 / EU), that legislates the protection of animals used for scientific purposes. Researchers received suitable training (FELASA-certified course) and certification from Portuguese authorities (Direcgao Geral de Veterinaria) to perform the experiments.

[0094] In the present disclosure, seven-week-old C57BL / 6 mice were anesthetized by intraperitoneal administration of a mixture of xylazine / ketamine (4 / 80 mg / Kg body weight; Rompun, Bayer, Leverkusen, Germany / Clorketam 1000, Vetoquinol, Lure, France). Mice were stereotaxically injected with viral vectors into the striatum, using the following coordinates calculated from bregma: anteroposterior: +0.6 mm; lateral: ±1.8 mm; ventral: -3.3 mm.

[0095] In the present disclosure, for the evaluation of editing efficiency, animals were bilaterally coinjected with i) concentrated lentiviral vectors, LV-PGK-A TX / V3 72Q. (300 ng of p24 antigen), ii) AAV1 / 2- SpCas9 (2xl09vg) and iii) AAVl / 2-sgCTRL: SEQ ID NO14 (2xl09vg, left hemisphere) or AAVl / 2-sgK0.2: SEQ ID NO2 (2xl09vg, right hemisphere), in a total volume of 2 pL.

[0096] Concentrated viral stocks were thawed on ice and diluted using sterile 0.5% BSA / PBS (mixes containing AAV and lentiviral vectors). Injections were performed using a 10-pL Hamilton syringe at an infusion rate of 0.25 pL / min. After the infusion, the syringe was allowed to remain in place for 5 minutes and then retracted 0.3 mm, where it was held for an additional 2 minutes prior to its complete removal, in order to allow viral diffusion and minimize backflow.

[0097] After surgery mice were maintained in their home cages and sacrificed 4 weeks later for tissue processing.

[0098] In the present disclosure, genomic DNA from in vitro and in vivo samples was extracted using the GeneJET Genomic DNA purification kit (Thermo Fisher Scientific, Waltham, USA) accordingly with the manufacturer's instructions. HEK293T cell cultures were rinsed with PBS before extraction.

[0099] For the extraction of genomic DNA from mice tissue, animals were sacrificed with a lethal dose of xylazine / ketamine (8 / 160 mg / Kg body weight) and brains quickly removed from the heads. Mice striata were dissected and stored at -809C until genomic DNA isolation. Each striatum was subsequently placed on iced patches and sliced into several pieces, using a clean scalpel. Only half of the striatal tissue was homogenized and digested for further genomic DNA purification.

[0100] In all cases, genomic DNA concentration and purity were determined using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

[0101] To evaluate CRISPR-Cas9 efficiency in indel (insertions / deletions) generation, a Surveyor nuclease assay (Integrated DNA Technologies, Coralville, USA) was performed. The first step of this assay involves the genomic PCR amplification of the region surrounding the CRISPR target sites. In an embodiment, forward and reverse primers used to amplify exon 2 of the human ATXN3 gene (SEQ ID NO18 and SEQ ID NO19) and ATXN3 cDNA (SEQ ID NO20 and SEQ ID NO21) were designed and validated in the present disclosure (Table 1). The design was based on the annotated reference sequence of the ATXN3 gene (SEQ ID NO15) available at the National Center for Biotechnology Information (NCBI) database (accession code NG_008198.2 - https: / / www.ncbi.nlm.nih.gov / gene / 4287, date of last access 19thApril 2023) using online PrimerBlast softwareThe pair of primers used to amplify exon 10 of ATXN3 gene for Surveyor nuclease assay (Table 1, SEQ ID NO22 and SEQ ID NO23) has been previously designed and validated by us in a prior study (DOI: 10.1016 / j.jmoldx.2020.03.003). Briefly, each PCR reaction (50 pl) was prepared on ice, using the following components: lOpI of 5x Phusion HF Buffer (Thermo Fisher Scientific, Waltham, USA), 1 pl dNTPs lOmM (Thermo Fisher Scientific, Waltham, USA), forward and reverse primers, with a final concentration of 0.5 pM each, 50 ng of DNA template and 0.5 pl (one unit) of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, USA). The PCR-amplification was performed in a Veriti 96-Well thermal cycler (Applied Biosystems, Waltham, USA) with the following protocol: one cycle at 989C for 30 seconds (initial denaturation), and 35 cycles at 989C for 10 seconds (denaturation), X9C for 10 seconds (annealing temperature for pair of primers is available in Table 1), 729C for 30 seconds (extension), with a final extension at 729C for 10 minutes.

[0102] Table 1 - Primer sequences used for PCR-amplification of the CRISPR-edited regions of the human ATXN3 gene (Exon 2 and Exon 10) and ATXN3 cDNA of a lentiviral-based mouse model (Exon 2). Annealing temperatures and expected amplicon sizes are displayed.

[0103] PCR amplicons were purified with the NucleoSpin Gel and PCR clean-up (Macherey-Nagel, Duren, Germany), according to the manufacturer's recommendations and the concentrations were furtherassessed using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, USA). For DNA heteroduplex formation, 420 ng of each purified PCR product were used, following the recommended parameters. The Surveyor nuclease S (Integrated DNA Technologies, Coralville, USA) digestion was performed in a Veriti 96-Well thermal cycler (Applied Biosystems, Waltham, USA), at 429C for 60 minutes and the reaction was stopped with the addition of Stop Solution (Integrated DNA Technologies, , Coralville, USA) on ice.

[0104] The resulting fragments were separated by electrophoresis on a 10% polyacrylamide gel prepared with lx Tris-borate-ethylenediaminetetraacetic acid (TBE, Bio-Rad, Hercules, USA), along with the GeneRuler 100 bp DNA ladder (Thermo Fisher Scientific, Waltham, USA) or the GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific, Waltham, USA), at 60 Volts until the bromophenol blue dye had migrated to the bottom of the gel. Gels were stained with Gelstar dye (Lonza, Basel, Switzerland) diluted in lx TBE (1:10,000), for 30 minutes at room temperature, and visualized with the Gel Doc EZ imaging system (BioRad, Hercules, USA).

[0105] Estimation of cleavage intensity was performed through the measurement of the integrated intensity of the band corresponding to the undigested PCR amplicon (a) and the bands corresponding to the cleaved fragments (b and c), using the Image Lab software, version 5.1 (Bio-Rad, Hercules, USA). The fraction of the cleaved PCR product (fcut) was determined [fcut = (b + c) / a + b + c)] and indel frequency was calculated based on the formula: indel (%) = 100 x (1 — ^ / l — fcut)). Expected fragment sizes for each ATX7V3-edited region are defined in Table 2.

[0106] Table 2 - Expected sizes for the Surveyor nuclease-cleaved products for each ATXN3 region

[0107] To evaluate the capacity of the generated CRISPR-Cas9 and CRISPR-Cas9n systems to delete the polyQ-encoding region of the human ATXN3 gene, in the present disclosure this region was PCR-amplified in modified and unmodified HEK293T cells, using the pair of primers available in Table 1 (SEQ ID NO22 and SEQ ID NO23). Each PCR reaction (50 pl) was prepared on ice, as previously described. To check for the existence of PCR amplicons with lower molecular weigh in multiplexed targeting conditions, 10 pL of the PCR products were electrophoresed in 1.5% agarose gels, adjacent to the GeneRuler lkb DNA ladder (Thermo Fisher Scientific, Waltham, USA). Agarose gels were prepared with lx TAE buffer (Sigma-Aldrich, St. Louis, USA). PCR products (15 pl) were further purified using Exonucleasel (0.5 pl Exol, Thermo Fisher Scientific, Waltham, USA) and FastAP thermosensitive alkaline phosphatase (1 pl Exol, Thermo Fisher Scientific, Waltham, USA) in a Veriti 96-Well thermal cycler (Applied Biosystems, Waltham, USA) as follows: 379C for 20 minutes, followed by a step of 809C, 20 min. The purified PCR amplicons were characterized by Sanger sequencing (GATC Biotech, Konstanz, Germany).

[0108] In the present disclosure, proteins were extracted from HEK293T cells and mice striata with a radio-immunoprecipitation assay (RIPA) buffer: 50 mM Tris-base, pH 8.0 (Fisher Scientific, Pittsburgh, USA); 150 mM sodium chloride (NaCI, Acros Organics, Antwerp, Belgium); 5 mM ethylene glycol tetraacetic acid (EGTA, Sigma-Aldrich, St. Louis, USA); 1% Triton X-100 (Fisher Scientific, Pittsburgh, USA); 0.5% sodium deoxycholate (Sigma-Aldrich, St. Louis, USA); 0.1% sodium dodecyl sulphate (SDS, Fisher Scientific, Pittsburgh, USA), supplemented with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), 0.2 mM phenylmethylsulphonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, USA), 1 mM dithiothreitol (DTT, Sigma-Aldrich, St. Louis, USA), 1 mM sodium orthovanadate (Sigma-Aldrich, St. Louis, USA) and 5 mM sodium fluoride (Sigma-Aldrich, St. Louis, USA).

[0109] After rinsing cellular cultures with PBS, HEK293T cells were scraped into supplemented RIPA buffer. The lysate was further sonicated by 2 series of 4 seconds ultra-sound pulses (1 pulse / sec) and centrifuged at 13,400 ref for 20 minutes at 49C. The supernatant was collected, and protein concentration determined using Bradford reagent (Bio-Rad, Hercules, USA).

[0110] For the extraction of proteins from in vivo samples, mice were sacrificed as previously described and mice striata were dissected and subsequently sliced into several pieces, using a clean scalpel. Half of the striatal tissue was used for genomic DNA purification, while the second half was lysed with RIPA buffer and homogenized. After sonication (2 series of 4 pulses), protein concentration was assessed using Bradford reagent (Bio-Rad, Hercules, USA).

[0111] Protein samples were denatured with 6x sample buffer (0.5 M Tris-HCI / 0.4% SDS, pH 6.8, 9.3% DTT, 10% SDS, 30% glycerol and 0.012% bromophenol blue) and incubated for 5 minutes at 959C. Thirty to sixty micrograms of total denatured protein were loaded into a SDS-polyacrylamide gel (4% stacking,10% resolving, Bio-Rad, Hercules, USA) for subsequent electrophoretic separation. After protein transfer to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, USA), each membrane was blocked in 5% non-fat milk diluted in TBS-T (0.1% Tween 20 in Tris buffered saline, Fisher Scientific, Pittsburgh, USA) for 1 hour at room temperature. The following primary antibodies (diluted in blocking solution) were used for the overnight incubation at 49C: mouse monoclonal anti-ATXN3 antibody (1H9; 1:5000; Merck Millipore, Burlington, USA); mouse monoclonal anti-FLAG antibody (M2; 1:1000; Sigma- Aldrich, St. Louis, USA); mouse monoclonal anti-p-actin antibody (AC74; 1:10,000; Sigma-Aldrich, St. Louis, USA); mouse monoclonal anti- -tubulin 1 antibody (SAP.4G5; 1:10,000; Sigma-Aldrich, St. Louis, USA). After three 15 min-washes in TBS-T membranes were finally incubated with the goat anti-mouse alkaline phosphatase-conjugated secondary antibody (1:10,000; Thermo Fisher Scientific, Waltham, USA) for 2 hours at room temperature. Membranes were washed (three washes of 15 min each) and revealed with enhanced chemifluorescence substrate (ECF, GE Healthcare, Chicago, USA) and protein bands were detected by chemifluorescence imaging (Chemidoc imaging system, Bio-Rad, Hercules, USA).

[0112] Semi-quantitative analysis was carried out based on the optical density of the scanned membranes using Image Lab analysis software (version 5.1, Bio-Rad, Hercules, USA). Normalization of the total amount of protein loaded in each lane was performed based on the amount of either p-tubulin or p- actin signals. In cellular experiments in which HEK293T cells were transfected with lentiCRISPRv2 expression plasmid (#52961, Addgene Watertown, USA), FLAG signals were also taken into consideration for the normalization of transfection efficiencies.

[0113] In the present disclosure, mice were sacrificed 4 weeks after stereotaxic injection with an overdose of xylazine / ketamine (8 / 160 mg / Kg body weight) and transcardially perfused with ice-cold PBS, followed by 4% PFA (Sigma-Aldrich, St. Louis, USA). Brains were collected and post-fixed in 4% PFA for 24 hours, at 49C. Cryoprotection was mediated by the immersion of brains in a 25% sucrose / PBS solution for 48 hours at 49C, allowing tissue dehydration. Brains were frozen at -809C and sliced into coronal sections with 25 pm in thickness, using a cryostat (LEICA CM3050S, Leica Microsystems, Wetzlar, Germany) at - 219C. The slices were collected in anatomical series, being stored at 49C as free-floating sections in PBS supplemented with 0.05% (m / v) sodium azide (Sigma-Aldrich, St. Louis, USA).

[0114] In the present disclosure, free-floating brain coronal sections were incubated for 30 minutes, at 379C, with a 0.1% phenylhidrazyne (Merck, Rahway, USA) / PBS solution, to block endogenous peroxidases. After permeabilization and blocking of nonspecific staining in PBS / 0.1% Triton X-100 with 10% NGS (Gibco, Invitrogen, Waltham, USA) for 1 hour at room temperature, sections were incubated overnight at 49C with primary antibodies diluted in blocking solution: rabbit polyclonal anti-ubiquitin antibody (1:300, Enzo Life Sciences, Farmingdale, USA) or rabbit polyclonal anti-dopamine and cyclic AMP- regulated neuronal phosphoprotein 32 (DARPP-32) antibody (1:1000, Merck Millipore, Burlington, USA). Sections were then washed and incubated with the biotinylated secondary goat anti-rabbit antibody(1:250, Vector Laboratories, Newark, USA) diluted in blocking solution, for 2 hours at room temperature. Bound antibodies were visualized using the Vectastain ABC kit (Vector Laboratories, Newark, USA), using the 3,3'-diaminobenzidine tetrahydrochloride (DAB, Vector Laboratories, Newark, USA) as substrate. Sections were then placed in slides, previously coated with a gelatine solution. After a dehydration process with ethanol solutions with increasing concentrations, and xylene substitute (Sigma-Aldrich, St. Louis, USA), sections were finally coverslipped with Eukitt quick-hardening mounting medium (Sigma-Aldrich, St. Louis, USA).

[0115] Images were captured with a slide scanner Zeiss Axio Scan.Zl microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany), equipped with a Hitachi HV-F202SCL camera, using the Plan-Apochromat 20x / 0.8 objective and the ZEN software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

[0116] In the present disclosure, free-floating brain coronal sections were incubated for 1 hour at room temperature in blocking / permeabilization solution (PBS / 0.1% Triton X-100 containing 10% NGS) and then incubated overnight at 49C with primary antibodies diluted in blocking solution: rabbit polyclonal antiionized calcium binding adaptor molecule 1 (lba-1) antibody (1:1000, Wako-Chemicals, Richmond, USA) and mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (GA5; 1:500; Merck Millipore, Burlington, USA). After a three-washing step, sections were incubated at room temperature for 2 hours, with secondary antibodies coupled to fluorophores: goat anti-rabbit Alexa Fluor 568 and goat anti-mouse Alexa Fluor 647 (1:250; Molecular Probes; Invitrogen, Waltham, USA), diluted in blocking solution. Sections were washed and further incubated with DAPI (1:5000, Sigma-Aldrich, St. Louis, USA), before being placed in gelatine-coated slides and coverslipped with fluorescence mounting medium (Dako, Glostrup, Denmark).

[0117] Images were captured with a slide scanner Zeiss Axio Scan.Zl microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany), equipped with a Hamamatsu ORCA-Flash 4.0 camera, using the Plan- Apochromat 20x / 0.8 objective and the ZEN software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

[0118] In the present disclosure, twelve stained sections per animal, distanced by 200 pm from each other, were used to perform the quantitative analysis of the total number of ubiquitin-positive inclusions as well as the extent of DARPP-32 loss in the striatum. The total number of inclusions was manually counted in all 12 sections and further multiplied by 8 to account for the intermediate sections. The quantification of DARPP-32 depleted areas in mice striatal sections was performed using an imageanalysis software (Image J software). The calculation of DARPP-32-depleted volume was based on the following formula: Volume = d(al + a2 + — F al2), where d is the distance between serial sections and al to al2 represent the individual areas for serial sections.

[0119] In the present disclosure, the quantitative analysis of the lba-1 and GFAP immunoreactivity was performed with ZEN 2 Blue edition software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) in the transduced striatal regions relative to their corresponding non-affected cortex (defined as background). Twelve stained-sections per animal, distanced by 200 pm from each other, were used for the purpose. Images were taken under the same image acquisition conditions and uniform adjustments of brightness and contrast were made to all images.

[0120] In the present disclosure, data are presented as mean ± standard error of mean (SEM). Statistical comparisons were performed using either one-way ANOVA followed by Dunnett's post hoc test or paired Student's t-test. Significance thresholds were set at *p<0.05 and **p<0.01 and considered non-significant (ns) when p>0.05. Calculations were performed using GraphPad Prism version 6.00 for Windows. Outliers were excluded from analysis using Grubb's test.ResultsGeneration and validation of sgRNA sequences for the human ATXN3 gene editingPermanent inactivation of the ATXN3 gene in HEK293T cells

[0121] Upon the induction of a DSB and the subsequent activation of the NHEJ repair pathway, short insertions and deletions (indels) can be typically found in the region surrounding the break site. When introduced into the coding sequence of a gene, these indels often result in frameshift mutations that create premature stop codons, leading to mRNA degradation by nonsense-mediated decay or to the production of non-functional truncated proteins. Therefore, this repair pathway can be exploited to induce gene knock-outs, which is likely to occur when targeting a gene near its 5' -end.

[0122] Aiming at permanently blocking the human ATXN3 gene expression, in this disclosure CRISPR- associated nuclease SpCas9 was programmed to target the exon 2 of this gene. In an embodiment, four 20-nucleotide guide sequences were designed for this purpose (sgKO.l-SEQ. ID NO1, sgKO.2-SEQ ID NO2, sgKO.3-SEQ ID NO3, sgKO.4-SEQ ID NO4, relative positions indicated in Figure 1A) and cloned into a lentiviral backbone (lentiCRISPRv2, plasmid #52961, Addgene), co-expressing both SpCas9 (FLAG-tagged) and the sgRNA scaffold.

[0123] In this disclosure, and in order to evaluate the gene editing efficiency of the generated guide sequences, HEK293T cells were transfected with the sgRNA-expressing plasmids, maintained in culture under puromycin selection conditions and harvested 72 hours after transfection (Figure IB). Cells transfected with a guide sequence targeting the bacterial lacZ gene (sgCTRL-SEQ. ID NO14) were used as a negative control.

[0124] In this disclosure, the ability of each sgRNA to promote the generation of indels was assessed by the Surveyor nuclease assay as follows. After PCR-amplification of the sgRNA-target sites, amplicons wereslowly reannealed to generate heteroduplexes that were subsequently cleaved by Surveyor nuclease, while leaving homoduplexes intact. The visualization of the Surveyor products upon electrophoretic separation (Figure 1C) demonstrated the ability of all the designed sgRNAs to induce a DSB at the target site, consequently modifying the ATXN3 gene in this cellular line. Locus modification efficiencies (percentage of indels) were determined in four independent experiments (Figure ID; SEQ ID NO1: 53.38 ± 0.54%; SEQ ID NO2: 49.33 ± 2.28%; SEQ ID NO3: 56.26 ± 2.22%; SEQ ID NO4: 56.04 ± 1.59%) showing the high editing efficiency of each of the sgRNAs.

[0125] As a consequence of the expression of the four guide sequences in HEK293T cells, a significant reduction of ATXN3 protein levels in CRISPR-edited cells was observed, as determined by western blot (Figure IE and F; SEQ ID NO14: 1.00 ± 0.17 vs SEQ ID NO1: 0.50 ± 0.08, SEQ ID NO2: 0.43 ± 0.13, SEQ ID NO3: 0.44 ± 0.10, SEQ ID NO4: 0.48 ± 0.09), suggesting that the developed gene editing approach is effective in the inactivation of ATX N 3 gene as a result of NHEJ-mediated DSB repair.Although SpCas9 binding specificity is determined by the Watson-Crick base-pairing interactions of a 20-nucleotide guide sequence with its target DNA, SpCas9 tolerates mismatches throughout the guide sequence, especially at PAM distant sites. Moreover, off-target sites followed by a 5'-NAG PAM sequence, have also been reported. In this disclosure, the Benchling web tool (https: / / benchling.com / ) was used to computationally predict potential off-targets for each sgRNA in the human genome. The screen identified 50 putative off- targets per guide, scored in accordance with the likelihood of off-target binding (Figure 2A). Full complementarity (zero mismatches) was exhibited by all guides only to the ATXN3 gene, while the majority of the retrieved off-targets carry a total of four mismatches (Figure 2B). Off-target genomic locations in the human genome were determined using the UCSC genome browser and mapped to intergenic, intronic and exonic regions (Figure 2C).

[0126] Considering that the four generated guide sequences demonstrated the ability to modify the ATXN3 gene, consequently reducing the expression of ATXN3 protein in this cellular line in the same order of magnitude ("'0.5 fold-change), the focus was on the sgKO.2 guide sequence (SEQ ID NO2) for subsequent in vivo experiments. Co-expression of SpCas9 and sgCTRL (SEQ ID NO14) or sgKO.2 (SEQ ID NO2), will be henceforth designated as CRISPR-CTRL and CRISPR-ATXN3, respectively.CRISPR-Cas9 system mediates ATXN3 gene disruption in the brain of a MJD / SCA3 mouse model, leading to the consequent reduction of mutant ATXN3 protein levels

[0127] Having demonstrated the efficacy of this gene editing system in vitro, in the present disclosure it was sought to evaluate a viral delivery strategy of CRISPR-Cas9 components into the mammalian brain.

[0128] Viral vectors based on lentivirus and AAVs have been described as efficient tools for in vivo gene delivery in disorders of the central nervous system. Differences in the safety profile of these two vectors, particularly the absence of pathogenicity of the wild-type AAVs, have attracted much attention to the latter. Low immunogenicity and very low rate of integration and potential insertional mutagenesis, inaddition to the efficient and sustainable neuronal transduction have contributed to the investment in AAV vectors for in vivo gene therapy, now a reality in clinical practice.

[0129] Considering the advantages of AAVs over lentivirus, the previously described SEQ ID NO14 (sgCTRL) and SEQ ID NO2 (sgKO.2) guide sequences were cloned into AAV vectors for in vivo delivery. Due to limits related with the packaging capacity of AAVs (~4.5 kb), a dual-vector system was used in which the SpCas9 (AAV-SpCas9, plasmid #60957, pX551, Addgene) and the sgRNA expressing cassettes (AAV- SpGuide, plasmid #60958, pX552, Addgene) are packaged in two distinct viral vectors.

[0130] In order to evaluate the therapeutic potential of the developed CRISPR-Cas9 system in vivo, the lentiviral-based mouse model of MJD / SCA3 was used. The generation of this model involves the stereotaxic injection, in the mouse striatum, of lentiviral vectors encoding for the human mutant ATXN3 protein with 72 glutamines, under the control of PGK promoter (LV-PGK-ATX7V3 72Q). The expression of mutant ATXN3 in the brain parenchyma results in its accumulation and aggregation, in parallel with neurodegeneration of transduced brain regions. Protein levels, number and size of ATXN3 aggregates, as well as the loss of neuronal markers can be precisely quantified, providing a particularly adequate quantitative pre-clinical model of MJD / SCA3.

[0131] In the present disclosure, stereotaxically co-injected LV-PGK-A TXN3 72Q and AAV1 / 2 vectors encoding SpCas9 and either the sgCTRL-SEQ ID NO14 (CRISPR-CTRL system, left hemisphere) or the sgKO.2-SEQ ID NO2 (CRISPR-ATXN3 system, right hemisphere) were stereotaxically co-injected in the striatum of seven-week-old mice (Figure 3A).

[0132] Surveyor analysis of CRISPR-ATXN3 (SEQ ID NO2) transduced striatal tissues showed an effective ATXN3 gene disruption, 4 weeks after viral injection. Locus modification efficiencies were determined in a total of five treated hemispheres (Figure 3B and C; 39.2% ± 1.46 efficiency in indel formation).

[0133] Immunoblotting of total protein extracts (Figure 3D and E) revealed a robust reduction in the levels of ATXN3 aggregates (CRISPR-CTRL: 1.00 ± 0.22 vs CRISPR-ATXN3: 0.08 ± 0.03, SEQ ID NO14 vs SEQ ID NO2, respectively). Reduction in soluble levels of the mutant form of the protein did not reach statistical significance, possibly as a result of study underpowerment (CRISPR-CTRL: 1.00 ± 0.18 vs CRISPR-ATXN3: 0.44 ± 0.1; n=5). No differences in the mouse endogenous Atxn3 levels were observed in treated hemispheres (CRISPR-CTRL: 1.00 ± 0.04 vs CRISPR-ATXN3: 0.96 ± 0.05).

[0134] Overall, data presented in the present disclosure indicate that CRISPR-ATXN3 optimized system (SEQ ID NO2) is highly efficient on the disruption of the human ATXN3 gene in vivo, preventing the formation of mutant ATXN3 protein, observations that are in accordance with the in vitro results.In vivo delivery of CRISPR-ATXN3 system alleviates neuropathology in a mouse model of MJD / SCA3

[0135] The lentiviral-based mouse model of MJD / SCA3 used in this disclosure is characterized by neuropathological deficits, as a result of mutant ATXN3 expression in the brain parenchyma. Apart fromthe development of neuronal intranuclear inclusions, containing the aggregated protein, this model is also characterized by an early neuronal dysfunction, detected by the large depleted area of the marker DARPP- 32, a regulator of dopamine receptor signalling. Therefore, in the present disclosure the potential of the developed CRISPR-ATXN3 (SEQ ID NO2) to prevent the appearance of MJD / SCA3-associated neuropathology in this model of the disease was evaluated.

[0136] In an embodiment, immunohistochemical analysis of coronal sections obtained from injected mice showed a drastic reduction in the total number of ubiquitin-positive inclusions in CRISPR-ATXN3 (SEQ ID NO2) injected hemispheres when compared with the contralaterally injected CRISPR-CTRL (SEQ ID NO14) hemispheres (Figure 4A, B and C; CRISPR-CTRL: 25410 ± 4583 aggregates vs CRISPR-ATXN3: 1061 ± 371.9 aggregates). Moreover, immunostaining for DARPP-32 showed a robust reduction of the depletion volume after ATXN3 gene disruption (Figure 4D, E and F; CRISPR-CTRL: 0.30 ± 0.05 mm3vs CRISPR-ATXN3: 0.05 ± 0.02 mm3), which is in accordance with the previous observations.

[0137] Mutant ATXN3 expression in the brain parenchyma also induces a local increase of neuroinflammatory markers, such as lba-1 and GFAP, revealing microglial recruitment and astrocytic activation at the injection site. Comparing to non-treated hemispheres (SEQ ID NO14, Figure 5A and D), lba-1 immunoreactivity was reduced upon CRISPR-ATXN3 (SEQ ID NO2) delivery (right hemispheres, Figure 5B-C), although no statistically significant differences between hemispheres were observed in GFAP immunoreactivity (Figure 5 E-F).

[0138] Overall, these results suggest that, in the lentiviral-based mouse model of MJD / SCA3, CRISPR- mediated ATXN3 gene disruption led to a marked phenotypic amelioration of neuropathological signs. ATXN3 inactivation caused by one hand, the clearance of ubiquitin-positive inclusions that are a hallmark of MJD / SCA3, while avoiding the appearance of the striatal neuronal degeneration and neuroinflammation, key neuropathological markers characteristic of this model of MJD / SCA3.CRISPR-Cas9-mediated deletion of the CAG repeat motif of the ATXN3 gene

[0139] A different gene editing approach for repeat disorders relies on the elimination of nucleotide repeat expansions. This might be accomplished through the simultaneous introduction of two DSBs flanking the repeat tract at either end. The two broken ends are re-joined by NHEJ repair pathway, while the repeat-containing fragment is eliminated.

[0140] In this disclosure, to remove the polyQ-encoding sequence of ATXN3 gene using CRISPR-Cas9, four sgRNAs targeting this region (relative positions in Figure 6A) were designed. Due to the CAG localization in the upstream region of exon 10, the design was expanded to the prior intron, in which a good sgRNA candidate was found (sgl9Tl-SEQ ID NO5). The other three candidates were designed to bind to the region downstream of the CAG tract: sgElOTl-SEQ ID NO6, sgE10T2-SEQ ID NO7 and sgE10T3-SEQ ID NO8.

[0141] These sequences were cloned into the lentiCRISPRv2 backbone (plasmid #52961, Addgene) and HEK293T cells were transfected for functional testing. Cells were maintained in culture under puromycin selection conditions and harvested 72 hours after transfection (Figure 6B). As a negative control, cells were transfected with a guide sequence targeting the bacterial lacZ gene (SEQ ID NO14). The Surveyor assay was used to determine the efficiency of each individual sgRNA to generate indels (Figure 6C; SEQ ID NO5: 29%; SEQ ID NO6: 40%; SEQ ID NO7: 28%; SEQ ID NO8: 32%). Locus modification efficiencies (percentage of indels) were determined on the basis of DNA cleaved fraction (indicated by arrowheads).

[0142] Given the ability of each individual sgRNA to mediate DSBs, the possibility of using these sequences in combination for CAG ablation was explored in the present disclosure (Figure 6D). HEK293T cells were co-transfected with the SEQ ID NO5 guide sequence, targeting the region upstream of the CAG tract, in combination with one of the sgRNA guides designed to the region downstream of the repeat motif (SEQ ID NO6, SEQ ID NO7 or SEQ ID NO8). Since the referred sequences were cloned in different plasmids, the expression of SpCas9 is mediated by both constructs. As such, in order to maintain the levels of SpCas9 equivalent, SEQ ID NO14 was used in combination with each of the active guides (SEQ ID NO5, SEQ ID NO6, SEQ ID NO7 or SEQ ID NO8) for the evaluation of individual outcomes.

[0143] The region encompassing the exon 10 of the ATXN3 gene was amplified by PCR and the generated amplicons electrophoresed in an agarose gel (Figure 6E). The designed multiplexed strategy targeting both ends of the CAG tract, led to the visualization of lower size PCR products (Figure 6E, lanes 6 to 8), with base lengths that are compatible with the ablation of the DNA sequence existing between the two expected DSB sites, including the CAG tract. Upon characterization of these PCR products with lower sizes by Sanger sequencing, the elimination of the repeat-containing fragment was confirmed.

[0144] In an embodiment, western blot analysis of HEK293T extracts revealed the presence of bands of decreased molecular weight in several conditions (Figure 6F), possibly corresponding to truncated versions of the ATXN3. Nonetheless, a more evident band is observed in conditions SEQ ID NO5 + SEQ ID NO7 and SEQ ID NO5 + SEQ ID NO8 at approximately 35 kDa (Figure 6F), which can be consistent with the expression of a modified allele.

[0145] Overall these results show the high potency of the generated guides to direct the SpCas9 nuclease to the ATXN3 gene, promoting the elimination of the CAG expansion in this human cellular line and the consequent formation of a truncated ATXN3 protein.CRISPR-Cas9 targeted nickases induced the excision of the CAG repeat motif of the ATXN3 gene

[0146] Although in the present disclosure putative off-targets of each of the sgRNAs targeting the vicinity of exon 10 (SEQ ID NO5-SEQ ID NO8) were not experimentally assessed, SpCas9 can mediate genomic off- target cleavage. Importantly, since a dual cleavage strategy is necessary for the repeat excision (DSBinsertion upstream and downstream of the CAG tract), the number of potential off-targets associated with the concomitant usage of two sgRNAs might be higher.

[0147] Therefore, in order to minimize potential off-targeting, in the present disclosure a SpCas9 nickase strategy for the precise excision of the repeat tract in the ATXN3 gene was also developed.

[0148] SpCas9 variants, developed by the mutation of each of the two catalytic domains (D10A or H840A point mutations into the RuvC or HNH, respectively) retained DNA-binding specificity, cutting either the DNA strand that is complementary to the sgRNA (D10A nickase) or the non-complementary strand (H840A nickase)4-10-X1. This approach brings a considerable advantage, since a DSB will only be generated if a pair of opposite oriented Cas9 nickases are in close proximity12, while individual single-stranded breaks inserted in potential off-target sites are repaired with high-fidelity.

[0149] In the present disclosure, a SpCas9 D10A variant has been employed.

[0150] To achieve CAG tract excision four different pairs of sgRNAs have been designed to target the upstream and downstream region of the repeat tract (relative positions in Figure 7A). Each pair of sgRNA guide sequences was designed to target opposite DNA strands, comprising a distance between nick sites in the range of 37-68 base pairs, since this has been reported as the optimal spacing for D10A nickase variants. Moreover, in all cases PAM sites are located outside the target region, since editing has also been reported to be much higher in this design in comparison with "PAM-in" configurations.

[0151] Due to the CAG localization in the upstream region of exon 10, once again the design was expanded to the prior intron, in which a good pair of sgRNA candidates was found (SEQ ID NO9 and SEQ ID NO5). The other three sgRNA pairs were designed to bind to the region downstream of the CAG tract, being paired as follows: SEQ ID NO8 and SEQ ID NQ10; SEQ ID NO11 and SEQ ID NO12; SEQ ID NO13 and SEQ ID NO12.

[0152] With the intent of using these sequences in in vivo models, in the present disclosure these sequences were cloned into an AAV-SpGuide_Plus vector adapted in the scope of the present disclosure to accomplish the simultaneous delivery of each pair of sgRNA molecules. To test the functionality of the designed system, HEK293T cells were transfected with the generated sequences and maintained in culture for 48 hours after transfection (Figure 7B).

[0153] In the present disclosure, to validate the capacity of the generated sequences to excise the CAG tract in the ATXN3 gene through the recruitment of a nickase variant, HEK293T cells were co-transfected with a three vector system: i) an AAV backbone expressing the SpCas9 D10A nickase (plasmid #112719, Addgene), ii) an AAV backbone expressing two sgRNAs designed to target opposite DNA strands in intron 9 and iii) an AAV backbone expressing two sgRNAs designed to target opposite DNA strands in exon 10 (Figure 7C). As a negative control, cells were transfected with AAV-SpCas9 D10A nickase and a guide sequence targeting the bacterial lacZ gene (SEQ ID NO14).

[0154] The region encompassing exon 10 of the ATXN3 gene was amplified by PCR and the generated amplicons electrophoresed in an agarose gel (Figure 7D). The designed multiplexed strategy targeting both ends of the CAG tract, led to the visualization of PCR products with lower sizes, as indicated by arrowheads (Figure 7D, lanes 7 to 9). These lower size amplicons display base lengths that are compatible with the ablation of the DNA sequence existing between the two expected DSB sites, demonstrating the effectiveness of the designed system to excise the CAG tract of the ATXN3 gene.

[0155] In an embodiment, western blot analysis of HEK293T extracts revealed the presence of bands of decreased molecular weight in conditions in which sgRNA guide sequences targeting intron 9 (SEQ ID NO9 and SEQ ID NO5) were concomitantly transfected with sequences targeting exon 10 (SEQ ID NO8 and SEQ ID NQ10; SEQ ID NO11 and SEQ ID NO12; SEQ ID NO13 and SEQ ID NO12; Figure 7E, lanes 6 to 8), possibly corresponding to truncated versions of the ATXN3.

[0156] Overall, these results show the efficiency of the generated guides to direct the SpCas9 D10A nickase to the ATXN3 gene, promoting the elimination of the CAG expansion in this human cellular line and the consequent formation of a truncated ATXN3 protein.

[0157] In the last years, CRISPR-Cas9 system turned into the most popular method for gene editing research. This widespread adoption is mainly due to the greater simplicity with which this system can be designed and assembled, allied to its high editing efficiency and the ability of multiplexing (insertion of multiple DSBs at once in the same cell). Therefore, in the present disclosure the possibility of using the CRISPR-Cas9 system to target the ATXN3 gene was investigated, causing either i) its permanent inactivation (gene knock-out) or ii) the excision of the CAG tract.

[0158] Since the identification of the causative mutation of MJD / SCA3, a stunning progress has been made on the development of potential pharmacological agents for this condition. Strategies aimed at reducing ATXN3 mRNA levels with resource to RNA interference and antisense oligonucleotides are developing rapidly and have proven efficient to slowdown the neurodegenerative cascade in several MJD / SCA3 models. Different approaches, focused on the reduction of the expanded polyQ-associated toxicity, have also been explored using antisense oligonucleotide-mediated exon skipping, in an attempt to maintain ATXN3 protein levels and its functional domains unaltered. Nonetheless, some challenges still remain. Despite limiting the disease progression, the aforementioned strategies may only produce an incomplete and / or transient therapeutic effect in target cells or tissues and therefore may fail to achieve a disease's cure.

[0159] The advent of novel gene editing approaches offers the possibility to permanently inactivate or correct deleterious mutations in several disease-causing genes at the most upstream level of the pathogenic cascade. Targeted gene knock-outs can be generated upon the introduction of a DSB in a codifying exon near the 5' -end of the gene, followed by NHEJ-induction of frameshift mutations and the consequent appearance of an early stop codon. On the other hand, the elimination of deleteriousmutations, as is the case of nucleotide repeat expansions, can also be achieved through the simultaneous introduction of two DSBs flanking the repeat tract at either end. The two broken ends are re-joined by NHEJ repair pathway, while the repeat-containing fragment is eliminated.

[0160] Considering that the RNA-guided SpCas9 nuclease targets DNA sites in immediate vicinity of a 5'- NGG PAM sequence, this constraint had to be considered while screening potential SpCas9 cleavage sites in the human ATXN3 gene. In the present disclosure, for the ATXN3 gene knock-out experiments, four sgRNAs (sgKO.l-SEQ ID NO1, sgKO.2-SEQ ID NO2, sgKO.3-SEQ ID NO3, sgKO.4-SEQ ID NO4) to target exon 2 of the human ATXN3 gene were designed. At the same time, in the present disclosure sgRNA sequences targeting the repeat-containing fragment located at the exon 10 of the human ATXN3 gene and able to recruit either SpCas9 (sgl9Tl-SEQ ID NO5, sgElOTl-SEQ ID NO6, sgE10T2-SEQ ID NO7 and sgE10T3-SEQ ID NO8) or a SpCas9 D10A nickase variant (sgNickl9TlA-SEQ ID NO9, sgNickl9TlB-SEQ ID NO5, sgNickElOTlA- SEQ ID NO8, sgNickElOTIB-SEQ ID NO10, sgNickE10T2A-SEQ ID NO11, sgNickE10T2B-SEQ ID NO12, sgNickE10T3A-SEQ ID NO13) were also designed, with the major goal of removing the region comprising the CAG tract. The non-targeting control sgRNA sequence used in the framework of the present disclosure, targets the lacZ gene from Escherichia coli (sgCTRL-SEQ ID NO14) and has previously been described elsewhere.

[0161] In the first approach described in the present disclosure, the designed knock-out sequences were tested in a human cellular line and proved to be effective in reducing the endogenous levels of the ATXN3 protein. From the panel of validated sequences, sgKO.2 guide sequence (CRISPR-ATXN3, SEQ ID NO2) was selected for knock-out experiments in an in vivo model of MJD / SCA3.

[0162] In order to evaluate the therapeutic potential of the developed CRISPR-Cas9 system in vivo, the lentiviral-based mouse model of MJD / SCA3 was used. The mutant ATXN3 gene was efficiently disrupted in CRISPR-ATXN3 treated hemispheres (SEQ ID NO2), as shown by the Surveyor analysis of transduced striatal tissues, 4 weeks after AAV delivery. Immunoblotting of total protein extracts revealed a significant reduction in the levels of ATXN3 aggregates and a reduction, although not statistically significant, of soluble levels of the mutant form of the protein. No differences in the mouse endogenous protein levels were observed in treated hemispheres.

[0163] Next, it was evaluated if the disruption of the ATXN3 gene could prevent the appearance of pathological markers characteristic of this animal model. Apart from a drastic reduction in the total number of ubiquitin-positive inclusions observed by immunohistochemical analysis of CRISPR-ATXN3 injected hemispheres (SEQ ID NO2), ATXN3 gene editing also preserved neuronal integrity, as expressed by the decreased loss of DARPP-32 immunoreactivity in treated striata. The tolerability of the selected sgKO.2 guide sequence (SEQ ID NO2) in the brain parenchyma was further assessed. In the present disclosure, it was observed a decreased microglial activation after treatment, suggesting the safety of SpCas9 and sgKO.2 guide (SEQ ID NO2) overexpression in the adult brain, 4 weeks after administration.

[0164] An important question to consider before the use of these systems in humans is the development of tools that can avoid the deleterious consequences from the long-term expression of editing machinery, namely i) the potential off-target events and ii) the immune responses elicited by the persistent expression of foreign proteins in edited cells. Therefore, to increase the safety of the developed approach, self-inactivating systems and / or non-viral delivery methods that allow a transient expression of SpCas9 in the brain parenchyma may constitute viable alternatives to consider in order to overcome these concerns.

[0165] In the second approach described in the present disclosure, four sgRNA sequences targeting the CAG tract were validated in the same human cellular line (HEK293T), demonstrating their activity in DNA cleavage. When HEK293T cells were co-transfected with the sgl9Tl guide (SEQ ID NO5), targeting the region upstream of the CAG tract, in combination with one of the sgRNA guides designed to the region downstream of the repeat motif (sgElOTl-SEQ ID NO6, gE10T2-SEQ ID NO7 or gE10T3-SEQ ID NO8), it was observed the formation of versions of the ATXN3 protein with decreased molecular weight. It is possible, that they correspond to edited protein products, lacking the polyQ tract and formed upon NHEJ-mediated resealing of the ablated region. Alternatively, they may instead correspond to truncated products, resulting from the appearance of a premature stop codon downstream the DSB sites.

[0166] Such an approach represents a potential therapeutic option for personalized cell transplantation, following genetic correction, mainly for late stages of the disease course. On the other hand, it can also constitute an attractive platform to generate isogenic cell lines, allowing a better understanding of MJD / SCA3's underlying mechanisms. Nonetheless, its translation to in vivo models will require some caution which is mainly related with i) the evaluation of the functionality and potential toxicity promoted by the generated truncated forms of the protein and with the ii) number of putative off-targets of these sequences, which might be increased due to the employment of a pair of sgRNA sequences at once.

[0167] In the third approach described in this disclosure, a SpCas9 D10A nickase variant was used to mediate the excision of the trinucleotide CAG located in exon 10 of the human ATXN3 gene, while increasing the safety of gene editing. Four sgRNA pairs targeting the vicinity of exon 10 were validated by transfection in HEK293T cells. When cells were co-transfected with both SpCas9 D10A nickase and sgRNA sequences targeting the upstream (SEQ ID NO9 and SEQ ID NO5) and downstream (SEQ ID NO8 and SEQ ID NQ10 or SEQ ID NO11 and SEQ ID NO12 or SEQ ID NO13 and SEQ ID NO12) of the CAGs, it was observed in the present disclosure the appearance of PCR amplicons with lower sizes and the formation of versions of ATXN3 protein with decreased molecular weight, as already observed in the second approach. Nonetheless, when comparing the obtained results with those from the second approach (excision of exon 10 with SpCas9), a lower efficiency of this nickase-based strategy seams to be accomplished. This might be due to i) the co-transfection with three different AAV-backbones in the nickase strategy versus the transfection of only one LV-backbone in the SpCas9-based experiment and ii) to the absence of an antibiotic selection gene in the AAV-based constructs used in the scope of this disclosure, hampering thepossibility to select transfected cells. As an embodiment, a combination of four sgRNA sequences can be accomplished in a single AAV-SpGuide vector, allowing the application of this multiplex system in an in vivo model, with minimized potential off-targets.

[0168] In summary, three different approaches based on CRISPR-Cas9 system were developed in the present disclosure. By targeting exon 2 of the ATXN3 gene, ATXN3 gene silencing was achieved in the adult mouse brain. This is, the first time that the effectiveness of CRISPR-Cas9 system is demonstrated in a MJD / SCA3 mouse model. Moreover, the CAG tract located at the exon 10 of the ATXN3 gene was excised (dual-cleavage strategy), using either SpCas9 or a SpCas9 D10A nickase-based systems. As a consequence of this excision, truncated variants of the protein were generated.

[0169] In this disclosure three different CRISPR-Cas9-based systems were validated to cause either the ATXN3 gene silencing or the removal of the deleterious mutation associated with MJD / SCA3. Despite achieving different outcomes with different efficiencies, they may all constitute promising candidates for the treatment of this devastating disorder.

[0170] As used herein the term "gene", refers to a defined region that is located within a genome and that may comprise regulatory, nucleic acid sequences responsible for the control of expression, i.e., transcription and translation of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

[0171] As used herein, the term "mutation" means any change in a polypeptide or nucleic acid molecule relative to a wild-type polypeptide or nucleic acid molecule from which the 'mutant' is derived and may, for example, comprise single or multiple amino acid or nucleotide changes, or both nucleotide and amino acid changes, including point mutations, null mutations, frame-shift mutations, and may comprise deletions, or insertions, or substitutions of one or more nucleic acids or amino acids, which may comprise naturally or non-naturally occurring nucleotides or amino acids or analogues thereof.

[0172] A "nucleic acid", as referred to herein, refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-, double-stranded or triplexed form. The term may encompass nucleic acids containing known analogues of natural nucleotides having similar binding properties as the reference nucleic acid. A particular nucleic acid sequence may also implicitly encompass conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences. The terms "nucleic acid", "nucleic acid sequence" or "polynucleotide" may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0173] In the context of the present disclosure, "G, "C", "A," "T," and "U", each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.

[0174] As used herein, the terms "treatment" or "treating" are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desiredresults include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0175] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0176] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0177] The following claims further set out particular embodiments of the disclosure.Sequence listingGuide sequences used for the generation of sgRNA constructs for gene knock-out purposesSEQ ID NO1: 5' - GATCCTCAATTGCACATCAGC - 3'SEQ ID NO2: 5' - GTGCCTGAATAACTTATTGCA - 3'SEQ ID NO3: 5' - GAATTGCACATCAGCTGGATG - 3'SEQ ID NO4: 5' - GATCAGCTGGATGAGGAGGAG - 3'Guide sequences used for the generation of sgRNA constructs for the excision of the CAG tract of the human ATXN3 geneSEQ ID NO5: 5' - GTCACGAATCAAAGTAGTCAC - 3'SEQ ID NO6: 5' - GCAGCAGCAGGGGGACCTATC - 3'SEQ ID NO7: 5' - GAGTGCTCCTGAACTGGTGGC - 3'SEQ ID NO8: 5' - GACTTGGGAGTGATCTAGGTA - 3'SEQ ID NO9: 5' - GCTTATGAATAGTTTTTCTCA - 3'SEQ ID NQ10: 5' - GCTGGTGGCTGGCCTTTCACA - 3'SEQ ID NO11: 5' - GCCACCAGTTCAGGAGCACTT - 3'SEQ ID N012: 5' - GATGTGAACTCTGTCCTGAT - 3'SEQ ID N013: 5' - GGAGCACTTGGGAGTGATCT - 3'Guide sequence used for the generation of a control non-targeting sgRNA constructSEQ ID NO14: 5' - GTGCGAATACGCCCACGCGAT - 3'Sequence of the human ATXN3 gene (accession code NG_008198.2)SEQ ID NO15: 5' -GTGGGGGCCGTTGGCTCCAGACAAATAAACATGGAGTCCATCTTCCACGAGAAAGTGAGTGTCCGCGTTCGGTGG GGAGCTGTCTGCCGCGCGGTGGCGGGCGTGGAGCGCGGCATCACCGCCTCTCGGAGGGCTGGGTGGGGCCCGA GTCGCCCCCATGCCGATCTCGCCCGGCGAGGGGCGACGCCGCAGCCTCCCGCCTCCTCGGCTCGAGGAGGGGAG CATCACCTACGCCCCTACTTCCCCCGCGGCCCCCGCCCTGGGAGCCGGGAGGGAGTATGGGCGGGGCCGGGGGC GTCTCGGGACACGGGAGTGGGGTGGCGCCCAGTGGGTTTGCTTCTGCCTTTCTCCGTCACTTTCCATCGCTTTTCG GAGGATTCCTTCACCCCTCCCCAATCCTTCCCTCTCCCTAGGGTCTAGCTAGAGTCATCTCTGGGACACCTCCCTCAA CCCCTCCTACCCTAATCCTGGCAGAATTAACTTTTCCTCCTCCGGACTGCTCAATTCTATATTGGAGTCTTCCCTACA CGTAGATCTTTGGGGTCTTGTTCGTGTCTTTCCCCTGCACTAGGTCCGCGAGCCTCCCGAGGGAGGAGACCTTGGC TCGCCCACTGTAGGGCCTGACATTTAGGAAGTGAAGTAGGAAACCCGGCGTGCCCCTAAACAGGGAAGTCGTCAC AAGAGTTTTTATTACGGGATGTTTGGGTTTGGTTTCTTTTGGTACTCCCATCTTTCCGGAGCAGGCGGCCAGCTTTG TTTTTAGGTATTAGGAGTGGACTGGGATGATTTTGTTGTAGTCTGCCTAGCCTGCTGTCCCTTTAACTCTTCCGTGA CCATGCACTTGAAGATACTGTTTGTGATATGTAAAGAAACTCCTCGTTTCTCTCATACTATTATCCAGCCATTTGTGT GTGAGTGAAGCCTTCCCCAGGACAGCTTTGGCACATGGTATCATGTTTCATAATAGTTTCGTGTTTGGAAAGAGTT GCTGGTAAGGCTGTTATTTAATAGGAGGAGCAAAGGGTTTTTGTTTTATTAAATACTTATAAATGATCATTTATCCC AGACATTTAAAATTCACACACACACAACAAATAAAGCAAAGACAAAAGAATACATTTACCAAATGTAAATCTGTAG CATAAATTTTTTTTAATTTTTATTTTAAAGATGGGGTCTCATTCTGTCACCCAGGCAGGTGTGCAATGGAGAGATCA TGGCTCACTGCAGCCTTGATCTCCTAGGCACAAGCGATCCTCCCGCCTCTGCCTCCAGAGTAGCTGGGACTACAGG TGCATATCGCCAGGGCCAGGTAATGTTTTTGGGAGAGACGGGGTCTCGCTGTGTTGCCCAGGCTGGTCTCGAACT CCTGGACTCAGGTGATTCTCCCACCTCGGCCTCTCGAAGTGCTGTGATTACAGGCGTGAGCCACTGTGCCTGGAAC AAATTGTTAAGTACAATGCTTTTCATTGTAGAAAACATCTCGGAAACTTTTGAAATAGGCTGATGTTCAGTGGGGGAGGAAGGACTCAGTCGTATAGTTGTCACTAATTTTTTGACTTGATTGACATGACTCGTAAATCATAGACAATAGAG ATTTGGTTGCTTGGCTGAGTAGAGTGCGTGAAAAATACACACGTACTTTTTTTTTTTTTTTTTTGAGATGGAGTTTG GCTCTTGTCACCCAGGCTGGAGTGCAATGGCGCCATCATGGCTCACTGCAACCTCCGCCTCCCCGTTCAAGCGATT CTCCTGCCTCAGTCTCCCCAGTAGCTGAGATTACAGGCGCCCGCCACCACGCCCAGCTAATTTTTGTATTTTTAGTA GAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCCAACTCCTGACAGGTGGTCCGCCCGCCTCGGCCTCCCAAA GTGCTGGGATTACAGGCGTGAGCCACCGCACCCGGCCATATTTTTGTTATTAATTTTCAAAGGCTTTGGTGTGGGA CCACATTTCAACATGGAAGGCCTTAAACATGTTCCACACTACTTCCTGAGAATTAGACAAGATTTTTAACAATATTG TTACCTAGTTGGGACACATTTGTACTGACCCATGGGATGAAAAAAAGCTGAGTGCTAGCCTAGTGAAAATCTACTT ACCCGAAAGAAATCCCTCTTAGTCTGGGTGCAGTGGCTCACACCAGTGCTTTGGGAGGCCCAGACGGGCGGATCA TGAGGTCAGTAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCA GGTGTGGTGGCAGGCGCCTGTAATCCCAGGTACTCTGGAGGCTGAGGCAGGAGAATTGCTTGAACCCGAGAGGC AGAGGTTGCAGTGAGCCGAGACCGTGCCACTGCACTTCAGCCTGGGCAACAGAGCGAGACTCCGTCTCAAAAAAAAGAAAAGGAAAAAAGAGTCCCTCTTAATTATCAGCATGTGTATAGGCCTACAGATACTTCAGGAATACCTTTACC ATTATCATCAACTTGTATCTACATAGCATGTGAAGATTCAACAATTTAGTTTTTTGGGCGTCCTCAAGAGTACGCAC CTATAACCATATGGCCCAATTGTTAATCTCCTATACAGTCCATTCTGGGAATGTTTGGGCTTACTGTGCCATTTTTCC GTTCACTGCCTTCCCCTCTGCAATATACCTTTAACCCTTGCTAGGTCCTGGGTTTGGAGAGCCAGAGAACCAACTTT GGCCCTAAAGAAGCTGTGTAGGTAGCAATATCTGCCTACGAAGGGCCTTGCAACCATTTCCTCTTGGAACCTTGGT TTCCTCTTTCTGAGTAGTCACTTTGAGTACCCTTTATTAAGTTAGAATGTAAAAACAGTTTCTCACTGATATATCTGC AGTGCCTGAGAGAGGGCCTGGCACAGAGTAAGTACTCAATAAATATTTGAATGGGGCCGGGCGTGGTGAGACCT GTCTCTACAAGAATGAACAAAATTAGCTGGGCGTGTTAGCACATGCCTGTAGACTTGGGAGGCTGAGGTGGGAGGATTGCATGAGTCTGGGAGGTCGAGGCTGTAGTGAGCCATGATCGCACCACTGCACTCCAGCCTAGGGGACAGA GCAAGATCCTGTCTCAAAAGAAAAAAATGTATATATTTGAATGGATAAAGAGATGGCTTTGAGTTTCTGAGATATA TATGGTGCTGTTTATCTAAAGTAAACAAGTTTTCTGTAAATATTTTAAGGCTTTGCAGGCCAGCTGTAGTCTCTGTC ACACATTCTTATTTGTGCATGTTTTTCCCAACCATGTAAAAATGTAAAGTGCATTCTTAGCTACTGGGGCAGGTTGA ATTTGGCCCATGGGCTAGAGTTTGCCAACCCCTAACTTAAACCTTTGTACTAACTTTATGACCACTACTGGATTTTTG TTGTTGTTTGTTTTAGTTCTGGTGCCTGCTTTGTTTTTTTTTTTTTTTTTAATCCTCTTGCTGATGTTTCTTGGTGCAGT TACTGTGCCATTTGTATTGGTGCTTTTAATGTAATGCAAACTGGTAATAATATCTAAACTTGCTGGGGTTGTACATA AAATTATTGAAAAGATTGAAAAGATGCTGAGCATTGACTCTGTGGCATTCATTATGCCCTTTTGTGATTGCTGGATT TTAGCCATCTTTAGGACATTTGAGCTTTAGGAGAAGCCAAATTCTGTATAAATGACTTGAAGTGCTAATAGCACAG GTTTTGAAACCTCTGCCTGGGTTTGAGTCTCAGCTCTGCCTTTTACTACCTGTGTGATCCTGAGCAAGTTACTTAGT ATCCCTGTCCTCTAGTTTCCTCCTCTGTAGTGTGGGGATAATAACATAGACATAACCTGAGAGTTAGAGTGTAGAG AAGGCTCCCTGGCAGATAGTGCTGTAGAAGTACTGGCCATTGCCATTACTCAGGTGCTTGTGTTTGCTGAACCTCA TAGTAAGGGCTCGGAGAGCACTAAGAGGAGGTGAGAAATGCTGCTAGATTGACAGCTTGTCCCCAGATAGCCCA TTCCCGAGAGCACCTTAGGTTTATACCTGATTTGTGTTGTAGTTAGTAGTGTCTCTGGTAATTTGAACTAGTTTCAG GTTGGTCTTGAAAACCTGGGGAGGTTGGGGGTAAATGATTTGGTAGCAGTTCTCTTTTGTGATTTTATACATTATCT TTGTAGAACTGCAGTTTGCTAATTCTCTGAGCCCAACACAATGAAGTCTGGGCCTAAAATCATAGAATTTCTTTTAT TTTTTTTTTTGTTTTTAATTTATTTATTCCCTCCCTCCCTCCTTTCTTCCTTTCTTCCTTTTCTTTCTTTCTTTCCTTCCTTC CTTCCTTCTTTCTTTTCTTTCTTTCTTTTCTTTCTTTGGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGCACGAA CTTTCTTCAGAGTCTCACTTTGTCACCAGGCTGGAGTGCAGTGGCGCGAACTCAGCTCACTGCAACCTCCGTCTCCT GAGTTCAAGAGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTATAGGCATGTGCCACCATGCCCAGCTAATT TTCTTATTTTTAGTAGAGACGAGGTTTCACCATGTTGGCCAGGATGGTCTTGATCTCTTGACCTCGTGATCCACCTG CCTCAGCCTCCCAAAGTGCGGGGATTACAGGCGTGAGCTACCACGCCCAGCCTATTTTTTATTTTTTGAGGCAGAG TCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGTGCAATCTCAGCTCACTGCAACCTCCGCCTCCTGGGTTCAGGT GATTCTCCTGCCTTAGCCTCCTGAGCACCTGGGACTACAGGCGCCTGCCACCACACCTGGCTAATTCTTATATTTTT AGTAGAGGCGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGATCTCAAGTGATCAACCTGCCTTGGCC TCCCAAAGTGCTGGAATTACAGCCATGAGCCACCATGCCCAGCCAAATCATGAGATTTCAATACCGCTGAACTTTG ATTATGGCAAAGTGAACTTCTGCTTTGATTAAAGCTTGATGAGAGAGGTGGCTGGGGATAGTTTGAGATAAGGGC AAGGCAGGAAAATGCATAATCTTACGTGGGCTCATTGTCATTGTACAATTCTTTTGGTCCATGTGGAATTTGATCC GTCCTATGACTTAAGTTATGTTTATTTTTGTTTTTATTTTTATTTATTTTGTGTCTTTTTGAGAGACATGATGTTGCTCT GTCACCTGGGCCAGAATACAGTGGCACAATCTTAGCTCCGTGTAGCCTTGAACTCCTGGGCTCAAGTGATCCTCCC ACCTCAGCCCCTCAAACAGTTGAGATTATAGTATGAACCACTGTGCCTAGCCTTAAGTGATTTTTAAATTTGTACTG AACAGTTTGTCCTTTCCTTCCATTAAATCATATTAGAAGTACAGAACTTGATATTTCCTGTAGCAATACAGTTTTTCT TTGATGAAGTTTGATTTCAAGTACTTATTTTTCATAATTTAAAGCTATTTTTTATAGAGAGAATTTTAATCAAATATTT GGATGTCACTATTGCTATATATGGTATTAAGTATGGTGACCATAGTTTGTAAACTCCAAACTGACAGCAAGACAGG AAATTTGTGTTAGCAAAGGCTTTTTTCTTACTGTTTGAATTTTTTAAAAATTAGATACAATACAGAGAGGAGCACAC AAATCATTAAGAGTACAGCTCAGCGAATTTTCACACAGTGAACATGTGTAAACAGCAAGTAACAAAAGATTTACCT GCATCCTATAACCTCCCATTATTCCCTTTTCTAGGTACTGTCTCTCCACTGCATTCCCACCAAATATAACCACTATGCT GAATTCTGACATCATAAATGAGTTTTGCCTGATTTTGAGCTTTTGTGACTGGAAGTGTACAGTGTATATACCCTTTC GATTCTGTCCTCTTTAGTTTACCATTGTTTGAGAAATTTATCCATACTGTTCCAGAATTAACTACTGTTAATTATTGTT AATTAACTACTGTTGTAGTTAATTCATCCTCATTGTTATCTAGTATTCTTTTGTGAGTAAACACAATTTCCATTCTACT GTGATCCCAGCTATCCATTTGGGTCGTTTCCAGTTTGGGGTCCATTACAAATAGTAATGCTATCTGTAATGCTATTT TGTATTACTACAAATAGTAATGCTATTTGTGGCACAAAAATACTGCTTTTGTGAACATTCTTATACATGTCTTTTGAT GAATGTATGTTTGCATTGCTGTTGTTTACATTATGTACCTAGTAATGGAATTGCTAGATCATAGGAGATGTATATAT TAAGCTTTAGTGGATGCATTACATAATTATTAGTTATTATTGGTTATACCAATTTATCCTCTCATCAGTAGTATACAA CAGTTTCTGTATCTCTAATCTCCAACATTTTAGCCATTTTAGAGTTTGTGTACTAACACATTGTGGTTTTAATTTACAT TTCCCTGATGACTAATAAAGTTGAGTACCTCTTTTGTGTTCTTTATAGCCATTTGACTGTCTTGTGAAGTGCTTGTTT GTCTTGCCTATTTTTCTTTTCmCTTTCTTTTTCTTCCTTCCTTCCTTTCmCTTTCTTCTTTCTTTCCTTCCTTCTTTTCT TTCTTTCTGTCTTTCTTTCTTGTCTTTCTTGTCTTTCTGTCTTTCTTGGTCTTGCCCTGTCACCCATGCTGGAGTGCAGT GGTGCAGTCTCAGCTTACTGTAGCCTCGACCTTTTTGGGGCTCAAGTTATCCTCCTTTCTCAGCCTCCCAAGAAGCT GGACTACAAGCACGCACCACCATGCTCAGTTAATTTTTTATTTTTTGTAGAAATGGGGTTTCACCATGTTGTCCAGG CTGGTCTCAAACTTCTGGGCTCAAGTAATCCTCCTGCCTTGGCCTCCCAAAATGCTGGGATTACAGGCATGAGCCA CCGCAGCCAGCCTTGGCTATTTTTCAAAAGGATATAAGTAGAACATCTGTATATCCCTTCAATTTGCATATTATTCA GTAAGAGTTGCACTCTGGTAGTAGAAATATATAAGGAGGAGAAAGAAGTGGAAACAAAAAGTCTATTCTCATGA GAAGACTTGGGGGATAGTGTTCTCTCTAGCTCCAAGCTACTTATTCCTTACGAAAAGTTGAAGATAAACTTATCTCAGACTGAGGCTGTCTCAATGTTGTCTTCCTATTCCATTATACACATATAACCCATATTTTTTTCACCAGCTGAATTTTG CTCCTAGAAAATTGATTCATCAGGAAAAATATCCGTCTTGCAAGGTGGTTCTCTTTAGAGTCTGCTGTGTGACATA GCTCAGGACAAATTGTGTGATGTCAGATAGGTTGGGTTAAGGAATAGACCTTATTGGGGAAAGAGAGAACTTGG AGGGCCAAGGTTAGCAGGAGAAGGAAATGTTCTCTCATCTGCCGTCAATTCAGGGAGGGGCAAACCTGGTGTCT GTGTTCACAGGGAGGGATCCATCCATCTGTGATTCTCCCTTCTTATCAGGTAGCATGGGAAAGCTACACTGTTGCG GGGAGGAGGGTCACACGCAGGCTACTTAGTACCAGGCACCCTGGACTTGGATTCAGGTTGCCAGTTGTGTGAGA AACTGCCCAGCACCTGAAGGCCCTGAACCCATGAGAAGTTGTACCTACCTCCCATGAGGAGGAATCCTGTCATCCC ATGGGAGCTGAGCTTGGGTGCAGTCCCTCTTGCTGGCTTGTCCAGGAGTGAGCTCCAGGGTTGTTTGGGACAGTT CTGCTCATTGCTTTACACTGTGTATACATTATCTGTAGAGTTCCATGAAGAGAACTTCAGCACTGTAACTGCAAGTT TTAACATGGAACAGAATTTTTCTCACCTGTATTAATTCTTAAGATTTGAAGTTCTATCAACAAGCATTTAGATTGTGT GGAGATTTTTTTATTTTTATTTTTGGAGACAGAGTCTTGCTCTGTTACCCAGACTGGAGTGGCAGTGGCATGGTCTT GGCTCACTGCAGGCTCTACTTCCTGGGTTCAAGCGATTCTCATGCCTCAGTGTCCTGATTAGCTAGGACTACAGGT ACACACCACCATGCTGGCTAATTTTTGTATTTTTAGTAGAGACGAGGTTTCACCGTATTGGTCAGGCTGGTCTCGAA CTCCCAGCCTCAAGCAGTCCACCCACCTCGGCCTCCCAAACTGCTGGGATTACAGGTGTGAGCCACCATGCTTGAC TGACATCATCATGTTAAAAGAATAAATGTTCTAGGGAGCTGGGCACAGTGTCATGTTTCTGTAGTTCTAGCTGCTC GGGAGGCTGAGGCAGGAAGATCCCTTGAGCCCTGGAGTTCAAGTCCAGCCTGGGCAACATAGTGAGATCTCTTTT TTTAAATAAATAAATAACTGTTCTAGGGACTAAAATTTCCTTTCACCATTAGTAATTTACTGTAGAATCTCCAAGAAT GAACTTATTTTAGGTACTGAAAATGAGGGAGACTAAATGTTTTATACAGTAGTTTTTAGTAAAATATGAGATTTGA TGCATTTGATAGATGATGTTTGTTTAAAATAATTCTTAAATTTTTGATCATGTAATTATAGTTTCATTAATGGTAGAT TTGTAAAATAAATGTTACCAAATGAAAATGCATGTACCTATGTTAATTATCCTTATCTAAAGCTGAAAGTTCAGTTCAACTATGTTAAAACATAGTAGGGGCCTGGCAGGGTGGCTCTTGCCTGTAATCCCAGAACTTAGGGAGGCCAAGGT GGGCAGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACATTGTGAAACCGTATCGCTACTAAAAATACAA AAAATTAGCCGGGCATGGCGGTGGGCACCTGTAGTCGCAGCTACTTGGTAGGCTGAGGCAGGAGAATGGCGTGA ACTCAGGAGGCAGAGCTTACAGTGAGCCGAGATCATGCCACTGCACTCCAGGCTGGGTGACAGAGCAAGACTCC ATCTCAAAAAAAAAAAAAAAGTTGGCCAGGTGTGGCGGCTCACACCTGTAATCCCAGCACTTTTGGAGGCCGAGG CAGGCGGATCACAAGATCAGGAGTTTGAGACCAGCCTGGCTAACAGAGTGAAACCCTGTATATACTAAAAATACA AAAATTAGCCAGGCATGGTGGTGCATGCCTGTAGTCCCAGCTACTTGAGAGGCTGAGGCAGGAGAATCACTTGAA CCCGGGAGGCGGAGGTTGTGGTAAGCTGAGATTGCTCCACTGCACTCCAGCCTGGACAACAGAGCAAGACTCTGT CTCAAAAAAAAAAAAAATTAATGATTAAATTATTTAGGGGAGCCGGGCGCAGTGGCTCACGCCTGTAATCCCAGC ACTTTGGGAGGCCAAGGCGGGCGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCTAACACAGGATGAAACC CCGTCTCTACTAAAAATACAAAAATTTAGCCGGGCGTGGTGGCGGGTGCCTGTAGTACCAGCTACTCGGGAGGCT GAGGCAGGAGAATGGCATGAACCCGGGTGGCGGAGCTTGCAGTGAGCCAAGATAGCGCCACTGCACTCCGGCCT GGGTGAAAGAGTGAGACTCCGTCTCAAAAAAAAAAAAAAATTATTTAGGGGAAGATACTATACAATTCTGTTTAA CAAGTCACATTTTAATTTTTTCTTTTGGAAATATTAGCAAGAAGGCTCACTTTGTGCTCAACATTGCCTGAATAACTT ATTGCAAGGAGAATATTTTAGCCCTGTGGAATTATCCTCAATTGCACATCAGCTGGATGAGGAGGAGAGGATGAG AATGGCAGAAGGAGGAGTTACTAGTGAAGATTATCGCACGTTTTTACAGGTACTGATTTTAAACTCACTAAGTCAC ATTTC I I I I I I I I I I I I I I I I I I GAGACGGAGTCTCGCCCTGTTGCCCATGCTGGAGTGCAATGGCGCGATCTCGGCT CACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCACAC GGCACTATGCCCGGCTAATTTTTTGTATCTTTGTTAGAGATGGGGTTTCACCATGTTGGTCAGGTTGGTCTCAAACT CCTGACCTTATGATCCACCTGTCTTGGCCTCCCAAAGTGCTGGGATTATAGGTGTGAGCCACCACACCCGGCTTACATTTCTTTTAAAAATGTGGATACCATTTAGAAAAGGATGGGCCATTCTTCCTATAGGGATCTGACTGGTGAATTATA ACTGTGCTGTTAACTTTGGAAATGGGAATGCACAAGATATTGTTTTAAATATGCACGCTAATGACAGTTTGTATCCT TCTTTCCCCACCCCCACCCTTGCTTCAACTACCTGTCAAAATTAACAGCAGCCTTCTGGAAATATGGATGACAGTGG TTTTTTCTCTATTCAGGTAAGTAGTCACAAGCATGTACTATGTGTTGCTTACATCCCAGGCACCGTTTCACAGCCTTT CAATAGTCACTGTAACAAGGCGACCTTCGGAAGTTCTTCTGTCTACAGAGTATAGATTATACTCTAGAGTACTAGA TTTTTTTTTTCTTGAGACAGAGTCTCGTTCTGTCACCTAGGCTGGAGTGCAGTGGCGTGATCTTGGCTCACTGTAGC CTCTGCCTCCCGGGTTCAAGCGATCCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCACCCGCCACCACA CCAGTTAATATTTGTATTTTTAGTAGAGATAGTGGGGTTTCACCGTGTTGGCCAGTCTGGTCTCCAACTCCTGACCT CAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTGCACCTGGCCAACTAGAGTACTAGATTTTTATATAGA TAAACATGAAAGGATTGTAGAATCTTCATATTAGAGTGGGGCATTTAAAAATTCCTTCTTGAGAAAGATTAATTTG CATCTGGATGCTAATAATAACCTTAATTCTGGCCGGGCGCGGTGGCTCACACCTGTAATCCCAGCACTTTGGGGAG GCCGAGGTGGGCGGATCACGAGGTCAGGAGATTGAGACCATCCTGGCTAACATGGTGAAACCCCGTCTCTACTAA AAATACAAAAATTAGCTGGACGTGGTGACACGTGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAT CGCTTGAACCAGGGAGTCGTAGGTTGCAGTGAGCCAAGATCGCGCCACTGCACTCTAGCCTGGTGACAGAGCGAGACTCCATCTCAAAGAAAAAAAGAAATCCTTAATTCTAATAAGTCACAATGTCTCAAACTTACCATCTGTTGGGTAAATTTGAGAAAATGCAATACCTTGCTACCATCCTTTTAAATCAGCCTACCAGACTGGATTTCCTTATTATGGTTTGTGGCTTTTGATTTTTTTTTTTTAATGTATAGCTCTCTTTGAATTCTTTGGTGGTTATATATATATGTACTCGCAAGATTCTTTTATCTGTGGGTCTTTCATTCTTTTTCTAACACTGTGAGTTGTATCCAGAGTACTTTCGGAACCTCTCCTGAGCGACCTATCTCTGCAGATATCTTTGTTTATGTTTCCCTTGTACTGCCCTCCTGGACTCTTCCTCATCCACCAGCATTTCCATCTAGTGCTTTACCGTGCCACTGCTAACAGGTAATGGCTACTGCAGGGCTGAAATCAGAGGCCAGAGTAGGCCCAGCACTTGGCGTTTCCTATTTGTGCCTTGCTGCTCTTGGTGCCTGTTCATGTGTGCCCACTACCTTGCACTCAATTTCTGTCTTTGCTGGTACCTGGCTCACTTGCTTCTTTGTTGGCTACCTTGGAGGGCAGATAGTGAATTTTCAGAAATTTCCCTTTTTTTGTCAGACAGATTGAAATAAACAGGTTTGCATTTTGTTTTTTCTACAAGCGGCAAGCCCATGACCCTAGAAGTCTGACATCTATGGAACCTTCAGTTTAAATGCCCAGGGAGAACTTATTTTGGTAGATATGATTTCTGACATTGCAGGTAGCAAGTTGAATATAATTTTTCTAAAGTAGCACCCACAGCAGCCAAATTATCAGATGTATATAGTAGACTAGTTTTAAGAAAAGCACTTATGGGTAGAATATACATCTGGATTTTTGAGGCAGTTTTATTTAGGAATTGTGTGGTTTTCTGGAACATCTCAGAGACCTGGTATGAAAAGCACTCTTCTAATATATATGTGTTTTTTTTTATGGATTTAGTGATATATCTATACACACACACTTTTTAAAACCTATAGCCGGCTGGGCGTGGTGGCTCATGCCTGTAATCCCAGTACTTTGGGAGGCCCAGGCGGGTGGATCACAAGGTCAGGAGATTGAGACCAGCCTGGCCAACAAGGTGAAACCCTGTCTCTACTAAAAATACAAAAATAGCTGGGTGTGGTGGCGTGTGCTTGTAATCCCAGCTACTCGGGAGCCTCAGGAGGAGAATCGCTTGAACCTGGGAGGCGGAGGTTGCAGCGAGCCGAGATCGTGCCACTATACTCCAGCCTGGGCGACAGAGCAAGACTCTGTCACAAAAAAAAAAAAAAAAACCTATAGCCTTCTAGAGAAATTTATATATGAAGTACACAACTAACATAGCTACACTTCCTAAATTTGGAATGGAGTGGTTTAGCTTATGAAAAGTTGCTATTTTTCTTAACAGGTTATAAGCAATGCCTTGAAAGTTTGGGGTTTAGAACTAATCCTGTTCAACAGTCCAGAGTATCAGAGGCTCAGGATCGATCCTATGTAAGATTCTGTTTTGCATTTCATACATTTCTTTTCCCAAATTTGATTTTTAAAGTTGTAATTTCTTAAAGAAGAGAAATACATTTTGAATACTTTTGTTTTGATGTTCCCTGTTTCATTCACTCAGACTTTCCTATTTCACCTTTGTGATGTCCATGAGCATCTGCCCTGTAGCCTTCCTGGCACCCCAGTGTCTGTGGCAGCACAGAGCTGACCCCATAAGTGGTGCATGAGGCCATCTTGTGGCACAGCATCACTAAGCTGCTGCAGAGACGTTCATATGGTTGTGTGATCTTTTAAAAACATCAGTGACACTTAACTATAAATATAATCTTAAATTATCACAAATTTTATATAATATTTGCCAGTAGACAACATAAATATGAATTCAATATTTCAAGTTAATATTGTCTGTTTTCTTTTTTAGAAATGAAAGATCATTTATATGCAATTATAAGGAACACTGGTTTACAGTTAGAAAATTAGGAAAACAGGTAACATTTCTTACCCTTCCTTGTCTTTTTTTCTTATATTGTACCCCATTTAAAACTAAAATGTGGGCCAGGTGTGGTGGCTCATGCCAACAGTTTGGGAGGCTGAGGTGGGGGGATCACTTGAAGCCAGGAGTTTGAGACCAGCCTGGGCAACAAAGGGAGGTCCTGTCTCTTAAAAAAAAAATAAAAATAAAAATAAAAATAAATAAAAAAAAAAACAAAGAGCCAGGCATGGTGGCTCACATCTGTAATTCCAGCTTACTTGGAAGGCTGAGTCAGAAGGATCACTTGAGCTCAGGAGTTTGAGGCTGCAGTGAACTATGATTTTGTCACTGTACCCCAGCCTGGGTGACAGAGTAAGACTGTTCTATAAAACATAAAAATAAAAAAAATATATTTAAAAATTAAAAAAAAAAAAGGATTGCTGACTTTAAAATTAGGAAACTGACCAGTAATGTGTGTGTGTGTAGCATGGTTTATCCTTCTTGATAGATAGAAATTGTCATTTTAAAAGATAATATCAGTTTTCCTTATAAATTTATTTGTGACAAGTATATGCAATTTAACTATATCATAAGAAAAATTCTATATTAAAGATAATACAAATGTGGTTACTTTTAAGTGGGTTTTTATGTGATGACTATGTTCTGTCAGTTAATTATTACATTTATAGATTTGTATTTAGCATAGTGCTGTCACAAAGCCTGAAATAGTGTCAAGCATGAATAAAGCATTCAATTATGTTTGCTTTAGTGTAAGATTATTCATTATGATTCCAAAAGCCATGTAATACGTACGTCTACAGAAAATCACTTCTATTTTTTAAATAAAACATGAAATATGTCTTGAGCAAGCTATTTTAAGAAACAATCATTTAACGTCCTTGTTATTAGAATTTTGAATCTTTGAAAGAGGGTTATTGAAAACCAGCTAGGACAGTAAAAAAGAATAAACTAGTGATACATGCAGCAATATGGATGAATCTCAAAATAATTATGCTGAAAGAATAACCCACAAACAAAATACTACCTGCTGTATGGTATCATTTATTAAAAGTCTAGAAAAGTGCAGATTCATCTGTAGTGATGGAAAGCAGATTGACCAGCGGTTGCCTGGGGACGAGAAGGCTATGGAGGAGTGAGAGGGGAGGGTTACAGAGAGGCACGGGAAACATGGCAATGAGGAATGTGTTCACTATCTTGGTTGTAGTAATGGTTTCATGGGAGTACAGTATACAAATGTGAAAACATTTCAGAGGCCAGATGCAGTGGCTCATGCCTGTAATCCCAGCACTTTTGGAGGCCAAGGCAGGAGGATTGCTTGAGCTCAAGGAGTTCAGGACCAGCCTGGGCAATGGCACAAGACCCCATCTCTAAAAAAAAAATGAAAGAAAAAAAAATTGGCTAGGCGTGGTGATGCATGGCCGTAGTCCCAGGTGCTAGGGAGGCTGAGGAGGGAGCACAGAGGTCAAGCCTGCAGTGAATCATGATCGTGCTACTGCACTCCAGCTTGGGTGACAGAAGGAGATCCTGTCTCAAAAAAAAAGTTTCAAATTATACACTTTAAATATGTGCAGTTTATTATATGTCACTTATACCCCAATAAATCTGTTTTTTTTAAAATGTAAATACAAGCCAAAAAAGGTATAAGTCAAGAAAATATATTGAATTAAATCTGTAAGAGATAATTCAAAAACAAAAACCCTATTGTTATCTTTTAAGTCACCCAAATCAAATTTGGGAAAAGTCACCTACTTAGCTTCATCCTAAGTTGGTTCTTTCTTTCTTTCTTTCCTTCTTTTGAGACGGATTCTTGCTCTATCGCCCAGGCTGGATTGCAGTGGCGGGATCTTGGCTCCCTGCAACCTCCGCCACCTGGGTTCAAGCAATTCTCTTGTCTCAGCCTCCCAAATAGCTGTGTCTACAGCCACGCACCACCACACCCAGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCGCCATGTTGGTCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCGTCCGTCTCTGCCTCTCAAAGTGCTGGGGTTACAGGCGTGAGCCACCATGCCGAGCCCTAAGTTGGTTCTTTCTTAAAGTTCTTCCTGAGGAGCCAAGAGCAAGTTAAGGAGATGTAACCTAGAAGCTTACAGTGGAGGCTAGCTGGGTGCAGT GGTTCACGCCTGTAATCCCAGCACTTTAGGAGGCTGAGGCAGGGAGATCACTGAGGCCAGGAGCTTGAGAGCAG CTTGGCCCAACACAGTGACACCTTGTCTCTACAAAAAAAAAAAAAAAAAAAGGCAGCTTACAGCAGTAGAGGCTG ATGCGAGTGGGAATCACCTCTAGGTAAAAACCAGTGTAGCGTACTGCTGAGATTATTTAACCTCTGGGTTTTATTT ATGTGTTTTTAAAAATTATGATCCAGTATTTTTTACTTTTTTTTGTATAAAGTAAGCACTGAATTTTTAAGGTTGTATT AATTTGCAAATAAATGTCTATCTTATTATTTTGAGAGATTTAAAAAATTTTAGTTCTTCAAAATTGCATTTTCACATTT TGAATTACGTTATCTTTGACAAATACAGAAGATGTCAAATTTTGGTTTATTTTCTTTGGTTCTAATTTATATTTTTGTT TAAAACTATATTTTTCACTATAGACTCTTTCTGTCTCTCGAGGTCCCTGTATAATGAAAAAGAAGGCTGGAAAAAGT ATTAACATTGTCAAAATCCAGGAAAAGTAGTTGGTCATGATATTGATCGTTAACTTTAGAAACTTTTTGTATCTTGT GGGTTAAATTAGGATTACTATGTGGTAGTGATAAATGATGTTAATTAGGGCCGAGTGCAGTGGCTAACACCTGTA ATTCCAGCATGTAGGGAGGCTGAGGTGGGAGGATGTCTTGAATCCAGGAGTTTGAGACCAGCCTGTACAACATA GTGTAAGACCCCTTCTCCACACAAAAAAATTAGAAAATTTGTCAAGCATCTTGGTGCACACCTGTAGTCCCAGCTG CTTGGGAGGATGAAGCGAGAGAATCACTTAAGCCCAGGTGTTCGAGGCTGCAGTGAGCTATGATTGCACCACTGC ACTCCAGACTAGATGACCATCTCTTTTAAAAAAATGTGTTTATATGTTATATGTGATAGTGCTTTTTAAAAACATTTT TAAATTATAGAGACAGGGTCTCACTATGTTACAGCCCAGGCTGGTCTCAAATTCCTGGGCTCAAGCAATCCTCCCA CCTTAGCTAACCTCCCAAAGTGCTCGGATTATAGGCATGAGCTGCATGCCCAGCTAATTTAGTGATTTTTAAAAACT GAGCTGGTAATTATAAATTCTCTTCCTGGAACTTCTGACTTTCTCACAATTGGAATCTTTTGACAAAAATTATCAGTA ATGGGAAAACTTTGTGTAGTTGTCATTTTTCCTCCCATCAGTGTGATAGATATGATTGGAGTTATGTTGGACTGATA TTTTGAAAAAAGATTTAATTATAGCTATTAATAAAGACATTTAAACTACTGACTATGCATTTTTATTCTTTTGGGAGG GTTTAATGTTTATAGTTTAAAGCAAACTGTTGTTTTTAAAAAAGTATCTAACAGGGCCGGGCGCGGTGGCTCACAC CTGTAATCCCAGCACTTTGGGAGGCCTAGGCGGGCGGATCACAAGGTCAAGAGATCAAGACCATCCTGGCTAACA TGGTGAAACCCTGTCTCTACTAAAAATACAAAAAAATAGCTGGGTGTGGCGGCGTGCGCCTGTAGTCCCAGCTACT CGGGAGGCTGAGGCAGGAGGATGGCATGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGC ACTCCAGCCTGGGCGACAGAGCAATACTCTGTCTAAAAAAAAAAAAAAAAAAAAAAAAAGAGTATTTAGCAGAG GCCAGGTGCAGTGGCTCATGTTTGTAATCCCAGAACTTTGGGAGGCTGAGGCGGGCGGATCATTTGAGGTCAGG AGTTTGAGACCAGCCTGGCCAATGTGGCAAATGTGCTGTCTCTAACTAAAAATACAAAAATTAGCTGGGTGTGGT GGTGCAGACCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTG CAGTGATCCGAGATCATGCCACTGCACTCCAGCCTGGGTTACAGAGTGAGACTCTTCTCAAAAAAAAAAAAAAGT ATTTAATAGTGATAAATCTGCAGTATTCTCTTGTAGTTTTTAAGATCATATTATTCAGTCAAAGAAAAGAGCTCAAC TTGAAATATTTCCAGAGTTTAAACAATCTTACTAAGCTTTGATGGGTTGTATCTATTCTTAACATGTGAAACTTCCTT ATTACCTATAATATACACTAACTTAAATATTGACAATTTTTTTCCAGTGGTTTAACTTGAATTCTCTCTTGACGGGTC CAGAATTAATATCAGATACATATCTTGCACTTTTCTTGGCTCAATTACAACAGGAAGGTAAGTAACGGCTGAACATT TTGTAATGTTACCTTTCGAAGTAGTTAAATAACCAGGCACATTAGATGACAGTGTGATAAAACTGTTTTTCTGGCAG TGGCAGTGAAACAATCTTTAGTTTTGACGTGGTGATAGGCTGTGATTTGGGTGACGCTGTTCAGTTAGAGTTCTCA CTGACACCTGGCCCTTCCTCTTCTGAGGATGCTGCTTTCTTTGCAGCCCTTCTAAGTAATGGCTTTTTCTTTTATACAT CACATATCACACGGCTGAGAGGAGGGATAGATGTTTTTCTTCTTTGCCTCTTCTAGGCCACTGTTCTTCCTTATAAA CTCCAGTTTCTTTGAAATACATGCCCCTAACGGCTGGGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAG GCTGAGGCAGGCGGATCACGATGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAATCCTGTCTCTACTAA AAATAACAAAAAATTAGCCGGGGTGTGGTGGCGGACGCCTGTAGTCCGAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCAGGAGGCGGAGCTTGCAGTGAGCTGAGATCGCGCCACTGCCCTCCAGCCTGGGCGACAG AGCGAGACTCCGTCTCAAAAAAAAAAAGAAAAGAAAAAAAAAAGAAATACATGCCCCTAGATTAAACTATCCCTT GTCCTTTTGCACTCATCCACAAGTCTCTTTTCATCAGTGATTTTAGGATCTGACTCGTTGTCTTTTTCTCTACTTCAAC TACTTTTATCATTCTTAATTATTTCTGTATCGTCAATCAATCCAGTACCTGCCTCTTAGTTTCAAAATCACTTACTCTT GCTTAGCTATTACCAGTAATCATAACCACTGTCAAATCTCAATTGCAAGCATATTACTCTTTAACTACCACCTCCTAT CTTTAAACCATGTTTTGTCTGTTTTTTTATTCCAGCCATTCTTTAAACCCTACTGTGGGGCCCAAGCATTTCCTTTATA CGCATTCTTCCTTTCTTCTACTGCTTATTTTCTGTAATCCGTCATCATAATCACTCCATTGCATTCTTCAACGTGTTTCC CCTCTCTCCCTCCATCATACTTGAATGACAAAAATCTCAACCCTGGTTAAACCACATCTTGGCCTTGTCCATTCCTGT ACCAGAGTAGCTGGACGTGGCTAAAAAATAACATAAAACATGATGATTGGTTTTACTTTTTTCTTAAATGATCTATC CATCCATTCACCCATCCATCTATCAAAGTGACTAGGCCTATTTCTGAAGCCCAGGCTGGAGTGCAGCAGCATAATC ACAGCTCATTGCAGCTCCAAACTCCTGGGCTCAAGTGATTCTCTTGCCTTAGCCTGTTGAGTAGCTGGGACTACAG GCTTGTGCTACCACACCTAGCTAAGGTTTTACTTTAAATTTATTATAATCACAAAATTCAGATGAGCCTTTAGTGCTG TCTGATATTTCTACTATGTTTTCTTAGTGATGTACCACCCTCCAAGGTGTTTATAAAAAATTATGTACCACTCTCCAA GAAGTTTATAAAAAATAATGTGCCACCCTCCAAGGTGACTAATTTCACAGCTTATGTCTTTAAACCTTTAAGCACTT TCCTCTCCCTTACACACCTTCCTTGTGGCTTTCCGTTACATTCTGCTGAGAACATAGAAGCAATTAAAATTATGTTCTTTCTACCAGCAAATTTATCAATTTGCTTATATCTTCACCTGTGCTTTGAGCCTATTTAAATAGATGAATGGTCCCCTA CCTCTAACCAAAACCAGTCCCTCACTTGTGGGCTGGATCCCAGCTCTTCTCACCTACTCAAGATGTTCCTGCTTTCAT CTCTCCACTCTCTTATATAATCAGTTCCCCCCCCCTTTTTTTGTAATATTCCTATAAGCAGTAAAATAAGCTTTTTATTT CCATTGATTAAAAATAAAAATCCTCTCTTAATTCCATGAAACTCCAGCTGCCTCCCCATTTTTATTTTTTCCTTAGGAT TGTCTCTAGTGTGCCTTCTCCTTTTCTTGAACTCTGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAACCTCCCGAGT AGCTGGGATTACAGGCGTGCACCACCATGACCGGCTAATTTTTTTTTTTTTTTTTTGAGATGGAGTTTCCCTCTTGTT GCTCCGGCTGGAGTGCAATGGCGTGATCTCGGCTCACCGTAACTTCTGCCTCCTGGGTTCAAGCGATTTTCTTGCCT CAGCCTCCCGAGTAGCTGGATTTACAGGCATGTGCCACCATGCCTGGCTAATTTTGTATTTTAGTAGAGATGGAAG GGGTTTCTCCATGTTTGTTAGGCTGGTCTCCAACTCCTGACCTCAGGTGAGCCGCCCACCTCGGCCCCCTAAAGTGC TGGGATTACAGGCATGAGCCACTGCGCCTGGCCCCGGCTAAATTTTTTTTTTTTTTTTTTGTATTTTTAGTAGAGACA GGGTTTCACCATATTGGCCAGGTTGGTCTCGAATTCCTGGCCTCGAGTGATCCACCTGCCTCAGCCTCCCAAAGTG CTGGGATTACAGGCGTGAGTCACCTTGCCTAGCCATCTTTTAGTAATGGTATTTGGAGATCACAATTTGAGTGCTG GCATGCTTATTGCTGCTGGGTTTGTTATGTAGTTATTGTGAATTCACATTTAGGAATATAGGGTTTTTAATTCTTTGA TTTTAGATACTTGTATCTTTTTTCTTTTATATTTAAAACCTTGGTTCCTGATGATATCCCTTCTTAGAAACCCTGTCTAC CTTTGGCCTTCAGCCCACCATGCTGTGGTTTTCCTAACTTGCTGCCTGCACTTTTCAGATTCCTTTCATGGATCTTAA ATATCATCTGTAAATAAGATCTATGTGTCAATAATTACCAAACTTTTATCTTTAGTCTTGACATCTACCCTGAACACC TAGCTTTGACTAACTCCTAGCTTTGGCATCTCCACTTGGAAATCCAAAAAGTGTTTCAAACTGAACATGTCTATGAA AGACTTATTTTTTTCTCTCTATCCATGCTATCCATCAGGTTTTCCATTTCCATAAGGGTGACTCTTGTACTCTGGTTCC TATATATTATACCGACAGAGCAGCCCAGAGTGCTTCTTAACCAGTGTAAGGCCTGTTATGTCCCACCCTCACTCTTT GTCCTTCAGTGGCTTCCCAGCACACTTAGAATAAAATCTGAAGTCTTAGGCCGGGCTTGGTGGCTCATGCCTGCAA TCCCAGCACTTTGGGAGGATGAGGGGGCAGATCACTTGAGGTCAGGAGTTGATGAGACCAGCCTGGCCAACATG GTGAAACCCTGTCTCTACCAAAAAATACAAAAATTAACTGGGTGTGGTGTTGTGCACCTGTAGTCCCAGCTACTCG GGAGGCTGAGATAGGAGAATCACTTGAACCCGGGAGGCAGAGGTTACAGCGAGCCAAGATCATACCACTGCACT CCAGCCTGGGTGACAGAACGAGACTCTCAAAAAAAAATTAAAAAAAAAAAATATGTGAAGTCTTGAATAAAACCC AAGATCTTTACCATGGCCCCTGAACAGGGCAGAGTATCCATTCTTCAGACACTCTTCATAGAATACCATGGTGAGC TGGCATATTTATTATACAATACAGAAACAATTTTACTGGCAGAAAACACATTAAACCGTCTAAACTCTGAATACAGT TGTCCTCATAAAAAATGTTCAACATACTATTTTGAGGTTTTCCATTAATAGTTCTTATAATCTTTGTCCCATTATGTGT TAATCCAACAAAGGATATCCAATAACAAACACCAAAGTTTAAGAAAAATGTGCTAGGCGCGGTGGCTCACACCTG TAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGCCCAGCCAACA TGGTGAAACCCTGCCTCTCCTAAAAATACAAACATTAACTGGGTGTGGTGGTGGGTGCCTGTAATCCCAGCTACTC AGGAGGCTGAGGCAGGAGAATCGCTTGAACCTCCTGGGAGGCAGAGGTTGCAGTGAGCTAATATTGCACCACTG CACTCCAGCCTGGGTGACAGAGTGAGACTCCATCTCAAATTAAAAAAAAAAAAAAATTAATGATAGAGAAACTTA AATCAGTTAGATTGTTTTAGGTATAGCCCATCCTTGGTTTTTGTGTGTAGCATCTAGCTTGGGGAAACCCTGGATTT CTGGAATCATATTTAGACACAGTCACACTAGACTAATGTAATTCTTTTGGGATGCAAACCACACGTTTGACACCTTA AATAGCTTTTAGGTATTTGGCTTCCCAGCCCCTATTTTTAGTTACAAGGGGTGTACATGTGTGGGTCAGGGTGGGG GTAGCTCTTTCCGCAGATGATTAGTTTTAGCCATGTTACTAGTTATTGCACACATTATCTGTGTCCTCACAGCAGCCC TGTGAGTAAGTGTATTAGGGTTCTCTAGAGGGACAGAACTAATAAGGTAGATGTATATATGAAGGGTAATGTATT AAGGAGTATCGACTCGTATGATCACAAGGTGAAGTCCCACAATAGGCTCTCTGCAGGCTGAGGAACCAGGAAGCC AGTCCAAGTCCCAAAACCTCAAAAGTAGGGAAGCTGACAGTGCAGCCTTCAGTCTGTGGCAAAAGGCCTGAGAGC CCCTGGCAAACCACTGGTGTAAGTTCAAGAGTCCAAAAGATGAAGAACTTGGAGTCTGATGTTTGAGGGCAGGAA GCATCCAGCATGGGAGAAAGATGAAGGCTCAGCAAGTCTAGTACTTCCACACTCTTATTTCTGCCTGCTTTATTCTA GCTGAGCTGGCAGCTGATTAGATGGTGACCACCCAGTTTGAGGGTGGGTCTACCTCTCCCAGTTCACTGGCTTAAA TGTTAATCTCCTTTGGCAACACCCTCGCAGACACACCCAGAAACAATAATTTGTAGCCTTCAATCCAATCAAGTTGA TAATATTAACCATCACAGGAAGGTACTAGTATCATATGTTTAACAGTAGAAACCAAGACAAATGCAGCTAGGAAGT GGGAGAACTGGGATCAGATGCAGGCAGTCTGATTCTAAATCAGTTGCTGTTACCCACTCTGACAACAGTAAGTGA GTAGCCTGCTCAGTCAAGTACTATATTAGTAGGGCCCTTTACAGACATATTTATTTCTCACAGTCACTCAATGAGAC GGCTCTTCCAGTCTTACAATGGAGAAAGTGAGGCTCAGAGACTTTAAGTAACTTACCTTAGACGACTTTACTAGTA AGTATAAGAATCATTATTTGGACTAAAGTCTTTCTGAATCCTCAGCTTGTATTTTTTTCCAGTGTTCTGTGCTGCCTTTTTATCTACTAGTGTTTTACATCAATTTTGAATCTCTTTACTAACTGGTTAGGTTGATTTTTGCCTTTTTTTTTTAGGTT ATTCTATATTTGTCGTTAAGGGTGATCTGCCAGATTGCGAAGCTGACCAACTCCTGCAGATGATTAGGGTCCAACA GATGCATCGACCAAAACTTATTGGAGAAGAATTAGCACAACTAAAAGAGCAAAGGTAAAAATGAGGCCTGCAGT ATGGAATATATGGTAGTATTTCATTATGAGAATTAAATTTTCATGCTTAGATTGAATATGTGGTCCTTGTGTTGTTG GCGACTCTATTTTGGACCTTATATTTTAGTGAAGTTTATTAGTTTAAACTTGAATCAACTCTTTGAAATACTTAAATA TATTAACTTAGTTAGCTGGTATGGTATATTCCTAGCACTTCGGGAGGCTGAGGCAGGCTGATTGCTTCAACCCAGGAGTTCGAGACCAGCCTGGGCAACATGGCAAAACCTCATCTCTACAAATAGTACAAAAATTAGCCAGATGTGGTGG TGTATGCCTATAGTCCCAGCTACTTGGGAGGCAGAGGAAGAAGGATCACCTGAAACTGGGGAGGTAGAGACTAC AGTGAGCCATAATCACACTACCGCACTCCAGCCTGGTCGAGAGAGTCAGACCCTGTCTCAAAAAAAAAAAAAAAA AGAAACGGAAAAAAAAAACTTAGTTGGATTCAAATTGCAACACAATCATTATATTACTAGAGCTTATTTGCCAGAA AACATTTTAAGTTTTGACTTACTTAAAGCCTTTACATTACAAATGCCTTTATGTTATGTCTAAAATAGAAGATTGGTT GCAGTTATTACCAGTGCTTTTGTTCTTTAGAGTCCATAAAACAGACCTGGAACGAGTGTTAGAAGCAAATGATGGC TCAGGAATGTTAGACGAAGATGAGGAGGATTTGCAGAGGGCTCTGGCACTAAGTCGCCAAGAAATTGACATGGA AGATGAGGAAGCAGATCTCCGCAGGGCTATTCAGCTAAGTATGCAAGGTAAAGACATTCTGATGTGTGTTGTATT CATTGCTGAAGAATTGATTCCAATTATTCTTAGATTTCATGGAAGTTAATGTACTCTTAGAGGTGTTTTGACAATTA CTGCAGAAGCAATAGCTATATAGTGGGCTTTCCCTTTAGATTTCTTATAATGGAAATCACTTTTTACAACCTATATTT TATTAGGAGTAGTTATATTTTTACTCCTGGTTATTTTATTTGGTTTCAACACTGTACTAACACAATAGTAAATTGTGG TTTTAATCTTTGTGGGTATCAGTTGACCCTTATCCAAATCAGCTGTTACATAAATATGTGCCATTAGACACTATGGA AGGGCCTGGACAGGGAATATAAACTGATTTTACAAAAACCCAACATTTATTGGCTATGCAACTTAAACCGTAAGCC CACTTTGGTGGGCCCAGTTTTTTAGTGATATAAACTATCAATAGAGAAAAGCGAAAACATATCCCCTAGACAATCT AGGCAAAGAAAAATGTTAAGACATAGCTCAAAGTAGCTTAATTAAAAGTTTGAAGTGGGTTTTTTGTTTTATTTTTT TCTAACTCATATGTATTTGCTTCTACTTTCTAATGAAATTATTTATCAGTTGATTTCCTTAGATATCTAAATAAAATTG AAATTTCATTAATGGGAAGATTATTTTTATCCTGAACTTTTCTTGCCTCTATGCATGCCTCTGAGTACTCCATATGGT GTGCAATCCCATTTTTGATTAATAGAGTCCTGCTGGATTAGCAGGGACAGAAATCAGCTTTAGATTTCTTTCTTTTTT TTTTTTCTTTCTTTTTTTTTTTTTTTTTTTTTGAGTCAGAGTCTCACTGTCGCCCAGCCTGGAGTGCAGTGATCTTGGC TCACTGCAACCCCTGCCTCCGAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCGCC TACCACCACGCCCAGCTAATTTTTTGTACTTTTAGTAGAGATAGGGTTTTGCCCTTTTGGCCAGGCTGGTCTTGAAC TCCTGACCTCAGGTGATCCACCTGCCTTGGCCTCCCAAAGTGCTGGGATTACATGTGTGAGCCACCACGCCCAGCC AGAAGAGTAGAATATTCTTAAAGAGAAAACGTTTTAAAGGCTTACTCAAATGAGTATAAACAAACATATTGTTGCT TGAATTGGTAAATACAGTGATTGGTTTTTGTTGTGTTGTGTTTTGTTTTCAGGTAGTTCCAGAAACATATCTCAAGA TATGACACAGACATCAGGTACAAATCTTACTTCAGAAGAGCTTCGGAAGAGACGAGAAGCCTACTTTGAAAAGTA AAGTAGTTGGTACAAGTTAAAGTAGCATGTTTAATATTTGCTTTGGCTATTTTGTCTATTTGTAAATGGTTACTGCCT GAATCCTGTGAATATTTGAATGTATTTTTTAAAAATTTACAGCAAATAGGACGGGCACGGTGGCTTACGCCTGTGA TGCTAGCAGTTTGGGAGGCCAAGGCGGGCAGATTGCCTGAGGTCAGGAGTTCGAGACCAGCCTGGGCAACACAG TGAAACCCCATCTCTACTAAAAATACAAAAGAATCAGCTGGGCATGGAAGCGTGCGCCTGTAGTCCCAGCTGCTTG GGAGGCTGAGCCAGGAGAATTGCTTGAACCCGGGACGTGGAGGTTGCAGTGAGCCGAGATCGCACCACTGCCCT CCAGACTGGGTGACAGAGTGAGACTCCGTCTCCAAAAATATATGTATATATATATAAATAAAAATAAAAATTTACG GCAAATAACATGAAACAAAAAAACCTTGCCCCAATACTGGATAAATTTTTTAAACTGAGTGAAGGAAACCTTATAA AATTTCATTTATTAAAAGAAAAATGAAATTAGGACAAGACAAGAAGAATGCCAATTGATCCTTTGGATGTACTTCT TGCTTACCTGATTAACCCTGCAAAATTCCTCTACCAATCAGTACGAAAAACAGCTTTGGAGGTATGGGAGCGCATT CCCAAATAGACGTGGTAGTTCATTTAGCTGCTCATGGCCGCTTCAGGCAGTCCTGTAAGCCTGTTAGCATCAGGGG AATGGATGCAAACCATAAATCTGGATCAACTCCTAAAACCTTACCTTGTGCCCAGCCTTGTAAGTGCTTGCTAAATA GGAATTCCACCATATGAAAATACATTCTTTTCAAGTAACTATCATTCAGACTTTTGTCCCCCACTTTTTTTTTTTAAAG AAAAATAAAAGGCTGGGCACGGTGGCTTACGTCTGTAATCCCACCATTTTAGGAGGCCAAGGCAGGTGGATCACC TGAGGTCAGGAATTCAAGACCAGCCTGACCAACATGGTGAAACCTCATCTCTACTAAAAATACAAAAATTAGCCGG GCATGGTGGTGGGTGCCTGTAATCCCAGCTACTTGGGAGGCTCAGACAGGAGAATCGCTTGAATCTGGGAGGCA GAAGTTGCAGTGAGCTGAGATAACGCCATTGCACTCCAGCCTGGGGGACAAGAGCGAGACTTCGTCTCAAAAAA AAAGAGAAAGAAAACTTCATGTTAAAGATTACAAGATAAATAATCAGACCCACTGATCCTAGGTCAGAAAACAGA GTCATAGCTCAATCTGACTTACTATTTGCTGTATTTCATCCATTCTGAGATGCACATAGTTTCACATTTCAATGTCTCT GAAATTGAGAAGCATCTTACAGTCATAATTGACAGTATATTAGCAGCACCTATAAATATTGGCTCATTTTACATTTG ATGGTATAATGAAGAAAATATTTACCTTTTTTTCTGTTTTGTTTTTAAGTCACAACTCAGAAGTAGATGAAGGAAAA TTCTGATCAGCTGACATCCTCTTAATGTGAGATATTTCTAGTCTTTATTCAGTATAGATTAATGGCTAATTATATGTT AAATTTCAAAGTAGTGCTTATTAGTGCTTTTTACTTTTAAGTTTCAAAATTAACTTTTTTATTATAATAAACTCCAAAT TTATACAAAAGTAGAAAAACTAGCATACTCCTGTTTATGACCCAGATTCAACAAATACTAGCACACGGCCAATCTTGCTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAATGGCACAATTTCTGCTC ACTGCAACCTCTGCCTCCTGAGTTCAAGCGATTCTCCCACTTCAGCCTCCCAAGTAGCTGGGATTACAGGTACACAC CACCATGCCTGGCTAATTCTTGTATTTTTAGTAGACACGGGATTTCACCATGTCGTCCAGGCTGGCCTTAAACTCCT GACCTCAAGTGATCCACCTGCCTCGGCCTCCCAGAGTGCTGGGATTACAGGCATGAGCCACTGAGCCCGGCCCAA TCTCGTTTTATAATACTCCCATCTCCCATTCTTTCCACTGTCCCACCTGCAAGTTTGGATTATTTTGTAACAAATCTCA ATCATCATATTATTCTATAACCATTTTAATATGTGTCTCTAAAATATATTAGCTTTATTTTTAACATAGTTAAATGCTATTGTCATAAAATAATAATCATAATAATTAATTGTAATTCTATATCATCAATTATCTAGTTAATGTAAAAAATAAATCT AAGGCCAGGCGCGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCAGATCACCTGAGATC AGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCCCATCTCTACTAAAAATACAAAAAATTAGCCAGGCGTG GTGGCGCATGCTTGTAATCCCAGCTACTTGAGAGGCTGAGGCAGGAGAATCACTTGAACCCGGGAGGCGAGGTT GCGGTGAGCCGAGATCGTGCCATTGCACTCTAGCCTGGGCAAAAAGAGTGAAACTCCATCTCAAATAAATAAATA AATAAATAATAAAAAATAACTTAAATCTACTTAATTAGAAAAACTAACATTCTAAAAATTTTATTTTAAGAAATATC AAAATTGGCTGGGCACGGTGGCTCACGCCTCTAATCCCTGCACTTTGGAAGGCTGAGGTGGGCGGATCACCTGAG GTCAGGAGGGTCAGGAGTACAAGACCAGCCTGGCCAACATGGCGAAACCCTGTCTCCACTAAAAATACAAAAATT AGCCAGGCATGATGATGGGCACCTGTAATCCCAGCTACTCAGGAGGCTGAGACAGAAGAATCGCTTGAACCCAG GAGGTAGAGGTTGCAGTGAGCTGAGATCACCCCACTGCACTCCAGCCTGGGTGACAGAGTGAAACTCCGCCTCAA AAAAAAAAAAAAGAGAAAAGAAATATAGAAATTAAAGCATACATGGCCAGGCGTAGTGGCTCATGTCTGTAATCC CAGCACTTTGGGAGGCTGAGGCAGGCAGATCACTTGAGGCCATGAGTTCAAGACCAACCTGGCCAACATGGCGA AAGCCTGTCTCTACTAAAAATACAAAAAAATTAGTTGGGCATGGTGGTGCACACCTGTAATCACAGCTACTTTGGA GGCTGAGGCAGGAGAATCGTTTGAACCCAGAGGTGGAGGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCTAT CCTGGGTGACAGAGCGAGATACTGTCTCAAAAAGAAAAAAAAAAGGCTGGGCGCGGTAGTTCATGCCTGCAATC CCAGCACTTTGGGAGGCCGAGGCAGGCAGATTACGAAGTCAGGAGATGGAGACCATCCTGGCTAATACAGTGAA ACCCCGTCTCTACTAAAAAATACACAAAAATTAGCTGGGTGTGGTGGCAGGCACCTGTAGTCCCAGCTACTCTGGA GGCTGAGGCAGGAGAATGGCATGAACCCGGGAGGTGGAGCTTGCAGTGAGCAGAGATCACACCACTGCACTCCA GTCTGGGCGACAGAGCGAGGCTCTGTCTCAAAAAAAAAAAAGAAAGCATACTCTCACCTCCTTCAGTGACTGATG TTAGTATTTTGGCACATTCTTTTTCTGTGACATATACACACTTACCTTGTAAGTGTTGTACTCATTTCCTATGACAGTA AATAGTCTTTGTAACAGGCTGCATGATATTTCATAAAATGAATGGATGTGGCATAATTTATATGTGAGCCTTTTGAA TTCTGCTATTATAATTAATATTGCAATGAACAATTCTTATATTGCCTCTACACCTCAAATGTCTTATCATTTCTTCTAG TTTTTCTGAGGATGTCAGATTATTGGGTTAAAGGATATGAACATTTTTAAGGCCTTGGAACAGATTTCTAAATTGCT TTCCAGAATAATTCCCATGTGATACTTTCACCATGTTTATTTCAGACTT I I I I I I I T I I I I I I I I I I GAGACGAAATCTC ACTCTGTCACCCAGGCTGGAGTGTAGTGGCATGATCTCGGCTCACTGCAACCTCCGCCTCCTGAGTTTAAGCGATT ATTCTGCCTCAGCCTCCCAAGTAGCTGCGGTTACAGGCAAGTGCCTCCATGCCTGGCTAATTTTTGTGTCTTTTGTA GACATGGGGTTTCACCATGTTGCCCAGGCTGGTTTCGAACTCCTGAGCTCAGGCAATCTGCCTACCTCGGCCTCCC AAAGTTCTGGGATTACAGGCGTGCACCACCGCGCCCAGCCATCAGAGTCTTTTTTGTCAAAATAAAATGGTCTAAA GACATACATCATAGAGAAACTATAATACAAAATTTACAGGTATATCTAAGAAAAGAAAAGTATATTTAAAGCATAA AAATAAACTGCTCTTTTACTTAAAATTTTTTAAAAACTGGATTAAAAATATGAAACTTCCAACAAATTGAGCTTTTTT TTTTTTTTTTTTCTTTTTTGAGACGAGGTCTCGCTTTTGTCACCCAGTCTGGAGTGCAGTGGCGCGATCTCGGCTCAC TGCAACCTCCACCTCCCTGGTTCAAGCAATTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCGCATGCCA CCACGTCGGGCTAATTTTTTTGTATTTTTAGTAGAGAGGGGGTTTCACCATGTTGGCCAGACTGGTCTCGAACTCCT GATCTCAGGCAATCTGCCAGCCTGGGTCTCCCAACATGCTGGGATTACAGGCATGAGCCACTGCACTCGGCCTGAACTTTTTATAGTAGTAACGATAATTCAGTAATGTCCAATAATGACTAAGTAAGTTATAACAAGTACAATGTCAGCAA TAACTAGTGCTTTTTAGTAAACAGGGTCAGGCAACCTTGTACCCTTTTAAAAATGTTCGAATATCGATATACCTCCT TCCTACTTGGTGGAGGATTGATTGAGGAGGAAAGTGTGCAGTGATGGTTACCAGCTTCAGCCTCTTGGCTTGACTT TGCAAATACTGGTGAGAATTTGGAAAGAGCTTGAGAATATCTTACATAGTCACATGTTGCTGAGAAGAGTTAAGA ACTAACTTCTTGATGTTCATTTTTAACAATGGCTTGCATTCAAAACCTTGTAGAGCTCATTAGTAGGAGCTAAGAAG CTAATATTTGCCTTTCACTAAAATTCCTGATTACTTAGCCTAGGTAGTTCGTTGTCTCTCTAGGTTCTGTCTTTGGGA GCTTGGGTCTAAGGTTATCAAGCTAACTCTTTCTTCCCTCTCACCCTTCCCAAATTGACCCTGGTGCTGATTTGTTAT TCATACGATTTTCTAGTTTTTCTTTTCCCTTTTTGAGTATTTGAAGCTTCATACTGAATATAGTAATCATAGTATTCAT GCATAAAGAAAATCATAAAGTAATTGCATAAATGCATAAAGTAATCATAGTTTTCATGCATTAAAAAAACTAGTTTT GGCTGGGCGCTATGGCTCACGCTTGTAATCCCAGCACTTTCGGAGGCCAAGGCAGGCGAATCATCTGAGGTCAGG AGTTCGAGACTAGCCTGGCCAACATGGCGAAACCTCTTCTCTACTAAAAATACAAAAAAATTAGCCGAGTATGGTG GCGGGCGCCTGTAATCCTAGCTATTTGGCAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCAGAGGTTGC AGTGAGCCGAGGTTGTGCCATTGCACTACAGCCTAGGCGACAAGAGCAAGACTCCATCTCAAAAAAAAAAAAAAA AAAAAAAAAACTCCCTATTACAGATTCATAATTTATGAGTCATTAAATAATATTTTCAAGCCATGACATTTTTTCCAG CAGTAGTCTCTAAATCTGTTTTACCATCATAAAACCCCAAGCAAAACTCTACTACATCAGCTGTGTCACTGTAAAAC CTGCCTTAACTCACAGAAGCATGAAATTAAGCAATGTGTGTGAAACTATTTTATAAACTGTAAAGTATTCCATACAT ACATGTTGGCAGTTATTAATGTCTTCTCTAGGTGTGGCTTTGAAATGGATGCAGATGCTTTCTGTTACAAAAAACAT AAGTTGCAAATGTTCTATAACAAGGAGAGACACAAATATCTTCATGGACATGGATTGCTATGAGTGTTTGATTGCC TAATACTTGAGCCACCACTTCAGTGATATGGTATAATTTATCAAACAGTGTTGAGAAACAGAAACTACTGGGGATG TTTTAAAGAGGAAAATACTTAATATAGAAATTAGGGGTTTACATAATCTTAAGAAAGGATGAAGGTGCAGCTCTTAGCCAGGCCTCCACAGTACCACAAACCAACTTGCAGGAAGAGCTGTAACCACTGCCCCAGTTGGGACAATGGGTAA TGAGGATATTAAATTTAAGAACATACTGCTATAGCAATGATCCTTGGCATAGAAAGCTGCCACCACAATTGCCTAG AGATGGGAACATGAAGTCTGGCCCCCATTGCAACAGCAGTGAAGCAGAATTTTGGGACTGGCATCTCCCAAATGG CTTTGCTTGCCACCAGAGAACAACCAAAGTGGAGGGAGATGGCTAGGCCTCATTTCTGCCTATTTTATTTTATTTTT TGAGACGGAGTCTTGTCTGTCGCCCAGGCTGGAGTGCAGTAGTGTGATCTCGGCTCACTGCAGCCTCCGCCTCCCA GCTTCAAACAATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCACCCGCCACTGTGCCCAGCCAATTTT CTTATTTTTAGTAGAGGTGGGGTTTTGCCACGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCTGCCC GCCTCAGCCTCCCAAAGTGTTGTGATTACAGGTATGAGCCACCATGCCTGGCCCATTTCTCCC I I I I I I I I I I I I I I I I TTTTTTGAGGTGGAGTCTCACTCTGTTGCCCAGACTGGAGTGCAGTGGTGCAATCTTGGCGCATTGCAACCTCTGC CTCCCAGTTTCAAGCAATTCTTCTGCTTCAGCCTCCTGAGTAGCTGGGACTACAGGTGTGTAGCACCACACCTGGCT AATTTTTGTTTTTGTTTTGTTTTTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGCATGATCTG GGCTCACTACAACCTCCGCCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCAGAGTAGCTGGGATTACAGGT GTGCGCCAACACACCTGGCTAATTTTTTTGTATTTTTAATAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTC GAACTCCTGACCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCCAG ACAAGGTTTGTATTTTTAGTAGAGACAGTTTTGCCATGTTGGCCAGGCTGGTCTTGAACTCCTCACCTCAGGTGATC CGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGCAAGCCACTGTGCCTGACCCGTTTCTGCTTTTTAAAG CTCATGTGAGCACTTAATTTGTAACCAGAATCCTACTTGTAAAATAATCTAAGACATGTAGCTTTTAGCTTTGTAAC CTCTATAATATTGATGGCACAGTGGGAGTGGATGCTGAGTACCACTTGAACATGTTCCACCTCAGTGTCTTCACAG CTGGAAGGTGTCTACATTGTTTCAAGGTGGACAATTGATTTACTTCTCATTTTTCATAAACTAAAAGTAGAATAAAG GCTATTCCTCTAAAATTGCTATCTCACCTGTCACTCCCTTGCATTCTCACATACCTTCTTGAGTGGAGGGGCAGAGG GCATGGAGTGATAGCAGATGTGCCAGGAATTCTCCATAACTCAGTCCGTCCCTCTTGTGCTATGTTGCAGCATCAG GATTTGCTAATGGGAGGATACTGCCCTTACGTGCATCATTAGCCATGCACACTAAGGTCTTACACCTACACACAGG TCAGTATTCTGGCTCAGAGACCAACAGGGAGAAATTGCAGTTCTCATTAGTTGAACTTTCTTTATTGTTCACAGTTT TAAAACACAAAATTGAGAGGAACTCTATAAAAAATGTGCCATTCTATTAATAATTGTTGCTGGTAATTTAAAAATCC TTGTTCCTTTTCAAATTCTTATATACCTTTTTTTTTTAAACACTTGATCTTAGCCAAAAGACCGAGAAGCAATCTTTTT TTTTTTTTTTTTTTTTTTTAACCTATAGCTTCTCACTGAGATTGTCAGCTGTTTGTAAGTTTTGGTTTTTGGTTTTCTGT GTTTGTATTTACATATATGAAATACAGATTGAGTATCCCTTATCCAAAATGCTTAAGACTGGAAGTGTTTTAGATTT GGGGTTTTTTAGGATTTGTGAATATTTGCACTATACTTACCAGTTAAGCATTCCAAATCCAAAATTTCAAATCTGAA GTGTTCCACTGAGCACCTCTTTTGAGTATCATGTTGGTGCTCAAAAAGTTTCTGATTTTGGAGCATTTGGATTTCTG ATTCTCGGATTTAGGATGCTTGACCTGTAATTTCAGATTTACATAAAAGCAGAAATAGTACACAGAGCTCCTTATAT CCTTCACCCAGATTCCCCAATTATTGGCCTTTCTGAACCATTTGGGAATAATATGCAGATATGATTTTCCATTATGTC TCAGTTGTTCAGTGTATATTTTCTAAGTACAAGAATATATTCCTACATATTTACATGATAACCGTCATGTTTAAACAT TTTAAAATGGGGATTTGTATTACATTGTTTCTCTTTTTGAAAAAATTACAGAGGAGCTTAATGCAATCAGTATTACT TAAAATCTGATAATGTGTGTTAAATAGTAGTTTTCATTTATTTCATTTATCAGGTGTTCAGTGAATGCTTACTATGTA ACAGCACAGTTATCAGCACTGGGGAAATAGATGAGTAAGATAAGATTTGCACTTTCATTAGCTTACATGCCATAAA GAGGGAAATAAAGAGAACACCAGATGATGATAAGTTTATGCTGAGAATTAAAATGAAGTGATGAAATAATGGGA ATGTCAGGTGGCTACTTTTGGTGGGATGGTCAGGAAAGGCATCTCTGGGGAGATAAATTTTAAGCTCAGACCTGA GTGAAAAGAATGAGCCAGCCATGGAAACATTATGTTAACTCACATGGTAGTTTGAAATGCTTTATCTGATCAAAGG TACTTATTTTTGGTGACTTTCAACAATATTAAGGGTCTATAAACCAACACTCATTTGCATAAGAATAACTACCAGTG AATCTTTTTGTATGATAGGTTTTTTGTTTGTTGTTTTTTTGAGACAGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGC AGTGGCGCGATCTTGGCTCACTGCAACCTCTACCTCCCCGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAAGTAG CTGGGATTACAGGTGCCTGCCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCGTGTTGTC CAGGCTCGTGTCAAACTTCTGACCTCAAGCCATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGA GCCACCACTCCTGGCCATGATAGGTTATTTTGTGATGAAAATACCTACCTCTTAATTTGTCTGATAAATTTAAATTTT ATGTCTAGATTTCCTAAGATCAGCACTTCCATATTTTAAAGTAATCTGTATCAGACTAACTGCTCTTGCATTCTTTTA ATACCAGTGACTACTTTGATTCGTGAAACAATGTATTTTCCTTATGAATAGTTTTTCTCATGGTGTATTTATTCTTTTA AGTTTTGTTTTTTAAATATACTTCACTTTTGAATGTTTCAGACAGCAGCAAAAGCAGCAACAGCAGCAGCAGCAGC AGCAGCAGGGGGACCTATCAGGACAGAGTTCACATCCATGTGAAAGGCCAGCCACCAGTTCAGGAGCACTTGGGAGTGATCTAGGTAAGGCCTGCTCACCATTCATCATGTTCGCTACCTTCACACTTTATCTGACATACGAGCTCCATGT GATTTTTGCTTTACATTATTCTTCATTCCCTCTTTAATCATATTAAGAATCTTAAGTAAATTTGTAATCTACTAAATTTC CCTGGATTAAGGAGCAGTTACCAAAAGAAAAAAAAAAAAAAAAGCTAGATGTGGTGGCTCACATCTGTAATCCCA GCACTTTGGGAAACCAAGGCAGGAGAGGATTGCTAGAACATTTAATGAATACTTTAACATAATAATTTAAACTTCA CAGTAATTTGTACAGTCTCCAAAAATTCCTTAGACATCATGGATATTTTTCTTTTTTTGAGATGGAGTCTTGCTCTGT CACCCAGGCTGGAGTGCAGTGTCGCGATCTCGGCTCACTGCAAGCTCTGCTTCCTGGGTTCATGGCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCGCCCGCCACATCGCCTGGCTAATTTTTTGTATTTTTAGTAGAGAC AGGGTTTCACCATGTTAGCCAGGATGGTCTCAATCTCCTGACCTCATGATCCGCCCGCCTCGGCCTCCCAAAGTGCT GGGATTACAGGCGTGAGCCATCACGTCCGGCCAGAAATCATGAATATTAGTAGGTGAAAAATAAACACATTTTAC CACCTGGAAAATGAAAAATACTTGAGTATAATCTAAATAACAATGGGAAGTGCAGAGTTACTTTCCAGGTCTCGGT TTAAATATGTCTTAAACTTTGGCCAATTAGTAGTAGAAGTTGAGAGAAAAAGTAACTATCTGACAAAGAAATTATA AGCAGAATATATAAAGAACTCTTAAAACTGAATAATCAGAAAACAACTCAATAAAAAGGTGAAGGATTTGAAAAG ATATTTCACCAAATAAGACATAGGGATGACAAATAAGCACATGAAAAGACTCTCAGCATCACTAGTCACAGGGAA ATGCACGATAAAACCACAGTGAGACACCATGGCACCCCTGTAGGTATGGCTTTAATGAAGAAATAAAACTGACAA TACCAAGTGTTGGCAAGGATCCAAGCAGCTGAGACTCATATACTGTTAATGGGAATGTAAAAGTGTACAGCTTTG GAAAACAGTTTGGCATTTTTTTGATAAATGTATACTTAGCCATGTGATCCAGCAGTCCCAATCATGTATATATAACC AAAAGAAAAGAAAACTTAGGTTCACATAAAAACTTATATCAAATGCTTATAGCTGACCAGGCATGGTGGCCCATGC CTATAATCCCAGCACTTTGGGAGGCCGAGGTTGGCAGATACCTGAAGTCAAGTGTTCGAGACCAGCCTGGCCAAC ATGGCAAAACCCTGTCTCTACTTAAAATACAAAAATTAGCCAGGCGTGATGGCAGGCACCTGTAGTCCAGCTATTC AGGAGGCTGAGGCAGGAGAATCACGTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCGTGCCACTATA CTCCAGCCTGGGTGACAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAAAAAAAGGGCTGGACACGGTGGCTTA CGCCTGTTATCCCGGCACTTTGGGAGGCCAAGGCTGATGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGC CAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAATTTGCTGGGCATGGTGGTGGGCACCTGTAATCCCAGG AGGCTGAGGCAGGAGAATCACTTGAACCCGGGAGGCGGAGATTGCAGTGAGCCAAGATTGTGCCATTGAACTCC AGCCTGGGTGACAAGACCAAAACTCCTTCTCAAAAAAAAAAAAGATTATAGCATCTTTATTCATCATTGCCCAAAAT TACAAACTGCCTAAATGTAGACCTTCATTTAGTTAATGAATGCACAAACTGTGGTATATCCAAACAATTGAATAAAA AAAGGAATGAACTGGTACTTTTTTCTATTCCTCCTGTTTAAGTACAGCCAAAACACCTCAACATTTGTATAAAACAT GAGCTGGGCTGGGTGCGGTGGCTCACACGTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTGGATCACCTAA GGTTGGGAGTTCAAGACCGGTCTGACCAACATGGAGAAACCCTGTCTCAACTAAAAATACAAGATTAGTCGGGCA TGGTGGCGCATGCCTGTAATCCCAGCTTCTTGGGAGGCTGAGGCAGGAGAATTGCTTGATCCCGGGAAGCGAAG GTTGCAGTAAGCTGAGATTGCACCATTGCACTCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAGAAAA AAAAAACCATTCAGCTGAATCTCAAAGGCAGAGAGAAGACAGACTGGCTAGGGACCTTGGAACCAGAGGAGCAG TGTGGTGGGGAGTGGACTGGATTTTCTTTTTGCCTCATTTATCCTGGACTTGGTGCTGGAGAAGCTATGGGTTCAG ACCAAGAGAAAACCCCATGAAAAGCCTGCTCTCTCTAGCCAAAAGAGGCAACCTAGCAAGATAAAAACCTTTAGA TAATAAGCACTTGACTCCAGTCAAACAAAACAGAATAAACTGGCCCCATTCACCCCTGTCAGCAAAGGCCAAGTGG GAGCCAAGATATGTACCCCAACCTGGAAGTCATAAGGTACACTTCTCCCCTTTCCCAGCCAAGGTGGTGTTAGAGA AGGCTGACTGGGGAGCTGGGATTCTCATTCCCTCCAGGAGGTGATAACACTCCTTTCACATGGTGTCAGTGGTCAC AGGGAGGCTGAACTTCCACCCAGTAATACATAGGCATCTCTCTGGCTCCTATATGGGTGATGTTGGAGAAGAGGC CGAGTAGAGAATCCAGACTGTTGCTGACACCCAGCAGTAACAAGGACACCTCCACAATGTCCGTGGAGGCCATGT GGAGATCAGTAACAAGGCACTGCTCTCCCTCCCAGTCAGAGAGATGTCAGTGGAGGACTAGGGGGCTAGAACTC CCATGTGCGTTCAGCAGTAATCCCCATGACCGCCACTCCTTGACATCACAGGCCTTGAAGAAACCTGGACTTTCACT CCCCTCTGGTTGTAGCGAGGTGGCACTCCCTTTTCCCTGTTGCCAGTGCTGTGTCAGTGGAGGCTTGCTAAATTGG AAGATGTAAATAAGATTCACATTCTCATAACATAATACCCCAAATTTTCAGGATTTAATTGAAAATCACTAAGCTGG GCATGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGCCAAACACTTAAGGTCAGGAATTCA AGACCAGCCTGGCCAGCATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTGGCACATG CCTGTAATCCCAGCTACTGGGAAGGCTAAGGCAGGAAAATCACTGGAACCTGGGAGACGGAGGTTGCAGTGATC CAAGATCGCACTAGTGTACTGCAGCCTGGGCAACAGAGCAAGACTCCATCTAAATTTGTGTCAGGATTCCCAGAA GGAGATGAGAAAGGGTGGGGCTGAAAAAAATTGAGGAAGAAGTCATGGCTGAAAATTTCCCAAATTTGGCAAAA GTCAGAAACCTACAGATTGAAAAAGCTGAATGAAGCTCAAATATGATAAACTCAAAGAAGTTCACACAGAGACAC ATCACAGTCAGATTTCTGAACACTGCAGACAAAAAATGAAGATCTCGAAATTAGCAAGAAATGACCTTACCTAAGC AATTTGAATGACAGCAGATTTCCCATCAGAGATCATAAAGGCCAGAAGGAAGGGGTACATACAACATTTTTTCTAG TGCTGAAAGACAAAAACTCTAGGCTGGGCACGGTGGCACACACCTGTAATCCCAGCACTTTTGGAGGCTGAGGCA GGCAGATCACCTGAAGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGGGAAACCCTGTCTCTACTAAAAATACA AAAATTAGCCAGGTGTGGTGGCACGCACCTATAATCCTAGCTACTTGGGAGGCTGAGGCAGGGGAATCGCTTGAA CCTGGGAGGCGACGGTTGCAGTGAGCCAAGGTCGCGCCACTGCACTCCAGCCTGGGCAGTTGAGCGAGACTCCA TCTCAAAAAAAAAAAAATTATCCAGGCTTGGTGGTGGGCGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCAAG AGAATTGGTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCCAAGCTCATGCCACTGTACTCCAGCCTGGGTGACA GAGCGAGACCTTGTCTCAAAAAAAAAAAAAAAAAAAAAAAACAAGAAAAAAACTCTAAACCCAGAGTTACATATC CAGTGAAATATCCTTCAGGAGTGAAGGGAAAATTAACGATTTGTCTTCAGGAGACCTACCCTAAAAGAATGGCTA AAGGAATTTCTCTAAACAGAAAAGAAATGATAAAAGAAGTAATTTTGGAACATCAGGAAGGAAGAAAGAACAATAAAAAGAGTAAAATATGGGTAAACACAATAGACTTTCCCCTCCTTTTGAATTTTCTAAATTGTATGATGGTTGAAGC AAGAATTATAGCACTGATTTGGTTTTCAGTATATATATTGGAAATATTTAAGGCATTATGTTACAGATGAAGGAGG GTCAAAGGATATAAAGGGAGGTAACCTTTCTATATTTCTTTTGTACTGATGCAGGCACTTTGGAAAATAATTTCACT ATTTGTTTAAAAACTGAACATACCCTGACCATATGACATAGCATCTATACTCCTGGGCATTTATCCCAGAGAAACAG AAATTTATTTATTTTTTTTTTAGTATTACACTCCGTAAGTGCTGTAATACTAGCACTTAGGGAGGCTGAGGCAAGCA GATTGCTTGAGCCCAGGAGTTCAAGACCAGCCTGGGCAATGCTGCACAGTCAAAAAAGAAAAACAAACATTTAGA AAACTATTTTAAAAGTCTTTAATTGCTGAATGCCTCTTTGGCTAATATTTGGAAGATCATTATTATTATTTTTCTTTTT TAGGCAGAGTCTTGCTCTGTCACTGAGGCTGGAGTGCAGTGGCGCCATCTCGGCTTACTGCAACCTCTGCCTCCCG GGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGTGTGCCACCATGCCCGGCTAATTT TTTGTGTTTTTAGTAGAGATGGGGTTTCACTATGTTAGTCAGGATGGTCTCCATCTCCTAACCTCGTGATCCGCCCA CCTCGGCTTCCCAAAATGCTGGGATTACAGGCGTGAGCCACTGTGCCCAGCCTGGAAGATCATTATTTAGTCCTAC AACTGACACATTGTTCCACTGACGCAATTGCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCAATCTGCCTGCCTC GGCCTCCCTAAGTGCTAGTATTACAGGCTTGAGCCACTGTGCCCAGCCAAAAATAGAAATTTATATTCTCACAAAA ACATGTACATGAATGTTTATAGCAGCTTTACTTGTCATAATCAAAAACTGGAAACAACCAAAATGTCCTACAGTGA AACAAACTGTAGTACATCCATAGCATGTAATACTCTACTGTCAGGATTAAAAAGAAACCCACTGTTGGCACAGGCA GCACCGTGGCTGGATCTCAGGGGCATTATGCTGAGTGCAAAAAAGCCTCAAAGGGTCTTACACTGTATGATTCCA CTTGTTCAACTAAAAATGACAGCTGTATAGAGATAGAGAACATATTAGTGGTTTCCACTAGTTAGAGAAAGTGGGT AAAAGATAGGTGGGTGGGAATATAAATCGATAGCAGGGAGATCTTTGTGGTATTATAACACTTCTATGTCTTGATT GTAGTGGTGGTGGTTACATGAATACACGTGTGATAAAATGCCATGTAGAACTACATATAACGTTGTGCCAATGTCA ATATCTAGGTTTTAGTTTGATCTTTAGTTACATAAGATGTAACTATTGGGTGAAATTGGGCAAAAGAGTACACGAA ACCTCTCTTAAATATCTTTACAACTTCCTTTGAATTGACAGTTTTTCAAAATAGAAAGTTGGGTTTTTGTAAATACAT GAATTGTTGATATACACAACAAATCTCAAATGCATTATGCTACGTGAAAGAAGCCATATTCAAAAGGCTACATACCTACTGATGCCTTTTATATGACGTGCAGGAAAAGATAAAACTGTAGGACAGAGAATATACTGGTGGCTATCTGGGA TTAGGAAATGGGGATCGACCACAAAGGGGCAGCATGGGGGAATTTTCTGGGGCAATGGAATGGTTGTGTATCTT GATGGTGTATTTGTCAAAATATATAGAACTATAAAAGTAAATTTTGCTTTATATGTATTAAATCAAAAAAAGAAACT CGTGCTCAAATAGAAATACATTTTCTGAGAACTTGCCTTTTGATGACTTTGAGAATTTTCTGGAAATTTTAAAGAAA TGTGGTTTTGTTTCCCAACAGGTGATGCTATGAGTGAAGAAGACATGCTTCAGGCAGCTGTGACCATGTCTTTAGA AACTGTCAGAAATGATTTGAAAACAGAAGGAAAAAAATAATACCTTTAAAAAATAATTTAGATATTCATACTTTCC AACATTATCCTGTGTGATTACAGCATAGGGTCCACTTTGGTAATGTGTCAAAGAGATGAGGAAATAAGACTTTTAG CGGTTTGCAAACAAAATGATGGGAAAGTGGAACAATGCGTCGGTTGTAGGACTAAATAATGATCTTCCAAATATT AGCCAAAGAGGCATTCAGCAATTAAAGACATTTAAAATAGTTTTCTAAATGTTTCTTTTTCTTTTTTGAGTGTGCAAT ATGTAACATGTCTAAAGTTAGGGCATTTTTCTTGGATCTTTTTGCAGACTAGCTAATTAGCTCTCGCCTCAGGCTTTT TCCATATAGTTTGTTTTCTTTTTCTGTCTTGTAGGTAAGTTGGCTCACATCATGTAATAGTGGCTTTCATTTCTTATTA ACCAAATTAACCTTTCAGGAAAGTATCTCTACTTTCCTGATGTTGATAATAGTAATGGTTCTAGAAGGATGAACAGT TCTCCCTTCAACTGTATACCGTGTGCTCCAGTGTTTTCTTGTGTTGTTTTCTCTGATCACAACTTTTCTGCTACCTGGT TTTCATTATTTTCCCACAATTCTTTTGAAAGATGGTAATCTTTTCTGAGGTTTAGCGTTTTAAGCCCTACGATGGGAT CATTATTTCATGACTGGTGCGTTCCTAAACTCTGAAATCAGCCTTGCACAAGTACTTGAGAATAAATGAGCATTTTT TAAAATGTGTGAGCATGTGCTTTCCCAGATGCTTTATGAATGTCTTTTCACTTATATCAAAACCTTACAGCTTTGTTG CAACCCCTTCTTCCTGCGCCTTATTTTTTCCTTTCTTCTCCAATTGAGAAAACTAGGAGAAGCATAGTATGCAGGCAA GTCTCCTTCTGTTAGAAGACTAAACATACGTACCCACCATGAATGTATGATACATGAAATTTGGCCTTCAATTTTAA TAGCAGTTTTATTTTATTTTTTCTCCTATGACTGGAGCTTTGTGTTCTCTTTACAGTTGAGTCATGGAATGTAGGTGT CTGCTTCACATCTTTTAGTAGGTATAGCTTGTCAAAGATGGTGATCTGGAACATGAAAATAATTTACTAATGAAAAT ATGTTTAAATTTATACTGTGATTTGACACTTGCATCATGTTTAGATAGCTTAAGAACAATGGAAGTCACAGTACTTA GTGGATCTATAAATAAGAAAGTCCATAGTTTTGATAAATATTCTCTTTAATTGAGATGTACAGAGAGTTTCTTGCTGGGTCAATAGGATAGTATCATTTTGGTGAAAACCATGTCTCTGAAATTGATGTTTTAGTTTCAGTGTTCCCTATCCCT CATTCTCCATCTCCTTTTGAAGCTCTTTTGAATGTTGAATTGTTCATAAGCTAAAATCCAAGAAATTTCAGCTGACAA CTTCGAAAATTATAATATGGTATATTGCCCTCCTGGTGTGTGGCTGCACACATTTTATCAGGGAAAGTTTTTTGATC TAGGATTTATTGCTAACTAACTGAAAAGAGAAGAAAAAATATCTTTTATTTATGATTATAAAATAGCTTTTTCTTCG ATATAACAGATTTTTTAAGTCATTATTTTGTGCCAATCAGTTTTCTGAAGTTTCCCTTACACAAAAGGATAGCTTTAT TTTAAAATCTAAAGTTTCTTTTAATAGTTAAAAATGTTTCAGAAGAATTATAAAACTTTAAAACTGCAAGGGATGTT GGAGTTTAGTACTACTCCCTCAAGATTTAAAAAGCTAAATATTTTAAGACTGAACATTTATGTTAATTATTACCAGT GTGTTTGTCATATTTTCCATGGATATTTGTTCATTACCTTTTTCCATTGAAAAGTTACATTAAACTTTTCATACACTTG AATTGATGAGCTACCTAATATAAAAATGAGAAAACCAATATGCATTTTAAAGTTTTAACTTTAGAGTTTATAAAGTT CATATATACCCTAGTTAAAGCACTTAAGAAAATATGGCATGTTTGACTTTTAGTTCCTAGAGAGTTTTTGTTTTTGTTTTTGTTTTTTTTTGAGACGGAGTCTTGCTATGTCTCCCAGGCTGGAGGGCAGTGGCATGATCTCGGCTCACTACAACTTCCACCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCAGAGTAGCTGGGATTACAGGCGCCCACCACCACACCCGGCAGATTTTTGTATTTTTGGTAGAGACGCGGTTTCATCATGTTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCGCCTGCCTTGGCCTCCCAAAGTGTTGGGATTACAGGCATGAGCCACTGCGCCTGGCCAGCTAGAGAGTTTTTAAAGCAGAGCTGAGCACACACTGGATGCGTTTGAATGTGTTTGTGTAGTTTGTTGTGAAATTGTTACATTTAGCAGGCAGATCCAGAAGCACTAGTGAACTGTCATCTTGGTGGGGTTGGCTTAAATTTAATTGACTGTTTAGATTCCATTTCTTAATTGATTGGCCAGTATGAAAAGATGCCAGTGCAAGTAACCATAGTATCAAAAAAGTTAAAAATTATTCAAAGCTATAGTTTATACATCAGGTACTGCCATTTACTGTAAACCACCTGCAAGAAAGTCAGGAACAACTAAATTCACAAGAACTGTCCTGCTAAGAAGTGTATTAAAGATTTCCATTTTGTTTTACTAATTGGGAACATCTTAATGTTTAATATTTAAACTATTGGTATCATTTTTCTAATGTATAATTTGTATTACTGGGATCAAGTATGTACAGTGGTGATGCTAGTAGAAGTTTAAGCCTTGGAAATACCACTTTCATATTTTCAGATGTCATGGATTTAATGAGTAATTTATGTTTTTAAAATTCAGAATAGTTAATCTCTGATCTAAAACCATCAATCTATGTTTTTTACGGTAATCATGTAAATATTTCAGTAATATAAACTGTTTGAAAAGGCTGCTGCAGGTAAACTCTATACTAGGATCTTGGCCAAATAATTTACAATTCACAGAATATTTTATTTAAGGTGGTGCTTTTTTTTTTTGTCCTTAAAACTTGATTTTTCTTAACTTTATTCATGATGCCAAAGTAAATGAGGAAAAAAACTCAAAACCAGTTGAGTATCATTGCAGACAAAACTACCAGTAGTCCATATTGTTTAATATTAAGTTGAATAAAATAAATTTTATTTCAGTCAGAGCCTAAATCACATTTTGATTGTCTGAATTTTTGATACTATTTTTAAAATCATGCTAGTGGCGGCTGGGCGTGGTAGCTCACGCCTGTAATCCCAGCATTTTGGGAGGCCGAAGTGGGTGGATCACGAGGTCGGGAGTTCGAGACCAGCTTGGCCAAAATGGTGAAACCCCATCTGTACTAAAAACTACAAAAATTAGCTGGGCGCGGTGGCAGGTGCCTGTAATCCCAGCTACCTGGGAGTCTGAGGCAGGAGAATTGCTTGAACCCTGGCGACAGAGGATGCAGTGAGCCAAGATGGTGCCACTGTACTCCAGACTGGGCGACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAATCATGCTAGTGCCAAGAGCTACTAAATTCTTAAAACCGGCCCATTGGACCTGTACAGATAAAAAATAGATTCAGTGCATAATCAAAATATGATAATTTTAAAATCTTAAGTAGAAAAATAAATCTTGATGTTTTAAATTCTTACGAGGATTCAATAGTTAATATTGATGATCTCCCGGCTGGGTGCAGTGGCTCACGCCTGTAATCCCAGCAGTTCTGGAGGCTGAGGTGGGCGAATCACTTCAGGCCAGGAGTTCAAGACCAGTCTGGGCAACATGGTGAAACCTCGTTTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTTGCACACACTTGTAATCCCAGCTACTCAGGAGGCTAAGAATCGCATGAGCCTAGGAGGCAGAGGTTGCAGAGTGCCAAGGGCTCACCACTGCATTCCAGCCTGCCCAACAGAGTGAGACACTGTTTCTGAAAAAAAAAAATATATATATATATATATATATGTGTGTATATATATATGTATATATATATGACTTCCTATTAAAAACTTTATCCCAGTCGGGGGCAGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCTGAGGCAGGTGGATCACCTGAAGTCCGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAACTTAAGCCAGGTATGGTGGCGGGCACCTGTAATCCCAGTTACTTGGGAGGCTGAGGCAGGAGAATCGTTTAAACCCAGGAGGTGGAGGTTGCAGTGAGCTGAGATCGTGCCATTGCACTCTAGCCTGGGCAACAAGAGTAAAACTCCATCTTAAAGGTTTGTTTGTTTTTTTTTAATCCGGAAACGAAGAGGCGTTGGGCCGCTATTTTCTTTTTCTTTCTTTCTTTCTTTCTTTTTTTTTTTTTCTGAGACGGAGTCTAGCTCTGCTGCCCAGGCTGGAGTACAATGACACGATGTTGGCTCACTGCAACCTCCACCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTACCTGGGATTACAGGCACCTGCCACTACACCTGGCGAATATTTGTTTTTTTTAGTAGAGACGGGCTTTTACCATGTTAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCTGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGCAGGCCACCACACCCGGCCTTGGGCCACTGTTTTCAAAGTGAATTGTTTGTTGTATCGAGTCCTTAAGTATGGATATATATGTGACCCTAATTAAGAACTACCAGATTGGATCAACTAATCATGTCAGCAATGTAAATAACTTTATTTTTCATATTCAAAATAAAAACTTTCTTTTATTTCTGGCCCCTTTATAACCAGCATCTTTTTGCTTTAAAAAATGACCTGGCTTTGTATTTTTTTAGTCTTAAACATAATAAAAATATTTTTGTTCTAATTTGCTTTCATGAGTGAAGATTATTGACATCGTTGGTAAATTCTAGAATTTTGATTTTGTTTTTTAATTTGAAGAAAATCTTTGCTATTATTATTTTTTCCAAGTGGTCTGGCATTTTAAGAATTAGTGCTAATAACGTAACTTCTAAATTTGTCGTAATTGGCATGTTTAATAGCATATCAAAAAACATTTTAAGCCTGTGGATTCATAGACAAAGCAATGAGAAACATTAGTAAAATATAAATGGATATTCCTGATGCATTTAGGAAGCTCTCAATTGTCTCTTGCATAGTTCAAGGAATGTTTTCTGAATTTTTTTAATGCTTTTTTTTTTTTTGAAAGAGGAAAACATACATTTTTAAATGTGATTATCTAATTTTTACAACACTGGGCTATTAGGAATAACTTTTTAAAAATTACTGTTCTGTATAAATATTTGAAATTCAAGTACAGAAAATATCTGAAACAAAAAGCATTGTTGTTTGGCCATGATACAAGTGCACTGTGGCAGTGCCGCTTGCTCAGGACCCAGCCCTGCAGCCCTTCTGTGTGTGCTCCCTCGTTAAGTTCATTTGCTGTTATTACACACACAGGCCTTCCTGTCTGGTCGTTAGAAAAGCCGGGCTTCCAAAGCACTGTTGAACACAGGATTCTGTTGTTAGTGTGGATGTTCAATGAGTTGTATTTTAAATATCAAAGATTATTAAATAAAGATAATGTTTGCTTTTCTA - 3'Oligonucleotide primers to verify the correct insertion of each sgRNA-codifying oligonucleotide pairs through Sanger sequencingSEQ ID NO16: 5' - GACTATCATATGCTTACCGT - 3'SEQ ID N017: 5' - CGCGTGAGGGCCTATTTCC - 3'Oligonucleotide primers for PCR amplification of the CRISPR-edited regions of the human ATXN3 gene (Exon 2 and Exon 10) and cDNA (Exon 2) of a lentiviral-based mouse modelSEQ ID NO18: 5' - TTTAGGGGAAGATACTATACAATTC - 3'SEQ ID NO19: 5' - TTTGACAGGTAGTTGAAGCAAG - 3'SEQ ID NQ20: 5' - ACTAGTGGATCCGTCGACAT - 3'SEQ ID NO21: 5' - CCCGTCAAGAGAGAATTCAAGT - 3'SEQ ID NO22: 5' - TTTCCTAAGATCAGCACTTCCA - 3'SEQ ID NO23: 5' - ACTGCTCCTTAATCCAGGGAA - 3'Bibliographic References1. Takiyama Y, Nishizawa M, Tanaka H, Kawashima S, Sakamoto H, Karube Y, Shimazaki H, Soutome M, Endo K, Ohta S, et al.: The gene for Machado-Joseph disease maps to human chromosome 14q. Nature genetics 1993, 4:300-304.2. Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, Kawakami H, Nakamura S, Nishimura M, Akiguchi I, et al.: CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nature genetics 1994, 8:221-228.3. Hsu PD, Lander ES, Zhang F: Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014, 157:1262-1278.4. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F: Multiplex genome engineering using CRISPR / Cas systems. Science 2013, 339:819-823.5. Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S: The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010, 468:67-71.6. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM: RNA-guided human genome engineering via Cas9. Science 2013, 339:823-826.7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E: A programmable dual-RNA- guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337:816-821.8. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F: DNA targeting specificity of RNA-guided Cas9 nucleases. Nature biotechnology 2013, 31:827-832.9. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS: Analysis of off-target effects of CRISPR / Cas- derived RNA-guided endonucleases and nickases. Genome research 2014, 24:132-141.10. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F: Genome engineering using the CRISPR- Cas9 system. Nature protocols 2013, 8:2281-2308.11. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM: CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology 2013, 31:833-838.12. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC: Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods 2014, 11:399-402.

Claims

C L A I M S1. Combination of guide RNAs for use in the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3, wherein said combination comprises: a first guide RNA for targeting intron 9 of ataxin-3 (ATXN3) gene, wherein said guide RNA comprises a sequence identical to the following list: SEQ ID NO: 5, and a second guide RNA for targeting exon 10 of ATX N 3 gene, wherein said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or combinations thereof; or a first and a second guide RNA for targeting intron 9 of ATXN3 gene, wherein said pair of guide RNAs comprises a sequence identical to a sequence of the following list: SEQ ID NO: 5 and SEQ ID NO: 9, or combinations thereof, and a first and a second guide RNA for targeting exon 10 of ATXN3 gene, wherein said pair of guide RNAs comprises a sequence identical to a sequence of the following list: SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or combinations thereof.

2. Combination of guide RNAs for use according to the previous claim, wherein the first and a second guide RNA for targeting intron 9 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to the combination of sequences of the following list: SEQ ID NO: 5 and SEQ ID NO: 9.

3. Combination of guide RNAs for use according to any of the previous claim, wherein the first and a second guide RNA for targeting exon 10 of ATXN3 gene, wherein said pair of guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 8 and SEQ ID NO: 10; or SEQ ID NO: 11 and SEQ ID NO: 12; or SEQ ID NO: 12 and SEQ ID NO: 13.

4. Guide RNA for the use in the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3, wherein said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 13, or combinations thereof or a guide RNA consisting in a sequence identical SEQ ID NO: 2.

5. Guide RNA for use according to any of the previous claims, for targeting exon 2 of ATXN3 gene, wherein said guide RNA comprises a single sequence identical to a sequence of the following list: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or combinations thereof.

6. Guide RNA for use according to any of the previous claims, for targeting exon 2 of ATXN3 gene, wherein said guide RNA consist in a single sequence identical to sequence SEQ ID NO: 2.

7. Guide RNA for use according to the previous claims, for targeting exon 10 of ATXN3 gene, wherein said guide RNA comprises a sequence identical to a sequence of the following list: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 13, or combinations thereof.

8. Guide RNA or a combination of guide RNAs according to any of the previous claims 1-6, or a CRISPR-Cas system according to any of the claims 7-10, or vector according to any of the claims 12-13, or a composition according to claim 14 for the use in the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3, wherein the guide RNA or combination of guide RNAs, or CRISPR-Cas system, or vector, or composition is administrated systemically, intravenously, intratumorally, orally, intranasally, intraperitoneally, intramuscularly, intravertebrally, intracerebrally, intracerebroventriculally, intracisternally, intrathecally, intraocularly, intracardiacally, intradermally, or subcutaneously, preferably intravenously, intracisternally, intrathecally or, in situ, by intracerebral administration.

9. CRISPR-Cas system for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 comprising a human codon-optimized Cas protein, a guide RNA or a combination of guide RNAs according to any of the previous claims, and a protospacer adjacent motif, wherein each guide RNA has a complementary sequence encompassing the 5' sequence adjacent to the protospacer- adjacent motif in the ATXN3 gene.

10. CRISPR-Cas system for use according to the previous claim, wherein human codon-optimized Cas protein, comprises a Cas protein.

11. CRISPR-Cas system for use according to any of the previous claims 9-10, wherein the mutated Cas is a Cas9 nickase.

12. CRISPR-Cas system for use according to any of the previous claims 9-11, wherein the Cas9 nickase is Streptococcus pyogenes Cas9 (SpCas9) D10A nickase.

13. CRISPR-Cas system for use according to any of the previous claims 9-12, wherein the Cas protein is selected from a list comprising: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus Cas9 (StlCas9), Neisseria meningitidis Cas9 (NmCas9), Campylobacter jejuni Cas9 (CjCas9), Francisella novicida Cas9 (FnCas9), Streptococcus canis Cas9 (ScCas9), or Staphylococcus simulans Cas9 (SsCas9), Geobacillus stearothermophilus Cas9 (GeoCas9), Acidaminococcus sp. Casl2a (AsCasl2a), Lachnospiraceae bacterium Casl2a (LbCasl2a), Bacillus hisashii, Casl2b (BhCasl2b), Deltaproteobacteria Casl2e (DpbCasl2e), Planctomycetes Casl2e (PlmCasl2e), and Unultured archeon Casl2f.

14. Vector or vector combination for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 comprising the CRISPR-Cas system according to any of the previous claims 9-13.

15. Vector or vector combination for use according to the previous claim, wherein the vector is selected from the group consisting of lipid nanoparticles, polymeric nanoparticle, liposome, extracellular vesicle, plasmid or viral vector; preferably a plasmid or a viral vector; more preferably adenovirus, lentivirus, retrovirus, or adeno-associated virus vector.

16. Composition for the treatment of Machado-Joseph disease or Spinocerebellar ataxia type 3 comprising a guide RNA or a combination of guide RNAs according to any of the previous claims 1-8, or a CRISPR-Cas system according to any of the previous claims 9-13, or a vector or a vector combination according to the previous claims 14-15.

17. In vitro or ex vivo method for editing the ATXN3 gene in a cell by a CRISPR-Cas genome editing system, the method comprising: a step of introducing the vector according to any of the previous claims 14-15 into the cell to achieve either: the excision of the CAG tract, by targeting by intron 9 and exon 10 of the ATXN3 gene; or the gene knock-out, by targeting exon 2 of the ATXN3 gene.

18. Method according to the previous claim, wherein the cell is an induced pluripotent stem cell, or a cell of the central nervous system; preferably neurons, or oligodendrocytes, or glial cells.

19. The use of guide RNA or a combination of guide RNAs according to any of the previous claims 1- 8, or a CRISPR-Cas system according to any of the previous claims 9-13, or vector according to any of the previous claims 14-15, or a composition according to the previous claim 16 for the manufacture of a medicament for the treatment of a neurodegenerative disease, wherein the neurodegenerative disease is an ATXN3 gene-related condition or disorder; preferably the ATXN3 gene-related condition or disorder is Machado-Joseph disease or Spinocerebellar ataxia type 3.

0. A method for treating or preventing ATXN3 gene-related condition or disorder in a subject, the method comprising administering guide RNA or a combination of guide RNAs according to any of the previous claims 1-8, or a CRISPR-Cas system according to any of the previous claims 9-13, or vector according to any of the previous claims 14-15, or a composition according to the previous claim 16 to the subject.

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