WO2022109890A1 - Modèle de souris dmd pdx, son procédé d'élaboration et son utilisation - Google Patents

Modèle de souris dmd pdx, son procédé d'élaboration et son utilisation Download PDF

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WO2022109890A1
WO2022109890A1 PCT/CN2020/131620 CN2020131620W WO2022109890A1 WO 2022109890 A1 WO2022109890 A1 WO 2022109890A1 CN 2020131620 W CN2020131620 W CN 2020131620W WO 2022109890 A1 WO2022109890 A1 WO 2022109890A1
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dmd
seq
cas9
mdscs
crispr
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PCT/CN2020/131620
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English (en)
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Xiaoping Li
Peng XIANG
Weiqiang Li
Qiong KE
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Sun Yat-Sen University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C12N5/0659—Satellite cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C07K14/4707—Muscular dystrophy
    • C07K14/4708—Duchenne dystrophy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • DMD Duchenne muscular dystrophy
  • DMD Duchenne muscular dystrophy
  • DGC dystrophin glycoprotein complex
  • Loss of functional dystrophin leads to the degradation of DGC and muscle fiber membrane fragility, resulting in progressive muscle degeneration [3] that leaves patients wheelchair-bound at around the age of 12 [4] , with premature death when the patients are in their twenties. To date there are no effective treatments for DMD.
  • Efforts have been made to rescue dystrophin expression in DMD by overexpressing a truncated but functional micro-dystrophin-utilizing lentivirus or adeno-associated virus (AAV) [6-8] or by inducing the skipping of out-of-frame exons with antisense oligonucleotides [9-11] and the read-through of nonsense mutation with small molecules [12] .
  • AAV adeno-associated virus
  • FIG. 3 shows restoration of dystrophin expression by CRISPR/Cas9; wherein, (A) CRISPR/Cas9-mediated gene editing restored dystrophin mRNA level in myotubes differentiated from DMD-MDSCs; illustration of primer binding sites (black arrow) for RT-PCR (top row) ; all detected bands were of the expected size; red arrows indicate bands that were verified by Sanger sequencing in (B) ; Cr, edited; M, marker; Un, unedited; (B) the successful reframing of cDNA in DMD-MDSCs subjected to three different gene editing strategies was confirmed by Sanger sequencing; (C) Western blot analysis of dystrophin expression in targeted ( ⁇ 45–55, ⁇ 46–54, and INDEL50) or untargeted myotubes differentiated from DMD-MDSCs; WT MDSCs served as a positive control; expected molecular weights were 427 kDa for WT and INDEL50, 361 kDa for ⁇
  • FIG. 6 shows in vivo differentiation of DMD-MDSCs into muscle fibers; wherein, (A) DMD-MDSCs infected with lentivirus expressing luciferase were transplanted into recipients and engraftment was monitored by imaging over a period of 4 weeks; bioluminescence images of representative injected mice were acquired on the indicated days; (B) qPCR analysis for human mitochondrial DNA revealed the presence of human cells in PDX DMD mouse following injection of DMD-MDSCs over a period of 4 weeks. Mouse DNA samples (M) and human DNA samples (H) , as well as a series of mouse-human cell dilution samples, were run as negative and positive controls to estimate the degree of human cell contribution.
  • A DMD-MDSCs infected with lentivirus expressing luciferase were transplanted into recipients and engraftment was monitored by imaging over a period of 4 weeks; bioluminescence images of representative injected mice were acquired on the indicated days;
  • B qPCR analysis for
  • FIG. S5 shows that targeted NGS detects the ratio of indels generated by gRNAs.
  • FIG. S17 (A) shows a heat map revealed the genes with off-target sites (sum of SNVs and indels) ; the names of genes were indicated in the right column, the shades of red varied according to the numbers of off-target sites in a single gene.
  • FIG. S17 (B) shows the ratio of indels types in off-target analysis.
  • gRNAs for the three targeting strategies were separately introduced along with Cas9 into DMD-MDSCs by electroporation. After 3 days, genomic DNA was extracted from the transfected DMD-MDSCs for analysis. Genomic deletions and indel mutations were assessed by end-point PCR and the Surveyor nuclease assay. The embodiments detected the expected deletion products of 708 kb and 334 kb in ⁇ 45-55 and ⁇ 46-54 targeted cells by PCR of the genomic DNA. Sanger sequencing confirmed the reframing of mutant DMD (FIG. 2D, FIG. S3A, B) .
  • the embodiment performed a reported highly sensitive quantitative PCR assay based on the detection of human mitochondrial DNA (hmtDNA) , which allows for the detection of as few as one human cell in 10,000 mouse cells [34, 35] .
  • the presence of human cells in TA injected with DMD-MDSCs was approximately 2.5% (FIG. 6B) .
  • human spectrin and lamin A/C were detected in muscle sections at the end of 4 weeks (FIG. 6C) , indicating that human MDSCs were able to differentiate into mature muscle fibers in mice. As shown in FIG.
  • Expression cassettes for human codon-optimized Cas9 and Cas12a nuclease and a panel of gRNAs were designed and constructed as previously described.
  • the dual fluorescence reporter vector was constructed based on the pCMV-tdTomato vector. Briefly, the embodiment added the EGFP coding sequence and an internal ribosome entry site (IRES) into the pCMV-tdTomato vector to obtain the intermediate vector pCMV-EGFP-IRES-TdTomato.
  • the cassette harboring introns 45 and 54 with targeting sites which flanked a multi-polyA sequence were inserted into pCMV-EGFP-IRES-TdTomato downstream of EGFP and upstream of the IRES.
  • CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors.
  • Dystrophin expression levels were increased by over 10%in human DMD muscle fibers.
  • the restored dystrophin in vivo was functional, as demonstrated by the expression of the dystrophin glycoprotein complex member ⁇ -dystroglycan.

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Abstract

L'invention concerne un procédé d'élaboration d'un modèle murin DMD PDX, un modèle murin DMD PDX, l'utilisation du modèle murin DMD PDX, et un procédé d'édition génomique pour restaurer l'expression de la dystrophine dans les fibres musculaires d'un patient atteint de la DMD.
PCT/CN2020/131620 2020-11-26 2020-11-26 Modèle de souris dmd pdx, son procédé d'élaboration et son utilisation WO2022109890A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017072590A1 (fr) * 2015-10-28 2017-05-04 Crispr Therapeutics Ag Matériaux et méthodes pour traiter la dystrophie musculaire de duchenne
CN106714845A (zh) * 2014-08-11 2017-05-24 得克萨斯州大学系统董事会 通过crispr/cas9介导的基因编辑预防肌营养不良
CN109863241A (zh) * 2016-11-18 2019-06-07 雀巢产品技术援助有限公司 肌肉干细胞的体外制备

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106714845A (zh) * 2014-08-11 2017-05-24 得克萨斯州大学系统董事会 通过crispr/cas9介导的基因编辑预防肌营养不良
WO2017072590A1 (fr) * 2015-10-28 2017-05-04 Crispr Therapeutics Ag Matériaux et méthodes pour traiter la dystrophie musculaire de duchenne
CN109863241A (zh) * 2016-11-18 2019-06-07 雀巢产品技术援助有限公司 肌肉干细胞的体外制备

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BENCHAOUIR R; MEREGALLI M; FARINI A; D'ANTONA G; BELICCHI M; GOYENVALLE A; BATTISTELLI M; BRESOLIN N; BOTTINELLI R; GARCIA L; TORR: "Restoration of Human Dystrophin Following Transplantation of Exon-Skipping-Engineered DMD Patient Stem Cells into Dystrophic Mice", CELL STEM CELL, ELSEVIER, vol. 1, no. 6, 13 December 2007 (2007-12-13), AMSTERDAM, NL , pages 646 - 657, XP009107565, ISSN: 1934-5909, DOI: 10.1016/j.ste.2007.09.016 *
MENG JINHONG, CHUN SOYON, ASFAHANI ROWAN, LOCHMÜLLER HANNS, MUNTONI FRANCESCO, MORGAN JENNIFER: "Human Skeletal Muscle–derived CD133+ Cells Form Functional Satellite Cells After Intramuscular Transplantation in Immunodeficient Host Mice", MOLECULAR THERAPY, vol. 22, no. 5, 1 May 2014 (2014-05-01), US , pages 1008 - 1017, XP055932355, ISSN: 1525-0016, DOI: 10.1038/mt.2014.26 *
MENG JINHONG, MUNTONI FRANCESCO, MORGAN JENNIFER: "CD133+ cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability", STEM CELL RESEARCH, vol. 30, 1 July 2018 (2018-07-01), NL , pages 43 - 52, XP055932353, ISSN: 1873-5061, DOI: 10.1016/j.scr.2018.05.004 *
MOTOHASHI，N. ET AL.: "Isolation， culture， and transplantation of muscle satellite cells.", J VIS EXP., vol. 86, 8 April 2014 (2014-04-08) *
TORRENTE Y; BELICCHI M; MARCHESI C; D'ANTONA G; COGIAMANIAN F; PISATI F; GAVINA M; GIORDANO R; TONLORENZI R; FAGIOLARI G; LAMPERTI: "Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.", CELL TRANSPLANTATION, vol. 16, no. 6, 1 January 2007 (2007-01-01), US , pages 563 - 577, XP009193750, ISSN: 0963-6897, DOI: 10.3727/000000007783465064 *
ZHANG SHU;WU SHI-WEN: "Therapeutic progress of Duchenne muscular dystrophy", PRACTICAL PHARMACY AND CLINICAL REMEDIES, vol. 22, no. 9, 26 September 2019 (2019-09-26), pages 897 - 903, XP055932361, DOI: 10.14053/j.cnki.ppcr.201909001 *

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