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WO 2012/138901 Al 11 October 2012 (11.10.2012) P O P C T

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WO 2012/138901 Al 11 October 2012 (11.10.2012) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/138901 Al 11 October 2012 (11.10.2012) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 9/22 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/US2012/032386 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (22) International Filing Date: HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, 5 April 2012 (05.04.2012) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/472,053 5 April 201 1 (05.04.201 1) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant (for all designated States except US): CEL- GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, LECTIS SA [FR/FR]; 8 Rue De La Croix Jarry, F-75013 UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, Paris (FR). TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (75) Inventors/Applicants (for US only): SILVA, George, H. SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, [US/FR]; 68 Avenue Jean Kiffer, F-94420 Le Plessis Tre- GW, ML, MR, NE, SN, TD, TG). vise (FR). BERTONATI, Claudia [ΓΓ/FR]; 18 Rue Des Gravilliers, F-75003 Paris (FR). Published: (74) Agents: SHIER, Vincent, K. et al; Obion, Spivak, M c — with international search report (Art. 21(3)) Clelland, Maier & Neustadt, L.L.P., 1940 Duke Street, A l — before the expiration of the time limit for amending the exandria, VA 223 14 (US). claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) [Continued on next page] (54) Title: METHOD FOR ENHANCING RARE-CUTTING ENDONUCLEASE EFFICIENCY AND USES THEREOF (57) Abstract: The present invention relates to a meth od for enhancing the efficiency of rare-cutting endonu- cieases such as meganuc leases. More specifically, the present invention concerns a method for the creation of fusion proteins that consist of one or more "enhan cer" domains fused to at least one rare-cutting endo- nuclease-derived scaffold in a single polypeptide chain for simple and efficient vectorization. The present in vention also relates to engineered chimeric rare-cutting endonucieases, vectors, compositions and kits used to implement the method and use of said chimeric rare- cutting endonucieases according to the invention for various applications ranging from homologous gene targeting to targeted mutagenesis and sequence remov Removal insertion Correction Inactivation al. or replacement J L J T Homologous Recombination (HR) Non-Homologous End Joining (NHEJ) Figure 1 w o 2012/138901 Al III 11 II II 11 III lllll I I III I III III il II I II — with sequence listing part of description (Rule 5.2(a)) METHOD FOR ENHANCING RARE-CUTTING ENDONUCLEASE EFFICIENCY AND USES THEREOF The present application claims priority t o U.S. Serial number 61/472,053 filed April 5, 2011, the entire contents of which are incorporated herein by reference. Field of the invention The present invention relates t o a method for enhancing the efficiency of rare-cutting endonucleases such as meganucleases. More specifically, the present invention concerns a method for the creation of fusion proteins that consist of one o r more "enhancer" domains fused t o at least one rare-cutting endonuclease-derived scaffold in a single polypeptide chain for simple and efficient vectorization. The present invention also relates t o engineered chimeric rare-cutting endonucleases, vectors, compositions and kits used t o implement the method and use of said chimeric rare-cutting endonucleases according t o the invention for various applications ranging from homologous gene targeting to targeted mutagenesis and sequence removal. Background of the invention Mammalian genomes constantly suffer from various types of damage, of which double-strand breaks (DSBs) are considered the most dangerous (Haber 2000). Repair of DSBs can occur through diverse mechanisms that can depend o n cellular context. Repair via homologous recombination (HR) is able t o restore the original sequence at the break. Because of its strict dependence o n extensive sequence homology, this mechanism is suggested t o be active mainly during the S and G2 phases of the cell cycle where the sister chromatids are in close proximity (Sonoda, Hochegger et al. 2006). Single-strand annealing (SSA) is another homology-dependent process that can repair DSBs between direct repeats and thereby promotes deletions (Paques and Haber 1999). Finally, non-homologous end joining (NHEJ) of D I A is a major pathway for the repair of DSBs that can function throughout the cell cycle and does not depend o n homologous recombination (Moore and Haber 1996; Haber 2008). NHEJ seems to comprise at least two different components: (i) a pathway that consists mostly in the direct re-joining of DSB ends, and which depends on the XRCC4, Lig4 and Ku proteins, and; (ii) an alternative NHEJ pathway, which does not depend on XRCC4, Lig4 and Ku, and is especially error- prone, resulting mostly in deletions, with the junctions occurring between micro-homologies (Frank, Sekiguchi et al. 1998; Gao, Sun et al. 1998; Guirouilh-Barbat, Huck et al. 2004; Guirouilh-Barbat, Rass et al. 2007; Haber 2008; McVey and Lee 2008). Homologous gene targeting (HGT), first described over 25 years ago (Hinnen, Hicks et al. 1978; Orr- Weaver, Szostak et al. 1981; Orr-Weaver, Szostak et al. 1983; Rothstein 1983), was one of the first methods for rational genome engineering and remains t o this day a standard for the generation of engineered cells or knock-out mice (Capecchi 2001). An inherently low efficiency has nevertheless prevented it from being used as a routine protocol in most cell types and organisms. To address these issues, an extensive assortment of rational approaches has been proposed with the intent of achieving greater than 1% targeted modifications. Many groups have focused on enhancing the efficacy of HGT, with two major disciplines having become apparent: (i) so-called "matrix optimization" methods, essentially consisting of modifying the targeting vector structure t o achieve maximal efficacy, and; (ii) methods involving additional effectors t o stimulate HR, generally sequence-specific endonucleases. The field of matrix optimization has covered a wide range of techniques, with varying degrees of success (Russell and Hirata 1998; Inoue, Dong et al. 2001; Hirata, Chamberlain et al. 2002; Taubes 2002; Gruenert, Bruscia et al. 2003; Sangiuolo, Scaldaferri et al. 2008; Bedayat, Abdolmohamadi et al. 2010). Stimulation of HR via nucleases, on the other hand, has repeatedly proven efficient (Paques and Duchateau 2007; Carroll 2008). For DSBs induced by biological reagents, e.g. meganucleases, ZFNs and TALENs (see below), which cleave DNA by hydrolysis of two phosphodiester bonds, the DNA can be rejoined in a seamless manner by simple re-ligationof the cohesive ends. Alternatively, deleterious insertions or deletions (indels) of various sizes can occur at the breaks, eventually resulting in gene inactivation (Liang, Han et al. 1998; Lloyd, Plaisier et al. 2005; Doyon, McCammon et al. 2008; Perez, Wang et al. 2008; Santiago, Chan et al. 2008; Kim, Lee et al. 2009; Yang, Djukanovic et al. 2009). The nature of this process, which does not rely on site-specific or homologous recombination, gives rise t o a third t argeted approach based on endonuclease-induced mutagenesis. This approach, as well as the related applications, may be simpler than those based on homologous recombination in that (a) one does not need t o introduce a repair matrix, and; (b) efficacy will be less cell-type dependant (in contrast t o HR, NHEJ is probably active throughout the cell cycle (Delacote and Lopez 2008). Targeted mutagenesis based on NEHJ has been used t o trigger inactivation of single or even multiple genes in immortalized cell lines (Cost, Freyvert et al. 2010; Liu, Chan et al. 2010). In addition, this method opens new perspectives for organisms in which the classical HR-based gene knock-out methods have proven inefficient, or at least difficult t o establish (Doyon, McCammon et al. 2008; Geurts, Cost et al. 2009; Shukla, Doyon et al. 2009; Yang, Djukanovic et al. 2009; Gao, Smith et al. 2010; Mashimo, Takizawa et al. 2010; Menoret, Iscache et al. 2010). Over the last 15 years, the use of meganucleases t o successfully induce gene targeting has been well documented, starting from straightforward experiments involving wild-type l-Scel t o more refined work involving completely re-engineered enzymes (Stoddard, Scharenberg et al. 2007; Galetto, Duchateau et al. 2009; Marcaida, Munoz et al. 2010; Arnould, Delenda et al. 2011). Meganucleases, also called homing endonucleases (HEs), can be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box and PD-(D/E)XK (Stoddard 2005; Zhao, Bonocora et al.
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