WO2012010976A2 - Meganuclease variants cleaving a dna target sequence in the tert gene and uses thereof - Google Patents

Abstract

Meganuclease variants cleaving DNA target sequences of the TERT gene, vectors encoding such variants, and cells expressing them. Methods of using meganuclease variants recognizing TERT gene sequences for modifying the TERT gene sequence, including gene correction and gene inactivation, or for incorporating a gene of interest or therapeutic gene using the TERT gene as a landing pad and a safe harbor locus.

Description

TITLE

MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE IN THE TERT GENE AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT (not applicable)

REFERENCE TO MATERIAL ON COMPACT DISK

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BACKGROUND OF THE INVENTION

Field of the Invention

The present invention concerns a process to generate new classes of induced Pluripotent Stem (iPS) cells and their derivatives characterized as clean and/or safe and/or secure by using endonucleases such as meganucleases and particularly the meganucleases of the present invention that recognize TERT polynucleotide sequences.

Description of the Related Art

TERT, or telomerase reverse transcriptase, is a catalytic protein component of the telomerase complex.

The telomerase complex is involved in the addition in specific cell types of telomeres which are repetitive, non coding DNA [TTAGGG] elements located at the end of chromosomes. In cells where telomerase is not active (i.e. somatic cells), with each cell division telomeres are getting shorter, due to incomplete lagging DNA strand synthesis and oxidative damage. Cells are thereby further committed to senescence and limited lifespan as they divide. This mechanism is active in somatic cells to limit the outgrowth of abnormal cells such as tumours. Telomere shortening, telomere attrition, has been hypothesized to be fundamental to normal senescence of cells, tissues and organisms. In germ lines cells, which are passed over through all the generations, and thereby immortal, telomeres are maintained in size despite cell division, thanks to the expression and activity of the telomerase complex in which TERT plays a crucial role. Telomere length is sensed by the cells to decide whether a cell should undergo senescence or not (Garcia et al.).

The telomerase complex is a ribonucleic complex composed of:

- hTR functional RNA, ubiquitously expressed, encoded by TERC gene;

- hTERT reverse transcriptase protein component which is highly expressed in germ line, stem cells and immortal cancer cells, encoded by TERT gene;

- Dyskerin/ NOP10/NHP2/GAR1 proteins;

While hTR is expressed ubiquitously, hTERT is only highly expressed in specific germ line cells, proliferative stem cells of renewal tissue and immortal cancer cells (Garcia et al). Overexpression of hTERT induces telomerase activity and telomerase elongation (Garcia et al). Mice have longer telomeres than human, express telomerase activity in most tissues and might not be optimal animal models. However telomerase deficient mice do have phenotypes and only late generations of mTR-/- mice, telomerase deficient mice, have defects in cell viability of highly proliferative tissues (Garcia et al).

Telomerase and its enzymatic component hTERT are thus involved in a large scope of phenomena covering ageing, senescence, maintenance of stem cell pools, control of cell fate, immortalization and cancer.

PLURIPOTENT STEM CELLS

Pluripotent cells have the capacity to differentiate into cells forming all three of the basic germ cell layers, endoderm, mesoderm and ectoderm and to cells subsequently differentiating from these layers. The TERT gene is expressed in pluripotent stem cells which possess a high tumorigenic potential due to their high proliferative potential. Even induced pluripotent stem cells (iPS cells) which are generated from somatic stem cells by the introduction of four genes (OCT4, SOX2, C-MYC and LF4 corresponding to two pluripotent stem cells markers and two oncogenes; Yamanaka et al, 2007) show the re- expression of TERT leading to the increase of telomere length respect to the somatic parental cells (Suhr at al, 2009). The expression of TERT in iPS cells is criteria for complete reprogramming validation and it can be also used to increase the reprogramming efficiency (Park et al, 2007). In this context TERT seems to be an important gene for pluripotent stem cells.

Recently, it has been shown that embryonic stem cells (ES) even if they are differentiated into precursor populations they maintained a tumorigenic potential characterized by an abnormal overgrowth after engraftment into animals (Tabar et al, 2005, Roy et al, 2006, Aubry et al, 2008). These data suggest a potential similar behavior for iPS cells which share ES cells properties. This high tumorigenic potential represents an obstacle for the use of pluripotent stem cells in cell therapy approaches for which they represent good candidate. In fact, due to their ability to proliferate indefinitely and to be differentiated into all cell types of the body they can represent an unlimited source of cells for transplantation. As described before, with the presence of telomerase, each dividing cell can replace the lost bit of DNA, and any single cell can then divide unbounded. Moreover telomerase activation has been observed in -90% of all human tumors, suggesting that the immortality conferred by telomerase plays a key role in cancer development.

Inhibition of telomerase has been viewed as a promising anticancer approach due to its specificity for cancer cells (for this purpose meganucleases targeting TERT can also be suitable, see next paragraph). Studies so far have shown that telomerase inhibition can inhibit the proliferation of cancer cells or cause apoptosis while it has no effect on most normal cells (Chen et al, 2009; Ohishi et al 2007).

CANCER

In the early stages of carcinogenesis, decreased activity of telomerase, leading to shortening of the telomeres at the end of chromosomes may contribute to genetic instability, chromosomal rearrangements and thus participate to carcinogenesis. Such role of shortened telomeres has for example been reported in colorectal carcinogenesis (Rampazzo E et al.). hTERT gene being localized close to the telomere on chromosome 5p has also been reported to be affected by such chromosomal rearrangement. The localization at the tip of 5p can explain the amplification of hTERT observed in 31 % of tumour cell lines and 30% of primary tumours (Mergny JL et al.). Rearrangement of sequences upstream of TERT gene and has been hypothesized to be a major mechanism of TERT promoter activation. Such translocations might also allow the hTERT promoter to escape from the native condensed chromatin environment (Zhao Y et al). A more relaxed chromatin structure around TERT gene in cancer cells might contribute to enhanced accessibility for a meganuclease to its site and ultimately increased meganuclease activity in cancer cells. Telomerase has been described has a common hallmark of cancer (Liu JP, et al.).

Indeed, at later stages of carcinogenesis enhanced activity of telomerase (and TERT) has been reported as an important factor in the immortalization of cancer cells (Garcia et al. Diaz de Leon et al plosone may 2010 ). Human telomerase is active in over 85% of primary cancers and its activity correlates closely with human telomerase reverse transcriptase (hTERT) expression (Fujiwara et al). The ability to modulate or restore telomerase function might be of therapeutic benefit to treat cancer. Some diseases involving telomere shortening described below include cancers such as acute myelogenous leukemia (AML) and non cancer symptoms.

Some TERT mutations have been monitored for prediction of the risks of development of breast cancer and might be associated with a reduced risk in individuals with a family history of breast cancer (SA Savage*, 1 ,2, SJ Chanock2 et al.).

TERT or TERT promoter are currently considered to develop treatments against cancer. Since telomerase activity is increased in cancer cells, telomerase inhibition is currently considered as a therapeutic target. In cancer, telomerase inhibition is sought by use of chemical inhibitors (including reverse transcriptase inhibitor AZT) or of RNAi for instance (Min XJ et al.). Recent approaches to telomerase inhibition include the use of Antisense oligonucleotides, RNA interference, ribozymes (Cunningham et al., tumours , Mergny JL et al.).

Telomerase being expressed at higher level in cancer cells than in healthy cells, anti- telomerase cancer immunotherapies are attempted, using human telomerase reverse transcriptase (hTERT) as a tumor antigen to produce anticancer vaccines (Liu JP et al.). The telomerase promoter, considered as a cancer-specific promoter, is currently being tested in cancer treatment to drive the expression of suicide genes specifically in cancer cells or to develop lytic viruses targeting cancer cells (Fujiwara et al.).

Supplementary solutions to block undesired telomerase activity in pathologic cells such as cancer cells are needed.

PATHOLOGIES OTHER THAN CANCER

Mutations in TERT have also been associated with other pathologies than cancer. Mutations in TERT have notably been associated with Pulmonary Fibrosis such as Autosomal Dominant Pulmonary Fibrosis/IPF, Bone Marrow Failure (BMF) and Dyskeratosis Congenita (DC or DKC, ref Savage SA, Garcia et al.). Telomere defects have also been associated with predisposition to hematologic malignancy and epithelial tumours.

Dyskeratosis congenita (DKC)

Dyskeratosis congenita (DKC) is characterized by symptoms including reticulated skin pigmentation, nail dystrophy and leukoplakia. This pathology is also associated to increased risks of development of progressive bone marrow failure (BMF) myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML) (ref Savage SA) .The median age of death is 16 years old and this disease affects 1/10⁶ individuals (Garcia et al). The patients appear healthy at birth and develop symptoms while ageing (i.e. as telomeres become shorter due to cell divisions). 40% of patients are affected in a gene associated with telomere maintenance (Garcia et al). At least six genes DKC1 , TERC, TERT, TI F2, NHP2 and NOP 10 have been found to cause DC (ref Savage SA). In the case of Recessive X linked DKC, the Dyskerin gene, interacting with telomerase protein component is mutated. In the case of dominant forms, mutations in TERC and TERT are found (Garcia et al). Missense mutations in TERT have been reported in DKC patients (Garcia et al). Dominant forms might be related to haploinsufficiency implying that the restoration of a WT functional allele might be of therapeutic benefit (Garcia et al). Families bearing TERT or TERC mutations display a worsening of symptoms and an earlier age of onset with successive, probably due to progressive shortening of telomeres in the germ line cells at each generation. TERT mutations have been reported in autosomal recessive and autosomal dominant forms of DKC. TERC mutations are linked with autosomal dominant forms of DKC with absence of mucocutaneous features, acute myelogenous leukemia, paroxysmal nocturnal hemoglobinuria, thrombocytosis. TERT mutations are similar. TERT and TERC mutations can have cumulative effects leading to haploinsuffiency.

Bone Marrow failure (BMF)

In over 80% of patient DKC is associated with Bone Marrow failure (BMF), which has also been related with mutations in TERC and TERT (Garcia et al). This latter pathology results in aplastic anemia, a reduced number of all three blood cell lineages. A link with the depletion of the pool of hematopoietic stem cells has been hypothesized.

Idiopathic pulmonary fibrosis (IPF)

Adult onset pulmonary fibrosis/ Idiopathic pulmonary fibrosis (IPF) has also been associated with mutations in TERT. Adult onset pulmonary fibrosis/ Idiopathic pulmonary fibrosis (IPF), is a pathology which prevalence is 4/100Ό00 in the general population, and 227/100Ό00 in people 75 years old and older. This represents 89Ό00 diagnosed patients and 34Ό00 new cases diagnosed per year in the united states (Garcia et al.). The symptoms are a progressive fibrotic disease, scarring of the lung which causes chronic cough and shortness of breath. This pathology mostly affects patients after the fifth decade. The age of onset is around 55 years in the familial forms while it is over 67 years in sporadic cases. The mean duration of survival is 3 years after diagnosis. The pathology is always lethal, with no spontaneous remission. There is currently no cure, no therapy to prolong life expectancy. Lung transplantation may be considered only for younger patients. The penetrance might incomplete in the case of dominant negative mutation (Garcia et al). This pathology is thus a very significant health issue. Garcia et al. found that almost 70% of telomerase mutation carriers over 40 had pulmonary disease of some disease. Heterozygous mutations in TERC or TERT were seen in 12% of the cohort of families affected by familial IPF. Mutations of TERT found in IPF patients were different from the ones found in DKC or BMF patients. In IPF several mutations have been identified as frameshift mutations such as V747fs missing half of the reverse transcriptase domain, with undetectable activity in vitro (Garcia et al). Other mutations such as PI 12fs and El 1 16fs delete most of the protein or the terminal 17 amino acids of the protein containing the conserved E-IV domain, respectively. The other identified mutations in TERT patients are missense and splice site mutations. The missense mutations span all the functional domains of the protein. Several mutations or exon skipping seem to involve motif C region of the reverse transcriptase domain. Inherited TERT mutations cause an earlier age related onset of disease, more severe phenotypes and patients mutated in TERT or TERC did present shorter telomeres than age-matched controls in circulating white blood cells (Garcia et al). The absence of telomerase activity is particularly exposed in cell types undergoing frequent division such as circulating cells, cultured fibroblasts, lymphoblasts, leukocytes , epithelium, and bone marrow. Lung tissue is very dependent upon renewal of telomerase expressing stem cells. A premature exhaustion of the pool of stem cells results in impaired lung scars repair.

Garcia et al., reviewed the TERT mutations associated with DKC, BMF and IPF. IPF is associated with mutations including L55Q, P33S, IVS l + l g>a, P 1 12fs, V144M, R486C, V747fs, IVS9-2a>c, R865C, R865H, E1 1 16fs, T1 1 10M. BMF is associated with mutations including A202T, H412Y, K570N, V694M, G682D, Y772C, V1090M. DKC is associated with mutations including P721 R, K902D, R979W, F1 127L, hence mostly in the C terminal half of the protein.

While TERC mutations mostly cause DKC and bone marrow failure, TERT mutations mostly cause IPF and DKC without classic skin features (Garcia et al/de Leon ). However some patients have shortened telomere length; TERT mutations (inactive telomerase) but do not present lung disease, which probably highlights the role of cumulative (age) environmental factors & smoking.

Despite sharing the common fact that TERT mutations have been reported in all of these pathologies, they still present some very distinct features. For instance DKC is usually a disease of childhood, while bone marrow failure due to TERT mutations can affect individuals in a wide range of ages and pulmonary fibrosis presents a phenotype that is age- dependent, more particularly in older individuals. DKC patients also generally posses shorter telomeres than IPF patients (Diaz de Leon et al). Attempts to cure DKC via telomerase function restoration have been made. Westin et al. (ref) have attempted to restore telomeres in Autosomal Dominant Dyskeratosis congenita skin fibroblasts by retroviral expression of TER (TERC) and/or telomerase reverse transcriptase (TERT)fibroblasts. Exogenous TER expression, without TERT, could not activate telomerase in AD DC skin fibroblasts. Transduction of TERT alone, however, provided AD DC cells with sufficient telomerase activity to extend average telomere length and proliferative capacity. Interestingly, they found that expression of TER and TERT together resulted in extension of lifespan and higher levels of telomerase and longer telomeres than expression of TERT alone in both AD DC and normal cells.

Telomerase and its enzymatic component hTERT are thus involved in a large scope of phenomena covering ageing, senescence, maintenance of stem cell pools, control of cell fate, immortalization and cancer. The ability to engineer hTERT gene or hTERT promoter is thus of a great importance.

Overexpression of telomerase is a key component of the transformation process in many malignant cancer cells. Although the beneficial evidence of telomerase inhibition is obvious, limitations of strategies remain to be resolved to increase the feasibility of clinical application.

Homologous gene targeting strategies have been used to knock out endogenous genes (WO90/1 1354; Capecchi M.R., Science, 1989, 244, 1288-1292; Smithies O., Nat Med, 2001 , 7, 1083-1086) or knock-in exogenous sequences into the genome. To enhance the efficiency of gene targeting, another strategy to enhance its efficiency is to deliver a DNA double-strand break (DSB) in the targeted locus, using an enzymatically induced double strand break at or around the locus where recombination is required (WO96/14408). A strategy known as "exon knock-in" involves the use of a meganuclease cleaving a targeted gene sequence to knock-in a functional exonic sequences. Meganucleases have been identified as suitable enzymes to induce the required double-strand break. Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (Thierry, A. and B. Dujon, Nucleic Acids Res., 1992, 20, 5625-5631). 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. U.S.A., 1996, 93, 5055-5060 ; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-277; Cohen- Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448 ; Donoho, et al., Mol. Cell. Biol., 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101 ).

Although several hundred natural meganucleases, also referred to as "homing endonucleases" have been identified (Chevalier, B.S. and B.L. Stoddard, Nucleic Acids Res., 2001 , 29, 3757-3774), the repertoire of cleavable target sequences is too limited to allow the specific cleavage of a target site in a gene of interest or GOI as there is usually no cleavable site in a chosen gene of interest. For example, there is no cleavage site for known naturally occurring I-Crel or I-Scel meganucleases in human TERT.

Theoretically, the making of artificial sequence-specific endonucleases with chosen specificities could alleviate this limit. To overcome this limitation, an approach adopted by a number of workers in this field is the fusion of Zinc-Finger Proteins (ZFPs) with the catalytic domain of Fokl, a class IIS restriction endonuclease, so as to make functional sequence- specific endonucleases (Smith et al., Nucleic Acids Res., 1999, 27, 674-681 ; Bibikova et al., Mol. Cell. Biol, 2001 , 21 , 289-297; Bibikova et al, Genetics, 2002, 161 , 1 169-1 175; Bibikova et al., Science, 2003, 300, 764; Porteus, M.H. and D. Baltimore, Science, 2003, 300, 763-; Alwin et al., Mol. Ther., 2005, 12, 610-617; Urnov et al., Nature, 2005, 435, 646-651 ; Porteus, M.H., Mol. Ther., 2006, 13, 438-446). Such ZFP nucleases have been used for the engineering of the IL2RG gene in human lymphoid cells (Urnov et al., Nature, 2005, 435, 646-651 ).

The binding specificity of Cys2-His2 type Zinc-Finger Proteins, is easy to manipulate because specificity is driven by essentially four residues per zinc finger (Pabo et al., Annu. Rev. Biochem., 2001 , 70, 313-340; Jamieson et al., Nat. Rev. Drug Discov., 2003, 2, 361 - 368). Studies from the Pabo laboratories have resulted in a large repertoire of novel artificial ZFPs, able to bind most G/ AN G/ AN G/ ANN sequences (Rebar, E.J. and CO. Pabo, Science, 1994, 263, 671 -673; Kim, J.S. and CO. Pabo, Proc. Natl. Acad. Sci. U S A, 1998, 95, 2812-2817), Klug (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91 , 1 1 163- 1 1 167; Isalan M. and A. Klug, Nat. Biotechnol., 2001 , 19, 656-660) and Barbas (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 1 1 163-1 1 167; Isalan M. and A. Klug, Nat. Biotechnol., 2001 , 19, 656-660).

Nevertheless, ZFPs have serious limitations, especially for applications requiring a very high level of specificity, such as therapeutic applications. It was shown that Fokl nuclease activity in ZFP fusion proteins can act with either one recognition site or with two sites separated by variable distances via a DNA loop (Catto et al., Nucleic Acids Res., 2006, 34, 171 1 -1720). Thus, the specificities of these ZFP nucleases are degenerate, as illustrated by high levels of toxicity in mammalian cells and Drosophila (Bibikova et al., Genetics, 2002, 161 , 1 169-1 175; Bibikova et al., Science, 2003, 300, 764-; Hockemeyer et al., Nat Biotechnol. 2009 Sep;27(9): 851 -7). The inventors have identified a new approach which circumvents these problems using engineered endonucleases, such as meganucleases recognizing TERT gene sequences.

In the wild, meganucleases are essentially represented by homing endonucleases. Homing Endonucleases (HEs),a widespread family of natural meganucleases including hundreds of proteins families (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. Given their exceptional cleavage properties in terms of efficacy and specificity, they could represent ideal scaffold to derive novel, highly specific endonucleases.

Homing Endonucleases belong to four major families. The LAGLIDADG family, named after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Seven structures are now available. Whereas most proteins from this family are monomelic and display two LAGLIDADG motifs, a few have only one motif, but dimerize to cleave palindromic or pseudo-palindromic target sequences.

Although the LAGLIDADG peptide is the only conserved region among members of the family, these proteins share a very similar architecture. The catalytic core is flanked by two DNA-binding domains with a perfect two-fold symmetry for homodimers such as I-Crel (Chevalier, et al., Nat. Struct. Biol., 2001 , 8, 312-316) and I-Msol (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269) and with a pseudo symmetry for monomers such as I-Scel (Moure et al., J. Mol. Biol., 2003, 334, 685-69, I-Dmol (Silva et al., J. Mol. Biol., 1999, 286, 1 123- 1 136) or I-Anil (Bolduc et al., Genes Dev., 2003, 17, 2875-2888). Both monomers or both domains of monomelic proteins contribute to the catalytic core, organized around divalent cations. Just above the catalytic core, the two LAGLIDADG peptides play also an essential role in the dimerization interface. DNA binding depends on two typical saddle-shaped αββαββα folds, sitting on the DNA major groove. Other domains can be found, for example in inteins such as PI-PfuI (Ichiyanagi et al., J. Mol. Biol., 2000, 300, 889-901 ) and PI-SceI (Moure et al., Nat. Struct. Biol., 2002, 9, 764-770), which protein splicing domain is also involved in DNA binding. The making of functional chimeric meganucleases by fusing the N-terminal I-Dmol domain with an I-Crel monomer have demonstrasted the plasticity of meganucleases (Chevalier et al., Mol. Cell., 2002, 10, 895-905; Epinat et al., Nucleic Acids Res, 2003, 31 , 2952-62; International PCT Applications WO 03/078619 and WO 2004/031346).

Different groups have used a semi-rational approach to locally alter the specificity of I-Crel (Seligman et al., Genetics, 1997, 147, 1653-1664; Sussman et al., J. Mol. Biol., 2004, 342, 31 -41 ; International PCT Applications WO 2006/097784 and WO 2006/097853; Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Rosen et al., Nucleic Acids Res., 2006, 34, 4791 -4800 ; Smith et al., Nucleic Acids Res., 2006, 34, el49), I-Scel (Doyon et al., J. Am. Chem. Soc, 2006, 128, 2477-2484), PI-SceI (Gimble et al., J. Mol. Biol., 2003, 334, 993- 1008 ) and I-Msol (Ashworth et al., Nature, 2006, 441 , 656-659).

In addition, hundreds of I-Crel derivatives with locally altered specificity were engineered by combining the semi-rational approach and High Throughput Screening:

- Residues Q44, R68 and R70 or Q44, R68, D75 and 177 of I-Crel were mutagenized and a collection of variants with altered specificity at positions ± 3 to 5 of the DNA target (5NNN DNA target) were identified by screening (International PCT Applications WO 2006/097784 and WO 2006/097853; Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Smith et al., Nucleic Acids Res., 2006, 34, el49).

- Residues 28, N30 and Q38 or N30, Y33, and Q38 or 28, Y33, Q38 and S40 of I- Crel were mutagenized and a collection of variants with altered specificity at positions ± 8 to 10 of the DNA target (10NNN DNA target) were identified by screening (Smith et al., Nucleic Acids Res., 2006, 34, el 49; International PCT Applications WO 2007/060495 and WO 2007/049156).

Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of a different half of each variant DNA target sequence (Arnould et al., precited; International PCT Applications WO 2006/097854 and WO 2007/034262). Interestingly, the novel proteins had kept proper folding and stability, high activity, and a narrow specificity.

Furthermore, residues 28 to 40 and 44 to 77 of I-Crel were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease half- site (Smith et al. Nucleic Acids Res., 2006, 34, el49; International PCT Applications WO 2007/049095 and WO 2007/057781).

The combination of mutations from the two subdomains of I-Crel within the same monomer allowed the design of novel chimeric molecules able to cleave a palindromic combined DN A target sequence comprising the nucleotides at positions ± 3 to 5 and ± 8 to 10 which are bound by each subdomain (Smith et al., Nucleic Acids Res., 2006, 34, el 49; International PCT Applications WO 2007/060495 and WO 2007/049156), as illustrated on figure 2b.

The combination of the two former steps allows a larger combinatorial approach, involving four different subdomains. The different subdomains can be modified separately and combined to obtain an entirely redesigned meganuclease variant (heterodimer or single- chain molecule) with chosen specificity. In a first step, couples of novel meganucleases are combined in new molecules ("half-meganucleases") cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such "half-meganuclease" can result in a heterodimeric species cleaving the target of interest. The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from different genes has been described in the following patent applications: XPC gene (WO2007093918), RAG gene (WO2008010093), HPRT gene (WO2008059382), beta-2 microglobulin gene (WO2008102274), Rosa26 gene (WO2008152523), Human hemoglobin beta gene (WO2009013622) and Human Interleukin-2 receptor gamma chain (WO2009019614).

These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields including gene therapy.

However, even though the base-pairs ± 1 and ± 2 do not display any contact with the protein, it has been shown that these positions are not devoid of content information (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269), especially for the base-pair ±1 and could be a source of additional substrate specificity (Argast et al., J. Mol. Biol., 1998, 280, 345-353; Jurica et al., Mol. Cell., 1998, 2, 469-476; Chevalier, B.S. and B.L. Stoddard, Nucleic Acids Res., 2001 , 29, 3757-3774). In vitro selection of cleavable I-Crel target (Argast et al., precited) randomly mutagenized, revealed the importance of these four base-pairs on protein binding and cleavage activity. It has been suggested that the network of ordered water molecules found in the active site was important for positioning the DNA target (Chevalier et al., Biochemistry, 2004, 43, 14015-14026). In addition, the extensive conformational changes that appear in this region upon I-Crel binding suggest that the four central nucleotides could contribute to the substrate specificity, possibly by sequence dependent conformational preferences (Chevalier et al., 2003, precited).

The inventors have identified and developed novel endonucleases, such as meganucleases, targeting TERT gene sequences, such as TERT target sites TERT2, a site within exon 2 of the TERT gene, and TERT5, a site within intron of the TERT gene, just upstream of exon 12 of the TERT gene, as non limiting examples. The novel endonucleases and particularly the meganucleases of the invention introduce double stranded breaks within the TERT gene offering the opportunities to modify, modulate, and control TERT gene expression, to detect TERT gene expression, or to introduce transgenes into the TERT gene locus.

BRIEF SUMMARY OF THE INVENTION

TERT mutations are widespread on the gene and protein domains. Some mutations are dominant, even if they might be linked with haploinsufficiency (cumulative/dose effect). This implies that the use of a meganuclease targeting sites present in the first exons of the gene such as tert2.1 , located in exon2, might be suitable to treat many patients when coupled with an exon knock in strategy, restoring the expression of a functional protein. Such an exon KI might even be performed while keeping control of expression by endogenous tert promoter. Tert2 meganuclease is located in exon 2 at a distance of about l OOObp from the ATG and could thus potentially be used to engineer tert promoter and allow to modulate telomerase expression and activity. Artificial promoters including inducible promoters might be introduced to allow control on cell fate, either commitment towards senescence or immortalization/sustainable cell culture capacity.

The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization (Sealey et al). Since Tert2 is located in the first exons of hTERT (exon2) it might also be used to impair hTERT function by using strategies based on nockOut by NHEJ (Non Homologous End Joining). In human tissue culture short telomeres produce end-to-end chromosome fusions, reciprocal translocations and aneuploidy (Calado et al.). Control of telomerase complex activity via TERT gene engineering, and subsequent control of telomere length may provide a powerful tool to limit chromosomal aberrations, enhance genetic stability and help maintain the ploidy constant in cells or tissues of interest.

A variety of human cell types have been immortalized by over expression of TERT and can be used for organotypic culture. Such bioengineered tissues do express normal differentiation markers suggesting TERT does not inhibit the normal differentiation of cells (Garcia et al). Autografts of cells rejuvenated by augmentation of the telomere length via temporary hTERT overexpression can be envisioned, for bone marrow cells or lymphocytes for instance (Garcia et al).

The possibility to modulate telomere length might be useful to generate human cell models or animal models of early carcinogenesis for example. Such models could be coupled with models modulated (either Knock out or inducible models) for other important proteins in cell cycle control such as p53. Indeed it is known that when the absence of telomerase activity is coupled with p53 mutations a lack of cell cycle checkpoint might occur which might accelerate telomere shortening, lead to translocations, genomic instability and ultimately cancer.

Since a large number of pathologies are age-related, it might be discovered overtime that telomere size or even TERT itself are an important contributing factors in an expanding set of age-related pathologies. The use of meganucleases targeting TERT might thus potentially be extended to these pathologies. For example patients with atherosclerotic heart disease have been reported to have significantly shorter telomeres compared with healthy age- matched controls (Garcia et al.). Shortened telomeres in cryopreserved blood cells have been retrospectively associated with higher mortality due to infectious disease. Shortened telomere lengths have also been associated with reduced bone mineral density, osteoporosis, obesity, and cigarette smoking (Garcia et al).

Whenever the target sites recognized by the TERT meganuclease is present in a genome either naturally (site present in homolog genes in other species or site present in other locations in a genome) or artificially introduced (i.e. use of a landing pad containing the target sequence), the described TERT meganucleases might be used in applications even unrelated to telomerase activity.

On the most exploratory point of view, since TERT is involved in mechanisms controlling senescence and its counterpart immortalization, the control of TERT level might be a key component in reaching immortality.

The development of a strategy of Exon Knock In might allow to restore (haploinsufficiency) sufficient telomerase activity for a large number of different mutations, hence a large cohort of patients. The Tert2 target described below being located in the first exons of TERT might the most favorable target for such strategy. Tert5 might be used to correct mutations in its vicinity or all mutations located downstream of the cleavage site (exon Kl of downstream exons).

Moreover, the role of hTERT mutations has been reported in a number of pathologies including including Autosomal Dominant Pulmonary Fibrosis, Idiopathic Pulmonary Fibrosis (IPF), Bone Marrow Failure (BMF) and Dyskeratosis Congenita. Diminished or abolished telomerase activity resulting from TERT mutations cause shortening of the telomeres and associated symptoms notably in cells of tissue displaying high proliferative rate (depletion of the pool of stem cells). The restoration of the expression of a functional TERT protein by use of TERT specific meganucleases might trigger a therapeutic effect either by precise correction of mutations (gene correction) or by introduction of a sequence coding for the functional protein for example ("Exon Knock In" strategy, well suited since mutations are widespread on gene/protein).

However TERT mutations are diverse and widespread on the gene, making precise gene correction of every single mutation, implying the development of as many several meganucleases targeting theses different regions difficult. The use of Exon Knock In (Exon KI) strategy in contrast might be a favored approach, especially for those meganucleases whose targeting sites are at the beginning of the gene such as tert2. Indeed exon KI allows the correction of mutations in the vicinity of the site as well as a functional compensation of mutations located downstream (introduction of a functional construct, shunting the mutated sequences). The present invention concerns a process to generate new classes of induced

Pluripotent Stem (iPS) cells and their derivatives characterized as clean and/or safe and/or secure by using endonucleases such as meganucleases and particularly the meganucleases of the present invention.

Key issues of current protocols to generate iPS by introducing the four transcription factors Oct3/4, Sox2, KLF4 and c-myc are that : - these introductions are not controlled and lead to heterogeneous populations of iPS cells where transgenes are not inserted at the same locus and / or not with the same copy number, iPS cells express these four transgenes permanently leading to problems for further differentiation steps. Endonucleases of the present invention are a tool of choice to overcome these classical issues allowing: stable, robust and single copy targeted insertion of the four transgenes at a defined locus allowing a controlled generation of homogenous iPS populations in high quantity. - the possibility to remove the four transgenes once iPS have been generated without any scar on the genome ("pop-out"), for obtaining clean iPS in further re- differentiation steps and therapeutic uses.

Another issue addressed by endonucleases of the present invention is the possibility to generate secured iPS and to standardize well-defined but still empirical current protocols. By using meganucleases inducing the targeting and the disruption of TERT gene as a non- limiting example, at a defined step of differentiation process, the progression of iPS toward differentiation states is made irreversible and safe since infinite self-renewable property of these cells is lost.

Targeting of the TERT gene by knock-out (directly by NHEJ i.e. Non Homologous End Joining or knock-in experiment (knock-in a resistance gene or a DNA sequence to disrupt the open reading frame) using specific custom meganucleases could be valuable to reduce the tumorigenicity of both cancer and pluripotent stem cells (ES and iPS). TERT knockout by means of meganucleases might provide a supplementary solution to block undesired telomerase activity in pathologic cells such as cancer cells. It can even be imagined to put meganuclease expression under the control of TERT promoter to perform knockout only in cancer cells. Pluripotent stem cells TERT-/- will present a reduced proliferative potential which will increased during passages and differentiation to reach the Hayflick limit (Hayflick & Moorhead, 1961 ). Thus, progenitor cells derived from these pluripotent stem cells will have a reduced tumorigenic potential and they will be safe to use for cell therapies.

Also, by using endonucleases to insert at a safe locus of the genome, genes of interest and particular inducible genes defined as essential for progression of iPS toward differentiated cells (growth factors, transcription factors), it is possible to standardize the differentiation steps of an iPS.

This endonuclease approach of iPS generation and differentiation open new avenues for screening molecules and / or genes in vitro: in order to secure, make safe, and standardize the iPS differentiation process, gene candidates from an expression library responsible or implicated in a defined differentiation step can be inserted at a safe locus of an iPS genome locus, by using meganucleases. to screen chemical libraries for compounds on primary cells carrying or not a genetic defect. in order to evaluate drug response at a single patient scale in pharmacogenomic approaches. to confirm or invalidate strategies or chemicals derived from predictive methods and algorithms in predictive toxicology measures.

Also, endonucleases can be the ideal tool to create reporter cell lines integrating at a safe locus, reporter gene fused to a promoter specific of a defined reprogrammation step in order to validate the iPS reprogrammation process. The same approach can be envisioned during the re-differentiation process, allowing to precisely control this process and create progenitor cells bank, still able to divide a limited number of times and known to be able to move through the body and migrate towards the tissue where they are needed; they are particularly useful for adult organisms therapy as they act as a repair system for the body without presenting the known transplantation problem of compatibility.

Regarding therapeutic uses, endonucleases are the ideal tool to target and correct in clean and safe iPS cells pathological gene defects before their reinjection in patient organisms as suggested above (Paques F. and Duchateau P., Current Gene Therapy, 2007, 7, 49-66).

Any gene involved in the reprogrammation of iPS cells is part of the present invention and is a useful target of endonucleases according to the invention. The present invention also concerns a new type of iPS; clean and/or safe and/or secure iPS cells as a new product will not anymore express the product of any gene of interest targeted for the process of cleaning and securization of such iPS cells, after the process of cleaning and securization occurs in said iPS cells.

In particular, the invention involves meganuclease variants that target and cleave TERT gene sequences, vectors encoding these variants, cells transformed with vectors encoding these meganuclease variants and methods for making a meganuclease variant through by expressing a polynucleotide encoding it. Methods for designing meganuclease variants recognizing the TERT gene, including meganuclease variants recognizing the TERT2 and TERT5 DNA sequences. These variant meganucleases may be used to investigate the function of the TERT gene, follow its expression in undifferentiated or pluripotent cells as well as in differentiated cells by introducing knock out mutations into the TERT gene or by introducing reporter genes or other genes of interest at the TERT locus, possibly for the production of proteins. The meganuclease variants of the invention may also be used to modulate TERT expression in a cell by interaction of this gene sequence with a meganuclease, for example, to control its phenotype, to knock down or control expression of TERT in a cell such as a tumor cell, or in various other therapeutic or diagnostic applications. A particular aspect of the invention is a meganuclease that can induce double stranded breaks in any gene involved in the reprogrammation process and particularly in the TERT gene.

Another aspect of the invention involves using such a meganuclease recognizing TERT sequences to knock out or modulate TERT expression. Fig. 1 illustrates TERT gene correction and gene inactivation strategies. Another aspect of the invention is the use of a meganuclease recognizing TERT to introduce a gene of interest into the TERT gene or locus. The gene of interest may be a reporter gene that permits the expression of TERT to be determined or followed over time, said reporter gene being associated or not to a nucleotidic sequence which is introduced into the genome in order to add new potentialities or properties to targeted cells. Methods for determining the effects of non-TERT genes or drug compounds on TERT expression or activity may be evaluated using assays employing a reporter gene. Such methods are particularly valuable when applied to tumor or cancer cells that have been modified to incorporate a TERT gene associated with a reporter. Alternatively, the gene of interest may be a therapeutic transgene other than TERT which uses the TERT locus as a safe harbor. Such therapeutic genes may be those that when coexpressed with TERT provide a particular cell phenotype to maintain or promote a particular phase or stage of cellular differentiation.

Thus, a third associated aspect of the invention relates to the use of the TERT gene locus as a "landing pad" to insert or modulate the expression of genes of interest.

BRIEF DESCRIPTION OF THE FIGURES

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-Crel meganuclease variants and their uses according to the invention, as well as to the appended drawings. A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below.

Figure 1 : Illustration of three different strategies for restoring a functional gene with meganuclease-induced recombination. A. Gene correction. A mutation occurs within the TERT gene. Upon cleavage by a meganuclease and recombination with a repair matrix the deleterious mutation is corrected. B. Gene inactivation by mutagenesis, this strategy being based on the Non Homologous End Joining (NHEJ) mechanism that can take place upon DNA cleavage in absence of matrix. The NHEJ can produce mutagenesis at the site of cleavage which can result in inactivation of the allele. C. Exonic sequences knock-in. A mutation occurs within the TERT gene. The mutated mRNA transcript is featured below the gene. In the repair matrix, all exons necessary to reconstitute a complete cDNA are fused in frame, with a polyadenylation site to stop transcription in 3 '. Introns and exons sequences can be used as homologous regions. Exonic sequences knock-in results into an engineered gene, transcribed into an mRNA able to code for a functional TERT protein.

Figures 2 a and b illustrate the combinatorial approach, described in International PCT applications WO 2006/097784 and WO 2006/097853 and also in Arnould, et al. (J. Mol. Biol., 2006, 355, 443-458) and Smith et al. (Nucleic Acids Res., 2006). This approach was used to entirely redesign the DNA binding domain of the I-Crel protein and thereby engineer novel meganucleases with fully engineered specificity.

Figure 3: Tert2 and Tert2 derived targets. The Tert2.1 target sequence (SEQ ID NO: 8) and its derivatives 10TGA_P (SEQ ID NO: 4), 10GAG P (SEQ ID NO: 7), 5GGC P (SEQ ID NO: 5) and 5TCC P (SEQ ID NO: 6), P stands for Palindromic) are derivatives of CI 221 (SEQ ID NO: 2), found to be cleaved by previously obtained I-Crel mutants. CI 221 , 10TGA P, 10GAG P, 5GGC P and 5TCC P were first described as 24 bp sequences, but structural data suggest that only the 22 bp are relevant for protein/DNA interaction. Consequently, positions ±12 are indicated in parenthesis. Tert2.1 (SEQ ID NO: 8) is the DNA sequence located in the human TERT gene at position 1 121 -1244 on NC000005.9. Tert2.2 (SEQ ID NO: 9) differs from Tert2.1 at positions -2;-l ;+l ;+2 where I-Crel cleavage site (GTAC) substitutes the corresponding Tert2.1 sequence. Tert2.3 (SEQ ID NO: 10) is the palindromic sequence derived from the left part of Tert2.2, and Tert2.4 (SEQ ID NO: 1 1 ) is the palindromic sequence derived from the right part of Tert2.2. Tert2.5 (SEQ ID NO: 12) is the palindromic sequence derived from the left part of Tert2.1 , and Tert2.6 (SEQ ID NO: 13) is the palindromic sequence derived from the right part of Tert2.1.

Figure 4: Activity cleavage in CHO cells of single chain SCOH-Ter2-bl - A(pCLS3713), SCOH-Ter2-bl -C(pCLS3714), SCOH-Ter2-bl-E(pCLS3715) compared to IScel (pCLS 1090) and SCOH-RAG-CLS (pCLS2222) meganucleases as positive controls. The empty vector control (pCLS1069) has also been tested on each target. Plasmid pCLS1728 contains control RAG 1.10.1 target sequence. In figure 4, the correspondence of the line graphs at their right ends to the legend (graph: legend) on the right is as follows: graph 1 (top) : 2; 2: 4; 3 : 7; 4: 6; 5: 8; 6: 1 ; 7: 5 and 8: 3.

Figure 5: Activity cleavage in CHO cells of single chain SCOH-Ter2-bl - C(pCLS3714) and SCOH-Ter2-bl -C_V2(pCLS4333) compared to IScel (pCLS 1090) and SCOH-RAG-CLS (pCLS2222) meganucleases as positive controls. The empty vector control (pCLS1069) has also been tested on each target. Plasmid pCLS 1728 contains control RAGl .10.1 target sequence. In figure 5, the correspondence of the line graphs at their right ends to the legend (graph: legend) on the right is as follows: graph 1 (top) : 2; 2: 6; 3 : 4; 4: 7; 5: 5; 6: 1 and 7: 3. Figure 6: Tert5 and Tert5 derived targets. The Tert5.1 target sequence (SEQ ID NO:

18) and its derivatives. 10TCC P (SEQ ID NO: 14), 10TAG P (SEQ ID NO: 17), 5CCT P (SEQ ID NO: 15) and 5GAG P (SEQ ID NO: 16), P stands for Palindromic) are derivatives of CI 221 (SEQ ID NO: 2), found to be cleaved by previously obtained I-Crel mutants. CI 221 , 10TCC_P, 10TAG_P, 5CCT_P and 5GAG_P were first described as 24 bp sequences, but structural data suggest that only the 22 bp are relevant for protein/DNA interaction. Consequently, positions ±12 are indicated in parenthesis. Tert5.1 (SEQ ID NO: 18) is the DNA sequence located in the human TERT gene at position 34429-34452 on NC000005.9. Tert5.2 (SEQ ID NO: 19) differs from tert5.1 at positions -2;-l ;+l ;+2 where I-Crel cleavage site (GTAC) substitutes the corresponding tert2.1 sequence. Tert5.3 (SEQ ID NO: 20) is the palindromic sequence derived from the left part of tert5.2, and tert5.4 (SEQ ID NO: 21 ) is the palindromic sequence derived from the right part of tert5.2. Tert5.5 (SEQ ID NO: 22) is the palindromic sequence derived from the left part of tert5.1 , and tert5.6 (SEQ ID NO: 23) is the palindromic sequence derived from the right part of tert5.1.

Figure 7: Activity cleavage in CHO cells of single chain SCOH-ter5bl -C (pCLS3479) compared to IScel (pCLS 1090) and SCOH-RAG-CLS (pCLS2222) meganucleases as positive controls. The empty vector control (pCLS1069) has also been tested on each target. Plasmid pCLS1728 contains control RAG 1.10.1 target sequence. In figure 7, the correspondence of the line graphs at their right ends to the legend (graph:legend) on the right is as follows: graph 1 (top) : 2; 2: 6 and 4; 4: 5; 5: 1 and 6: 3. Figure 8: Vector Map of pCLS1072

Figure 9: Vector Map of pCLS1090

Figure 10: Vector Map of pCLS2222

Figure 1 1 : Vector Map of pCLS 1853

Figure 12: Vector Map of pCLS l 107 Figure 13: Vector Map of pCLS 1069 Figure 14: Vector Map of pCLS 1058 Figure 15: Vector Map of pCLS1055 Figure 16: Vector Map of pCLS0542 Figure 17: Vector Map of pCLS 1728

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a process to generate new classes of induced Pluripotent Stem (iPS) cells and their derivatives characterized as clean and/or safe and/or secure by using endonucleases such as meganucleases and particularly the meganucleases of the present invention that recognize TERT gene sequences.

Key issues of current protocols to generate iPS by introducing the four transcription factors Oct3/4, Sox2, KLF4 and c-myc are that : these introductions are not controlled and lead to heterogeneous populations of iPS cells where transgenes are not inserted at the same locus and / or not with the same copy number, iPS cells express these four transgenes permanently leading to problems for further differentiation steps.

Endonucleases of the present invention are a tool of choice to overcome these classical issues allowing: stable, robust and single copy targeted insertion of the four transgenes at a defined locus allowing a controlled generation of homogenous iPS populations in high quantity. the possibility to remove the four transgenes once iPS have been generated without any scar on the genome ("pop-out"), for obtaining clean iPS in further re- differentiation steps and therapeutic uses. Another issue addressed by endonucleases of the present invention is the possibility to generate secured iPS and to standardize well-defined but still empirical current protocols. By using meganucleases inducing the targeting and the disruption of TERT gene as non limiting examples, at a defined step of differentiation process, the progression of iPS toward differentiation states is made irreversible and safe since infinite self-renewable property of these cells is lost.

Also, by using endonucleases to insert at a safe locus of the genome, inducible genes defined as essential for progression of iPS toward differentiated cells (growth factors, transcription factors), it is possible to standardize the differentiation steps of an iPS. This endonuclease approach of iPS generation and differentiation open new avenues for screening molecules and / or genes in vitro: in order to securize and standardize the iPS differentiation process, gene candidates from an expression library responsible or implicated in a defined differentiation step can be inserted at a safe locus of an iPS genome locus, by using endonucleases. to screen chemical libraries for compounds on primary cells carrying or not a genetic defect. in order to evaluate drug response at a single patient scale in pharmacogenomic approaches. - to confirm or invalidate strategies or chemicals derived from predictive methods and algorithms in predictive toxicology measures.

Also, endonucleases can be the ideal tool to create reporter cell lines integrating at a safe locus, reporter gene fused to a promoter specific of a defined reprogrammation step in order to validate the iPS reprogrammation process. The same approach can be envisioned during the re-differentiation process, allowing to precisely control this process and create progenitor cells bank, still able to divide a limited number of times and known to be able to move through the body and migrate towards the tissue where they are needed; they are particularly useful for adult organisms therapy as they act as a repair system for the body without presenting the known transplantation problem of compatibility. Regarding TERT function, the targeting of this gene will be useful to better understand the pluripotency properties of pluripotent stem cells by knock-in and knock-out experiments in ES and iPS cells. For this purpose TERT recognizing meganucleases are the tool of choice because they can be designed to target specifically this gene. Thus, it will be possible to knock-out the gene specifically but also to knock-in reporter gene which will be expressed under TERT regulators element. Thus, TERT expression could be followed both at the undifferentiated and differentiated stages. Such approach will also allow to monitor the process of de-differentiation of differentiated cells.

Another application of TERT designed meganucleases will be the study of the reprogramming process and the identification of new factors able to play a role in this process. In fact, although huge work has been made by the scientific community, the reprogramming process remains still largely inefficient (<0.1%) and not well controlled. Moreover strategy based on transgene integration are presently the most efficient, but they suffer major drawbacks. The integration site for transgenesis remains unpredictable and irreproducible, which can affect endogenous cellular gene functions or promote tumorigenesis. In addition, although integrated reprogramming factors become transcriptionally silenced over time through de novo DNA methylation, they can be spontaneously reactivated during cell culture and differentiation. The development of new strategy to improve the reprogramming process is therefore required.

Taking advantage of TERT meganucleases, it will be possible to knock-in into somatic cells a reporter gene under the control of the endogenous TERT regulatory sequences and control elements to monitor reprogramming efficiency through the expression of the reporter gene that will mimic the activation of the pluripotency gene TERT.

Finally, TERT meganucleases could be also useful to reduce the tumorigenic potential of pluripotent stem cells by knocking down this gene. In fact, recent work on ES cells has highlighted the presence of abnormal overgrowth after engraftment into animals of differentiated precursors derived from ES cells (Tabar et al, 2005, Roy et al, 2006, Aubry et al, 2008). Choice of TERT as a candidate for this purpose is also based on the fact that recently TERT has been described for its potential role in human tumor development (Jeter et al, 2009; You et al, 2009; Ji et al, 2009). In this context, the knock-out of hTERTwill inhibit tumor formation by reducing proliferation and clonogenic growth. Pluripotent stem cells are useful for cell therapy (Brignier at al, The Journal of Allergy Clinical Immunology) and drug screening (Phillips et al, Biodrugs 2010) because they give access to all cell types of the body as neurons for example. They have also a human origin; they can be obtained in unlimited quantities. In fact, cell therapy or drug screening studies are performed using primary cells which are obtained in limited quantities and have few proliferative potential. Another source is adult stem cells but compared to pluripotent stem cells they are still limited due to their access and their culture conditions. Moreover, regarding transplantation, problem of compatibility are still present; this problem could be overcome using iPS cells which can be derived directly from the patient to graft.

For drug screening studies iPS cells are valuable for a given disease, iPS cells could be generated for several patients and their unaffected parents, given thus access to the human diversity. Moreover, the mutation causal of the pathology is not induced in the original one. Then the effect of the mutation can be studied in different tissues to identify the effect of a potential drug on the affected tissue but also on others tissues to check the absence of secondary effects.

Meganucleases directed against TERT will therefore represent a tool of choice for several applications which will permit to better understand pluripotent stem cells and thus may be overcome actual problems lead by these cells for cell therapy and drug screening studies.

As mentioned above certain aspects of the invention reflect different strategies for modulating, modifying or controlling TERT gene expression that can be implemented with the TERT recognizing meganucleases of the invention.

Meganucleases that recognize TERT target sequences

Table I below shows target nucleotide sequences within the TERT locus recognized by meganucleases of the invention. Target sites inside (TERT2) and outside (TERT5) of the TERT coding sequence are useful for different procedures. As non limiting examples, insertion into TERT2 is useful in producing knock-out mutations of TERT and cleavage into TERT5 can be used to introduce regulatory or reporter sequences. Table I: sequences and location of the targeted sites in the TERT gene

Endonucleases that recognize TERT target sequences

Table Ibis below shows target nucleotide sequences within the TERT locus recognized by endonucleases of the invention.

Table Ibis: sequences of targeted sites in the TERT gene

Methods for knocking-out (KO) TERT gene expression Different strategies can be implemented for correcting or inactivating TERT gene (Fig. 1 ). The coding sequence can be corrected upon meganuclease cleavage and recombination with a repair matrix (Fig. 1 A). The TERT gene can be inactivated by non homologous end joining (NHEJ) using a meganuclease targeting a sequence without a repair matrix (Fig. I B). Meganuclease targeting the TERT2 sequence is such an enzyme. In that case, no matrix is needed. Some exons can be deleted by the action of one meganuclease supplied by a Knocking In DNA matrix (not shown). Meganucleases recognizing TERT2 or TERT5 sequences can be used in that sense. A second sub-type of knock-out strategy consists in the replacement of a large region within TERT gene by the action of two meganucleases (example: TERT2 + TERT5) and a KO matrix can be used for the deletion of large sequences (not shown). Such a KO matrix can be built using sequences deleted of the targeted exon as well as some mutated exons.

Knocking In ("ΚΓ) a gene of interest KI at the TERT locus

Since the TERT locus can be used for the expression of reporter and genes of interest, some meganuclease targeting sequences in exons (Fig. I B) or in introns (Fig. 1 C) are useful for the integration of knock in matrix by homologous recombination. Such a KI matrix can be built using sequences homologous to the targeted locus added of the gene of interest with or without regulation elements (Fig. 1 C).

I-Crel variants of the present invention were created using the combinatorial approach illustrated in Figure 2b and described in International PCT applications WO 2006/097784 and WO 2006/097853, and also in Arnould et al. (J. Mol. Biol., 2006, 355, 443-458) and Smith et al. (Nucleic Acids Res., 2006), allowing to redesign the DNA binding domain of the I-Crel protein and thereby engineer novel meganucleases with fully engineered specificity.

The cleavage activity of the variant according to the invention may be performed by any well-known, in vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736; Epinat et al., Nucleic Acids Res., 2003, 31 , 2952-2962; Chames et al., Nucleic Acids Res., 2005, 33, el 78; Arnould et al., J. Mol. Biol., 2006, 355, 443-458, and Arnould et al, J. Mol. Biol., 2007, 371 , 49-65. 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 (non-palindromic) DNA target sequence within the intervening sequence, cloned in yeast or in a mammalian expression vector. Usually, the genomic DNA target sequence comprises one different half of each (palindromic or pseudo-palindromic) parent homodimeric I-Crel meganuclease target sequence. Expression of the heterodimeric 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 an appropriate assay. The cleavage activity of the variant against the genomic DNA target may be compared to wild type I-Crel or I-Scel activity against their natural target.

Possibly or not, at least two rounds of selection/screening are performed according to the process illustrated Arnould et al., J. Mol. Biol., 2007, 371 , 49-65. In the first round, one of the monomers of the heterodimer is mutagenised, co-expressed with the other monomer to form heterodimers, and the improved monomers Y⁺ are selected against the target from the gene of interest. In the second round, the other monomer (monomer X) is mutagenised, co- expressed with the improved monomers Y⁺ to form heterodimers, and selected against the target from the gene of interest to obtain meganucleases (X⁺ Y⁺) with improved activity. The mutagenesis may be random-mutagenesis or site-directed mutagenesis on a monomer or on a pool of monomers, as indicated above. Both types of mutagenesis are advantageously combined. Additional rounds of selection/screening on one or both monomers may be performed to improve the cleavage activity of the variant. In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of I-Crel are at positions 44, 68, 70, 75 and/or 77.

In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 28 to 40 of I-Crel are at positions 28, 30, 32, 33, 38 and/or 40.

In another preferred embodiment of said variant, it comprises one or more mutations in I-Crel monomer(s) at positions of other amino acid residues that contact the DNA target sequence or interact with the DNA backbone or with the nucleotide bases, directly or via a water molecule; these residues are well-known in the art (Jurica et al., Molecular Cell., 1 98, 2, 469-476; Chevalier et al., J. Mol. Biol, 2003, 329, 253-269). In particular, additional substitutions may be introduced at positions contacting the phosphate backbone, for example in the final C-terminal loop (positions 137 to 143; Prieto et al., Nucleic Acids Res., Epub 22 April 2007). Preferably said residues are involved in binding and cleavage of said DNA cleavage site. More preferably, said residues are at positions 138, 139, 142 or 143 of I-Crel. Two residues may be mutated in one variant provided that each mutation is in a different pair of residues chosen from the pair of residues at positions 138 and 139 and the pair of residues at positions 142 and 143. The mutations which are introduced modify the interaction(s) of said amino acid(s) of the final C-terminal loop with the phosphate backbone of the I-Crel site. Preferably, the residue at position 138 or 139 is substituted by a hydrophobic amino acid to avoid the formation of hydrogen bonds with the phosphate backbone of the DNA cleavage site. For example, the residue at position 138 is substituted by an alanine or the residue at position 139 is substituted by a methionine. The residue at position 142 or 143 is advantageously substituted by a small amino acid, for example a glycine, to decrease the size of the side chains of these amino acid residues.

More preferably, said substitution in the final C-terminal loop modify the specificity of the variant towards the nucleotide at positions ± 1 to 2, ± 6 to 7 and/or ± 1 1 to 12 of the I- Crel site.

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 from the TERT gene. The additional residues which are mutated may be on the entire I-Crel sequence, and in particular in the C-terminal half of I-Crel (positions 80 to 163). Both I-Crel monomers are advantageously mutated; the mutation(s) in each monomer may be identical or different. For example, the variant comprises one or more additional substitutions at positions: 2, 7, 8, 19, 43, 54, 61 , 80, 81 , 96, 105 and 132. Said substitutions are advantageously selected from the group consisting of: N2S, 7E, E8 , G19S, F43L, F54L, E61R, E80 , 18 IT, K96E, VI 05 A and II 32V. More preferably, the variant comprises at least one substitution selected from the group consisting of: N2S, K7E, E8K, G19S, F43L, F54L, E61 R, E80K, 18 IT, K96E, VI 05 A and 1132V. The variant may also comprise additional residues at the C-terminus. For example a glycine (G) and/or a proline (P) residue may be inserted at positions 164 and 165 of I-Crel, respectively.

According to a preferred embodiment, said additional mutation in said variant further impairs the formation of a functional homodimer. More preferably, said mutation is the G19S mutation. The G19S mutation is advantageously introduced in one of the two monomers of a heterodimeric I-Crel variant, so as to obtain a meganuclease having enhanced cleavage activity and enhanced cleavage specificity. In addition, to enhance the cleavage specificity further, the other monomer may carry a distinct mutation that impairs the formation of a functional homodimer or favors the formation of the heterodimer. In 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, V, L, M, F, I and W. In particular, the variant is selected from the group consisting of SEQ ID NO: 25 to 35.

The variant of the invention may be derived from the wild-type I-Crel (SEQ ID NO: 1 ) or an I-Crel scaffold protein having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity with SEQ ID NO: 1 , such as the scaffold called I-Crel N75 (167 amino acids; SEQ ID NO: 3) having the insertion of an alanine at position 2, and the insertion of AAD at the C-terminus (positions 164 to 166) of the I-Crel sequence. In the present Patent Application all the I-Crel variants described comprise an additional Alanine after the first Methionine of the wild type I-Crel sequence (SEQ ID NO: 1). These variants also comprise two additional Alanine residues and an Aspartic Acid residue after the final Proline of the wild type I-Crel sequence. These additional residues do not affect the properties of the enzyme and to avoid confusion these additional residues do not affect the numeration of the residues in I-Crel or a variant referred in the present Patent Application, as these references exclusively refer to residues of the wild type I-Crel enzyme (SEQ ID NO: 1 ) as present in the variant, so for instance residue 2 of I-Crel is in fact residue 3 of a variant which comprises an additional Alanine after the first Methionine.

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 variant may also comprise a nuclear localization signal (NLS); said NLS is useful for the importation of said variant into the cell nucleus. The NLS may be inserted just after the first methionine of the variant or just after an N-terminal tag.

The variant according to the present invention may be a homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence. Alternatively, said variant is a heterodimer, resulting from the association of a first and a second monomer having different substitutions at positions 28 to 40 and 44 to 77 of I-Crel, said heterodimer being able to cleave a non-palindromic DNA target sequence from the TERT gene.

In particular said heterodimer variant is composed by one of the possible associations between variants constituting N-terminal and C-terminal monomers of single chain molecules from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 28 and SEQ ID NO: 29 to SEQ ID NO: 35.

The DNA target sequences are situated in the TERT Open Reading Frame (ORF) and these sequences cover all the TERT ORF. In particular, said DNA target sequences for the variant of the present invention and derivatives are selected from the group consisting of the SEQ ID NO: 4 to SEQ ID NO: 23, as shown in figures 3 and 6 and Table I. Said DNA targets of the endonucleases or endonuclease variants according to the present invention can also be selected from the group consisting of SEQ ID NO: 54 to 64.

The sequence of each I-Crel variant is defined by the mutated residues at the indicated positions. The positions are indicated by reference to I-Crel sequence (SEQ ID NO: 1 ) ; I- Crel has N, S, Y, Q, S, Q, R, R, D, I and E at positions 30, 32, 33, 38, 40, 44, 68, 70, 75, 77 and 80 respectively.

Each monomer (first monomer and second monomer) of the heterodimeric variant according to the present invention may also be named with a letter code, after the eleven residues at positions 28, 30, 32, 33, 38, 40, 44, 68 and 70, 75 and 77 and the additional residues which are mutated, as indicated above. For example, the mutations 7E33T38R40Q43I44D50R70S75R77T80K96E153G in the N-terminal monomer constituting a single chain molecule targeting the TERT2 target of the present invention (SEQ ID NO: 25), this single chain molecule SCOH-Ter2-bl -A being encoded by the expression plasmid pCLS3713, said plasmid entire sequence being given by SEQ ID NO:36.

In the present invention, for a given DNA target, ".2" derivative target sequence differs from the initial genomic target at positions -2, -1 , +1 , +2, where I-Crel cleavage site (GTAC) substitutes the corresponding sequence at these positions of said initial genomic target. ".3" derivative target sequence is the palindromic sequence derived from the left part of said ".2" derivative target sequence. ".4" derivative target sequence is the palindromic sequence derived from the right part of said ".2" derivative target sequence. ".5" derivative target sequence is the palindromic sequence derived from the left part of the initial genomic target. ".6" derivative is the palindromic sequence derived from the left part of the initial genomic target.

In the present invention, a "N-terminal monomer" constituting one of the monomers of a single chain molecule, refers to a variant able to cleave ".3" or ".5" palindromic sequence. In the present invention, a "C-terminal monomer" constituting one of the monomers of a single chain molecule, refers to a variant able to cleave ".4" or ".6" palindromic sequence.

The heterodimeric variant as defined above may have only the amino acid substitutions as indicated above. In this case, the positions which are not indicated are not mutated and thus correspond to the wild-type I-Crel (SEQ ID NO: 1 ).

The invention encompasses I-Crel variants having at least 85 % identity, preferably at least 90 % identity, more preferably at least 95 % (96 %, 97 %, 98 %, 99 %) identity with the sequences as defined above, said variant being able to cleave a DNA target from the TERT gene.

The heterodimeric variant is advantageously an obligate heterodimer variant having at least one pair of mutations corresponding to residues of the first and the second monomers which make an intermolecular interaction between the two I-Crel monomers, wherein the first mutation of said pair(s) is in the first monomer and the second mutation of said pair(s) is in the second monomer and said pair(s) of mutations prevent the formation of functional homodimers from each monomer and allow the formation of a functional heterodimer, able to cleave the genomic DNA target from the TERT gene.

To form an obligate heterodimer, the monomers have advantageously at least one of the following pairs of mutations, respectively for the first monomer and the second monomer: a) the substitution of the glutamic acid at position 8 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at position 7 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine residues at positions 7 and 96, by an arginine,

b) the substitution of the glutamic acid at position 61 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at position 96 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine residues at positions 7 and 96, by an arginine,

c) the substitution of the leucine at position 97 with an aromatic amino acid, preferably a phenylalanine (first monomer) and the substitution of the phenylalanine at position 54 with a small amino acid, preferably a glycine (second monomer); the first monomer may further comprise the substitution of the phenylalanine at position 54 by a tryptophane and the second monomer may further comprise the substitution of the leucine at position 58 or lysine at position 57, by a methionine, and

d) the substitution of the aspartic acid at position 137 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the arginine at position 51 with an acidic amino acid, preferably a glutamic acid (second monomer).

For example, the first monomer may have the mutation D137R and the second monomer, the mutation R51 D. The obligate heterodimer meganuclease comprises advantageously, at least two pairs of mutations as defined in a), b), c) or d), above; one of the pairs of mutation is advantageously as defined in c) or d). Preferably, one monomer comprises the substitution of the lysine residues at positions 7 and 96 by an acidic amino acid (aspartic acid (D) or glutamic acid (E)), preferably a glutamic acid ( 7E and K96E) and the other monomer comprises the substitution of the glutamic acid residues at positions 8 and 61 by a basic amino acid (arginine (R) or lysine ( ); for example, E8 and E61 R). More preferably, the obligate heterodimer meganuclease, comprises three pairs of mutations as defined in a), b) and c), above.

The obligate heterodimer meganuclease consists advantageously of a first monomer (A) having at least the mutations (i) E8R, E8K or E8H, E61 R, E61 K or E61 H and L97F, L97W or L97Y; (ii) 7R, E8R, E61 R, 96R and L97F, or (iii) 7R, E8R, F54W, E61 R, 96R and L97F and a second monomer (B) having at least the mutations (iv) K.7E or 7D, F54G or F54A and 96D or K96E; (v) K7E, F54G, L58M and 96E, or (vi) K7E, F54G, K.57M and 96E. For example, the first monomer may have the mutations K.7R, E8R or E8 , E61 R, 96R and L97F or K7R, E8R or E8K, F54W, E61 R, K96R and L97F and the second monomer, the mutations K7E, F54G, L58M and 96E or K7E, F54G, K57M and K96E. The obligate heterodimer may comprise at least one NLS and/or one tag as defined above; said NLS and/or tag may be in the first and/or the second monomer. The subject-matter of the present invention is also a single-chain chimeric meganuclease (fusion protein) derived from an I-Crel variant as defined above. The single- chain meganuclease may comprise two I-Crel monomers, two I-Crel core domains (positions 6 to 94 of I-Crel) or a combination of both. Preferably, the two monomers/core domains or the combination of both, are connected by a peptidic linker.

More preferably the single-chain chimeric meganuclease is composed by one of the possible associations between variants from the group consisting of N-terminal monomers and C-terminal monomers, given in Tables II and III, respectively for a given DNA target, at the TERT2 and TERT5 loci, said monomer variants being connected by a linker. More preferably the single-chain chimeric meganuclease according to the present invention is one from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 28 and SEQ ID NO: 29 to SEQ ID NO: 35. Regarding TERT2.1 target at TERT2 locus, the single-chain chimeric meganuclease according to the present invention is one from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 28. Regarding TERT4.1 target, the single-chain chimeric meganuclease according to the present invention is one from the group consisting of SEQ ID NO: 29 to SEQ ID NO: 35.

It is understood that the scope of the present invention also encompasses the I-Crel variants per se, including heterodimers, obligate heterodimers, single chain meganucleases as non limiting examples, able to cleave one of the target sequences in TERT gene.

It is also understood that the scope of the present invention also encompasses the I- Crel variants as defined above that target equivalent sequences in TERT gene of eukaryotic organisms other than human, preferably mammals, more preferably a laboratory rodent (mice, rat, guinea-pig), or a rabbit, a cow, pig, horse or goat, those sequences being identified by the man skilled in the art in public databank like NCBI.

It is also understood that the scope of the present invention also encompasses endonucleases derived from a TALE-nuclease (TALEN) as explained below. Particularly, endonucleases according to the invention can be a fusion between a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one or two catalytic domains having endonuclease activity. Such endonucleases can target sequences within TERT gene locus according to the present invention selected from the group consisting of SEQ ID NO: 54 to 64 as non-limiting example. 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 a homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease. It is understood that the subject-matter of the present invention is also a polynucleotide fragment encoding one of the variant species as defined above, obtained by any method well-known in the art.

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 a 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 nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those skilled 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), rhabdo virus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e. 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 (particularly self inactivacting lentiviral vectors), 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 , URA3 and LEU2 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 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 polypeptide is expressed. Preferably, when said variant is a heterodimer, the two polynucleotides 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-/3-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), a-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 cleavage site as defined above.

For instance, said sequence sharing homologies with the regions surrounding the genomic DNA cleavage site of the variant is a fragment of the TERT gene. Alternatively, the vector coding for an I-Crel variant/single-chain meganuclease and the vector comprising the targeting construct are different vectors.

More preferably, the 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) or included in 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. Therefore, the targeting DNA construct is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. 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 may be any sequence used to alter the chromosomal DNA in some specific way including a sequence used to repair a mutation in the TERT gene, restore a functional TERT gene in place of a mutated one, modify a specific sequence in the TERT gene, to attenuate or activate the TERT gene, to inactivate or delete the TERT gene 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/recombinant cell lines) or genome therapy (gene correction or recovery of a functional gene). The targeting construct comprises advantageously a positive selection marker between the two homology arms and eventually a negative selection marker upstream of the first homology arm or downstream of the second homology arm. The marker(s) allow(s) the selection of cells having inserted the sequence of interest by homologous recombination at the target site.

The sequence to be introduced is a sequence which repairs a mutation in the TERT gene (gene correction or recovery of a functional gene), for the purpose of genome therapy. For correcting the TERT gene, cleavage of the gene occurs in the vicinity of the mutation, preferably, within 500 bp of the mutation. The targeting construct comprises a TERT gene fragment which has at least 200 bp of homologous sequence flanking the target site (minimal repair matrix) for repairing the cleavage, and includes a sequence encoding a portion of wild- type TERT gene corresponding to the region of the mutation for repairing the mutation. Consequently, the targeting construct for gene correction comprises or consists of the minimal repair matrix; it is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Preferably, when the cleavage site of the variant overlaps with the mutation the repair matrix includes a modified cleavage site that is not cleaved by the variant which is used to induce said cleavage in the TERT gene and a sequence encoding wild-type TERT gene that does not change the open reading frame of the TERT gene.

Alternatively, for the generation of knock-in cells/animals, the targeting DNA construct may comprise flanking regions corresponding to TERT gene fragments which has at least 200 bp of homologous sequence flanking the target site of the I-Crel variant for repairing the cleavage, an exogenous gene of interest within an expression cassette and eventually a selection marker such as the neomycin resistance gene.

For the insertion of a sequence, DNA homologies are generally located in regions directly upstream and downstream to the site of the break (sequences immediately adjacent to the break; minimal repair matrix). However, when the insertion is associated with a deletion of ORF sequences flanking the cleavage site, shared DNA homologies are located in regions upstream and downstream the region of the deletion.

Alternatively, for restoring a functional gene cleavage of the gene occurs in the vicinity or upstream of a mutation. Preferably said mutation is the first known mutation in the sequence of the gene, so that all the downstream mutations of the gene can be corrected simultaneously. The targeting construct comprises the exons downstream of the cleavage site fused in frame (as in the cDNA) and with a polyadenylation site to stop transcription in 3'. The sequence to be introduced (exon knock-in construct) is flanked by introns or exons sequences surrounding the cleavage site, so as to allow the transcription of the engineered gene (exon knock-in gene) into a mRNA able to code for a functional protein. For example, the exon knock-in construct is flanked by sequences upstream and downstream of the cleavage site, from a minimal repair matrix as defined above.

The subject matter of the present invention is also a targeting DNA construct as defined above.

The subject-matter of the present invention is also a composition characterized in that it comprises at least one meganuclease as defined above (variant or single-chain chimeric meganuclease) and/or at least one expression vector encoding said meganuclease, as defined above. Preferably, said composition is a pharmaceutical composition. In a preferred embodiment of said composition, it comprises a targeting DNA 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 meganuclease according to the invention. The subject-matter of the present invention is further the use of a meganuclease as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for reparing mutations of the TERT gene.

The subject-matter of the present invention is also further a method of treatment of a genetic disease caused by a mutation in TERT gene comprising administering to a subject in need thereof an effective amount of at least one variant encompassed in the present invention.

According to an advantageous embodiment of said use, it is for inducing a double- strand break in a site of interest of the TERT gene comprising a genomic DNA target sequence, 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 in the TERT gene, modifying a specific sequence in the TERT gene, restoring a functional TERT gene in place of a mutated one, attenuating or activating the TERT gene, introducing a mutation into a site of interest of the TERT gene, introducing an exogenous gene or a part thereof, inactivating or deleting the TERT gene or a part thereof, translocating a chromosomal arm, or leaving the DNA unrepaired and degraded.

Given the fact that TERT gene is only expressed in iPS cells or cancer cells, therefore, one can consider the TERT locus as a safe harbor in cells that do not normally express TERT, provided the insert can be expressed from this locus. In cells that do normally express TERT, provided the insertion does not affect the expression of TERT, or provided there remain a functional allele in the cell. For example insertion in introns can be made with no or minor modification of the expression pattern.

However, in this approach, the TERT gene itself can be disrupted.

Therefore, in another aspect of the present invention, the inventors have found that endonucleases variants targeting TERT gene can be used for inserting therapeutic transgenes other than TERT at TERT gene locus, using this locus as a safe harbor locus. In other terms, the invention relates to a mutant endonuclease capable of cleaving a target sequence in TERT gene locus, for use in safely inserting a transgene, wherein said disruption or deletion of said locus does not modify expression of genes located outside of said locus.

The subject-matter of the present invention is also further a method of treatment of a genetic disease caused by a mutation in a gene other than TERT gene comprising administering to a subject in need thereof an effective amount of at least one variant encompassed in the present invention.

The skilled in the art can easily verify whether disruption or deletion of a locus modifies expression of neighboring genes located outside of said locus using proteomic tools. Many protein expression profiling arrays suitable for such an analysis are commercially available. By "neighboring genes" is meant the 1 , 2, 5, 10, 20 or 30 genes that are located at each end of the TERT gene locus.

In a derived main aspect of the present invention, the inventors have found that the TERT locus could be used as a landing pad to insert and express genes of interest (GOIs) other than therapeutics. In this aspect, inventors have found that genetic constructs containing a GOI could be integrated into the genome at the TERT gene locus via meganuclease-induced recombination by specific meganuclease variants targeting TERT gene locus according to a previous aspect of the invention. The subject-matter of the present invention is also further a method for inserting a transgene into the genomic TERT locus of a cell, tissue or non-human animal wherein at least one variant of the invention is introduced in said cell, tissue or non-human animal.

In a preferred embodiment, the TERT locus further allows stable expression of the transgene. In another preferred embodiment, the target sequence inside the TERT locus is only present once within the genome of said cell, tissue or individual.

In another preferred embodiment meganuclease variants according to the present invention can be part of a kit to introduce a sequence encoding a GOI into at least one cell. In a more preferred embodiment, the at least one cell is selected form the group comprising: CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-1 16 cells; Hu-h7 cells; Huvec cells; Molt 4 cells. The subject-matter of the present invention is also a method for making a TERT gene knock-out or knock-in recombinant cell, comprising at least the step of: (a) introducing into a cell, a meganuclease as defined above (I-Crel variant or single- chain derivative), so as to induce a double stranded cleavage at a site of interest of the TERT gene comprising a DNA recognition and cleavage site for said meganuclease, simultaneously or consecutively,

(b) introducing into the cell of step (a), 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, so as to generate a recombinant cell having repaired the site of interest by homologous recombination,

(c) isolating the recombinant cell of step (b), by any appropriate means.

The subject-matter of the present invention is also a method for making a TERT gene knock-out or knock-in animal, comprising at least the step of:

(a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease as defined above, so as to induce a double stranded cleavage at a site of interest of the TERT gene comprising a DNA recognition and cleavage site for said meganuclease, simultaneously or consecutively,

(b) introducing into the animal precursor cell or embryo of step (a) 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, so as to generate a genetically modified animal precursor cell or embryo having repaired the site of interest by homologous recombination,

(c) developing the genetically modified animal precursor cell or embryo of step (b) into a chimeric animal, and

(d) deriving a transgenic animal from the chimeric animal of step (c).

Preferably, step (c) comprises the introduction of the genetically modified precursor cell generated in step (b) into blastocysts so as to generate chimeric animals.

The targeting DNA is introduced into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest. For making knock-out cells/animals, the DNA which repairs the site of interest comprises sequences that inactivate the TERT gene.

For making knock-in cells/animals, the DNA which repairs the site of interest comprises the sequence of an exogenous gene of interest, and eventually a selection marker, such as the neomycin resistance gene.

In a preferred embodiment, said targeting DNA construct is inserted in a vector.

The subject-matter of the present invention is also a method for making a TERT- deficient cell, comprising at least the step of:

(a) introducing into a cell, a meganuclease as defined above, so as to induce a double stranded cleavage at a site of interest of the TERT gene comprising a DNA recognition and cleavage site of said meganuclease, and thereby generate genetically modified TERT gene- deficient cell having repaired the double-strands break, by non-homologous end joining, and

(b) isolating the genetically modified TERT gene-deficient cell of step (a), by any appropriate mean. The subject-matter of the present invention is also a method for making a TERT gene knock-out animal, comprising at least the step of:

(a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease, as defined above, so as to induce a double stranded cleavage at a site of interest of the TERT gene comprising a DNA recognition and cleavage site of said meganuclease, and thereby generate genetically modified precursor cell or embryo having repaired the double-strands break by non-homologous end joining,

(b) developing the genetically modified animal precursor cell or embryo of step (a) into a chimeric animal, and

(c) deriving a transgenic animal from a chimeric animal of step (b). Preferably, step (b) comprises the introduction of the genetically modified precursor cell obtained in step (a), into blastocysts, so as to generate chimeric animals.

The cells which are modified may be any cells of interest as long as they contain the specific target site. For making knock-in/transgenic mice, the cells are pluripotent precursor cells such as embryo-derived stem (ES) cells, which are well-known in the art. For making recombinant human cell lines, the cells may advantageously be PerC6 (Fallaux et al., Hum. Gene Ther. 9, 1909-1917, 1998) or HEK293 (ATCC # CRL- 1573) cells.

The animal is preferably a mammal, more preferably a laboratory rodent (mice, rat, guinea-pig), or a rabbit, a cow, pig, horse or goat.

Said meganuclease 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.

For making recombinant cell lines expressing an heterologous protein of interest, the targeting DNA comprises a sequence encoding the product of interest (protein or RNA), and eventually a marker gene, flanked by sequences upstream and downstream the cleavage site, as defined above, so as to generate genetically modified cells having integrated the exogenous sequence of interest in the TERT gene, by homologous recombination.

The sequence of interest may be any gene coding for a certain protein/peptide of interest, included but not limited to: reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, disease causing gene products and toxins. The sequence may also encode a RNA molecule of interest including for example an interfering RNA such as ShRNA, miRNA or siRNA, well-known in the art.

The expression of the exogenous sequence may be driven, either by the endogenous TERT gene promoter or by a heterologous promoter, preferably an ubiquitous or tissue specific promoter, either constitutive or inducible, as defined above. In addition, the expression of the sequence of interest may be conditional; the expression may be induced by a site-specific recombinase such as Cre or FLP (Akagi K, Sandig V, Vooijs M, Van der Valk M, Giovannini M, Strauss M, Berns A (May 1997). " Nucleic Acids Res. 25 (9): 1766-73.; Zhu XD, Sadowski PD ( 1995). J Biol Chem 270).

Thus, the sequence of interest is inserted in an appropriate cassette that may comprise an heterologous promoter operatively linked to said gene of interest and one or more functional sequences including but not limited to (selectable) marker genes, recombinase recognition sites, polyadenylation signals, splice acceptor sequences, introns, tag for protein detection and enhancers.

The subject matter of the present invention is also a kit for making TERT gene knockout or knock-in cells/animals comprising at least a meganuclease and/or one expression vector, as defined above. Preferably, the kit further comprises a targeting DNA comprising a sequence that inactivates the TERT gene flanked by sequences sharing homologies with the region of the TERT gene surrounding the DNA cleavage site of said meganuclease. In addition, for making knock-in cells/animals, the kit includes also a vector comprising a sequence of interest to be introduced in the genome of said cells/animals and eventually a selectable marker gene, as defined above.

The subject-matter of the present invention is also the use of at least one meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing a pathological condition caused by a mutation in the TERT gene as defined above, in an individual in need thereof. The use of the meganuclease may comprise at least the step of (a) inducing in somatic tissue(s) of the donor/ individual a double stranded cleavage at a site of interest of the TERT gene comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into said somatic tissue(s) 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 TERT gene upon recombination between the targeting DNA and the chromosomal DNA, as defined above. The targeting DNA is introduced into the somatic tissues(s) under conditions appropriate for introduction of the targeting DNA into the site of interest.

According to the present invention, said double-stranded cleavage may be induced, ex vivo by introduction of said meganuclease into somatic cells from the diseased individual and then transplantation of the modified cells back into the diseased individual.

The subject-matter of the present invention is also a method for preventing, improving or curing a pathological condition caused by a mutation in the TERT gene, 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 can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into mouse cells, by any convenient means well-known to those in the art, which are 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: - liposomes, polyethyleneimine (PEI); in such a case said association is administered and therefore introduced into somatic target cells.

- membrane translocating peptides (Bonetta, The Scientist, 2002, 16, 38; Ford et al., Gene Ther., 2001 , 8, 1 -4 ; Wadia and Dowdy, Curr. Opin. Biotechnol, 2002, 13, 52-56); in such a case, the sequence of the variant/single-chain meganuclease is fused with the sequence of a membrane translocating peptide (fusion protein).

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, electroporation). 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.

Since meganucleases recognize a specific DNA sequence, any meganuclease developed in the context of human gene therapy could be used in other contexts (other organisms, other loci, use in the context of a landing pad containing the site) unrelated with gene therapy of TERT in human as long as the site is present.

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. 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, electroporation). 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").

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. (US 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—polypropylene glycol copolymer are described in Saifer et al. (US 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 a 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 also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example, further rounds of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel meganucleases.

The different uses of the meganuclease and the methods of using said meganuclease according to the present invention include the use of the I-Crel variant, 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 meganuclease, as defined above.

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, WO 2004/031346 and WO 2009/095793). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention. In particular, the invention encompasses also the I-Crel variants defined in the tables II and III.

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 I-Crel variant or single-chain derivative as defined in the present invention are produced by expressing the polypeptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed (in the case of the variant only) in a host cell or a transgenic animal/plant modified by one expression vector or two expression vectors (in the case of the variant only), under conditions suitable for the expression or co-expression of the polypeptide(s), and the variant or single-chain derivative is recovered from the host cell culture or from the transgenic animal/plant.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuIIis et al. U.S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Definitions

- Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, in which, for example, Q means Gin or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.

- Amino acid substitution means the replacement of one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a peptide sequence is an amino acid substitution. - Altered/enhanced/increased cleavage activity, refers to an increase in the detected level of meganuclease cleavage activity, see below, against a target DNA sequence by a second meganuclease in comparison to the activity of a first meganuclease against the target DNA sequence. Normally the second meganuclease is a variant of the first and comprise one or more substituted amino acid residues in comparison to the first meganuclease. - iPS or iPSC refer to induced Pluripotent Stem Cells.

- by "clean iPS" cells is intended iPS cells in which transgenes that have been first inserted in their genomes for their reprogrammation toward said iPS, have been secondarily removed without any scar in their genome for obtaining such clean iPS, avoiding problems in further re-differentiation steps and therapeutic uses due to the permanent expression of these transgenes in classical approach. - by "safe iPS" is intended iPS cells that have lost self-renewable property for example by knocking-out at least a gene conferring or implicated in said self-renewable cellular property.

- by "secure iPS" cells is intended iPS cells in which, at a defined step of differentiation process, the progression of iPS cells toward more differentiated cell types is made irreversible.

- by "clean and/or safe and/or secure" iPS is intended iPS cells comprising one or more of the previously-described properties.

- by reprogrammation process is intended the process of dedifferentiation of a somatic cell toward iPS cells.

- 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.

- by "endonuclease" is intended any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within of a DNA or RNA molecule, preferably a DNA molecule. Endonucleases do not cleave the DNA or RNA molecule irrespective of its sequence, but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as "target sequences" or "target sites" and significantly increased HR by specific meganuclease-induced DNA double-strand break (DSB) at a defined locus (Rouet et al, 1994; Choulika et al, 1995). Endonucleases can for example be a homing endonuclease (Paques et al. Curr Gen Ther. 2007 7:49-66), a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus et al. Nat Biotechnol. 2005 23:967-973) or a chemical endonuclease (Arimondo et al. Mol Cell Biol. 2006 26:324-333; Simon et al. NAR 2008 36:3531 -3538; Eisenschmidt et al. NAR 2005 33 :7039-7047; Cannata et al. PNAS 2008 105:9576-9581 ). In chemical endonucleases, a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences (Kalish and Glazer Ann NY Acad Sci 2005 1058: 151 -61). Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention. In the scope of the present invention is also intended any fusion between molecules able to bind DNA specific sequences and agent/reagent/chemical able to cleave DNA or interfere with cellular proteins implicated in the DSB repair (Majumdar et al. J. Biol. Chem 2008 283, 17: 1 1244-1 1252; Liu et al. NAR 2009 37:6378-6388); as a non limiting example such a fusion can be constituted by a specific DNA-sequence binding domain linked to a chemical inhibitor known to inhibate religation activity of a topoisomerase after DSB cleavage. Endonuclease can be a homing endonuclease, also known under the name of meganuclease. By "meganuclease", is intended an endonuclease having a double-stranded DNA target sequence of 12 to 45 bp. Such homing endonucleases are well-known to the art (see e.g. Stoddard, Quarterly Reviews of Biophysics, 2006, 38:49-95). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease. Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomenc enzyme comprising the two domains on a single polypeptide.

Endonucleases according to the invention can also be derived from TALENs, a new class of chimeric nucleases using a Fokl catalytic domain and a DNA binding domain derived from Transcription Activator Like Effector (TALE), a family of proteins used in the infection process by plant pathogens of the Xanthomonas genus(Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 201 1 ). The functional layout of a Fokl-based TALE-nuclease (TALEN) is essentially that of a ZFN, with the Zinc-finger DNA binding domain being replaced by the TALE domain. As such, DNA cleavage by a TALEN requires two DNA recognition regions flanking an unspecific central region. Endonucleases encompassed in the present invention can also be derived from TALENs. An endonuclease according to the present invention can be derived from a TALE-nuclease (TALEN), i. e. a fusion between a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one or two catalytic domains.

- by "meganuclease domain" is intended the region which interacts with one half of the DNA target of a meganuclease and is able to associate with the other domain of the same meganuclease which interacts with the other half of the DNA target to form a functional meganuclease able to cleave said DNA target.

- by "meganuclease variant" or "variant" it is intended a meganuclease obtained by replacement of at least one residue in the amino acid sequence of the parent meganuclease with a different amino acid. - by "peptide linker" it is intended to mean a peptide sequence of at least 10 and preferably at least 17 amino acids which links the C-terminal amino acid residue of the first monomer to the N-terminal residue of the second monomer and which allows the two variant monomers to adopt the correct conformation for activity and which does not alter the specificity of either of the monomers for their targets. - by "subdomain" it is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half- site.

- by "targeting DNA construct/minimal repair matrix/repair matrix" it is intended to mean a DNA construct comprising a first and second portions which are homologous to regions 5' and 3 ' of the DNA target in situ. The DNA construct also comprises a third portion positioned between the first and second portion which comprise some homology with the corresponding DNA sequence in situ or alternatively comprise no homology with the regions 5' and 3 ' of the DNA target in situ. Following cleavage of the DNA target, a homologous recombination event is stimulated between the genome containing the TERT gene and the repair matrix, wherein the genomic sequence containing the DNA target is replaced by the third portion of the repair matrix and a variable part of the first and second portions of the repair matrix.

- by "functional variant" is intended a variant which is able to cleave a DNA target sequence, preferably said target is a new target which is not cleaved by the parent meganuclease. For example, such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.

- by "selection or selecting" it is intended to mean the isolation of one or more meganuclease variants based upon an observed specified phenotype, for instance altered cleavage activity. This selection can be of the variant in a peptide form upon which the observation is made or alternatively the selection can be of a nucleotide coding for selected meganuclease variant.

- by "screening" it is intended to mean the sequential or simultaneous selection of one or more meganuclease variant (s) which exhibits a specified phenotype such as altered cleavage activity.

- by "derived from" it is intended to mean a meganuclease variant which is created from a parent meganuclease and hence the peptide sequence of the meganuclease variant is related to (primary sequence level) but derived from (mutations) the sequence peptide sequence of the parent meganuclease. - by "I-Crel" is intended the wild-type I-Crel having the sequence of pdb accession code l g9y, corresponding to the sequence SEQ ID NO: 1 in the sequence listing.

- by "I-Crel variant with novel specificity" is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease. The terms "novel specificity", "modified specificity", "novel cleavage specificity", "novel substrate specificity" which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence. In the present Patent Application all the I-Crel variants described comprise an additional Alanine after the first Methionine of the wild type I-Crel sequence (SEQ ID NO: 49). These variants also comprise two additional Alanine residues and an Aspartic Acid residue after the final Proline of the wild type I-Crel sequence. These additional residues do not affect the properties of the enzyme and to avoid confusion these additional residues do not affect the numeration of the residues in I-Crel or a variant referred in the present Patent Application, as these references exclusively refer to residues of the wild type I- Crel enzyme (SEQ ID NO: 1 ) as present in the variant, so for instance residue 2 of I-Crel is in fact residue 3 of a variant which comprises an additional Alanine after the first Methionine. - by "I-Crel site" is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by I-Crel. I-Crel sites include the wild-type non-palindromic I-Crel homing site and the derived palindromic sequences such as the sequence 5'- t_i₂c.i 1 a.10a. a_8a.7C.6g-5t-4c.3g.2t. ia₊ i C₊2g+3a₊4C₊5g₊₆t+7 8t+ t+iog+i ia+ i 2 (SEQ ID NO: 2), also called C1221 (Figures 3 and 6).

- by "domain" or "core domain" is intended the "LAGLIDADG homing endonuclease core domain" which is the characteristic αιβιβ₂α₂β3β₄α3 fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues. Said domain comprises four beta-strands (βιβ₂β₃β₄) folded in an anti-parallel beta- sheet which interacts with one half of the DNA target. This domain is able to associate with another LAGLIDADG homing endonuclease core domain which interacts with the other half of the DNA target to form a functional endonuclease able to cleave said DNA target. For example, in the case of the dimeric homing endonuclease I-Crel (163 amino acids), the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94. - by "subdomain" is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half- site.

- by "chimeric DNA target" or "hybrid DNA target" it is intended the fusion of a different half of two parent meganuclease target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).

- by "beta-hairpin" is intended two consecutive beta-strands of the antiparallel beta- sheet of a LAGLIDADG homing endonuclease core domain (βιβ₂ 0Γ,β₃β₄) which are connected by a loop or a turn,

- by "single-chain meganuclease", "single-chain chimeric meganuclease", "single- chain meganuclease derivative", "single-chain chimeric meganuclease derivative" or "single- chain derivative" is intended a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic spacer. The single-chain meganuclease is able to cleave a chimeric DNA target sequence comprising one different half of each parent meganuclease target sequence. - by "DNA target", "DNA target sequence", "target sequence" , "target-site", "target" , "site", "site of interest", "recognition site", "recognition sequence", "homing recognition site", "homing site", "cleavage site" is intended a 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease such as I-Crel, or a variant, or a single-chain chimeric meganuclease derived from I-Crel. These terms refer to a distinct DNA location, preferably a genomic location, at which a double stranded break (cleavage) is to be induced by the meganuclease. The DNA target is defined by the 5' to 3' sequence of one strand of the double-stranded polynucleotide, as indicate above for CI 221. Cleavage of the DNA target occurs at the nucleotides at positions +2 and -2, respectively for the sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an I-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target.

- by "DNA target half-site", "half cleavage site" or half-site" is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain.

- by "chimeric DNA target" or "hybrid DNA target" is intended the fusion of different halves of two parent meganuclease target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).

- by " gene" is intended the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which encodes for a specific protein or segment of protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region. The gene may further comprise a terminator, enhancers and/or silencers, by "gene" is also intended one or several part of this gene, as listed above.

- by "TERT gene", is preferably intended a TERT gene of a vertebrate or part of it, more preferably the TERT gene or part of it of a mammal such as human. TERT gene sequences are available in sequence databases, such as the NCBI/GenBank database. This gene has been described in databanks as NC00005.9 entry (NCBI (Sequence update 10-JUN- 2009).

- by "DNA target sequence from the TERT gene", "genomic DNA target sequence", " genomic DNA cleavage site", "genomic DNA target" or "genomic target" is intended a 22 to 24 bp sequence of the TERT gene as defined above, which is recognized and cleaved by a meganuclease variant or a single-chain chimeric meganuclease derivative.

- by "parent meganuclease" it is intended to mean a wild type meganuclease or a variant of such a wild type meganuclease with identical properties or alternatively a meganuclease with some altered characteristic in comparison to a wild type version of the same meganuclease. In the present invention the parent meganuclease can refer to the initial meganuclease from which a series of variants are derived from.

- by "vector" is intended a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

- by "homologous" is intended a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95 % identity, preferably 97 % identity and more preferably 99 % or 99.5%. - "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.

- by "mutation" is intended the substitution, deletion, insertion of one, two, three, four, five, six, ten or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence. Said mutation can affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

- "gene of interest" or "GOI" refers to any nucleotide sequence encoding a known or putative gene product.

- As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome. The term "locus" usually refers to the specific physical location of an endonuclease' s target sequence on a chromosome. Such a locus, which comprises a target sequence that is recognized and cleaved by an endonuclease according to the invention, is referred to as "locus according to the invention".

- by "safe harbor" locus of the genome of a cell, tissue or individual, is intended a gene locus wherein a transgene could be safely inserted, the disruption or deletion of said locus consecutively to the insertion not modifying expression of genes located outside of said locus, TERT gene being a good safe harbor locus because this gene is silent in normal cells and only expresses in germline cells, proliferative stem cells of renewal tissue, in iPS cells or cancer cells.

- As used herein, the term "transgene" refers to a sequence encoding a polypeptide. Preferably, the polypeptide encoded by the transgene is either not expressed, or

expressed but not biologically active, in the cell, tissue or individual in which the transgene is inserted. Most preferably, the transgene encodes a therapeutic polypeptide useful for the treatment of an individual.

The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

As used above, the phrases "selected from the group consisting of," "chosen from," and the like include mixtures of the specified materials. Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

The following non-limiting examples illustrate some aspects of the invention.

EXAMPLES Protein design

I-Crel variants were created using a combinatorial approach, to redesign the DNA binding domain of the 1-Crel protein and thereby engineer novel meganucleases with fully engineered specificity for the desired TERT gene target. Some of the DNA targets identified by the inventors to validate their invention are given in Table I above. Derivatives of these DNA targets are given in Figs. 4 & 7.

The combinatorial approach, as illustrated in Fig. 2 and described in International PCT applications WO 2006/097784 and WO 2006/097853, and also in Arnould et al. (J. Mol. Biol., 2006, 355, 443-458) and Smith et al. (Nucleic Acids Res., 2006, 34, el 49) was used to redesign the DNA binding domain of the I-Crel protein and thereby engineer novel meganucleases with fully engineered specificity.

Example 1 : Engineering meganucleases targeting the Tert2 site A. Construction of variants targeting the Tert2 site

Tert2 is an example of a target for which meganuclease variants have been generated. The Tert2 target sequence or Tert2.1 (CTGACTGGCGCTCGGAGGCTCG SEQ ID NO : 8) is located in exon 2 of the human telomerase reverse transcriptase TERT gene at position 1221 -1244 on NC00005.9 sequence update 10-JUN-2009.

The Tert2 sequence or Tert2.1 (SEQ ID NO: 8) is partially a combination of the 10TGA P (SEQ ID NO : 4 ), 5GGC P (SEQ ID NO: 5 ), 10GAG P (SEQ ID NO: 7) and 5TCC_P (SEQ ID NO: 6) target sequences which are shown on Figure 3. These sequences are cleaved by meganucleases obtained as described in International PCT applications WO 2006/097784 and WO 2006/097853, Arnould et al. (J. Mol. Biol., 2006, 355, 443-458) and Smith et al. (Nucleic Acids Res., 2006, 34 el49).

Two palindromic targets, Tert2.3 (SEQ ID NO: 10) and Tert2.4 (SEQ ID NO: 1 1 ), and two pseudo palindromic targets, Tert2.5 (SEQ ID NO: 12) and Tert2.6 (SEQ ID NO: 13), were derived from Tert2.1 (SEQ ID NO: 8) and Tert2.2 (SEQ ID NO: 9) (Figure 3). Since Tert2.3 and Tert2.4 are palindromic, they are cleaved by homodimeric proteins. Therefore, homodimeric I-Crel variants cleaving either the Tert2.3 palindromic target sequence of SEQ ID NO: 10 or the Tert2.4 palindromic target sequence of SEQ ID NO: 1 1 were constructed using methods derived from those described in Chames et al. (Nucleic Acids Res., 2005, 33, el 78), Arnould et al. (J. Mol. Biol., 2006, 355, 443-458), Smith et al. (Nucleic Acids Res., 2006, 34, el 49) and Arnould et al. (Arnould et al. J Mol Biol. 2007 371 :49-65).

Single chain obligate heterodimer constructs were generated for the I-Crel variants able to cleave the Tert2 target sequences when forming heterodimers. These single chain constructs were engineered using the linker RM2

(AAGGSDKYNQALS YNQALSKYNQALSGGGGS) (SEQ ID NO: 24). During this design step, mutations 7E, K96E were introduced into the mutant cleaving Tert2.3 (monomer 1 = monomer Nter) and mutations E8 , G19S,E61 R into the mutant cleaving Tert2.4 (monomer 2 = monomer Cter) to create the single chain (SC) molecules: monomerl(K7E K96E)-RM2-monomer2 (E8 G19S E61 R) that is further called SCOH-Ter2-bl scaffold (Table II). Additional amino-acid substitutions have been found in previous studies to enhance the activity of I-Crel derivatives: some of these mutations correspond to the replacement of Phenylalanine 54 with Leucine (F54L), Glutamic acid 80 with Lysine (E80K), Valine 105 with Alanine (V105A) and Isoleucine 132 with Valine (I132V). Some combinations (either one, both or none) were introduced into the coding sequence of N-terminal and C-terminal protein fragment, and some of the resulting proteins were assayed for their ability to induce cleavage of the Tert2 target.

In the SCOH-Ter2-bl-C_V2 single chain mutations at position 70 and 75 have been reverted to I-Crel WT 70r 75d residues in the first part (first monomer) as well as on the second part of the single chain (second monomer). The resulting proteins are shown in Table II below.

Table II: Examples of SCOH-Ter2-bl useful for Tert2 targeting

B. Validation of some SCOH-Ter2-bl variants in a mammalian cells extrachromosomal assay.

The activity of the single chain molecules against the Tert2 target was monitored using the described CHO assay along with our internal control SCOH-RAG and I-Sce I meganucleases. All comparisons were done from 0.7 to 25ng transfected variant DNA

(Figures 4 & 5). All the single molecules displayed Tert2 target cleavage activity in CHO assay as listed in Table II. Variants shared specific behavior upon assayed dose depending on the mutation profile they bear (Figures 4 & 5). For example, pCLS3714 -SCOH-ter2-bl -C displayed a high activity at low doses comparable to SC-Rag on Rag target. All of the described variants are active and can be considered for inserting transgenes or performing gene modification (including Knock Out and mutagenesis) into the tert2 locus or in DNA sequences containing Tert2 target sequence.

Example 2 : Engineering meganucleases targeting the Tert5 site

A) Construction of variants targeting the Tert5 site

Tert5 is an example of a target for which meganuclease variants have been generated.

The Tert5 target sequence or Tert5.1 (ATTCCCCCCTGTGTCTCAGCTATG SEQ ID NO :

18) is located in the intron just upstream of exonl2 of the human telomerase reverse transcriptase TERT gene at position 34429-34452 on NC00005.9 sequence update 10-JUN- 2009.

The Tert5 sequence or Tert5.1 (SEQ ID NO: 18) is partially a combination of the 10TCC_P (SEQ ID NO : 14 ), 5CCT P (SEQ ID NO : 15 ), 10TAG P (SEQ ID NO : 17 ) and 5GAG_P (SEQ ID NO : 16 ) target sequences which are shown on Figure 6. These sequences are cleaved by meganucleases obtained as described in International PCT applications WO 2006/097784 and WO 2006/097853, Arnould et al. (J. Mol. Biol, 2006, 355, 443-458) and Smith et al. (Nucleic Acids Res., 2006, 34 el49). Two palindromic targets, Tert5.3 (SEQ ID NO: 20) and Tert5.4 (SEQ ID NO: 21 ), and two pseudo palindromic targets, Tert5.5 (SEQ ID NO: 22) and Tert5.6 (SEQ ID NO: 23), were derived from Tert5.1 (SEQ ID NO: 18) and Tert5.2 (SEQ ID NO: 19) (Figure 6). Since Tert5.3 and Tert5.4 are palindromic, they are cleaved by homodimeric proteins. Therefore, homodimeric I-Crel variants cleaving either the Tert5.3 palindromic target sequence of SEQ

ID NO: 20 or the Tert5.4 palindromic target sequence of SEQ ID NO: 21 were constructed using methods derived from those described in Chames et al. (Nucleic Acids Res., 2005, 33, el 78), Arnould et al. (J. Mol. Biol., 2006, 355, 443-458), Smith et al. (Nucleic Acids Res., 2006, 34, el49) and Arnould et al. (Arnould et al. J Mol Biol. 2007 371 :49-65).

Single chain obligate heterodimer constructs were generated for the I-Crel variants able to cleave the Tert5 target sequences when forming heterodimers. These single chain constructs were engineered using the linker RM2 (AAGGSDKYNQALSKYNQALSKYNQALSGGGGS) (SEQ ID NO: 24). During this design step, mutations K7E, K96E were introduced into the mutant cleaving Tert5.3 (monomer 1 = monomer Nter) and mutations E8K, G 19S,E61 R into the mutant cleaving Tert5.4 (monomer 2 = monomer Cter) to create the single chain molecules: monomer 1 (K7E K96E)- RM2-monomer2(E8K G19S E61 R) that is further called SCOH-Ter5-b l scaffold (Table III). The same strategy was applied to a second scaffold, termed SCOH-Ter5-b56 scaffold (Table III).

Additional amino-acid substitutions have been found in previous studies to enhance the activity of I-Crel derivatives: some of these mutations correspond to the replacement of

Phenylalanine 54 with Leucine (F54L), Glutamic acid 80 with Lysine (E80K), Valine 105 with Alanine (VI 05 A) and Isoleucine 132 with Valine (1132V). Some combinations (either one, both or none) were introduced into the coding sequence of N-terminal and C-terminal protein fragment, and some of the resulting proteins were assayed for their ability to induce cleavage of the Tert5 target.

B. Validation of some SCOH-Ter5-bl variants in a mammalian cells extrachromosomal assay.

The activity of the single chain molecules against the Tert5 target was monitored using the described CHO assay along with our internal control SCOH-RAG and I-Sce I meganucleases. All comparisons were done from 3 to 25ng transfected variant DNA (Figure 7). All the single molecules displayed Tert5 target cleavage activity in CHO assay as listed in Table III. Variants shared specific behavior upon assayed dose depending on the mutation profile they bear (Figure 7). For example, SCOH-ter5-bl -C shown on figure 7 displays an activity level comparable to the activity level of I- Scel. All of the variants described are active and can be considered for inserting transgenes or performing gene modification (including Knock Out and mutagenesis) into the tert5 locus or in DNA sequences containing Tert5 target sequence.

Name of Single N-terminal C-terminal SC Chain (SC)- monomer monomer

Protein sequence

encoding mutations mutations SEQ I D plasmid in SC in SC NO:

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSSKFKHYLQLTFRVTQKTQRRW

SCOH-Te r5- LLDKLVDEIGVGYVRDSGSVSNYDLSEIKPLHNFLTQLQPF LELKRKQAN LVLKII

EQLPSAKESPDKFLEVCTWVDQVAALNDS KTRKTTSETVRAVLDG LSE KKSS

b56- B PAAGGSDKYNG3ALS YNC5ALSKYNQALSGGGGSNKKF LLYLAGFVDSDGSI IA 29 /pCLS31 84 QIKPNQTCKF HQLSLTFNVTQ TQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

(SEQ ID : 40) SKIKPLHNFLTQLQPFLKLKQ QAN LVLXII EQLPSAKESPDKFLEVCTWVDQIA

ft.L DSKTRKTTSETVRAVLDSLSEK KS SP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSSKFKHYLQLTFRvTQKTQRRW LLDKLVDEIGVGYVRDSGSVSNYDLSEIKPLHNFLTQLCJPF LELKRKQAN LVLKII

SCOH-Te r5- EQLPSAKESPDKFLEVCTWVDQIAALNDS KTRKTTSETVRAVLDG LSE KKKSS

b56-A PAAGGSDKYNQALSKYNQALSKYNQALSGGGGSNKKF LLYLAGFVDSDGSI IA 30

QIKPNQTCKFKHQLSLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

/pCLS32 95)

SKIKPLHNFLTQLQPFLKLKQKQAN LVLKII EQLPSAKESPDKFLEVCTWVDQIA ALNDSKTRKTTSETVRAVLDSLSEKKKSSP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSS KFKHYLQLTFRVTQKTQRRW

S COH-Te r5- LLDKLVDEIGVGYVRDSGSVSNYDLSEIKPLHNFLTQ LQPF LELKRKQAN LVLKII

EQLPSAKESPDKFLEVCTWVDQIAALN DS KTRKTTSETVRAVLDG LSE KKKSS

b 56- D PAAGGSDKYNQALSKYNQALSKYNQALSGGG GSNKKF LLYLAGFVDSDGSI IA 31 /pCLS33 48 QIKPNQTCKFKHQLSLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

(SEQ ID : 42) SKIKPLHNFLTQLQPFLKLKQKQAN LVLKII EQLPSA KES PDKFLE VCTWVDQVA

ALN DSKTRKTTSETVRAVLDSLSEKKKS SP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSS KFKHYLQLTFRVTQKTQRRW

SCOH-Te r5- LLDKLVDEIGVGYVADSGSVSNYD LSE IKP LHNFLTQLQ PFLELKQ KQAN LVLKII

EQLPSAKESPDKFLEVCTWVDQIAALNDS KTRKTTSETVRA VLDS LSE KKKSSP

b l-A AAGGSD KYN QALSKYNQALSKYN Q AL SGGGG SN KKF L LY LAGFVD SDGSII AQI 32 /pCLS34 77 KPNQSCKFKHQLRLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNLS

(SEQ ID : 43) KIKPLHNFLTQLQPFLKLKQKQANLVLKIIEQLPSAKESPDKF LEVCTWVDQIAAL

NDSKTRKTTSETVRAVLDSLSEKKKSSP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSS KFKHY LQLTFRVTQKTQRRW

S COH-Te r5- LLD KLVD EIGVGYVADSGSVSN YD LSE KP LH N FLTQLQ PF LE LKQ KQAN LVL Kl

EQLPSAKESPDKFLEVCTWVDQVAALNDS KTRKTTSETVRA VLDS LS EKKKSS

b l-B PAAGGSDKYNQALSKYNQALSKYNQALSGGG GSNKKF LLYLAGFVDSDGSI IA 33 /pCLS34 78 QIKPNQSCKFKHQLRLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

(SEQ ID : 44) SKIKPLHNFLTQLQPFLKLKQKQAN LVLKII EQLPSAKESPDKFLEVCTWVDQIA

M-N DSKTRKTTSETVRAVLDSLSEKKKS SP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSSKFKHYLQLTFRVTQKTQRRW

SCOH-Te r5- LLDKLVDEIGVGYVADSGSVSNYD LSE IKP LH N FLTQLQ PFLE LKQ KQAN LVL KM

EQLPSAKESPDKFLEVCTWVDQVAALNDS KTRKTTSETVRA VLDS LS EKKKSS

b l-C PAAGGSD KYN QALSKYNQALSKYN OA LSGGG GSNKKF LLYLAGFVDSDGSI IA 34 /pCLS34 79 QIKPNQSCKFKHQLRLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

(SEQ ID : 45) SKIKPLHNFLTQLQPFLKLKQKQAN LVLKII EQLPSA KES PDKFLE VCTWVDQVA

ALN DSKTR KTTSETVR AVLDSLSEKKKS SP

MANTKYNEEFLLYLAGFVDGDGSIIAKIRPNQSS KFKHY LQLTFRVTQKTQRRW

SCOH-Te r5- LLDKLVDEIGVGYVADSGSVSNYD LSK IKP LH N FLTQLQ PFLE LKQ KQAN LVL Kll

EQLPSAKESPDKFLEVCTWVDQVAALN DS KTRKTTSETVRA VLDS LS EKK KSS

b l-E PAAGGSD KYN QALSKYNQALSKYN OA LSGGG GSNKKF LLYLAGFVDSDGSIIA 35 /pCLS34 80 QIKPNQSCKFKHQLRLTFNVTQKTQRRWFLDKLVDRIGVGYVHDSGSVSYYNL

(SEQ ID : 46) SKIKPLHNFLTQLQPFLKLKQKQAN LA LKII EQLPSA KES PDKFLE VCTWVDQVA

ALNDSKTRKTTSETVRAVLDSLSEKKKSSP

Table III: Examples of SCOH-Ter5-bl and SCOH-Ter5-b56 useful for Tert5 targeting Example 3 : Cloning and extrachromosomal assay in mammalian cells.

A. Cloning of Tert2 and Tert5 targets in a vector for CHO screen

The targets were cloned as follows using oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence; the following oligonucleotides were ordered from PROLIGO.

These oligonucleotides have the following sequences:

Tert2:

5 ' -TGGC ATAC A AGTTTCCTGACTGGCGCTCGG AGGCTCGTC AATCGTCTGTC A -3 ' (SEQ ID NO: 47), Tert5:

5'- TGGC ATAC AAGTTTATTCCCCCCTGTGTCTCAGCTATGC AATCGTCTGTC A -3' (SEQ ID NO: 48),

Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into CHO reporter vector (pCLS 1058). Target was cloned and verified by sequencing (MILLEGEN).

B. Cloning of the single chain molecules

A series of synthetic gene assembly was ordered to Gene Cust. Synthetic genes coding for the different single chain variants targeting TERT gene were cloned in pCLS 1853 using Ascl and Xhol restriction sites. C. Extrachromosomal assay in mammalian cells

CHO l cells were transfected as described in example 1.2. 72 hours after transfection, culture medium was removed and 150μ1 of lysis/revelation buffer for b - galactosidase liquid assay was added. After incubation at 37°C, OD was measured at 420 nm. The entire process is performed on an automated Velocityl 1 BioCel platform. Per assay, 150 ng of target vector was cotransfected with an increasing quantity of variant DNA from 0.7 to 25 ng or 3 to 25 ng. The total amount of transfected DNA was completed to 175ng (target DNA, variant DNA, carrier DNA) using an empty vector (pCLS0002).

Example 4: Deposited Biological Materials The present invention also concerns the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris) deposits n° CNCM 1-4347 and CNCM 1-4346 as well as the inserts respectively encoding TERT2 and TERT5 variants (respectively SEQ ID NO : 26 and SEQ ID NO : 34) in the plasmids contained in the E. coli strains deposited under the respective deposit numbers above.

Modifications and other embodiments

Various modifications and variations of the described meganuclease products, compositions and methods as well as the concept of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not intended to be limited to such specific embodiments. Various modifications of the described modes for carrying out the invention which are obvious to those skilled in the medical, biological, chemical or pharmacological arts or related fields are intended to be within the scope of the following claims. Unless specifically defined herein below, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

List of references cited in the description*

1. Christine Kim Garcia, et al. Nucleic Acids Res. 2007 December; 35(22): 7406-7416.

Published online 2007 December.

2. Takahashi K, Tanabe et al. Cell 2007; 131 : 861 -872.

3. Suhr ST, et al. JB. PLoS One. 2009 Dec 2;4(12):e8124. Erratum in: PLoS One.

2010;5(3) doi: 10.1371/annotation/c786dl41 -fd0f-45fd-80ec-96d80be620dc.

4. Park et al. 2008 Nature. 10;451 (7175): 141 -6

5. Aubry L, et al. PNAS. 2008 Oct 28; 05(43): 16707-12. Epub 2008 Oct 15

6. Tabar V, et al. Nat Biotechnol. 2005 May;23(5):601 -6. Epub 2005 Apr 24.

7. Roy NS, et al. Nat Med. 2006 Nov;12(l l ): 1259-68. Epub 2006 Oct 22. Erratum in:

Nat Med. 2007 Mar; 13(3):385.

8. Chen X, et al. 2009 Mol Biotechnol/, *41 *, 194-199.

9. Ohishi et al. 2007 Methods Mol Biol/, *405*, 133-146

10. Rampazzo E, et al. Br J Cancer. 2010 Apr 13; 102(8): 1300-5.

1 1. Mergny JL, et al. Nucleic Acids Res. 2002 Feb 15;30(4):839-65. Review.

12. Zhao Y, et al. Genes Chromosomes Cancer. 2009 Nov;48(l l ):963-74.

13. Liu JP, et al. Biochim Biophys Acta. 2010 Jan;1805(l ):35-42. Epub 2009 Sep 12.

14. Alberto Diaz de Leon et al. PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 I el 0680

15. Fuiiwara T. et al. Expert Opin Biol Ther. 2009 Mar;9(3):321 -9.

16. SA Savage et al. British Journal of Cancer (2007) 97, 832 - 836

17. Min XJ, et al. Zhonghua Yan Ke Za Zhi. 2009 Dec;45(l 2): 1 1 1 1 -7.

18. A.P. Cunninghama, et al. Curr Med Chem. 2006 ; 13(24): 2875-2888.

19. Capecchi MR.Science. 1989 Jun 16;244(4910): 1288-92. Review.

20. Smithies et al. Nat Med 2001 7(10): 1083-6

21. Thierry and Dujon Nucleic Acids Res 1992 20: 5625-5631

22. Puchta et al. Nucleic Acids Res 1993 21 : 5034-5040

23. Rouet et al. Mol Cell Biol 1994 14 : 8096-8106

24. Choulika et al. Mol Cell Biol 1995 15 : 1968-1973

25. Puchta et al. Proc Natl Acad Sci U.S.A 1996 93 : 5055-5060

26. Sargent et al. Mol Cell Biol 1997 17: 267-277

27. Cohen-Tannoudji et al. Mol Cell Biol 1998 18: 1444-1448

28. Donoho et al. Mol Cell Biol 1998 18 : 4070-4078

29. Elliott et al. Mol Cell Biol 1998 18 : 93-101

30. Chevalier and Stoddard Nucleic Acids Res 2001 29 : 3757-3774 31. Smith et al. Nucleic Acids Res 1999 27: 674-681

32. Bibikova et al. Mol Cell Biol 2001 21 : 289-297

33. Bibikova et al. Genetics 2002 161 : 1 169-1 175

34. Bibikova et al. Science 2003 300 : 764

35. Porteus and Baltimore Science 2003 300 : 763

36. Alwin et al. Mol Ther 2005 12 : 610-617

37. Urnov et al. Nature 2005 435 : 646-651

38. Porteus M.H. Mol Ther 2006 13: 438-446

39. Pabo et al. Annu Rev Biochem 2001 70 : 313-340

40. Jamieson et al. Nat Rev Drug Discov 2003 2 : 361 -368

41. Rebar and Pabo Science 1994 263 : 671-673

42. Kim and Pabo Proc Natl Acad Sci U S A 1998 95 : 2812-2817

43. Klug et al. Proc Natl Acad Sci USA 1994 91 : 1 1 163-1 1 167

44. Isalan and Klug Nat Biotechnol 2001 19 : 656-660

45. Catto et al. Nucleic Acids Res 2006 34 : 171 1 -1720

46. Hockemeyer et al., Nat Biotechnol. 2009 Sep;27(9): 851 -7).

47. Chevalier et al. Nat Struct Biol 2001 8 : 312-316

48. Chevalier et al. J Mol Biol 2003 329 : 253-269

49. Moure et al. J Mol Biol 2003 334 : 685-693,

50. Silva et al. J Mol Biol 1999 286 : 1 123-1 136

51. Bolduc et al. Genes Dev 2003 17 : 2875-2888

52. Ichiyanagi et al. J Mol Biol 2000 300 : 889-901

53. Moure et al. Nat Struct Biol 2002 9 : 764-770

54. Chevalier et al. Mol Cell 2002 10 : 895-905

55. Epinat et al. Nucleic Acids Res 2003 31 : 2952-62

56. Seligman et al.Genetics 1997 147 : 1653-1664

57. Sussman et al. J Mol Biol 2004 342 : 31-41

58. Arnould et al. J Mol Biol 2006 355 : 443-458

59. Rosen et al. Nucleic Acids Res 2006 34 : 4791 -4800

60. Smith et al. Nucleic Acids Res 2006 34 el 49

61. Doyon et al. J Am Chem Soc 2006 128 : 2477-2484

62. Gimble et al. J Mol Biol 2003 334 : 993-1008 63. Ashworth et al. Nature 2006 441 : 656-659

64. Argast et al. J Mol Biol 1998 280 : 345-353

65. Sealev DC et al. Nucleic Acids Res. 2010 Apr;38(6):2019-35. Epub 2009 Dec 23.

66. Calado RT. et al. Hematology Am Soc Hematol Educ Program. 2009:338-43.

67. Jeter CR et al. Stem Cells. 2009 May;27(5):993- 1005.

68. Roy NS et al. Nat Med. 2006 Nov; 12( 1 1): 1259-68. Epub 2006 Oct 22. Erratum in: Nat Med. 2007 Mar; 13(3):385.

69. You JS et al. Cancer Res. 2009 Jul 15;69(14):5716-25. Epub 2009 Jun 30.

70. Ji J et al. PLoS One. 2009 Nov 30;4(1 l):e8065.

71. Ji L et al. J Cell Physiol. 2009 Oct;221 (l ):54-66.

72. Brignier AC and Gewirtz AM. J Allergy Clin Immunol. 2010 Feb; 125(2 Suppl 2):S336-44. Epub 2010 Jan 12. Review.

73. Phillips BW, Crook JM. BioDrugs. 2010 Apr 1 ;24(2):99-108. doi: 10.2165/1 1532270- 000000000-00000. Review.

74. Jurica et al. Mol Cell 1998 2 : 469-476

75. Chevalier et al. Biochemistry 2004 43 : 14015-14026

76. Paques F. and Duchateau P., Current Gene Therapy, 2007, 7, 49-66

77. Chames et al, Nucleic Acids Res., 2005, 33, el 78

78. Arnould et al., J. Mol. Biol., 2007, 371 , 49-65

79. Prieto et al., Nucleic Acids Res., Epub 22 April 2007

80. Boch, J., H. Scholze, et al. (2009). "Breaking the code of DNA binding specificity of TAL-type III effectors." Science 326(5959): 1509-12.

81 . Capecchi, M. R. (1989). "Altering the genome by homologous recombination." Science 244(4910): 1288-92.

82. Christian, M., T. Cermak, et al. (2010). "Targeting DNA double-strand breaks with TAL effector nucleases." Genetics 186(2): 757-61.

83. Li, T., S. Huang, et al. (2010). "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and Fokl DNA-cleavage domain." Nucleic Acids Res 39(1 ): 359-72.

84. Moscou, M. J. and A. J. Bogdanove (2009). "A simple cipher governs DNA recognition by TAL effectors." Science 326(5959): 1501.

85. Smithies, O. (2001 ). "Forty years with homologous recombination." Nat Med 7(10):

1083-6.

Claims

1. A method to generate clean and/or safe and/or secure induced pluripotent stem cells ("iPS") and their derivatives at various differentiation stages by using at least one endonuclease.

2. A method to generate clean and/or safe and/or secure iPS according to claim 1 wherein said at least one endonuclease induces a double-strand break in a TERT gene.

3. A method to generate clean and/or safe and/or secure iPS according to claim 1 using two endonucleases wherein one endonuclease induces a double-strand break in the TERT gene and wherein one endonuclease induces a double-strand break in another gene.

4. A method to generate clean and/or safe and/or secure iPS according to claim 1 wherein said endonuclease is a meganuclease.

5. A method to generate clean and/or safe and/or secure iPS according to claim 1 wherein said endonuclease is derived from a Transcription Activator Like Effector (TALE).

6. A method according to claim 5 wherein said endonuclease is a fusion between a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one or two catalytic domains having endonuclease activity.

7. A meganuclease that induces a double-strand break in a TERT gene and variants.

8. The meganuclease of claim 7, which recognizes the TERT2 sequence (SEQ ID NO:

8).

9. The meganuclease of claim 7, which recognizes the TERT2 sequence (SEQ ID NO:

8) and which comprises the variant I-Crel amino acid sequence of any one of SEQ ID NOS: 25 to 28.

10. The meganuclease of claim 7, which recognizes the TERT5 sequence (SEQ ID NO: 18).

1 1 . The meganuclease of claim 7, which recognizes the TERT5 sequence (SEQ ID

NO: 18) and which comprises the variant I-Crel amino acid sequence of any one of SEQ ID NOS: 29 to 35.

12. The meganuclease variant of claim 7, which is a homodimer.

13. The meganuclease variant of claim 7, which is a heterodimer.

14. The meganuclease variant of claim 7, which is a obligate heterodimer.

15. The meganuclease variant of claim 7, which is a single chain.

16. A pharmaceutical composition comprising the meganuclease of claim 7 and optionally a pharmaceutically acceptable carrier or excipient.

17. A polynucleotide that encodes the meganuclease of claim 7 or that encodes a fragment thereof having meganuclease activity.

18. A pharmaceutical composition comprising the polynucleotide of claim 17.

19. A vector comprising the polynucleotide of claim 17.

20. A host cell containing the vector of claim 19.

21. A method for producing a meganuclease comprising culturing the host cell of claim 20.

22. A method for knocking out the expression of the TERT gene comprising inducing at least a double strand break in a TERT gene using the meganuclease of claim 7.

23. A method for inserting a gene or polynucleotide of interest or part of it into the TERT gene by inducing at least a double strand break in the TERT gene using the meganuclease of claim 7.

24. The method of claim 23, wherein the gene or polynucleotide of interest is a reporter gene or reporter sequence.

25. The method of claim 23, wherein the gene or polynucleotide of interest is a therapeutic transgene or a modulator gene.

26. A method for determining the level of expression of TERT in a cell comprising inserting a reported gene into the TERT gene using the meganuclease of claim 7 in a manner that the reporter gene is coexpressed with the TERT gene or part of it and measuring the level of reporter gene expression.

27. A method for determining the level of expression of TERT in a cancer cell comprising inserting a reported gene into the TERT gene of a cancer cell using the meganuclease of claim 7 in a manner that the reporter gene is coexpressed with the TERT gene and measuring the level of reporter gene expression.

28. A cancer or tumor cell into which a reporter gene has been operatively inserted so as to coexpress with the TERT gene using the meganuclease of claim 7.

29. A method for screening an agent for its ability to modulate TERT activity or cellular phenotype comprising contacting said agent with a cell into which a reporter gene has been operatively inserted so as to coexpress with the TERT gene using the meganuclease of claim 7.

30. A recombinant cell containing the TERT gene or part of it and the nucleotidic gene of interest.

31. A cell bank containing cells in which TERT is knocked-out by an endonuclease.

32. A cell bank containing cells in which TERT is knocked-out by a meganuclease

33. A process to regulate expression of TERT in an eukaryotic cell comprising the introduction in TERT gene of nucleotidic sequences of interest by using the endonuclease of claim 5 further comprising the selection of said eukaryotic cell.

34. A deposit of biological material containing meganuclease variants having the CNCM accession numbers CNCM 1-4346 and CNCM 1-4347.

35. DNA inserts of SEQ ID NO : 26 and SEQ ID NO : 34 which are part of the biological material deposits of claim 34.

36. Purified iPS cells culture wherein said iPS cells are clean and/or safe and/or secure.

37. Purified iPS cells culture according to claim 36 wherein the TERT gene of said iPS cells is not functional.

38. A purified differentiated cell culture selected from purified iPS cells culture according to claims 36 or 37.

39. A method for eliminating or reducing expression of TERT in a cell comprising genetically engineering a TERT gene of the cell using the meganuclease of claim 7.

40. The method of claim 39 comprising:- eliminating or reducing the expression of TERT in a tumor or cancer cell, or in a embryonic stem (ES) cell or a induced pluripotent cell (iPS), and

- recovering a tumor or cancer cell, or an ES or iPS, having no or reduced expression of TERT.

41. A method of treating a subject having cancer or a tumor comprising administering a tumor or cancer cell obtained by the method of claim 40.

42. A method for treating a subject with an ES or an iPC obtained by the method of claim 38, or with a progenitor cell derived from said ES or iPC.

43. A method for causing or increasing the expression of TERT in a cell comprising genetically engineering, introducing or modifying a TERT gene in the cell using the meganuclease of claim 7.

44. The method of claim 43 comprising causing or increasing the expression of TERT in a cell in need of increased expression of telomerase.

45. The method of claim 44, wherein said cell has a mutation in the telomerase gene associated with a disorder or disease.

46. The method of claim 44, wherein said cell produces a suboptimal amount of TERT, a suboptimal amount of telomerase, or is a senescent cell.

47. A method for treating a subject with a cell obtained by the method of claim 43.

48. A method for detecting a TERT gene or a TERT gene variant comprising cleaving a target DNA sequence suspected of containing a TERT gene with the endonuclease of claim 7.