[go: up one dir, main page]

WO2024235844A1 - Procédés de prévention de la génotoxicité sur cible induite par des nucléases - Google Patents

Procédés de prévention de la génotoxicité sur cible induite par des nucléases Download PDF

Info

Publication number
WO2024235844A1
WO2024235844A1 PCT/EP2024/062916 EP2024062916W WO2024235844A1 WO 2024235844 A1 WO2024235844 A1 WO 2024235844A1 EP 2024062916 W EP2024062916 W EP 2024062916W WO 2024235844 A1 WO2024235844 A1 WO 2024235844A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
loh
nuclease
editing
Prior art date
Application number
PCT/EP2024/062916
Other languages
English (en)
Inventor
François MOREAU-GAUDRY
Aurélie BEDEL
Sabrina FAYET
Original Assignee
Institut National de la Santé et de la Recherche Médicale
Chu De Bordeaux
Université De Bordeaux
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Santé et de la Recherche Médicale, Chu De Bordeaux, Université De Bordeaux filed Critical Institut National de la Santé et de la Recherche Médicale
Publication of WO2024235844A1 publication Critical patent/WO2024235844A1/fr

Links

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/87—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention is in the field of medicine, in particular, gene therapy.
  • Targeted nucleases and in particular the CRISPR-Cas9 system, are a breakthrough that has propelled gene therapy into a new era 1 ' 4 .
  • Important advances are illustrated by several ongoing preclinical and clinical studies in fields such as immunotherapy, virology, and monogenic diseases. Nevertheless, a major concern is the potential genotoxicity of DNA double-strand breaks (DSB), which arise from incorrect or ineffective DNA repair and DNA damage response.
  • the risk of genomic instability seems to be the Achilles heel of CRISPR-Cas9.
  • Detailed genotyping of edited cells revealed that the full spectrum of Cas9-induced outcomes might be more complex than the sole induction of insertion and deletions InDeis at the targeted locus (ON-target genotoxicity) 5 .
  • Chromosome truncations associated with megabasic loss-of-heterozygosity with copy losses have been reported not only in cancer cell lines 11,12 but also in p53 -proficient human induced pluripotent stem cells (hiPSC) and embryos 9,13 .
  • Terminal megabase-scale copy-neutral LOHs (CN-LOHs) without loss of genetic material have also been observed in cancer cell lines 14 , human embryonic stem cells (hESC) and hiPSC 9 ' 15 .
  • HSPC human hematopoietic stem/progenitor cells
  • the present invention is defined by the claims.
  • the present invention relates to methods of preventing the ON-target genotoxicity induced by a nuclease.
  • the CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has impelled gene editing into clinical applications.
  • Comprehensive analysis of gene editing products at the targeted cut-site revealed a complex spectrum of outcomes.
  • ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and sensitive detection methods.
  • the inventors developed two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH).
  • FMReD Fluorescence-Assisted Megabase-scale Rearrangements Detection
  • the first object of the present invention relates to a method of preventing the ON- target genotoxicity induced by a nuclease in population of cells comprising the steps of i) inducing the G0/G1 phase cell cycle arrest in the population of cells and ii) editing the population of cells of step i) set with the nuclease.
  • the term “genotoxicity” has its general meaning in the art and refers to a damage to the genetic material of a live cell.
  • the damage to the genetic material of a cell may include, for example, damage resulting from nucleotide or polynucleotide deletion, addition, point mutation, dimerization or recombination, and DNA breakage or degradation.
  • the damage may also include that which is evidenced by chromosomal numerical abnormalities, such as polyploidy or aneuploidy.
  • the term “genotoxicity” describes a deleterious action of an agent (e.g. a nuclease) on a cell's genetic material affecting its integrity. Said genotoxicity can be potentially mutagenic or carcinogenic, specifically those capable of causing genetic mutation and of contributing to the development of tumors.
  • ON-target genotoxicity refers to the presence of unwanted genomic modifications at the targeted locus by the nuclease.
  • ON-target genotoxicity includes insertion/deletion of a few bases to megabase-scale rearrangements (“ON-target megabase- scale genotoxicity”).
  • the method of the present invention is particularly suitable for preventing ON-target megabase-scale genotoxicity. More particularly, the method of the present invention is particularly suitable for reducing LOH frequency.
  • LOH loss of heterozygosity
  • LOH has its general meaning in the art and refers to a type of genetic abnormality in diploid organisms in which one copy of an entire gene and its surrounding chromosomal region are lost.
  • the method of the present invention is particularly suitable for reducing the nuclease-induced megabase-scale LOHs that include megabase-scale loss-of-heterozygosity with copy losses (CL-LOH) and megabase-scale copyneutral loss-of-heterozygosity (CN-LOH).
  • CL-LOH megabase-scale loss-of-heterozygosity with copy losses
  • CN-LOH megabase-scale copyneutral loss-of-heterozygosity
  • the term “reduce” or “prevent” or grammatical variations thereof means, respectively, lessening the effects or keeping the effects from occurring completely.
  • the term “reduce” means a reduction by a statically significant amount, for example, an increase of at least 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • the term “editing” refers to a type of genetic engineering in which a nucleic acid sequence is inserted, replaced, or removed from a target nucleic acid sequence, e.g., the genome of a cell, using one or more nucleases according to the present invention.
  • the nucleases create specific double-strand breaks (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) (e.g., homologous recombination).
  • HDR homology-directed repair
  • the term "editing” refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., a naturally-occurring wild type nucleic acid sequence or a naturally-occurring mutated nucleic acid sequence by introducing a change to a specific genomic target; the genomic target may include a chromosomal region, a coding polynucleotide (e.g., a gene), a promotor, a non-coding polynucleotide, or any nucleic acid sequence.
  • the changes to a nucleic acid may include deletion, addition and other changes to the nucleic acid sequence in the genome.
  • target nucleic acid sequence means a specific sequence or the complement thereof that one wishes to alter.
  • nuclease includes an enzyme that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.
  • the nuclease is a TALEN.
  • TALEN has its general meaning in the art and refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene.
  • TALENs are produced artificially by fusing a TAL effector (“TALE”) DNA binding domain, e.g., one or more TALEs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TALEs to a DNA-modifying domain, e.g., a FokI nuclease domain.
  • TALEs Transcription activator-like effects
  • TALEs can be engineered to bind any desired DNA sequence (Zhang (2011), Nature Biotech.
  • TALE Transcription activator-like effector
  • DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition.
  • TALEN TALEN
  • N nuclease
  • FokI FokI endonuclease
  • Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity (Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity (Miller et al. (2011) Nature Biotech. 29: 143-8).
  • TALEN can be used inside a cell to produce a double-strand break in a target nucleic acid, e.g., a site within a gene.
  • a mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining (Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16). For example, improper repair may introduce a frame shift mutation.
  • foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene via the homologous direct repair pathway, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene.
  • homologous direct repair pathway e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene.
  • the nuclease of the present invention is a Zinc-Finger Nuclease (ZFN).
  • ZFN Zinc-Finger Nuclease
  • a ZFN comprises a DNA-modifying domain, e.g., a nuclease domain, e.g., a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain.
  • the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers (Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160).
  • a zinc finger is a small protein structural motif stabilized by one or more zinc ions.
  • a zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence.
  • Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences.
  • Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art (Sera (2002), Biochemistry, 41 :7074-7081; Liu (2008) Bioinformatics, 24: 1850-1857).
  • a ZFN using a FokI nuclease domain or other dimeric nuclease domain functions as a dimer.
  • a pair of ZFNs are required to target non-palindromic DNA sites.
  • the two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5).
  • a ZFN can create a DSB in the DNA, which can create a frame-shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell.
  • the nuclease is a CRISPR/Cas nuclease.
  • CRISPR/Cas nuclease has its general meaning in the art and refers to segments of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes.
  • CRISPR/Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements.
  • CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA).
  • the CRISPR/Cas nucleases Cas9 and Cpfl belong to the type II and type V CRISPR/Cas system and have strong endonuclease activity to cut target DNA.
  • Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a trans-activating small RNA (tracrRNA) that also serves as a guide for ribonuclease Ill-aided processing of pre-crRNA.
  • the crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA.
  • Cas9 recognizes a trinucleotide (NGG for S. Pyogenes Cas9) protospacer adjacent motif (PAM) to specify the cut site (the 3 rd or the 4 th nucleotide upstream from PAM).
  • NVG trinucleotide
  • PAM protospacer adjacent motif
  • the CRISPR/Cas nuclease is the Cas9.
  • the term “Cas9” or “Cas9 endonuclease” refers to a CRISPR- associated protein with two nuclease domains that uses a crRNA:tracRNA duplex for sitespecific double- stranded cleavage of DNA.
  • the terms may refer to a wild-type Cas9 protein, or any variant, including mutants, homologs, orthologs, that mediate RNA-guided double-stranded or single- stranded cleavage of DNA.
  • Cas9 refers wild type Cas9 from Streptococcus pyogenes (NCBI Reference Sequence:NC_017053.1), Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria.
  • NCBI Refs NC_016782.1, NC_016786.1
  • Spiroplasma syrphidicola NC 021284.1
  • Prevotella intermedia NCBI Ref: NC_017861.1
  • Spiroplasma taiwanense NCBI Ref: NC_021846.1
  • Streptococcus iniae NCBI Ref: NC_021314.1
  • Belliella baltica NCBI Ref: NC_018010.1
  • Psychroflexus torquisl NCBI Ref: NC 018721.1
  • Streptococcus thermophilus NCBI Ref: YP 820832.1
  • Listeria innocua NCBI Ref: NP_472073.1
  • Campylobacter jejuni NCBI Ref: YP_002344900.1
  • Neisseria, meningitidis NCBI Ref: YP_002342100.1
  • the nuclease involves the use of one or more guide RNA(s).
  • guide RNA or “gRNA” has its general meaning in the art and is a particular type of guide nucleic acid which associates with a CRISPR/Cas nuclease (e.g. Cas9), directing the nuclease to a specific sequence in a DNA molecule that includes complementarity to protospacer sequence of the guide RNA.
  • CRISPR/Cas nuclease e.g. Cas9
  • a further object of the present invention relates to a method of altering a target sequence of a nucleic acid molecule in a population of cells for which a G0/G1 phase cell cycle arrest is induced that comprises the step consisting in contacting the target nucleic acid sequence of said population of cells with (a) the nuclease and (b) one or more guide RNA(s).
  • the method of the present invention is used to alter a target polynucleotide sequence of interest in the population of cells for any purpose.
  • the population of cells can be a population of cells isolated from any multicellular organism, e.g., a plant cell (e.g., a rice cell, a wheat cell, a tomato cell, an Arabidopsis thaliana cell, a Zea mays cell, and the like), a cell from a multicellular protist, a cell from a multicellular fungus, an animal cell such as a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.) or a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal, etc.), a cell from a human, a cell from a healthy human, a cell from a human patient, a cell from a cancer patient, etc.
  • the cell with induced gene regulation can be transplanted to a subject (e.g., patient).
  • the cell can be derived from the subject
  • the cell is an eukaryotic cell.
  • a stem cell e.g., embryonic stem cell, induced pluripotent stem cell, adult stem cell (e.g., mesenchymal stem cell, neural stem cell, hematopoietic stem cell, organ stem cell), a progenitor cell, a somatic cell (e.g., fibroblast, hepatocyte, heart cell, liver cell, pancreatic cell, muscle cell, skin cell, blood cell, neural cell, immune cell), and any other cell of the body, e.g., human body.
  • a stem cell e.g., embryonic stem cell, induced pluripotent stem cell
  • adult stem cell e.g., mesenchymal stem cell, neural stem cell, hematopoietic stem cell, organ stem cell
  • a progenitor cell e.g., fibroblast, hepatocyte, heart cell, liver cell, pancreatic cell, muscle cell, skin cell, blood cell, neural cell, immune cell
  • the cells can be primary cells or eukaryotic cell cultures derived from a subject, e.g., an animal subject or a human subject, and allowed to grow in vitro for a limited number of passages.
  • the cells are disease cells or derived from a subject with a disease.
  • the cells can be cancer or tumor cells.
  • the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)). More preferably the eukaryotic cell is a hematopoietic stem cell.
  • HSCs hematopoietic stem cells
  • ES embryonic stem cells
  • iPS induced pluripotent stem cells
  • the cells are p53 -proficient cells or p53-deficient cells.
  • p53 has its general meaning in the art and refers the tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The p53 protein responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism.
  • p53-proficient cell refers to a cell has a "wild type TP53 gene" i.e. a gene encoding p53 protein which does not harbor a p53 dominant negative mutation.
  • p53-deficient cells are typically characterized by the presence of at least one p53 dominant negative mutation.
  • p53 dominant negative mutation has its general meaning in the art and refers to any mutation which results in to a dysfunction of the protein leading to the loss of its transcriptional activity associated with a negative effect on the wild type protein in heterozygous status.
  • p53 loss of function mutations have fully been exemplified in the prior art and thus the skilled man in the art can easily identifies p53 dominant negative mutations (Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M.
  • p53 dominant negative mutations include somatic and germline mutations indentified in previous studies are mainly missense mutations. Examples of p53 dominant negative mutations include but are not limited to p.R175H, p.R248W and p.R273H.
  • the target polynucleotide sequence of interest in the eukaryotic cell is used to generate a mutate cell, which results in a genotype that differs from its original genotype.
  • the target polynucleotide sequence of interest in the eukaryotic cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell).
  • the target polynucleotide sequence of interest in the eukaryotic cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).
  • the alteration may be a homozygous alteration or a heterozygous alternation.
  • the alteration may be an insertion, deletion, or the combination thereof. As will be appreciated by those skilled in the art, an insertion/deletion in a coding region of a genomic sequence will result in a frameshift mutation or a premature stop codon.
  • the alteration may be a point mutation. In some embodiments, the alteration consists in introducing a plurality of point mutations.
  • the method of the present invention of the present invention is used to generate a knock-out of a target polynucleotide sequence.
  • the knocking out of a selected polynucleotide sequence can be useful for many applications, such as knocking out a target polynucleotide sequence of interest in the eukaryotic cell clone in vitro for research purposes; and knocking out a target polynucleotide sequence ex vivo for treating or preventing a disorder associated with increased expression of the target polynucleotide sequence.
  • the term "knock out” includes deleting all or a portion of the target polynucleotide sequence in a way that mutes the function of the target polynucleotide sequence.
  • the alternation may result in a change of the target polynucleotide sequence of interest from an undesired sequence to a desired sequence.
  • the method of the present invention is used to correct any type of mutation or error in a target polynucleotide sequence of interest, including but not limited to inserting a nucleotide sequence that is missing from a target polynucleotide sequence due to a deletion, deleting a nucleotide sequence from a target polynucleotide sequence due to an insertion mutation, and replacing an incorrect nucleotide sequence with a correct nucleotide sequence.
  • the alteration results in reduced or increased expression of a target polynucleotide sequence of interest.
  • the term “cell cycle” has its general meaning and refers to the biological process through which cells replicate and make two new cells.
  • Cell cycle has different stages called GO, Gl, S, G2, and M. Resting cells that are not dividing are in a phase called GO.
  • Gl, S, G2, and M mitosis
  • These periods of activity are separated by regulatory checkpoints at major transition phases. These checkpoints include G0/G1 which regulates the entry of a quiescent cell back into the cycle, Gl/S, and G2/M.
  • G0/G1 phase cell cycle arrest indicates that population of cells cannot undergo cell division and are blocked in the G0/G1 phase of the cell cycle.
  • the G0/G1 phase cell cycle arrest is induced by any agent that selectively induces Gl cell cycle arrest.
  • the percentage of cells in the Gl phase increase, while the percentage of cells in the G2/M phase and S phase decrease.
  • the agent is a compound that induces substantially pure (i.e., “clean”) G1 cell cycle arrest in the population of cells (e.g., wherein treatment with the agent induces cell cycle arrest such that the majority of cells are arrested in G1 as defined by standard methods (e.g., propidium iodide staining or others) and with the population of cells in the G2/M and S phases combined being 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1% or less of the total cell population).
  • cleaning substantially pure
  • the term “selective CDK4/6 inhibitor compound” refers to a compound that selectively inhibits at least one of CDK4 and CDK6 or whose predominant mode of action is through inhibition of CDK4 and/or CDK6.
  • the term “cyclin- dependent kinase 4” or “CDK4” has its general meaning in the art and refers the cell division protein kinase 4 that is an enzyme that in humans is encoded by the CDK4 gene.
  • the term “cyclin-dependent kinase 6” or “CDK6” has its general meaning in the art and refers the cell division protein kinase 6 that is an enzyme that in humans is encoded by the CDK6 gene.
  • selective CDK4/6 inhibitors are compounds that generally have a lower 50% inhibitory concentration (IC50) for CDK4 and/or CDK6 than for other kinases.
  • the selective CDK4/6 inhibitor can have an IC50 for CDK4 or CDK6 that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower than the compound's IC50s for other CDKs (e.g., CDK1 and CDK2).
  • the selective CDK4/6 inhibitor can have an IC50 for CDK4 or CDK6 that is at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 times lower than the compound's IC50s for other CDKs.
  • the selective CDK4/6 inhibitor can have an IC50 that is more than 100 times or more than 1000 times less than the compound's IC50s for other CDKs.
  • the selective CDK4/6 inhibitor compound is a compound that selectively inhibits both CDK4 and CDK6.
  • the CDK4/6 inhibitor is a poor inhibitor (e.g., >1 pM in vitro IC50) of one or more tyrosine kinases.
  • the CDK4/6 inhibitor is a high potency inhibitor of serine and/or theonine kinases.
  • the CDK4/6 inhibitor is a poor CDK1 inhibitor (e.g., (e.g., >1 pM in vitro IC50).
  • the CDK4/6 inhibitor is characterized by having a 10-fold or 50-fold or 100-fold or greater relative potency for inhibiting CDK4 or CDK6 as compared to CDK1.
  • Selective CDK4/6 inhibitors that can be used according to the presently disclosed methods include any known small molecule (e.g., ⁇ 1000 Daltons, ⁇ 750 Daltons, or less than ⁇ 500 Daltons), selective CDK4/6 inhibitor, or pharmaceutically acceptable salt thereof.
  • the inhibitor is a non-naturally occurring compound (i.e., a compound not found in nature).
  • Several classes of chemical compounds have been reported as having CDK4/6 inhibitory ability (e.g., in cell free assays).
  • CDK4/6 inhibitors useful in the presently disclosed methods can include, but are not limited to, pyrido[2,3-d]pyrimidines (e.g., pyrido[2,3-d]pyrimidin-7-ones and 2-amino-6- cyano-pyrido[2,3-d]pyrimidin-4-ones), triaminopyrimidines, aryl[a]pyrrolo[3,4-d]carbazoles, nitrogen-containing heteroaryl-substituted ureas, 5-pyrimidinyl-2-aminothiazoles, benzothiadiazines, acridinethiones, and isoquinolones.
  • pyrido[2,3-d]pyrimidines e.g., pyrido[2,3-d]pyrimidin-7-ones and 2-amino-6- cyano-pyrido[2,3-d]pyrimidin-4-ones
  • triaminopyrimidines
  • the pyrido[2,3- d]pyrimidine is a pyrido[2,3-d]pyrimidinone. In some embodiments the pyrido[2,3- d]pyrimidinone is pyrido[2,3-d]pyrimidin-7-one. In some embodiments, the pyrido[2,3- d]pyrimidin-7-one is substituted by an aminoaryl or aminoheteroaryl group. In some embodiments, the pyrido[2,3-d]pyrimidin-7-one is substituted by an aminopyridine group.
  • the pyrido[2,3-d]pyrimidin-7-one is a 2-(2-pyridinyl)amino pyrido[2,3- d]pyrimidin-7-one.
  • the pyrido[2,3-d]pyrimidin-7-one compound can have a structure of Formula (II) as described in U.S. Patent Publication No. 2007/0179118 to Barvian et al., herein incorporated by reference in its entirety.
  • the pyrido[2,3- d]pyrimidine compound is 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-l-yl-pyridin-2- ylamino)-8H-pyrido-[2,3-d]pyrimidin-7-one (i.e., PD 0332991 or palbociclib) or a pharmaceutically acceptable salt thereof. See Toogood et al., J. Med. Chem., 2005, 48, 2388- 2406.
  • the pyrido[2,3-d]pyrimidinone is a 2-amino-6-cyano-pyrido[2,3- d]pyrimidin-4-ones.
  • CDK4/6 inhibitors comprising a 2-amino-6-cyano-pyrido[2,3- d]pyrimidin-4-one are described, for example, by Tu et al. See Tu et al., Bioorg. Med. Chem. Lett., 2006, 16, 3578-3581.
  • Suitable 5-pyrimidinyl-2-aminothiazole CDK4/6 inhibitors are described by Shimamura et al. See Shimamura et al., Bioorg. Med. Chem. Lett., 2006, 16, 3751- 3754.
  • Useful benzothiadiazine and acridinethiones compounds include those, for example, disclosed by Kubo et al. See Kubo et al., Clin.
  • the benzothiadiazine is substituted by one or more halo, haloaryl, or alkyl group.
  • the benzothiadiazine is selected from the group consisting of 4- (4-fluorobenzylamino)-l,2,3-benzothiadiazine-l,l -di oxide, 3-chloro-4-methyl-4H- benzofe] [ 1 ,2,4]thiadiazine- 1 , 1 -dioxide, and 3 -chloro-4-ethyl-4H-benzo[e] [ 1 ,2,4]thiadiazine- 1,1 -di oxide.
  • the acridinethione is substituted by one or more amino or alkoxy group.
  • the acridinethione is selected from the group consisting of 3-amino-10H-acridone-9-thione (3ATA), 9(10H)-acridinethione, 1,4-dimethoxy-lOH- acridine-9-thione, and 2,2 Z -diphenyldiamine-bis-[N,N z -[3-amido-N-methylamino)-10H- acridine-9-thione]] .
  • 3-amino-10H-acridone-9-thione (3ATA)
  • 9(10H)-acridinethione 1,4-dimethoxy-lOH- acridine-9-thione
  • 2,2 Z -diphenyldiamine-bis-[N,N z -[3-amido-N-methylamino)-10H- acridine-9-thione]] 2,2 Z -diphenyldiamine-bis-[N,N z -[3
  • Abemaciclib N-[5-[(4- ethylpiperazin- 1 - yl)methyl]pyridin-2-yl] -5 -fluoro-4-(7-fiuoro-2-methyl-3 -propan-2- ylbenzimidazol-5- yl)pyrimidin-2-amine
  • U.S. Pat. No. 7,855,211 incorporated herein.
  • Palbociclib (6-Acetyl-8-cyclopentyl-5 -methyl-2- ⁇ [5 -( 1 -piperazinyl)-2- pyridinyl]amino ⁇ pyrido[2,3- d]pyrimidin-7(8H)-one) is described in U.S. Pat. No. 7 208,489 incorporated herein.
  • Ribociclib (7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-l-ylpyridin-2- yl)amino]pyrrolo[2,3- d]pyrimidine-6-carboxamide) is described in U.S. Pat. Application No. 2010/0105653 incorporated herein.
  • the editing of the population of cells and the G0/G1 phase cell cycle arrest can occur simultaneously.
  • the agent directly and selectively induces the G0/G1 cell cycle arrest in cells, without the need for prolonged (e.g., 48 hour or longer) treatment with the agent prior to exposure with the nuclease.
  • Ex vivo therapy can comprise administering a composition (e.g., a cell) generated or modified outside of an organism to a subject (e.g., patient).
  • the composition e.g., a cell
  • ex vivo therapy can comprise administering a cell generated or modified outside of an organism to a subject (e.g., patient), wherein the primary cell has been cultured in vitro in accordance with the methods of the present invention.
  • the composition e.g., a cell
  • the composition can be derived from the subject (e.g., patient) to be treated by ex vivo therapy.
  • ex vivo therapy can include cell-based therapy, such as adoptive immunotherapy.
  • a further object of the present invention relates to a method of therapy in a patient in need thereof, the method comprising transplanting a therapeutically effective amount of a population of edited eukaryotic cells obtained by the methods herein disclosed.
  • a further object of the present invention relates to a method of therapy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the nuclease in combination with a therapeutically effective amount of the agent that induces the G0/G1 cell cycle arrest.
  • the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third%) drug.
  • the drugs may be administered simultaneous, separate or sequential and in any order.
  • Drugs administered in combination have biological activity in the subject to which the drugs are delivered.
  • a combination thus comprises at least two different drugs, and wherein one drug is the agent that induces the G0/G1 cell cycle arrest and wherein the other drug is the nuclease.
  • the term "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above.
  • the drugs of the present invention are administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • kits containing reagents for performing the abovedescribed methods including all component for performing the edition as described herein.
  • one or more of the reaction components e.g. the nucleases and the agent that induces the G0/G1 cell cycle arrest for the methods disclosed herein can be supplied in the form of a kit for use.
  • the kit comprises (a) at least one nuclease or a polynucleotide encoding thereof, and (b) one or more guide RNA molecules designed for guiding the nuclease(s) and (c) and one agent that induces the G0/G1 cell cycle arrest.
  • the kit can include one or more other reaction components.
  • an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate.
  • additional components of the kits include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA or nucleases or verifying the target nucleic acid's status, and buffers or culture media for the reactions.
  • the kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detection.
  • the components used can be provided in a variety of forms.
  • the components e.g., enzymes, RNAs, probes and/or primers
  • the components can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead.
  • the components when reconstituted, form a complete mixture of components for use in an assay.
  • the kits of the invention can be provided at any suitable temperature.
  • for storage of kits containing protein components or complexes thereof in a liquid it is preferred that they are provided and maintained below 0° C., preferably at or below -20° C., or otherwise in a frozen state.
  • the kits can also include packaging materials for holding the container or combination of containers.
  • kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).
  • the kits may further include instructions recorded in a tangible form for use of the components.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Cell division affects Cas9-induced LOH frequency. Fluorescent cells correspond to cells with LOH induced by CRISPR-Cas9. Cells (TP53-WT and TP53 z ) were exposed to palbociclib to synchronize cells in G0/G1 phase before and during editing. Cell cycle analysis was performed by flow cytometry to confirm synchronization. Fluorescent cell quantification after Cas9: SORCS1 editing (results normalized on fluorescent cell rate in non-synchronized cells, n>3 independent experiments, mean ⁇ SD). InDeis were quantified by sequencing and ICE. P: Palbociclib.
  • Figure 3 Low cell division rate prevents LOH targeting b-globin region in hCD34 + cells
  • a Left Schematic representation of globin region targeting in hCD34 + cells from cord blood, and H 19/IGF2/CDKN 1C imprinting center in chrl Ip.
  • Right Identification of three SNPs by allele-specific qPCR in H 19/IGF2/KCNQ1 imprinting center, telomeric to globin region in Chrl Ip.
  • FIG. 4 Synchronization of hCD34+ cells from cord blood by Palbociclib exposure during 48 hours prevents the formation of micronuclei (small DNA-containing structures isolated from the main nucleus evoking a massive DNA rearrangement called chromothripsis).
  • a Effect of palbociclib on the HSPCs cell cycle. At day 0 at day 2 without or with Ipmol palbociclib and at day 7 demonstrating the efficient blockade of CD34 + cells in G0G1 cells.
  • b Effect of palbociclib on micronuclei frequency measured by flow cytometry (In Vitro MicroFlow R ). Control without transfection or 48 hours post transfection targeting HBG1/HBG2 human promoters without or with Ipmol palbociclib exposure.
  • hFF hTERT Human foreskin fibroblasts immortalized with hTERT (hFF hTERT) used in hFAMReD system were from ATCC® (CRL 4001, BJ-5ta). They were partially invalidated for UROS and totally invalidated for TP53 using an RNP made of Cas9 protein complexed with a gRNA targeting UROS exon 4 and TP 53 exon 4, respectively.
  • hFFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM with glucose (4.5 g.L' x ), L-Glutamine (1 g.L' 1 ) and pyruvate supplemented with 20% fetal bovine serum, MEM non- essential amino acids 100X (Gibco® by ThermoFisher scientific, Carlsabad, CA, USA), 100 U/mL penicillin, and lOOpg/mL streptomycin (all from Eurobio, Courtaboeuf, France).
  • DMEM Dulbecco’s modified Eagle’s medium
  • MEM non- essential amino acids 100X Gibco® by ThermoFisher scientific, Carlsabad, CA, USA
  • penicillin 100 U/mL penicillin
  • streptomycin all from Eurobio, Courtaboeuf, France.
  • CD34 + HSPCs were isolated from the cord blood of healthy donors from Bagatelle Hospital, according to the ethical institutional review board of Bagatelle Hospital, (Maison de Sante Cypruse de Bordeaux, Talence, France) and with the mother’s informed consent. Briefly, mononuclear cells were isolated by Ficoll gradient. CD34 + cells were purified according to the manufacturer’s instructions (Human CD34-Positive Selection kit II ref 17865 from Stem Cell Technologies) and purity was analyzed by flow cytometry using PE-conjugated anti-CD34 antibody (clone 561 Biolegend (San Diego CA, USA) 25 pg/mL, lot # B2044487, 5 pL/test).
  • CD34 + cells were thawed and cultured in expansion medium consisting in Stem Span SFEM (Stem Cell Technologies) supplemented with hFlt3-L (50 ng/mL), SCF (50 ng/mL), human TPO (50 ng/mL), human IL3 (20 ng/mL) and human IL6 (10 ng/mL) (all from Peprotech), StemRegenin 1 (SRI) (1 pM) (Stem Cell Technologies), VPA valproic acid (500 pM) (Sigma-Aldrich), 100 U/mL penicillin, and lOO pg/mL streptomycin (Eurobio).
  • Stem Span SFEM Stem Span SFEM
  • hFlt3-L 50 ng/mL
  • SCF 50 ng/mL
  • human TPO 50 ng/mL
  • human IL6 10 ng/mL
  • CD34 + cells Two days after thawing, CD34 + cells were transfected with Cas9 RNP (see below). After cell tracking staining and sorting, CD34 + cells were plated in 35-mm tissue culture dishes at 50 and 1000 cells/mL with 1 mL of methylcellulose medium (Stemcell Technologies, MethoCult H4034 Optimum). After 3 weeks, individual colonies were subsequently picked from plates and washed in PBS to remove all the methylcellulose.
  • Cells were digested with proteinase K in lysis buffer (10 mmol/L Tris-Cl, pH 8.0, 50 mmol/L KC1, 2.5 mmol/L MgC12, 0.5% Tween 20, 100 mg/mL proteinase K) at 56 °C for 1 h, followed by a 10-min exposure at 95 °C.
  • lysis buffer 10 mmol/L Tris-Cl, pH 8.0, 50 mmol/L KC1, 2.5 mmol/L MgC12, 0.5% Tween 20, 100 mg/mL proteinase K
  • All cells were cultured in a standard humidified 37 °C, 5% CO2 incubator.
  • fibroblasts (murine and human) and hCD34 + cells, respectively.
  • 200,000 cells were nucleofected with 16.9 pg Cas9 RNP and 5 pM of Alt-R® Cas9 Electroporation Enhancer.
  • Alt-R® S.p.Cas9 Nuclease V3 protein (or either HiFi Cas9, Cas9 D10A or dCas9 when specified) was complexed to crRNA:tracrRNA according to the manufacturer’s instructions. Then complexes were incubated for 20 min at room temperature before electroporation.
  • Cas9 proteins and crRNA were purchased from Integrated DNA Technologies, Coralville, USA.
  • PCR products were purified with Nucleospin® Gel and PCR Clean-up (Macherey-Nagel).
  • Sanger sequencing was done on purified PCR products and sequenced by LIGHTRUN (GATC Biotech, Konstanz, Germany).
  • ICE v2 CRISPR Analysis tool
  • hFFs or hCD34 + cells were stained immediately after editing. To do so, cells were plated and stained for 3 hours with 5 pL of Cell Tracking Red Dye Kit (Abeam, reference ab269446, Cambridge, UK) per 100 pl of medium. After 3 hours of incubation in the staining medium, cells were PBS-washed three times by centrifugation at 500 g for 10 min. After 48 hours, cells were FACS-sorted to isolate 10% cell tracking hlgh and 10% cell tracking low cells and expanded (FACS Aria, BD). Comparison of initial and 48h cell tracking curves was performed using FlowJo Software (BD biosciences). hFAMReD fluorescent cell quantification and sorting.
  • dbSNP dbSNP
  • UROS DOCK1
  • MGMT Sanger sequencing to confirm ChrlOq LOH.
  • the genomic regions flanking SNPs were amplified by PCR (HotStarTaq Plus DNA polymerase, Qiagen®, Venlo, Netherlands) with adequate primers (data not shown).
  • PCR products were purified with Nucleospin® Gel and PCR Clean-up (Macherey -Nagel) and sequenced.
  • SNP genotyping was performed by real-time quantitative PCR analysis (CFX Connect device, Biorad®) of genomic DNA with a common reverse primer and two SNP allele-specific forward primers. Only curves with Ct ⁇ 37 were included for the analysis. To be considered as an SNP loss (homozygous), the profile can be only one or two curves with a delta Ct > 6.
  • Array CGH was performed on 8 x 60k oligonucleotide microarrays (Agilent Technologies, CA). DNA was labeled (cyanine 3 or cyanine 5) using the Genomic DNA ULS Labeling Kit from Agilent Technologies and hybridized onto the microarrays according to the manufacturer’s instructions (Agilent). Scanning of the microarrays was performed using a G5761 A scanner (Agilent). Data analysis was carried out with Agilent Technologies software, namely Feature Extraction for Cytogenomics V5.0 to calculate the fluorescence ratio and Agilent CytoGenomics 5.2 to visualize chromosomal imbalances.
  • Deletions and duplications in the heterozygous state were characterized by values of the log2 ratio of fluorescence intensities (cyanine5/cyanine3) below -0.5 and above + 0.3, respectively, with the statistical algorithm ADM2 used at a threshold of 5.
  • Combined SNP/CGH array was performed on Genetisure Cyto 180 K CGH/SNP arrays (Agilent Technologies, Santa Clara, USA). DNA was labeled (cyanine 3 or cyanine 5) using the Genomic DNA ULS Labeling Kit from Agilent Technologies and hybridized onto the microarrays according to the manufacturer’s instructions (Agilent). For SNP+ CGH arrays, tested DNAs were hybridized against male control DNA obtained from Agilent. Microarrays were scanned with a G2565CA scanner (Agilent).
  • hFAMReD relies on a cell phenotype switch, from non-fluorescent to fluorescent, induced by a megabase-scale LOH (data not shown). This switch is due to the accumulation of fluorescent red porphyrins occurring in URO S -deficient cells 22 , which is readily detectable by flow cytometry upon ALA (5-amino-levulinic acid) precursor exposure (data not shown).
  • hFFs immortalized human foreskin fibroblasts
  • RNP ribonucleoprotein
  • UROS ⁇ ' ribonucleoprotein
  • Editing efficiency was high (98% of InDeis), resulting in 86.2% of fluorescent cells.
  • FISH did not reveal a significant level of megabasic CL-LOH in edited WT (p 53 -profi ci ent) hFFs 11 .
  • FISH sensitivity may not be sufficient to detect rare deletions.
  • Targeting ABRAXAS2 (10q26.13), 1 Mb centromeric to UROS (data not shown) we observed a slight increase in the level of fluorescent cells (PE-CyA5 + : 0.09 ⁇ 0.05%) 15 days after editing, suggesting the occurrence of LOH at the UROS locus.
  • CRISPR-Cas9 nuclease induces megabase-scale LOHs in p53-proficient fibroblasts (at a rate of around one cell with extra-large rearrangement per 1000 edited cells), which is undetectable by FISH. Additionally, no significant increase in fluorescent cell numbers was evidenced using the catalytically inactive deadCas. Using the Cas9 D10A nickase, the LOH rate was not significantly different from controls (data not shown). We cannot exclude the possibility of rare LOH with single-strand breaks (SSB) induced by nickase (as observed in ref 11).
  • SSB single-strand breaks
  • clone #1 presented a terminal CL-LOH starting from the cut-site while clone #2 had a CN-LOH starting from the cut-site, with an extra-large interstitial lOq duplication detected in the bulk analysis.
  • these genomic abnormalities were not detectable by cGH or by single nucleotide polymorphism (SNP) array without cell sorting (without hFAMRed LOH enrichment, data not shown).
  • TP53 was invalidated with an RNP Cas9:gRNA targeting TP53 in the hFAMReD UROS ' hFFs, using the same RNP previously reported 11 .
  • TP53' 1 ' a loss of function variant in TP53
  • p53‘ ' fibroblasts ABRAXAS2 and SORCS1 targeting (located 1 Mb and 19 Mb centromeric to UROS, respectively) led to the occurrence of 5.6% ⁇ 0.8 and 5.56% ⁇ 0.3 of fluorescent cells, respectively (data not shown), a 60-fold increase compared to p53-proficient-hFFs.
  • p53 plays a critical role in both the Gl/S and G2/M cell cycle checkpoints 31 .
  • cell cycle is arrested by activation of these cell-cycle checkpoints to facilitate DSB repair by non-homologous end-joining (NHEJ) or homologous recombination (HR).
  • NHEJ non-homologous end-joining
  • HR homologous recombination
  • a DSB during replication (S) and mitosis (M) could be deleterious because it can trigger replication fork collapse and induce missegregation of acentric fragments 32 .
  • S replication
  • M mitosis
  • telomeric SNPs in the H 19/IGF2/KCNQ1 imprinting center 2.5 Mb telomeric to the cut-site (heterozygous SNP in parental cells).
  • allelic loss in at least two telomeric SNPs was necessary to establish the existence of a telomeric megabase-scale LOH.
  • CFC colony -forming cell
  • hFAMReD showed that megabase-LOH genotoxicity is cell-division- dependent. Importantly, we confirmed this new concept in clinically relevant cells for gene therapy protocols using CRISPR-Cas9 nuclease.
  • Palbociclib prevent the formation of micronuclei (small DNA-containing structures isolated from the main nucleus).
  • hFAMReD is able to detect the appearance of persistent events by red fluorescence and to quantify them (only after day 15) but with high sensitivity (0.02%) in a cell bulk.
  • FISH, array CGH, and SNP array without subcl oning/single cell analysis) all lack sensitivity to detect these rare rearrangements present in p53 -proficient cells.
  • nucleotide e.g. NGS, long-range PCR
  • chromosome levels e.g. CAST-seq, FAMReDs, single-cell SNP analysis
  • G0/G1 synchronized cells and low-divided cells were protected from LOHs.
  • Cells may be protected by enhanced canonical-NHEJ 38,39 .
  • the c-NHEJ repair pathway could limit LOH by joining both chromosome parts after DSB. This would be in accordance with two studies 40,41 showing that when cNHEJ is deficient, DSBs are repaired by genotoxic pathways and induce kilobasic rearrangements.
  • a low proliferation rate strongly reduces the occurrence of LOHs without compromising NHEJ editing efficiency (in two human cell models, targeting two different chromosomes and using two methods to quantify LOH).
  • FAMReD offer another advantage in the possibility of isolating cells with stable LOHs for in-depth analysis.
  • CL-LOH LOH
  • CN-LOH CN-LOH
  • the rearrangement type profile was distinct when targeting different loci. In all cases, CN-LOH were predominant.
  • CL-LOH was only observed in the event of DSB at the closest target to the telomere (8.5 Mb).
  • the rearrangement type profile was not modified by TP53 invalidation for a defined locus.
  • CN-LOHs which are not detectable by FISH or array CGH, have a functional impact, depending on the genes in the chromosomal region of interest. These CN-LOH are probably due to a loss of chromosome extremity and to a secondary duplication of the remaining allele by break-induced replication (BIR) 45 to avoid CNV. These large LOHs could contribute to tumorigenesis by activating potential oncogenes or by unmasking mutated tumor suppressor genes. In this study, LOH was sometimes associated with centromeric extra-large duplication, again illustrating the diversity of ON-target rearrangements induced by CRISPR-Cas9 nuclease.
  • Vassilev LT et al. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci U S A. 103, 10660-10665 (2006).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Le système CRISPR-Cas9 a révolutionné notre aptitude à modifier avec précision le génome et présente une édition de gènes poussée dans des applications cliniques. Une analyse complète de produits d'édition de gènes au niveau du site de coupe ciblé a révélé un spectre complexe de résultats. Une génotoxicité sur cible est sous-estimée avec des procédés basés sur la PCR standard et nécessite des procédés de détection appropriés et sensibles. Ici, les inventeurs ont développé deux systèmes de détection de réarrangements à l'échelle de la mégabase assistés par fluorescence (FAMReD) complémentaires qui garantissent la détection, la quantification et le tri cellulaire de cellules éditées avec une perte d'hétérozygosité (LOH) à l'échelle de la mégabase. Ils ont révélé des réarrangements chromosomiques complexes rares provoqués par la nucléase Cas9 et ont montré que la fréquence LOH dépend du taux de division cellulaire lors de l'édition et de l'état p53. L'arrêt du cycle cellulaire lors de l'édition a supprimé l'apparition de LOH sans compromettre l'édition. Ces données ont été confirmées dans des cellules souches/progénitrices humaines, suggérant que des essais cliniques doivent tenir compte de l'état p53 et du taux de prolifération cellulaire lors de l'édition pour limiter ce risque et concevoir des protocoles plus sûrs.
PCT/EP2024/062916 2023-05-12 2024-05-10 Procédés de prévention de la génotoxicité sur cible induite par des nucléases WO2024235844A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23305760.3 2023-05-12
EP23305760 2023-05-12

Publications (1)

Publication Number Publication Date
WO2024235844A1 true WO2024235844A1 (fr) 2024-11-21

Family

ID=86760357

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/062916 WO2024235844A1 (fr) 2023-05-12 2024-05-10 Procédés de prévention de la génotoxicité sur cible induite par des nucléases

Country Status (1)

Country Link
WO (1) WO2024235844A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040006074A1 (en) 1998-04-28 2004-01-08 The Government Of The United States Of America Cyclin dependent kinase (CDK)4 inhibitors and their use for treating cancer
US7208489B2 (en) 2002-01-22 2007-04-24 Warner-Lambert Company 2-(pyridin-2-ylamino)-pyrido [2,3-d]pyrimidin-7-ones
US20100105653A1 (en) 2008-08-22 2010-04-29 Novartis Ag Pyrrolopyrimidine compounds and their uses
US7855211B2 (en) 2008-12-22 2010-12-21 Eli Lilly And Company Protein kinase inhibitors
US20180298392A1 (en) * 2014-11-07 2018-10-18 Editas Medicine, Inc. Methods for improving crispr/cas-mediated genome-editing
US20210371855A1 (en) * 2018-06-15 2021-12-02 Ideaya Biosciences, Inc. Methods of inhibiting proliferative cells

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040006074A1 (en) 1998-04-28 2004-01-08 The Government Of The United States Of America Cyclin dependent kinase (CDK)4 inhibitors and their use for treating cancer
US7208489B2 (en) 2002-01-22 2007-04-24 Warner-Lambert Company 2-(pyridin-2-ylamino)-pyrido [2,3-d]pyrimidin-7-ones
US20070179118A1 (en) 2002-01-22 2007-08-02 Warner-Lambert Company 2-(pyridin-2-ylamino)-pyrido [2,3 d]pyrimidin-7-ones
US20100105653A1 (en) 2008-08-22 2010-04-29 Novartis Ag Pyrrolopyrimidine compounds and their uses
US7855211B2 (en) 2008-12-22 2010-12-21 Eli Lilly And Company Protein kinase inhibitors
US20180298392A1 (en) * 2014-11-07 2018-10-18 Editas Medicine, Inc. Methods for improving crispr/cas-mediated genome-editing
US20210371855A1 (en) * 2018-06-15 2021-12-02 Ideaya Biosciences, Inc. Methods of inhibiting proliferative cells

Non-Patent Citations (74)

* Cited by examiner, † Cited by third party
Title
ADIKUSUMA, F. ET AL.: "Large deletions induced by Cas9 cleavage.", NATURE, vol. 560, 2018, pages E8 - E9, XP036563461, DOI: 10.1038/s41586-018-0380-z
ALANIS-LOBATO, G. ET AL.: "Frequent loss of heterozygosity in CRISPR-Cas9- edited early human embryos.", PROC. NATL ACAD. SCI. USA, vol. 118, 2021, pages e2004832117
ANZALONE, A. V.KOBLAN, L. W.LIU, D. R.: "Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors.", NAT. BIOTECHNOL., vol. 38, 2020, pages 824 - 844, XP037622140, DOI: 10.1038/s41587-020-0561-9
ARLT, M.F.: "Replication stress induces genome-wide copy number changes in human cells that resemble polymorphic and pathogenic variants", AM J HUM GENET., vol. 84, 2009, pages 339 - 350
BITINAITE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, 1998, pages 10570 - 5
BOUTIN JULIAN ET AL: "ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected", vol. 5, no. 1, 1 February 2022 (2022-02-01), pages 19 - 30, XP093082198, ISSN: 2573-1599, Retrieved from the Internet <URL:http://dx.doi.org/10.1089/crispr.2021.0120> DOI: 10.1089/crispr.2021.0120 *
BOUTIN, J. ET AL.: "CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.", NAT COMMUN., vol. 12, 2021, pages 4922
BOUTIN, J. ET AL.: "ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected", CRISPR J., vol. 5, 2022, pages 19 - 30, XP093082198, DOI: 10.1089/crispr.2021.0120
CARROLL ET AL., GENETICS SOCIETY OF AMERICA, vol. 188, 2011, pages 773 - 782
CERMAK ET AL., NUCL. ACIDS RES., vol. 39, 2011, pages e82
CONG, L. ET AL.: "Multiplex genome engineering using CRISPR/Cas systems.", SCIENCE, vol. 339, 2013, pages 819 - 823, XP055871219, DOI: 10.1126/science.1231143
CULLOT G. ET AL: "Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9", vol. 14, no. 1, 10 July 2023 (2023-07-10), XP093082195, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-023-39632-w> DOI: 10.1038/s41467-023-39632-w *
CULLOT, G. ET AL.: "CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.", NAT. COMMUN., vol. 10, 2019, pages 1136
DAVIS, L. ET AL.: "POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.", PROC. NATL ACAD. SCI. USA, vol. 15, 2020, pages 22900 - 22909
DOUDNA, J. A.CHARPENTIER, E.: "Genome editing. The new frontier of genome engineering with CRISPR-Cas9", SCIENCE, vol. 346, 2014, pages 1258096, XP055162699, DOI: 10.1126/science.1258096
DOYON ET AL., NATURE METHODS, vol. 8, 2010, pages 74 - 79
ENACHE, O.M. ET AL.: "Cas9 activates the p53 pathway and selects for p53-inactivating mutations.", NAT GENET., vol. 52, 2020, pages 662 - 668, XP037525537, DOI: 10.1038/s41588-020-0623-4
GUO ET AL., J. MOL. BIOL., vol. 200, 2010, pages 96
HAAPANIEMI, E. ET AL.: "CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.", NAT. MED., vol. 24, 2018, pages 927 - 930, XP036542072, DOI: 10.1038/s41591-018-0049-z
HALAZONETIS TDGORGOULIS VGBARTEK J.: "An oncogene-induced DNA damage model for cancer development.", SCIENCE, vol. 319, 2008, pages 1352 - 1355
HER, J.BUNTING, S.F.: "How cells ensure correct repair of DNA double-strand breaks.", J BIOL CHEM., vol. 293, 2018, pages 10502 - 10511, XP055830556, DOI: 10.1074/jbc.TM118.000371
HERMETZ, K.E. ET AL.: "Large inverted duplications in the human genome form via a fold-back mechanism", PLOS GENET., vol. 10, 2014, pages e1004139
HOCKEMEYER ET AL., NATURE BIOTECH., vol. 29, 2011, pages 731 - 734
HUERTAS, P., NAT. STRUCT. MOL. BIOL., vol. 17, 2010, pages 11 - 16
IHRY, R. ET AL.: "p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.", NAT. MED., vol. 24, 2018, pages 939 - 946, XP036542073, DOI: 10.1038/s41591-018-0050-6
IKEDA, K. ET AL.: "Efficient scarless genome editing in human pluripotent stem cells.", NAT METHODS., vol. 15, 2018, pages 1045 - 1047, XP036648015, DOI: 10.1038/s41592-018-0212-y
JASIN, M.HABER, J.E.: "The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair", DNA REPAIR (AMST)., vol. 44, 2016, pages 6 - 16, XP029645039, DOI: 10.1016/j.dnarep.2016.05.001
JENNIFER HOLLYWOOD: "Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools", 1 January 2013 (2013-01-01), pages 1 - 193, XP055209264, Retrieved from the Internet <URL:https://cora.ucc.ie/bitstream/handle/10468/1407/JH thesis.pdf?sequence=3> [retrieved on 20150824] *
JIANG, L.: "CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies.", CANCER RES., vol. 82, 2022, pages 36 - 45
KIM, PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 1156 - 1160
KNOPP YVONNE ET AL: "Transient retrovirus-based CRISPR/Cas9 All-in-One Particles for efficient, targeted gene knock-out", MOLECULAR THERAPY-NUCLEIC ACIDS, CELL PRESS, US, vol. 13, 1 January 2018 (2018-01-01), pages 256 - 274, XP002806692, ISSN: 2162-2531, DOI: 10.1016/J.OMTN.2018.09.006 PUBLISHER: *
KONDRATOVA, A.: "Replication fork integrity and intra-S phase checkpoint suppress gene amplification.", NUCLEIC ACIDS RES., vol. 43, 2015, pages 2678 - 2690
KOSICKI, M. ET AL.: "Cas9-induced large deletions and small InDels are controlled in a convergent fashion.", NAT COMMUN., vol. 13, 2022, pages 3422
KOSICKI, M.TOMBERG, K.BRADLEY, A.: "Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.", NAT. BIOTECHNOL., vol. 36, 2018, pages 765 - 771, XP036929645, DOI: 10.1038/nbt.4192
KRAMARA, J.OSIA, B.MALKOVA, A.: "Break-induced replication: the where, the why, and the how.", TRENDS GENET., vol. 34, 2018, pages 518 - 531
KUBO ET AL., CLIN. CANCER RES., vol. 5, 1999, pages 4279 - 4286
LEIBOWITZ, M. L. ET AL.: "Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.", NAT. GENET., vol. 53, 2021, pages 895 - 905, XP037475892, DOI: 10.1038/s41588-021-00838-7
LEVINE, A.J.: "p53: 800 million years of evolution and 40 years of discovery.", NAT REV CANCER, vol. 20, 2020, pages 471 - 480, XP037201231, DOI: 10.1038/s41568-020-0262-1
LEVINE, M.S.HOLLAND, A.J.: "The impact of mitotic errors on cell proliferation and tumorigenesis", GENES DEV., vol. 32, 2018, pages 620 - 638
LIU, BIOINFORMATICS, vol. 24, 2008, pages 1850 - 1857
MACIEJOWSKI, J. ET AL.: "Chromothripsis and Kataegis Induced by Telomere Crisis.", CELL, vol. 163, 2015, pages 1641 - 1654, XP029360515, DOI: 10.1016/j.cell.2015.11.054
MIYAHARA: "Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9", HUMAN GENE THERAPY, vol. 28, no. 12, 1 December 2017 (2017-12-01), GB, pages A36, XP055742223, ISSN: 1043-0342, DOI: 10.1089/hum.2017.29055.abstracts *
MOSCOU ET AL., SCIENCE, vol. 326, 2009, pages 3501 - 12
MURNANE, J.P: "Telomere dysfunction and chromosome instability", MUTAT RES., vol. 730, 2012, pages 28 - 36, XP028436148, DOI: 10.1016/j.mrfmmm.2011.04.008
NAHMAD, A.D. ET AL.: "Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage.", NAT BIOTECHNOL., vol. 12, 2022, pages 1807 - 1813
PAPATHANASIOU, S. ET AL.: "Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing.", NAT. COMMUN., vol. 12, 2021, pages 5855
PESCH, A.M. ET AL.: "Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers", CLIN CANCER RES., vol. 26, 2020, pages 6568 - 6580
PETITJEAN AMATHE EKATO SISHIOKA CTAVTIGIAN SVHAINAUT POLIVIER M.: "Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database", HUM MUTAT., vol. 28, no. 6, June 2007 (2007-06-01), pages 622 - 9, XP071974067, Retrieved from the Internet <URL:http://p53.iarc.fr> DOI: 10.1002/humu.20495
PUY, H.GOUYA, L.DEYBACH, J.C.: "Porphyrias.", LANCET., vol. 375, 2010, pages 924 - 937, XP026978225, DOI: 10.1016/S0140-6736(09)61925-5
RAYNER, E. ET AL.: "CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods", CRISPR J., vol. 2, 2019, pages 406 - 416
REES, H. A.LIU, D. R.: "Base editing: precision chemistry on the genome and transcriptome of living cells.", NAT. REV. GENET., vol. 19, 2018, pages 770 - 788
REZZA, A. ET AL.: "Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in", SCI REP., vol. 9, 2019, pages 3486
ROUET, P.SMIH, F.JASIN, M.: "Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.", PROC. NATL ACAD. SCI. USA, vol. 91, 1994, pages 6064 - 6068, XP002005205, DOI: 10.1073/pnas.91.13.6064
ROY, S. ET AL.: "p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLO pathways", ELIFE, vol. 7, 2018, pages e31723
SCHIROLI, G. ET AL.: "Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response.", CELL STEM CELL., vol. 24, 2019, pages 551 - 565
SCHMIDT M.: "Palbociclib - from Bench to Bedside and Beyond", BREAST CARE., vol. 11, 2016, pages 177 - 181
SCHWARTZ DROTTER V.: "p53-dependent cell cycle control: response to genotoxic stress", SEMIN CANCER BIOL., vol. 8, 1998, pages 325 - 336
SERA, BIOCHEMISTRY, vol. 41, 2002, pages 7074 - 7081
SHIMAMURA ET AL., BIOORG. MED. CHEM. LETT, vol. 16, 2006, pages 3751 - 3754
SHIN, H. Y. ET AL.: "CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome.", NAT. COMMUN., vol. 8, 2017, pages 15464, XP055821911, DOI: 10.1038/ncomms15464
SZCZEPEK ET AL., NATURE BIOTECH., vol. 25, 2007, pages 786 - 793
TOOGOOD ET AL., J. MED. CHEM., vol. 48, 2005, pages 2388 - 2406
TRAN, N.T.: "Precise CRISPR-Cas-mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells", SCI ADV., vol. 8, 2022, pages eabm9106
TU ET AL., BIOORG. MED. CHEM. LETT., vol. 16, 2006, pages 3578 - 3581
VAKULSKAS, C.A.: "Behlke, M.A. Evaluation and Reduction of CRISPR Off-Target Cleavage Events.", NUCLEIC ACID THER., vol. 29, 2019, pages 167 - 174, XP055728865, DOI: 10.1089/nat.2019.0790
VASSILEV LT ET AL.: "Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.", PROC NATL ACAD SCI USA., vol. 103, 2006, pages 10660 - 10665, XP055590963, DOI: 10.1073/pnas.0600447103
VOUSDEN, K.H.LANE, D.P.: "p53 in health and disease.", NAT REV MOL CELL BIOL., vol. 8, 2007, pages 275 - 283
WEISHEIT, I. ET AL.: "Detection of deleterious on-target effects after HDR mediated CRISPR editing", CELL REP., vol. 26, 2020, pages 107689
WEN WEI ET AL: "CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells", EXPERIMENTAL HEMATOLOGY, ELSEVIER INC, US, vol. 110, 15 March 2022 (2022-03-15), pages 13 - 19, XP087060191, ISSN: 0301-472X, [retrieved on 20220315], DOI: 10.1016/J.EXPHEM.2022.03.002 *
WOOD ET AL., SCIENCE, vol. 333, 2011, pages 307
ZHOU B.P. ET AL.: "Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells.", NAT CELL BIOL., vol. 3, 2001, pages 245 - 252
ZHOU B.P. ET AL.: "HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.", NAT. CELL. BIOL., vol. 11, 2001, pages 973 - 982
ZUCCARO, M. V. ET AL.: "Allele-specific chromosome removal after Cas9 cleavage in human embryos.", CELL, vol. 29, 2020, pages 31389 - 31391
ZUO, E. ET AL.: "CRISPR/Cas9-mediated targeted chromosome elimination", GENOME BIOL., vol. 18, 2017, pages 224, XP055943547, DOI: 10.1186/s13059-017-1354-4

Similar Documents

Publication Publication Date Title
Cullot et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
JP6993063B2 (ja) ゲノムエンジニアリング
US10557151B2 (en) Somatic human cell line mutations
Li et al. Base pair editing in goat: nonsense codon introgression into FGF 5 results in longer hair
JP2020510443A (ja) 細胞ゲノムにおける、相同組換え修復（ｈｄｒ）の効率を上昇させるための方法
Dan et al. Genetic studies on mammalian DNA methyltransferases
Yue et al. Xist RNA repeat E is essential for ASH2L recruitment to the inactive X and regulates histone modifications and escape gene expression
JP2022512773A (ja) 加齢関連クローン性造血およびそれに関係する疾患の予防
US20190330698A1 (en) Diabetes polygenic risk score
Acosta et al. Use of two gRNAs for CRISPR/Cas9 improves bi‐allelic homologous recombination efficiency in mouse embryonic stem cells
WO2019111258A1 (fr) Traitement de patients parkinsoniens avec des mutations dans le gène lrrk2
WO2019003193A1 (fr) Méthodes pour le traitement d&#39;une maladie à l&#39;aide de systèmes d&#39;édition de gènes
Scott et al. Targeted genome regulation and modification using transcription activator‐like effectors
Karagyaur et al. Practical recommendations for improving efficiency and accuracy of the CRISPR/Cas9 genome editing system
JPWO2019017321A1 (ja) 遺伝子変異導入方法
WO2019194751A1 (fr) Utilisation d&#39;une population de cellules souches mésenchymateuses essentiellement pures de la membrane amniotique du cordon ombilical pour générer une cellule souche mammifère portant un transgène
Ray et al. Genome editing with CRISPR-Cas9: A budding biological contrivance for colorectal carcinoma research and its perspective in molecular medicine
Sargent et al. Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs)
Yao et al. Epigenetic regulation of bovine spermatogenic cell-specific gene boule
Do et al. Zinc finger nuclease induced DNA double stranded breaks and rearrangements in MLL
US20190341125A1 (en) Inflammatory bowel disease polygenic risk score
WO2024235844A1 (fr) Procédés de prévention de la génotoxicité sur cible induite par des nucléases
JP2022553855A (ja) 長さが２１～３０ヌクレオチドのガイド配列を使用したヘテロ接合ｅｌａｎｅ遺伝子の対立遺伝子のさまざまなノックアウト
US20250102493A1 (en) Compositions and methods for treatment of aneuploid cancers
Umbach CRISPR-Cas strategies for the correction of Cornelia de Lange Syndrome and Cystic Fibrosis mutations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24723913

Country of ref document: EP

Kind code of ref document: A1