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WO2018128587A1 - Mutant smchd1 pour thérapie - Google Patents

Mutant smchd1 pour thérapie Download PDF

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Publication number
WO2018128587A1
WO2018128587A1 PCT/SG2018/050008 SG2018050008W WO2018128587A1 WO 2018128587 A1 WO2018128587 A1 WO 2018128587A1 SG 2018050008 W SG2018050008 W SG 2018050008W WO 2018128587 A1 WO2018128587 A1 WO 2018128587A1
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Prior art keywords
smchdl
residue
amino acid
human
peptide
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Inventor
Bruno Reversade
Jeanne AMIEL
Marnie BLEWITT
James Murphy
Bernd WOLLNIK
Asif Javed
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Walter and Eliza Hall Institute of Medical Research
Universitatsmedizin Gottingen
Georg August Universitaet Goettingen
Institut National de la Sante et de la Recherche Medicale INSERM
Agency for Science Technology and Research Singapore
Original Assignee
Walter and Eliza Hall Institute of Medical Research
Universitatsmedizin Gottingen
Georg August Universitaet Goettingen
Institut National de la Sante et de la Recherche Medicale INSERM
Agency for Science Technology and Research Singapore
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Publication of WO2018128587A1 publication Critical patent/WO2018128587A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to a peptide or fragment thereof.
  • the present invention relates to a variant peptide for use in therapy.
  • Facioscapulohumeral muscular dystrophy is a disorder characterized by progressive weakness and wasting of muscles of face, neck, and shoulder girdle.
  • FSHD has variable age onset of between 10 to 30 years of age and the severity of the condition varies from milder cases where dystrophy is not observed until later in life to rare severe cases where dystrophy is apparent in infancy and early childhood. In some subjects, muscle weakness can spread to abdominal muscles and sometimes hip muscles.
  • FSHD type 1 is an autosomal dominant inherited dystrophy and, in most cases, an affected subject inherits the altered chromosome from one affected parent. However, there were subject who have no history of the disorder in the family.
  • FSHD type 2 is a digenic inherited dystrophy where the subject must inherit a mutation in the SMCHD1 gene and a copy of the "permissive" chromosome 4.
  • the present disclosure aims to provide a therapy for treating subjects having FSHD.
  • an isolated variant of SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) peptide or fragment thereof, comprising one or more mutation(s) that increases SMCHDl activity in a cell compared to a cell with a wild-type SMCHDl peptide.
  • the mutation is comprised in one or more regions selected from the group consisting of: the N-terminal motif I, which is the highly conserved region of GHKL-ATPases and participates in the coordination of the Mg2+-ATP complex during ATP hydrolysis, and the C-terminal to ATPase domain.
  • the N-terminal motif I which is the highly conserved region of GHKL-ATPases and participates in the coordination of the Mg2+-ATP complex during ATP hydrolysis, and the C-terminal to ATPase domain.
  • the mutation is comprised in region between amino acid corresponding to residue 111 to amino acid corresponding to residue 702 of SMCHDl.
  • the mutation is a substitution mutation.
  • the mutation is at one or more position(s) selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl, amino acid residue corresponding to residue 135 of human SMCHDl, amino acid residue corresponding to residue 136 of human SMCHDl, amino acid residue corresponding to residue 342 of human SMCHDl, amino acid residue corresponding to residue 348 of human SMCHDl, amino acid residue corresponding to residue 420 of human SMCHDl, amino acid residue corresponding to residue 518 of human SMCHDl, and amino acid residue corresponding to residue 552 of human SMCHDl (wherein each residue position is referenced to SMCHDl human protein UniProtKB identifier: A6NHR9).
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with an amino acid with a polar uncharged side chain, amino acid residue corresponding to residue 135 of human SMCHDl has been substituted with an amino acid with polar uncharged side chain and/or an amino acid with sulfur side chain, amino acid residue corresponding to residue 136 of human SMCHDl has been substituted with an amino acid with an aliphatic side chain, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with an amino acid with polar uncharged side chain, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an amino acid with positively charged side chain, amino acid residue corresponding to residue 420 of human SMCHDl has been substituted with an amino acid with hydrophobic side chain, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with an amino acid with negatively charged side chain, and amino acid residue residue corresponding to residue 134 of
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 135 of human SMCHDl has been substituted with a cysteine and/or a asparagine acid residue, amino acid residue corresponding to residue 136 of human SMCHDl has been substituted with a glycine residue, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an arginine residue, amino acid residue corresponding to residue 420 of human SMCHDl has been substituted with a valine residue, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with a glutamic acid residue, and amino acid residue corresponding to residue 552 of human SMCHDl has been substituted with a glutamine residue (wherein each residue position is
  • the present invention provides an isolated nucleic acid or fragment thereof encoding the isolated variant SMCHDl peptide as described herein, wherein the nucleic acid comprises one or more mutation(s) that increases SMCHDl protein activity in a cell compared to a cell with a wild-type SMCHDl peptide.
  • the SMCHDl mutation is a missense mutation.
  • the SMCHDl mutation is at one or more position(s) selected from the group consisting of: c.407, c.403, c.404, c.1043, c.1259, c.1655, c.1552, c.1025, and C.400 (nucleic acid residue referenced to NCBI Reference Sequence: NM_015295.2).
  • the SMCHDl mutation is one or more selected from the group consisting of: c.407A>G, c.403A>T, c.404G>A, c.l043A>G, c.l259A>T, c. l655G>A, c.l552A>G, c. l025G>C, and c.400G>T (nucleic acid residue referenced to referenced to NCBI Reference Sequence: NM_015295.2).
  • the present invention provides a vector comprising the nucleic acid as described herein.
  • the present invention provides an isolated variant of SMCHDl as described herein, or the isolated nucleic acid as described herein, or the vector as described herein for use in therapy.
  • the present invention provides a pharmaceutical composition comprising the isolated variant SMCHDl peptide or fragment thereof as described herein, and/or an isolated nucleic acid or fragment thereof as described herein, or a vector as described herein, and a pharmaceutically acceptable excipient thereof.
  • the present invention provides a gene-therapy composition comprising an isolated nucleic acid as described herein or a vector as described herein.
  • the present invention provides a host cell comprising the vector as described herein.
  • the present invention provides a method of treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof, comprising administering a therapeutically effective amount of an agent capable of increasing the expression level or activity of SMCHDl peptide and/or nucleic acid to the subject in need thereof.
  • FSHD facioscapulohumeral muscular dystrophy
  • the agent is one or more selected from the group consisting of an isolated variant SMCHDl peptide or fragment thereof as described herein, a transgene encoding the isolated variant SMCHDl peptide as described herein, an isolated nucleic acid or fragment thereof as described herein, a vector as described herein, a pharmaceutical composition as described herein, and a gene-therapy composition as described herein.
  • the FSHD is a type 1 and/or type 2 FSHD.
  • the present invention provides a method of screening an agent (or a nucleotide mutation) capable of increasing SMCHDl activity, comprising: a. introducing the agent (or nucleotide mutation) to an assay for determining SMCHDl activity, b. determining the SMCHDl activity the agent (or nucleotide mutation) elicits, and c.
  • the agent (or nucleotide mutation) elicits with the SMCHDl activity elicited by one or more selected from the group consisting of an isolated variant SMCHDl peptide or fragment thereof as described herein, and/or a transgene encoding the isolated variant SMCHDl peptide as described herein, and/or an isolated nucleic acid or fragment thereof as described herein, and/or a vector as described herein, wherein when the SMCHDl activity of the agent (or nucleotide mutation) is the same or more than the SMCHDl activity of the one or more selected from the group consisting of the isolated variant SMCHDl peptide or fragment thereof as described herein, and/or the transgene encoding the isolated variant SMCHDl peptide as described herein, and/or the isolated nucleic acid or fragment thereof as described herein, and/or the vector as described herein, the agent (or nucleotide mutation) is considered a suitable agent (or nucleo
  • the present invention provides a use of one or more selected from the group consisting of an isolated variant SMCHDl peptide as described herein, a transgene encoding the isolated variant SMCHDl peptide as described herein, an isolated nucleic acid as described herein, a vector as described herein, a pharmaceutical composition as described herein, and a gene-therapy composition as described herein in the manufacture of a medicament for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof.
  • FSHD facioscapulohumeral muscular dystrophy
  • the present invention provides a kit for screening an agent capable of increasing SMCHDl activity, comprising a reference control comprising one or more selected from the group consisting of an isolated variant SMCHDl peptide as described herein, transgene encoding the isolated variant SMCHDl peptide as described herein, an isolated nucleic acid as described herein, and a vector as described herein; and a reagent for determining S MCHD 1 activity .
  • Figure 1 shows a series of photograph of patients having BAMS.
  • Figure 1 SMCHDl is mutated in Bosma arhinia microphthalmia syndrome (BAMS) and isolated arhinia.
  • Figure 1 shows the physical manifestation of BAMS.
  • FIG. 2 shows graphs of the results of ATPase assays using the purified recombinant N-terminal region harboring BAMS or FSHD2 mutations, (a-e) ATPase assays performed using recombinant protein encompassing amino acids 111-702 of Smchdl.
  • the amount of ADP produced at each protein concentration 0.1, 0.2, 0.4 and 0.6 ⁇
  • ATP concentration (1, 2.5, 5 and 10 ⁇
  • Figure 3 shows the results of in vivo functional assays in Xenopus embryos indicate that BAMS mutations behave as gain-of-function alleles
  • (a) qPCR shows that smchdl is zygotically transcribed. Expression levels are shown relative to 18S rRNA.
  • stage 45 tadpoles injected with SMCHD1A134S display craniofacial anomalies and smaller eyes compared to control and SMCHD1WT injected tadpoles
  • (h) The eye diameter is significantly reduced in embryos overexpressing BAMS mutants (blue) relative to SMCHD1WT overexpressing siblings (black), or embryos overexpressing an FSHD2 mutant (open circles), n at least 15 embryos for each condition
  • mRNAs injected for this panel did not contain a poly A signal and were polyadenylated in vitro, hence requiring higher RNA concentration to produce a phenotype (in other panels in this figure, and in Figure 13, the mRNAs contain a poly A signal allowing polyadenylation in vivo).
  • Biological variation between clutches of frog tadpoles is seen in the data presented in panels h and k.
  • n 20 embryos for each condition, n.s. not significant, ***p ⁇ 0.001, ** p ⁇ 0.01.
  • Figure 3 are in vivo results that partially recapitulate the microphthalmia and facial hypoplasia seen in severe BAMS pateints, further support the notion that, in contrast to FSHD2 alleles, BAMS -associated missense mutations exhibit gain- of-function or neomorphic activity.
  • Figure 3 provides support that the identification of gain-of-function mutations in SMCHD1 can be used in gene therapy approach for treatment of FSHD.
  • Figure 4 shows photographic images of computed tomography and magnetic resonance imaging in BAMS, (a-c) Controls and (d-f) patient 1 at four years.
  • Patient 1 displays maxillary hypoplasia and absent nasal bones (d and e).
  • Olfactory bulbs and sulci (labelled with red and white arrows, respectively, on the left side in the control in c) are absent in patient 1 (f).
  • Skeletal imaging of patients 14 (g,h) and 11 (i) indicates similar midface hyploplasia.
  • Figure 5 shows BAMS pedigrees and Sanger sequencing chromatograms of SMCHD1 mutations. Individuals submitted for exome sequencing are indicated by a red asterisk. A question mark indicates no phenotypic information was available. Note Sanger sequencing was unavailable for individual 13.
  • FIG. 6 shows multiple sequence alignment of vertebrate SMCHDlorthologues and yeast Hsp90. Residues mutated in BAMS are indicated by pink arrows. Residues mutated in FSHD are indicated by purple arrows.
  • Hs Homo sapiens
  • Mm Mus musculus
  • Bt Bos taurus
  • Gg Gallus gallus
  • Md Monodelphis domestica
  • Cm Chelonia mydas
  • Xt Xenopus tropicalis
  • Dr Danio rerio
  • Sc Saccharomyces cerevisiae.
  • FSHD mutation reference LOVD SMCHD1 variant database, http://databases.lovd.nl/shared/variants/SMCHDl/unique.
  • Figure 7 shows photographic images of the results of X-gal staining of mouse embryos expressing lacz from the Smchdl locus.
  • E embryonic day. gt/+, embryos heterozygous for the Smchdlgt allele expressing lacz. +/+, wildtype embyros. hf, head folds, npl, nasal placode, ov, optic vesicle, npi, nasal pit.
  • ne nasal epithelium, f-i, coronal sections, r and s, transverse sections.
  • An asterisk in panel p indicates deep nasal staining.
  • Figure 8 shows bar graphs of results of sodium bisulfite sequencing in BAMS patients, a, individuals 1-6. b, individuals 8-11 and 14. The position of the three different regions analyzed within D4Z4 is indicated above the corresponding column (left, DR1; middle, 5'; right, Mid). For each sample, at least 10 cloned DNA molecules were analyzed by Sanger sequencing. Each histogram column corresponds to a single CpG. Black corresponds to the global percentage of methylated CpGs and white to the global percentage of unmethylated CpGs. The percentage of methylated CpGs among the total CpGs in each individual analyzed are given in Table 2.
  • the level of methylation is statistically significantly different between controls and FSHD2 patients for the DR1 (** ; p ⁇ 0.001) and the 5' (*** ; p ⁇ 0.0001) regions.
  • the level of methylation is significantly different between controls and BAMS patients for the 5' region (*, p ⁇ 0.05) and between BAMS patients and their relatives for the DR1 (*, p ⁇ 0.05) and 5' (**, p ⁇ 0.001) regions.
  • Figure 10 shows Smchdl structure modelling, based on the structure of Hsp90.
  • Residues mutated in BAMS are indicated with an underline (i.e. W342, H346, R552, D420, K518, A134, E136, and S 135).
  • Residues mutated in FSHD are indicated in boxes (i.e. Y283, H263, L194, Y353, G425, G478, R479, T537, G137, P690, V615, and C492).
  • FIG 11 shows fibroblasts derived from BAMS patients show no defects in NHEJ or in H2AX activation.
  • MMEJ microhomology mediated end joining
  • WT wildtype
  • XRCC4-deficient fibroblasts show multiple smaller DNA bands after BstXI digestion indicating defects in NHEJ-mediated DNA repair and leading to preferential use of MMEJ- mediated DNA double strand repair
  • BAMS patient fibroblasts show no defects in NHEJ- mediated DNA repair pathways compared to wildtype.
  • Figure 12 shows ATPase assays performed using recombinant wildtype or mutant Smchdl protein in the presence of radicicol. Data are displayed as mean + s.d. of technical triplicates. The data are representative of at least two independent experiments using different batches of protein preparation. Figure 12 indicates that BAMS-associated mutations elevate the catalytic activity of SMCHDl.
  • Figure 13 shows SMCHDl overexpression in Xenopus causes dose dependent craniofacial anomalies.
  • Figure 14 shows SDS/PAGE gels showing the Purity of proteins used for ATPase assays. Purified recombinant wild type or mutant proteins were resolved by 4-20% (w/v) Tris-Glycine reducing SDS/PAGE and were stained with SimplyBlue SafeStain. Protein quantities loaded: left gel, 1.4 ⁇ g; middle gel, 1.05 ⁇ g; right gel, 0.7 ⁇ g. Molecular weight (MW) markers are as indicated on the left-hand side.
  • Bosma arhinia microphthalmia syndrome is an extremely rare and striking condition characterized by complete absence of the nose with or without ocular defects.
  • the present disclosure shows that missense mutations in the extended ATPase domain of the epigenetic regulator SMCHD1 causes BAMS in all 14 cases studied. All mutations were de novo where parental DNA was available. Biochemical tests and in vivo assays in Xenopus embryos suggest that these mutations behave as gain-of-function alleles. This is in contrast to loss-of-function mutations in SMCHD1 that have been associated with facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • loss-of-function mutations in SMCHD1 are associated with FSHD2, BAMS and FSHD2 represent a rare example of gain- versus loss-of-function mutations in the same gene leading to vastly different human disorders, in terms of the affected tissue and age of onset.
  • FSHD is caused in part by a loss of SMCHD1, augmentation of the expression or activity of SMCHD1 in affected muscles can surprisingly be developed as a form of treatment.
  • the identification of gain-of-function mutations in SMCHD1 in the present disclosure can inform gene therapy approaches, or in combination with future structural studies on the effect of these mutations on the ATPase domain, aid the design of drugs that reproduce the gain-of-function effect, for treatment of FSHD. Therefore, the present disclosure establishes SMCHD1 in its enzymatic function that can be exploited for the development of therapeutics for FSHD.
  • a method of treatment for facioscapulohumeral muscular dystrophy comprising administering a polypeptide or nucleotide sequence encoding SMCHD1 or a functional fragment thereof into the subject, or administering to the subject a compound capable of modifying the expression levels or activity of SMCHDl .
  • the inventors of the present invention have identified mutations that can increase SMCHDl activity. Therefore mimicking these mutations, or targeting these amino acids directly can be used as a way of increasing SMCHDl activity.
  • an isolated variant of SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) peptide or fragment thereof, comprising one or more mutation(s) that increases SMCHD1 activity in a cell compared to a cell with a wild-type SMCHD1 peptide.
  • isolated is used herein to refer to polynucleotides, polypeptides and proteins that are extracted or obtained from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • isolated and recombinant are interchangeable and means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
  • An isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
  • mutant as used herein is meant to include all kinds of nuclear and/or mitochondrial gene mutations, including any alteration in the base sequence of a DNA strand compared to the wild-type reference strand.
  • the mutation is comprised in one or more regions selected from the group consisting of: the N-terminal motif I, which is the highly conserved region of GHKL-ATPases and participates in the coordination of the Mg 2+ -ATP complex during ATP hydrolysis, and the C-terminal to ATPase domain.
  • the mutation is comprised on both of the regions of the N- terminal motif I, which is the highly conserved region of GHKL-ATPases and participates in the coordination of the Mg 2+ -ATP complex during ATP hydrolysis, and the C-terminal to ATPase domain.
  • the mutation is comprised in region between amino acid corresponding to residue 111 to amino acid corresponding to residue 702 of SMCHD1 (wherein each residue position is referenced to SMCHD1 human protein UniProtKB identifier: A6NHR9, or SEQ ID NO: 1).
  • the wildtype SMCHD1 has the sequence SEQ ID NO: 1, as follows:
  • the term "mutation" is intended to include not only naturally occurring mutations, but also artificially introduced mutations.
  • the mutation can include random or site-specific mutations, including, but is not limited to, missense mutation, nonsense mutation, insertion, deletion, duplication, frameshift mutation, repeat expansion, and the like.
  • the mutation is missense mutation.
  • the mutation leads to a substitution mutation.
  • the mutation is a substitution mutation.
  • the mutation as described herein is at one or more position(s) selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl, amino acid residue corresponding to residue 135 of human SMCHDl, amino acid residue corresponding to residue 136 of human SMCHDl, amino acid residue corresponding to residue 342 of human SMCHDl, amino acid residue corresponding to residue 348 of human SMCHDl, amino acid residue corresponding to residue 420 of human SMCHDl, amino acid residue corresponding to residue 518 of human SMCHDl, and amino acid residue corresponding to residue 552 of human SMCHDl (wherein each residue position is referenced to SMCHDl human protein UniProtKB identifier: A6NHR9 or SEQ ID NO: 1).
  • the variant SMCHDl as described herein may comprise at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight mutations as described herein.
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with an amino acid with a polar uncharged side chain, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with an amino acid with polar uncharged side chain, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an amino acid with positively charged side chain, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with an amino acid with negatively charged side chain, and amino acid residue corresponding to residue 552 of human SMCHDl has been substituted with an amino acid with a negative charged side chain (wherein each residue position is referenced to SMCHDl human protein UniProtKB identifier: A6NHR9; SEQ ID NO: 1).
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an arginine residue, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with a glutamic acid residue, and amino acid residue corresponding to residue 552 of human SMCHDl has been substituted with a glutamine residue (wherein each residue position is referenced to SMCHDl human protein UniProtKB identifier: A6NHR9; SEQ ID NO: 1).
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with an amino acid with a polar uncharged side chain, amino acid residue corresponding to residue 135 of human SMCHDl has been substituted with an amino acid with polar uncharged side chain and/or an amino acid with sulfur side chain, amino acid residue corresponding to residue 136 of human SMCHDl has been substituted with an amino acid with an aliphatic side chain, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with an amino acid with polar uncharged side chain, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an amino acid with positively charged side chain, amino acid residue corresponding to residue 420 of human SMCHDl has been substituted with an amino acid with hydrophobic side chain, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with an amino acid with negatively charged side chain, and amino acid residue residue corresponding to residue 134 of
  • the mutation is one or more selected from the group consisting of: amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 135 of human SMCHDl has been substituted with a cysteine and/or a asparagine acid residue, amino acid residue corresponding to residue 136 of human SMCHDl has been substituted with a glycine residue, amino acid residue corresponding to residue 342 of human SMCHDl has been substituted with a serine residue, amino acid residue corresponding to residue 348 of human SMCHDl has been substituted with an arginine residue, amino acid residue corresponding to residue 420 of human SMCHDl has been substituted with a valine residue, amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with a glutamic acid residue, and amino acid residue corresponding to residue 552 of human SMCHDl has been substituted with a glutamine residue (wherein each residue position is referenced
  • amino acid residue corresponding to residue 134 of human SMCHDl has been substituted with a serine residue is SEQ ID NO: 2 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHDl comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 2.
  • amino acid residue corresponding to residue 135 of human amino acid residue corresponding to residue 135 of human
  • SMCHDl has been substituted with a cysteine residue is SEQ ID NO: 3 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHDl comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 3.
  • amino acid residue corresponding to residue 135 of human SMCHDl has been substituted with a asparagine residue is SEQ ID NO: 4 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHDl comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 4.
  • amino acid residue corresponding to residue 136 of human amino acid residue corresponding to residue 136 of human
  • SMCHDl has been substituted with a glycine residue is SEQ ID NO: 5 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHD1 comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 5.
  • amino acid residue corresponding to residue 342 of human SMCHD1 has been substituted with a serine residue is SEQ ID NO: 6 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHD1 comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 6.
  • amino acid residue corresponding to residue 348 of human amino acid residue corresponding to residue 348 of human
  • SMCHD1 has been substituted with a arginine residue is SEQ ID NO: 7 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHD1 comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 7.
  • the amino acid residue corresponding to residue 420 of human SMCHDl has been substituted with a valine residue is SEQ ID NO: 8 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHDl comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 8.
  • amino acid residue corresponding to residue 518 of human SMCHDl has been substituted with a glutamic acid residue is SEQ ID NO: 9 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHDl comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 9.
  • amino acid residue corresponding to residue 552 of human SMCHDl has been substituted with a glutamine residue is SEQ ID NO: 10 (substituted residue underlined and in italics), having the following sequence:
  • the isolated variant of SMCHD1 comprises an amino acid having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or identical to SEQ ID NO: 10.
  • gain-of- function mutation in SMCHD1 as disclosed herein can be useful in treating subjects having
  • the present invention provides an isolated variant of
  • SMCHD1 as described herein for use in therapy or medicine.
  • the present invention also provides an isolated nucleic acid or fragment thereof encoding the isolated variant SMCHD1 peptide as described herein, wherein the nucleic acid comprises one or more mutation(s) that increases SMCHD1 protein activity in a cell compared to a cell with a wild-type SMCHD1 peptide.
  • the SMCHD1 mutation that caused the substitution mutation is a missense mutation.
  • the SMCHD1 mutation is a missense mutation.
  • the SMCHD1 mutation is at one or more position(s), including, but is not limited to c.407, c.403, c.404, c.1043, c.1259, c.1655, c.1552, c.1025, and c.400 (nucleic acid residue referenced to NCBI Reference Sequence: NM_015295.2).
  • the SMCHD1 mutation may be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the mutations such as c.407, c.403, c.404, c.1043, c.1259, c.1655, c.1552, c.1025, and c.400 (nucleic acid residue referenced to NCBI Reference Sequence: NM_015295.2).
  • SMCHD1 mutation may be one or more including, but not limited to, c.407A>G, c.403A>T, c.404G>A, c.l043A>G, c. l259A>T, c.l655G>A, c.l552A>G, c. l025G>C, c.400G>T, and the like (nucleic acid residue referenced to referenced to NCBI Reference Sequence: NM_015295.2). As would be understood by the person skilled in the art, the mutation is denoted by the alphabet recitation.
  • c.406A>G refers to a missense mutation at position 406 of the nucleic acid residue referenced at NCBI reference sequence NM_015295.2 and the nucleic acid adenine (A) has been replaced by guanine (G).
  • the term "nucleic acid” is used in accordance to the common use in the art and is referred to include the various nucleic acids known in the art.
  • the nucleic acid includes, but is not limited to, a DNA, an mRNA, and an RNA.
  • the present disclosure also provides for an isolated nucleic acid as described herein for use in therapy (such as gene-therapy).
  • the present disclosure provides for one or more vector(s) comprising the nucleic acid as described herein.
  • the vectors as used herein refer to any vectors that would be recognized by the person skilled in the art to be capable of being used in gene therapy, nucleic acid, or peptide expression. Accordingly, in some examples, the vector may include, but is not limited to, an adeno-associated virus based vector, a retro- associated virus based vector, and the like.
  • a pharmaceutical composition comprising the isolated variant SMCHD1 peptide or fragment thereof as described herein, and/or an isolated nucleic acid or fragment thereof as described herein, or a vector as described herein, and a pharmaceutically acceptable excipient thereof.
  • the present invention provides a gene-therapy composition comprising an isolated nucleic acid as described herein or a vector as described herein.
  • the present invention also provides one or more host cell(s) comprising the vector as described herein.
  • an SMCHDl mutein comprising an amino acid change of at least one of the following: 134Ser, 135Asn, 135Cys, 136Gly, 342Ser, 348Arg, 420Val, 518Glu, and 552Gln.
  • the variant SMCHDl peptide or fragment as described herein has been illustrated to be useful increasing the expression of SMCHDl in vivo.
  • the experimental data provides proof that the variant SMCHDl peptide could be used to treat FSHD.
  • a method of treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof comprising administering a therapeutically effective amount of an isolated variant SMCHDl peptide or fragment thereof as described herein, and/or a transgene encoding the isolated variant SMCHDl peptide as described herein, and/or an isolated nucleic acid or fragment thereof as described herein, and/or a vector as described herein, and/or a pharmaceutical composition as described herein, or a gene-therapy composition as described herein, to the subject in need thereof.
  • FSHD facioscapulohumeral muscular dystrophy
  • FSHD facioscapulohumeral muscular dystrophy
  • the isolated peptide, and/or transgene, and/or nucleic acid, and/or vector, and/or pharmaceutical composition may be delivered via a route known in the art.
  • the route of delivery or administration includes, but is not limited to, intravascularly, intramuscularly, intradermally, intraperitoneally, intraarterially, and the like.
  • the present invention also provides a method of treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof, comprising administering a therapeutically effective amount of an agent capable of increasing the expression level or activity of SMCHDl peptide and/or nucleic acid to the subject in need thereof.
  • FSHD facioscapulohumeral muscular dystrophy
  • the agent capable of increasing the expression level or activity of SMCHDl peptide and/or nucleic acid to the subject may be the variant SMCHDl peptide and/or nucleic acid as described herein.
  • the isolated peptide, and/or transgene, and/or nucleic acid, and/or vector, and/or pharmaceutical composition, and/or agent capable of increasing the expression level or activity of SMCHDl peptide and/or nucleic acid to the subject may be delivered via a route known in the art.
  • the route of delivery or administration includes, but is not limited to, intravascularly, intramuscularly, intradermally, intraperitoneally, intraarterially, and the like.
  • the FSHD as discussed in the present disclosure may be a type 1 and/or type 2 FSHD. In some examples, the FSHD as discussed in the present disclosure may be a type 2 FSHD.
  • the subject is an animal such as, but is not limited to, a human and a non-human mammal. In some examples, the subject is a human.
  • nucleotide mutations or muteins as disclosed herein can be used to screen compounds that can modulate SMCHDl.
  • a method of screening using the nucleotide mutations or muteins as described herein to identify compounds that can modulate SMCHDl activity is provided.
  • the gain of function (GOF) mutations or muteins as described herein may be used as a) positive controls in drug screens for increasing SMCHDl activity; b) possible therapeutic in the form of introducing these gain of function (GOF) SMCHDl protein or gene in FSHD patients; c) tools in developing molecules that mimic the gain of function (GOF) activity of these mutations or increase the activity of endogenous SMCHDl.
  • a method of screening an agent (or a nucleotide mutation) capable of increasing SMCHDl activity comprising: a. introducing the agent (or nucleotide mutation) to an assay for determining SMCHDl activity, b. determining the SMCHDl activity the agent (or nucleotide mutation) elicits, and c.
  • the agent (or nucleotide mutation) elicits with the SMCHDl activity elicited by an isolated variant SMCHDl peptide or fragment thereof as described herein, and/or a transgene encoding the isolated variant SMCHDl peptide as described herein, and/or an isolated nucleic acid or fragment thereof as described herein, and/or a vector as described herein, wherein when the SMCHDl activity of the agent (or nucleotide mutation) is the same or more than the SMCHDl activity of the isolated variant SMCHDl peptide or fragment thereof as described herein, and/or the transgene encoding the isolated variant SMCHDl peptide as described herein, and/or the isolated nucleic acid or fragment thereof as described herein, and/or the vector as described herein, the agent (or nucleotide mutation) is considered a suitable agent (or nucleotide mutation) for increasing SMCHDl activity.
  • the assay for determining SMCHDl activity may include, but is not limited to, an ATPase activity assay, assays capable of determining and/or measuring the expression and activity of a downstream target of SMCHDl (such as DUX4; and such assays may be a cell-based assay and/or a reporter-based assay), assays capable of determining the in vivo activity of SMCHDl (for example microinjection of the agent into Xenopus embryos, wherein mutant agent that causes increase in SMCHDl activity may cause craniofacial abnormalities in Xenopus embryos during development), and the like.
  • an ATPase activity assay assays capable of determining and/or measuring the expression and activity of a downstream target of SMCHDl (such as DUX4; and such assays may be a cell-based assay and/or a reporter-based assay
  • assays capable of determining the in vivo activity of SMCHDl for example microinjection
  • the present disclosure also provides a kit for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof, comprising a therapeutically effective amount of an isolated variant SMCHDl peptide as described herein, and/or a transgene encoding the isolated variant SMCHDl peptide as described herein, and/or an isolated nucleic acid as described herein, and/or a vector as described herein, and/or a pharmaceutical composition as described herein, or a gene-therapy composition as described herein.
  • FSHD facioscapulohumeral muscular dystrophy
  • the present disclosure also provides a kit for screening an agent capable of increasing SMCHDl activity, comprising a reference control comprising an isolated variant SMCHDl peptide as described herein, and/or a transgene encoding the isolated variant SMCHDl peptide as described herein, and/or an isolated nucleic acid as described herein, and/or a vector as described herein, and a reagent for determining SMCHDl activity.
  • the reagent for determining SMCHDl activity as used herein are reagents that are commonly used in the art of determining the activity of a gene in a human or animal body.
  • the reagents may include primers as used in the present disclosure, for example primers described in the Experimental section.
  • the reagents for determining SMCHDl activity may include at least one or more of primers selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.
  • the methods of using the primer/primer pairs as disclosed herein are commonly known in the art and one exemplary method is discussed in the Experimental section of the present disclosure.
  • a peptide includes a plurality of peptides (or polypeptides), including mixtures and combinations thereof.
  • the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale.
  • the term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to the same trait or characteristic in the population as a whole. However, this term is without valuation of the difference seen.
  • the term "about" in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the mean depth of coverage obtained for the three samples from case 1 was 123-, 149- and 150- fold, and 98% of the exome was covered by at least 15-fold.
  • Downstream processing was performed using the Genome Analysis Toolkit (GATK), SAMtools and Picard.
  • GATK Genome Analysis Toolkit
  • Variant calls were made with the GATK Unified Genotyper. All calls with read coverage ⁇ 2-fold or a Phred-scaled S P quality score of ⁇ 20-fold were removed from consideration.
  • Variant annotation was based on Ensembl release 71. Variants were filtered against publicly available SNPs plus variant data from more than 7,000 in-house exomes (Institut Imagine).
  • SNVs and small indels were annotated with the ANNOVAR software package using the following datasets and programs: gene information from GENCODE (version 19); allele frequencies of the 1000 Genome Project (version August, 2015), ExAC (version 0.3; see URLs), EVS (release ESP6500SI-V2; see URLs) and an in-house database; and predictions of protein damage by PolyPhen-2 and SIFT via dbNSFP (version 3.0).
  • DNA methylation was analyzed at single base resolution after sodium bisulfite modification, PCR amplification, cloning and Sanger sequencing. Briefly, 2 ⁇ g of genomic DNA was denatured for 30 minutes at 37°C in NaOH 0.4N and incubated overnight in a solution of sodium bisulfite 3M pH 5 and 10 mM hydroquinone using a previously described protocol. Converted DNA was then purified using the Wizard DNA CleanUp kit (Promega) following the manufacturer's recommendations and precipitated by ethanol precipitation for 5 hours at -20°C. After centrifugation, DNA pellets were resuspended in 20 ⁇ of water and stored at -20°C until use.
  • Wizard DNA CleanUp kit Promega
  • Converted DNA was then amplified using primer sets (Table 3) designed with the MethPrimer software avoiding the presence of CpGs in the primer sequence in order to amplify methylated and unmethylated DNA with the same efficiency.
  • Amplification was carried out using High Fidelity Taq polymerase (Roche) according to the manufacturer's instructions. After initial denaturation at 94°C for two minutes, amplification was done at 94°C for 20 seconds, 54°C for 30 seconds and 72°C for one minute for 10 cycles, then at 94°C for 20 seconds, 54°C for 30 seconds, followed by an extension step of 4 minutes and 30 seconds for the first cycle and an increment of 30 seconds at each subsequent cycle for 25 cycles.
  • PCR products were then purified using the Wizard SV gel and PCR Purification system (Promega), resuspended in 50 ⁇ of water and cloned using the pGEM®-T Easy Vector cloning kit (Promega). Colonies were grown overnight at 37°C with ampicillin selection and randomly selected colonies were PCR amplified directly using T7 or SP6 primers. For each sample and region, at least ten randomly cloned PCR products were sequenced according to Sanger's method by Eurofins MWG Operon (Ebersberg, Germany) with either SP6 or T7 primers. Sequences were analyzed using the BiQ Analyser software and the average methylation score was calculated as the number of methylated CpGs for the total number of CpGs in the reference sequence.
  • a homology model of the N-terminal region of Smchdl was generated using the online server Phyre2 (Protein Homology/ Analogy Recognition Engine 2). Protein sequence of 111-702 aa of mouse Smchdl was submitted as the input sequence and intensive modelling mode was selected. The second highest scoring model with the most sequence alignment coverage based on the crystal structure of yeast Hsp90 (PDB: 2CG9) was elected for further evaluation. The model was visualized in PyMOL. The multiple sequence alignment was generated using CLUSTAL W (via the PBIL server) and ESPript 3.0.
  • a 12-point 10 ⁇ ADP/ ATP standard curve was set up in parallel. Reactions were incubated at room temperature for 1 hour in the dark before addition of 10 ⁇ of detection mix (IX Stop & Detection Buffer B, 23.6 ⁇ g/ml ADP2 antibody) for a further hour of incubation. Fluorescence polarization readings were performed with an Envision plate reader (PerkinElmer Life Sciences) following the manufacturer's instructions. The amount of ADP present in each reaction was estimated by using the standard curve following the manufacturer's instructions.
  • Tyr353Cys Forward cat att tat cat tac tGt att cat gga cca aaa g 27 p.
  • Tyr353Cys Reverse c ttt tgg tec atg aat aCa gta atg ata aat atg 28 p.
  • Thr527Met Forward c age aca aat aaa ctg aTG ttt atg gat ctt gag ctg 29 p.
  • Thr527Met Reverse cag etc aag ate cat aaa CAt cag ttt att tgt get g 30
  • mice were housed and mouse work approved under the Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee approval (AEC 2014.026).
  • Embryos were produced from C57BL/6 Smchdl ⁇ congenic strain sires mated with C57BL/6 dams, with embryo ages ranging from embryonic day 8.5 to embryonic day 12.55. All embryos analyzed were female. No randomization or blinding was used during the experimental procedure.
  • Embryos were briefly fixed in 2 % paraformaldehyde/0.2 % glutaraldehyde and stained in 1 mg/ml X-gal for several hours. Cryosections were cut at 12 ⁇ .
  • Xenopus laevis were used according to guidelines approved by the Singapore National Advisory Committee on Laboratory Animal Research. Protocols for fertilization, injections and whole mount in situ hybridization are according to methods known in the art.
  • Human SMCHDl Origene was cloned into expression vector pCS2+, linearized with NotI and transcribed with mMESSAGE mMachine SP6 transcription kit (Thermo Fisher). Transcribed mRNA was column purified and its concentration measured using a Nanodrop. The mRNA contains a poly A signal that allows for polyadenylation in vivo.
  • the two dorsal-animal blastomeres were injected at the 8-cell stage with the synthesized mRNA. Embryos were allowed to develop at room temperature until stage 45-46 (4 days post fertilization) and fixed. Eye diameter was measured using a Leica stereomicroscope with a DFC 7000T digital camera. No statistical method was used to predetermine sample size. No randomization or blinding was used. Embryos that died before gastrulation were excluded. Statistics were calculated using 1 way ANOVA, followed by Dunn's multiple comparison test. A p value of less than 0.05 was considered significant. Results are given as means + s.d. Injections were performed on multiple clutches to reduce clutch- specific bias.
  • Embryonic extracts were prepared by lysing Stage 12 embryos in CelLytic Express (Sigma) on ice, followed by centrifugation to remove yolk proteins. Extracts were analyzed by Western blot with anti- SMCHD1 (Atlas HPA039441) and anti-p-Actin antibodies (clone AC-74, Sigma). cDNA was made from RNA extracted from Xenopus laevis embryos of various stages using iScript reverse transcriptase (Bio-Rad).
  • qPCR was performed using the following primers, xsmchdl qPCR F 5'- CAGTGGGTGTCATGGATGCT (SEQ ID NO: 31), xsmchdl qPCR R 5'- TCCATGGCTAGACCACTTGC (SEQ ID NO: 32), XL 18S F 5'- GCAATTATTTCCCATGAACGA (SEQ ID NO: 33), XL 18S R 5'- ATCAACGCGAGCTTATGACC (SEQ ID NO: 34).
  • In situ hybridization probe for smchdl was amplified from stage 20 cDNA using primers 5 ' -CGAATGCAAAGTCCTTGGGC (SEQ ID NO: 35) and 5 ' -GCATCC ATGAC ACCC ACTGA (SEQ ID NO: 36), cloned into pGEM- T, linearized and transcribed using DIG-labelling mix (Roche) according to manufacturer's guidelines.
  • XRCC4-deficient cells and primary fibroblast cell lines established from cases 1 and 2 were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Gibco), and antibiotics. Testing for mycoplasma contamination was negative.
  • DMEM Dulbecco's modified Eagle medium
  • FCS fetal calf serum
  • Testing for mycoplasma contamination was negative.
  • H2AX activation cells were either irradiated with 100 J/m 2 UV-C or treated with 50 ⁇ etoposide (Sigma-Aldrich, USA) for 1 hour. Drugs were then washed out, fresh media was added, and cells were incubated for 6 hrs and then subjected to Western blot analysis.
  • MMEJ Microhomology-mediated End- Joining
  • the olfactory placode ectoderm thickens and invaginates to form the olfactory epithelium within the nasal cavity, a process that depends on cross-talk between the placodal epithelium and the underlying cranial neural crest-derived mesenchyme.
  • ablation of the nasal placode epithelium in chick embryos disrupts development of adjacent nasal skeletal elements.
  • Strong X-gal staining was observed in the developing face of mouse embryos expressing lacz from the Smchdl locus, including in the nasal placodes and optic vesicles at E9.5 and nasal epithelium at E12.5 ( Figure 7).
  • Eurexpress in situ hybridization data indicates regional expression of Smchdl in the nasal cavity at E14.5, while transcriptional profiling of post-natal olfactory epithelium demonstrated that Smchdl is specifically expressed in immature olfactory sensory neurons. These data are consistent with roles for SMCHDl during early nasal development.
  • Gonadotropin-releasing hormone (GnRH) neurons migrate from the olfactory placode along olfactory axon tracts to the hypothalamus, where they regulate reproductive hormone release from the pituitary gland.
  • Smchdl was identified as a modifier of transgene silencing in mice and was subsequently shown to be involved in X chromosome inactivation, being required for CpG island (CGI) methylation on the inactive X (Xi), CGI-independent silencing of some X chromosome genes, and Xi compaction.
  • CGI CpG island
  • Xi inactive X
  • Xi CpG island
  • mice null for Smchdl display midgestation lethality due to derepression of inactive X chromosome genes
  • male mutant mice display perinatal lethality of undescribed causes in certain strains or viability on the FVB/n background. Strikingly, craniofacial abnormalities have not been documented in Smchdl loss-of-function mice regardless of their sex.
  • FSHD facioscapulohumeral muscular dystrophy
  • FSHD2 facioscapulohumeral muscular dystrophy
  • FSHD1 FSHD1
  • FSHD2 FSHD2
  • FSHD1 OMIM 158900
  • D4Z4 repeat contraction leads to hypomethylation of the locus and derepression of DUX4 expression on a permissive haplotype (4qA) that harbors a stabilizing polyadenylation signal for DUX4 mRNA.
  • FSHD2 occurs in individuals harboring loss-of-function SMCHDl mutations and the permissive 4qA allele, without the requirement for D4Z4 repeat contraction, although SMCHDl mutations can also modify the severity of FSHD1.
  • SMCHDl is thought to function as a silencer at the 4q locus via binding to the D4Z4 repeats.
  • SMCHD1 variants Over 80 unique, putatively pathogenic SMCHD1 variants have been reported in FSHD2 patients (LOVD SMCHD1 variant database). These mutations, which include clear loss-of-function alleles, occur throughout the protein, and are not clustered in specific domains. Several loss- of-function mutations have also been reported in ExAC ( Figure lm), and over 60 deletions affecting SMCHD1 have been reported in the DECIPHER database (available phenotypic information does not indicate arhinia). The methylation status of D4Z4 repeats was analyzed in peripheral blood leukocytes in BAMS patients by sodium bisulphite sequencing (Table 2 and Figures 8 and 9).
  • SMCHD1 Proteins of the SMC family are involved in chromatid cohesion, condensation of chromosomes and DNA repair.
  • SMCHD1 is considered a non-canonical member of the family, with a C-terminal chromatin-binding hinge domain and an N-terminal GHKL (gyrase, Hsp90, histidine kinase, and MutL) ATPase domain21 ( Figure lm).
  • GHKL gyrase, Hsp90, histidine kinase, and MutL
  • SMCHD1 uses energy obtained from ATP hydrolysis to manipulate chromatin ultrastructure and interactions.
  • the purified Smchdl ATPase domain and an adjacent C-terminal region have been shown to adopt a structural conformation similar to Hsp9021. Consistent with this, the Hsp90 inhibitor radicicol decreased the ATPase activity of Smchdl.
  • ATPase assays were conducted using the purified recombinant N-terminal region harboring BAMS or FSHD2 mutations. Compared to wildtype, hydrolysis of ATP was increased for the N-terminal region containing the mutations p.Alal34Ser, p.Serl35Cys or p.Glul36Gly, strongly or slightly decreased for the FSHD2 mutations p.Tyr353Cysl5 or p.Thr527Metl8, respectively, and unchanged for the BAMS mutation p.Asp420Val (Figure 2a-f).
  • the present inventors have surprisingly identified de novo missense gain-of-function mutations restricted to the extended ATPase domain of SMCHD1 as the cause of isolated arhinia and BAMS. It will be of great interest to explore the epistatic relationships between SMCHD1 and known regulators of nasal development, such as PAX6 and FGF and BMP signaling, as well as to uncover other potential human- specific nasal regulators. Nose shape and size vary greatly between human populations and even more drastically among animal species, the elephant's trunk being an extreme example. As such, it will be interesting to determine the role of SMCHD1 in controlling nose size from an evolutionary perspective.
  • loss-of-function mutations in SMCHD1 are associated with FSHD2
  • BAMS and FSHD2 represent a rare example of gain- versus loss-of-function mutations in the same gene leading to vastly different human disorders, in terms of the affected tissue and age of onset.
  • FSHD is caused in part by a loss of SMCHD1
  • augmentation of the expression or activity of SMCHD1 in affected muscles can surprisingly be developed as a form of treatment.
  • SMCHD1 gain-of-function mutations in SMCHD1 in the present disclosure can inform gene therapy approaches, or in combination with future structural studies on the effect of these mutations on the ATPase domain, aid the design of drugs that reproduce the gain-of-function effect, for treatment of FSHD. Importantly for such an approach, the deleterious consequences of SMCHD1 gain-of- function appear restricted to a narrow window of human embryonic development.

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Abstract

L'invention concerne un variant isolé du peptide SMCHD1, ou un fragment de celui-ci, comprenant une ou plusieurs mutations qui augmentent l'activité SMCHD1 dans une cellule par comparaison à une cellule avec un peptide SMCHD1 de type sauvage. L'invention concerne également un acide nucléique isolé codant pour le peptide SMCHD1 variant de l'invention, un vecteur comprenant l'acide nucléique décrit, une composition pharmaceutique comprenant le peptide, l'acide nucléique, ou le vecteur de l'invention, et une cellule hôte comprenant le vecteur déjà décrit. L'invention concerne également un procédé de traitement de la dystrophie musculaire facio-scapulo-humérale (FSHD) chez un sujet en ayant besoin et un procédé de criblage d'un agent capable d'augmenter l'activité SMCHD1.
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