WO2022058295A1 - Procédé de diagnostic de maladies induites par expansion de répétition à l'aide d'une cartographie optique - Google Patents

Procédé de diagnostic de maladies induites par expansion de répétition à l'aide d'une cartographie optique Download PDF

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WO2022058295A1
WO2022058295A1 PCT/EP2021/075172 EP2021075172W WO2022058295A1 WO 2022058295 A1 WO2022058295 A1 WO 2022058295A1 EP 2021075172 W EP2021075172 W EP 2021075172W WO 2022058295 A1 WO2022058295 A1 WO 2022058295A1
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repeat expansion
nucleic acid
repeat
length
target locus
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Georg Haas
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Aix Marseille Universite
Institut National de la Sante et de la Recherche Medicale INSERM
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Aix Marseille Universite
Institut National de la Sante et de la Recherche Medicale INSERM
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention relates to a method for diagnosing a disease induced by repeat expansion by determining the length of said repeat expansion using optical mapping technology and a kit for determining repeat expansion length.
  • DM1 and DM2 myotonic dystrophy
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • Huntington disease eight other polyglutamine disorders including the most common forms of dominantly inherited ataxia, the most common recessive ataxia (Friedreich ataxia), and the most common heritable mental retardation (Fragile X Syndrome).
  • G4C2 GGGGCC
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the expanded G4C2 repeats account for about 40% of familial ALS, up to 7 % of sporadic ALS and 20 % of familial FTD (Renton A. E. et al., 2011, Neuron, 72, 257-68; DeJesus-Hernandez M. et al., 2011, Neuron, 72, 245-56).
  • ALS/FTD patients with C9orf72-linked ALS/FTD typically carry between several hundred to more than ten thousand G4C2 repeats in their germline DNA, as compared to less than 23 and most often only two repeats in healthy subjects (Beck J. et al., 2013, American journal of human genetics, 92, 345-53).
  • expanded G4C2 repeats in the C90rf72 are also the most frequent genetic cause of Huntington disease (HD) phenocopies (Hensman Moss D. J. et al., 2014, Neurology, 82, 292-9).
  • HD Huntington disease
  • expanded C9 repeats have been observed in spinocerebellar ataxias (Aydin G.
  • NGS Next Generation Sequencing
  • Nanopore or PacBio next generation sequencing remain however afflicted by high error rates and Nanopore sequencing fails to quantify the number of G4C2 repeats in plasmids carrying more than 70 repeats.
  • PacBio sequencing also proved largely unsuccessful in C9 repeat analysis and is not able to accurately determine the number of expanded G4C2 repeats in plasmids or in human genomic DNA (Ebbert M. T. W. et al., 2018, Mol Neurodegener, 13, 46).
  • W02017/40813 describes a method for diagnosing ALS using CRISPR/dCas labeling system. Repeat sequences targeted by dCas/gRNA complex can be visualized in microscopy but the number of repeat sequences cannot be accurately determined.
  • the inventors use CRISPR/dCas labeling system by contacting dCas with two guide RNAs to label DNA in each region that flanks repeat expansion.
  • This particular labeling in combination with optical genome mapping represents an efficient tool to quantify precisely the length of the repeat sequence expansion in patient.
  • the method developed here allows the diagnostic detection of pathogenic repeat expansions in patient derived clinically relevant biomaterials such as whole blood and fibroblasts with unprecedented sensitivity.
  • the present invention relates to a method for diagnosing a disease induced by repeat expansion in a subject by determining the length of said repeat expansion within a target locus in a subject sample comprising the steps of: i) labeling nucleic acid from said subject sample in each region that flanks repeat expansion of said target locus by contacting said nucleic acid with a dCas protein and at least a first and a second guide RNA comprising a sequence complementary respectively to the first and second regions that flank repeat expansion of the target locus, said guide RNA and/or dCas comprising a detection label, preferably fluorescent molecule such as Atto647, ii) linearizing labeled nucleic acid in a fluidic nanochannel, iii) determining the length of repeat expansion by detecting the signal of said detection label within linearized nucleic acid, iv) comparing the length of repeat expansion within target locus in nucleic acid from subject sample with a reference of repeat expansion length in said target locus derived from a normal subject.
  • said labeled nucleic acid is isolated after step ii) prior to be linearized in a fluidic nanochannel.
  • said guide RNA comprises a capture tag, said capture tag is used to isolate said labeled nucleic acid. More preferably, said capture tag is biotin and labeled nucleic acid is isolated using streptavidin magnetic bead.
  • said DNA is marked with non-specific backbone marker, preferably YOYO- 1 marker.
  • the length of repeat expansion is determined by detecting signal of detection label, preferably the signal of label is detected by microscopy.
  • said disease induced by repeat expansion is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) and said target gene is C9orf72.
  • said first gRNA preferably comprises a sequence selected from the group consisting of SEQ ID NO: 1 to 3 and/or said second gRNA preferably comprises a sequence selected from the group consisting of SEQ ID NO: 4 to 6.
  • said subject sample is blood sample.
  • the method as described above is carried out in multiplex by labeling nucleic acid in each region that flanks repeat expansion of different target loci with different detection labels.
  • Said multiplex method allows to diagnosis diseases induced by repeat expansion selected from the group consisting of myotonic dystrophy such as DM1 and DM2, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington disease, spinocerebellar ataxia (SCA), Friedreich ataxia, Fragile X syndrome and Fragile X tremor ataxia syndrome, CAG polyglutamine diseases, spinal and bulbar muscular atrophy (SBMA), myoclonic epilepsies linked to pentanucleotide repeats.
  • myotonic dystrophy such as DM1 and DM2
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • SCA spinocerebellar ataxia
  • Friedreich ataxia Fragile X syndrome and Fragile
  • the present invention relates to a kit for determining repeat expansion length in a target locus in a sample comprising a dCas protein and at least a first and a second guide RNAs comprising each a sequence complementary respectively to the first and second regions that flank repeat expansion of the target locus, each guide RNAs and/or dCas comprising a detection label, preferably a fluorescent label and optionally each guide RNA comprising a capture marker.
  • Figure 1 Bionano optical mapping of C9 repeat expansions.
  • A The normal C90rf72 (C9) gene (Hg38) is well covered by DLE-1 sites (small vertical bars). Normal C9 allele contains 2 repeat units in a segment of 6.35 kb delimited by two DLE-1 sites (arrowheads).
  • B Size of normal C9 alleles (ref., arrowheads) and of expanded mutant C9 alleles (arrows) in iPS cells of an ALS patient.
  • C Size of normal C9 alleles (ref, arrowheads) and expanded mutant C9 alleles (arrows) in skin fibroblasts of an ALS patient.
  • Figure 2 Optical mapping of C9 repeat expansions.
  • D. DNA backbone is stained with YOYO-1. DETAILED DESCRIPTION
  • CRISPR/dCas labeling system in combination with nucleic acid capture and optical mapping to determine precisely the length of the repeat expansion.
  • the present disclosure relates to a method for determining the length of repeat expansion within target locus of a nucleic acid.
  • nucleic acid refers to RNA and DNA.
  • the method according to the present disclosure is particularly suitable for being carried out on genomic DNA, particularly on isolated genomic DNA.
  • the nucleic acid can be any size, including several nucleotides in length to several million nucleotides in length.
  • the nucleic acid molecule is the length of a chromosome.
  • the method according to the present disclosure may be performed in the absence of prior nucleic acid amplification in vitro and the nucleic acid molecule is directly harvested and isolated from a biological sample. Harvest and isolation of nucleic acids are routinely performed in the art and suitable methods can be found in standard molecular biology textbooks.
  • the nucleic acid molecule may be harvested from a biological sample such as a tissue or a biological fluid.
  • repeat expansion refers to tandem repeats in nucleic acid which is a sequence of two or more nucleotides that is repeated in nucleic acid in such a way that the repeats are directly adjacent to each other.
  • the increase in copy number of repeats induces instability and depending on where it is located (target locus) may cause as non-limiting examples defects in protein encoded by a gene, change the regulation of gene expression, produce a toxic RNA or lead to chromosome instability.
  • Tandem repeat can be a tri-, tetra-, penta-, hexa-, dodeca-nucleotide or other type of nucleotide repeat.
  • the method according to the present disclosure comprises the labeling of nucleic acid in each region that flanks repeat expansion of the target locus.
  • the inventors use CRISPR- based technique (CRISPR/dCas) for specifically labeling said regions.
  • CRISPR/dCas CRISPR- based technique
  • the CRISPR/dCas system has a strong and stable affinity for its target DNA and enables to visualize accurately target sequence.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas protein CRISPR-associated protein
  • Cas protein is a DNA endonuclease that uses single guide RNA sequence as a guide to recognize and generate double-strand breaks in DNA that is complementary to the single guide RNA sequence.
  • Cas protein comprises two active cutting sites namely HNH nuclease domain and RuvC-like nuclease domain.
  • CRISPR-based imaging technique (CRISPR/dCas labeling system) is carried out with a modified Cas protein that lacks nuclease activity.
  • Said modified Cas protein also named dead Cas protein (dCas) lacks nuclease activity which can be inhibited or prevented by at least one mutation and/or deletion in the HNH and/or RuvC-like catalytic domains of the Cas protein.
  • the resulting dCas protein lacks nuclease activity but is able to use guide RNA as a guide to recognize DNA target sequence.
  • the dCas protein can be one type of the dCas proteins known in the art; preferably dCas protein is dCas9 protein.
  • dCas is contacted with a guide RNA (gRNA) designed to comprise a complementary sequence of the target nucleic acid sequence to specifically recognize said target sequence.
  • gRNA guide RNA
  • a “guide RNA” or “single guide RNA” refers to a nucleic acid that promotes the specific targeting or homing of a gRNA/Cas complex to a target nucleic acid.
  • gRNA refers to a RNA that comprises a transactivating crRNA (tracrRNA) and a crRNA.
  • said guide RNA corresponds to a crRNA and tracrRNA which can be used separately or fused together.
  • the complementary sequence pairing with the target sequence recruits dCas to bind the DNA at the target sequence.
  • crRNA is engineered to comprise a complementary sequence to a portion of each region that flanks repeat expansion, such that it is capable of targeting said regions.
  • the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 12 nucleotides which is complementary to the target sequence.
  • complementary sequence refers to the sequence part of polynucleotide (e.g. part of crRNa or tracRNA) that can hybridize to another part of polynucleotides under standard low stringent conditions.
  • sequences are complementary to each other pursuant to the complementarity between two nucleic acid strands relying on Watson-Crick base pairing between the strands, i.e. the inherent base pairing between adenine and thymine (A-T) nucleotides and guanine and cytosine (G-C) nucleotides.
  • A-T adenine and thymine
  • G-C guanine and cytosine
  • Said gRNA can be designed by any methods known by one of skill in the art in view of the present disclosure.
  • the guide RNAs comprise complementary sequence to each region that flanks repeat expansion of the target locus.
  • region that flanks repeat expansion it is intended the regions adjacent to the 5’ and 3’ end of the repeat expansion, respectively.
  • the regions adjacent to the 5’ and 3’ end of the repeat expansion corresponds to sequences located between 0.1 kb and 5 kb, preferably between 0.5 kb and 2 kb, more preferably at about 1 kb upstream and downstream of the repeat expansion.
  • Two gRNA comprising complementary sequence to each region that flanks the repeat expansion can be used simultaneously.
  • said target locus is C9orf72 gene and the gRNA is designed to comprise complementary sequences to each region that flanks repeat expansion of the C9orf72 gene.
  • Said first gRNA preferably comprises a sequence selected from the group consisting of SEQ ID NO: 1 to 3 and/or said second gRNA preferably comprises a sequence selected from the group consisting of SEQ ID NO: 4 to 6.
  • nucleic acid is contacting with said dCas and guide RNA as described above to label each region that flanks repeat expansion of the target locus.
  • Said guide RNA hybridizes to said region that flanks the repeat expansion.
  • dCas and/or gRNA comprises a detection label.
  • said detection label such as fluorescent molecule can be linked to dCas as previously described in Chen et al. Cell. 2013.155(7): 1479-91, Ma H, et al. Proc Natl Acad Sci U S A. 2015, 112(10):3002-7.
  • Labeled dCas protein may be produced by recombinant DNA technology from an appropriately designed DNA plasmid according to any methods well-known in the art.
  • labeled gRNA can be obtained by modifying guide RNA to bind to a detection label.
  • said labeled gRNA can be obtained by the method described in Shao S. et al. 2016, NAR 44(9):e86, Wang S.Y. et al. 2016.
  • said guide RNA bind a detection label at a stem loop sequence
  • said gRNA can be labelled by inserting binding RNA hairpin such as MS2, PP7 or BoxB aptamer hairpins, which recruit MCP, PCP or N22 fused to detection label.
  • binding RNA hairpin such as MS2, PP7 or BoxB aptamer hairpins, which recruit MCP, PCP or N22 fused to detection label.
  • Said labeled gRNA may be produced by in vitro transcription from a suitable constructed DNA plasmid according to any methods well-known in the art
  • said labeled gRNA is chemically synthesized and fluorescent label is included during gRNA synthesis.
  • Said detection label refers to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • detection labels include but are not limited to biotin for staining with streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabel, enzymes and calorimetric label.
  • the detection label is a fluorescent protein or enzyme provoking the appearance of colored product.
  • said detection label is a fluorescent protein.
  • fluorescent proteins can be any fluorescent proteins that emit in optical spectrum wavelengths.
  • Said fluorescent protein can be selected from the group consisting of: BFPs (such as Sirius, EBFP, SBFP2, EBFP2, Azurite, mKalamal proteins), TagBFP and mBlueberry2, CFPs (e.g. CUPE3A, mTurquoise, mseCFP, Cerulean, ECFP, CyPet, TagCFP, mTFPl, Midori- ishi Cyan, and mUKG), GFPs (e.g.
  • TurboGFP AceGFP, Azami Green, ZsGreen, TagGFP2, EGFP, mEGFP, mWasabi, Emerald, Superfolder GFP, Sapphire, T-Sapphire, and SGFP2), YFPs (such as TagYFP, mAmetrine, EYFP, Topaz, SYFP2, Venus, mCitrine, Ypet, PhiYFP, and ZsYellowl), OFPs (such as mBanana, mOrange, Kusabira Orange2, mOrange2, OFP, and TurboRFP), RFPs (such as tdTomato, DsRed2, DsRed, DsRed- Express, TagRFP, TagRFP-T, mTangerine, DsRed-Express2, DsRed-Max, AsRed2, m Apple, mStrawberry, mRuby, mRFPl, tdRFP611 and mCherry), or FRFP
  • said labeled nucleic acid can be then captured.
  • Said labeled nucleic acid can be isolated by using a gRNA which comprises a capture tag to permit the capture of the labeled nucleic acid with a capture agent bound to a solid support.
  • capture tag refers to molecular entity that is one member of a binding pair that interacts with a capture agent.
  • the tag is typically sufficiently small to allow attachment of the tag to gRNA without interfering with the structure or function of the gRNA.
  • capture agent refers to a material able to bind to the capture tag and intended to be immobilized onto a solid support, for example a surface of a solid substrate or a bead.
  • solid support is used in reference to any solid or stationary material or object to which reagents such as capture agents may be attached.
  • solid supports include beads, particles, resin, cell culture flasks microscope slides, wells of microtiter plates, coverslips, as well as many other suitable items.
  • Solid supports may be magnetic, paramagnetic, or non-magnetic. In a preferred embodiment, said solid support is magnetic bead.
  • capture tag and capture agent include, but are not limited to, streptavidin and biotin, histidine tags and nickel metal ions, glutathione- S- transferase and glutathione.
  • said capture tag is contained in guide RNA, and gRNA/dCas- nucleic acid complex is isolated by the bound of capture tag to capture agent which is attached onto solid support.
  • said capture tag is biotin and capture agent streptavidin, preferably attached to magnetic bead.
  • the nucleic acid is labeled with a nonspecific label, for example a backbone label such as an intercalating dye.
  • a backbone label such as an intercalating dye.
  • said DNA backbone label includes YOYO marker, TOTO-3, SyberGreen, ethidium bromide
  • the method may comprise labeling the nucleic acid by an additional chemistry, for example direct enzymatic labeling using an enzyme such as DLE- 1 and optionally further including a stain in addition to the enzymatic labeling (e.g., BIONANO GENOMICS “DLS” technology), or nicking followed by nick labeling and repair (e.g., BIONANO GENOMICS “NLRS” technology) to produce a nucleic acid with two or more different specificity motifs with different labels.
  • an additional chemistry for example direct enzymatic labeling using an enzyme such as DLE- 1 and optionally further including a stain in addition to the enzymatic labeling (e.g., BIONANO GENOMICS “DLS” technology), or nicking followed by nick labeling and repair (e.g., BIONANO GENOMICS “NLRS” technology) to produce a nucleic acid with two or more different specificity motifs with different labels.
  • BIONANO GENOMICS “DLS” technology
  • the inventors showed that the flexible and efficient labeling of target sequences with CRISPR/dCas system enables to determine accurately repeat expansion length with optical mapping, particularly in fluidic nanochannel such as the Bionano Genomics IRYS or SAPHYR system.
  • labeled nucleic acid is linearized in a fluidic nanochannel in which said nucleic acid remains intact in a linearized, stretched conformation that permits the determination of the positions of detection labels along the length of the linearized nucleic acid.
  • Fluidic nanochannels can be used for the linearization of long nucleic acid molecules (e.g., kilobase, or megabase-length) as well as short nucleic acid molecules.
  • said fluidic nanochannels used in the method according to the present disclosure can be nanofluidic nanochannels as described in WO2008/121828, W02010/002883, and WO2009/149362.
  • Suitable fluidic nanochannel according to the present disclosure has a diameter of less than about twice the radius of gyration of the DNA in its extended form.
  • a nanochannel of such can exert entropic confinement of the freely extended, fluctuating DNA coils to extend and elongate the nucleic acid.
  • the nanochannel has a characteristic cross-sectional dimension of at least about the persistence length of the nucleic acid molecule.
  • the fluidic nanochannel can have a length of at least 10 nm and a cross-sectional diameter of less than 200 nm, more preferably less than 100 nm.
  • the signal of the detection label on the linearized nucleic acid can be detected to determine the distance between the two detection labels and thus the length of repeat expansion.
  • the detection of the label can be carried out by any techniques known to the skilled person in the art such as non-limiting examples fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atom force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, a total internal reflection (TIR) system, and an electromagnetic detection system.
  • fluorescent detection system an electrical detection system
  • a photographic film detection system a chemiluminescent detection system
  • an enzyme detection system an atom force microscopy (AFM) detection system
  • STM scanning tunneling microscopy
  • optical detection system a nuclear magnetic resonance (NMR) detection system
  • NMR nuclear magnetic resonance
  • TIR total internal reflection
  • the measurement of the fluorescence intensity can be performed by any fluorescence techniques known to the skilled person, such as fluorescence microscopy, fluorescence spectroscopy, preferably by fluorescence microscopy.
  • said optical mapping in fluidic nanochannel is realized in nanochannel arrays such as the Bionano Genomics IRYS or SAPHYR system.
  • a microscopic device scans chips containing flow cells which each contain hundreds of thousands of parallel nanochannels generated. Each nanochannel of the chip allows only a single linearized nucleic acid molecule to travel through while preventing the molecule from tangling or folding back on itself.
  • Hardware and software analysis such as Bionano Data Solutions can be used to determine the length of the repeat expansion.
  • the length of repeat expansion may be determined by using as standard a set of DNA containing multiple repeats of known size.
  • the method according to the present disclosure is useful to determine simultaneously the length of repeat expansion in different target loci.
  • Said method can be carried out in multiplex using different labels for each target locus, preferably different fluorescent molecules.
  • the different fluorescent molecules emit each at sufficiently different wavelengths to be distinguishable using conventional method of fluorescence detection.
  • the first fluorescent molecule can emit in red (emission wavelength between about 605 nm and 780 nm) and the second fluorescent molecule can emit in green (emission wavelength between about 510 nm and 570 nm).
  • the first fluorescent molecule can emit in blue (emission wavelength between about 460 nm and 480 nm) and the second fluorescent molecule can emit in orange (emission wavelength between about 585 nm and 605 nm).
  • the person skilled in the art knows how to select several different fluorescent molecules and combinations thereof to develop a multiplex method according to the present disclosure.
  • Labeled nucleic acids can be linearized in Saphyr chip which each contains hundreds of thousands of parallel nanochannels.
  • the nanofluidic environment of the flow chamber allows nucleic acid to move swiftly through hundreds of thousands of parallel nanochannels simultaneously, enabling high-throughput fluorescence microscope imaging to determine the length of repeat expansion in different regions and/or on different samples.
  • the method as described above is suitable for diagnosing a disease induced by repeat expansion in a nucleic acid of subject sample.
  • said “subject” refers to a mammal including a non-primate (e.g. a cow, pig, horse, cat, dog, rat and mouse) and a primate (e.g. a monkey and a human), and more preferably a human.
  • a non-primate e.g. a cow, pig, horse, cat, dog, rat and mouse
  • a primate e.g. a monkey and a human
  • Said nucleic acid, in particular genomic DNA may be isolated from subject sample using conventional methods.
  • cells from subject sample are lysed to release nucleic acid.
  • Said cells can be lysed by using lysis buffer well-known by one skilled in the art.
  • said cells are lysed using the DNA isolation protocol of 30268 Rev C Bionano Prep SP Frozen Cell Pellet or 30246 Rev C Bionano Prep SP Frozen Human Blood DNA Isolation Protocol (Bionano Genomics).
  • sample of a subject refers to any biological sample comprising cells of said patient.
  • it is a blood sample or tissue sample. More preferably it is a blood sample.
  • diagnosis disease induced by repeat expansion it is intended a method of assessing whether a subject has or is likely to develop said disease induced by repeat expansion. Such a method may be performed when a subject has already exhibited clinical symptoms of said disease or alternatively the method may be performed as a means of assessing whether the subject has a predisposition towards developing said disease. This enables a medical practitioner to take appropriate action to prevent or lessen the likelihood of onset of the disease or to allow appropriate treatment of the disease.
  • Repeat expansion diseases are for example neurodegenerative diseases including as nonlimiting examples myotonic dystrophy such as DM1 and DM2 , amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington disease, dominantly inherited ataxia (spinocerebellar ataxia (SCA)), recessive ataxia (Friedreich ataxia), Fragile X syndrome and Fragile X tremor ataxia syndrome, CAG polyglutamine diseases, spinal and bulbar muscular atrophy (SBMA), Unverricht-Lundborg myoclonic epilepsy (EPM1).
  • myotonic dystrophy such as DM1 and DM2 , amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington disease, dominantly inherited ataxia (spinocerebellar ataxia (SCA)), recessive ataxia (Friedreich ataxia), Fragile X syndrome and Fragile X tremor at
  • Disease induced by repeat expansion refers to disorder in which repeats of nucleotide repeats increase in copy numbers until they cross a threshold above which they become unstable. In several diseases, the larger the expansion the faster the onset of disease, and the more severe the disease becomes. Examples of normal and disease repeat expansion unit ranges within respective target locus for several diseases are indicated in the Table 1 below.
  • the method for diagnosing a disease induced by repeat expansion in a subject comprises determining the length of the repeat expansion within a target locus as described above in nucleic acid of a subject sample, wherein the length of repeat expansion in said target locus is compared with a reference of repeat expansion length in said target locus derived from a healthy subject.
  • the reference can be the number of repeat sequences within the target locus from a healthy subject that does not have a disease induced by repeat expansion and preferably that has no family history of developing such diseases.
  • the reference length of repeat expansion in the target locus derived from a healthy subject can already be known by one skilled in the art as exemplified in the Table 1.
  • the reference of normal length of repeat expansion in subject can be determined by the method as described in the present disclosure using healthy subject sample.
  • the method according to the present disclosure is particularly suitable for diagnosing a disease induced by repeat expansion by determining precisely the length of the repeat expansion in target locus and comparing said length with the reference.
  • the number of repeat units is at least 2 fold expanded but usually even more, 3, 5, 10 fold, up to 100 or even more than 1000 fold expanded in subject having or being likely to develop the disease induced by repeat expansion.
  • the method according to the present disclosure is particularly suitable for diagnosing frontotemporal dementia (FTD) and/or amyotrophic lateral sclerosis (ALS) by determining precisely the length of the GGGGCC repeat in C9orf72 gene that can be greater than around 60 repeats units, ranging up to more than 5000 units in allele of subject having or being likely to develop ALS and/or FTD and between 2 and 30 in normal subject.
  • FTD frontotemporal dementia
  • ALS amyotrophic lateral sclerosis
  • the method as described above may also be used for diagnosing several diseases induced by repeat expansion in a subject by determining simultaneously the length of repeat expansion within different target loci in a subject sample by labeling nucleic acid in each region that flanks repeat expansion of different target loci with different detection labels.
  • Labeled nucleic acid can be linearized in Saphyr chip which each contains hundreds of thousands of parallel nanochannels.
  • the nanofluidic environment of the flow chamber allows nucleic acid to move swiftly through hundreds of thousands of parallel nanochannels simultaneously, enabling high-throughput fluorescence microscope imaging to determine the length of repeat expansion in different regions and/or different samples.
  • the present disclosure also relates to a kit for determining repeat expansion length in a target locus in a sample comprising a dCas protein and at least a first and a second guide RNA as described above, guide RNA and/or dCas comprising a detection label, preferably a fluorescent label and optionally guide RNA comprising a capture tag.
  • said kit comprises containers each comprising one or more compounds at a concentration or in an amount that facilitates the reconstitution and/or the use of the dCas and gRNA and the implementation of the method according to the invention.
  • the kit may also comprise instructions indicating the methods for preparing and/or using the dCas and gRNA for determining the length of repeat expansion within target locus according to the method of the present disclosure.
  • the inventors measured the size of C9 repeat alleles in various tissues, organs and cells of human patients with C9-linked ALS or FTD and in healthy controls.
  • the inventors first isolated high molecular weight DNA from blood, skin fibroblasts and fibroblast-derived IPSC lines by using the Bionano prep SP protocol. The inventors then performed genome-wide labeling of CTTAAG sequences by fluorescent DLE-1 labeling (DLS technology), counterstained the DNA with YOYO-1 and resuspended the genomic DNA. The inventors then loaded the labeled DNA on the flow chambers of a Saphyr chip and performed optical imaging for 24 to 48 hours. Using Bionano Saphyr analysis, the inventors detected the normal C9 alleles at sizes of 5.97 and 6.35 kb, perfectly aligned to the human reference sequences ref9 and Hg38 (Fig. 1A).
  • the inventors decreased the input of genomic DNA to 12.1 ng. To determine the accuracy of the technique, the inventors successfully analyzed repeat size in DNA preparations that were engineered to carry expanded G4C2 repeats.
  • the inventor developed a highly original gene-targeted approach for the accurate analysis of pathogenic expanded and normal C9 repeats in ALS, FTD and other neurodegenerative diseases.
  • the principle of the invention is the fluorescent labeling of C9 repeat alleles by modified CRISPR/dCas9 (dead Cas9) technology, the biochemical capture of the labeled C9 alleles and the optical imaging of the captured C9 alleles.
  • C9 guide RNAs to the fluorochrome Alexa 647 which emits in the far red. This should ensure minimal fluorescence leaking into other channels when and optimal compatibility with the other fluorochromes e.g. YOYO-1 for DNA backbone counterstaining (emitting in blue) and Atto’ 532 (emitting in green) for combination with DLE-1 mediated genome-wide DNA labelling.
  • fluorochromes e.g. YOYO-1 for DNA backbone counterstaining (emitting in blue) and Atto’ 532 (emitting in green) for combination with DLE-1 mediated genome-wide DNA labelling.
  • the inventors have designed several guide RNAs to target specific sequences located in the first intron/promoter region of the human C9 gene at about 1 kb upstream and downstream of the G4C2 repeat (Fig. 2B) (Table 2).
  • Table 2 sgRNAs at positions 5’ and 3’ of the GGGGCC repeat expansion in the C9Orf72 gene. This strategy allows accurate quantification of G4C2 repeat expansion at the low end of 20 to 100 units together with accurate quantification and at the upper end to repeat units from 1.000 or 100.000 units.
  • the inventors To capture the fluorescently labeled C9 alleles, the inventors have chosen to couple the guide RNA with Biotin and to use Streptavidin magnetic beads (Fig. 2C). To be able to detect guide RNA/C9 DNA complexes with the built-in microscope system of Bionano Saphyr, the inventors counterstain the DNA backbone with YOYO-1 (Fig. 2D).
  • IPSCs IPBMCs are clonal, minimizing the risk of genomic mosaicism
  • G4C2 repeat number seems quite stable during cellular proliferation and passaging S. Almeida et al., 2013, Acta Neuropathol, 126, 385-99
  • IPSC cultures represent a scalable source for the preparation of large quantities of high molecular weight DNA which is crucial for Bionano analyses
  • the IPSC collection contains lines with G4C2 repeat numbers of 2, 70, 641 and 3.000 G4C2 repeats. This range of repeats allows to define the upper/lower limits for Bionano-based repeats sizing.
  • PBMCs peripheral blood mononuclear cells
  • immortalized lymphoblastoid cells In addition to mutant C9 IPSC lines, the inventors use frozen whole blood, peripheral blood mononuclear cells (PBMCs) and immortalized lymphoblastoid cells.
  • PBMCs peripheral blood mononuclear cells
  • the DNA concentration is then measured by Qubit, and 10 pg of DNA are used for labeling with CRISPR/dCas9 and subsequent Biotin/Streptavidin capture.
  • the C9 alleles are imaged by the Saphyr2 system and high resolution images analyzed by dedicated software. The inventors measure the molecular distances between the CRISPR/dCas9 sites.

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Abstract

La présente invention concerne un procédé pour diagnostiquer une maladie induite par une expansion de répétition par détermination de la longueur de ladite expansion de répétition à l'aide d'une technologie de cartographie optique et un kit pour déterminer une longueur d'expansion de répétition.
PCT/EP2021/075172 2020-09-15 2021-09-14 Procédé de diagnostic de maladies induites par expansion de répétition à l'aide d'une cartographie optique Ceased WO2022058295A1 (fr)

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