WO2014102688A1 - Nouvelle matrice de conception pour l'amélioration du ciblage génique dirigé par homologie - Google Patents
Nouvelle matrice de conception pour l'amélioration du ciblage génique dirigé par homologie Download PDFInfo
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Classifications
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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
- C12N15/87—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
-
- 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
Definitions
- the present invention is in the field of genome engineering and particularly pertains to the use of a new design of repair/donor matrix for targeted integration into the genome of a cell.
- Homology-directed gene targeting allows achieving stable disruption, replacement or modification of a gene present in a genome. It is typically performed by transfecting a cell with an exogenous nucleic acid, called donor matrix, having homology to a selected endogenous sequence, by a process called "homologous recombination". This process is based on the natural ability of cells to repair their genetic defects by exchanging genetic material between allele sequences, thereby allowing genetic recombination between endogenous and exogenous homologous nucleic acid sequences (Capecchi 1989). This process needs extensive homology between the exogenous nucleic acid sequence and the endogenous target locus for exchanges to occur at a useful frequency. Usually, more than 80% identity is required between the sequences over 40 nucleotides.
- donor matrices consist of large double stranded DNA constructs, which include one or two sequences homologous to the endogenous target locus, an exogenous genetic sequence for insertion, and usually a gene encoding a selectable marker (selection of phenotypic marker).
- Such donor matrices are generally produced under the form of plasmids from 1 to 10 kb in size. Such plasmids may be linearized or single stranded prior to cell transfection.
- the exogenous nucleic acid is integrated in the genome through this unique sequence of homology.
- this integration is less precise than by using two homologous sequences.
- precise insertion can be obtained at a locus straight between the two homologous genomic sequences, thereby allowing gene replacement at the targeted locus (see WO 2010/1 1354).
- oligonucleotides that partially hybridize with each other, enables to form a donor matrix that can drive transgene integration with success. So far, oligonucleotides had not been considered as useful donor matrices because they are too short in size. They are indeed routinely synthesized no longer than about 200 bp, which is not enough for performing gene insertion. On another hand, partially hybridized oligonucleotides were not expected to allow efficient homologous recombination within the cell genome.
- the present invention thus opens the way to the use of combined oligonucleotides as donor matrix for homologous recombination.
- the inventors have thus designed a new type of donor matrix that has unexpectedly proven to be at least as efficient as the former design formed by linear single or double DNA strands.
- Such matrix is composed of at least two single-stranded oligonucleotides that partially hybridize together over a complementary sequence. The hybridization of these oligonucleotides allows generating a matrix that is only partially double-stranded with single strand extremities.
- the partially hybridized oligonucleotides can span as twice as the length of each single strand oligonucleotides.
- the new designed matrix may span an insertion sequence of more than 200 bp to several kbs.
- Such oligonucleotides that form the new designed matrix are advantageous because they are quicker to synthesize and easier to transfect than plasmids, which are larger and require initial cloning steps.
- the present invention more particularly relates to this new design of donor matrix useful for targeted genetic modification by homologous recombination which comprises at least two single-strand oligonucelotides wherein (i) the first linear single-strand oligonucleotide comprises a nucleic acid sequence homologous to a targeted genetic sequence of interest and (ii) the second linear single-strand oligonucleotide comprises complementary sequence to at least one portion of the first single-strand oligonucleotide such that the two single-strand oligonucleotides can hybridize together and recombine with said targeted genetic sequence of interest.
- the present invention also encompasses methods for targeted genetic modification by using the new designed matrix. These methods cover the different aspects of designing, synthesizing and hybridizing the oligonucleotides as a donor matrix, and of transfecting cells with said matrix according to the invention to perform homologous recombination at a selected genomic locus.
- the present invention more particularly relates to a new design of donor matrix useful for targeted genetic modification by homologous recombination.
- Said donor matrix more particularly comprises at least two single-strand oligonucelotides, wherein (i) the first linear single-strand oligonucleotide comprises a nucleic acid sequence homologous to a targeted genetic sequence of interest and (ii) the second linear single-strand oligonucleotide comprises a hybridization sequence to at least a portion of the first single-strand oligonucleotide such that the two single-strand oligonucleotides can hybridize together and recombine with said targeted genetic sequence of interest.
- homologous recombination refers to a conserved DNA maintenance pathway involved in the repair of double-stand breaks and other DNA lesions.
- an endogenous genetic sequence i.e. nucleic acid sequence initially present into the cell
- a donor matrix i.e. nucleic acid sequence initially present into the cell
- the genes present on the endogenous genetic sequence can be knocked-out, knocked-in, replaced, corrected or mutated, in a rational, precise and efficient manner.
- the process requires sequence homology between one sequence present on the donor matrix, referred to as homologous sequence, and the endogenous targeted genetic sequence of interest.
- homologous recombination is performed using two flanking sequences having identity with the endogenous sequence in order to make more precise integration as described in WO9011354.
- transfer can involve mismatch correction of heteroduplex nucleic acid that forms between the cleaved target and the donor matrix, and/or "synthesis-dependent strand annealing", in which the donor matrix is used to re-synthesize the genetic information that will become part of the target, and/or related processes.
- nucleic acid homologous sequence a nucleic acid sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 80% identity, preferably at least 90% identity and more preferably at least 95%, and even more preferably 98 % identity.
- Identity refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
- Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. It is recommended that the homologous sequence be at least 10 nucleotides in length.
- the oligonucleotides of the present invention are deemed to be at least 20 nucleotides, preferably at least 50, more preferably at least 100, and even more preferably, at least 150 nucleotides in length, while it is recommended the homologous sequence to be at least 30, preferably at least40 and more preferably at least 50 nucleotides in length.
- genetic sequence of interest any endogenous nucleic acid sequence, such as, for example a gene or a non-coding sequence within or adjacent to a gene, in which it is desirable modify by targeted cleavage and/or targeted homologous recombination.
- the sequence of interest can be present in a chromosome, an episome, an organellar genome such as mitochondrial or chloroplast genome or an infecting viral genome, for example.
- a sequence of interest can be within the coding sequence of a gene, within transcribed non-coding sequence such as, for example, leader sequences, trailer sequence or introns, or within non- transcribed sequence, either upstream or downstream of the coding sequence.
- each oligonucleotide that forms the donor matrix comprises a sequence that is complementary to one a portion of another single-strand oligonucleotide such that the two single-strand oligonucleotides can partially hybridize together.
- hybridization sequence is meant the sequence part of the oligonucleotide that can hybridize to one of the other oligonucleotides under standard low stringent conditions.
- Such conditions can be for instance at room temperature for 2 hours by using a buffer containing 25% formamide, 4x SSC, 50 mM NaH2P04 / Na2HP04 buffer; pH 7.0,5x Denhardt's, 1 mM EDTA,1 mg/ml DNA + 20 to 200 ng/ml probe to be tested (approx. 20 - 200 ng/ml)).
- This can be also predicted by standard calculation of hybridization using the number of complementary bases within the sequence and the content in G-C at room temperature as provided in the literature.
- the hybridization sequences are complementary to each other pursuant to the complementarity between two nucleic acid strands relying on Watson-Crick base pairing between the strands, i.e. the inherent base pairing between adenine and thymine (A-T) nucleotides and guanine and cytosine (G-C) nucleotides.
- A-T adenine and thymine
- G-C guanine and cytosine
- Accurate base pairing equates with Watson-Crick base pairing includes base pairing between standard and modified nucleosides and base pairing between modified nucleosides, where the modified nucleosides are capable of substituting for the appropriate standard nucleosides according to the Watson-Crick pairing.
- the complementary sequence of the single-strand oligonucleotide can be any length that supports specific and stable hybridization between the two single-strand oligonucleotides under the reaction conditions.
- the hybridization sequence generally authorizes a partial double stranded overlap between the two hybridized oligonucleotides over more than 10 bp, preferably more than to 20 bp, more preferably more than 40 bp, while the homologous sequences are generally less than 150 bp, preferably less than 100 bp, more preferably less than 50 bp in length.
- the hybridization sequence is advantageously selected not to be homologous to any sequence in the genome to avoid off-target recombination or recombination not involving the whole donor matrix (i.e. only one oligonucleotide).
- one oligonucleotide can hybridize another oligonucleotide to a limited portion thereof, which means that the oligonucleotides cannot hybridize to each other over more than 70% of their length, preferably no more than 60 %, even more preferably no more than 50%.
- the hybridized oligonucleotides generally overlap over less than 80% of their length, preferably over less than 70%, more preferably over less than 50%, even more preferably less than 40%, allowing a single strand extremity of at least 5 bp, preferably at least lObp, more preferably at least 20 bp, even more preferably more than 40 bp.
- modified nucleotide refers to a nucleotide that differs in structure from the standard or “unmodified” nucleotides 2 '-deoxy- adenosine, 2'-deoxy-thymidine, 2'-deoxy-cytidine and 2'-deoxy-guanosine, and that is capable of pairing with an unmodified nucleotide or a modified nucleotide.
- Non limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines).
- Modified nucleotide also include dideoxy nucleotides, 2'-0-methyl nucleotides, locked nucleic acids (LNA), peptides nucleic acids (PNA), and morpholinos.
- the nucleotides of the single-stranded nucleic acids may be linked by phosphodiester, phosphorodiamate bonds, or combinations thereof. Such modified nucleotides are useful for instance to introduce markers, tag, epitopes or reactive groups into the genome.
- the linear single-strand oligonucleotide can also comprise one or more phosphorothioatephosphodiester bonds between terminal base pairs to protect the linear donor polynucleotide from exonucleolytic degradation. These bonds may be in two or more positions at the 5' and/or 3' ends of the molecule and may be added during isolation or synthesis using standard methodology. See, e.g. (Ciafre, Rinaldi et al. 1995).
- Said single- strand oligonucleotide may comprise phosphodiester linkages, phosphorothiorate linkages, phosphoramidite linkages, phosphorodiamidate linkages, or combinations thereof.
- the donor matrix preferably comprises two homologous sequences, such that homologous recombination occurs at the genome locus of interest between the corresponding targeted genetic sequences.
- the donor matrix is advantageously composed of (1) at least one first single-strand oligonucleotide that comprises a first nucleic acid sequence homologous to a first endogenous sequence and (2) a second single-strand oligonucleotide that comprises a second nucleic acid sequence homologous to a second endogenous sequence, said second single-strand oligonucleotide comprising a sequence that can partially hybridize with the first single-strand oligonucleotide.
- the first and/or second single-strand oligonucleotides comprise a mutation of at least one nucleotide relative to the targeted genetic sequence of interest.
- Said mutation can be a substitution, a deletion, an insertion of at least one nucleotide, or combination thereof.
- the single-strand oligonucleotide can comprise one, two, three, four, five or more nucleotides changes. This is particularly useful for gene correction or repair as described in standard homologous recombination techniques.
- one of the linear single-strand oligonucleotide of said donor matrix comprises an exogenous sequence that comprises a coding sequence.
- the two or more linear single-strand oligonucleotides of said donor matrix comprise an exogenous sequence.
- the exogenous sequence may be included in one oligonucleotide or divided on several oligonucleotides to be reconstituted by hybridization of said oligonucleotides together forming the donor matrix. Accordingly the exogenous sequence can be encoded by the hybridization and/or the homologous sequences of the donor matrix.
- the exogenous sequence in the matrix is advantageously flanked by two homologous sequences to direct its targeted insertion.
- the exogenous sequence may comprise a sequence encoding a polypeptide (e.g., cDNA), a promoter sequence, an enhancer sequence, marker genes, cleavage enzyme recognition site, epitope tags.
- Marker genes can be sequences encoding protein that mediate antibiotic resistance, sequence encoding colored, fluorescent or luminescent proteins. The marker(s) allow(s) the selection of cells having inserted the exogenous sequence by homologous recombination at the targeted genetic sequence of interest.
- the new designed matrix has for advantages to be easily synthesized without resorting to cloning steps.
- Oligonucleotides sequences can be designed and chemically produced up to 200 nucleotides in a cost-effective way using a computer (Reese, Colin B. (2005). Oligo- and poly-nucleotides: 50 years of chemical synthesis. Organic & Biomolecular Chemistry3 (21): 3851. They are also deemed easier to transfect by the methods known in the art.
- the resulting matrix is not limited in length and may span an insertion exogenous DNA of more than 1 kb. Other advantages also arise with respect to therapeutic applications.
- the matrix according to the invention cannot self-replicate and barely contain sequences other than the sequences useful for insertion.
- the matrix is transiently present into the cells and does not allow gene expression by itself until it is inserted into the genome. This reduces potential side-effects, in particular random or off-target integration into the genome.
- oligonucleotides are more easily produced under GMP (good manufacturing practices) than plasmids, since these later require cloning and host cell techniques.
- the present invention has therefore therapeutic interest, especially in the field of gene therapy.
- the present invention relates to methods for targeted genetic modification by homologous recombination by using a donor matrix as previously described.
- Such methods more particularly comprise one or several of the following steps of: a) Synthesizing two single-strand oligonucleotides, wherein the first linear single- strand oligonucleotide comprises nucleic acid sequence homologous to a targeted endogenous genetic sequence and the second linear single-strand oligonucleotide comprises a complementary sequence to a portion of the first linear single-strand oligonucleotide;
- step b) of hybridizing the two single- strand oligonucleotides can take place after step c) of transfecting the cell with the oligonucleotides.
- a further step of induction of DNA cleavage by a rare-cutting endonuclease into the targeted endogenous nucleic acid sequence can be performed before the above step d) to improve the frequency of homologous recombination.
- the induced nucleic acid break can be repaired by homologous recombination using the matrix resulting into efficient targeted genetic modification.
- the present invention also relates to a method for targeted genetic modification using the matrix as previously described that further comprises expressing in the cell an engineered rare-cutting endonuclease being able to cleave the targeted genetic sequence of interest in the genome of a cell.
- the expression of a rare-cutting endonuclease in a cell can result from delivery of the rare- cutting endonuclease protein to a cell or by delivery of a nucleic acid encoding the rare- cutting endonuclease to a cell, wherein the nucleic acid is transcribed, and/or the transcript is translated to generate the rare-cutting endonuclease.
- Trans-splicing, nucleic acid cleavage, and nucleic acid ligation can also be involved in expression of a protein in a cell.
- the nucleic acid encoding the rare-cutting endonuclease can be DNA or RNA and is co-transfected along with the matrix.
- nucleic acid(s) or molecule(s) into a cell can be realized using any suitable methods known in the art.
- Suitable delivery means include microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids and delivery via liposomes, immunoliposomes, virosomes or artificial virions.
- rare-cutting endonuclease By “rare-cutting endonuclease” according to the present invention, it is referred to any wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule.
- any wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule.
- engineered endonucleases to successfully induce gene targeting has been well documented starting from straightforward experiments involving wild-type I-Scel to more refined work involving completely re-engineered enzyme (Stoddard, Monnat et al. 2007; Marcaida, Prieto et al. 2008; Galetto, Duchateau et al. 2009; Arnould, Delenda et al. 201 1).
- the endonuclease according to the present invention recognizes
- the rare-cutting endonuclease according to the invention can for example be a homing endonuclease also known as meganuclease (Paques and Duchateau 2007).
- homing endonucleases are well-known to the art (see e.g. (Stoddard, Monnat et al. 2007).
- Homing endonucleases recognize a nucleic acid target sequence and generate a single- or double- strand break.
- Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length.
- the homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease.
- Examples of such endonuclease include I-Sce I, I-Chu I, I-Cre I, I-Csm I, PI-Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, Pi-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I, PI- Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI- Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I,
- the homing endonuclease according to the invention is a LAGLIDADG endonuclease such as I-Scel, I-Crel, I-Ceul, I-Msol, and I-Dmol.
- said LAGLIDADG endonuclease is I-Crel. Wild-type I-Crel is a homodimeric homing endonuclease that is capable of cleaving a 22 to 24 bp double-stranded target sequence.
- I-Crel variants may be homodimers (meganuclease comprising two identical monomers) or heterodimers (meganuclease comprising two non-identical monomers). It is understood that the scope of the present invention also encompasses the I- Crel variants per se, including heterodimers (WO2006097854), obligate heterodimers (WO2008093249) and single chain meganucleases (WO03078619 and WO2009095793). The invention also encompasses hybrid variant per se composed of two monomers from different origins (WO03078619).
- Said rare-cutting endonuclease can also be an engineered nucleic acid binding domain fused to a nuclease catalytic domain.
- nuclease catalytic domain is intended the protein domain comprising the active site of an endonuclease enzyme.
- Such nuclease catalytic domain can be, for instance, a cleavage domain or a nickase domain.
- cleavage domain is intended a protein domain whose catalytic activity generates a Double Strand Break (DSB) in a DNA target.
- nickase domain is intended a protein domain whose catalytic activity generates a single strand break in a DNA target sequence.
- the catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, like for instance I-Tevl, ColE7, NucA and Fok-I.
- said rare-cutting endonuclease is a monomeric TALEN.
- a monomeric TALEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-Tevl described in WO2012138927.
- said rare-cutting endonuclease can be a "TALE-nuclease" resulting from the fusion of nucleic acid binding domain derived from a Transcription Activator like Effector (TALE) and to such endonuclease catalytic domain able to cleave a target genetic sequence of interest.
- TALE Transcription Activator like Effector
- Said Transcription Activator like Effector (TALE) corresponds to an engineered TALE comprising a plurality of TALE repeat sequences, each repeat comprising a RVD specific to each nucleotide base of a TALE recognition site.
- each TALE repeat sequence of said TALE is made of 30 to 42 amino acids, more preferably 33 or 34 wherein two critical amino acids (the so-called repeat variable dipeptide, RVD) located at positions 12 and 13 mediates the recognition of one nucleotide of said TALE binding site sequence; equivalent two critical amino acids can be located at positions other than 12 and 13 specially in TALE repeat sequence taller than 33 or 34 amino acids long.
- RVD repeat variable dipeptide
- RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A.
- critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity.
- other amino acid residues is intended any of the twenty natural amino acid residues or unnatural amino acids derivatives.
- said TALE of the present invention comprises between 8 and 30 TALE repeat sequences. More preferably, said TALE of the present invention comprises between 8 and 20 TALE repeat sequences; again more preferably 15 TALE repeat sequences.
- said TALE comprises an additional single truncated TALE repeat sequence made of 20 amino acids located at the C-terminus of said set of TALE repeat sequences, i.e. an additional C-terminal half- TALE repeat sequence.
- said TALE of the present invention comprises between 8.5 and 30.5 TALE repeat sequences, ".5" referring to previously mentioned half- TALE repeat sequence (or terminal RVD, or half- repeat). More preferably, said TALE of the present invention comprises between 8.5 and 20.5 TALE repeat sequences, again more preferably, 15.5 TALE repeat sequences.
- said half- TALE repeat sequence is in a TALE context which allows a lack of specificity of said half- TALE repeat sequence toward nucleotides A, C, G, T.
- said half- TALE repeat sequence is absent.
- said TALE of the present invention comprises TALE like repeat sequences of different origins.
- said TALE comprises TALE like repeat sequences originating from different naturally occurring TAL effectors.
- TALE-nuclease have been already used to stimulate gene targeting and gene modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010).
- Such engineered TAL-nucleases are commercially available under the trade name TALENTM (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).
- MBBD modular base-per-base specific nucleic acid binding domains
- endonucleases such as Fokl and Tevl .
- Said MBBD scan be engineered, for instance, from newly identified proteins, namely EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH from the recently sequenced genome of the endosymbiont fungi Burkholderia Rhizoxinica (Lackner, Moebius et al. 201 1).
- MBBBD proteins comprise modules of about 31 to 33 amino acids that are base specific.
- modules display less than 40 % sequence identity with Xanthomonas TALE common repeats, whereas they display more polypeptide sequence variability. When they are assembled together, these modular polypeptides can although target specific nucleic acid sequences in a quite similar fashion as Xanthomonas TAL-nucleases.
- the rare-cutting endonuclease according to the invention can also be a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the nuclease catalytic domain of an endonuclease such as Fokl (Porteus and Carroll, 2005)(Eisenschmidt, Lanio et al. 2005 ; Arimondo, Thomas et al. 2006; Simon, Cannata et al. 2008; Cannata, Brunei et al. 2008 ).
- ZFN Zinc-Finger nuclease
- said rare-cutting endonuclease according to the present invention can be a Cas9 endonuclease.
- RNA-guided Cas9 nuclease Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013
- CRISPR Clustered Regularly Interspaced Short palindromic Repeats
- CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid.
- CRISPR-mediated genome engineering first proceeds by the selection of target sequence often flanked by a short sequence motif, referred as the proto-spacer adjacent motif (PAM).
- PAM proto-spacer adjacent motif
- a specific crRNA complementary to this target sequence is engineered.
- Trans-activating crRNA (tracrRNA) required in the CRISPR type II systems paired to the crRNA and bound to the provided Cas9 protein.
- Cas9 acts as a molecular anchor facilitating the base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al. 201 1).
- the dual tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9 to the cognate target sequence.
- Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA.
- DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif - PAM).
- Cas9 subsequently introduces a blunt double strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).
- the invention encompasses both wild-type and variant rare-cutting endonucleases. It is understood that, rare-cutting endonuclease according to the present invention can also comprise single or plural additional amino acid substitutions or amino acid insertion or amino acid deletion introduced by mutagenesis process well known in the art. In the frame of the present invention, such variant endonucleases remain functional, i.e. they retain the capacity of recognizing and specifically cleaving a target sequence.
- rare-cutting endonuclease variants which present a sequence with high percentage of identity or high percentage of homology with sequences of rare-cutting endonuclease described in the present application, at nucleotidic or polypeptidic levels.
- high percentage of identity or high percentage of homology it is intended 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95, more preferably 97%, more preferably 99% or any integer comprised between 70% and 99% identity.
- Cells can be any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.
- primary cell or “primary cells” are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. These cells thus represent a more valuable model to the in vivo state they refer to.
- fungus refers to a fungal, plant, algal or animal cell or a cell line derived from the organisms listed below and established for in vitro culture. More preferably, the fungus is of the genus Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces or Pichia; More preferably, the fungus is of the species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum, Penicillium citrinum, Acremonium Chrysogenum, Trichoderma reesei, Mortierella alpine, Chrysosporium lucknowense, Kluyveromyceslactis, Pichia pastoris or Pichia ciferrii.
- the plant is of the genus Arabidospis, Nicotiana, Solanum, lactuca, Brassica, Oryza, Asparagus, Pisum, Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis, Citrus, Sorghum; More preferably, the plant is of the species Arabidospis thaliana, Nicotiana tabaccum, Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Solanum esculentum, Lactuca saliva, Brassica napus, Brassica oleracea, Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus officinalis, Pisumsativum, Medicago sativa, zea mays, Hordeum vulgare, Secale cereal, Triticuma estivum, Triticum durum, Capsicum sativus, Cucur
- the animal cell is of the genus Homo, Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus, Meleagris, Drosophila, Caenorhabditis; more preferably, the animal cell is of the species Homo sapiens, Rattus norvegicus, Mus musculus, Sus scrofa, Bos taurus, Danio rerio, Canis lupus, Felis catus, Equus caballus, Salmo salar, Oncorhynchus mykiss, Gallus gallus, Meleagris gallopavo, Drosophila melanogaster, Caenorhabditis elegans.
- the cell is preferably a plant cell, a mammalian cell, a fish cell, an insect cell or cell lines derived from these organisms for in vitro cultures or primary cells taken directly from living tissue and established for in vitro culture.
- cell lines can be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-1 16 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
- All these cell lines can be modified by the method of the present invention to provide cell line models to produce, express, quantify, detect, study a gene or a protein of interest; these models can also be used to screen biologically active molecules of interest in research and production and various fields such as chemical, biofuels, therapeutics and agronomy as non- limiting examples.
- a particular aspect of the present invention relates to an isolated cell as previously described transfected with the oligonucleotides that form the donor matrix according to the invention.
- said isolated cell comprises a donor matrix comprising at least two hybridized single-strand oligonucleotides of at least 20 nucleotides wherein the first linear single-strand oligonuclotide comprises nucleic acid sequence homologous to a targeted genetic sequence of interest and the second linear single-strand oligonucleotide comprises a complementary sequence to a portion of the first linear single-strand oligonucleotide.
- said cell according to the invention comprises at least one modified targeted genetic sequence of interest, wherein said targeted genetic sequence of interest is modified by said matrix according to the method previously described.
- the resulting modified cell can be used as a cell line for a diversity of applications ranging from bioproduction, animal transgenesis (by using for instance stem cells), plant transgenesis (by using for instance protoplasts), to cell therapy (by using for instance T-cells).
- the present invention thus expands to the cells lines, transgenic animals or plants resulting from the transformation of such cells according to the invention, as well as to the therapies that may be applied therefrom.
- the method of targeted genetic modification disclosed herein can have a variety of applications.
- the method can be used for clinical or therapeutic applications.
- the method can be used to repair or correct disease-causing genes, as for example a single nucleotide change in sickle-cell disease.
- the method can be used to correct splice junction mutations, deletions, insertions, and the like in other genes or chromosomal sequences that play a role in a particular disease or disease state.
- the present invention also encompasses transgenic animals or plants which comprises modified targeted genetic sequence of interest by the methods described above.
- the present invention also concerns a kit to perform the targeted genetic modification by homologous recombination in a cell according to the present invention.
- This kit comprises the donor matrix as previously described, which is typically formed by at least two single-strand oligonucleotides of at least 20 nucleotides wherein the first linear single-strand oligonuclotide comprises nucleic acid sequence homologous to a targeted genetic sequence of interest and the second linear single-strand oligonucleotide comprises a complementary sequence to a portion of the first linear single-strand oligonucleotide.
- first and second oligonucleotides according to the invention may be any of those described in the present application.
- said kit further comprises a nucleic acid encoding for a rare- cutting endonuclease which is able to cleave the genetic sequence of interest and activate the homologous recombination.
- This nucleic acid can be under a suitable form for transfection and expression in the selected host cell.
- Said nucleic acid may encode any rare-cutting endonuclease described in the present application.
- the kit according to the invention can be used for therapeutic purposes, in particular for treating genetic diseases.
- Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine.
- r represents g or a (purine nucleotides)
- k represents g or t
- s represents g or c
- w represents a or t
- m represents a or c
- y represents t or c (pyrimidine nucleotides)
- d represents g, a or t
- v represents g, a or c
- b represents g, t or c
- h represents a, t or c
- n represents g, a, t or c.
- nucleic acid refers to deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
- the terms can encompass known modified nucleotides.
- gene is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein.
- a gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region.
- the gene may further comprise a terminator, enhancers and/or silencers.
- exogenous sequence is meant a sequence encoding a polypeptide intended to be introduced into a cell, tissue or organism by recombinant technologies.
- the polypeptide encoded by the exogenous sequence is either not expressed, or expressed but not biologically active, in the cell, tissue or organism in which the exogenous sequence is inserted.
- mutation is intended the substitution, the deletion, and/or the addition of one or more nucleotides/amino acids in a nucleic acid/amino acid sequence.
- Identity refers to sequence identity between two nucleic acid molecules or polypeptides.
- a polynucleotide having a sequence at least, for example, 95% “identical” to a query sequence of the present invention it is intended that the sequence of the polynucleotide is identical to the query sequence except that the sequence may include up to five nucleotide alterations per each 100 nucleotides of the query sequence.
- up to 5% (5 of 100) of the nucleotides of the sequence may be inserted, deleted, or substituted with another nucleotide.
- the « needle » program which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch 1970) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used.
- the needle program is for example available on the ebi.ac.uk world wide web site.
- the percentage of identity in accordance with the invention is preferably calculated using the EMBOSS ::needle (global) program with a "Gap Open” parameter equal to 10.0, a "Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.
- delivery vector or “ delivery vectors” is intended any delivery vector which can be used in the present invention to put into cell contact or deliver inside cells or subcellular compartments agents/chemicals and molecules (proteins or nucleic acids) needed in the present invention. It includes, but is not limited to, transducing vectors, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other appropriate transfer vectors. These delivery vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids. These delivery vectors are molecule carriers.
- vector refer more particularly to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- a “vector” in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids.
- Preferred 5 vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
- Viral vectors include retrovirus, adenovirus, parvovirus (e. g., adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.
- parvovirus e. g., adenoassociated viruses
- coronavirus e. g., negative strand RNA viruses
- negative RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and
- Herpes Simplex virus types 1 and 2 Epstein-Barr virus, cytomegalovirus
- poxvirus e.g., vaccinia, fowlpox and canarypox
- Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
- retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retro viridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. 20 N.
- One type of preferred vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication.
- Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.
- a vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolatereductase, hygromycinphosphotransferase, herpes simplex, virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthineguanine, phosphoribosyltransferase for eukaryotic cell culture; TRP1 for S. cerevisiae; tetracyclin, rifampicin or ampicillin resistance in E. coli.
- selectable markers for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolatereductase, hygromycinphosphotransferase, herpes simplex, virus thymidine kinase, adenosine deaminase,
- said vectors are expression vectors, wherein a sequence encoding a polypeptide of interest is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said polypeptide. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome binding site, a RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer or silencer elements. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Suitable promoters include tissue specific and/or inducible promoters.
- inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-p-d-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature.
- tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), a-antitrypsin protease, human surfactant (SP) A and B proteins, ⁇ -casein and acidic whey protein genes. Delivery vectors and vectors can be associated or combined with any cellular permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques facilitating contact with or entry inside cells of the molecules needed in the present invention.
- FIG. 1 Targeted genetic modification by homologous recombination using new design of donor matrix.
- the donor matrix according to the invention is composed of the 1 hybridization of two linear single-strand oligonucleotides.
- the first and the second oligonucleotides comprise an exogenous sequence (white box) that is flanked by two regions sharing homology with targeted endogenous gene (black and grey regions).
- the oligonucleotides of the donor matrix are partially hybridized and single stranded at their extremities, homologous recombination occurs between endogenous gene and the donor matrix resulting into the integration of exogenous sequence in the targeted endogenous gene.
- FIG. 2 Example of donor matrix design: The donor matrix is composed of the hybridization of two oligonucleotides of 161 nucleotides (SEQ ID NOS: 1 and 2), sharing 48 nucleotides of complementary sequence (framed). Inside of complementary sequence, there is an exogenous sequence (bold underlined) allowing the identification of homologous gene targeting event.
- Each oligonucleotide is partially homologous to the targeted sequence.
- the hybridization of these oligonucleotides allows us to generate a molecular partially double-stranded but with a homology sequence almost twice larger than the single strand oligonucleotides.
- 293 H cells were co-transfected with both TALE nucleases RAGT2 encoding by the plasmids pCLS12957 (SEQ ID NO: 4) and pCLS12958 (SEQ ID NO: 5) designed to target the DNA sequence 5 '-TATATTTAAGCACTTATatgtgtgtaacaggtATAAGTAACCATAAACA-3 ' (SEQ ID NO: 6) and the donor matrix described above.
- the percentage of homologous gene targeting (HGT) was evaluated using specific PCR.
- oligonucleotides of 161 nucleotides were used: RAGT2_01igo_F (SEQ ID NO: 1) and RAGT2_01igo_R (SEQ ID NO: 2).
- Each oligonucleotide consists of 132 nucleotides of nucleic acid sequence homologous to the RAG1 locus and 29 nucleotides of exogenous sequences.
- the complementary sequence contains 48 nucleotides including the 29 nucleotides of exogenous sequences ( Figure 1).
- the oligonucleotides were resuspended in H20 at the concentration of 20pmoles ⁇ l and diluted 1 :50 in TE buffer (Tris lOmM, EDTA ImM).
- ⁇ of each oligonucleotide was mixed in presence of 20 ⁇ 1 of annealing buffer 10X (Tris lOmM, EDTA ImM and NaCl 0.1M) and heated to 95°C. The solution was then cooled at 25 °C on the bench.
- annealing buffer 10X Tris lOmM, EDTA ImM and NaCl 0.1M
- the human 293H cells were plated at a density of 1.2 x 10 6 cells per 10 cm dish in complete medium (DMEM supplemented with 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 ⁇ g/ml), amphotericin B (Fongizone: 0.25 ⁇ g/ml, Invitrogen-Life Science) and 10% FBS).
- complete medium DMEM supplemented with 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 ⁇ g/ml), amphotericin B (Fongizone: 0.25 ⁇ g/ml, Invitrogen-Life Science) and 10% FBS.
- DNA extraction was performed with the ZR-96 genomic DNA kit (Zymo research) according to the supplier's protocol.
- the detection of targeted integration is performed by specific PCR amplification using a primer located within the heterologous insert of the donor matrix (SEQ ID NO: 9), and another one located on the genomic sequence outside of the homology (SEQ ID NO: 10).
- the percentage of HGT event was then determined by the number of positive wells corrected by the plating efficiency (0.33) and transfection efficacy comprises between 14 and 48%.
- RNA and host factor RNase III Nature 471(7340): 602-7.
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Abstract
La présente invention concerne une nouvelle conception d'une matrice de donneurs pour effectuer une recombinaison homologue dans des cellules, ladite matrice étant constituée d'oligonucléotides à brun unique qui s'hybrident partiellement les uns avec les autres. L'invention concerne également des méthodes pour la conception et l'utilisation d'une telle matrice pour obtenir des cellules génétiquement modifiées.
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Cited By (5)
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WO2017205650A1 (fr) * | 2016-05-27 | 2017-11-30 | Life Technologies Corporation | Compositions et méthodes pour améliorer la recombinaison homologue |
WO2018108987A1 (fr) | 2016-12-14 | 2018-06-21 | Biological Research Centre, Hungarian Academy Of Sciences | Mutagénisation d'acides nucléiques intracellulaires |
EP3781677A4 (fr) * | 2018-04-16 | 2022-01-19 | University of Massachusetts | Compositions et méthodes pour l'édition génétique améliorée |
WO2022243531A1 (fr) * | 2021-05-20 | 2022-11-24 | Cellectis S.A. | Thérapie génique pour le traitement d'une immunodéficience combinée sévère (scid) liée à rag1 |
US11873487B2 (en) | 2019-06-20 | 2024-01-16 | University Of Massachusetts | Compositions and methods for improved gene editing |
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WO2017205650A1 (fr) * | 2016-05-27 | 2017-11-30 | Life Technologies Corporation | Compositions et méthodes pour améliorer la recombinaison homologue |
US10428327B2 (en) | 2016-05-27 | 2019-10-01 | Life Technologies Corporation | Compositions and methods for enhancing homologous recombination |
EP3872177A1 (fr) * | 2016-05-27 | 2021-09-01 | Life Technologies Corporation | Compositions et méthodes pour améliorer la recombinaison homologue |
WO2018108987A1 (fr) | 2016-12-14 | 2018-06-21 | Biological Research Centre, Hungarian Academy Of Sciences | Mutagénisation d'acides nucléiques intracellulaires |
US10669537B2 (en) | 2016-12-14 | 2020-06-02 | Biological Research Centre | Mutagenizing intracellular nucleic acids |
EP3781677A4 (fr) * | 2018-04-16 | 2022-01-19 | University of Massachusetts | Compositions et méthodes pour l'édition génétique améliorée |
US11981892B2 (en) | 2018-04-16 | 2024-05-14 | University Of Massachusetts | Compositions and methods for improved gene editing |
US11873487B2 (en) | 2019-06-20 | 2024-01-16 | University Of Massachusetts | Compositions and methods for improved gene editing |
WO2022243531A1 (fr) * | 2021-05-20 | 2022-11-24 | Cellectis S.A. | Thérapie génique pour le traitement d'une immunodéficience combinée sévère (scid) liée à rag1 |
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