Patent Application
20090222937
The invention relates to a meganuclease variant cleaving a DNA target sequence from a xeroderma pigmentosum gene (XP gene), to a vector encoding said variant, to a cell, an animal or a plant modified by said vector and to the use of said meganuclease variant and derived products for genome therapy, in vivo and ex vivo (gene cell therapy), and genome engineering.
Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disease characterized by a hypersensitivity to exposure to ultraviolet A (UV) rays, a high predisposition for developing skin cancers on sunlight exposed areas, and in some cases neurological disorders (Hengge, U. R. and W. Bardenheuer, Am. J. Med. Genet. C. Semin. Med. Genet., 2004, 131: 93-100; Magnaldo, T. and A. Sarasin, Cells Tissues Organs, 2004, 177: 189-198; Cleaver, J. E., Nat. Rev. Cancer, 2005, 5: 564-573; Hengge U. R., Clin. Dermatol., 2005, 23: 107-114). Cells of XP patients present a reduced capacity to eliminate UV induced DNA lesions (Cordonnier, A. M. and R. P. Fuchs, Mutat. Res., 1999, 435, 111-119). Such abnormality results from a defect in the Nucleotide Excision Repair (NER) process, a versatile mechanism conserved among eukaryotes and implicated in the correction of the damaged DNA by excision of the damaged nucleotides and re-synthesis. Defect in this process leads to a persistence of UV damage in the DNA, resulting in mutagenesis and tumour development in the UV exposed skin area. The three major types of skin cancers, squamous cell carcinomas, basal cell carcinomas and malignant melanomas already appear in childhood. XP Patients were assigned to 7 complementation groups (XP-A to XP-G) by cell fusion experiments, and each complementation group turned out to result from mutations in a distinct NER gene. The human genes, and the encoded proteins were often named after the complementation group. For example, the XPC gene (
Until now the only treatment available to XP patients is either full protection against sun exposure (as well as against certain common lamps producing long-wavelength UV) or repeated surgery to remove appearing skin cancers. Several attempts of autologous graft have been made to replace such cancerous area with skin from unexposed parts of the patient's body. However, since the grafted cells are also sun-sensitive the benefits for the patients are at best, limited to a few years, and the majority of patients die before reaching adulthood because of metastases. Skin engraftment can be made locally, but with the general limitations of grafts, in term of immunological tolerance.
Thus gene and cell therapy represent a huge hope for this kind of disease. Since cells from the skin lineage can be easily manipulated in vitro, a possibility would be to manipulate patient cells and correct their genetic defect, before grafting them back at the site of the tumour. Compared to other XP complementation groups, XP-C seems to be the best candidate for corrective gene transfer. In Europe and North Africa, XP-C is involved in more than half of the XP patients and although XPC expression is ubiquitous, XP-C patients remain free of neurological problems observed in other XP groups. Preliminary studies aimed at tissue therapy of XP patients have shown that in vitro retroviral transduction of XP fibroblasts from various complementation groups (XP-A, XP-B, XP-C, XP-D) and of XP-C primary keratinocytes with the XP cloned genes result in the recovery of full DNA repair capacity (Arnaudeau-Begard et al., Hum. Gene Ther., 2003, 14, 983-996; Armelini et al., Cancer Gene Ther., 2005, 12, 389-396). Furthermore, cells from the skin lineage can be easily manipulated, and then used to reconstruct functional skin (Amaudeau-Begard et al., Hum. Gene Ther., 2003, 14, 983-996; Armelini et al., Cancer Gene Ther., 2005, 12, 389-396). Thus, a rationale and promising alternative for long term tissular therapy would then consist in an ex vivo gene correction of the XP-C locus in keratinocytes before grafting back a reconstructed skin to the patient.
Homologous recombination is the best way to precisely engineer a given locus. Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M. R., Science, 1989, 244: 1288-1292; Smithies O., Nat. Med., 2001, 7: 1083-1086) or knock-in exogenous sequences in the chromosome.
It can as well be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases, such as XP. However, this application is in fact difficult, due to the low efficiency of the process (10−6 to 10−9 of transfected cells). In the last decade, several methods have been developed to enhance this yield. For example, chimeraplasty (De Semir et al. J. Gene Med., 2003, 5: 625-639) and Small Fragment Homologous Replacement (Goncz et al., Gene Ther, 2001, 8: 961-965; Bruscia et al., Gene Ther., 2002, 9: 683-685; Sangiuolo et al., BMC Med. Genet., 2002, 3: 8-; De Semir and Aran, Oligonucleotides, 2003, 13: 261-269; U.S. Pat. No. 6,010,908) have both been used to try to correct CFTR mutations with various levels of success.
Another strategy to enhance the efficiency of recombination is to deliver a DNA double-strand break in the targeted locus, using meganucleases. Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (12 to 45 bp). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21: 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-5060; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-277; Donoho et al., Mol. Cell. Biol, 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448). Recently, I-SceI was used to stimulate targeted recombination in mouse hepatocytes in vivo. Recombination could be observed in up to 1% of hepatocytes (Gouble et al., J. Gene Med., 2006, 8, 616-622).
However, the use of this technology is limited by the repertoire of natural meganucleases. For example, there is no cleavage site for a known natural meganuclease in human XP genes. Therefore, the making of meganucleases with tailored specificities is under intense investigation, and several laboratories have tried to alter the specificity of natural meganucleases or to make artificial endonuclease.
Recently, fusion of Cys2-His2 type Zinc-Finger Proteins (ZFP) with the catalytic domain of the Type IIS FokI endonuclease were used to make functional sequence-specific artificial endonucleases (Smith et al., Nucleic Acids Res., 1999, 27: 674-681; Bibikova et al., Science, 2003, 300: 764; Porteus M. H. and D. Baltimore, Science, 2003, 300: 763). The binding specificity of ZFPs is relatively easy to manipulate, and a repertoire of novel artificial ZFPs, able to bind many (g/a)nn(g/a)nn(g/a)nn sequences is now available (Pabo et al., Annu. Rev. Biochem., 2001, 70, 313-340; Segal, D. J. and C. F. Barbas, Curr. Opin. Biotechnol., 2001, 12, 632-637; Isalan et al., Nat. Biotechnol., 2001, 19, 656-660). This last strategy allowed recently for the engineering of the IL2RG gene in vitro (Urnov et al., Nature, 2005, 435, 646-651). Nevertheless, preserving a very narrow specificity is one of the major issues for genome engineering applications, and presently it is unclear whether ZFPs would fulfill the very strict requirements for therapeutic applications.
Homing Endonucleases (HEs) are a widespread family of natural meganucleases including hundreds of proteins (Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). These proteins are encoded by mobile genetic elements which propagate by a process called “homing”: the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus (Kostriken et al., Cell; 1983, 35, 167-174; Jacquier, A. and B. Dujon, Cell, 1985, 41, 383-394). Given their natural function and their exceptional cleavage properties in terms of efficacy and specificity, HEs provide ideal scaffolds to derive novel endonucleases for genome engineering. Data have been accumulated over the last decade, characterizating the LAGLIDADG family, the largest of the four HE families (Chevalier and Stoddard, precited). LAGLIDADG refers to the only sequence actually conserved throughout the family and is found in one or (more often) two copies in the protein. Proteins with a single motif, such as I-CreI, form homodimers and cleave palindromic or pseudo-palindromic DNA sequences, whereas the larger, double motif proteins, such as I-SceI are monomers and cleave non-palindromic targets. Seven different LAGLIDADG proteins have been crystallized, and they exhibit a very striking conservation of the core structure, that contrasts with the lack of similarity at the primary sequence level (Jurica et al., Mol. Cell., 1998, 2, 469-476; Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier et al., J. Mol. Biol., 2003, 329, 253-269; Moure et al., J. Mol. Biol, 2003, 334, 685-695; Moure et al., Nat. Struct. Biol., 2002, 9, 764-770; Ichiyanagi et al., J. Mol. Biol., 2000, 300, 889-901; Duan et al., Cell, 1997, 89, 555-564; Bolduc et al., Genes Dev., 2003, 17, 2875-2888; Silva et al., J. Mol. Biol., 1999, 286, 1123-1136). In this core structure, two characteristic αββαββα folds, also called LAGLIDADG Homing Endonuclease Core Domains, contributed by two monomers, or by two domains in double LAGLIDAG proteins, are facing each other with a two-fold symmetry. DNA binding depends on the four β strands from each domain, folded into an antiparallel β-sheet, and forming a saddle on the DNA helix major groove (
Two approaches have been used to derive novel endonucleases with new specificities, from Homing Endonucleases:
Protein Variants
Seligman and co-workers used a rational approach to substitute specific individual residues of the I-CreI αββαββα fold (Sussman et al., J. Mol. Biol., 2004, 342, 31-41; Seligman et al., Genetics, 1997, 147, 1653-64); substantial cleavage was observed for few I-CreI variants (Y33C, Y33H, Y33R, Y33L, Y33S, Y33T, S32K, S32R) and only for a target modified in position ±10.
In a similar way, Gimble et al. modified the additional DNA binding domain of PI-SceI (J. Mol. Biol., 2003, 334, 993-1008); they obtained protein variants with altered binding specificity but no altered specificity and most of the variants maintained a lot of affinity for the wild-type target sequence.
The semi-rational approach used in theses studies permits the identification of endonucleases with altered specificity; however, it does not allow the direct production of endonucleases with predicted specificity.
Hybrid or Chimeric Single-Chain Proteins
New meganucleases could be obtained by swapping LAGLIDADG Homing Endonuclease Core Domains of different monomers (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). These single-chain chimeric meganucleases wherein the two LAGLIDADG Homing Endonuclease Core Domains from different meganucleases are linked by a spacer, are able to cleave the hybrid target corresponding to the fusion of the two half parent DNA target sequences.
The construction of chimeric and single chain artificial HEs has suggested that a combinatorial approach could be used to obtain novel meganucleases cleaving novel (non-palindromic) target sequences: different monomers or core domains could be fused in a single protein, to achieve novel specificities. These results mean that the two DNA binding domains of an I-CreI dimer behave independently; each DNA binding domain binds a different half of the DNA target site (
The generation of collections of novel meganucleases, and the ability to combine them by assembling two different monomers/core domains considerably enriches the number of DNA sequences that can be targeted (
To reach a larger number of sequences, it would be extremely valuable to be able to identify smaller independent subdomains that could be combined (
However, a combinatorial approach is much more difficult to apply within a single monomer or domain than between monomers since the structure of the binding interface is very compact and the two different ββ hairpins which are responsible for virtually all base-specific interactions do not constitute separate subdomains, but are part of a single fold. For example, in the internal part of the DNA binding regions of I-CreI, the gtc triplet is bound by one residue from the first hairpin (Q44), and two residues from the second hairpin (R68 and R70; see
A semi rational design assisted by yeast high throughput screening method allowed the Inventors, to identify and isolate thousands of I-CreI variants in positions 28, 30, 33, 38 and 40 with altered specificities in positions ±8 to 10 (10NNN DNA target). These new proteins were designed to cleave one of the 64 targets degenerate at nucleotides ±10, ±9, ±8 (10NNN DNA target) of the I-CreI original target site (
The capacity to combine four sub-domains considerably increases the number of DNA sequences that can be targeted (
Thus, it was not clear if mutants identified on 10NNN and 5NNN DNA targets as homodimers cleaving a palindromic sequence with the four central nucleotides being gtac, would allow the design of new endonucleases that would cleave targets containing changes in the four central nucleotides.
The Inventors have identified hundreds of DNA targets in the XP genes that could be cleaved by I-CreI variants. The combinatorial strategy described in
Even though the combined variants were initially identified towards nucleotides 10NNN and 5NNN respectively, and a strong impact of the four central nucleotides of the target on the activity of the engineered meganuclease was observed, functional meganucleases with a profound change in specificity regarding the other base-pairs of the target were selected. Furthermore, the activity of the engineered protein could be significantly improved by two successive rounds of random mutagenesis and screening, to compare with the activity of the I-CreI protein. Finally, the extensive redesign of the DNA binding domain is not made at the expense of the level of specificity, the novel endonucleases keeping a very narrow numbers of cleavable cognate targets.
These I-CreI variants which are able to cleave a DNA target sequence from a XP gene can be used for repairing the mutations associated with Xeroderma pigmentosum. Other potential applications include genome engineering at the XP genes loci.
The invention relates to an I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 26 to 40 and 44 to 77 of I-CreI, and is able to cleave a DNA target sequence from a xeroderma pigmentosum (XP) gene.
The cleavage activity of the variant according to the invention may be measured by any well-known, in vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736 or in Arnould et al., J. Mol. Biol., 2006, 355: 443-458. For example, the cleavage activity of the variant of the invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic DNA target sequence within the intervening sequence, cloned in a yeast or a mammalian expression vector. Expression of the variant results in a functional endonuclease which is able to cleave the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene, whose expression can be monitored by appropriate assay.
Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.
According to the present invention, the positions of the mutations are indicated by reference to the I-CreI amino acid sequence SEQ ID NO: 217.
In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of I-CreI are in positions 44, 68, 70, 75 and/or 77.
In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 26 to 40 of I-CreI are in positions 28, 30, 32, 33, 38 and/or 40.
In another preferred embodiment of said variant, said substitution(s) are in the subdomains situated from positions 28 to 40 and 44 to 70 of I-CreI, preferably in positions 28, 30, 32, 33, 38, 44, 68 and/or 70.
In another preferred embodiment of said variant, it comprises the substitution of the aspartic acid in position 75 by an uncharged amino acid, preferably an asparagine (D75N) or a valine (D75V).
In another preferred embodiment of said variant, it comprises one or more substitutions at additional positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. The I-CreI interacting residues are well-known in the art. The residues which are mutated may interact with the DNA backbone or with the nucleotide bases, directly or via water molecule.
In another preferred embodiment of said variant, it comprises one or more additional mutations that improve the binding and/or the cleavage properties of the variant towards the DNA target sequence of the XP gene. The additional residues which are mutated may be on the entire I-CreI sequence. These mutations may be substitutions in positions 19, 24, 42, 69, 80, 85, 87, 87, 109, 133 and 161. These mutations may affect the active site (position 19), the protein-DNA interface (for example, position 69), the hydrophobic core (for example, positions 85, 87 or 109) or the C-terminal part (for example, position 161).
In yet another preferred embodiment of said variant, said substitutions are replacement of the initial amino acids with amino acids selected from the group consisting of: A, D, E, G, H, K, N, P, Q, R, S, T, Y, C, W, L and V.
The variant according to the present invention may be an homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence. Alternatively, said variant is an heterodimer, resulting from the association of a first and a second monomer having different mutations in positions 26 to 40 and 44 to 77 of I-CreI, preferably in positions 28 to 40 and 44 to 70, said heterodimer being able to cleave a non-palindromic DNA target sequence from a XP gene.
The DNA target sequence which is cleaved by said variant may be in an exon or in an intron of the XP gene. Preferably, it is located, either in the vicinity of a mutation, preferably within 500 bp of the mutation, or upstream of a mutation, preferably upstream of all the mutations of said XP gene.
In another preferred embodiment of said variant, said DNA target sequence is from a human XP gene (XPA to XPG genes).
DNA targets from each human XP gene are presented in Tables IX to XV and
For example, the sequences SEQ ID NO: 1 to 24 are DNA targets from the XPC gene; SEQ ID NO: 1 to 23 are situated in or close to one of the exons and these sequences cover all the exons of the XP gene (Table XI and
Hererodimeric variants which cleave each DNA target are presented in Tables I to VIII and
The sequence of each variant is defined by its amino acid residues at the indicated positions. For example, the first heterodimeric variant of Table I consists of a first monomer having K, S, R, D, K, R, G and N in positions 28, 33, 38, 40, 44, 68, 70 and 75, respectively and a second monomer having R, D, R, K, A, S, N and I in positions 28, 30, 38, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I-CreI sequence SWISSPROT P05725, SEQ ID NO: 217 or pdb accession code 1 g9y; I-CreI has G, I, Q, K, N, S, Y, Q, S, A, Q, R, D, R, D, I, E, H, F, I, A and S, in positions 19, 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 68, 69, 70, 75, 77, 80, 85, 87, 109, 133 and 161, respectively. The variant may consist of an I-CreI sequence having the amino acid residues as indicated in the Table. In this case, the positions which are not indicated are not mutated and thus correspond to the wild-type I-CreI sequence. Alternatively, the variant may comprise an I-CreI sequence having the amino acid residues as indicated in the Table. In the latter case, the positions which are not indicated may comprise mutations as defined above, or may not be mutated. For example, the variant may be derived from an I-CreI scaffold protein encoded by SEQ ID NO: 26, said I-CreI scaffold protein (SEQ ID NO: 218) having the insertion of an alanine in position 2, the substitutions A42T, D75N, W110E and R111Q and three additional amino acids (A, A and D) at the C-terminus. In addition, said variant, derived from wild-type I-CreI or an I-CreI scaffold protein, may comprise additional mutations, as defined above.
The target which is cleaved by each heterodimeric variant is indicated in the last column of the Table.
In addition, the variants of the invention may include one or more residues inserted at the NH2 terminus and/or COOH terminus of the sequence. For example, a tag (epitope or polyhistidine sequence) is introduced at the NH2 terminus and/or COOH terminus; said tag is useful for the detection and/or the purification of said variant.
The subject-matter of the present invention is also a single-chain chimeric meganuclease derived from an I-CreI variant as defined above. The single-chain chimeric meganuclease is a fusion protein comprising two I-CreI monomers, two I-CreI core domains (positions 6 to 94 of I-CreI) or a combination of both. Preferably, the two monomers/core domains or the combination of both are connected by a peptidic linker.
The subject-matter of the present invention is also a polynucleotide fragment encoding a variant or a single-chain chimeric meganuclease as defined above; said polynucleotide may encode one monomer of an homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease.
The subject-matter of the present invention is also a recombinant vector for the expression of a variant or a single-chain meganuclease according to the invention. The recombinant vector comprises at least one polynucleotide fragment encoding a variant or a single-chain meganuclease, as defined above. In a preferred embodiment, said vector comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant.
A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semi-synthetic or synthetic DNA. Preferred vectors are those capable of autonomous repli-cation (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), para-myxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.
Preferably said vectors are expression vectors, wherein the sequence(s) encoding the variant/single-chain meganuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said variant. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome-binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the poly-peptide is expressed. Preferably, when said variant is an heterodimer, the two poly-nucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-β-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), α-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes.
According to another advantageous embodiment of said vector, it includes a targeting construct comprising sequences sharing homologies with the region surrounding the genomic DNA target cleavage site as defined above.
Alternatively, the vector coding for a I-CreI variant and the vector comprising the targeting construct are different vectors.
In both cases, the targeting construct comprises a sequence to be introduced flanked by sequences sharing homologies with the regions surrounding the genomic DNA cleavage sites of the variant as defined here after.
More preferably, said targeting DNA construct comprises:
a) sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above, and
b) a sequence to be introduced flanked by sequences as in a).
Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be located between the two arms. The sequence to be introduced is preferably a sequence which repairs a mutation in the gene of interest (gene correction or recovery of a functional gene), for the purpose of genome therapy. Alternatively, it can be any other sequence used to alter the chromosomal DNA in some specific way including a sequence used to modify a specific sequence, to attenuate or activate the endogenous gene of interest, to inactivate or delete the endogenous gene of interest or part thereof, to introduce a mutation into a site of interest or to introduce an exogenous gene or part thereof. Such chromosomal DNA alterations are used for genome engineering (animal models).
For correcting the XP gene, cleavage of the gene occurs in the vicinity of the mutation, preferably, within 500 bp of the mutation (
For example, the target which is cleaved by each of the variant (Tables I to VIII) and the minimal matrix for repairing the cleavage with each variant are indicated in Tables IX to XV and in
For example, for correcting some of the mutations in the XPC gene found in Xeroderma pigmentosum (XP), as indicated in
ARG579TER (Exon 4; Premature Stop Codon):
variant: 28Q,33S,38R,40K,44Q,68Y,70S,75N,77Y (first monomer)/28T,33T,38Q,40R,44T,68E,70S,75R,77R (second monomer), and a targeting construct comprising at least positions 9887 to 10086 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant 30D,33R,38T,44N,68R,70S,75Q,77R (first monomer)/28R,33A,38Y,40Q,44D,68Y,70S,75S,77R (second monomer) and a targeting construct comprising at least positions 10173 to 10372 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 6: Substitution PRO218HIS:
variant: 28K,33N,38Q,40Q,44R,68Y,70S,75E,771 (first monomer)/28K,33T,38A,40Q,44K,68Q,70S,75N,77R (second monomer), and a targeting construct comprising at least positions 13051 to 13250 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 9: deletion DEL1132AA or insertion insVAL580:
*variant: 28K,30N,38Q,44Q,68R,70S,75R,77T,80K (first monomer)/28T,33T,38Q,40R,44T,68Y,70S,75R,77V (second monomer) and a targeting construct comprising at least positions 19580 to 19779 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant: 28K,30N,38Q,44A,68Y,70S,75Y,77K (first monomer)/28K,33R,38E,40R,44T,68R,70S,75Y,77T,133V (second monomer) and a targeting construct comprising at least positions 20303 to 20502 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variants: 28K,33R,38Q,40A,44Q,68R,70N,75N (first monomer)/28K,33R,38A,40Q,44Q,68R,70S,75N,77K (second monomer) or 33H,75N (first monomer) and 33R,38A,40Q,44K,70N,75N (second monomer), and a targeting construct comprising at least positions 20349 to 20548 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
variant: 30N,33H,38Q,44K,68A,70S,75N,771 (first monomer)/28K,33N,38Q,40Q,44R,68Y,70S,75E,77V (second monomer), and a targeting construct comprising at least positions 20389 to 20588 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Exon 14: Substitution LYS822GLN:
variant: 28Q,33S,38R,40K, 42T,44K,70S,75N,77Y (first monomer)/28R,33A,38Y,40Q,44A,68R,70S,75E,77R (second monomer), and a targeting construct comprising at least positions 30416 to 30615 of the XPC gene, for efficient repair of the DNA double-strand break, and all sequences between the meganuclease cleavage site and the mutation site, for efficient repair of the mutation.
Alternatively, for restoring a functional gene (
The subject-matter of the present invention is also a composition characterized in that it comprises at least one variant, one single-chain chimeric endonuclease and/or at least one expression vector encoding said variant/single-chain molecule, as defined above.
In a preferred embodiment of said composition, it comprises a targeting DNA construct comprising a sequence which repairs a mutation in the XP gene, flanked by sequences sharing homologies with the genomic DNA cleavage site of said variant, as defined above. The sequence which repairs the mutation is either a fragment of the gene with the correct sequence or an exon knock-in construct, as defined above.
Preferably, said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the variant/single-chain chimeric endonuclease according to the invention.
In the case where two vectors may be used, the subject-matter of the present invention is also products containing an I-CreI variant or single-chain chimeric meganuclease expression vector as defined above and a vector which includes a targeting construct as defined above, as a combined preparation for simultaneous, separate or sequential use in Xeroderma pigmentosum.
The subject-matter of the present invention is also the use of at least one meganuclease variant/single-chain chimeric meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing Xeroderma pigmentosum in an individual in need thereof, said medicament being administrated by any means to said individual.
In this case, the use of the meganuclease (variant/single-chain derivative) comprises at least the step of (a) inducing in somatic tissue(s) of the individual a double stranded cleavage at a site of interest comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into the individual a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA. The targeting DNA is introduced into the individual under conditions appropriate for introduction of the targeting DNA into the site of interest.
According to the present invention, said double-stranded cleavage is induced, either in toto by administration of said meganuclease to an individual, or ex vivo by introduction of said meganuclease into somatic cells (skin cells) removed from an individual and returned into the individual after modification.
The subject-matter of the present invention is also a method for preventing, improving or curing Xeroderma pigmentosum in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means.
The meganuclease (variant/single-chain derivative) can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into somatic cells of an individual, by any convenient mean well-known to those in the art, which is appropriate for the particular cell type, alone or in association with either at least an appropriate vehicle or carrier and/or with the targeting DNA.
According to an advantageous embodiment of the uses according to the invention, the meganuclease (polypeptide) is associated with:
According to another advantageous embodiment of the uses according to the invention, the meganuclease (polynucleotide encoding said meganuclease) and/or the targeting DNA is inserted in a vector. Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.
Once in a cell, the meganuclease and if present, the vector comprising targeting DNA and/or nucleic acid encoding a meganuclease are imported or translocated by the cell from the cytoplasm to the site of action in the nucleus.
For purposes of therapy, the meganucleases and a pharmaceutically acceptable excipient are administered in a therapeutically effective amount. Such a combination is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality.
In one embodiment of the uses according to the present invention, the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response. A variety of methods for ameliorating or eliminating deleterious immunological reactions of this sort can be used in accordance with the invention. In a preferred embodiment, the meganuclease is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate meganucleases to polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”) (preferably of 500 to 20,000 daltons average molecular weight (MW)). Conjugation with PEG or PPG, as described by Davis et al., (U.S. Pat. No. 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble endonuclease conjugates with anti-viral activity. Similar methods also using a polyethylene-poly-propylene glycol copolymer are described in Saifer et al. (U.S. Pat. No. 5,006,333).
The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector.
The invention also concerns a non-human transgenic animal or a transgenic plant, characterized in that all or part of their cells are modified by a polynucleotide or a vector as defined above.
As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or an eukaryotic cell, such as an animal, plant or yeast cell.
The subject-matter of the present invention is further the use of a meganuclease (variant or single-chain derivative) as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for genome engineering (animal models generation: knock-in or knock-out), for non-therapeutic purposes.
According to an advantageous embodiment of said use, it is for inducing a double-strand break in the gene of interest, thereby inducing a DNA recombination event, a DNA loss or cell death.
According to the invention, said double-strand break is for: repairing a specific sequence, modifying a specific sequence, restoring a functional gene in place of a mutated one, attenuating or activating an endogenous gene of interest, introducing a mutation into a site of interest, introducing an exogenous gene or a part thereof, inactivating or deleting an endogenous gene or a part thereof, translocating a chromosomal aim, or leaving the DNA unrepaired and degraded.
According to another advantageous embodiment of said use, said variant, polynucleotide(s), vector are associated with a targeting DNA construct as defined above.
In a first embodiment of the use of the meganuclease (variant/single-chain derivative) according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at the genomic locus comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease; 2) providing a targeting DNA construct comprising the sequence to be introduced flanked by sequences sharing homologies to the targeted locus. Said meganuclease variant can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell. This strategy is used to introduce a DNA sequence at the target site, for example to generate knock-in or knock-out animal models or cell lines that can be used for drug testing.
The subject-matter of the present invention is also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example a third round of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel, third generation homing endonucleases.
The different uses of the I-CreI variant and the methods of using said I-CreI variant according to the present invention include also the use of the single-chain chimeric meganuclease derived from said variant, the polynucleotide(s), vector, cell, transgenic plant or non-human transgenic mammal encoding said variant or single-chain chimeric endonuclease, as defined above.
The I-CreI variant according to the invention may be obtained by a method for engineering I-CreI variants able to cleave a genomic DNA target sequence from a gene of interest, for example a mammalian gene, comprising at least the steps of:
(a) constructing a first series of I-CreI variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 26 to 40 of I-CreI, preferably from positions 28 to 40 of I-CreI,
(b) constructing a second series of I-CreI variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of I-CreI, preferably from positions 44 to 70 of I-CreI,
(c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions −10 to −8 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions −10 to −8 of said genomic target and (ii) the nucleotide triplet in positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions −10 to −8 of said genomic target
(d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions −5 to −3 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions −5 to −3 of said genomic target and (ii) the nucleotide triplet in positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions −5 to −3 of said genomic target (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions +8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (ii) the nucleotide triplet in positions −10 to −8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +8 to +10 of said genomic target
(f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet in positions +3 to +5 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +3 to +5 of said genomic target and (ii) the nucleotide triplet in positions −5 to −3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target
(g) combining in a single variant, the mutation(s) in positions 28 to 40 and 44 to 70 of two variants from step (c) and step (d), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet in positions −10 to −8 is identical to the nucleotide triplet which is present in positions −10 to −8 of said genomic target, (ii) the nucleotide triplet in positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions −10 to −8 of said genomic target, (iii) the nucleotide triplet in positions −5 to −3 is identical to the nucleotide triplet which is present in positions −5 to −3 of said genomic target and (iv) the nucleotide triplet in positions +3 to +5 is identi-cal to the reverse complementary sequence of the nucleotide triplet which is present in positions −5 to −3 of said genomic target
(h) combining in a single variant, the mutation(s) in positions 28 to 40 and 44 to 70 of two variants from step (e) and step (f), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet in positions +3 to +5 is identical to the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (ii) the nucleotide triplet in positions −5 to −3 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (iii) the nucleotide triplet in posi-tions+8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (iv) the nucleotide triplet in positions −10 to −8 is identical to the reverse complementary sequence of the nucleo-tide triplet in positions +8 to +10 of said genomic target.
(i) combining the variants obtained in steps (g) and (h) to form heterodimers.
(j) selecting and/or screening the heterodimers from step (i) which are able to cleave said genomic DNA target situated in a mammalian gene.
Steps (a), (b), (g), (h) and (i) may further comprise the introduction of additional mutations in order to improve the binding and/or cleavage properties of the mutants. Additional mutations may be introduced at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. This additional step may be performed by generating a library of variants as described in the International PCT Application WO 2004/067736.
The method for engineering I-CreI variants of the invention advantageously comprise the introduction of random mutations on the whole variant or in a part of the variant, in particular the C-terminal half of the variant (positions 80 to 163) to improve the binding and/or cleavage properties of the mutants towards the DNA target from the gene of interest. The mutagenesis may be performed by generating random mutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art and commercially available. Preferably, the mutagenesis is performed on the entire sequence of one monomer of the heterodimer formed in step (i) or obtained in step (j), advantageously on a pool of monomers, preferably on both monomers of the heterodimer of step (i) or (j).
Preferably, two rounds of selection/screening are performed according to the process illustrated by
The combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques.
The combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), so as to allow the formation of heterodimers. For example, host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the variant(s), so that heterodimers are formed in the host cells.
The selection and/or screening in steps (c), (d), (e), (f) and/or G) may be performed by using a cleavage assay in vitro or in vivo, as described in the International PCT Application WO 2004/067736 or in Arnould et al., J. Mol. Biol., 2006, 355(3): 443-58.
According to another advantageous embodiment of said method, steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break.
The polynucleotide sequence(s) encoding the variant as defined in the present invention may be prepared by any method known by the man skilled in the art. For example, they are amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.
The recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.
The variant of the invention is produced by expressing the poly-peptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed in a host cell modified by one or two expression vector(s), under conditions suitable for the expression or co-expression of the polypeptides, and the variant is recovered from the host cell culture.
Single-chain chimeric meganucleases able to cleave a DNA target from the gene of interest are derived from the variants according to the invention by methods well-known in the art (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention.
In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the I-CreI meganuclease variants and their uses according to the invention, as well as to the appended drawings in which:
The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity, are described in the International PCT Application WO 2004/067736, Arnould et al., J. Mol. Biol., 2006, 355, 443-458, Epinat et al., N.A.R., 2003, 31, 2952-2962 and Chames et al., Nucleic Acids Res., 2005, 33, e178). These assays result in a functional LacZ reporter gene which can be monitored by standard methods.
I-CreI wt and I-CreI D75N open reading frames were synthesized, as described previously (Epinat et al., N.A.R., 2003, 31, 2952-2962). Mutation D75N was introduced by replacing codon 75 with aac. Three combinatorial libraries (Ulib4, Ulib5 and Lib4) were derived from the I-CreI D75N protein by replacing three different combinations of residues, potentially involved in the interactions with the bases in positions ±8 to 10 of one DNA target half-site. The diversity of the meganuclease libraries was generated by PCR using degenerated primers harboring a unique degenerated codon (coding for 10 or 12 different amino acids), at each of the selected positions.
The three codons at positions N30, Y33 and Q38 (Ulib4 library) or K28, N30 and Q38 (Ulib5 library) were replaced by a degenerated codon VVK (18 codons) coding for 12 different amino acids: A,D,E,G,H,K,N,P,Q,R,S,T). In consequence, the maximal (theoretical) diversity of these protein libraries was 123 or 1728. However, in terms of nucleic acids, the diversity was 183 or 5832. Fragments carrying combinations of the desired mutations were obtained by PCR, using a pair of degenerated primers (Ulib456for and Ulib4rev; Ulib456for and Ulib5rev,
In Lib4, ordered from BIOMETHODES, an arginine in position 70 was first replaced with a serine (R70S). Then positions 28, 33, 38 and 40 were randomized. The regular amino acids (K28, Y33, Q38 and S40) were replaced with one out of 10 amino acids (A,D,E,K,N,Q,R,S,T,Y). The resulting library has a theoretical complexity of 10000 in terms of proteins.
The C1221 twenty-four bp palindrome (tcaaaacgtcgtacgacgttttga, (SEQ ID NO: 25) is a repeat of the half-site of the nearly palindromic natural I-CreI target (tcaaaacgtcgtgagacagtttgg, SEQ ID NO: 38). C1221 is cleaved as efficiently as the I-CreI natural target in vitro and ex vivo in both yeast and mammalian cells. The 64 palindromic targets were derived from C1221 as follows: 64 pairs of oligonucleotides ((ggcatacaagtttcnnnacgtcgtacgacgtnnngacaatcgtctgtca (SEQ ID NO: 39) and reverse complementary sequences) were ordered form Sigma, annealed and cloned into pGEM-T Easy (PROMEGA) in the same orientation. Next, a 400 bp PvuII fragment was excised and cloned into the yeast vector pFL39-ADH-LACURAZ, also called pCLS0042, and the mammalian vector pcDNA3 derivative (pcDNA3.1-LAACZ), both described previously (Epinat et al., 2003, precited), resulting in 64 yeast reporter vectors (target plasmids).
Alternatively, double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotides, was cloned using the Gateway protocol (INVITROGEN) into yeast and mammalian reporter vectors.
The library of meganuclease expression variants was transformed into the leu2 mutant haploid yeast strain FYC2-6A: alpha, trp1Δ63, leu2Δ1, his3Δ200. A classical chemical/heat choc protocol that routinely gives us 106 independent transformants per μg of DNA derived from (Gietz and Woods, Methods Enzymol., 2002, 350, 87-96), was used for transformation. Individual transformant (Leu+) clones were individually picked in 96 wells microplates.13824 colonies were picked using a colony picker (QpixII, GENETIX), and grown in 144 microtiter plates.
The 64 target plasmids were transformed using the same protocol, into the haploid yeast strain FYBL2-7B: a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202, resulting in 64 tester strains.
Meganuclease expressing clones were mated with each of the 64 target strains, and diploids were tested for beta-galactosidase activity, by using the screening assay illustrated on FIG. 2 of Arnould et al., 2006, precited. I-CreI variant clones as well as yeast reporter strains were stocked in glycerol (20%) and replicated in novel microplates. Mating was performed using a colony gridder (QpixII, GENETIX). Mutants were gridded on nylon filters covering YPD plates, using a high gridding density (about 20 spots/cm2). A second gridding process was performed on the same filters to spot a second layer consisting of 64 different reporter-harboring yeast strains for each variant. Membranes were placed on solid agar YPD rich medium, and incubated at 30° C. for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (2%) as a carbon source (and with G418 for coexpression experiments), and incubated for five days at 37° C., to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37° C., to monitor β-galactosidase activity. After two days of incubation, positive clones were identified by scanning. The β-galactosidase activity of the clones was quantified using appropriate software. The clones showing an activity against at least one target were isolated (first screening). The spotting density was then reduced to 4 spots/cm2 and each positive clone was tested against the 64 reporter strains in quadruplicate, thereby creating complete profiles (secondary screening).
The open reading frame (ORF) of positive clones identified during the first and/or secondary screening in yeast was amplified by PCR on yeast colonies using primers: PCR-Gal10-F (gcaactttagtgctgacacatacagg, SEQ ID NO:40) and PCR-Gal10-R (acaaccttgattgcagacttgacc, SEQ ID NO:41) or 5′ggggacaagtttgtacaaaaaaggcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc3′ (SEQ ID NO: 225) and 5′ggggaccactttgtacaagaaagctgggtttagtcggccgccggggaggatttcttcttctcgc 3′ (SEQ ID NO: 226), from PROLIGO. Briefly, yeast colony is picked and resuspended in 100 μl of LGlu liquid medium and cultures overnight. After centrifugation, yeast pellet is resuspended in 10 μl of sterile water and used to perform PCR reaction in a final volume of 50 μl containing 1.5 μl of each specific primers (100 pmol/μl). The PCR conditions were one cycle of denaturation for 10 minutes at 94° C., 35 cycles of denaturation for 30 s at 94° C., annealing for 1 min at 55° C., extension for 1.5 min at 72° C., and a final extension for 5 min. The resulting PCR products were then sequenced.
The open reading frames (ORFs) of positive clones identified during the primary screening were recloned using the Gateway protocol (Invitrogen). ORFs were amplified by PCR on yeast colonies, as described in e). PCR products were then cloned in: (i) yeast gateway expression vector harboring a galactose inducible promoter, LEU2 or KanR as selectable marker and a 2 micron origin of replication, (ii) a pET 24d(+) vector from NOVAGEN, and (iii) a CHO gateway expression vector pcDNA6.2 from INVITROGEN. Resulting clones were verified by sequencing (MILLEGEN).
I-CreI is a dimeric homing endonuclease that cleaves a 22 bp pseudo-palindromic target. Analysis of I-CreI structure bound to its natural target has shown that in each monomer, eight residues establish direct interactions with seven bases (Jurica et al., 1998, precited). According to these structural data, the bases of the nucleotides in positions +8 to 10 establish specific contacts with I-CreI amino-acids N30, Y33 and Q38 (
An exhaustive protein library vs. target library approach was undertaken to engineer locally this part of the DNA binding interface. First, the I-CreI scaffold was mutated from D75 to N. The D75N mutation did not affect the protein structure, but decreased the toxicity of I-CreI in overexpression experiments.
Next the Ulib4 library was constructed: residues 30, 33 and 38, were randomized, and the regular amino acids (N30, Y33, and Q38) replaced with one out of 12 amino acids (A,D,E,G,H,K,N,P,Q,R,S,T). The resulting library has a complexity of 1728 in terms of protein (5832 in terms of nucleic acids).
Then, two other libraries were constructed: Ulib5 and Lib4. In Ulib5, residues 28, 30 and 38 were randomized, and the regular amino acids (K28, N30, and Q38) replaced with one out of 12 amino acids (ADEGHKNPQRST). The resulting library has a complexity of 1728 in terms of protein (5832 in terms of nucleic acids). In Lib4, an Arginine in position 70 was first replaced with a Serine. Then, positions 28, 33, 38 and 40 were randomized, and the regular amino acids (8, Y33, Q38 and S40) replaced with one out of 10 amino acids (A,D,E,K,N,Q,R,S,T,Y). The resulting library has a complexity of 10000 in terms of proteins.
In a primary screening experiment, 20000 clones from Ulib4, 10000 clones from Ulib5 and 20000 clones from Lib4 were mated with each one of the 64 tester strains, and diploids were tested for beta-galactosidase activity. All clones displaying cleavage activity with at least one out of the 64 targets were tested in a second round of screening against the 64 targets, in quadriplate, and each cleavage profile was established. Then, meganuclease ORF were amplified from each strain by PCR, and sequenced, and 141 different meganuclease variants were identified.
The 141 validated clones showed very diverse patterns. Some of these new profiles shared some similarity with the wild type scaffold whereas many others were totally different. Results are summarized in
However, a lot of proteins display very different patterns. With a few variants, cleavage of a unique sequence is observed. For example, protein I-CreI K28 R30G33 T38 S40 R70N75 is active on the “ggg” target, which was not cleaved by wild type protein, while I-CreI Q28 N30 Y33 Q38 R40 S70 N75 cleaves AAT, one of the targets cleaved by I-CreI N75. Other proteins cleave efficiently a series of different targets: for example, I-CreI N28 N30 S33 R38K40 S70 N75 cleaves ggg, tgg and tgt, CreI K28 N30H33 Q38 S40 R70 N75 cleaves aag, aat, gac, gag, gat, gga, ggc, ggg, and ggt. The number of cleaved sequences ranges from 1 to 10. Altogether, 37 novel targets were cleaved by the mutants, including 34 targets which are not cleaved by I-CreI and 3 targets which are cleaved by I-CreI (aag, aat and aac,
A first series of I-CreI variants having at least one substitution in positions 44, 68 and/or 70 of I-CreI and being able to cleave mutant I-CreI sites having variation in positions ±3 to 5 was identified previously (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). The cleavage pattern of the variants is presented in
A second series of I-CreI variants having at least one substitution in positions 28, 30, 33 or 28, 33, 38 and 40 of I-CreI and being able to cleave mutant I-CreI sites having variation in positions ±8 to 10 was identified as described in example 1. The cleavage pattern of the variants is presented in
Positions 28, 30, 33, 38 and 40 on one hand, and 44, 68 and 70, on another hand are on a same DNA-binding fold, and there is no structural evidence that they should behave independently. However, the two sets of mutations are clearly on two spatially distinct regions of this fold (
This hypothesis was verified by using targets situated in a gene of interest, the XPC gene. The targets cleaved by the I-CreI variants are 24 bp derivatives of C1221, a palindromic sequence cleaved by I-CreI. However, the structure of I-CreI bound to its DNA target suggests that the two external base pairs of these targets (positions −12 and 12) have no impact on binding and cleavage (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774; Chevalier et al., 2003, J. Mol. Biol., 329, 253-269) and in this study, only positions −11 to 11 were considered. Consequently, the series of targets identified in the XPC gene were defined as 22 bp sequences instead of 24 bp.
Xa.1, Xb.1 and Xc. 1 are 22 bp (non-palindromic) targets located at position 9119, 13521 and 20438, respectively of the human XPC gene (
Xa.1 is partly a patchwork of the 10TGC_P, 10AGG_P, 5TTT_P and 5CCT_P targets (
Xb.1 is partly a patchwork of the 10GGG_P, 10TGT_P, 5GGG_P and 5TAC_P targets (
Xc.1 is partly a patchwork of the 10GAG_P, 10GTA_P and 5TCT_P targets (
Therefore, to verify this hypothesis, two palindromic targets, corresponding to the left (Xx.3) and right half (Xx.4) sequences of the identified targets (Xx. 1) were produced (
This example shows that I-CreI mutants can cut the Xx.3 DNA target sequences derived from the left part of the Xx.2 targets in a palindromic form (
Xa.3 is similar to 5TTT_P in positions ±1, ±2, ±3, ±4, ±5 and ±111 and to 10TGC_P in positions ±1, ±2, ±8, ±9, ±10 and ±11.
Xb.3 is similar to 5GGG_P in positions ±1, ±2, ±3, ±4, ±5 and ±11 and to 10GGG_P in positions ±1, ±2, ±8, ±9, ±10 and ±11
Xc.3 is similar to C1221 in positions ±1, ±2, ±3, ±4, ±5, ±7 and ±11 and to 10GAG_P in positions ±1, ±2, ±7, ±8, ±9, ±10 and ±11
The wild-type I-CreI is known to be tolerant for nucleotide substitutions at positions ±1, ±7 and ±6 (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269; Jurica et al., Mol. Cell., 1998, 2, 469-476). Thus, it was hypothesized that positions +6 and +7 would have little effect on the binding and cleavage activity.
Mutants able to cleave the 5TTT_P and 5GGG_P targets were previously obtained by mutagenesis on I-CreI N75 at positions 44, 68 and 70 or I-CreI S70 at positions 44, 68, 75 and 77, as described in Arnould et al., J. Mol. Biol., 2006, 355, 443-458 (
Some sets of proteins are both mutated at position 70. However, it was hypothesized that two separable functional subdomains exist in I-CreI. That implies that this position has little impact on the specificity in bases 10 to 8 of the target.
Therefore, to check whether combined mutants could cleave the Xa.3 and Xb.3 targets, mutations at positions 44, 68, 70 and/or 75 from proteins cleaving the 5NNN region of the target (5TTT_P and 5GGG_P targets) were combined with the 28, 30, 32, 33, 38 and/or 40 mutations from proteins cleaving the 10NNN region of the targets (10TGC_P and 10GGG_P targets), as illustrated in
Xc.3 which is identical to C1221 in positions ±3 to 5 should be cleaved by previously identified mutants cleaving the 10 GAG_P target (no combination of mutations).
The C1221 derived target was cloned as follows: oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence was ordered from Proligo (as example: 5′ tggcatacaagtttctgccttttgtacaaaaggcagacaatcgtctgtca 3′ (SEQ ID NO: 42, for the Xa.3 target). Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the GatewayR protocol (INVITROGEN) into yeast reporter vector (pCLS1055,
I-CreI mutants cleaving 10TGC_P, 10GGG_P, 5TTT_P or 5GGG_P were identified as described in example 1 and
Thus, to generate an intact coding sequence by in vivo homologous recombination in yeast, approximately 25 ng of each of the two overlapping PCR fragments and 25 ng of the pCLS0542 vector DNA linearized by digestion with NcoI and EagI or 25 ng of the pCLS1107 vector DNA linearized by digestion with DraIII and NgoMIV were used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200) using a high efficiency LiAc transformation protocol (Gietz, R. D. and R. A. Woods, Methods Enzymol, 2002, 350, 87-96). Combinatorial mutants can advantageously be generated as libraries: PCR reactions were pooled in equimolar amounts and transformed into yeast together with the linearized plasmid. Transformants were selected on either synthetic medium lacking leucine (pCLS0542) or rich medium containing G418 (PCLS1107). Colonies were picked using a colony picker (QpixII, Genetix), and grown in 96 well microtiter plates.
The experimental procedure is as described in example 1, except that a low gridding density (about 4 spots/cm2) was used.
To recover the mutant expressing plasmids, yeast DNA was extracted using standard protocols and used to transform E. coli. Sequencing of mutant ORF was then performed on the plasmids by MILLEGEN SA. Alternatively, the ORFs of positive clones identified during the primary screening in yeast were amplified by PCR on yeast DNA extract from colonies (Akada et al., Biotechniques, 2000, 28(4): 668-70, 672, 674) using primers 5′ ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc 3′ (SEQ ID NO: 225) and 5′ggggaccactttgtacaagaaagctgggtttagtcggccgccgggaggatttcttcttctcgc 3′ (SEQ ID NO: 226) from PROLIGO and sequencing was performed directly on PCR product by MILLEGEN. PCR products were cloned either in (i) yeast gateway expression vectors harboring a galactose inducible promoter, LEU2 or KanR as selectable marker and a 2 micron origin of replication, (ii) CHO gateway expression vector pcDNA6.2 from INVITROGEN. Resulting clones were verified by sequencing (MILLEGEN).
I-CreI N75 mutants cutting the 10TGC_P (ctgcacgtcgt_P) target and I-CreI N75 (Q44, R68, R70) mutants cutting the 5TTT_P (caaaactttgt_P) target were combined, resulting in combinatorial mutants that were screened against the Xa.3 target (ctgccttttgt_P).
I-CreI N75 mutants cutting the 10GGG_P target and I-CreI N75 mutants cutting the 5GGG_P target were combined, resulting in combinatorial mutants that were screened against the Xb.3 target.
At least twice the diversity of each library was screened. No correlation was observed between the number of active mutants identified and the number of combinations tested.
The Xc.3 target sequence contained the wild type sequence GTC at positions ±5, ±4 and ±3; therefore the combinatorial approach was not necessary to generate specific variants towards this target. I-CreI variants previously identified with altered substrate specificity towards bases ±10, ±9 and ±8 were directly screened against the Xc.3 DNA target.
Eight combinatorial mutants were found cleave the Xa.3 target (Table XVI). Two of the mutants cleaving the Xa.3 target have the following sequence:
Thirty combinatorial mutants were found to cleave the Xb.3 target (Table XVI).
Among the mutants cutting the 10GAG_P target which were tested, twenty one were found to cleave the Xc.3 target (Table XVI).
Results were confirmed in a secondary screen (
This example shows that I-CreI variants can cleave the Xx.4 DNA target sequence derived from the right part of the Xx.2 target in a palindromic form (
Xa.4 is similar to 5CCT_P in positions ±1, ±2, ±3, ±4, ±5 and ±7 and to 10AGG_P in positions ±1, ±2, ±7, ±8, ±9 and ±10. It was hypothesized that positions 16 and +11 would have little effect on the binding and cleavage activity.
Xb.4 is similar to 5TAC_P in positions ±1, ±2, ±3, ±4, ±5, ±6 and ±11 and to 10TGT_P in positions ±1, ±2, ±6, ±8, ±9, ±10, and ±11. It was hypothesized that positions ±7 would have little effect on the binding and cleavage activity.
Xc.4 is similar to 5TCT_P in positions ±1, ±2, ±3, ±4, ±5, ±7 and ±11 and to 10GTA_P in positions ±1, ±2, ±7, ±8, ±9, ±10, and ±11. It was hypothesized that positions +6 would have little effect on the binding and cleavage activity.
Mutants able to cleave the 5CCT_P, 5TAC_P and 5TCT_P targets were previously obtained by mutagenesis on I-CreI N75 at positions 44, 68 and 70 or I-CreI S70 at positions 44, 68, 75 and 77, as described in Arnould et al., J. Mol. Biol., 2006, 355, 443-458 (
Some sets of proteins are both mutated at position 70. However, it was hypothesized that I-CreI comprises two separable functional subdomains. That implies that this position has little impact on the specificity in base 10 to 8 of the target.
Therefore, to check whether combined mutants could cleave the Xx.4 target, mutations at positions 44, 68, 70 and/or 75 from proteins cleaving 5CCT_P, 5TAC_P and 5TCT_P targets were combined with the 28, 30, 32, 33, 38, and/or 40 mutations from proteins cleaving 10AGG_P, 10TGT_P, and 10GTA_P targets, as illustrated in
See example 3.
I-CreI combined mutants were constructed by associating mutations at positions 44, 68 and 70 with the 28, 30, 33, 38 and 40 mutations on the I-CreI N75 scaffold. Combined mutants were screened against the Xx.4 DNA targets At least twice the diversity of each library was screened. No correlation was observed between the number of active mutants identified and the number of combinations tested. Two percent of the hybrid mutants appear to be functional for the Xb.4 DNA targets while as many as 55% were active against Xa.4. After secondary screening and sequencing, 104, 8 and 4 different cleavers were identified, for the Xa.4, Xb.4 and Xc.4 target, respectively (Table XVII).
I-CreI N75 mutants cutting the 10AGG_P (caggacgtcgt_P; SEQ ID NO: 32) target (amino acids at positions 28, 30, 33, 38 and 40 are indicated) and I-CreI N75 mutants cutting the 5CCT_P (caaaaccctgt_P; SEQ ID NO: 34) target (amino acids at positions 44, 68 and 70 are indicated) are listed in Table XVIII. 39 of the 104 positives are presented in
I-CreI mutants able to cleave each of the palindromic Xx.2 derived targets Xx.3 and Xx.4, were identified in examples 3 and 4. A subset of pairs of such mutants (one cutting Xx.3 and one cutting Xx.4), were co-expressed in yeast. Upon co-expression, there should be three active molecular species, two homodimers, and one heterodimer. It was assayed whether the heterodimers that should be formed could cleave the Xx.1 and Xx.2 targets as depicted in
In order to co-express two I-CreI mutants in yeast, mutants cutting the Xx.3 sequence were subcloned in a kanamycin resistant yeast expression vector (PCLS1107,
Mutants were amplified by PCR reaction using primers common for leucine vector (pCLS0542) and kanamycin vector (pCLS1107) (Ga110F and Ga110R). Approximately 25 ng of PCR fragment and 25 ng of vector DNA (pCLS1107) linearized by digestion with DraIII and NgoMIV are used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200) using a high efficiency LiAc transformation protocol. An intact coding sequence for the I-CreI mutant is generated by in vivo homologous recombination in yeast.
Yeast strain expressing a mutant cutting the Xx.4 target was transformed with DNA coding for a mutant cutting the Xx.3 target in pCLS1107 expression vector. Transformants were selected on −L Glu+G418 medium.
The experimental procedure is as described in example 1, except that a low gridding density (about 4 spots/cm2) was used.
The Table XIX summarizes the number of total and active heterodimers tested by co-expression in yeast against the targets Xx.2 and Xx.1.
In all cases, heterodimers with cleavage activity for the target.2 were identified (
Examples of functional combinations for the Xa.1 target are presented in Tables XX and XXI. As a general rule, functional heterodimers cutting Xx.1 sequence were always obtained when the two expressed proteins gave a strong signal as homodimer. Moreover, while many mutants are still very active against Xc.1, the mutants capable of cleaving the Xa.1 target displayed a weak activity
Error-prone PCR was used to introduce random mutations in a pool of 4 chosen mutants. Libraries were generated by PCR using either Mn2+, or by two-steps process using dNTPs derivatives 8-oxo-dGTP and dPTP as described in the protocol from JENA BIOSCIENCE GmbH for the JBS dNTP-Mutagenesis kit. Primers used are: preATGCreFor (5′-gcataaattactatacttctatagacacgcaaacacaaatacacagcggccttgccacc-3′, SEQ ID NO: 227) and ICreIpostRev (5′-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgc-3′, SEQ ID NO: 228). For the first round of activity improvement, the new libraries were cloned in vivo in yeast in the linearized kanamycin vector (pCLS1107,
Since, the decrease in the cleavage activity between the target.2 and the target .1 (
The 6 combinations giving the strongest cleavage activity were selected and the ORFs were sequenced. Interestingly, the 6 most active heterodimers resulted from 6 different combinations of 3 independent mutants cleaving Xa.3 with 2 different mutants cleaving Xa.4. The
The protein sequence analysis of the best cutters does not reveal any particular protein domains affected by the mutation process. As compared with the original sequence, the error-prone PCR introduced mutations at positions 19, 69 and 87 in the ORF of active mutants cleaving Xa.3 target and mutations at positions 32, 85 and 109 in the coding sequence of the mutants cleaving Xa.4 target. The positions 33 and 38 were also reverted in the protein sequence of the M2 and M5 proteins. Interestingly, the amino acid at position 19 was mutated in all proteins with activity towards the Xa.3 target. This position is part of the catalytic site (Chevalier et al., Biochemistry, 2004, 43, 14015-14026) and with the adjacent Asp20, is involved in the metal cation binding. This is the only mutation which can be directly linked to improvement of the catalytic mechanism. The mutations in positions 32 and 69 affect the protein-DNA interface and the mutations in positions 85 and 87 affect the hydrophobic core. The other mutations affect mainly the core protein indicating that a mechanism of propagated conformational change is responsible for the improved activity. This long range effect could, for example, improve the binding affinity of the mutant and therefore increase its cleavage activity.
These results demonstrate that the combinatorial approach associated to random mutagenesis allows the rapid and efficient production of custom-designed endonucleases for specific DNA substrates.
CHO cells were transfected with Polyfect® transfection reagent according to the supplier (QIAGEN) protocol. 72 hours after transfection, culture medium was removed and 150 μl of lysis/revelation buffer added for β-galactosidase liquid assay (1 liter of buffer contains 100 ml of lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton X100 0.1%, BSA 0.1 mg/ml, protease inhibitors), 10 ml of Mg 100× buffer (MgCl2 100 mM, β-mercaptoethanol 35%), 110 ml ONPG (8 mg/ml) and 780 ml of sodium phosphate 0.1 M pH7.5). After incubation at 37° C., OD was measured at 42 0 nm. The entire process is performed on an automated Velocity11 BioCel platform.
The hybrid meganucleases active in yeast were tested in mammalian cells by transient co-transfection of CHO cells with a target vector and meganuclease expression vectors. For this purpose, subsets of mutants and their corresponding targets (Xa.1 and Xc.1) were cloned into appropriate vectors as described in example 1, and the meganuclease-induced recombination efficiency was measured by a standard, quantitative ONPG assay that monitors the restoration of a functional β-galactosidase gene, as described previously (International PCT Application WO 2006/097853; Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Smith et al., Nucleic Acids Res., Epub 27 November 2006).
The degeneracy at individual positions of the I-CreI target has been previously assayed using in vitro site selection in which variant DNA targets cleavable by wtI-CreI could be recovered (Argast et al., J. Mol. Biol., 1998, 280, 345-353). It indicates that most nucleotide positions in the site can be mutated without loss of binding or cleavage. However no exhaustive study was done. In order to compare the improved mutants towards Xa.1 target with I-CreI homing endonuclease, we have generated all possible targets carrying individual mutation were generated for Xa.3, Xa.4 and the palindromic I-CreI target C1221. The protein scaffold used to generate all the mutants carries a D to N mutation at position 75. This mutation was introduced in order to decrease the energetic strains caused by the replacement of the basic residues at positions 68 and 70 in the libraries. It was shown previously that the D75N mutation decreased the toxicity of over-expressed I-CreI protein without affecting the protein basic folding properties and activity (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). The extent of degeneracy of base-pair recognition of wild type I-CreI (wt I-CreI), the initial protein scaffold I-CreI(N75) and the combinatorial mutants was assayed by measuring their cleavage efficiency in yeast on their respective palindromic targets (C1221, Xa.3 and Xa.4) carrying individual site mutations. I-CreI D75 (wt) and I-CreI N75
As shown in
The
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/IB2006/000589 | Feb 2006 | IB | international |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2007/000924 | 2/13/2007 | WO | 00 | 10/9/2008 |
