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US 2012/0244131 A1 Delacote Et Al US 2012O244131A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0244131 A1 Delacote et al. (43) Pub. Date: Sep. 27, 2012 (54) METHOD FORMODULATING THE CI2N IS/II (2006.01) EFFICIENCY OF DOUBLE-STRAND A6II 35/00 (2006.01) BREAK-INDUCED MUTAGENESIS C40B 30/06 (2006.01) CI2N 5/10 (2006.01) (75) Inventors: Fabien Delacote, Paris (FR), (52) U.S. Cl. ....... 424/93.21; 506/10; 435/6.13:435/.440; Philippe Duchateau, Livry Gargan 435/320.1; 435/.414; 435/417; 435/411; 435/412: (FR), Christophe Perez-Michaut, 435/419:435/254.3:435/254.5; 435/254.6; Paris (FR) 435/254.23; 435/254.2:435/254.11: 435/325: 435/348; 435/366; 435/353; 435/354; 435/351; (73) Assignee: CELLECTISSA, Romainville 435/350; 435/349; 536/24.5; 536/23.1: 514/44A; Cedex (FR) 514744 R (21) Appl. No.: 13/367,098 (57) ABSTRACT (22) Filed: Feb. 6, 2012 A method for modulating double-strand break-induced mutagenesis at a genomic locus of interest in a cell, thereby Related U.S. Application Data giving new tools for genome engineering, including thera (60) Provisional application No. 61/439,739, filed on Feb. peutic applications and cell line engineering. A method for 4, 2011. modulating double-strand break-induced mutagenesis, con s cerns the identification of effectors that modulate double O O Strand break-induced mutagenesis by use of interfering Publication Classification agents; these agents are capable of modulating double-strand (51) Int. Cl. break-induced mutagenesis through their respective director A6 IK3I/7088 (2006.01) indirect actions on said effectors. Methods of using these GOIN 2L/76 (2006.01) effectors, interfering agents and derivatives, respectively, by CI2N 15/63 (2006.01) introducing them into a cell in order to modulate and more CI2N 15/79 (2006.01) particularly to increase double-strand break-induced A6IP35/00 (2006.01) mutagenesis. Specific derivatives of identified effectors and CI2N I/19 (2006.01) interfering agents, vectors encoding them, compositions and CI2N L/15 (2006.01) kits comprising Such derivatives for modulating or increasing C7H 2L/02 (2006.01) double-strand break-induced mutagenesis. 38;& 8::cis bikiig. 8:8 3:338c:38 $38.8, 8:8s 3:38:888:g :38: 88s $38888 Patent Application Publication Sep. 27, 2012 Sheet 1 of 12 US 2012/0244131 A1 Figure if ercis binding and protection xix. 8 is processing if &rcis ligation K. ix.88 eterocities xx-xes 88.8 yatex xxcrax 8:8xx {x}rgix Patent Application Publication Sep. 27, 2012 Sheet 2 of 12 US 2012/0244131 A1 igure 8& ::$8883: : -8:::::::::::::::::: 8xxx;8:38 : {yeire serine stretc: -8 Act . x 8.8 xxxix.88 Kia exiors . x: i: 88888 - 88. xx 3' xxxixty & ... :8: ::::: :::::: :::::::::::::::::: 888; 8.x: x: 8x8:8 pie ori 88.88: 888:88: 88: 88x8: Patent Application Publication Sep. 27, 2012 Sheet 3 of 12 US 2012/0244131 A1 rigue 3 Patent Application Publication Sep. 27, 2012 Sheet 4 of 12 US 2012/0244131 A1 88: Patent Application Publication Sep. 27, 2012 Sheet 5 of 12 US 2012/0244131 A1 Rix * : {xx $38: 88: 3:38: Patent Application Publication Sep. 27, 2012 Sheet 6 of 12 US 2012/0244131 A1 Figure 8 38 i.888: 8 -&xi ix3.88883: 8xxx:ix: 8:8 {{yxix. $xists 8:8 2 8: 888x388 : 8: 88.tiers . 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Kak sv early promoter Figure 9 cx expire :::::::::::::: c promoter : xxxx k: xii. xii: Patent Application Publication Sep. 27, 2012 Sheet 9 of 12 US 2012/0244131 A1 08: 8888 Sv4.0 polyA 8x8 xxx; Figure 1 Patent Application Publication Sep. 27, 2012 Sheet 10 of 12 US 2012/0244131 A1 ::::::::::: 888 Patent Application Publication Sep. 27, 2012 Sheet 11 of 12 US 2012/0244131 A1 8388x cow are prise 8. 8: 88: ::::::888 : :::::::::::: K 88 rex sex 8x8 as Kg8. 8:8: is 88: 8v8 eary 8x Figure is a8: 88: a prox 88:8; 8x 8x888 88: 888y 8888 Patent Application Publication Sep. 27, 2012 Sheet 12 of 12 US 2012/0244131 A1 8 8 - : 8. & &: 38 38 38 38 & *igure 8 :::::::::::::: 888 $888 8 : 88::::::::8x88 US 2012/0244131 A1 Sep. 27, 2012 METHOD FOR MODULATING THE Terada, Urawa et al. 2002; Endo, Osakabe et al. 2006; Endo, EFFICIENCY OF DOUBLE-STRAND Osakabe et al. 2007). Typically, GT events occur in a fairly BREAK-INDUCED MUTAGENESIS small proportion of treated mammalian while GT efficiency is extremely low in higher plant cells and range between 0.01 CROSS-REFERENCE TO RELATED 0.1% of the total number of random integration events APPLICATION (Terada, Johzuka-Hisatomietal. 2007). The low GT frequen 0001. This application claims priority under 35 U.S.C. cies reported in various organisms are thought to result from S119(e) to U.S. Provisional Application No. U.S. 61/439,739, competition between HR and non homologous end joining filed Feb. 4, 2011, which is hereby incorporated by reference (NHEJ) for repair of dsDNA breaks (DSBs). As a conse in its entirety. quence, the ends of a donor molecule are likely to be joined by NHEJ rather than participating in HR, thus reducing GT FIELD OF THE INVENTION frequency. There is extensive data indicating that DSBs repair by NHEJ is error-prone. Often, DSBs are repaired by end 0002 The present invention relates to a method for modu joining processes that generate insertions and/or deletions lating double-strand break-induced mutagenesis at a genomic (Britt 1999). Thus, these NHEJ-based strategies might be locus of interestina cell, thereby giving new tools for genome more effective than HR-based strategies for targeted engineering, including therapeutic applications and cell line mutagenesis into cells. Indeed, expression of I-Sce I, a rare engineering. More specifically, the method of the present cutting restriction enzyme, has been shown to introduce invention for modulating double-strand break-induced mutations at I-Sce I cleavage sites in Arabidopsis and tobacco mutagenesis (DSB-induced mutagenesis), concerns the iden (Kirik, Salomon et al. 2000). Nevertheless, the use of restric tification of effectors that modulate said DSB-induced tion enzymes is limited to rarely occurring natural recogni mutagenesis by uses of interfering agents; these agents are tion sites or to artificial target sites. To overcome this prob capable of modulating DSB-induced mutagenesis through lem, meganucleases with engineered specificity towards a their respective direct or indirect actions on said effectors. chosen sequence have been developed. Meganucleases show The present invention also concerns the uses of these effec high specificity to their DNA target, these proteins being able tors, interfering agents and derivatives, respectively, by intro to cleave a unique chromosomal sequence and therefore do ducing them into a cell in order to modulate and more par not affect global genome integrity. Natural meganucleases are ticularly to increase DSB-induced mutagenesis. The present essentially represented by homing endonucleases, a wide invention also relates to specific derivatives of identified spread class of proteins found in eukaryotes, bacteria and effectors and interfering agents, vectors encoding them, com archae (Chevalier and Stoddard 2001). Early studies of the positions and kits comprising such derivatives in order to I-Sce I and HO homing endonucleases have illustrated how modulate and more particularly to increase DSB-induced the cleavage activity of these proteins can be used to initiate mutagenesis. HR events in living cells and have demonstrated the recombi nogenie properties of chromosomal DSBs (Dujon, Colleaux BACKGROUND OF THE INVENTION et al. 1986; Haber 1995). Since then, meganuclease-induced 0003 Mutagenesis is induced by physical and chemical HR has been successfully used for genome engineering pur means provoking DNA damages when incorrectly repaired poses in bacteria (Posfai, Kolisnychenko et al. 1.999), mam leading to mutations. Several chemicals are known to cause malian cells (Sargent, Brenneman et al. 1997: Cohen-Tan DNA lesions and are routinely used. Radiomimetic agents noudji, Robine et al. 1998; Donoho, Jasin et al. 1998), mice work through free radical attack on the Sugar moieties of (Cbuble, Smith et al. 2006) and plants (Puchta, Dujon et al. DNA (Povirk 1996). A second group of drugs inducing DNA 1996; Siebert and Puchta 2002). Meganucleases have damage includes inhibitors of topoisomerase I (Topol) and II emerged as scaffolds of choice for deriving genome engineer (Topol I) (Teieher 2008) (Burden and N. 1998). Other classes ing tools cutting a desired target sequence (Paques and of chemicals bind covalently to the DNA and form bulky Duchateau 2007). adducts that are repaired by the nucleotide excision repair 0005 Combinatorial assembly processes allowing to (NER) system (Nouspikel 2009). Chemicals inducing DNA engineer meganucleases with modified specificities has been damage have a diverse range of applications and are widely described by Arnould et al. (Arnould, Chames et al. 2006; used. However, although certain agents are more commonly Smith, Grizot et al. 2006; Arnould, Perez et al. 2007: Grizot, applied in studying a particular repair pathway (e.g. cross Smith et al. 2009). Briefly, these processes rely on the iden linking agents are favored for NER studies), most drugs tifications of locally engineered variants with a substrate simultaneously provoke a variety of lesions (Nagy and Sou specificity that differs from the substrate specificity of the toglou 2009).
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