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WO2016166268A1 - Engineering animal or plant genome using dna-guided argonaute interference systems (dais) from mesophilic prokaryotes - Google Patents

Engineering animal or plant genome using dna-guided argonaute interference systems (dais) from mesophilic prokaryotes Download PDF

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WO2016166268A1
WO2016166268A1 PCT/EP2016/058320 EP2016058320W WO2016166268A1 WO 2016166268 A1 WO2016166268 A1 WO 2016166268A1 EP 2016058320 W EP2016058320 W EP 2016058320W WO 2016166268 A1 WO2016166268 A1 WO 2016166268A1
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cell
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Julien Valton
Philippe Duchateau
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Cellectis SA
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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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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

Definitions

  • DAIS DNA-guided Argonaute interference systems
  • This patent application relates to materials and methods for gene editing in mammalian cells, and more particularly to methods for gene editing using DNA-guided Argonaute (Ago) interference systems (DAIS) from mesophilic prokaryotes in animal cells such as T-cells or in plant cell.
  • Ago DNA-guided Argonaute
  • DAIS DNA-guided Argonaute interference systems
  • Argonaute proteins from bacteria such as Thermus thermophilus (strain HB27) have been recently described in bacteria to act as a barrier for the uptake and propogation of foreign DNA (Swarts D.C, et al. Nature 507: 258-261 )
  • Tt Ago is loaded with 5' phosphorylated DNA guides, from 13 to 25 base pairs that are mostly plasmid derived and have a strong bias for a 5'-end deoxycytidine.
  • the argonaute (Ago) gene family generally encodes proteins comprising four characteristic domains: N- terminal, PAZ, Mid and a C-terminal catalytic domain referred to as PIWI domain (Meister et al., Molecular Cell 15 (2): 185-197).
  • Ago proteins refer to any heterologous polypeptide or polynucleotide sequence comprising at least such PIWI domain sequence.
  • Multiple sequence alignment of core motifs of PIWI domains indicate an active site comprising the motif (D/E)-(D/E)XK, X being any standard amino acid.
  • the PIWI domain is believed to contribute to recognition of base pairing with double stranded nucleic acids.
  • the small interfering DNAs guide TtAgo cleave complementary DNA strands at high temperature (75°C).
  • T-cells are mammalian cells known to be very sensitive to foreign DNA and refractory to DNA transfection.
  • thermophilus isolated from diverse species other than Thermus thermophilus which are not thermophile could be heterologously expressed in mammalian cells.
  • These Ago proteins have the particularity, and can be optimized, to be active in a wide temperature range, including a temperature at around 37°C and below, allowing their use in different function (such as gene editing), in animal cell as well as in plant cell. Based on this finding they have set up in particular a strategy of gene editing using DNA-guided Ago to engineer T-cells suitable for immunotherapy.
  • DAIS DNA-guided Argonaute interference system
  • DAIS can be used for targeted mutagenesis, targeted chromosomal deletions, targeted gene inversion, translocation or insertion and for multiplexed genome modifications.
  • Such technology can be used to engineer living cells for specific applications such as cellular immunotherapy, gene therapy, generation of genetically modified animals, as well as cells for bioproduction as non-limiting examples.
  • This DAIS by using appropriate Ago proteins from mesophilic prokaryotes also allows targeted modifications of genomic DNA in plant cell.
  • this document presents a method for modifying the genomic material of mammalian cells, especially T-cells.
  • the method includes introducing one, two or multiple short DNA molecules (referred herein as DNA-guides) into the mammalian cells, along with the prokaryotic DAIS that is known to catalyze single strand DNA break at the sequences targeted by DNA-guide.
  • the DAIS can be delivered as DNA, mRNA and purified apo or holo protein (prebound to DNA guide) or via lentivirus.
  • the DAIS coding sequence can be regulated by a constitutive or inducible promoter.
  • the mammalian cells may be primary or immortalized cells, somatic or stem cells including induced Pluripotent Stem Cells (iPSC).
  • Figure 1 Schematic representation of the method for inducing double strand cleavage in a nucleic acid target sequence according to the present invention through the heterologous expression of Ago from a mesophilic prokaryote in a cell in the presence of oligonucleotides that act as specific guides to the selected locus.
  • the 2 cleavage events presented in this figure can be performed in a sequentially or concomitantly manner.
  • Figure 2 Schematic representation of the method according to the invention using distant cleavage sites, which may either lead to a significant deletion of the locus region (Nx) between the two sites or to a cohesive end cleavage profile.
  • Figure 3 Schematic representation showing strategy to inactivate TCR locus in T-cells.
  • Figure 4 Schematic representation showing strategy to modify TCR locus in T-cells by homologous recombination using an insertion matrix (donor DNA).
  • Figure 5 Use a chimeric fusion of Ago from mesophilic prokaryote in which the catalytic domain is inactivated by replacement of several amino acids to a catalytic domain from an enzyme involved for instance in gene editing.
  • This DAIS can be used along with one or multiple DNA guide and coupled to a catalytic domain from another enzyme (ex: nucleases such as FOKI, iTevl, meganuclease; transcription activator such as VP64); to the locus of interest.
  • the catalytic head may be used in various ways such as cutting, methylation, phosphorylation or inactivation.
  • Figure 6 Schematic representation of the method according to the invention using the DAIS presented in Figure 5 (with an Ago catalytically dead from mesophilic prokaryote coupled to, here, FOKI). This may either lead to a significant deletion of the locus region (Nx) between the two sites or to a cohesive end cleavage profile.
  • Figure 7 Amino acid alignment obtained using BLAST on PIWI catalytic domains from diverse mesophilic prokaryotes listed in Table 1. In particular, this alignment shows the conserved amino acids for all these species, and allows the identification of several to be mutated in order to inactivate the catalytic domain. DESCRIPTION
  • the present invention broadly relates to a method of modifying the genetic material of n animal cell or in plant cell, through the expression of an Ago protein or an Ago protein variant from mesophilic prokaryote into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus.
  • DNA guide exogenous oligonucleotide
  • Ago protein variant in the present invention an Ago protein from mesophilic prokaryote in which at least one amino acid is being replaced by another one.
  • said one or several amino acids are essential in the catalytic function. Therefore, when these amino acids are mutated, the catalytic function is inactivated or also called “catalytically dead”.
  • one or several amino acids may be changed to optimized Ago proteins in their performances at a specific range of temperatures.
  • the Ago protein or the Ago protein variant from mesophilic prokaryote has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the Ago proteins from prokaryotic bacteria T Synechococcus sp. PCC 7002 (SEQ ID NO.
  • Synechococcus elongates (SEQ ID NO.3), Synechocystis sp, (SEQ ID NO.4 ), Aromatoleum aromaticum EbN1 (SEQ ID NO.5), Pseudomonas luteola (SEQ ID NO.6), Marinospirillum minutulum (SEQ ID NO.7 ), Haloferax denitricans (SEQ ID NO.8), Halorubrum kocurii (SEQ ID NO.9), Halobacterium satilanarum (SEQ ID NO.10), Natronobacterium gregoryi (SEQ ID NO.1 1 ), Clostridium butyricum (SEQ ID NO.12), Clostridium sartagoforme (SEQ ID NO.13), Halobacterium sp. DL1 (SEQ ID NO.14), Halorubrum lacusprofundi (SEQ ID NO.15 ), and Natrialba asiatica (SEQ ID NO.16
  • Table 1 Temperature range of Ago activity for a set of mesophilic prokaryotes (identified with their ATCC number unless stated)
  • Synechocysti SEQ ID NO.4 VFGLIRKKLDDRLKESKATAKYIISQVYGIPRETEPLRV s sp DGYVMPKAKLLALNGQEVDIVAKVRYKGCAKIGETKF
  • Halorubrum EQGRDIDRLCIMRDGKISEDIDAVREGLSGIEAEIDIVGI kocurii SEQ ID NO.9 RKSGQPRIAEFDGTRFRIAEKGVGFVDADRSQSIIHAF
  • Natronobacte SEQ ID NO.1 1 HSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTT rium gregoryi YRPRRGHIVWGLRDECATDSLNTLGNQSWAYHRNN
  • the Ago protein or the Ago protein variant is chosen among Natronobacterium gregoryi, Synechococcus sp. PCC 700, Halobacterium satilanarum and Natrialba asiatica.
  • the present disclosure provides for the engineering of site-directed polypeptides to recognize a desired target nucleic acid sequence with desired enzyme specificity and/or activity. Modifications to a site-directed polypeptide can be performed through protein engineering. Protein engineering can include fusing functional domains to such engineered site-directed polypeptide which can be used to modify the functional state of the overall site-directed polypeptide or the actual target nucleic acid sequence of an endogenous cellular locus.
  • the site-directed polypeptide of the disclosure can be used to regulate endogenous gene expression, both through activation and repression of endogenous gene transcription.
  • the site-directed polypeptide-fusions can also be linked to other regulatory or functional domains, for example nucleases, transposases or methylases, to modify endogenous chromosomal sequences.
  • the site- directed polypeptide may be linked to at least one or more regulatory domains.
  • regulatory or functional domains include transcription factor repressor or activator domains such as KRAB and VP16, co-repressor and co-activator domains, DNA methyl transferases, histone acetyltransferases, histone deacetylases, and DNA cleavage domains such as the cleavage domain from the endonuclease Fokl.
  • the catalytic domain of the Ago protein from mesophilic prokaryotes is inactivated by replacement of at least one amino acid, and wherein said modified Ago protein is coupled to a catalytic domain from an enzyme having a function in gene editing, modification of gene expression or epigenetic modulation.
  • a schematic representation is given in Figure 6.
  • gene editing is means genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases.
  • the nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end-joining (NHEJ).
  • HR homologous recombination
  • NHEJ non-homologous end-joining
  • modulation of gene expression it is meant in particular enzymes which are involved as activator or repressor during the transcription process.
  • the Ago protein catalytic domain from mesophilic prokaryotic is inactivated by the mutations of the 2 aspartic acids in positions 478 and 546, and optionally the glutamic acid in position 512. These amino acids are usually changed into non-charged and short-chain amino acids such as alanine (A).
  • said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain from a type IIS restriction enzyme, and as an example, Fok-I.
  • said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain of a meganuclease such as l-Scel or l-Crel or one of their variants, or another homing endonuclease such as l-Tevl.
  • the percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (BI2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14 (Basic Local Alignment Search Tool provided by the NCBI at http://ncbi.nlm.nih.gov.) BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • Such analysis was performed using the AA sequence from the PIWI catalytic domain of the Thermophilus Ago.
  • Figure 7 shows the alignment of catalytic domain from 15 mesophilic prokaryotes.
  • Optimized Ago proteins from a mesophilic prokaryote can be derived from such a protein, or from an Ago protein from other species, by directed evolution in order to optimize its performance at a range of temperature comprised between 30°C and 40°C.
  • One method according to the invention for optimizing an Ago protein from a mesophilic prokaryote to have it induce more cleavage activity at a temperature below 40°C can comprise the steps of:
  • oligonucleotides introducing oligonucleotides into a cell , said oligonucleotides being selected to hybridize a toxic gene, resistance gene or a reporter gene present into said cell;
  • expression of said Ago protein from a mesophilic prokaryote may be placed under the control of an inducible promoter to reduce potential genotoxicity of the Ago protein into said cell.
  • the above method can further comprise the step of cultivating the cells in which cleavage by Ago from a mesophilic prokaryote has occurred at the preselected locus, recovering and isolating said cells in which cleavage by said Ago has occurred at the preselected locus.
  • the cells obtained by this method are a further object of the present invention.
  • the present invention aims more particularly to engineer immune cells, in particular T cells, and most preferably human T cells from patients or donors, for their use in immunotherapy.
  • the present invention provides with a method for modifying the genetic material of a primary T-cell, comprising at least one of the following steps:
  • DNA guide providing specificity of cleavage to said Ago protein to a preselected locus.
  • the oligonucleotide used as a DNA guide in the method according to the present invention is generally 10 to 50 nucleotides in length, preferably 15 to 30 nucleotides, more preferably 20 to 25 nucleotides, which confer a high target specificity to the method of the invention.
  • Such oligonucleotide is preferably phosphorylated at its 5' terminus, and has also preferably a CA doublet at its 5' terminus. This is believed to improve the interaction between the guide and the Ago protein.
  • At least 2 oligonucleotides are selected to respectively hybridize each strand of a double-strand DNA at sites that are closed enough to each other to obtain double strand break.
  • the 2 oligonucleotides will be designed to hybridize each strand at the same locus so that Ago from a mesophilic prokaryote will create a blunt double strand break.
  • the present invention contemplates also the possibility to target at multiple preselected loci of a particular gene (i.e. drug resistance) by using a library of DNA guides. These are synthetized beforehand and are designed to hybridize to different parts of the gene of interest.
  • This set of DNA guides are subcloned together in a plasmid bearing the polymerase such as T7 and can be transfected into a pool of cells to be genetically modified along with the Ago protein. In the cells, said DNA guides are released and can intervene in the DAIS process. Then, it will be possible to screen phenotypically each isolated cell (i.e. for instance their drug resistance).
  • the method comprises the step of performing homologous recombination at the preselected locus by bringing into the cell a donor DNA comprising a sequence homologous to that of the preselected locus into contact with said genetic material.
  • a schematic representation is given in Figure 4 using the modified TRAC locus as an example.
  • Various homologous recombination techniques have been described in the prior art, especially in US 6,528,313 and US 8,921 ,332, but so far have never been practiced using Ago proteins from mesophilic prokaryotes.
  • This method allows donor DNA comprising a transgene, a promoter, an expression cassette or a repairing sequence to be inserted at a preselected locus. Said method can be practiced in gametes and oocytes in view of obtaining cells to develop a transgenic animal.
  • repair mechanism like non homologous end joining (NHEJ) may also be used to introduce transgenes into cell genome upon cleavage by the Ago protein from a mesophilic prokaryote as per the invention.
  • NHEJ non homologous end joining
  • oligonucleotides targeting different loci can be carried out to inactivate said loci simultaneously, producing a multiplex genome engineering method.
  • Said additional loci which may be targeted in said immune cells alone or in combination, are more particularly genes that confer resistance to chemotherapy drugs (ex: fludarabine, chlorofarabine...), such as those genes encoding deoxycytidine kinase (dCk) or encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene thereby conferring resistance to 6-thioguanine (6TG) as described in PCT/EP2014/075317.
  • Resistance to lymphodepleting agents can also be achieved by inactivating certain genes such as those encoding glucocorticoid receptors (GR) and CD52 (target for alemtuzumab).
  • MHC major histocompatibility complex
  • immune checkpoints may also be targeted with the effect of reducing the elimination of the engrafted allogeneic immune-cells by the host's defense system, such as PD1 , CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1 , LAG 3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1 , SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , F0XP3,
  • a further aspect of the invention concerns the polynucleotide vectors that are used for the genome engineering of the cells, and the cells transfected with such vectors, prior or after the step of gene inactivation.
  • oligonucleotides are transfected into the cells ex-vivo using electroporation, whereas the polynucleotide encoding the Ago protein from a mesophilic prokaryote is transduced using a retroviral or lentiviral vector.
  • the invention encompasses a kit for genetic engineering of cells comprising a polynucleotide encoding Ago protein from a mesophilic prokaryote, preferably introduced into a lentiviral or retroviral vector and at least one oligonucleotide.
  • Another kit is composed of a prokaryotic cell in which the Ago protein from a mesophilic prokaryote is stably introduced, along with at least one oligonucleotides designed to hybridize and inactivate a genomic locus within said cell.
  • Another aspect of the invention is related to a method for regenerating a modified animal comprising the steps of:
  • the present invention encompasses also a method wherein said Ago or Ago variant protein is optimized to be more active at a temperature below 30 °C, and preferably between 20 and 25°C, and wherein said genetic material is a plant cell.
  • Another aspect of the invention concerns a method for regenerating a modified plant comprising the steps of:
  • transgenic animal or plant, or tissue, organ thereof, which have been modified according to the method of the invention is well known the person skilled in the art, we can refer to some of reference books such as "Regenerative Biology and Medicine”, D.L Stocum, 2012, Academic press, or “Transgenic Animal Technology: a Laboratory Handbook", C. Pinker, 2014, Elsevier, “Transgenic Plants: Methods and Protocols", L. Pena, 2015, Springer Science & Business Media.
  • the methods and compositions described herein allow for novel therapeutic applications, (e.g., prevention and/or treatment of: genetic diseases, cancer, fungal, protozoal, bacterial, and viral infection, ischemia, vascular disease, arthritis, immunological disorders, etc.), novel diagnostics (e.g. prediction and/or diagnosis of a condition) as well as providing for research tools (e.g. kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening), and means for developing plants with altered phenotypes, including but not limited to, increased disease resistance, and altering fruit ripening characteristics, sugar and oil composition, yield, and color.
  • the methods and compositions described herein allow for novel epigenetic studies.
  • Still another aspect of the invention is relative to an animal or a plant cell transfected by an Ago protein or an Ago variant protein from a mesophilic prokaryote, wherein said Ago protein or Aga variant protein shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.2 to SEQ ID NO.16; or a polypeptide vector shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.17 to SEQ ID N0.21
  • DAIS could be used to process endogenous locus according to different architectures described in Figure 1.
  • DAIS could be used in combination with 2 DNA guide oligonucleotides as illustrated in Figure 1.
  • DNA guide could be designed to bind to the forward and reverse strand of the locus to process in a complimentary fashion ( Figure 1 ). They could also be designed to bind to two different DNA targets located on the forward and reverse strand of the locus to process in an uncomplimentary fashion ( Figure 2). Both architectures would catalyze DNA nicking on the reverse and forward strands (figure 1 , dashed arrows). The nick positions will depend on the DNA guide location.
  • the cleavage is expected to occur between the 10 th and the 1 1 th bp of the locus-DNA guide duplex (Sheng et al. 2014; Swarts et al. 2014. For the sake of clarity, the DNA guide orientation and nucleotide numbering are indicated and the 10 th nucleotide is displayed in bold ( Figure 1 and 2).
  • a second plasmid bearing the same coding sequences downstream the pT7 promoter was also constructed to allow in vitro preparation of the corresponding polyadenylated mRNA.
  • Different DNA guides oligonucleotides SEQ ID NO 26-37) complementary to the forward and reverse strand of the TRAC locci (SEQ ID NO 22-25, Figure 3) were chemically synthesized.
  • Each DNA guide oligonucleotide consisted in 21 bp DNA oligonucleotide harboring a 5' phosphate groupment.
  • Figure 7 represents the alignment of aminoacid for the PIWI catylitic domain from mesophilic prokaryote.
  • Figure 5 shows a schematic representation of the optimized DAIS from mesophilic prokaryote to render the catalytic domain "dead", and which is coupled to another enzyme (here in the example: FOK-I).
  • the catalytic aminoacid D478, E512 and D546 were mutated to alanine and DAIS and the overall sequence was fused to the endonuclease domain of the type IIS FOKI (SEQ ID NO 21 , following Table 3).
  • the resulting DNA sequence encoding the chimeric DAIS (split-DAIS) was then used to assess the endonucleases activity of the system in primary T cells according to the experimental protocol described in Example 2 and to the architecture described in figure 5.

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Abstract

This invention relates to materials and methods for gene editing in mammalian cells, and more particularly to methods for gene editing using DNA-guided Argonaute (Ago) interference systems (DAIS) from mesophilic prokaryotes in eukaryotic cell such as T-cells or plant cell.

Description

Engineering animal or plant genome using DNA-guided Argonaute interference systems (DAIS) from mesophilic prokaryotes
Technical field
This patent application relates to materials and methods for gene editing in mammalian cells, and more particularly to methods for gene editing using DNA-guided Argonaute (Ago) interference systems (DAIS) from mesophilic prokaryotes in animal cells such as T-cells or in plant cell.
Background
Argonaute proteins (Ago) from bacteria such as Thermus thermophilus (strain HB27) have been recently described in bacteria to act as a barrier for the uptake and propogation of foreign DNA (Swarts D.C, et al. Nature 507: 258-261 ) In vivo, Tt Ago is loaded with 5' phosphorylated DNA guides, from 13 to 25 base pairs that are mostly plasmid derived and have a strong bias for a 5'-end deoxycytidine. The argonaute (Ago) gene family generally encodes proteins comprising four characteristic domains: N- terminal, PAZ, Mid and a C-terminal catalytic domain referred to as PIWI domain (Meister et al., Molecular Cell 15 (2): 185-197). According to the present invention, Ago proteins refer to any heterologous polypeptide or polynucleotide sequence comprising at least such PIWI domain sequence. Multiple sequence alignment of core motifs of PIWI domains indicate an active site comprising the motif (D/E)-(D/E)XK, X being any standard amino acid. The PIWI domain is believed to contribute to recognition of base pairing with double stranded nucleic acids.
The small interfering DNAs guide TtAgo cleave complementary DNA strands at high temperature (75°C).
On another hand, T-cells are mammalian cells known to be very sensitive to foreign DNA and refractory to DNA transfection.
Here, the inventors surprisingly found that a set of Ago proteins isolated from diverse species other than Thermus thermophilus which are not thermophile could be heterologously expressed in mammalian cells. These Ago proteins have the particularity, and can be optimized, to be active in a wide temperature range, including a temperature at around 37°C and below, allowing their use in different function (such as gene editing), in animal cell as well as in plant cell. Based on this finding they have set up in particular a strategy of gene editing using DNA-guided Ago to engineer T-cells suitable for immunotherapy.
Summary of invention
As per the present invention, the inventors have established that DNA-guided Argonaute interference system (DAIS) from diverse mesophilic prokaryotic organisms provides an efficient and easy-to-implement tool for generating targeted modifications of genomic DNA. Among them, DAIS can be used for targeted mutagenesis, targeted chromosomal deletions, targeted gene inversion, translocation or insertion and for multiplexed genome modifications. Such technology can be used to engineer living cells for specific applications such as cellular immunotherapy, gene therapy, generation of genetically modified animals, as well as cells for bioproduction as non-limiting examples. This DAIS by using appropriate Ago proteins from mesophilic prokaryotes also allows targeted modifications of genomic DNA in plant cell.
In a more specific aspect, this document presents a method for modifying the genomic material of mammalian cells, especially T-cells. The method includes introducing one, two or multiple short DNA molecules (referred herein as DNA-guides) into the mammalian cells, along with the prokaryotic DAIS that is known to catalyze single strand DNA break at the sequences targeted by DNA-guide. The DAIS can be delivered as DNA, mRNA and purified apo or holo protein (prebound to DNA guide) or via lentivirus. When supplied as DNA, the DAIS coding sequence can be regulated by a constitutive or inducible promoter. The mammalian cells may be primary or immortalized cells, somatic or stem cells including induced Pluripotent Stem Cells (iPSC).
The different features of the invention are detailed in the examples, figures and claims provided hereafter as non-limiting features.
FIGURES
Figure 1 : Schematic representation of the method for inducing double strand cleavage in a nucleic acid target sequence according to the present invention through the heterologous expression of Ago from a mesophilic prokaryote in a cell in the presence of oligonucleotides that act as specific guides to the selected locus. The 2 cleavage events presented in this figure can be performed in a sequentially or concomitantly manner.
Figure 2: Schematic representation of the method according to the invention using distant cleavage sites, which may either lead to a significant deletion of the locus region (Nx) between the two sites or to a cohesive end cleavage profile.
Figure 3: Schematic representation showing strategy to inactivate TCR locus in T-cells.
Figure 4: Schematic representation showing strategy to modify TCR locus in T-cells by homologous recombination using an insertion matrix (donor DNA).
Figure 5: Use a chimeric fusion of Ago from mesophilic prokaryote in which the catalytic domain is inactivated by replacement of several amino acids to a catalytic domain from an enzyme involved for instance in gene editing. This DAIS can be used along with one or multiple DNA guide and coupled to a catalytic domain from another enzyme (ex: nucleases such as FOKI, iTevl, meganuclease; transcription activator such as VP64...) to the locus of interest. The catalytic head may be used in various ways such as cutting, methylation, phosphorylation or inactivation.
Figure 6: Schematic representation of the method according to the invention using the DAIS presented in Figure 5 (with an Ago catalytically dead from mesophilic prokaryote coupled to, here, FOKI). This may either lead to a significant deletion of the locus region (Nx) between the two sites or to a cohesive end cleavage profile.
Figure 7: Amino acid alignment obtained using BLAST on PIWI catalytic domains from diverse mesophilic prokaryotes listed in Table 1. In particular, this alignment shows the conserved amino acids for all these species, and allows the identification of several to be mutated in order to inactivate the catalytic domain. DESCRIPTION
Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I . Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
The present invention broadly relates to a method of modifying the genetic material of n animal cell or in plant cell, through the expression of an Ago protein or an Ago protein variant from mesophilic prokaryote into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus.
By "Ago protein variant" is meant in the present invention an Ago protein from mesophilic prokaryote in which at least one amino acid is being replaced by another one. In particular, said one or several amino acids are essential in the catalytic function. Therefore, when these amino acids are mutated, the catalytic function is inactivated or also called "catalytically dead". In other purposes, one or several amino acids may be changed to optimized Ago proteins in their performances at a specific range of temperatures.
According to a preferred aspect of the invention, the Ago protein or the Ago protein variant from mesophilic prokaryote has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the Ago proteins from prokaryotic bacteria T Synechococcus sp. PCC 7002 (SEQ ID NO. 2), Synechococcus elongates (SEQ ID NO.3), Synechocystis sp, (SEQ ID NO.4 ), Aromatoleum aromaticum EbN1 (SEQ ID NO.5), Pseudomonas luteola (SEQ ID NO.6), Marinospirillum minutulum (SEQ ID NO.7 ), Haloferax denitricans (SEQ ID NO.8), Halorubrum kocurii (SEQ ID NO.9), Halobacterium satilanarum (SEQ ID NO.10), Natronobacterium gregoryi (SEQ ID NO.1 1 ), Clostridium butyricum (SEQ ID NO.12), Clostridium sartagoforme (SEQ ID NO.13), Halobacterium sp. DL1 (SEQ ID NO.14), Halorubrum lacusprofundi (SEQ ID NO.15 ), and Natrialba asiatica (SEQ ID NO.16 ) such as presented in the following Tables 1 and 2.
Table 1 : Temperature range of Ago activity for a set of mesophilic prokaryotes (identified with their ATCC number unless stated)
ATCC number or
other Temperature
Organism identification range
TtAGO sequence blasted BAA-163 65°C
Synechococcus sp. PCC 7002 27264 38°C
Synechococcus elongatus 33192 10-25 °C
Synechocystis sp 27184 30-33°C
Aromatoleum aromaticum EbN1 29143 26°C
Pseudomonas luteola 43273 30 °C
Marinospirillum minutulum 19193 35-45°C
Haloferax denitricans 35960 37°C
Halorubrum kocurii JCM14978 25-55°C
Halobacterium satilanarum 33171 42°C
Natronobacterium gregoryi 43098 37°C
Clostridium butyricum 19398 35-37°C
Clostridium sartagoforme 25778 30-37°C
Halobacterium sp. DL1 751944NCBI 37°C
Halorubrum lacusprofundi 49239 37°C
Natrialba asiatica 700171 37°C
Table 2: Polypeptide sequences of Ago proteins and plasmid construction containing the same (or Ago protein variant) from diverse mesophilic prokaryotes
Name SEQ ID # Polypeptide/Polynucleotide sequences 5' > 3'
MNHLGKTEVFLNRFALRPLNPEELRPWRLEVVLDPPP
GREEVYPLLAQVARRAGGVTVRMGDGLASWSPPEV
LVLEGTLARMGQTYAYRLYPKGRRPLDPKDPGERSV
LSALARRLLQERLRRLEGVWVEGLAVYRREHARGPG
WRVLGGAVLDLWVSDSGAFLLEVDPAYRILCEMSLE
AWLAQGHPLPKRVRNAYDRRTWELLRLGEEDPKELP
LPGGLSLLDYHASKGRLQGREGGRVAWVADPKDPR
KPIPHLTGLLVPVLTLEDLHEEEGSLALSLPWEERRRR
Thermus TREIASWIGRRLGLGTPEAVRAQAYRLSIPKLMGRRA thermophilus SEQ ID N0.1 VSKPADALRVGFYRAQETALALLRLDGAQGWPEFLR
Argonaute RALLRAFGASGASLRLHTLHAHPSQGLAFREALRKAK
EEGVQAVLVLTPPMAWEDRNRLKALLLREGLPSQILN
VPLREEERHRWENALLGLLAKAGLQWALSGAYPAEL
AVGFDAGGRESFRFGGAACAVGGDGGHLLWTLPEA
QAGERIPQEVVWDLLEETLWAFRRKAGRLPSRVLLLR
DGRVPQDEFALALEALAREGIAYDLVSVRKSGGGRV
YPVQGRLADGLYVPLEDKTFLLLTVHRDFRGTPRPLK
LVHEAGDTPLEALAHQIFHLTRLYPASGFAFPRLPAPL
HLADRLVKEVGRLGIRHLKEVDREKLFFV
MICWQKTEADIRSDVSGWEIYRGFKLDIFVSSQGNVF
LEVDEHYKLFSAWTLTQWLEWYPDFSAKILRNTYDG
RTWKLQKITNEDPNNIDSGTGQTLATYHKNHQKPATD
QEINSSKVIYVKPYNSKKEESYPHLSSRVKPCLFLDTL
SELASQGDRKVKEVFNLIKPSIEDRFSRATEIAKQLAV
EIYNIDEEISDDIKVVQVQAQQSSQDKSILLAHSSKRID
Synechococc SEQ ID NO.2 KVFKSLDQGCFRVGEIKFGCINLLDKNQDGWSKFILQ us sp. PCC KLQKLATIHNINIDANLVKAAVDIPEKDFEIDQFWESWV 7002 EQDIKTILVISEWLDNKYKTKLTRDALEHGITLQFMLPL
KENVRRIESNKGVESKITCFTSDQYRANNILLGLLAKA
GWQAVGLPLLNNEYAADLVIGFDAGRNETLSYGTSSF
AVLADGQILGWELPEAQKGEILDPDHVRRTVRRIISQF
QKMNGKQSPKRILLMRDGLIQQQEFSLITEALEEGEIK
YDLISVRKSGAGRIGRRNSDGTYVDAPKGTVIFSGND
KFKIVTSEAKAGGSARPLFVVREQGDTPIEIIAEQFYRL
TQLHPVSGSFTSRLPMPLNYADKLAKKIQSLGSVGILQ
NLDRKKLFFV
MDLLSNLRRSSIVLNRFYVKSLSQSDLTAYEYRCIFKK
TPELGDEKRLLASICYKLGAIAVRIGSNIITKEAVRPEKL
QGHDWQLVQMGTKQLDCRNDAHRCALETFERKFLE
RDLSASSQTEVRKAAEGGLIWWWGAKGIEKSGNGW
Synechococc EVHRGRRIDVSLDAEGNLYLEIDIHHRFYTPWTVHQW us elongatus SEQ ID NO.3 LEQYPEIPLSYVRNNYLDERHGFINWQYGRFTQERP
QDILLDCLGMSLAEYHLNKGATEEEVQQSYVVYVKPI
SWRKGKLTAHLSRRLSPSLTMEMLAKVAEDSTVCDR
EKREIRAVFKSIKQSINQRLQEAQKTASWILTKTYGISS
PAIALSCDGYLLPAAKLLAANKQPVSKTADIRNKGCAK
IGETSFGYLNLYNNQLQYPLEVHKCLLEIANKNNLQLS LDQRRVLSDYPQDDLDQQMFWQTWSSQGIKTVLVV
MPWDSHHDKQKIRIQAIQAGIATQFMVPLPKADKYKA
LNVTLGLLCKAGWQPIQLESVDHPEVADLIIGFDTGTN
RELYYGTSAFAVLADGQSLGWELPAVQGGETFSGQA
IWQTVSKLIIKFYQICQRYPQKLLLMRDGLVQEGEFQQ
TIELLKERKIAVDVISVRKSGAGRMGQEIYENGQLVYR
DAAIGSVILQPAERSFIMVTSQPVSKTIGSIRPLRIVHE
YGSTDLELLALQTYH LTQLH PASGFRSCRLPWVLH LA
DRSSKEFQRIGQISVLQNISRDKLIAV
MNLLARIEKTSWLNHFFVKNLDKEDLCFHEYQCKFS
QPPQQGDEQRAISSICYKVGVTAVRLGSRIITKEEVSC
EKMKGSSWKLVKMSDRELSCEDNLQRKAIEIFERKIL
EKQLKYFNKTAIEKATEGGLIWWIRDQHGVEKFGNG
WEVHRGRKIDVVIDSDKNLYLEIDIHHRFYTPWTLHQ
WLENYPDIPINYVRNTYKDQNNNYISWQYEEVSEQSP
QETIVEGLGISLADYHRNNGASEEEISSIWYVRRADR
WNSKSVAHLSKRLSPSLTMEMLANISEQGKDSKEIKD
Synechocysti SEQ ID NO.4 VFGLIRKKLDDRLKESKATAKYIISQVYGIPRETEPLRV s sp DGYVMPKAKLLALNGQEVDIVAKVRYKGCAKIGETKF
GCLNLFNEKHQYPEEVSKCLLDIEKNSHTKIKINSYRA
QQDFPDSELEQQMFWQKWSDEGIKTVLVVMDWSPN
ERKQKIRVQALQAGIATQFIVPKPKADPYKALNWLGL
LCKAGWQPVRLAPLQYDETADLIIGFDTGTNRELYYG
TSAFAVLADGQSLGWELPDIQRGESFSGKAIWQTVS
KLVLKFHQSCKRYPKKLLLMRDGLVQEGEFQQTIEEL
VKSNIAVDWGVRKSGTARMGQEIIQDNGEVAYRDAR
VGTVIFARKERSFTIVTSQ P VTAQ I G S AK P L RW H E YG
NTSLDLLALQTYQLTQLHPASGFRSCRLPWVLHLADR
SSKEFQRLGQISILQNISREKLIAV
MPVYYYRLTFDDDHEDINPTQLTRRGARMLSGANGW
CAVTDGEPLRVLSLRPLKVLRHEWNGMCCTASDETA
GTLKSANPSEREAIQRLLNQCLMQGARKLAYSSDGG
LEAEQGAGNLVRLTACQPSERVSAGGDYLDAFQSVT
LIPDVLPDGTALVGFDVRHRLLPRPHLTLDWVIRKRPE
WMEGIRRVRHRYVSNGQYGTAELIGIATGRTALSTFP
TPKGEVSLLGYHESKGNLPEGEREAVAASHVVQVRY
Aromatoleum GRDTRAKPMEHLAALLQPMFGFDTLSQIDSRLLERIA aromaticum RSLKWPVGERLKAAIRLVKGLKVSELDACLQAVEDTQ
EbN1 SEQ ID NO.5 RFARDLKPEFRLLFRGAAVADSERAVLRHGAYQGMT
RKLVVPLVVGGTALEQAVAVRHFEAVERVCRQWHSD
LPSWKTAPPAGDAEALNQRLAQRKPENALLLIGLGRG
ADKRKIRNVAYRYGLATQFMRLDHPPRTYQSTYYNN
LAAGVFSKGGGVLCAIDDMPGETDLFIGLDLGGVSQR
APGLAFLFTREGAQLGWQLAEAQRGERVEDAVLGDL
LERSLQAYRQVHLGALPRRIALHRDGRLFESLDVIRNF
ERDYSVRVDVLEWKSGCPPLYRRGWAENKKAFRN
PEVGDAFELPGLDELIIATYSGEELGSSWGDKVTVRP
LRLRKRYGETDLHTLARQVVLLSRIHGASLYRHPRLP
VTTHHADRFATLRQECNLDDLSKMDRLCPVYL
MSKVGIAALQLERNLGSLPVYVYDLTFVSSPSDQEPI
QLTRRGASQLSWVNRQTAITDFGHFKLVTLRPLSAYE
ANWQGVHCVVQGERELTLSAENELHRETIKRLVNQC
Pseudomona LMQGTRQLANVSNGAMQAEYGQGYQVELIDCLASSR s luteola SEQ ID NO.6 VAVRNEFLDVFQCLRLVPEVLPDGSTLVSFMVRHRLL PREHITLDWVIKTRPEWLESIKRVRHRYSSQGKAPGS
ANFIAVEKSRSAQSQISTAQGKISLFDYHDQRGNILPG
EKSLAYDSSVVKVSYGRSKESFEHLATLLQPMFDFEA
LQGIDSKLLERIAKGLKWPIADRLEAAAQRIKNLKVGE
LACGLTTVRNLDERVQYLRPPIRLRFANDKIGDHEKLV
TKHGAFRGMQRELWPIVIGGTNEEQQAAKQHFYNV
EKICQLWSNESPRWKQVPSANDELELDARLSSKTLP
EALLFIALGRNAN KQAIRNVAYRYGLATQFMRLDHPA
RIYQQTYYNNLAAGLFSKAGGLICGLDQMPGDTDLFI
GLDLGGVGQRLPGTAFLFTRSGAQLGWQLAEAQKG
ERVENEVLHDLLERSLQAFVRANDGLLPQRITLHRDG
HLYESLDVIKRFEQKHNVGIDVLEVIKSGCPPLFRRSQ
GPEGKAIYSNPEVGDAFELSGLDELIVATYSSQDLGQ
AWGDKVTVRPLRLRKRYGLTDLHTLAKQVILLSRIHG
ASLYRHPRLPVTTHHADRFASLRQECHLDDLSRMDR
LCPVYL
MGLNKNLNQVTVYRYQWIESAVDEKDSSVQLTRKV
ARLTASKNGWQPVTDISSFSIISLQPLKLLTIQAFGLNF
TLKQDGQLALNAEKENERAAIERLLNQDLYSAVYRLS
VDRGRQGGKPFKAIRHRAGWAEIEETQPSERIRVNS
DYLDLFKTLRLIPELLPNGQVILGLSLGHKICARNGITL
DWVIKNRKGWLPNIKRVRHRYSNQGQAPAVADFYGL
VAD KTAE SLVPGTELSLYKYHASKNNFSPE QQ VS VS A
SQVVSVGYGPKHKYEHLAALLEPMFDFETLQKIDSPL
Marinospirillu LNRIAQGLKWPVMERLRTSGEMVKGLMLPSFAAQVI m minutulum SEQ ID NO.7 QAKPLEQAVENICPNFKLSFYQGRPGSSEKDVLRLKA
FQGMTLSQVVCLAVGTQASPANLSNHFIKLQAACQRL
SGEPLPEWRGVTSVQPLKDAMELDARLSKKPQKNTL
LVIAIDKTVN KAEVRDVAFRH KLACQFMLTDHQPKTY
QPSYYNNLAAGVFSKGGGLICGLEEMPGDVDLFIGLD
MGGVTQKAPGSAFLFTRNGAQLGWQLADLQKGERL
EDEALKNLLSKSIQEYARNHEGALPRKLWHRDGRFF
ES L D 1 VQAI E RQYG 1 N 1 S VL E VI KS GAP 1 LF RKYQQAG K
MQYRNPEVGDVYRYLGLDELIIATYSGQELGAWGEK
VSVRPLRLRKRYGDETLDNLAKQVLLLTRIHGASLYR
HPRLPVTTHHADRFATLRQSCSLEALSHMDRTCPVYL
MTNRSPQTNSRTPERQADINPGTYVLHGRGNRRLTD
VKVNRYELRVDGGVENHWDSQSFTSSAAYYLDRTH
GTPVASAGPLSVISVTGLSKPVNVWGKRVTPVKTETV
QLDPKKKRDREHLRAFVQSCLRRAVPDDTYAYKFIND
IVRNDPAFSTGTDGFAAHPKHEVKVQIASDGTVLAHV
ESGYSIKSKSTLDNLYSPGSQLPNMKVAHDTDRYAKE
GQGWLKGWSEFNYTDYIRDVGSSIAELHEGTADEDW
Haloferax RQRLIEENPRLVKVKYGNMVGNQLPHFLRLSPRPEQ denitricans SEQ ID NO.8 VQRQDYDFFSQFISRRAMMPDEKYDYSKSFFESLSR
LPVIDLEFEPGPTNHPYKRINVREQNTRLVFSDDQQS
NTPSGGLREYGVYASPGRYRVGLLMPSQWEETLQKL
TPLLVKGLNNIGAPAGVTGYHYGLGDISNYTPVAHEIR
SETDAWAWPNKGAADDFGIDDPHHELKRTLMRKGI
PTQMLQKSSAQELIKQRATPNNDKFLNILSAIVAKAGG
TPWQVDSLPGETDAFMGLDVTRDSESGQHSGASAS
VVLADGTTFAAESTTQQGGEKFAARHVEQFVRDLW
DFAEGQEREINRVCIMRDGKVHEDIDAVREGLSELDA
EIDIVGVRKRGQPRIADYNGTRFKIAYKGMAFVDADR DEAVIHGFGKPEIRDDNPVGTPQTFKLVRHSGPTDIET
LARQAYWLSEVHVGSPAKSPRLPI PI EYADKAAEYVR KEYVSPGKVIKGPAYI
MLAVVPNKGVAEDFGIDDPYKELKRTLLRKGIPTQMM
QKSTVDEIVGQKAGIGNDKFLNALSAWAKVGGTPW
QIDSLPGKTDAFMGLDVTYDESSEQHAGASASVVLA
DGTTFAAESTTQQGGEKFSARHVEQFVRDLVFDFAG
Halorubrum EQGRDIDRLCIMRDGKISEDIDAVREGLSGIEAEIDIVGI kocurii SEQ ID NO.9 RKSGQPRIAEFDGTRFRIAEKGVGFVDADRSQSIIHAF
GKPEIHDDNPVGTPRTFRLTKDSGPTDVETLTRQAY
WLSEIHFGSPVRSPRLPVPIEYADMAAEYVREEYVSP
GTVIEGPAYI
MQAEIDDAFADVPESATEQTGHALTTFRSEKSLSEFA
VHEYTLKAKDGYRPDDHQAALRDTYKARRDILGEFES
SSPPVIAVRDALALATPSPLPVEDLELKNFEMVNDGP
HTLDYTDYSDKAVTKGLI DASLRRI LDGDYEVRGI DTV
LSKRPVIQKQDFRLHERWNLSLSVTNAGVVYLSVDFR
HKTISEYTLDKFDLDKLYRGLRVNVTYKQSGKGAFVD
ELMDKTVNESIDDMGNQSWEYHEGAERIPDRVLKEI
ARADRRWKVTKQGSHNTEYYPQRLLALQGHPQNVK
AFAPRFNEATRGKTRLSAQRCLNRATTFVENLPPVIP
Halobacteriu SEQ ID NO.10 LGGATLSFDATPVNGDDTWEMSRLFETDAHILQFAEE m satilanarum QTGDHPRRVKHNEVYEAPEEFSVCLVHPSAGPHAER
WENLDQQLKDIGAGPEAVNRVQYDPFKTADEIYTDLL
VDVPDDHEYSAACVILPKADFSMGESSASDIYHEMKK
ALRQRRVDSQMAHIDTLATSYALPNVALGLVAAAGGI
PFTTEDAMPGETDLFIGIDVSHRYPRDTDERVHIAAST
TSIYGDGTILGYTSAKPQTGEKVPPKELKNLTRQSIAG
YKQEHGEYPDRIVIHRDGFMREDLDQVEEMLESMDIN
YDWEIRKQSPARVLNLADGVAKIPDKGIAALNREENR
AILATFGDPESQATSSNTGLPQPIQVERKAGDTDIKTL
TAQVYLLSQSHVGAMNSTARLPITTYYADRASEAAAE
GYLPETSKLRQNIGFI
MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQH
PRMSLAFEQDNGERRYITLWKNTTPKDVFTYDYATG
STYIFTNIDYEVKDGYENLTATYQTTVENATAQEVGTT
DEDETFAGGEPLDHHLDDALNETPDDAETESDSGHV
MTSFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTL
AYTVRQELYTDHDAAPVATDGLMLLTPEPLGETPLDL
DCGVRVEADETRTLDYTTAKDRLLARELVEEGLKRSL
WDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVG
Natronobacte SEQ ID NO.1 1 HSGRAYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTT rium gregoryi YRPRRGHIVWGLRDECATDSLNTLGNQSWAYHRNN
QTPINTDLLDAIEAADRRWETRRQGHGDDAVSFPQE
LLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEK
AQAFAERLDPVRLNGSTVEFSSEFFTGNNEQQLRLLY
ENGESVLTFRDGARGAHPDETFSKGIVNPPESFEVAV
VLPEQQADTCKAQWDTMADLLNQAGAPPTRSETVQ
YDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFA
DLASPTETYDELKKALANMGIYSQMAYFDRFRDAKIF
YTRN VALG LLAAAGGVAFTTEH AM PG DADM Fl G I DVS
RSYPEDGASGQINIAATATAVYKDGTILGHSSTRPQL
GEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGF
MNEDLDPATEFLNEQGVEYDIVEIRKQPQTRLLAVSD VQYDTPVKSIAAINQNEPRATVATFGAPEYLATRDGG
GLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHNST ARLPITTAYADQASTHATKGYLVQTGAFESNVGFL
MNNLTFEAFEGIGQLNELNFYKYRLIGKGQIDNVHQAI
WSVKYKLQANNFFKPVFVKGEILYSLDELKVIPEFENV
EVILDGNIILSISENTDIYKDVIVFYINNALKNIKDITNYRK
YITKNTDEIICKSILTTNLKYQYMKSEKGFKLQRKFKIS
PWFRNGKVILYLNCSSDFSTDKSIYEMLNDGLGWG
LQVKNKWTNANGNIFIEKVLDNTISDPGTSGKLGQSLI
DYYINGNQKYRVEKFTDEDKNAKVIQAKIKNKTYNYIP
QALTPVITREYLSHTDKKFSKQIENVIKMDMNYRYQTL
Clostridium SEQ ID NO.12 KSFVEDIGVIKELNNLHFKNQYYTNFDFMGFESGVLE butyricum EPVLMGANGKIKDKKQIFINGFFKNPKENVKFGVLYPE
GCMENAQSIARSILDFATAGKYNKQENKYISKNLMNIG
FKPSECIFESYKLGDITEYKATARKLKEHEKVGFVIAVI
PDMNELEVENPYNPFKKVWAKLNIPSQMITLKTTEKF
KNIVDKSGLYYLHNIALNILGKIGGIPWIIKDMPGNIDCF
IGLDVGTREKGIHFPACSVLFDKYGKLINYYKPTIPQS
GEKIAETILQEIFDNVLISYKEENGEYPKNIVIHRDGFS
RENIDWYKEYFDKKGIKFNIIEVKKNIPVKIAKVVGSNIC
NPIKGSYVLKNDKAFIVTTDIKDGVASPNPLKIEKTYGD
VEMKSILEQIYSLSQIHVGSTKSLRLPITTGYADKICKAI
EYIPQGVVDNRLFFL
MKEFNVITEFKNGINSKSIEIYIYKMMVRDFEKRHNEN
YDWKELINLNNNSTIVFYEQYIASFKEIEKWGNEQYIN
VEKRAINLESNEKKILERLLLKEIKNNIDNNKYKVVKDSI
YINKPVYNEKGIKIDRYFNLDINVESNGDIIIGFDISHNF
EYINTLEYEIKNNNIKIGDRVKDYFYNLTYEYVGIAPFTI
SEENEYMGCSIVDYYENKNQSYIVNKLPKDMKAILVK
NNKNSIFPYIPSRLKKVCRFENLPQNVLRDFNTRVKQ
KTNEKMQFMVDEVINIVKNSEHIDVKKKNMMCDNIGY
Clostridium KIEDLQQPDLLFGNARAQRYPLYGLKNFGVYENKRIEI sartagoforme SEQ ID NO.13 KYFIDPILAKSKMNLEKISKFCDELEQFSSKLGVGLNR
VKLNNIVNFKEIRMDNEDIFSYEIRKIVSNYNETTIVILS
EENLNKYYNIIKKTFSGGNEVPTQCIGFNTLSYTEKNK
DSIFLNILLGVYAKSGIQPWILNEKLNSDCFIGLDVSRE
NKVNKAGVIQWGKDGRVLKTKVISSSQSGEKIKLETL
REIVFEAINSYENTYRCKPKHITFHRDGINREELENLK
NTMTNLGVEFDYIEITKGINRRIATISEGEEWKTIMGRC
YYKDNSAYVCTTKPYEGIGMAKPIRIRRVFGTLDIEKIV
EDAYKLTFMHVGAINKIRLPITTYYADLSSTYGNRDLIP
TNIDTNCLYFI
MQAEIDDAFADVPESATEQTGHALTTFRSEKSLSEFA
VHEYTLKAKDGYRPDDHQAALRDTYKARRDILGEFES
SSPPVIAVRDALALATPSPLPVEDLELKNFEMVNDGP
HTLDYTDYSDKAVTKGLI DASLRRI LDGDYEVRGI DTV
LSKRPVIQKQDFRLHERWNLSLSVTNAGVVYLSVDFR
Halobacteriu SEQ ID NO.14 HKTISEYTLDKFDLDKLYRGLRVNVTYKQSGKGAFVD m sp. DL1 ELMDKTVNESIDDMGNQSWEYHEGAERIPDRVLKEI
ARADRRWKVTKQGSHNTEYYPQRLLALQGHPQNVK
AFAPRFNEATRGKTRLSAQRCLNRATTFVENLPPVIP
LGGATLSFDATPVNGDDTWEMSRLFETDAHILQFAEE
QTGDHPRRVKHNEVYEAPEEFSVCLVHPSAGPHAER
WENLDQQLKDIGAGPEAVNRVQYDPFKTADEIYTDLL VDVPDDHEYSAACVILPKADFSMGESSASDIYHEMKK
ALRQRRVDSQMAHIDTLATSYALPNVALGLVAAAGGI
PFTTEDAMPGETDLFIGIDVSHRYPRDTDERVHIAAST
TSIYGDGTILGYTSAKPQTGEKVPPKELKNLTRQSIAG
YKQEHGEYPDRIVIHRDGFMREDLDQVEEMLESMDIN
YDWEIRKQSPARVLNLADGVAKIPDKGIAALNREENR
AILATFGDPESQATSSNTGLPQPIQVERKAGDTDIKTL
TAQVYLLSQSHVGAMNSTARLPITTYYADRASEAAAE
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According to a more preferred embodiment, the Ago protein or the Ago protein variant is chosen among Natronobacterium gregoryi, Synechococcus sp. PCC 700, Halobacterium satilanarum and Natrialba asiatica.
The present disclosure provides for the engineering of site-directed polypeptides to recognize a desired target nucleic acid sequence with desired enzyme specificity and/or activity. Modifications to a site-directed polypeptide can be performed through protein engineering. Protein engineering can include fusing functional domains to such engineered site-directed polypeptide which can be used to modify the functional state of the overall site-directed polypeptide or the actual target nucleic acid sequence of an endogenous cellular locus. The site-directed polypeptide of the disclosure can be used to regulate endogenous gene expression, both through activation and repression of endogenous gene transcription. The site-directed polypeptide-fusions can also be linked to other regulatory or functional domains, for example nucleases, transposases or methylases, to modify endogenous chromosomal sequences. In some cases, the site- directed polypeptide may be linked to at least one or more regulatory domains. Non- limiting examples of regulatory or functional domains include transcription factor repressor or activator domains such as KRAB and VP16, co-repressor and co-activator domains, DNA methyl transferases, histone acetyltransferases, histone deacetylases, and DNA cleavage domains such as the cleavage domain from the endonuclease Fokl.
According to a further aspect of the invention, the catalytic domain of the Ago protein from mesophilic prokaryotes is inactivated by replacement of at least one amino acid, and wherein said modified Ago protein is coupled to a catalytic domain from an enzyme having a function in gene editing, modification of gene expression or epigenetic modulation. A schematic representation is given in Figure 6.
By "gene editing" is means genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases. The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and non-homologous end-joining (NHEJ).
By "modulation of gene expression", it is meant in particular enzymes which are involved as activator or repressor during the transcription process.
By "epigenetic modulation", it is meant in particular enzymes such as methylases. According to a preferred embodiment, the Ago protein catalytic domain from mesophilic prokaryotic is inactivated by the mutations of the 2 aspartic acids in positions 478 and 546, and optionally the glutamic acid in position 512. These amino acids are usually changed into non-charged and short-chain amino acids such as alanine (A).
According to another preferred embodiment, said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain from a type IIS restriction enzyme, and as an example, Fok-I.
This type of nuclease such as Fok-I needs to be dimerized in order to be active (i.e. to cleave). This particularity has the advantage to provide more specific and efficient targeted cleavages. According to another alternative which is contemplated within the invention, said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain of a meganuclease such as l-Scel or l-Crel or one of their variants, or another homing endonuclease such as l-Tevl.
The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (BI2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14 (Basic Local Alignment Search Tool provided by the NCBI at http://ncbi.nlm.nih.gov.) BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Such analysis was performed using the AA sequence from the PIWI catalytic domain of the Thermophilus Ago. Figure 7 shows the alignment of catalytic domain from 15 mesophilic prokaryotes.
Optimized Ago proteins from a mesophilic prokaryote can be derived from such a protein, or from an Ago protein from other species, by directed evolution in order to optimize its performance at a range of temperature comprised between 30°C and 40°C.
One method according to the invention for optimizing an Ago protein from a mesophilic prokaryote to have it induce more cleavage activity at a temperature below 40°C, can comprise the steps of:
a) Introducing oligonucleotides into a cell , said oligonucleotides being selected to hybridize a toxic gene, resistance gene or a reporter gene present into said cell;
b) Creating a variant of the gene encoding Ago protein from a mesophilic prokaryote and expressing said gene into said cell;
c) Cultivating said cell at a temperature below 40°C;
d) Recovering said variant encoding Ago protein from said cultured cells.
Such variant proteins obtainable form this method form a further object of the present invention.
According to one aspect of the invention expression of said Ago protein from a mesophilic prokaryote may be placed under the control of an inducible promoter to reduce potential genotoxicity of the Ago protein into said cell.
The above method can further comprise the step of cultivating the cells in which cleavage by Ago from a mesophilic prokaryote has occurred at the preselected locus, recovering and isolating said cells in which cleavage by said Ago has occurred at the preselected locus. The cells obtained by this method are a further object of the present invention.
The present invention aims more particularly to engineer immune cells, in particular T cells, and most preferably human T cells from patients or donors, for their use in immunotherapy. In particular, the present invention provides with a method for modifying the genetic material of a primary T-cell, comprising at least one of the following steps:
Providing a T-cell from a donor;
Expanding said T-cell;
- Transfecting said T-cell with a nucleic acid expressing a Ago Protein from a mesophilic prokaryote;
Further transfecting said T cell with at least one exogenous oligonucleotide
(DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus.
Reference is made to pages 28 to 34 of WO2013176915 by the applicant, which are incorporated herein, describing the steps of activating and expanding allogeneic T- cells from donors, which are transduced with nucleic acids, (retroviral or lentiviral vectors or mRNA) encoding chimeric antigen receptors, to result into so-called "CAR immune cells". This aspect of the invention is illustrated in the following examples with the inactivation of TCR in Jurkat cells and T-cells, in view of providing T-cells from donors that are made "universal" - i.e. suitable for their engraftment into patients, while reducing the risk for graft-versus-host disease.
The oligonucleotide used as a DNA guide in the method according to the present invention is generally 10 to 50 nucleotides in length, preferably 15 to 30 nucleotides, more preferably 20 to 25 nucleotides, which confer a high target specificity to the method of the invention. Such oligonucleotide is preferably phosphorylated at its 5' terminus, and has also preferably a CA doublet at its 5' terminus. This is believed to improve the interaction between the guide and the Ago protein.
According to a preferred embodiment of the invention, at least 2 oligonucleotides are selected to respectively hybridize each strand of a double-strand DNA at sites that are closed enough to each other to obtain double strand break. Preferably, the 2 oligonucleotides will be designed to hybridize each strand at the same locus so that Ago from a mesophilic prokaryote will create a blunt double strand break.
According to another embodiment, the present invention contemplates also the possibility to target at multiple preselected loci of a particular gene (i.e. drug resistance) by using a library of DNA guides. These are synthetized beforehand and are designed to hybridize to different parts of the gene of interest. This set of DNA guides are subcloned together in a plasmid bearing the polymerase such as T7 and can be transfected into a pool of cells to be genetically modified along with the Ago protein. In the cells, said DNA guides are released and can intervene in the DAIS process. Then, it will be possible to screen phenotypically each isolated cell (i.e. for instance their drug resistance).
According to a further aspect of the invention the method comprises the step of performing homologous recombination at the preselected locus by bringing into the cell a donor DNA comprising a sequence homologous to that of the preselected locus into contact with said genetic material. A schematic representation is given in Figure 4 using the modified TRAC locus as an example. Various homologous recombination techniques have been described in the prior art, especially in US 6,528,313 and US 8,921 ,332, but so far have never been practiced using Ago proteins from mesophilic prokaryotes. This method allows donor DNA comprising a transgene, a promoter, an expression cassette or a repairing sequence to be inserted at a preselected locus. Said method can be practiced in gametes and oocytes in view of obtaining cells to develop a transgenic animal.
Otherwise repair mechanism like non homologous end joining (NHEJ) may also be used to introduce transgenes into cell genome upon cleavage by the Ago protein from a mesophilic prokaryote as per the invention. A schematic representation is given in Figure 3 using the modified TRAC locus as an example.
According to a further aspect of the invention, several oligonucleotides targeting different loci can be carried out to inactivate said loci simultaneously, producing a multiplex genome engineering method. Said additional loci, which may be targeted in said immune cells alone or in combination, are more particularly genes that confer resistance to chemotherapy drugs (ex: fludarabine, chlorofarabine...), such as those genes encoding deoxycytidine kinase (dCk) or encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene thereby conferring resistance to 6-thioguanine (6TG) as described in PCT/EP2014/075317. Resistance to lymphodepleting agents can also be achieved by inactivating certain genes such as those encoding glucocorticoid receptors (GR) and CD52 (target for alemtuzumab).
Further genes may also be inactivated alone or in combination with the previous genes, such as those involved in the expression of major histocompatibility complex (MHC), in particular 32m and HLA genes,
Other genes encoding so-called "immune checkpoints" may also be targeted with the effect of reducing the elimination of the engrafted allogeneic immune-cells by the host's defense system, such as PD1 , CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1 , LAG 3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1 , SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , F0XP3, PRDM1 , BATF, GUCY1A2, GUCY1A3, GUCY1 B2 and GUCY1 B3.
A further aspect of the invention concerns the polynucleotide vectors that are used for the genome engineering of the cells, and the cells transfected with such vectors, prior or after the step of gene inactivation.
According to a preferred embodiment, oligonucleotides are transfected into the cells ex-vivo using electroporation, whereas the polynucleotide encoding the Ago protein from a mesophilic prokaryote is transduced using a retroviral or lentiviral vector. In this regard, the invention encompasses a kit for genetic engineering of cells comprising a polynucleotide encoding Ago protein from a mesophilic prokaryote, preferably introduced into a lentiviral or retroviral vector and at least one oligonucleotide. Another kit is composed of a prokaryotic cell in which the Ago protein from a mesophilic prokaryote is stably introduced, along with at least one oligonucleotides designed to hybridize and inactivate a genomic locus within said cell.
Another aspect of the invention is related to a method for regenerating a modified animal comprising the steps of:
(a) Modification of genetic material according to any one of claims 1 to 35, wherein said genetic material is an oocyte;
(b) Regeneration of a transgenic animal.
The present invention encompasses also a method wherein said Ago or Ago variant protein is optimized to be more active at a temperature below 30 °C, and preferably between 20 and 25°C, and wherein said genetic material is a plant cell.
Another aspect of the invention concerns a method for regenerating a modified plant comprising the steps of:
(a) Modification of genetic material according to any one of claims 1 to 35, wherein said genetic material is from vegetal origin such as isolated cell, plant fragment or callus;
(b) Regeneration of a plant.
The step of regeneration of transgenic animal or plant, or tissue, organ thereof, which have been modified according to the method of the invention is well known the person skilled in the art, we can refer to some of reference books such as "Regenerative Biology and Medicine", D.L Stocum, 2012, Academic press, or "Transgenic Animal Technology: a Laboratory Handbook", C. Pinker, 2014, Elsevier, "Transgenic Plants: Methods and Protocols", L. Pena, 2015, Springer Science & Business Media.
The methods and compositions described herein allow for novel therapeutic applications, (e.g., prevention and/or treatment of: genetic diseases, cancer, fungal, protozoal, bacterial, and viral infection, ischemia, vascular disease, arthritis, immunological disorders, etc.), novel diagnostics (e.g. prediction and/or diagnosis of a condition) as well as providing for research tools (e.g. kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening), and means for developing plants with altered phenotypes, including but not limited to, increased disease resistance, and altering fruit ripening characteristics, sugar and oil composition, yield, and color. The methods and compositions described herein allow for novel epigenetic studies.
Still another aspect of the invention is relative to an animal or a plant cell transfected by an Ago protein or an Ago variant protein from a mesophilic prokaryote, wherein said Ago protein or Aga variant protein shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.2 to SEQ ID NO.16; or a polypeptide vector shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.17 to SEQ ID N0.21
Some applications of the general principles of the invention described above are detailed in the following examples and claims. These examples are not limitative and may be combined with any of the previous aspects of the present invention.
Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
EXAMPLES
1 - Overall design and mechanism of DAIS
DAIS could be used to process endogenous locus according to different architectures described in Figure 1. To process an endogenous locus via error-prone NHEJ, DAIS could be used in combination with 2 DNA guide oligonucleotides as illustrated in Figure 1. DNA guide could be designed to bind to the forward and reverse strand of the locus to process in a complimentary fashion (Figure 1 ). They could also be designed to bind to two different DNA targets located on the forward and reverse strand of the locus to process in an uncomplimentary fashion (Figure 2). Both architectures would catalyze DNA nicking on the reverse and forward strands (figure 1 , dashed arrows). The nick positions will depend on the DNA guide location. When a given DNA guide is considered, the cleavage is expected to occur between the 10th and the 1 1 th bp of the locus-DNA guide duplex (Sheng et al. 2014; Swarts et al. 2014. For the sake of clarity, the DNA guide orientation and nucleotide numbering are indicated and the 10th nucleotide is displayed in bold (Figure 1 and 2).
2 - Heterologous expression in mammalian cells of TtAGO related proteins identified from Synechococcus sp. PCC 7002, Natronobacterium gregoryi, Halobacterium sp DL1 and Natrialba asiatica
Using the coding sequence of Tt. Ago, a BLASTP analysis was performed to identify closely related proteins from mesophyle prokaryotes. A series of mesophile Ago proteins were identified (SEQ ID NO 2-16).
To demonstrate the activity of the DAIS Ago proteins in mammalian cells, as the sequences shown in Table 2, 4 different plasmids wereconstructed to express non-Tt AGO proteinsisolated from Synechococcus sp. PCC 7002 (SEQ ID. NO 17), Natronobacterium gregoryi (SEQ ID. NO 18), Halobacterium spDL1 (SEQ ID. NO 19) and from Natrialba asiatica (SEQ ID. NO 20). Upstream a 2A-cis acting hydrolase element is linked to the coding sequence of BFP and under the control of pEF1 alpha or pCMV promoters in mammalian cells. A second plasmid bearing the same coding sequences downstream the pT7 promoter was also constructed to allow in vitro preparation of the corresponding polyadenylated mRNA. Different DNA guides oligonucleotides (SEQ ID NO 26-37) complementary to the forward and reverse strand of the TRAC locci (SEQ ID NO 22-25, Figure 3) were chemically synthesized. Each DNA guide oligonucleotide consisted in 21 bp DNA oligonucleotide harboring a 5' phosphate groupment. 3 - DAIS endonucleases activity from mesophilic prokaryotes in primary T cells
To test the ability of DAIS from mesophilic prokaryotes to promote error-prone NHEJ events at the TRAC locus (Figure 3), 20 μg of mRNA encoding DAIS were electroporated in the presence of ≥ 2 DNA guides chosen in the list described in Table 3 (SEQ ID NO 26-37) in Primary T cells using Pulse Agile technology according to the manufacturer protocol. Six days post transfection, cells were recovered and genomic DNA was extracted. PCR amplification of TRAC endogenous locus was then performed and the resulting amplicon was subjected to Endo T7 assay to determine the extent of targeted mutagenesis promoted by the DAIS at the TRAC locus. Our results showed a barely detectable endo T7 signal, indicating that DAIS is able to promote error-prone NHEJ at the TRAC locus although with a low efficiency.
4- Expression of triple mutant inactive DAIS fused to endonuclease catalytic head (split DAIS) proficient for gene editing in primary T cells
Figure 7 represents the alignment of aminoacid for the PIWI catylitic domain from mesophilic prokaryote. Figure 5 shows a schematic representation of the optimized DAIS from mesophilic prokaryote to render the catalytic domain "dead", and which is coupled to another enzyme (here in the example: FOK-I).
To generate a DAIS split nuclease, the catalytic aminoacid D478, E512 and D546 were mutated to alanine and DAIS and the overall sequence was fused to the endonuclease domain of the type IIS FOKI (SEQ ID NO 21 , following Table 3). The resulting DNA sequence encoding the chimeric DAIS (split-DAIS) was then used to assess the endonucleases activity of the system in primary T cells according to the experimental protocol described in Example 2 and to the architecture described in figure 5. Our results showed that the optimized split-DAIS display a significant nuclease activity in primary T cells at the locus considered.Table 3: Polynucleotide sequences of targets and pairs of oligos (DNA guides) used for inactivating TCRa gene in human T-cells Table 3: Polynucleotidic sequences for the TRAC targets and the corresponding pairs of oligos used
Figure imgf000026_0001
REFERENCES:
Sheng G, Zhao H, Wang J, Rao Y, Tian W, et al. (2014) Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc Natl Acad Sci U S A ^ ^ ^■. 652-657. Swarts DC, Jore MM, Westra ER, Zhu Y, Janssen JH, et al. (2014) DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507: 258-261 .
Makarova KS, Wolf Yl, van der Oost J, Koonin EV. (2009) Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements. Biol Direct 4: 29.
Swarts, DC, Jore, MM, Westra, ER, Zhu, Y, Janssen, JH, Snijders, AP et al. (2014). DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507: 258-61 .
Sheng, G, Zhao, H, Wang, J, Rao, Y, Tian, W, Swarts, DC et al. (2014). Structure- based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand- mediated DNA target cleavage. Proc Natl Acad Sci U S A 111 : 652-7.

Claims

1 . A method of modifying the genetic material of an animal or plant cell through expression of an Ago protein or an Ago protein variant from a mesophilic prokaryote into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus.
2. A method according to claim 1 , wherein said mesophilic prokaryotic organism is chosen among Synechococcus sp. PCC 7002, Synechococcus elongatus, Synechocystis sp,, Aromatoleum aromaticum EbN 1 , Pseudomonas luteola,, Marinospirillum minutulum, Haloferax denitricans Halorubrum kocurii, Halobacterium satilanarum, Natronobacterium gregoryi, Clostridium butyricum, Clostridium sartagoforme, Halobacterium sp. DL1 , Halorubrum lacusprofund, and Natrialba asiatica.
3. A method according to claim 1 or claim 2, wherein said mesophilic prokaryotic organism is chosen among Natronobacterium gregoryi, Synechococcus sp. PCC 700, Halobacterium satilanarum and Natrialba asiatica.
4. A method according to any one of claim 1 -3, wherein the catalytic domain of the Ago protein from mesophilic prokaryotes is inactivated by replacement of at least one amino acid, and wherein said modified Ago protein is coupled to a catalytic domain from an enzyme having a function in gene editing, modification of gene expression or epigenetic modulation.
5. A method according to claim 4, wherein the inactivation of the Ago protein catalytic domain from mesophilic prokaryotic is performed by the mutations of the 2 aspartic acids in positions 478 and 546, and optionally the glutamic acid in position 512
6. A method according to claim 4 or 5, wherein said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain from a type IIS restriction enzyme.
7. A method according to any one of claim 4 to 6, wherein said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain of FOK-1 .
8. A method according to claim 4 or claim 5, wherein said catalytic domain of the enzyme to be coupled to catalytically inactivated Ago protein from a mesophilic prokaryote is a catalytic domain of a meganuclease or another endonuclease such as I- Tevl.
9. A method according to claim 8, wherein said meganuclease l-Scel and I- Crel or one of their variants,
10. A method according to any one of claim 1 to 9, further comprising the step of cultivating the cells in which cleavage by Ago or Ago variant has occurred at the preselected locus.
1 1 . A method according to claim 10, further comprising the step of recovering the culture supernatant of said cultured cells to recover the molecules produced by the modified cells.
12. A method according to claim 10 or 1 1 , further comprising the step of recovering the cells in which cleavage by Ago or Ago variant has occurred at the preselected locus.
13. A method according to claim 12, further comprising the step of freezing or conditioning the cells in which cleavage by Ago or Ago variant has occurred at the preselected locus, for use as a therapeutic product.
14. A method according to any one of claims 1 to 13, wherein said cell is a mammalian cell.
15. A method according to any one of claims 1 to 14, wherein said cell is a human cell.
16. A method according to any one of claims 1 to 15, wherein said cell is a T- cell.
17. A method according to claim 16 wherein said locus in said T-cell is selected from the genes encoding T cell receptor (TCR), Glucocorticoid receptors (GR), dCK, 32m, HLA, HPRT, PD1 , CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1 , LAG 3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1 , SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , IL10RA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , F0XP3, PRDM1 , BATF, GUCY1A2, GUCY1A3, GUCY1 B2 and GUCY1 B3.
18. A method according to any one of claims 1 to 17, wherein said oligonucleotide is 10 to 50 nucleotides in length, preferably 15 to 30 nucleotides, more preferably 20 to 25 nucleotides.
19. A method according to any one of claims 1 to 18, wherein said oligonucleotide is phosphorylated, preferably at its 5' terminus.
20. A method according to any one of claims 1 to 19, wherein said oligonucleotide has a CA doublet at its 5' terminus.
21 . A method according to any one of claims 1 to 20, wherein several loci are cleaved using various specific oligonucleotides (multiplex).
22. A method according to any one of claims 1 to 21 , wherein at least 2 oligonucleotides are selected to respectively hybridize each strand of a double-strand DNA at sites that are closed enough to each other to obtain double strand break.
23. A method according to any one of claims 1 to 22, wherein the 2 oligonucleotides hybridize each strand at the same locus so that Ago will create a blunt double strand break.
24. A method according to any one of claims 1 to 22, further comprising the step of performing homologous recombination at the preselected locus by bringing a donor DNA comprising a sequence homologous to that of the preselected locus into contact with said genetic material.
25. A method according to claim 24, wherein said donor DNA comprises a transgene, a promoter, an expression cassette or a repairing sequence to be inserted at the preselected locus.
26. A method according to any one of claims 1 to 24, wherein said Ago protein is heterologously expressed from a polynucleotide introduced into said cell.
27. A method according to any one of claims 1 to 26, wherein said Ago or Ago variant protein has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.2 to SEQ ID NO.16 .
28. A method according to any one of claims 1 to 27, wherein said Ago or Ago variant protein is optimized to be more active at a temperature below 40 °C.
29. A method according to any one of claims 1 to 27, wherein said Ago or Ago variant protein is optimized to be more active at a temperature between 30 and 40 °C, preferably 37°C.
30. A method according to claim 26, wherein said polynucleotide encoding said Ago or Ago variant protein is transduced by a retroviral or lentiviral vector.
31 . A method according to claim 26, wherein said polynucleotide is mRNA.
32. A method according to claim 26, wherein said mRNA is introduced into said cell by electroporation.
33. A method according to any one of claims 1 to 32, wherein said Ago protein expression is under the control of an inducible promoter to reduce potential genotoxicity of the Ago protein into said cell.
34. A method according to any one claims 1 to 33, further comprising the step of introducing said modified genetic material into an animal stem cell to develop a transgenic animal.
35. A method according to any one of claims 1 to 27 or claims 30 to 34, wherein said Ago or Ago variant protein is optimized to be more active at a temperature below 30 °C, and preferably between 20 and 25°C, and wherein said genetic material is a plant cell.
A method for regenerating a modified animal comprising the steps of:
(a) Modification of genetic material according to any one of claims 1 to 35, wherein said genetic material is an oocyte;
(b) Regeneration of a transgenic animal.
A method for regenerating a modified plant comprising the steps of:
(a) Modification of genetic material according to any one of claims 1 to 35, wherein said genetic material is from vegetal origin such as isolated cell, plant fragment or callus;
(b) Regeneration of a plant.
38. Animal or vegetal cell transfected by an Ago protein or an Ago variant protein from a mesophilic prokaryote, wherein said Ago protein or Aga variant protein shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.2 to SEQ ID NO.16;
or a polypeptide vector shares at least has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.17 to SEQ ID N0.21
39. A polynucleotide vector comprising a gene encoding Ago or Ago variant protein from a mesophilic prokaryote.
40. A retrovirus or lentiviral vector comprising a polynucleotide encoding an Ago or Ago variant protein from a mesophilic prokaryote for transducing mammalian cells.
41 . Kit for genetic engineering of cells comprising a polynucleotide encoding Ago or Ago variant protein from a mesophilic prokaryote and at least one oligonucleotide.
42. A method for optimizing Ago protein to induce more cleavage activity at a temperature below 40°C, wherein said method comprises the following steps:
(a) Introducing oligonucleotides into a cell, said oligonucleotides being selected to hybridize a toxic gene, resistance gene or a reporter gene present into said cell; (b) Creating a variant of the gene encoding Ago protein and expressing said gene into said cell;
(c) Cultivating said cell at a temperature below 40°C;
(d) Recovering said variant encoding Ago protein from said cultured cells that do not express said toxic gene, resistance gene or reporter gene.
43. Method according to claim 42, wherein said Ago or Ago variant protein or an Ago variant has at least 70%, more preferably at least 75%, and even more preferably at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO.2 to SEQ ID NO.16
44. Method according to claim 42 or claim 43, wherein said temperature is between 30 and 40°C.
45. Method according to anyone of claim 42 to claim 44, wherein said cell is a mammalian cell.
46. Method according to anyone of claim 42 to claim 45, wherein said Ago or Ago variant protein from a mesophilic prokaryote is assayed for more stable expression in said cell at said temperature.
47. Optimized variant gene encoding Ago from a mesophilic prokaryote susceptible to be obtained by the method of any one of claims 42 to 46.
48. Ago or Ago variant protein from a mesophilic prokaryote encoded by the variant gene according to claim 47.
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