(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 2019/020811 Al 31 January 2019 (31.01.2019) W !P O PCT (51) International Patent Classification: C12N 9/10 (2006.01) C12N 9/14 (2006.01) C12N 9/ 2 (2006.01) (21) International Application Number: PCT/EP20 18/070479 (22) International Filing Date: 27 July 2018 (27.07.2018) (25) Filing Language: English (26) Publication Langi English (30) Priority Data: 17306006.2 27 July 2017 (27.07.2017) EP (71) Applicant: EUKARYS [FR/FR]; 4 rue Pierre Fontaine, 91058 Evry (FR). (72) Inventor: JAIS, Philippe; 58 avenue du Bas-Meudon, 92130 Issy-Les-Moulineaux (FR). (74) Agent: BRINGER IP; 1 Place du President Thomas Wil son, 31000 Toulouse (FR). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 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, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) o0 0 © (54) Title: NEW CHIMERIC ENZYMES AND THEIR APPLICATIONS © (57) Abstract: The present invention relates to a chimeric enzyme comprising or consisting of at least one catalytic domain of a capping enzyme and at least one RNA-binding domain of a protein-RNA tethering system as well as its application for the production of an RNA molecule with a 5'-terminal cap. NEW CHIMERIC ENZYMES AND THEIR APPLICATIONS The present invention relates to the field of expression systems, particularly in eukaryotic cells. In particular, the invention relates to a chimeric enzyme useful for the production of RNA molecules with 5'-term inal cap structures and preferably with a 3' poly(A) tail. In the eukaryotes, precursors of messenger RNA (m RNA) , i.e. the pre-m RNAs, are synthesized by the RNA polymerase II and then undergoes multiple post-transcriptional modifications, which are required for their biological activities including translation, stability or imm une response modulation. Two of these modifications are particularly critical for mRNA metabolism and its translation : the addition of a cap at their 5'-end and a polyadenylation tail at their 3'-end. The capping is a specialized structure found at the 5'-end of nearly all eukaryotic messenger RNAs. The simplest cap structure, cap-0, results of the addition of a guanine nucleoside methylated at N7 that is joined by 5'-5' triphosphate bound to the end of primary RNA (i.e. m7GpppN where N is any base, p denotes a phosphate and m a methyl group) . In the so called canonical pathway, the formation of the cap-0 involves a series of three enzymatic reactions: RNA triphosphatase (RTPase) removes the γ phosphate residue of 5' triphosphate end of nascent pre-m RNA to diphosphate, RNA guanylyltransferase (GTase) transfers GMP from GTP to the diphosphate 5' end of nascent RNA term inus, and RNA N7-guanine methyltransferase (N7-MTase) adds a methyl residue on azote 7 of guanine to the GpppN cap (Furuichi and Shatkin 2000) . In higher eukaryotes and some viruses, the 2'-hydroxyl group of the ribose of the first (i.e. cap-1 structures; m7GpppNm 2'- °pN) and second (i.e. cap2 structures; m7GpppNm 2' 0 pNm 2' 0 ) transcribed nucleotides can be methylated by two separate ribose-2'-0 MTases, respectively named capl - and cap2-specific MTases (Langberg and Moss 1981 ). However, In contrast to the cellular N7-MTase activity that is exclusively nuclear, cap-1 ribose-2'-0 MTase activity has been detected in both the cytoplasm and nucleus of HeLa cells, whereas cap2 MTase activity is exclusively found in their cytoplasm (Langberg and Moss 1981 ). The formation of the 5'-term inal m7GpppN cap is the first step of pre-m RNA processing. The m7GpppN cap plays important roles in mRNA stability and its transport from the nucleus to the cytoplasm (Huang and Steitz 2005, Kohler and Hurt 2007) . In addition, the 5'-term inal m7GpppN cap is important for the translation of mRNA to protein by anchoring the eukaryotic translation initiation factor 4F (el F4F) complex, which mediates the recruitment of the 16S portion of the small ribosomal subunit to mRNA (Furuichi, LaFiandra et al. 1977, Gingras, Raught et al. 1999, Rhoads 1999) . The 5'-terminal m7GpppN cap therefore enhances drastically the translation of mRNA both in vitro (Lo, Huang et al. 1998) , and in cellulo (Malone, Feigner et al. 1989, Gallie 1991 , Lo, Huang et al. 1998, Kozak 2005) .The cap-0, cap-1 and cap-2 modifications participate in the innate immune response, by distinguishing self from non-self RNA through the RNA sensor RIG-1 and MDA5, which in turn induce an interferon type-l response (Hornung, Ellegast et al. 2006, Daffis, Szretter et al. 201 0). Since they are widely used in the life sciences, biotechnology and medicines, many expression systems have been designed to efficiently produce proteins and/or RNAs particularly in eukaryotic cells. The inventor has developed in the past an artificial expression system (i.e. a chimeric enzyme) for efficient transgenesis in eukaryotic cells, which autonomously generates mRNA molecules, in particular in the cytoplasm of said cells (WO 201 1/ 128444) . Using this system , RNA chains are synthesized by RNA polymerase moiety of this chimeric enzyme and are capped at 5'-end by its mRNA capping enzyme moiety. In addition, a poly(A) tail can be produced at the 3'-end of transcripts by transcription of a polyadenosine track from DNA templates. This system has notably the advantage of not using the endogenous RNA transcription machinery of eukaryotic cells, e.g. RNA polymerase II and associated factors involved in transcription and post-transcription. Other attempts to couple capping to transcription and thus to improve the translatability of uncapped transcripts produced by the T7 RNA polymerase by fusing the carboxyl-terminal domain (CTD) of the largest subunit of the RNA polymerase II (POLR2A) , have to enhance the capping of both constitutively and alternatively spliced substrates in cellulo (Kaneko, Chu et al. 2007, Natalizio, Robson-Dixon et al. 2009) . The CTD comprises 25-52 heptapeptide repeats of the consensus sequence YSPTSPS 7, which is highly conserved throughout evolution and subject to reversible phosphorylation during the transcription cycle (Palancade and Bensaude 2003) . When phosphorylated, the CTD is thought to mediate the coupling of transcription and capping of nascent transcripts, by binding one or more subunits of the mRNA capping enzymes in yeast (Cho, Takagi et al. 1997, McCracken, Fong et al. 1997) and mammals (McCracken, Fong et al. 1997, Yue, Maldonado et al. 1997) . Noticeably, RNA polymerase II with Ser -phosphorylated CTD repeats undergoes promoter proximal pausing which is coincident with the co-transcriptional capping of the nascent transcripts (Komarnitsky, Cho et al. 2000, Schroeder, Schwer et al. 2000) . However, in contrast to what could be expected intuitively, the fusion of the CTD to the single-unit T7 RNA polymerase is not sufficient to enhance the capping of both constitutively and alternatively spliced substrates in vivo (Kaneko, Chu et al. 2007, Natalizio, Robson-Dixon et al. 2009) . There remains therefore a significant need in the art for new and improved expression systems, in particular in eukaryotic cells, which are appropriate for gene therapy and large-scale protein production without cytotoxicity or induced-cytotoxicity. The present inventor has made a significant step forward with the invention disclosed herein. The purpose of the invention is to fulfill this need by providing new chimeric enzymes, which make it possible to solve in whole or part the problems mentioned-above. Unexpectedly, the inventor has notably demonstrated that monomeric or oligomeric chimeric (non- natural) enzymes comprising catalytic domains of a capping enzyme, particularly a catalytic domain of a RNA triphosphatase, a catalytic domain of a guanylyltransferase, a catalytic domain of a N7-guanine methyltransferase, and a RNA-binding domain of a protein-RNA tethering system , said RNA-binding domain binding specifically to a RNA element consisting of a specific RNA sequence and/or structure, allows to highly increase the capping rate of specific mRNAs produced by a RNA polymerase. These results are surprising since the formation of the cap is a complex process and that the capping of exogenous transcripts cannot be achieved by most other approaches, such as the fusion enzyme CTD-T7 RNA polymerase (Kaneko, Chu et al. 2007, Natalizio, Robson-Dixon et al.