WO2015115903A1 - Édition du génome induite par rupture d'adn de restriction à l'aide de nucléases génétiquement modifiées - Google Patents

Édition du génome induite par rupture d'adn de restriction à l'aide de nucléases génétiquement modifiées Download PDF

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WO2015115903A1
WO2015115903A1 PCT/NL2015/050072 NL2015050072W WO2015115903A1 WO 2015115903 A1 WO2015115903 A1 WO 2015115903A1 NL 2015050072 W NL2015050072 W NL 2015050072W WO 2015115903 A1 WO2015115903 A1 WO 2015115903A1
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nucleic acid
dna
donor
construct
cells
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Manuel António Faria Viola GONÇALVES
Maarten HOLKERS
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Academisch Ziekenhuis Leiden H.O.D.N. Lumc
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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

Definitions

  • the invention relates to the fields of molecular biology and genetic engineering and gene therapy, in particular DNA break-induced genetic
  • HR Homologous recombination
  • HR is used by eukaryotes for, e.g. proper chromosomal segregation during meiotic division and repair of harmful double- strand DNA breaks.
  • HR is generally defined as an exchange of homologous segments between two DNA molecules.
  • HR provides methods for genetically modifying chromosomal DNA sequences by introducing e.g. small mutations or exogenous nucleotide sequences.
  • a microorganism or a eukaryotic cell is transformed with an exogenous DNA sequence.
  • the center of the exogenous nucleotide sequence contains the desired DNA sequence, which is flanked by segments of homology with the cell's chromosomal DNA.
  • the exogenous DNA is introduced into the cell and recombines into the cell's DNA.
  • chromosomal positions together with the introduction of donor DNA containing sequences similar to those flanking a genomic target sequence at which a site- specific DSB is induced, can increase gene targeting by several orders of
  • a problem with the present HR-based genome editing strategies is the number of unwarranted and unpredictable illegitimate recombination events that occur concomitantly with the, often fewer, precise and targeted DNA modifying events.
  • these illegitimate recombination-derived events such as those derived from the engagement of the error-prone non-homologous end-joining DNA repair pathway, the exogenous donor DNA molecule that was provided into the cell is inserted at a different (i.e. off-target) chromosomal position, or is introduced in the wrong way into the target site of the cell's genome.
  • Illegitimate recombination is not desired because it negates the safety advantage associated with the precision of the HR event.
  • the present inventors found that the nature of the HR vector influences parameters underlying reliable genome editing. It was shown that introducing donor DNA using a capped linear replication incompetent construct reduces the ratio of illegitimate to legitimate recombination. It also reduces the incidence of incorrect DNA inserts. In particular it was found that such a capped construct provides for exceptional levels of site-specific and accurate DNA editing. It was further found that this exceptional specificity and accuracy stems from the embedding of donor DNA sequences within capped vectors.
  • the present inventors assessed the specificity and the accuracy of exogenous DNA insertion using the standard viral vector for the delivery of donor DNA into mammalian cells using HR, the integrase- defective lentiviral vector (IDLV) as well as the specificity and the accuracy of exogenous DNA insertion resulting from widely used non-viral vector systems, i.e., both linear and circular donor DNA plasmids.
  • IDLV integrase- defective lentiviral vector
  • the effect of using three different types of designer sequence-specific nucleases to induce double-stranded DNA breaks was evaluated.
  • human cells exposed to IDLV- containing donor DNA Fig.
  • rAAV recombinant ade no- associated viral vectors
  • Illegitimate recombination causing both the occurrence of donor DNA insertion at a chromosomal site other than the target site and insertion of an incorrect donor DNA sequence at the target site, are disadvantageous and unpredictable events.
  • Disadvantages include disruption of open reading frames (ORFs) in the genomic sequence, inserts that do not restore endogenous ORFs or do not yield sufficient transgene expression and unpredictable and/or excess expression levels of transgenes.
  • Improper repair of DSBs may lead to chromosomal aberrations such as translocations, deletions, inversions, amplifications or to mutations. All these events may contribute to cell dysfunction, cell death, or tumor formation.
  • capped vectors are used for delivering exogenous DNA, specificity and accuracy is greatly improved.
  • Analysis of 110 randomly selected myoblast clones revealed that no illegitimate recombination had occurred at all. All clones contained the exogenous DNA insert at the AAVSl target site (Fig. 5d and Fig. 5e).
  • Gene targeting in protein-capped adenoviral vector-modified cells was also confirmed by Southern blot analyses (Fig. 6).
  • the inserts were correctly integrated as evidenced by HR- derived exogenous DNA-target site junctions at both termini (Fig. 7d and Fig. 7e).
  • adenoviral vector-modified myoblasts Fig. 5f
  • adenoviral vector-modified HeLa cell populations Fig. 5c
  • the present invention demonstrates that using such capped vector, a HR specificity and accuracy of 100% can be achieved.
  • the specific and accurate adenoviral vector-mediated gene targeting process stems from using capped linear donor DNA (Fig. 8 and Fig.9).
  • the high specificity and accuracy is observed both when TALENs and the RN A- guided nuclease (RGN) system are used for
  • the reduction in the frequency of illegitimate recombination obtained in accordance with the invention is important at least at two levels: (i) reducing the frequency of chromosomal off-target or random insertion events of the donor DNA and (ii) preventing or minimizing the formation of ORF- disruptive forms of the inserted exogenous DNA (e.g. donor-target site DNA junction(s) arising from illegitimate recombination as opposed to error-free HR events) or also ORF- disruptive concatemeric forms ("footprints”) that lead to heterogeneous and/or unpredictable expression levels in stably- transduced cell populations.
  • ORF- disruptive forms of the inserted exogenous DNA e.g. donor-target site DNA junction(s) arising from illegitimate recombination as opposed to error-free HR events
  • footprints ORF- disruptive concatemeric forms
  • the invention therefore provides a method for modifying a target sequence in a genome of a cell comprising:
  • a linear replication incompetent construct comprising: i) a first nucleic acid sequence which is homologous to a first region of said target sequence,
  • a molecule attached to at least one terminus of said construct, whereby said first region is located on one side of said site of interest and said second region is located on the other side of said site of interest.
  • the invention further provides a linear replication incompetent construct comprising:
  • a targeting region comprising:
  • iii) optionally a donor nucleic acid sequence located between said first and second nucleic acid sequences; and - a molecule attached to at least one terminus of said construct.
  • the invention further provides a set of constructs, comprising:
  • - a second construct comprising a nucleic acid sequence encoding an endonuclease capable of inducing a DNA break in a genome of a cell.
  • the invention further provides the use of a construct according to the invention for modifying a target sequence in a genome of a cell.
  • the invention further provides the use of a set of constructs according to the invention for modifying a target sequence in a genome of a cell.
  • a "target sequence” as used herein refers to a sequence in the genome of a cell of which the genomic sequence is to be modified.
  • a "site of interest” as used herein refers to a genomic location in which a DNA break is to be introduced. Said DNA break can be a single-stranded or a double-stranded DNA break.
  • a preferred site of interest is a genomic location in which a double-stranded DNA break is to be introduced.
  • the site of interest is also referred to as target site and preferably comprises a recognition site for an endonuclease which induces a DNA break at the target site.
  • the DNA break can be a single-stranded or double-stranded DNA break. In a preferred embodiment the DNA break is a double-stranded DNA break.
  • a preferred target sequence for modification in accordance with a method of the invention is a safe harbor locus.
  • Preferred, but non-limiting target loci are AAVS1, CCR5, CCR2, the ROSA26 locus, DMD21, FUT8 and SH6 as well as house-keeping genes such as HPRT1, GAPDH and DHFR.
  • safe harbor locus refers to a locus that is generally accepted to be a locus that allows safe and stable insertion and expression of a transgene. Such genomic safe harbors can be in intragenic as well as extragenic regions in a genome of a cell.
  • a preferred safe harbor locus is a genomic locus that fulfils the following criteria: i) a distance of > 50 kb from the 5' terminus of any gene, ii) a distance of >300 kb from cancer-related genes, iii) a distance of > 300 bp from any microRNA; iv) the locus is located outside a gene transcription unit, and v) the locus is located outside an ultra-conserved region. These criteria are described in ref. [2] .
  • Use of a safe harbor as target sequence is particularly suitable for use in targeted gene addition in the present invention, i.e. methods wherein a specific exogenous DNA molecule such as a gene is inserted in the target cell's genome.
  • genes underlying a genetic disease are genes underlying a genetic disease.
  • the term "genetic disease” or “genetic disorder” refers to a pathological condition that is directly or indirectly caused by or associated with at least one genetic mutation.
  • a mutation refers to a nucleotide change, such as a single or multiple nucleotide replacement, deletion or insertion, in a nucleotide sequence.
  • Genomic DNA that contains a mutation has a nucleotide sequence that is different in sequence from that of the corresponding wild- type genomic DNA.
  • the target sequence can be any gene or genomic sequence of a cell, such as a cell of a microorganism, plant or an animal.
  • a method of modifying a target sequence in a genome of a cell according to the invention can be performed in vitro, in vivo or ex vivo.
  • ex vivo as used herein is meant that a method of the invention is performed in cells that have been removed from the body of an individual.
  • Modifying as used herein is also referred to as "altering” and means a replacement of one or more nucleotides with one or more other nucleotides, the insertion of one or more nucleotides, and/or the deletion of one or more nucleotides, or a combination thereof, within the target sequence in the genome of a cell.
  • a modification of a genomic sequence is preferably a replacement of one or more nucleotides within a gene.
  • gene refers to a nucleic acid sequence that undergoes transcription and that includes coding sequences necessary for the production of a protein, (polypeptide or RNA.
  • a gene may encode a particular protein or (poly)peptide, or code for an RNA sequence that is of interest in itself, such as an antisense inhibitor.
  • Replacement of one or more nucleotides within a gene is particularly suitable for gene repair, preferably for gene repair of a genetic mutation, such as a genetic mutation that is directly or indirectly associated with a genetic disease.
  • Another preferred modification is the in situ editing of the sequence of an endogenous target gene to alter or to expand the function of that said gene, e.g.
  • an heterologous sequence coding for a tag such as a fluorescent poly(peptide) for the live-cell spatio-temporal tracing and quantification of endogenous gene expression and (ii) changing the biochemical properties of the product encoded by the said target gene.
  • Another preferred modification is insertion of one or more nucleotides, preferably of a gene.
  • Such application is particularly suitable for targeted gene addition, preferably for methods wherein a specific exogenous DNA molecule such as a gene or an artificially designed transcriptional unit is inserted in the target cell's genome in order to achieve expression of said exogenous DNA molecule.
  • transcriptional unit refers to a nucleic acid molecule comprising a sequence of at least a promoter, a protein- and/or RNA-coding sequence and a transcription termination signal such as a polyadenylation signal.
  • construct refers to an artificially constructed segment of nucleic acid, such as a vector or plasmid.
  • a circular plasmid can be linearized by treatment with a suitable restriction enzyme based on the nucleotide sequence of the plasmid.
  • suitable linear constructs are viral vectors based on adenoviruses, herpes viruses, adeno-associated viruses, retroviruses, vaccinia virus and bacteriophages, preferably bacteriophages that replicate via a protein-primed DNA replication mechanism, such as Phi29, Bam 35, Nf, PRD1 or Cp-1.
  • Non-limiting examples of suitable non-viral constructs are in vitro-cwpTped (synthetic or recombinant) linear nucleic acid molecules, including in vitro-cwpped linear nucleic acid molecules generated by terminal protein-primed DNA replication, and protein-capped linear plasmids such as recombinant linear plasmids derived from Streptomyces such as described in ref. [3].
  • Generation of linear nucleic acid molecules by terminal protein-primed DNA replication involves the use of a protein as primer for DNA synthesis.
  • the protein is generally named terminal protein (TP) and becomes covalently attached at the 5' termini of the DNA.
  • TP terminal protein
  • a preferred linear replication incompetent construct is a double-stranded linear replication incompetent construct.
  • a preferred linear vector is a linear replication incompetent viral vector, more preferably selected from the group consisting of an adenoviral vector, a herpes viral vector, an adeno-associated viral vector, a retroviral vector, a vaccinia viral vector and a bacteriophage viral vector, such as those based on Phi29, Bam 35, Nf, PRD1 or Cp-1.
  • a more preferred linear vector is a double -stranded linear replication incompetent viral vector.
  • a preferred viral vector is an adenoviral vector.
  • Adenoviruses are non- enveloped DNA viruses.
  • the adenovirus genome is a linear double-stranded DNA molecule of approximately 36 kilobases (kb).
  • the packaged adenoviral DNA molecule has a 55 kiloDalton (kDa) terminal protein covalently bound to the 5' terminus of each strand.
  • the adenoviral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication and the late phase which is the period during which viral DNA replication occurs.
  • the early gene products are expressed during the early phase. Functions of these early genes include the preparation of the host cell for synthesis of viral structural proteins.
  • the early gene products are encoded by regions El, E2, E3 and E4 in the adenoviral genome. Late gene products are expressed in addition to the early gene products during the late phase during which nucleic acid and protein synthesis of the host cell are turned off. The host cell thus becomes committed to the production of adenoviral DNA and adenoviral proteins.
  • Advantages of adenoviral vectors include their ability to infect both dividing and non-dividing cells, accommodation of up to 38 kb of foreign DNA and the fact that adenoviral DNA does not normally integrate into the host cell genome. As a result of the latter, the transferred gene effect will be transient because the adenoviral and foreign DNA will be lost with continued division of host/target cells.
  • Adenovirus -based vectors are being widely investigated for use in vaccination protocols and as anti-cancer gene therapies.
  • Such adenoviral vectors are based on recombinant adenoviruses that are either replication-incompetent or replication competent.
  • Replication-incompetent adenoviral vectors have a number of characteristics that are disadvantageous for use in therapy. For instance, the generation of replication-incompetent adenoviral vectors requires the use of a complementing cell line that provides the deleted protein or proteins in trans. Replication-competent adenoviral vectors lyse host cells that are infected by the vector. For use as anti-cancer agents, replication-competent adenoviral vectors are advantageous because replication and spreading of the viral vector throughout the tumor occurs. Similarly, replication and spreading of the adenoviral vector for expression of transgenes is advantageous in order to achieve high amounts of expressed foreign protein.
  • Tumor specific promoters can be used that cause the virus to replicate and consequently exert the cytotoxic effect of the therapeutic protein specifically in tumorous tissue.
  • the use of replication competent adenoviral vectors then serve to deliver the vector carrying the therapeutic gene to as many cells as possible while the expression of the therapeutic protein is restricted to cells having the tumor specific promoter.
  • a linear construct used in accordance with the invention is preferably replication incompetent.
  • replication incompetent also called replication defective or non-replicating, refers to a construct that is not capable of replicating itself.
  • the replication incompetent construct is preferably a vector, more preferably a viral vector, that as a result of gene deletions in its genome is incapable of replication by itself.
  • Replication incompetent adenoviruses typically lack one or more of the early region genes.
  • An adenoviral vector can for instance be made replication incompetent by deletions in the early-region 1 E1A and E1B genes, collectively referred to as El, of the adenoviral genome.
  • the E2A protein may induce an immune response and because it plays a key role in the switch to the synthesis of late adenovirus proteins and in the viral DNA replication process.
  • the E3 genes may also be deleted because they are not essential for virus replication in cultured packaging cells.
  • a preferred linear replication incompetent construct used in accordance with the invention is a linear replication incompetent adenoviral vector having the E1A and E1B genes deleted and optionally the E2 and/or the E3 genes deleted as well.
  • Particularly preferred is a so-called “minimal” adenoviral vector, which is also referred to as “gutless”, “gutted”, high-capacity, gene-deleted or helper-dependent adenoviral vector. From the parental wild-type adenovirus genome, such vector comprises exclusively the cis-acting inverted terminal repeats (ITR) and the packaging signal.
  • ITR inverted terminal repeats
  • Replication incompetent viral vectors can be produced in packaging cells.
  • "Packaging cell” as used herein refers to a cell that expresses in trans the required genes necessary for production of infectious viruses that are lacking in the viral backbone.
  • the in trans required proteins are preferably expressed from genetic elements that are integrated into the genome of the packaging cell.
  • the genetic elements typically comprise the coding region for the viral proteins.
  • the genetic elements preferably have essentially no overlapping nucleic acid sequences with the replication incompetent linear viral vector. This prevents the generation of replication competent viruses due to homologous DNA sequences present in the vector and in the packaging cells.
  • packaging cells for the production of replication incompetent constructs are 293, HepG2, CHO, BHK, Sf9, Sf 21 , 293, 293T-derived cpNX, BTI-Tn 5 B 1-4, COS, N1H/3T3, Vero, CV1, NSO, PER.C6 [5], N52.E6 [6] and PER.E2A [7] cells.
  • Packaging cells specifically designed to reduce or essentially prevent homologous recombination between the vector and packaging cell sequences are, for instance, PER.C6, N52.E6 and PER.E2A.
  • virus particle comprising a linear replication incompetent construct according to the invention.
  • Said viral particle preferably comprises a linear replication incompetent viral vector comprising a molecule, preferably a peptide, polypeptide or protein, more preferably a peptide, polypeptide or protein of between 1 and 100 kDa, attached to one or both termini of the said linear construct, e.g. the 3' terminus and/or the 5' terminus, preferably to the 5' terminus of each strand.
  • said virus particle is a double-stranded DNA-containing virus particle, more preferably an adenovirus particle.
  • Such virus particle is particularly suitable for use in a method in accordance with the invention, e.g.
  • exogenous nucleic acid sequence refers to a nucleic acid fragment that is introduced into a cell, preferably into the genome of a cell.
  • An exogenous nucleic acid sequence as used herein can be either a nucleic acid sequence that is not normally present in said cell or that is essentially identical to a nucleic acid sequence that is endogenous to said cell.
  • kit of parts comprising a virus particle comprising a linear replication incompetent construct according to the invention and instructions for use.
  • a linear replication incompetent construct used in a method of the invention comprises a first nucleic acid sequence which is homologous to a first region of said target sequence and a second nucleic acid sequence which is homologous to a second region of said target sequence.
  • first and second nucleic acid sequences are herein also referred to as "first and second homologous nucleic acid sequences”.
  • a nucleic acid sequence preferably comprises a chain of nucleotides, more preferably DNA and/or RNA, most preferably DNA.
  • the first and second regions of the target sequence are herein also referred to as "first and second homologous regions”.
  • homologous refers to a nucleic acid sequence with enough identity in nucleotides between the first or second nucleic acid sequence and the first or second regions of the target sequence, respectively, to enable HR between sequences.
  • homologous sequences have at least 95% sequence identity, more preferably at least 97% sequence identity, more preferably at least 98% sequence identity and more preferably at least 99% sequence identity.
  • a most preferred first nucleic acid sequence is identical to a first region of the target sequence and a most preferred second nucleic acid sequence is identical to a second region of the target sequence.
  • the percentage of identity of a nucleic acid sequence is defined herein as the percentage of residues in a nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and without introducing gaps. Methods and computer programs for the alignment are well known in the art, for example "Align 2".
  • the first and second homologous nucleic acid sequences enable HR to occur between the linear replication incompetent construct and the target sequence in a genome of a cell. If a donor nucleic acid is present, the first and second homologous nucleic acid sequences are located on opposite sides of the donor nucleic acid sequence, which means that the first homologous nucleic acid sequence is located on one side of the donor nucleic acid sequence and the second homologous nucleic acid sequence is located on the other side of the donor nucleic acid sequence.
  • the first and second homologous regions of the target sequence are preferably located on opposite sides of the site of interest in the target sequence, which means that the first homologous region is located on one side of the site of interest and the second homologous region is located on the other or opposite side of the site of interest, or the first and second homologous regions of the target sequence contain the site of interest. If located on opposite sides of the site of interest, the first and second homologous regions are preferably located adjacent to the site of interest in said target sequence and thus to the target site at which the single-stranded or double-stranded DNA break occurs. This is because the HR frequency decreases with incremental distance between the site-specific DNA break and the homologous nucleic acid sequences.
  • a region is located on opposite sides of the site of interest in the target sequence, which means that the first homologous region is located on one side of the site of interest and the second homologous region is located on the other or opposite side of the site of interest, or the first and second homologous regions of the target sequence contain the site of interest.
  • the first and second homologous regions are preferably located within 10 base pairs (bp) from the site of interest, preferably within 7 bp from the site of interest, more preferably within 5 bp from the site of interest, more preferably within 4 bp from the site of interest, more preferably within 3 bp from the site of interest, more preferably within 2 bp from the site of interest, more preferably within 1 bp from the site of interest.
  • said first and second homologous regions are located directly adjacent to the site of interest, which means that no nucleic acids are present between the homologous regions and the site of interest, or the first and second homologous regions of the target sequence contain the site of interest.
  • Said site of interest preferably only contains the nucleic acids that between which a DNA break is induced.
  • the total region of homology i.e. the total amount of nucleic acids of the first and second homologous nucleic acid sequences, comprises preferably at least 100 base pairs (bp).
  • the first homologous nucleic acid sequence comprises at least 50 bp and the second homologous nucleic acid sequence also comprises at least 50 bp.
  • the first and second nucleic acid sequences comprise an identical number of bp.
  • the total region of homology is at least 150 bp, more preferably at least 200 bp, more preferably at least 300 bp, more preferably at least 400 bp, more preferably at least 500 bp.
  • preferred total lengths of the total region of homology are between 500 bp and 20 kb, more preferably between 500 bp and 15 kb.
  • a linear replication incompetent construct used in a method of the invention optionally further comprises a donor nucleic acid sequence that is located between the first and second homologous nucleic acid sequences.
  • the donor nucleic acid sequence is preferably flanked by the first and second homologous nucleic acid sequences such that the linear replication incompetent construct used in a method of the invention comprises the first nucleic acid sequence homologous to a first region of the target sequence, followed by a donor nucleic acid sequence which is followed by the second nucleic acid sequence that is homologous to a second region in the target sequence.
  • donor nucleic acid sequence refers to a nucleic acid sequence that is to be inserted, or that has been inserted, into the target sequence via the HR process.
  • the donor nucleic acid sequence typically comprises the modification that is to be introduced into the target sequence.
  • the donor nucleic acid sequence is inserted into the target sequence in the genome of the cell.
  • a preferred donor nucleic acid sequence can be essentially identical to part of the target sequence to be modified, with the exception of one or more nucleotides that are different from a nucleotide at the same position in the sequence of the target sequence.
  • HR with such essentially identical donor nucleic acid results in the introduction of one or more point mutations, which is for instance useful in gene repair.
  • a donor nucleic acid sequence comprising the nucleic acid sequence of a gene and/or of an artificially designed transcriptional unit that is to be added to the target sequence.
  • the replacement of a target sequence with that of a donor sequence is yet another preferred embodiment.
  • a donor nucleic acid sequence need not be present in a construct used in accordance with the present invention. For instance, if the purpose of a HR event is to remove the target sequence or part of the target sequence without introducing a donor nucleic acid sequence.
  • An example of such application is deletion of a gene or part thereof from the genome of a cell.
  • a linear replication incompetent construct used in a method of the invention further comprises a molecule attached to at least one terminus of said construct.
  • a molecule can be any molecule that is attached to the terminus of a linear replication incompetent construct according to the invention.
  • Preferred but non limiting examples of such molecules are inorganic or organic molecules including, but not limited to, a protein, a peptide or polypeptide, a carbohydrate, a polysaccharide, a glycoprotein, a lipid, a hormone, a drug and a nanoparticle such as a quantum dot.
  • terminal refers to the region including the end of a stand of a nucleic acid molecule and may include the final nucleotide and up to 5 adjacent nucleotides.
  • 5' terminus refers to the region including the end of the strand of a nucleic acid molecule that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus. This region may include the final nucleotide of the nucleic acid strand and up to 5 adjacent nucleotides.
  • 3' terminus refers to the region including the end of the strand of a nucleic acid molecule which terminates at the hydroxyl group of the third carbon in the sugar-ring of the deoxyribose or ribose at its terminus. This region may include the final nucleotide of the nucleic acid strand and up to 5 adjacent nucleotides.
  • a molecule is preferably attached to one or more of the terminal five nucleotides of the 3' and/or 5' terminus or termini, more preferably to one or more of the terminal four nucleotides, more preferably to one or more of the terminal four nucleotides, more preferably to one or more of the terminal four nucleotides.
  • said molecule preferably a (polypeptide or protein, is attached to the terminal nucleotide of the 3' and/or 5' terminus or termini.
  • a linear HR construct with a molecule attached to at least one terminus thereof, preferably to both termini, blocks DNA-DNA interactions involving illegitimate recombination, such as between the construct and DNA sequences, such as genomic DNA, other vector DNA and cytoplasmatic DNA or between multiple copies of the HR construct.
  • Such construct with a molecule attached to at least one 5' or 3' terminus is also referred to as a capped construct or a protein-capped construct if the molecules are peptides, polypeptides or proteins.
  • a capped construct or protein-capped construct preferably comprises a molecule or protein attached to both 5' termini, or to both 3' termini, of a double-stranded nucleic acid molecule. Further preferred are capped constructs that comprise molecules or proteins attached to all termini, i.e. the two 5' and the two 3' termini of a double-stranded nucleic acid molecule.
  • Fig. 9 provides an overview of generic arrangements of possible terminally-capped linear nucleic acid constructs in accordance with the invention.
  • illegitimate recombination events including inaccurate recombination such as concatemerization (i.e. multi-copy end-to-end assembly of exogenous DNA) are minimized.
  • concatemerization i.e. multi-copy end-to-end assembly of exogenous DNA
  • present inventors found that the occurrence of illegitimate recombination can be reduced and HR accuracy is largely increased if a protein-capped HR construct is used.
  • the superiority of the use of a protein-capped HR construct is shown over the most commonly used viral (IDLV) and non-capped non-viral vectors.
  • IDLV most commonly used viral
  • non-capped non-viral vectors the reduced illegitimate recombination and increased HR accuracy appeared to be independent from the system used to introduce DSBs, as the effects were seen both with TALENs and with RGN complexes.
  • adenoviral vector was used as an HR construct.
  • the adenovirus contains a 55 kDa terminal protein that is covalently bound to the 5' ends of the linear double-stranded adenovirus genome.
  • the TP is attached to the 5' termini of the genome by a phosphodiester bond.
  • the 87 kDa precursor of the TP covalently binds to the first deoxycytidine nucleotide residue (dCMP) of a newly synthesized adenoviral DNA chain.
  • the protein-bound dCMP then functions as a primer for DNA synthesis.
  • a preferred molecule is a peptide, polypeptide or protein.
  • peptide' “polypeptide” and “protein” refer to proteinaceous molecules that comprise multiple amino acids.
  • the terms “peptide' "polypeptide” and “protein” are herein collectively referred to as "(polypeptide”.
  • Such peptide, polypeptide or protein preferably has a size of between 1 and 100 kDa, more preferably of between 10 and 80 kDa.
  • suitable molecules that can be used in accordance with the invention are Biotin, optionally attached to a binding partner of Biotin, such as Avidin, Streptavidin or Neutravidin, terminal proteins and precursor terminal proteins of adenoviruses, e.g.
  • phage terminal proteins such as Bacillus phage protein phi29, Bacillus phage protein Nf, Enterobacteria phage protein PRD1, Streptococcus phage protein Cp- 1 and
  • Actinomyces phage protein av- 1, and Streptomyces terminal proteins such as S. lividans TpgL, S. coelicolor TpgC, S. avermitilis TpgAl, S. rochei TpgRM and S. scabiei TpgL.
  • Amino acid sequences of suitable adenoviral, phage or Streptomyces terminal proteins and precursor terminal proteins are shown in Fig. 10 .
  • a preferred molecule is an adenoviral, phage or Streptomyces terminal protein or precursor terminal protein or a protein having at least 70% sequence identity thereto, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity thereto. More preferably, a molecule has an amino acid sequence that has least 70% sequence identity to a sequence from Fig. 10 , preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity thereto.
  • a molecule preferably a peptide, polypeptide or protein, is preferably attached to a 5' terminus or a 3' terminus or both of the construct by a covalent or non-covalent bond, preferably to a 3' and/or 5' terminal nucleotide of said construct.
  • a linear replication incompetent construct used in accordance with the invention comprises at least two molecules, preferably (polypeptides, one at each 5' or 3' terminus of said construct. Further preferred is a linear replication
  • a preferred linear replication incompetent construct for use in a method of the invention is a, preferably double-stranded, linear replication incompetent construct comprising at least two molecules attached to the 5' and/or 3' terminus of each strand.
  • said construct is a linear replication incompetent viral vector, preferably double-stranded, comprising two (polypeptides attached to the 5' terminus of each strand, preferably the 55 kDa adenoviral proteins.
  • a particularly preferred construct is a linear replication incompetent adenoviral vector, preferably double-stranded, comprising two, preferably inert, peptides, polypeptides or proteins attached to the 5' terminus of each strand, wherein said (polypeptides preferably are the 55 kDa adenoviral terminal protein or a protein similar thereto.
  • the molecule is preferably an inert molecule when present in the target cell.
  • inert molecule refers to a molecule that has little or no ability to react with other molecules, e.g. that, when attached to the terminus of a construct in accordance with the invention, does not have biological functionality in the cell into which the linear construct is introduced other than blocking DNA- DNA illegitimate recombination.
  • Linear replication incompetent constructs comprising one or more molecules attached to at least one of their termini can be obtained by producing in cell lines and bacterial strains viral vectors whose parental viruses replicate via a protein-primed DNA replication mechanism such as adenoviruses and
  • Linear replication incompetent constructs comprising one or more molecules attached to at least one of their termini can also be obtained by using in vitro and in bacteria DNA replication systems such as those based on the phage Phi29 and the Streptomyces replicons, respectively (see, for example, refs. [3,4]).
  • Linear replication incompetent constructs containing molecules attached to at least one of their termini can further be generated by in vitro capping of synthetic and/or recombinant nucleic acids. This can involve the direct chemical conjugation of molecules (e.g. polypeptides]) to linear replication incompetent constructs or, alternatively, first to oligonucleotide intermediates.
  • the resulting modified oligonucleotides can subsequently be ligated to an "acceptor" linear replication incompetent construct.
  • a non-limiting example of a modified oligonucleotide is provided by a 5' and/or 3' Biotin-modified oligonucleotide.
  • the ligation of the modified oligonucleotides to "acceptor" donor DNA molecules can entail Watson- Crick hybridizations and/or DNA ligase treatments.
  • the modified oligonucleotides can be used directly in the form of single-stranded or, after hybridization, double- stranded short linear replication incompetent donor DNA molecules.
  • the in vitro capping of synthetic and/or recombinant nucleic acids can also involve PCR amplification of a linear replication incompetent construct template by using as primers the aforementioned modified oligonucleotides, such as the 5' and/or 3' Biotin-modified oligonucleotide.
  • Biotin-modified primers can be used to generate Biotin-capped linear replication incompetent constructs by PCR amplification of a donor DNA molecule of choice.
  • the resulting products can be further modified, e.g. to introduce a bulkier "inert" moiety and/or an "effector" motif such as a nuclear localization signal.
  • Such modification can comprise the covalent binding of Biotin to a protein of interest by chemical conjugation or by the strong non-covalent coupling of Biotin to Biotin-binding partners such unmodified or fusion protein-modified Avidin, Streptavidin or Neutravidin.
  • terminally-capped linear nucleic acid constructs harboring the donor DNA resulting from the above-described procedures can be delivered into target cells by methods known in the art, which include, but are not limited to, viral vector particle-mediated transduction, microinjection, gene-gun or by standard nucleic acid transfection methods such as those based on calcium phosphate precipitation, liposomes, polycations, magnetofection and electroporation.
  • the invention provides a method for modifying a target sequence in a genome of a cell comprising:
  • a double-stranded linear replication incompetent construct preferably a viral vector, comprising
  • a second nucleic acid sequence which is homologous to a second region of said target sequence, iii) optionally a donor nucleic acid sequence located between said first and second nucleic acid sequences and
  • a double -stranded linear replication incompetent construct preferably a viral vector, comprising
  • a targeting region comprising:
  • said first construct being a double-stranded linear replication incompetent construct, preferably a viral vector, and
  • - a second construct comprising a nucleic acid sequence encoding an endonuclease capable of inducing a site-specific DNA break in a genome of a cell.
  • a method of the invention comprises inducing a DNA break at a site of interest in the target sequence.
  • Said DNA break is preferably a single-stranded or double-stranded DNA break, more preferably a double-stranded DNA break.
  • a single-stranded or double-stranded break is induced in a site of interest of a target sequence.
  • the site of interest preferably comprises a genetic mutation or is located in a safe harbor locus.
  • a single-stranded or double -stranded DNA break is preferably induced by a sequence-specific endonuclease that recognizes large DNA recognition target sites of approximately 12 to 60 bp. For instance, a typical ZFN monomer may have a recognition site of 12 bp, i.e.
  • TALEN dimers the total target size is typically around 17 bp x2 + 12/13 bp of a spacer.
  • homing endonucleases have target sites of 18-50 bps.
  • the I-Scel homing endonuclease and engineered derivatives have a target site of 18 bp.
  • the RNA-guided nucleases (RGNs) based on the CRISPR/Cas adaptive immune systems of bacteria have a target site of about 20 bp. The sequence-specific endonuclease specifically binds to the site of interest.
  • the term "specifically binds to the site of interest and introduces a single-stranded or double- stranded DNA break within the target sequence” means that the endonuclease is designed such that it binds to the particular sequence of a site of interest or near a site of interest and preferably does not bind to other sequences located in the genome. In a preferred embodiment said endonuclease essentially does not bind to such other sequences located in the genome.
  • a method of the invention comprising introducing into said cell an endonuclease capable of inducing said single-stranded or double-stranded break.
  • Said endonuclease is for instance introduced into target cells directly as unmodified protein, or as protein modified with a protein transduction domain, or by linking it to structural component(s) of a gene delivery system.
  • said endonuclease is for instance introduced into target cells as DNA or mRNA nucleic acid constructs encoding it.
  • a method of the invention comprising introducing into said cell a construct comprising a nucleic acid sequence encoding an endonuclease capable of inducing said site-specific DNA break.
  • a construct according to the invention comprising a nucleic acid sequence encoding an endonuclease capable of inducing a single-stranded or double-stranded break, preferably a double-stranded DNA break, in the target sequence in a genome of a cell.
  • an endonuclease capable of inducing a single-stranded or double-stranded break, preferably a double-stranded DNA break, in the target sequence in a genome of a cell.
  • Any nuclease able to cleave a genome at a specific position and induce a single- or double-stranded DNA break can be used in the present invention.
  • Non- limiting examples of nucleases that can be used in accordance with the present invention are homing endonucleases, which are also referred to as meganucleases, Transcription Activator- Like (TAL) effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and RNA-guided nucleases (RGNs) based on CRISPR/Cas adaptive immune systems of prokaryotes [8,9] .
  • TAL Transcription Activator- Like
  • ZFNs Zinc-finger nucleases
  • RGNs RNA-guided nucleases
  • Meganucleases are endonucleases having a large nucleotide recognition site. Meganucleases are also called rare-cutting or very rare-cutting endonucleases. They have a high specificity for their nucleic acid target and can cleave a predefined chromosomal target sequence of choice. Meganucleases are found in eukaryotes, bacteria and archaea.
  • Wild-type meganucleases such as I-Scel, 1-Crel and 1-Dmol, recognizing and cleaving their specific native DNA target sequences as well as custom engineered meganucleases which recognize and cleave novel DNA target sequences to which they were engineered to bind to can be used in accordance with the invention [8].
  • Suitable meganucleases that can be used in accordance with the invention are I-Scel, PI-SceI, I-Ceul, I-Crel, I-Chul, I-Csml, PI-Tlil, PI-MtuI, , I-SceII, I-Sce III, ⁇ -CivI, Pi-Ctrl, PI-Aael, P-Bsul, PI-Dhal, I- Dmol, PI-Dral, PI-FacI, PI-PhoI, I-Msol, PI-MavI, PI-MchI, PI-MfuI, PI-Mfll, PI- Mgol, PI-MinI, PI-Mkal, PI-Mlel, PI-Mmal, PI-MshI, PI-MsmI, PI-MthI, PI-MtuI, PI-NpuI, Pl-P
  • TALENs comprise a TALE (Transcription Activator -Like Effector) DNA- binding domain fused to a nuclease domain (e.g; that of the type IIS restriction enzyme Fokl).
  • TALEs Transcription Activator -Like Effector
  • Fokl the type IIS restriction enzyme
  • TALEs contain a central region of tandem direct repeats that are responsible for sequence-specific DNA binding. Most repeats are composed of 34 amino acids with the only distinguishing feature among different repeats being a hypervariable
  • RVD repeat-variable di-residue
  • TALENs operate in pairs of two monomers [9]. The directional binding of each TALEN monomer to its respective half-target site induces dimerization of the Fokl portions resulting in site-specific DNA cleavage. Therefore, TALENs can be engineered to recognize and cleave a DNA target of choice with high specificity.
  • ZFNs are yet another class of artificial endonucleases that can be designed to bind to a predefined genomic target site and thus induce a DNA break at this specific site [9].
  • ZFNs are artificial enzymes generated by fusing an array of zinc finger DNA-binding domains to a nuclease DNA-cleaving domain (e.g. that of Fokl). Like TALENs, ZFNs work as dimers inducting single- or double-stranded DNA breaks at predefined target sequences of choice.
  • RGNs are RNA-dependent DNA nucleases based on type II clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) adaptive immune systems of prokaryotes.
  • CRISPR type II clustered regularly interspaced short palindromic repeat
  • Cas CRISPR-associated
  • These RGN systems comprise transfecting cells with RNA Pol-II and RNA Pol-III expression cassettes encoding, respectively, the Cas9 nuclease and a chimeric single guide RNA (sgRNA).
  • the sequence-specific sgRNA module is engineered by fusing sequence-tailored CRISPR RNAs (crRNAs) to trans-acting CRISPR RNA (tracrRNA) scaffolds.
  • crRNAs sequence-tailored CRISPR RNAs
  • tracrRNA trans-acting CRISPR RNA
  • a method of the invention comprises introducing into the cell nucleases capable of inducing the single-stranded or double-stranded DNA break, preferably double-stranded DNA break.
  • Nucleases can be introduced into a target cell using both transgenic and transgene-free strategies. The nucleases can for instance be introduced into the target cells directly as unmodified proteins or as proteins modified with so-called protein transduction domains (PTDs), a.k.a cell- penetrating peptides (CPPs), of which RQIKIWFQNRRMKWKK (Antennapedia) and GRKKRRQRRRPPQ (HIV Tat) are but two examples.
  • PTDs protein transduction domains
  • CPPs cell- penetrating peptides
  • nucleases can be introduced in a target cell by linking or fusing them to structural components of gene delivery vehicles such as viral vectors or by delivering them packaged in vector particles in the form of "ready-for-expression" mRNA templates.
  • these nucleases are introduced into the target cells in the form of recombinant constructs comprising nucleic acid sequences encoding them (i.e. DNA or mRNA).
  • DNA constructs preferably comprise a coding sequence that encodes an endonuclease which is preferably coupled to an inducible promoter.
  • inducible promoter refers to a promoter of which the expression can be regulated. Inducible promoters are known to the skilled person.
  • Suitable inducible promoters depend on the type of cell that is targeted by HR in accordance with the invention, and are known to the skilled person.
  • suitable inducible promoters include, but are not limited to, RAmy3D and CaMV promoters for expression in plant cells, polyhedrin, p lO, IE-0, PCNA, OplE2, OplEl, and Actin 5c promoters for expression in insect cells, and beta-actin promoter, immunoglobin promoter, 5S RNA promoter, promoter/enhancer elements derived from the human genes EEF1A1, UBC and PGK1 (a.k.a.
  • virus derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV), and Simian virus 40 (SV40) promoters for expression in mammalian cells.
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • SV40 Simian virus 40
  • a preferred nuclease used in accordance with the invention is a Transcription Activator- Like (TAL) effector nuclease (TALEN).
  • TAL Transcription Activator- Like
  • introduction of DSBs in the target sequence with the use of a TALEN in combination with a capped DNA targeting construct results in particularly specific and accurate HR.
  • a method for modifying a target sequence in a genome of a cell comprising:
  • a DNA break preferably a double-stranded DNA break
  • a site of interest in said target sequence by introducing into said cell an endonuclease capable of inducing said DNA break or a construct comprising a nucleic acid sequence encoding an endonuclease capable of inducing said DNA break, wherein said endonuclease is a TALEN;
  • a linear replication incompetent construct preferably an adenoviral vector, comprising i) a first nucleic acid sequence which is homologous to a first region of said target sequence,
  • a molecule preferably a (poly)peptide, attached to at least one terminus of said construct, whereby said first region is located on one side of said site of interest and said second region is located on the other side of said site of interest.
  • a construct comprising a nucleic acid sequence encoding a TALEN is introduced into said cell.
  • said linear replication incompetent construct comprises a (poly)peptide attached to the 5' terminus of each strand of a double- stranded viral, preferably adenoviral, construct.
  • a molecule preferably a (poly)peptide, attached to at least one terminus of said construct,
  • said first construct preferably being a double-stranded adenoviral vector, and - a second construct comprising a nucleic acid sequence encoding a TALEN.
  • linear replication incompetent construct preferably a viral construct, more preferably an adenoviral construct, comprising:
  • a targeting region comprising:
  • a second nucleic acid sequence which is homologous to a second s region of said target sequence; and iii) optionally a donor nucleic acid sequence located between said first and second nucleic acid sequences;
  • TALEN Transcription Activator- Like effector nucleases
  • RNA-guided nuclease such as the Cas9 nuclease
  • Cas9 nuclease which is used in combination with a single guide RNA (sgRNA) addressing the RGN to a target site in the genome of a cell.
  • sgRNA single guide RNA
  • introduction of DSBs in the target sequence with the use of Cas9 and a single guide RNA (sgRNA) in combination with a capped DNA targeting construct results in particularly specific and accurate HR.
  • sgRNA single guide RNA
  • a DNA break preferably a double-stranded DNA break
  • a site of interest in said target sequence by introducing into said cell an endonuclease capable of inducing said DNA break or a construct comprising a nucleic acid sequence encoding an endonuclease capable of inducing said DNA break, wherein said endonuclease is a RGN, preferably Cas9;
  • a linear replication incompetent construct preferably a viral vector, more preferably an adenoviral vector, comprising
  • a construct comprising a nucleic acid sequence encoding an RGN, preferably Cas9, and a construct comprising a nucleic acid sequence encoding a sgRNA addressing the RGN, preferably Cas9, to the target site in the human genome, is introduced into said cell.
  • a single construct comprising a nucleic acid sequence encoding an RGN, preferably Cas9, and a nucleic acid sequence encoding a sgRNA addressing the RGN, preferably Cas9, to the target site in the genome of a cell, is introduced into said cell.
  • RGN preferably Cas9
  • a sgRNA target sequence is that in the AAVSl safe harbor locus shown in Fig. 11a.
  • said linear replication incompetent construct comprises a (polypeptide attached to the 5' terminus of each strand of a double-stranded viral, preferably adenoviral, construct.
  • a molecule preferably a (poly)peptide, attached to at least one terminus of said construct,
  • said first construct preferably being a double-stranded viral vector, more preferably an adenoviral vector,
  • - a second construct comprising a nucleic acid sequence encoding an RGN, preferably Cas9, and
  • the RGN and sgRNA are preferably introduced into said cell by transfecting cells with RNA Pol-II and RNA Pol-III expression cassettes encoding, respectively, the RGN and a chimeric single guide RNA (sgRNA).
  • said second construct preferably is a RNA Pol-II expression cassette encoding the RGN and said third construct is preferably a RNA Pol-III expression cassette encoding a chimeric single guide RNA (sgRNA).
  • a set of constructs comprising - a first linear replication incompetent construct comprising:
  • a molecule preferably a (poly)peptide, attached to at least one terminus of said construct,
  • said first construct preferably being a double-stranded viral vector, more preferably an adenoviral vector,
  • a second construct comprising a nucleic acid sequence encoding an RGN, preferably Cas9, and a nucleic acid sequence encoding a sgRNA addressing the RGN, preferably Cas9, to the target sequence in the genome of said cell.
  • linear replication incompetent construct preferably a viral construct, more preferably an adenoviral construct, comprising:
  • a targeting region comprising:
  • iii) optionally a donor nucleic acid sequence located between said first and second nucleic acid sequences
  • Said construct further optionally comprises a nucleic acid sequence encoding a single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • the construct comprising nucleic acid sequence encoding an endonuclease capable of inducing the single-stranded or double-stranded DNA break is preferably a vector, such as a linear or circular plasmid or minicircles non- viral vectors or a viral vector based on adenoviruses, herpes viruses, adeno- associated viruses, retroviruses, vaccinia virus, SV40, baculoviruses, alphaviruses, herpes simplex viruses, poxviruses and bacteriophages.
  • a method of the invention may comprise introducing into a cell two separate constructs, i.e.
  • a method of the invention comprises introducing into a cell a single construct, i.e.
  • a linear replication incompetent constructs as defined herein, comprising a nucleic acid sequence encoding an endonuclease capable of inducing a site-specific break, preferably a double-stranded DNA break, and regions of homology, a donor nucleic acid and a molecule attached to at least one terminus thereof.
  • Said endonuclease is preferably a TALEN or an RGN.
  • a linear replication incompetent construct according to the invention and, optionally, a construct comprising a nucleic acid sequence encoding an endonuclease can be introduced in a cell by various methods known in the art. Such methods are known to a skilled person, and include, but are not limited to, electroporation, calcium phosphate-mediated transfection and lipofection for non- viral constructs or by transducing cells with viral vectors containing the constructs. In the Examples a method to introduce constructs into the cells by using viral vectors is provided under the heading "transduction experiments”.
  • the nucleases can be introduced into the target cells in the form of mRNA or directly as unmodified proteins or as proteins containing so-called supercharged protein transduction domains (PTDs), a.k. a cell-penetrating peptides (CPPs), of which RQIKIWFQNRRMKWKK (Antennapedia) and GRKKRRQRRRPPQ (HIV Tat) are but two examples.
  • PTDs supercharged protein transduction domains
  • CPPs cell-penetrating peptides
  • Said nuclease is preferably a TALEN or an RGN.
  • the nucleic acid sequence of a linear replication incompetent construct according to the invention used for HR is preferably present in a HR cassette.
  • the HR cassette comprises the first nucleic acid sequence, second nucleic acid sequence and optionally a donor nucleic acid sequence as defined herein.
  • the cassette preferably further comprises a selection marker, and/or a nucleic acid sequence encoding an endonuclease that is capable of inducing site-specific breaks.
  • Said endonuclease is preferably a TALEN or an RGN.
  • the HR cassette can further comprise a marker sequence, such as a positive or negative selection maker, which can be used to identify cells which have undergone HR.
  • RNA polymerase II promoters e.g. from the HSV-1, SV40, RSV and CMV viral genomes and from the mammalian genes HPRT1, PGK1, EEF1A1 and UBC, from chimeric elements such as the CAG promoter and from artificially designed regulatory elements such as the doxy cycline -controllable promoter consisting of a minimal CMV promoter [mCMV] fused to 7 copies of the E.coli tetO sequence in tandem) and polyadenylation signals (e.g.
  • RNA polymerase III promoters e.g. U6, HI and 7SK
  • terminator sequences e.g. TTTTT
  • short RNAs e.g. microRNAs [miRNAs], short-hairpin RNAs [shRNAs] and single-guide RNAs [sgRNAs]
  • selection marker genes include those that confer resistance to Geneticin, Zeocin, Puromycin and Hygromycin as well as the genes HPRT1, DHFR, HSV-1 thymidine kinase, inosine monophosphate dehydrogenase 2 (IMPDH2) variants and the P140K mutant of the methylguanine methyltransferase gene (MGMV 140 ⁇ , or a gene compatible with FACS- and MACS- based selection/isolation such as the tNGFR (truncated nerve growth factor receptor).
  • Other elements that can be included within expression units present in linear replication incompetent constructs are those encoding heterologous tags (e.g.
  • RNA target sequences for tissue-specific gene expression and "self-cleaving" peptides e.g. the P2A, T2A, E2A, and F2A peptides from the porcine teschovirus-l, Thosea asigna virus, equine rhinitis A virus and foot-and-mouth disease virus, respectively.
  • a still further example of a genetic element that may be present in a HR assette are those which work at an epigenetic level by favoring an open chromatin structure for the exogenous DNA and by blocking its interaction with neighboring endogenous genes, e.g.
  • Scaffold/Matrix Attachment Regions such as those from the IFNB1 and immunoglobulin-] light chain (Igkc) loci and the human 1-68 MAR, and insulator elements such as the cHS4 and Locus Control Regions (LCRs) such as that of the human beta-globin gene.
  • constructs or (nucleic acid-containing) virus particles allow for a specific, accurate and controlled generation of cells having a donor nucleic acid sequence integrated into a specific site in the genome or having a deletion of genomic sequences for both therapeutic and non-therapeutic applications.
  • Such methods, constructs and (nucleic acid- containing) virus particles are particularly suitable for gene therapy, gene targeting and/or gene repair.
  • Gene therapy refers to the use of nucleic acid sequences, in particular DNA, as a pharmaceutical agent to treat disease, in particular genetic diseases, viral diseases or tumors.
  • Use of methods and/or constructs of the invention for gene therapy involve for instance introducing a nucleic acid sequence that encodes a functional gene to repair a mutated gene, correcting a genetic mutation, or introducing a nucleic acid sequence that encodes a therapeutic protein.
  • legitimate recombination refers to the integration of a donor nucleic acid sequence as defined herein in the target sequence in the genome of the targeted cell in the correct arrangement.
  • “illegitimate recombination” refers to the integration of a donor nucleic acid sequence as defined herein at a location in the genome of the cell other than within the target sequence or integration of said donor nucleic acid sequence in the target sequence of the genome in an incorrect structure or arrangement.
  • An incorrect structure or arrangement of a donor nucleic acid sequence includes for instance the chromosomal integration of the donor nucleic acid sequence in multiple copies in tandem (i.e. concatemers) and/or the unpredictable incorporation of unwarranted viral- or prokaryotic-derived sequences at the target site.
  • a construct, set of constructs or virus particle according to the invention for HR-mediated genome engineering wherein the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10.
  • Said virus particle is preferably a nucleic acid-containing virus particle.
  • homologous recombination refers to the
  • a sequence identity of at least 90%, more preferably of at least 95%, more preferably of at least 98%, more preferably of at least 99%.
  • Said sequence identity may be 100%.
  • Said HR preferably comprises a method for modifying a target sequence in a genome of a cell according to the invention. "The ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence" as used herein is defined as:
  • a method of the invention therefore preferably comprises modifying a target sequence in a genome of at least 10 cells, and/or introducing an exogenous nucleic acid sequence in the genome of at least 10 cells, more preferably of at least 100 cells, more preferably at least 10 3 cells, more preferably at least 10 4 cells.
  • a cell includes both references to a single cell as well as to more than one cell.
  • a construct, set of constructs or virus particle according to the invention for a method of HR-mediated genome engineering is provided wherein in said method the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10 and wherein said method comprises engineering the genome of at least 10 cells, more preferably of at least 100 cells, more preferably at least 10 3 cells, more preferably at least 10 4 cells.
  • a method for modifying a target sequence in a genome of a cell comprising:
  • a chromosomal DNA break preferably a single-stranded or double- stranded DNA break, more preferably a double-stranded DNA break, at a site of interest in said target sequence
  • Said method preferably comprises modifying a target sequence in a genome of at least 10 cells, more preferably of at least 100 cells, more preferably at least 10 3 cells, more preferably at least 10 4 cells.
  • the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence can for instance be determined by isolating and screening individual randomly- selected cellular clones genetically modified with the donor DNA. The isolation of these clones can be done by limiting dilution or can be assisted by using cell sorting methodologies such as Fluorescence -Activated Cell Sorting and
  • Magnetic-Activated Cell Sorting combined with fluorescent reporter proteins (e.g. enhanced green fluorescence protein) and cell-surface recombinant proteins (e.g. truncated nerve growth factor receptor), respectively (see, for example, refs.
  • fluorescent reporter proteins e.g. enhanced green fluorescence protein
  • cell-surface recombinant proteins e.g. truncated nerve growth factor receptor
  • Cellular clones can also be isolated by deploying marker gene/small- molecule drug combinations. Characterization of chromosomally inserted donor DNA in the various randomly-selected cellular clones (e.g. genomic position, copy number, and structure/arrangement) can be carried out by a multitude of complementary techniques known in the art which include: Southern blot analysis, PCR, inverted PCR and DNA sequencing of donor-chromosomal DNA junctions (centromeric- and telomeric-oriented). In addition, the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence can also be determined by direct analysis of a target cell population of interest by using for instance:
  • Also provided is a method for HR comprising providing a cell with a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention, wherein the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10.
  • a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention for the preparation of a pharmaceutical composition for HR wherein the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10.
  • a construct, set of constructs or (nucleic acid- containing) virus particle according to the invention for use in HR wherein the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10.
  • a construct, set of constructs or (nucleic acid- containing) virus particle according to the invention for use in a method of HR wherein in said method the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence is more than 10 and wherein said method comprises modifying a target sequence in a genome of at least 10 cells, more preferably of at least 100 cells, more preferably at least 10 3 cells, more preferably at least 10 4 cells.
  • the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence in accordance with a method or use of the invention is more than 20, more preferably more than 50, more preferably more than 100, more preferably more than 1000, more preferably more than 10.000.
  • Said HR-based genome editing preferably comprises a method for modifying a target sequence in a genome of a cell according to the invention.
  • a method or use of the invention therefore preferably comprises modifying a target sequence and/or introducing a donor or exogenous nucleic acid sequence in a genome of at least 10 cells, more preferably of at least 100 cells, more preferably at least 10 3 cells, more preferably at least 10 4 cells.
  • a method or use of the invention is particularly suitable for treating and/or preventing a genetic disease and/or for gene repair and/or for gene modification. Further provided is a method for modifying a target sequence in a genome of a cell according to the invention wherein said target sequence comprises a mutation, for treating or preventing a genetic disease in an individual.
  • an "individual” refers to a human or an animal, preferably an animal that can be affected by a genetic disease, such as humans, non-human primates, rodents, ovines, bovines, canines, felines, ruminants and other mammals, birds, insects, fish and reptiles, and other vertebrates.
  • a genetic disease such as humans, non-human primates, rodents, ovines, bovines, canines, felines, ruminants and other mammals, birds, insects, fish and reptiles, and other vertebrates.
  • said individual is a human.
  • treating a genetic disease includes counteracting, inhibiting or curing a genetic disease and/or alleviating, inhibiting or abolishing symptoms resulting from a genetic disease.
  • the target sequences for modification in accordance with a method of the invention is a gene underlying a genetic disease, said gene having one or more mutations.
  • the genetic disease may be the result of a point mutation in a gene in an individual or due to a frame-shift, deletion, duplication or recombination of said gene.
  • a single-gene or monogenetic disease is caused by mutation(s) in a single gene.
  • Genetic diseases may also be polygenic, meaning they are associated with the effects of one or more mutation in multiple genes.
  • Gene repair refers to a modification of a gene in the genome of a target cell to restore the correct function of said gene that comprises one or more mutations and as a result thereof has reduced or lost function or has gained an unwanted function.
  • Genetic diseases that can be treated or prevented with such method include, but are not limited to, diseases selected from the group consisting of hemophilia B, hemophilia A, Duchene muscular dystrophy, cystic fibrosis, thalassemia, sickle cell anemia, X-linked severe combined immunodeficiency (SCID), ADA-SCID, Wiskott-Aldrich syndrome, epidermolysis bullosa dystrophica, epidermolysis bullosa junctional, RAG- 1 deficiency SCID, RAG-2 deficiency SCID, metachromatic leukodystrophy, limb-girdle muscular dystrophy (type 2C), limb- girdle muscular dystrophy (type 2A), X-linked chronic granulomatous disease, and glycogen storage disease II.
  • the genes that are modified to repair a mutation in said gene with a method of the invention for treatment or prevention are the following F9 (hemophilia B); F8 (hemophilia A); DMD (Duchene muscular dystrophy); CFTR (cystic fibrosis); beta-globin (thalassemia); hemoglobin/HBB (sickle cell anemia); IL2RG (X-linked severe combined immunodeficiency [SCID]); ADA (ADA-SCID); WAS (Wiskott-Aldrich syndrome); COL7A1 (epidermolysis bullosa dystrophica); LAMC2, LAMB3, COL17A1 or ITGB4 (epidermolysis bullosa junctional); RAG- 1 (RAG-1 deficiency SCID); RAG-2 (RAG-2 deficiency SCID); ARSA (metachromatic leukodystrophy); SGCG (limb-girdle muscular dystrophy, type 2C); CAPN3 (limb-girdle
  • Also provided is a method for treatment or prevention of a genetic disease comprising administering to an individual in need thereof a therapeutic amount of a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention.
  • a construct, set of constructs or (nucleic acid-containing) virus particle for modifying a target sequence in a genome of a cell for treatment or prevention of a genetic disease.
  • a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention for the preparation of a pharmaceutical composition for modifying a target sequence in a genome of a cell and/or for treatment or prevention of a genetic disease.
  • constructs or (nucleic acid- containing) virus particle according to the invention for use in modifying a target sequence in a genome of a cell. Also provided is a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention for use in the treatment or prevention of a genetic disease.
  • the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence in accordance with a method or use of the invention is preferably more than 10, preferably more than 20, more preferably more than 50, more preferably more than 100, more preferably more than 1000, more preferably more than 10.000.
  • a method or use of the invention is particularly suitable for targeted gene addition.
  • Targeted gene addition has many applications in both gene therapy and cell engineering.
  • the invention therefore further provides a method for modifying a target sequence in a genome of a cell for targeted gene addition and/or for introducing an exogenous nucleic acid sequence of interest into the genome of said cell.
  • targeted gene addition comprises introducing a donor nucleic acid sequence, preferably DNA that encodes a protein, a therapeutic protein or a non-coding RNA molecule.
  • said target sequence for modification preferably is a (genomic) safe harbor locus.
  • Non-limiting examples of such safe harbor loci are AAVSl, CCR5, CCR2, the ROSA26 locus, FUT8, DMD21, SH6 and house-keeping genes such as HPRT1, GAPDH and DHFR.
  • a preferred (extragenic) safe harbor locus is a genomic locus that fulfils the following criteria: i) a distance of > 50 kb from the 5' terminus of any gene, ii) a distance of >300 kb from cancer-related genes, iii) a distance of > 300 bp from any microRNA; iv) the locus is located outside a gene transcription unit, and v) the locus is located outside an ultra-conserved region [2].
  • a method for targeted gene addition comprising administering to a cell a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention.
  • a method for targeted gene addition in an individual comprising administering to said individual, preferably to said individual's cells, a construct, set of constructs or (nucleic acid-containing) virus particle according to the invention.
  • Such method preferably comprises a method for modifying a target sequence in the genome of a cell according to the invention
  • constructs, set of constructs or (nucleic acid-containing) virus particle for modifying a target sequence in a genome of a cell for targeted gene addition.
  • constructs, set of constructs or (nucleic acid-containing) virus particle according to the invention for the preparation of a pharmaceutical composition for targeted gene addition.
  • constructs set of constructs or (nucleic acid- containing) virus particle according to the invention for use in targeted gene addition.
  • the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence in accordance with a method or use of the invention is preferably more than 10, preferably more than 20, more preferably more than 50, more preferably more than 100, more preferably more than 1000, more preferably more than 10.000.
  • Methods for modifying a target sequence in the genome of a cell according to the invention and constructs or sets of constructs according to the invention are also particularly suitable for research purposes.
  • Examples of such applications include the study of gene function which is for instance achieved by targeted gene inactivation by deleting or adding one or more nucleotides of a gene of interest or by overexpressing a gene of interest by gene addition, e.g. insertion of an exogenous gene into the genome of a cell, for instance at a safe harbor locus.
  • modifications of the target genome for research purposes include, but are not limited to, the introduction of mutations into a gene or the deletion of one or more parts of a gene or of an entire gene or regulatory elements of a gene for analysis of the effect on gene function, or the modification of mutations or polymorphisms in a gene to determine which mutation or polymorphism is causative of a physiological or biochemical effect(s) or phenotype(s).
  • Methods for modifying a target sequence in the genome of a cell according to the invention and constructs or sets of constructs according to the invention are also particularly suitable for engineering cell lines for biotechnology applications, such as for instance for enhanced production of proteins, metabolites and vaccines.
  • Methods for modifying a target sequence in the genome of a cell according to the invention and constructs or sets of constructs according to the invention are also particularly suitable for cell engineering to create synthetic gene circuits, modify metabolic pathways and reprogram cell phenotypes such as by controlling cellular
  • Methods for modifying a target sequence in the genome of a cell according to the invention and constructs or sets of constructs according to the invention are also particularly suitable for the generation of transgenic prokaryotes (e.g. Escherichia coli, Bacillus subtilis, Mycoplasm genitalium, Synechocystis and Pseudomonas), transgenic protists (e.g. Chlamydomonas,
  • transgenic fungi e.g. , Saccharomyces cerevisiae, Neurospora crassa and Schizosaccharomyces pombe
  • transgenic plants e.g. Arabidopsis, sorghum, rice. Lotus japonicus and
  • Brachypodium and transgenic animals (e.g. Drosophila, C. elegans, zebrafish, mice, rats, cows, pigs and non-human primates).
  • the invention further provides a method for modifying a target sequence in a genome of a cell, the method comprising providing a construct according to the invention or a set of constructs according to the invention to said cell.
  • the invention further provides a cell, comprising a genomic modification that is produced by a method of the invention.
  • a cell comprising a genomic modification that is produced by a method of the invention.
  • the ratio of legitimate versus illegitimate integration of a donor nucleic acid sequence as defined herein is more than 10, preferably more than 20, more preferably more than 50, more preferably more than 100, more preferably more than 1000, more preferably more than 10.000.
  • Most preferably said cell comprises only the modification that was present on the linear replication incompetent construct and that was introduced following recombination of said construct into the target sequence, such as an insertion of one or more base pairs into the target sequence or a deletion of one or more base pairs from the target sequence.
  • populations of cells can be obtained that have a higher level of legitimate integration of donor nucleic acid in their genome as compared to methods known in the art.
  • a population of cells comprising cells having a donor nucleic acid sequence in their genome, wherein the ratio of cells having legitimate integration of said donor nucleic acid in their genome versus cells having illegitimate integration of said donor nucleic acid in their genome is more than 10. Said donor nucleic acid sequence is introduced in said genome.
  • the ratio of cells having legitimate integration of said donor nucleic acid in the genome versus cells having illegitimate integration of said donor nucleic acid in the genome is more than 20, more preferably more than 50, more preferably more than 100, more preferably more than 1000, more preferably more than 10.000.
  • up to approximately 9% of the cells of a cell population of the invention may have illegitimate integration of donor nucleic acid in their genome.
  • a population of cells according to the invention comprising cells having a donor nucleic acid sequence introduced in their genome, wherein between 0.01% and 9% of said cells have illegitimate integration of said donor nucleic acid sequence in their genome, preferably between 0.01 and 5% of said cells, more preferably between 0.01% and 3% of said cells, more preferably between 0.01% and 1% of said cells, most preferably between 0.01% and 0.1% of said cells.
  • a population of cells of the invention is for instance a population of cells obtained directly after performing a HR-based genome engineering method, preferably a method of the invention.
  • at least 2% of said cells in said population have said donor nucleic acid sequence in their genome, more preferably at least 3% of said cells, more preferably at least 4% of said cells, more preferably at least 5% of said cells, more preferably at least 7% of said cells, more preferably at least 10% of said cells. It is also possible to isolate from a population of cells obtained directly after performing a HR-based genome engineering method of the invention cells that have a donor nucleic acid sequence in their genome.
  • a population of cells according to the invention wherein up to 100% of the cells have said donor nucleic acid sequence in their genome, preferably up to 100% of the cells of the population have legitimate integration of said donor nucleic acids sequence in their genome.
  • the methods of the invention allow for rapidly enriching for the desired genome-modified cell population.
  • the precision an accuracy of integration of donor nucleic acid of a method of the invention reduces the dependency on time-consuming screening of genetically modified populations to identify properly targeted cells.
  • the need for isolating, identifying and expanding monoclonal cell populations when using previously known methods, which generally have higher number of cells having illegitimate integration of donor nucleic acid in their genome, is by-passed.
  • a population of cells according to the invention is preferably a polyclonal cell population.
  • the term "polyclonal population of cells” refers to a population of cells that contains cells with a donor nucleic acid sequence in their genome integrated into their genome, originating from more than one, preferably more than 10, preferably more than 100 cells independent HR recombination events.
  • the donor nucleic acid sequence is preferably introduced in the genome of cells of a population of cells using a method of the invention. Further, said donor nucleic acid sequence is preferably introduced into the cells of said population of cells using a linear replication incompetent construct according to the invention. Cells of said population that contain the donor nucleic acid sequence in their genome may therefore comprise a residual linear replication incompetent construct according to the invention or part thereof.
  • a population of cells according to the invention is thus provided comprising cells having a donor nucleic acid sequence in their genome, wherein at least 1% of said cells comprise a linear replication incompetent construct according to the invention or part thereof, preferably at least 2% of said cells, more preferably at least 5% of said cells, more preferably at least 10% of said cells.
  • Said part for instance comprises the molecule attached to at least one terminus of said construct, and optionally further comprises said terminus.
  • Up to 100% of said cells having a donor nucleic acid sequence in their genome may contain said linear replication incompetent construct according to the invention or part thereof.
  • Said linear replication incompetent construct is preferably a linear replication incompetent viral vector, more preferably a double- stranded linear replication incompetent viral vector, more preferably a vector selected from the group consisting of an adenoviral vector, a herpes viral vector, an ade no- associated viral vector, a retroviral vector, a vaccinia viral vector and a bacteriophage vector, such as Phi29, Bam 35, Nf, PRD1 or Cp-1, most preferably an adenoviral vector.
  • a population of cells according to the invention is for instance an ex vivo population of cells.
  • a population of cells according to the invention is an in vitro population of cells.
  • a population of cells according to the invention is an in vivo population of cells.
  • a population of cells according to the invention is particularly suitable for use in transplantation into an individual.
  • a population of cells for use in a method of treatment or prevention of a disease whereby said cells are transplanted into said individual.
  • Said individual is preferably a human.
  • Said disease is preferably a genetic disease, such as a disease selected from the group consisting of hemophilia B, hemophilia A, Duchene muscular dystrophy, cystic fibrosis, thalassemia, sickle cell anemia, X-linked severe combined
  • SCID immunodeficiency
  • ADA-SCID Wiskott-Aldrich syndrome
  • epidermolysis bullosa dystrophica epidermolysis bullosa junctional
  • RAG-1 deficiency SCID RAG-2 deficiency SCID
  • metachromatic leukodystrophy limb-girdle muscular dystrophy (type 2C)
  • limb-girdle muscular dystrophy type 2A
  • X-linked chronic granulomatous disease and glycogen storage disease II.
  • the cells of said population are derived from said individual before a donor nucleic acid sequence is introduced in the genome of the cells.
  • Said population of cells that is transplanted into said individual is preferably enriched for cells containing the donor nucleic acid sequence in their genome after performing a HR based method of the invention.
  • at least 25% of said cells in said population have said donor nucleic acid sequence in their genome, more preferably at least 50% of said cells, more preferably at least 70% of said cells, more preferably at least 80% of said cells, more preferably at least 90% of said cells in said population have said donor nucleic acid sequence in their genome.
  • inventions essentially all cells of said population of cells contain said donor nucleic acid sequence in their genome. Also provided is a method of treating or preventing a disease, preferably a genetic disease, comprising administering to an individual in need thereof a population of cells according to the invention. Also provided is the use of a population of cells according to the invention for the preparation of a medicament for the treatment or prevention of a disease, preferably a genetic disease, in an individual. Said individual is preferably a human.
  • a population of cells according to the invention is further particularly suitable for expressing protein or (poly)peptide encoded by the donor nucleic acid sequence, in particular if it is essential to be able determine the exact location of the encoding nucleic acid that is introduced in the genome of the cells.
  • Cells of a population of cells according to the invention are particularly suitable for such application because it is possible to exactly and with high accuracy determine the location in the genome of the cells where the donor nucleic acid is introduced and the donor nucleic acid is introduced with high accuracy and specificity.
  • the donor nucleic acid encodes a protein, polypeptide or peptide of interest, for expressing said protein, polypeptide or peptide encoded by said donor nucleic acid.
  • a population of cells according to the invention is further particularly suitable for generating isogenic cellular substrates that genetically differ among each other exclusively in a well-defined genomic region or in a few nucleotides or in a single nucleotide.
  • Such isogenic cells which may include induced pluripotent stem cells (iPS) can, for instance, serve as model systems to study disease phenotypes in vitro and to screen libraries of small-molecule drugs to isolate compounds that revert or ameliorate said disease phenotypes.
  • iPS induced pluripotent stem cells
  • a few nucleotides for instance refers to up to 100 nucleotides, preferably up to 50, more preferably up to 25, more preferably up to 10, such as 2, 3, 4, 5, 6, 7, 8, 90 or 10 nucleotides.
  • a pharmaceutical composition comprising a construct according to the invention, a set of constructs according to the invention and/or a (nucleic acid-containing) virus particle according to the invention. Said
  • composition preferably further comprises a suitable
  • a pharmaceutical composition according to the invention is preferably suitable for human use.
  • the invention further provides a method for producing a cell comprising a modified genome, preferably at least one modified gene, the method comprising providing a construct according to the invention or a set of constructs according to the invention to said cell, and selecting a cell in which the genome has been modified at the target sequence and that functionally expresses a recombined selection marker.
  • Said method preferably comprises inducing an inducible promoter for expression of the endonuclease, thereby inducing a site-specific DNA break in the target sequence.
  • Figure 1 Diagram of the gene targeting strategies for the recombinant eGFP allele and the native AAVSl safe harbor locus whose sequence is embedded within that of PPP1R12C. Illustrations of designer ZFN and TALEN proteins composed of sequence-tailored zinc finger arrays and transcription activator-like repeats, respectively, fused to the nuclease domain of Fokl.
  • the ZFN and TALEN composed of sequence-tailored zinc finger arrays and transcription activator-like repeats, respectively, fused to the nuclease domain of Fokl.
  • EFla promoter human EEF1A1 regulatory sequences
  • SUR SV40 5'UTR (SU) and R region (R) from HTLV- 1
  • eGFP reporter-encoding ORF
  • GHpA human GH-1 polyadenylation signal
  • ⁇ HIV-1 packaging signal
  • white and grey boxes 5' and 3' retroviral long terminal repeat sequences, respectively.
  • IDLV.donor eGFP and IDLV.donor S1 genomes contain HR substrates consisting of a reporter expression unit flanked by sequences identical to those bracketing the ZFN and TALEN target sites, respectively.
  • the expression unit in IDLV.donor eGFP consists of the hybrid GAG promoter, the FP635 (a.k.a. Katushka) ORF and the bovine GH-1 polyadenylation signal, whereas that in IDLV.donor S1 comprises the human PGK-1 promoter, the eGFP ORF and the bovine GH-1 polyadenylation signal.
  • FIG. 1 Gene targeting of IDLV-delivered donor DNA by using ZFNs and TALENs.
  • Flow cytometry was performed at 35 days post-infection.
  • H27 cells genetically modified through eGFP-targeted exogenous DNA integration or via NHEJ-mediated eGFP disruption plus off-target exogenous DNA insertion acquire an eGFP7FP635 + phenotype (red bar), whereas those subjected exclusively to off- target insertion events become marked as eGFP + /FP635 + (orange bars).
  • Figure 3 Molecular characterization of myoblasts genetically modified by using TALENs and IDLV donor DNA.
  • FIG. 5 Gene targeting in human myoblasts with AdV-delivered donor DNA and chromosomal site-specific DSBs.
  • Target cells were transduced with AdV.Al.donor S1 alone or with AdV.Al.donor S1 mixed with AdV.TALEN-L S1 and AdV.TALEN-R S1 (L/R).
  • Flow cytometry was done at 24 days post-infection. Plotted data correspond to mean ⁇ s.d. (P ⁇ 0.0001).
  • Figure 8 Nuclease-mediated gene targeting of donor DNA associated with or segregated from AdV genomes, (a) Testing the effect of donor DNA eviction from AdV genomes on the frequency of HR-driven gene targeting.
  • Upper panel experimental strategy for the TALEN-mediated excision of HR templates from AdV DNA in transduced cells.
  • AdV.A2.donor sl/T TS AdV carrying donor sl DNA framed by target sequences for the AAVSl- specific TALENs (solid vertical arrowheads); Open oval, terminal protein (TP) covalently attached to the 5' termini of AdV DNA; ⁇ , AdV packaging signal; exo., exogenous DNA; HR, homologous recombination.
  • the precision of the genome editing process can be further compounded by the chromosomal insertion of concatemeric vector DNA forms.
  • protein-capped AdV genomes make interactions between donor templates and off-target DSBs less probable with the pairing of, and single-strand DNA invasion at, the shared endogenous and exogenous DNA sequences conferring the very high target site specificity of AdV-delivered donor DNA.
  • FIG. 9 Generic arrangements of possible terminally-capped linear nucleic acid constructs in accordance with the invention.
  • the oval indicates the capping molecule; the arrow indicates linear constructs with capping moieties at both termini.
  • Figure 10 Examples of terminal proteins and of precursor terminal proteins from adenoviruses, phage terminal proteins and Streptomyces terminal proteins that can be used as capping molecules in accordance with the invention and amino acid sequence thereof.
  • Potential nuclear localization signal (NLS) sequences in phage terminal proteins are highlighted as indicated in ref. [12] .
  • Potential NLS sequences in Streptomyces terminal proteins are highlighted as indicated in ref. [3] .
  • FIG. 11 Nuclease -induced gene targeting following AdV- versus plasmid- mediated delivery of HR substrates, (a) TALEN and RGN target sites at AAVSl. Recognition sequences for the nuclease complexes TALEN-L S1 :TALEN-R S1 and Cas9:gRNA S1 complexes drawn in relation to the PPP1R12C locus in which they are embedded. The target sites of the TALEN pair and the RNA-guided nuclease are shown in upper case and boxed, respectively. The protospacer adjacent motif (PAM) is shaded.
  • PAM protospacer adjacent motif
  • FIG. 11a Genotyping assay for validating AdV.Cas9 and AdV.gRNA S1 in HeLa cells.
  • PCR products spanning the AAVSl target region (Fig. 11a) were amplified from genomic DNA of HeLa cells transduced with AdV.Cas9 alone at an MOI of 300 TCIDso/cell (Cas9) or with 1: 1 mixtures of AdV.Cas9 and AdV.gRNA S1 at a total MOI of 60, 120, 180, 240 and 300 TCIDso/cell.
  • HeLa cells were either co-transduced with AdV.Cas9, AdV.gRNA sl and AdV.A2.donor sl (first panel) or were co-transfected with the plasmid pair encoding the AAVSl- specific TALENs plus Pacl-linearized pAdV.donor sl , covalently-closed
  • the frequency of eGFP + cells present in the various long-term HeLa cell cultures was determined by flow cytometry at 23 days after transgene delivery and is indicated within each dot plot. Pictograms of the various types of donor DNA templates deployed in these experiments are drawn next to their respective dot plots.
  • PCR screening for detecting HR- mediated targeting of donor DNA delivered in an in cellula linearized plasmid was carried out on DNA from eGFP + HeLa cell clones isolated from cultures subjected to the transfer of AVSi-specific TALENs and p AdV. donor S1/T TS .
  • amplifications targeting eGFP served as internal controls for the presence and integrity of DNA templates.
  • Vertical arrowheads indicate clones lacking the targeted exogenous ⁇ -AAVSl junction.
  • Nuclease-free water and genomic DNA of mock-transduced HeLa cells served as negative controls, whilst genomic DNA extracted from an AAVSl -targeted eGFP + HeLa cell clone provided for positive controls.
  • Marker GeneRuler DNA Ladder Mix molecular weight marker.
  • FIG. 16 Gene targeting analyses of HeLa cells genetically modified by deploying TALENs and covalently-closed circular donor plasmids.
  • PCR screening for detecting HR-mediated targeting of donor DNA delivered in a supercoiled plasmid was performed on DNA from eGFP + HeLa cell clones isolated from cultures exposed to A4VS2-specific TALENs and pAdV.donor S1 .
  • PCR amplifications targeting eGFP served as internal controls for the presence and integrity of DNA templates.
  • Vertical arrowheads mark clones lacking the targeted exogenous ⁇ -AAVSl junction.
  • FIG. 17 Gene targeting analyses of HeLa cells genetically modified by using the CRISPR/Cas9 system and protein-capped linear AdV DNA.
  • PCR screening for detecting HR-mediated targeting of donor DNA delivered in protein-capped AdV genomes was performed on DNA from eGFP + HeLa cell clones grown from cultures exposed to Cas9:gRNA S1 and AdV.A2.donor S1 .
  • PCR amplifications targeting eGFP served as internal controls for the presence and integrity of DNA templates.
  • FIG. 18 Prokaryotic DNA status of cell populations genome-edited by combining engineered nucleases with AdV or plasmid HR templates. Kan H - specific PCR analysis of genomic DNA extracted from eGFP + sorted HeLa cells initially transduced with AdV. ⁇ 2. donor S1 (AdV DNA) or transfected with pAdV.donor S1 (supercoiled), Pad -linearized pAdV.donor S1 (linear in vitro) or TALEN pair- susceptible pAdV.donor sl/T TS (linear in vivo). Targeted DSBs were induced in AdV- transduced and plasmid-transfected cells by using A4VS2-specific RGN
  • Plasmid pAdV.donor S1 served as positive control (+), whereas genomic DNA from parental HeLa cells (-) and nuclease-free water provided for negative controls. The integrity of the various DNA templates was controlled for by carrying out parallel eGFP- specific PCR amplifications. Lane M, GeneRuler DNA Ladder Mix molecular weight marker.
  • Figure 19 Testing TALEN-mediated release of donor DNA from AdV genomes in transduced cells, (a) Characterization of control AdV.A2.donor sl/FRT and test AdV.donor sl/T " TS DNA by restriction fragment length analysis. Left-hand panel, generic structures of AdV.A2.donor sl/FRT and AdV.A2.donor sl/T"TS recombinant genomes. FRT and T-TS, target sites for the yeast site-specific FLP
  • Cellular DNA was extracted from HeLa cells co-transduced with AdV.A2.donor sl/FRT and FLPe-encoding vector hcAd.FLPe.F50 and from HeLa cells transduced with a combination of AdV.A2.donorSi/T-TS ; AdV.A2.TALEN-L sl and AdV.A2.TALEN-R sl (L/R).
  • PCR amplifications with "outward-facing" primers (half arrows) on linear templates will result in specific products only upon donor DNA excision and circularization.
  • the HeLa cells (American Type Culture Collection) and its eGFP-positive H27 clone derivative [13] were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 5% fetal bovine serum (FBS; Invitrogen).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the 293T lentiviral vector producer cells were maintained in DMEM containing 10% FBS, whereas the AdV packaging cell lines PER.C6 [5] and PER.E2A [7] were cultured in DMEM supplemented with 10% FBS and 10 mM MgC (Sigma-
  • the complete and annotated DNA sequences of lentiviral vector transfer plasmids pLV.donor eGFP and pLV.donor S1 can be retrieved via GenBank accession numbers KF419293 and KF419294, respectively.
  • the AdV shuttle plasmid pSh.AAVSl.eGFP sl/T TS was constructed by inserting upstream and downstream of the donor sl DNA module in pSh.AAVSl.eGFP [16] two annealed oligodeoxyribonucleotides containing bipartite target sequences for the AAVSl- specific TALENs (T-TS).
  • T-TS AAVSl-specific TALENs
  • pSh.AAVSl.eGFP a direct repeat of FRT sites in place of the T-TS sequences, was pursued in parallel. These maneuvers resulted in pSh.AAVSl.eGFP sl/FRT .
  • the AdV molecular clones pAd.AEl.donor sl .F 50 and pAd.AElAE2A.donor sl .F 50 were assembled by HR in E. coli strains [17] BJ ⁇ S ⁇ 3 ⁇ - 1 50 and BJ5183P AdEas y- 2 50 respectively, transformed with Mssl-treated pSh.AAVSl.eGFP.
  • AdV molecular clones pAd.AElAE2A.donor sl/T TS .F 50 and pAd.AE lAE2A.donor sl/FRT .F 50 were built by HR following the transformation of the latter cells with Mssl-digested plasmids pSh.AAVSl.eGFP sl/T TS and pSh.AAVSl.eGFP sl/FRT , respectively.
  • the AdV shuttle plasmid AT25_pAdV.PGK.Cas9 contains a humanized ORF encoding the S. pyogenes nuclease Cas9 under the transcriptional control of the PGK-1 promoter and the SV40 polyadenylation signal, whereas the AdV shuttle plasmid E43_pAdV.U6.gRNA S1 encodes a U6 promoter-driven single guide RNA (herein referred to as gRNA S1 ) targeting the Cas9 protein to the human AAVSl locus.
  • gRNA S1 U6 promoter-driven single guide RNA
  • AD05_pAdV.AElAE2A.PGK.Cas9.F 50 and AD09_pAdV.AElAE2A.U6.gRNA sl .F 50 were generated by HR in BJ5183 pAdE sy 2 50 cells [17] following their transformation with Mssl-treated AT25_pAdV.PGK.Cas9 and E43_pAdV.U6.gRNA Si , respectively.
  • AdV.TALEN-L S1 and AdV.TALEN-R S1 have been detailed elsewhere [19].
  • AdV.A2.TALEN-L S1 and AdV.A2.TALEN-R S1 were initiated by transfecting PER.C6 and PER.E2A cells with Pad -linearized AL25_pAd.AEl.donor sl .F 50 and
  • the DNA transfection-mediated rescue of AdV particles in packaging cell lines as well as their subsequent propagation and purification were performed essentially as described previously [17, 19].
  • the isolation and restriction fragment length analysis procedures applied to AdV. ⁇ 2. donor S1/T TS and AdV.A2.donor sl/FRT DNA has been detailed elsewhere [17].
  • the titers of the various reporter-encoding AdV stocks, expressed in terms of transducing units (TU) per ml, were determined through limiting dilutions on HeLa indicator cells seeded at a density of 8xl0 4 cells per well of 24-well plates. At 3 days post-transduction, frequencies of reporter- positive cells were measured by reporter-directed flow cytometry.
  • the titers of the reporter-negative AdV preparations were established by TCID50 assays in complementing cells and by fluorometric quantification of genome -containing vector particles (VP) per ml as described elsewhere [17,20].
  • VSV-G vesicular stomatitis virus glycoprotein G
  • IDLV.donor eGFP and IDLV.donor S1 was carried out by transient transfections of 293T cells with transfer plasmids AP45_pLV.
  • H27 indicator cells were carried out as follows. Eighty-thousand cells were seeded in wells of 24-well plates (Greiner Bio-One). The next day, IDLV.donor eGFP was added at an multiplicity of infection (MOI) of 45 TU/cell together with the LV.ZFN- l eGFP and LV.ZFN-2 eGFP vectors each applied at an MOI of 8 TU/cell. Parallel H27 cultures that were either untreated or were incubated exclusively with
  • IDLV.donor eGFP at an MOI of 45 TU/cell, served as controls.
  • the frequencies of reporter-positive and reporter-negative H27 cell populations were monitored and quantified,
  • transgene expression parameters i.e. frequencies of reporter-positive and reporter-negative target cells, mean fluorescence intensities and coefficients of variation
  • the measurement of transgene expression parameters were determined by using a BD LSR II flow cytometer (BD Biosciences). Data were analyzed with the aid of BD FACSDiva 6.1.3 software (BD Biosciences). Mock- transduced target cells were used to set background fluorescence levels. Typically, 10,000 viable single cells were analyzed per sample.
  • the light microscopic analyses were carried out with an 1X51 inverse fluorescence microscope equipped with a XC30 Peltier-cooled digital color camera (Olympus). The images were processed with the aid of CelF 3.4 imaging software (Olympus).
  • HeLa cells were seeded at a density of 6.5 l0 4 cells per well of 24-well plates. The next day, the cells were transfected with DNA mixtures consisting of 100 ng of 1383.pVAX.AAVSl.TALEN.L-94 [19], 100 ng of
  • the targeting constructs were pAdV.donor S1 , Pad -linearized pAdV.donor S1 and pAdV. donor S1/T TS .
  • Controls were provided by transfecting HeLa cells with 200 ng of 1383.pVAX.AAVSl.TALEN.L-94 mixed together with 200 ng of pAdV.donor S1 , Pad -linearized pAdV.donor S1 or pAdV.donor sl/T TS .
  • each of the plasmid mixtures were diluted in 50 ⁇ of 150 mM NaCl and received 1.32 ⁇ of a 1 mg/ml polyethylenimine solution under vigorous shaking for about 10 sec. After a 20-min incubation period at room temperature, the resulting polycation-DNA complexes were directly added into the culture medium. After 7 hours, the transfection mixtures were removed and fresh culture medium was added. The resulting cell populations were subsequently subjected to sub-culturing for 3 weeks after which cells stably expressing eGFP in these populations were individually sorted by flow cytometry into wells of 96-well plates. Viable single cell-derived clones corresponding to the various experimental settings were randomly selected for transgene expression and integration status analysis.
  • RNA-guided nuclease Cas9 Gene targeting of AdV donor DNA by using the RNA-guided nuclease Cas9.
  • HeLa cells were seeded at a density of 8.0 l0 4 cells per well of 24-well plates. The following day, the cells were transduced with AdV.Cas9 (150
  • HeLa cells were either mock-transduced or were transduced with AdV.Cas9 (150 TCIDso/cell) and AdVA2. donor S1 (10 TU/cell).
  • AdV.Cas9 150 TCIDso/cell
  • AdVA2. donor S1 10 TU/cell.
  • mock- and vector-transduced HeLa cells started to be sub- cultured twice per week.
  • eGFP stably-expressing cells were individually sorted by flow cytometry into wells of 96-well plates. Viable single cell-derived clones isolated from cultures initially exposed to AdV.Cas9, AdV.gRNA S1 and AdV.A2.donor sl > were randomly selected for transgene expression and integration status analysis.
  • Genomic DNA extraction Genomic DNA was extracted from cell populations and clones essentially as described before [25]. In brief, the cells were collected and incubated overnight at 55°C in 500 ⁇ of lysis buffer (100 mM Tris-HCl [pH 8.5], 5 mM ethylenediaminetetraacetic acid [EDTA], 0.2% sodium dodecyl sulfate and 200 mM NaCl) supplemented with freshly added Proteinase K (Thermo Scientific) at a final concentration of 100 ng/ml. The cell lysates were extracted twice with a buffer-saturated phenol:chloroform:isoamyl alcohol mixture (25:24: 1) and once with chloroform.
  • lysis buffer 100 mM Tris-HCl [pH 8.5], 5 mM ethylenediaminetetraacetic acid [EDTA], 0.2% sodium dodecyl sulfate and 200 mM NaCl
  • Proteinase K Thermo Scientific
  • the genomic DNA was precipitated by the addition of 2.5 volumes of absolute ethanol and 0.5 volumes of 7.5 M ammonium acetate (pH 5.5). After washing with 70% ethanol, the DNA pellets were air-dried and dissolved in 100 ⁇ of Tris-EDTA buffer (10 mM Tris [pH 8.0] and 1 mM EDTA) supplemented with RNase A (Thermo Scientific) at a final concentration of 100 ⁇ g/ml.
  • Tris-EDTA buffer (10 mM Tris [pH 8.0] and 1 mM EDTA) supplemented with RNase A (Thermo Scientific) at a final concentration of 100 ⁇ g/ml.
  • the genomic DNA of eGFP-positive clones derived from cultures transfected with plasmid DNA as well as that derived from cultures co-transduced with AdV.Cas9, AdV.gRNA S1 and AdV.A2.
  • A4VS2-derived arm of the targeting donor DNA was obtained by digestion of plasmid pSh.AAVSl.eGFP with XmaJI (Thermo Scientific) followed by preparative agarose gel electrophoresis.
  • the probe was radiolabeled with [oc- 32 P]dATP (GE Healthcare Life Sciences) by using the DecaLabel DNA labeling Kit following the manufacturer's instructions (Thermo Scientific). Prior to its deployment, the radiolabeled probe were separated from unincorporated dNTPs through size -exclusion chromatography in Sephadex-50 columns (GE Healthcare Life Sciences).
  • a Storm 820 Phosphoimager was used for the detection of the probe-hybridized DNA. The images were acquired by using the Storm Scanner Control 5.03 software and were processed with the aid of
  • COBRA-FISH karyotyping The COBRA-FISH karyotyping of HeLa target cells was done according to a published protocol [26].
  • AdV.A2.donor sl/FRT (3 TU/cell). At 72 hours post-transduction extrachromosomal DNA was isolated essentially as described previously [28] after which 2- ⁇ 1 DNA samples were subjected to PCR.
  • the PCR mixtures consisted of 0.4 ⁇ of primer #997 (5'-GCACTGAAACCCTCAGTCCTAGG-3'), 0.4 ⁇ of primer #998 (5'- CGGCGTTGGTGGAGTCC-3'), 0.1 mM of each dNTP (Invitrogen), 1 mM MgC (Promega), lx Colorless GoTaq Flexi Buffer (Promega) and 2.5 U of GoTaq Flexi DNA polymerase (Promega).
  • IDLVs Integrase-defective lentiviral vectors
  • IDLVs represent one of the most commonly used viral vectors for the delivery of HR substrates into human cells
  • the eGFP expression unit in HeLa-derived H27 cells and the AAVSl safe harbor locus in human myoblasts were targeted by zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), respectively (Fig. 1).
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • the ZFNs [32] and TALENs [19] were introduced into target cells through lentiviral and adenoviral vectors (AdVs), respectively, together with the corresponding target site-matched HR substrates in IDLV.donor eGFP and IDLV.donor S1 (Fig. 1).
  • FIG. 3a revealed that 86.6% of the eGFP + cells underwent homology-directed chromosomal insertion of the exogenous DNA (Figs. 2c, 2d and Fig. 3a).
  • the resulting A4VS2-donor DNA junctions represented events involving the "telomeric” (6.7%), the “centromeric” (3.9%) or both ends of the targeting template (76.0%) (Fig. 2d).
  • Further characterization of IDLV donor DNA integrants revealed high frequencies of head-to-tail (H-T) concatemeric forms not only in the non-targeted but also in the three -targeted clonal fractions (38.5%) (Figs. 2c, 2d and Fig.
  • tandem repeats are expected to neither restore endogenous ORFs nor yield homogeneous transgene expression levels in the context of gene repair and gene replacement strategies, respectively.
  • An overview of the prevalent structures that can be acquired by chromosomally integrated IDLV donor DNA is depicted in Fig. 4.
  • recombinant adeno-associated viral (rAAV) vectors constitute the most commonly used types of viral vectors for the delivery of HR substrates into mammalian cells.
  • rAAV adeno-associated viral
  • free-ended rAAV genomes become inserted at sporadic genomic DSBs after being co-opted by illegitimate recombination pathways involved in the repair of chromosomal DNA breaks.
  • donor DNA delivered in the context of protein-capped AdV genomes instead, firstly, display a less promiscuous chromosomal DNA integration profile and secondly, yield a more precise
  • TALEN-dependent increase in the frequencies of stably transduced cells (Fig. 5a) with not only TALEN-induced but also residual exogenous DNA chromosomal integration rates being lower than those measured in their IDLV.donor S1 - transduced counterparts (Fig. 2b).
  • the degree of the TALEN-dependent increase in the frequencies of stably transduced cells (Fig. 5a) with not only TALEN-induced but also residual exogenous DNA chromosomal integration rates being lower than those measured in their IDLV.donor S1 - transduced counterparts (Fig. 2b).
  • the degree of the TALEN-dependent increase in the frequencies of stably transduced cells (Fig
  • IDLV.donor sl -transduced myoblasts (Fig. 2b).
  • AdV.A2.donor S1 and TALEN-encoding AdVs resulted in eGFP + populations whose narrow distribution of transgene expression levels (Fig. 5b, 2 nd bar) approached those of clones harboring A4VS2-targeted donor sl DNA (Fig. 5b, 1 st bar) and departed from those of IDLV donor sl -modified populations (Fig. 5b, 3 rd and 4 th bars).
  • donors- modified clones revealed, in addition, a clone that underwent bi-allelic gene targeting and a clone that, in addition to the typical A4VS2-targeted donor DNA, contained an integrant whose origin is consistent with an HR-independent DNA integration event (Fig. 6).
  • Fig. 6 HR-independent DNA integration event
  • AdV gene targeting is compatible with the RNA-guided CRISPR/Cas9 nuclease system
  • HeLa cells were co- transfected with expression constructs encoding the A4VS2-specific TALENs mixed with pAdV.donor S1 (supercoiled), Pacl-linearized pAdV.donor S1 (in vitro linearized) or TALEN-cleavable pAdV. donor S1/T TS (in vivo linearized).
  • HeLa cells co-transfected exclusively with the TALEN-L S1 expression construct and each of the donor DNA plasmid types, provided for negative controls.
  • Pelascini, L.P.L., Janssen, J.M. & Goncalves M.A.F.V. Histone deacetylase inhibition activates transgene expression from integration- defective lentiviral vectors in dividing and non-dividing cells. Hum. Gene Ther. 24, 78-96 (2013).

Abstract

L'invention concerne des procédés de modification d'une séquence cible dans un génome d'une cellule par recombinaison homologue, des constructions utilisées dans ces procédés et des applications thérapeutiques et non thérapeutiques de ceux-ci.
PCT/NL2015/050072 2014-02-03 2015-02-03 Édition du génome induite par rupture d'adn de restriction à l'aide de nucléases génétiquement modifiées WO2015115903A1 (fr)

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