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WO2025074350A2 - Outils basés sur le système crispr-cas9 : nouvelles approches thérapeutiques potentielles pour la maladie de machado-joseph - Google Patents

Outils basés sur le système crispr-cas9 : nouvelles approches thérapeutiques potentielles pour la maladie de machado-joseph Download PDF

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WO2025074350A2
WO2025074350A2 PCT/IB2024/059800 IB2024059800W WO2025074350A2 WO 2025074350 A2 WO2025074350 A2 WO 2025074350A2 IB 2024059800 W IB2024059800 W IB 2024059800W WO 2025074350 A2 WO2025074350 A2 WO 2025074350A2
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gene
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atxn3
crispr
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Sara Isabel MONTEIRO LOPES
Carlos Adriano ALBUQUERQUE ANDRADE DE MATOS
Rui Jorge GONÇALVES PEREIRA NOBRE
Luís Fernando MORGADO PEREIRA DE ALMEIDA
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Universidade de Coimbra
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2320/00—Applications; Uses
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    • C12N2320/33—Alteration of splicing
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/19—Omega peptidases (3.4.19)
    • C12Y304/19012—Ubiquitinyl hydrolase 1 (3.4.19.12)

Definitions

  • 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.
  • MJD Machado-Joseph disease
  • SCA3 spinocerebellar ataxia type 3
  • ATXN3 polyglutamine-codifying region in the ataxin-3
  • CRISPR bacterial clustered regularly interspaced short palindromic repeat
  • 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.
  • 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 protein 12 .
  • 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.
  • 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.
  • CRISPR bacterial clustered regularly interspaced short palindromic repeat
  • CRISPR-associated (Cas) nuclease 9 SpCas9
  • sgRNA short single guide RNA
  • PAM obligatory protospacer adjacent motif
  • 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 domains 4-7 .
  • DSB double-strand break
  • HNH and RuvC nuclease domains 4-7 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.
  • NHEJ double-strand breaks
  • 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.
  • 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 8
  • 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 DSB 7 Cas9 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 strands 4 - 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 proximity 12 .
  • a knock-out approach was initially developed by directing the CRISPR-Cas9 system to exon 2 of the ATXN3 gene.
  • 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.
  • 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.
  • 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
  • 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 ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4.
  • the guide RNA comprises SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4.
  • 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).
  • the guide RNAs comprise a combination of a guide targeting intron 9 (SEQ ID NO: 5) and a guide targeting exon 10
  • 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).
  • PAM protospace
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mutated Cas is a Cas9 nickase.
  • the Cas9 nickase is Streptococcus pyogenes Cas9 (SpCas9) D10A nickase.
  • the Cas9 protein further includes a functional domain.
  • 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.
  • SpCas9 Streptococcus pyogenes Cas9
  • SaCas9 Staphyloc
  • Casl2a (AsCasl2a), Lachnospiraceae bacterium Casl2a (LbCasl2a), Bacillus hisashii, Casl2b (BhCasl2b), Delta proteobacteria Casl2e (DpbCasl2e), Planctomycetes Casl2e (PlmCasl2e), and Unultured archeon Casl2f.
  • composition as described in the present disclosure, wherein Cas protein comprises one or more mutations to improve the efficiency and safety.
  • composition as described in the present disclosure, wherein said mutated Cas protein is a nickase.
  • composition as described in the present disclosure, wherein Cas protein further includes a functional domain.
  • composition as described in the present disclosure wherein Cas protein is a Cas9.
  • 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.
  • 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.
  • 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.
  • the gene delivery vector consists of a single vector or several vectors.
  • 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.
  • AAV adeno-associated vectors
  • non-viral vector e.g. a liposome, a nanoparticle, an exosome or a microvesicle.
  • 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.
  • the cell is an induced pluripotent stem cell, or a cell of the central nervous system; preferably neurons, or oligodendrocytes, or glial cells.
  • 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.
  • 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.
  • 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.
  • 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 sequences are 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.
  • a sequence targeting the bacterial lacZ gene sgCTRL-SEQ ID NO14
  • 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).
  • this vector also codifies for FLAG-tagged SpCas9 and a puromycin resistance cassette.
  • 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).
  • selection medium with puromycin 10 pg/mL for 48 hours.
  • Locus modification efficiencies were analysed using Surveyor nuclease assay.
  • FIG. 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.
  • FIG. 3 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.
  • Myc- tagged Lentivirus encoding for the human mutant ATXN3 protein with 72 glutamines
  • AAVs encoding for SpCas9 HA-tagged
  • AAVs encoding for the CTRL guide sequence SEQ ID NO14, EGFP-KASH co-expression
  • FIG. 4 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).
  • FIG. 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 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.
  • FIG. 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.
  • sgRNA guide sequence (SEQ ID NO6) is designed to target a DNA sequence that includes a known single-nucleotide polymorphism (G nucleotide is underlined)
  • G nucleotide is underlined
  • the change of this specific nucleotide might be of relevance for the development of an allele specific editing approach.
  • HEK293T cells were transfected with plasmids encoding the SpCas9 and each of the designed sgRNAs targeting the vicinity of the CAG tract.
  • sgRNA sequence targeting the bacterial lacZ gene sgCTRL-SEQ ID NO14.
  • FIG. 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).
  • sgRNAs 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.
  • 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) .
  • plasmids encoding the SpCas9 D10A nickase and each pair of the designed sgRNAs targeting the vicinity of the CAG tract
  • 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.
  • sgRNAs to target exon 2 of the human ATXN3 gene were designed and used these sequences for ATXN3 gene knock-out experiments.
  • sgRNA sequences were designed to remove the region comprising the CAG tract, located at the exon 10 of the human ATXN3 gene.
  • 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.
  • sgRNA guide SEQ ID NO6 is designed to target a DNA sequence that includes a known exonic single-nucleotide polymorphism (C 987 GG/G 987 GG: 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.
  • 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.
  • 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 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).
  • 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.
  • 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 19 th April 2023).
  • NCBI National Center for Biotechnology Information
  • the co-expression of SpCas9 and sgRNAs 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.
  • 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).
  • 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).
  • 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 37 9 C for 30 minutes, followed by an incubation at 95 9 C for 5 minutes and a ramp down to 4 9 C by 0.1 9 C/second.
  • 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.
  • T7 DNA ligase New England Biolabs, Ipswich, USA
  • 5 pL 2x T7 DNA ligase reaction buffer New England Biolabs, Ipswich, USA
  • 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 42 9 C and were immediately transferred to ice (2 minutes). Aseptic S.O.C. medium (Invitrogen, Waltham, USA) was added and competent bacteria were incubated at 37 9 C 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 37 9 C.
  • a pre-warmed selective plate 100 pg/mL ampicillin, Enzo Life Sciences, Farmingdale, USA
  • 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 Hindlll restriction 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).
  • 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
  • AAV adeno-associated viral
  • sequences used for knock-out experiments in the adult brain 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.
  • EGFP enhanced green fluorescent protein
  • 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.
  • Sapl Lgul
  • 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.
  • 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).
  • 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.
  • sequences designed to excise the CAG tract of ATXN3 gene using a nicking strategy were cloned into a AAV-SpGuide vector adapted to accomplish the simultaneous delivery of two sgRNA molecules.
  • 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).
  • 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).
  • 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.
  • ITRs invert terminal repeats'
  • 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.
  • Ajul Thermo Fisher Scientific, Waltham, USA
  • the resulting product was purified upon electrophoretic separation, using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany).
  • 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 annealed oligonucleotides 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)
  • 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).
  • 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).
  • 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.
  • HEK293T 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 37 9 C, under a humidified atmosphere containing 5% CO2.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin/streptomycin Gibco, Invitrogen, Waltham, USA
  • 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.
  • 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.
  • LV-SpCas9-sgl9Tl SEQ ID NO5
  • LV-SpCas9-sgE10Tl SEQ ID NO6
  • LV-SpCas9-sgE10T2 SEQ ID NO7
  • LV-SpCas9-sgE10T3 SEQ ID NO8
  • the non-targeting sequence LV-SpCas9-sgCTRL
  • 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.
  • 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/).
  • HIV-1 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).
  • 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).
  • BSA bovine serum albumin
  • PBS phosphate-buffered saline
  • 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.
  • AAV1/2 adeno-associated virus serotype 1/2
  • AAV-sgCTRL SEQ ID NO14
  • AAV-sgKO.2 SEQ ID NO2
  • 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.
  • AAVpro Titration kit for Real Time PCR
  • Ver.2 Titration kit
  • Ver.2 Ver.2
  • 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.
  • 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.
  • animals were bilaterally coinjected with i) concentrated lentiviral vectors, LV-PGK-A TX/V3 72Q. (300 ng of p24 antigen), ii) AAV1/2- SpCas9 (2xl0 9 vg) and iii) AAVl/2-sgCTRL: SEQ ID NO14 (2xl0 9 vg, left hemisphere) or AAVl/2-sgK0.2: SEQ ID NO2 (2xl0 9 vg, right hemisphere), in a total volume of 2 pL.
  • mice were maintained in their home cages and sacrificed 4 weeks later for tissue processing.
  • 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.
  • mice 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 -80 9 C 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.
  • xylazine/ketamine 8/160 mg/Kg body weight
  • genomic DNA concentration and purity were determined using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, USA).
  • 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.
  • 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 19 th April 2023) using online PrimerBlast software
  • NCBI National Center for Biotechnology Information
  • the 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).
  • 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 98 9 C for 30 seconds (initial denaturation), and 35 cycles at 98 9 C for 10 seconds (denaturation), X 9 C for 10 seconds (annealing temperature for pair of primers is available in Table 1), 72 9 C for 30 seconds (extension), with a final extension at 72 9 C for 10 minutes.
  • 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.
  • 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 further assessed 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 42 9 C for 60 minutes and the reaction was stopped with the addition of Stop Solution (Integrated DNA Technologies, , Coralville, USA) on ice.
  • Stop Solution Integrated DNA Technologies, , Coralville, 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: 37 9 C for 20 minutes, followed by a step of 80 9 C, 20 min.
  • the purified PCR amplicons were characterized by Sanger sequencing (GATC Biotech, Konstanz, Germany).
  • 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.
  • 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
  • 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 4 9 C.
  • the supernatant was collected, and protein concentration determined using Bradford reagent (Bio-Rad, Hercules, USA).
  • 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).
  • 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 95 9 C. 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.
  • 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
  • PVDF polyvinylidene difluoride
  • mice 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
  • SAP.4G5 mouse monoclonal anti- -tubulin 1 antibody
  • 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).
  • 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 4 9 C. Cryoprotection was mediated by the immersion of brains in a 25% sucrose/PBS solution for 48 hours at 4 9 C, allowing tissue dehydration.
  • Brains were frozen at -80 9 C and sliced into coronal sections with 25 pm in thickness, using a cryostat (LEICA CM3050S, Leica Microsystems, Wetzlar, Germany) at - 21 9 C.
  • the slices were collected in anatomical series, being stored at 4 9 C as free-floating sections in PBS supplemented with 0.05% (m/v) sodium azide (Sigma-Aldrich, St. Louis, 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).
  • 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.
  • CRISPR- associated nuclease SpCas9 was programmed to target the exon 2 of this gene.
  • 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.
  • lentiviral backbone lentiCRISPRv2, plasmid #52961, Addgene
  • 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.
  • 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 were slowly 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.
  • 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
  • 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, in addition 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.
  • 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.
  • 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).
  • 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 from the 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.
  • 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).
  • 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.
  • 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).
  • 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 proximity 12 , while individual single-stranded breaks inserted in potential off-target sites are repaired with high-fidelity.
  • sgRNAs 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the non-targeting control sgl9Tl
  • 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.
  • 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.
  • genes 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.
  • 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.
  • G, "C”, “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.
  • 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 desired results 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.
  • 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.
  • Oligonucleotide primers to verify the correct insertion of each sgRNA-codifying oligonucleotide pairs through Sanger sequencing SEQ ID NO16: 5' - GACTATCATATGCTTACCGT - 3'

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Abstract

La présente divulgation concerne la maladie de Machado-Joseph (MJD), ou l'ataxie spinocérébelleuse de type 3 (SCA3), une maladie neurodégénérative à transmission autosomique dominante causée par une répétition excessive de la région polyglutaminique codifiante dans le gène de l'ataxine-3 (ATX7V3). L'ATXN3 étendu est susceptible d'agréger et de perturber divers systèmes cellulaires, conduisant finalement à un dysfonctionnement des cellules et à la mort dans des populations neuronales spécifiques. Jusqu'à présent, aucun traitement apte à inverser ou à bloquer la progression MJD/SCA3 n'a été développé. Des stratégies basées sur la suppression des produits géniques délétères ont démontré des résultats prometteurs dans des études précliniques. Néanmoins, ces stratégies ne ciblent pas la cause racine de la maladie, produisant un effet thérapeutique incomplet et/ou transitoire dans des cellules ou des tissus cibles. Récemment, des agents thérapeutiques à base de gènes, y compris les systèmes de courte répétition palindromique groupée et régulièrement espacée (CRISPR) pour l'édition de gènes, ont été utilisés avec succès pour inactiver et corriger de manière permanente des gènes liés à la maladie, ce qui laisse espérer la mise au point d'un remède définitif pour les maladies héréditaires.
PCT/IB2024/059800 2023-10-06 2024-10-07 Outils basés sur le système crispr-cas9 : nouvelles approches thérapeutiques potentielles pour la maladie de machado-joseph Pending WO2025074350A2 (fr)

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