Functionalized circular RNA and methods and intermediates for their production

Abstract

The present invention relates to circular RNA molecules, methods of producing circular RNA molecules, circular and non-circular intermediates thereof, and methods of treating or preventing a disease. In particular, the present invention provides circRNAs that can be prepared via chemical circularization of RNA, regardless of the circRNA sequence, length or size. Due to chemical nature of the circularization process, it allows the attachment of tags, markers, etc. after the circularization at a position in close proximity to the position where the circRNA was circularized.

Description

[0001] New PCT-Patent Application

[0002] (based on Priority Application: EP 24461634.8)

[0003] Siec Badawcza Lukasiewicz -PORT Polski Osrodek Rozwoju Technologii

[0004] Vossius Ref.: AJ3700 PCT S3

[0005] Functionalized circular RNA and methods and intermediates for their production

[0006] Field of the invention

[0007] The present invention relates to circular RNA molecules, methods of producing circular RNA molecules, circular and non-circular intermediates thereof, and methods of treating or preventing a disease.

[0008] Background of the invention

[0009] Despite its inherent fragility, RNA is one of the most potent substances in the context of therapeutic gene delivery (NPTL 1). The potency of therapeutic messenger RNA (mRNA) stems from both advanced methods of delivery and chemical modification of the mRNA itself. Both in serum and in the cytosol, mRNA is primarily targeted by exonucleases (e.g. decapping complexes, deadenylases, exosomes) (NPTL 2). Circularization is one of the most protective modifications of mRNA, as it prevents exonucleolytic digestion (NPTL 3). Currently known methods of RNA circularization are based on chemical reactions (NPTL 4 and 5), enzymatic reactions, (NPTL 6 to 9) and reactions catalyzed by catalytic sequences embedded in circRNA precursors (NPTL 10 to 13) (pre-circRNA).

[0010] The reported methods of RNA circularization that are based on chemical reactions rely on the use of short RNA sequences, chemically synthesized via phosphoramidite method, such as 3' phosphorylated RNA or 3'-amino RNA, which undergo condensation induced by cyanogen bromide (BrCN) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (NPTL 4 and 5). The phosphoramidite solid phase synthesis of nucleic acids is a robust method of production of modified and unmodified oligonucleotides, however it is not practical for the production of long (> 100 nt) RNA sequences. In consequence, circRNA obtained with these methods encode relatively short sequences and has limited use in the context of RNAs encoding a protein or peptide, which are usually significantly longer.

[0011] Moreover, RNA circularization can be carried out with ligases, especially T4 RNA ligase I and T4 RNA ligase II (NPTL6, 7 and 14). Both enzymes require 5' monophosphorylated precursors, which are first adenylated and subsequently ligated with the 3' OH of the ribose at the 3' end of a different or the same RNA strand (NPTL 15). Both enzymes are capable of intramolecular ligation (circularization) of RNA (if reaction occurs with the 3' end of the same RNA strand); however, they have been found to be strongly dependent on target RNA length and sequence (NPTL 11, 12, 14, 16) This is detrimental, as it difficult to predict or improve performance of such enzymes.

[0012] The most recognized methods of RNA circularization involve catalytic nucleic acids (NPTL 8 to 12, 17) Wesselhoeft et al. have circularized RNA in vitro by using the permuted intron-exon (PIE) system and optimized self-splicing intronic sequences (NPTL 11, 12). The PIE system consists of fused partial exons flanked by half-intron sequences (NPTL 10, 17) During the selfsplicing reaction, two transesterification reactions occur. First, 3' hydroxyl group of a guanosine nucleotide engages 5' splice site. Second, the freed hydroxyl group at the end of the intermediate engages the 3' splice site, which results in circularization. Litke et al. have shown the Twister-optimized RNA durable overexpression (Tornado) system for RNA circularization (NPTL8) Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes that spontaneously undergo autocatalytic cleavage. This leaves 5'-hydroxyl and 2',3'-cyclic phosphate termini, which are then ligated by the RNA ligase RtcB.

[0013] The so-far-reported methods based on catalytic nucleic acid activity require sequence design and development, therefore cannot be used on pre-circRNA of any given sequence. Furthermore, pre-circRNA's ability to undergo circularization can be hindered by the presence of chemical modifications, such as natural and synthetic nucleosides and nucleobases (NPTL 12).

[0014] One of the biggest flaws of circRNA in the context of gene delivery and gene therapies is low protein expression. The main pipelines of eukaryotic translational machinery are based on cap-dependent mechanisms (NPTL 18, 19). Discovered or synthesized circular RNA (circRNA) generally does not contain elements such as 7-methylguanosine cap, or any kind of 5' nucleoside analog (NPTL 20, 21). Due to circular and "cap-less" structure, the translation of most of currently known circRNA occurs in an IRES (internal ribosome entry site)-dependent manner (NPTL 5, 7, 9, 11, 12, 19, 22, 23). In eukaryotic cells, cap-dependent translation is much more effective than IRES-dependent translation (NPTL 19) On the other hand, the stability of circRNA is higher, in consequence, circRNAs exhibit significantly longer half-lives and the protein expression from these mRNAs lasts longer (NPTL 12, 14).

[0015] NPTL34 reports on a LEGO method to generate capped circular mRNAs using an enzymatic approach. However, the scope of modifications possible to incorporate using this method is likely limited by the compatibility of the substrate structure with enzyme's scificity, so the method cannot be envisaged as a universal and straightforward tool to modify circRNAs.

[0016] PTL1 discloses circular RNAs and transfer vehicles, along with related compositions and methods of treatment. Pharmaceutical compositions comprising such circular RNAs as well as precursor RNAs and materials useful in producing the precursor or circular RNAs have been also disclosed in cited document. PTL2 describes a vector for making circular RNA, allowing the production of a circular RNA that is translatable or biologically active inside eukaryotic cells. PTL3 discloses a recombinant nucleic acid molecule of the transcriptional circular RNA, recombinant expression vector, pre-circularized RNA, circular RNA, recombinant host cell, pharmaceutical composition and protein preparing method. The transcription product of the recombinant nucleic acid molecule is a circular RNA which contains a specific IRES element. PTL4 discloses cyclic RNA and a method for production thereof which comprises the use of T4 DNA ligase.

[0017] Background Literature

[0018] Non-Patent Literature NPTL1: Qin, S.; Tang, X.; Chen, Y.; Chen, K.; Fan, N.; Xiao, W.; Zheng, Q.; Li, G.; Teng, Y.; Wu, M.; et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduction and Targeted Therapy 2022, 7 (1). DOI: 10.1038 / s41392-022-01007-w. Janowski, M.; Andrzejewska, A. The legacy of mRNA engineering: A lineup of pioneers for the Nobel Prize. Molecular Therapy - Nucleic Acids 2022, 29, 272-284. DOI: 10.1016 / j.omtn.2022.07.003 NPTL2: Garneau, N. L.; Wilusz, J.; Wilusz, C. J. The highways and byways of mRNA decay. Nature Reviews Molecular Cell Biology 2007, 8 (2), 113-126. DOI: 10.1038 / nrm2104. Houseley, J.; Tollervey, D. The Many Pathways of RNA Degradation. Cell 2009, 136 (4), 763-776. DOI: 10.1016 / j.cell.2009.01.019

[0019] NPTL3: Jeck, W. R.; Sharpless, N. E. Detecting and characterizing circular RNAs. Nature Biotechnology 2014, 32 (5), 453-461. DOI: 10.1038 / nbt.2890

[0020] NPTL4: Dolinnaya, N. G.; Blumenfeld, M.; Merenkova, I. N.; Oretskaya, T. S.; Krynetskaya, N.; Ivanovskaya, M. G.; Vasseur, M.; Shabarova, Z. A. Oligonucleotide circularization by template-directed chemical ligation. Nucleic Acids Research 1993, 21 (23), 5403-5407. DOI: 10.1093 / nar / 21.23.5403 (acccessed 10 / 7 / 2022). Fedorova, O. A.; Gottikh, M. B.; Oretskaya, T. S.; Shabarova, Z. A. Cyanogen Bromide-Induced Chemical Ligation: Mechanism and Optimization of the Reaction Conditions. Nucleosides and Nucleotides 1996, 15 (6), 1137-1147. DOI: 10.1080 / 07328319608007382.

[0021] NPTL5: Nakamoto, K.; Abe, N.; Tsuji, G.; Kimura, Y.; Tomoike, F.; Shimizu, Y.; Abe, H. Chemically synthesized circular RNAs with phosphoramidate linkages enable rolling circle translation. Chemical Communications 2020, 56 (46), 6217-6220, DOI: 10.1039 / D0CC02140G.

[0022] NPTL6: Rigden, J. E.; Rezaian, M. A. In Vitro synthesis of an infectious viroid: Analysis of the infectivity of monomeric linear CEV. Virology 1992, 186 (1), 201-206. DOI: https: / / doi.org / 10.1016 / 0042-6822(92)90074-Y.

[0023] NPTL7: Chen, C.-y.; Sarnow, P. Initiation of Protein Synthesis by the Eukaryotic Translational Apparatus on Circular RNAs. Science 1995, 268 (5209), 415-417. DOI: 10.1126 / science.7536344

[0024] NPTL8: Litke, J. L.; Jaffrey, S. R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature Biotechnology 2019, 37 (6), 667-675. DOI: 10.1038 / S41587-019-0090-6

[0025] NPTL9: Kumar, A.; Palmer, N.; Miyasaki, K.; Finburgh, E.; Xiang, Y.; Portell, A.; Dailamy, A.; Suhardjo, A.; Chew, W. L.; Kwon, E. J.; et al. Extensive in vitro and in vivo protein translation via in situ circularized RNAs. Cold Spring Harbor Laboratory: 2022.

[0026] NPTL10) Ford, E.; Ares, M. Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4. Proceedings of the National Academy of Sciences 1994, 91 (8), 3117-3121. DOI: 10.1073 / pnas.91.8.3117 NPTL11: Wesselhoeft, R. A.; Kowalski, P. S.; Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nature Communications 2018, 9 (1). DOI: 10.1038 / s41467-018-05096-6

[0027] NPTL12: Wesselhoeft, R. A.; Kowalski, P. S.; Parker-Hale, F. C.; Huang, Y.; Bisaria, N.; Anderson, D. G. RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo. Molecular Cell 2019, 74 (3), 508-520.e504. DOI: 10.1016 / j. molcel.2019.02.015 NPTL13: Ni, L.; Yamada, T.; Murata, A.; Nakatani, K. Mismatch binding ligand upregulated back-splicing reaction producing circular RNA in a cellular model. Chemical Communications 2022, 58 (22), 3629-3632. DOI: 10.1039 / dlcc06936e

[0028] NPTL14: Qu, L.; Yi, Z.; Shen, Y.; Lin, L.; Chen, F.; Xu, Y.; Wu, Z.; Tang, H.; Zhang, X.; Tian, F.; et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell 2022, 185 (10), 1728-1744.el716. DOI: 10.1016 / j.cell.2022.03.044

[0029] NPTL15: Nichols, N. M.; Tabor, S.; McReynolds, L. A. RNA Ligases. Current Protocols in Molecular Biology 2008, 84 (1), 3.15.11-13.15.14, DOI: https: / / doi.org / 10.1002 / 0471142727.mb0315s84

[0030] NPTL16: Kaufmann, G.; Klein, T.; Littauer, U. Z. T4 RNA ligase: Substrate chain length requirements. FEBS Letters 1974, 46 (1-2), 271-275. DOI: 10.1016 / 0014-5793(74)80385-6 NPTL17: Puttaraju, M.; Been, M. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Research 1992, 20 (20), 5357-5364. DOI: 10.1093 / nar / 20.20.5357

[0031] NPTL18: Ramanathan, A.; Robb, G. B.; Chan, S.-H. mRNA capping: biological functions and applications. Nucleic Acids Research 2016, 44 (16), 7511-7526. DOI: 10.1093 / nar / gkw551 (acccessed 10 / 7 / 2022). Topisirovic, I.; Svitkin Yuri, V.; Sonenberg, N.; Shatkin Aaron, J. Cap and cap-binding proteins in the control of gene expression. Wiley Interdisciplinary Reviews: RNA 2010, 2 (2), 277-298. DOI: 10.1002 / wrna.52. Mitchell, S. F.; Walker, S. E.; Algire, M. A.; Park, E.-H.; Hinnebusch, A. G.; Lorsch, J. R. The 5'-7-Methylguanosine Cap on Eukaryotic mRNAs Serves Both to Stimulate Canonical Translation Initiation and to Block an Alternative Pathway. Molecular Cell 2010, 39 (6), 950-962. DOI: 10.1016 / j.molcel.2010.08.021

[0032] NPTL19: Koch, A.; Aguilera, L.; Morisaki, T.; Munsky, B.; Stasevich, T. J. Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells. Nature Structural & Molecular Biology 2020, 27 (12), 1095-1104. DOI: 10.1038 / s41594-020-0504-7. NPTL20: Warminski, M.; Sikorski, P. J.; Kowalska, J.; Jemielity, J. Applications of Phosphate Modification and Labeling to Study (m)RNA Caps. Topics in Current Chemistry 2017, 375 (1), 16. DOI: 10.1007 / s41061-017-0106-y.

[0033] NPTL21: Sikorski, P. J.; Warminski, M.; Kubacka, D.; Ratajczak, T.; Nowis, D.; Kowalska, J.; Jemielity, J. The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells. Nucleic acids research 2020, 48 (4), 1607-1626.

[0034] NPTL22: Chen, C.-K.; Cheng, R.; Demeter, J.; Chen, J.; Weingarten-Gabbay, S.; Jiang, L.; Snyder, M. P.; Weissman, J. S.; Segal, E.; Jackson, P. K.; et al. Structured elements drive extensive circular RNA translation. Molecular Cell 2021, 81 (20), 4300-4318. e4313. DOI: 10.1016 / j. molcel.2021.07.042. Yang, Y.; Fan, X.; Mao, M.; Song, X.; Wu, P.; Zhang, Y.; Jin, Y.; Yang, Y.; Chen, L.-L.; Wang, Y.; et al. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Research 2017, 27 (5), 626-641. DOI: 10.1038 / cr.2017.31. Fan, X.; Yang, Y.; Chen, C.; Wang, Z. Pervasive translation of circular RNAs driven by short I RES-like elements. Nature Communications 2022, 13 (1). DOI: 10.1038 / s41467-022-31327-y

[0035] NPTL23: Lei, M.; Zheng, G.; Ning, Q.; Zheng, J.; Dong, D. Translation and functional roles of circular RNAs in human cancer. Molecular Cancer 2020, 19 (1). DOI: 10.1186 / sl2943-020-1135-7. Prats, A.-C.; David, F.; Diallo, L. H.; Roussel, E.; Tatin, F.; Garmy-Susini, B.; Lacazette, E. Circular RNA, the Key for Translation. International Journal of Molecular Sciences 2020, 21 (22), 8591. DOI: 10.3390 / ijms21228591

[0036] NPTL24: Martinez-Salas, E.; Francisco-Velilla, R.; Fernandez-Chamorro, J.; Embarek, A. M. Insights into Structural and Mechanistic Features of Viral IRES Elements. Frontiers in Microbiology 2018, 8, Review. Marques, R.; Lacerda, R.; Romao, L. Internal Ribosome Entry Site (IRES)-Mediated Translation and Its Potential for Novel mRNA-Based Therapy Development. Biomedicines 2022, 10 (8), 1865. DOI: 10.3390 / biomedicinesl0081865. Chen, R.; Wang, S. K.; Belk, J. A.; Amaya, L.; Li, Z.; Cardenas, A.; Abe, B. T.; Chen, C.-K.; Wender, P. A.; Chang, H. Y. Engineering circular RNA for enhanced protein production. Nature Biotechnology 2022. DOI: 10.1038 / s41587-022-01393-0.

[0037] NPTL25: Nowotny, M.; Gaidamakov, S. A.; Ghirlando, R.; Cerritelli, S. M.; Crouch, R. J.; Yang, W. Structure of human RNase Hl complexed with an RNA / DNA hybrid: insight into HIV reverse transcription. Molecular cell 2007, 28 (2), 264-276.

[0038] NPTL26: Jacques, J.-P.; Susskind, M. M. Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA. Nucleic Acids Research 1991, 19 (11), 2971-2977. DOI: 10.1093 / nar / 19.11.2971. Cebrian, J.; Kadomatsu-Hermosa, M. J.; Castan, A.; Martinez, V.; Parra, C.; Fernandez-Nestosa, M. J.; Schaerer, C.; Martinez-Robles, M.-L.; Hernandez, P.; Krimer, D. B.; et al. Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules. Nucleic Acids Research 2015, 43 (4), e24-e24. DOI: 10.1093 / nar / gkul255.

[0039] NPTL27: Abe, B. T.; Wesselhoeft, R. A.; Chen, R.; Anderson, D. G.; Chang, H. Y. Circular RNA migration in agarose gel electrophoresis. Molecular Cell 2022, 82 (9), 1768-1777. el763. DOI: 10.1016 / j. molcel.2022.03.008. NPTL28: Haque, F. M.; Grayson, S. M. The synthesis, properties and potential applications of cyclic polymers. Nature Chemistry 2020, 12 (5), 433-444. DOI: 10.1038 / s41557-020-0440-5 NPTL29: Vicens, Q.; Kieft, J. S. Thoughts on how to think (and talk) about RNA structure. Proceedings of the National Academy of Sciences 2022, 119 (17). DOI: 10.1073 / pnas.2112677119

[0040] NPTL30: Kibbe, W. A. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Research 2007, 35 (suppl_2), W43-W46. DOI: 10.1093 / nar / gkm234

[0041] NPTL31: Mamot, A.; Sikorski, P. J.; Siekierska, A.; de Witte, P.; Kowalska, J.; Jemielity, J.

[0042] Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation. Nucleic Acids Research 2022, 50 (1), e3-e3. DOI: 10.1093 / nar / gkab867

[0043] NPTL32: Warminski, M.; Warminska, Z.; Kowalska, J.; Jemielity, J. mRNA Cap Modification through Carb-amate Chemistry: Synthesis of Amino- and Carboxy-Functionalised Cap Analogues Suitable for Labelling and Bioconjugation. European Journal of Organic Chemistry 2015, 2015 (28), 6153-6169. DOI: 10.1002 / ejoc.201500672.

[0044] NPTL33: Warminski, M.; Trepkowska, E.; Smietanski, M.; Sikorski, P. J.; Baranowski, M. R.; Bednarczyk, M.; Kedzierska, H.; Majewski, B.; Mamot, A.; Papiernik, D.; et al. Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo. Journal of the American Chemical Society 2024, 146 (12), 8149-8163. DOI: 10.1021 / jacs.3cl2629. NPTL34: Chen, H. et al.; Chemical and topological design of multicapped mRNA and capped circular RNA to augment translation, Nature biotechnology 2024, 1546-1696, DOI: 10.1038 / s41587-024-02393-y

[0045] Patent Literature

[0046] PTL1: WO 2021 / 113777

[0047] PTL2: WO 2019 / 236673

[0048] PTL3: EP 4008336

[0049] PTL4: US 2015 / 0079630

[0050] Summary of the invention

[0051] The present invention relates to functionalized circular RNA and methods and intermediates for their production. An object of the disclosed invention are analogs of circular RNA (circRNA) that are obtained by the synthesis of 5'-modified precursors (pre-circRNAs) and their subsequent circularization via a chemical reaction. The circularization occurs due to the presence of two chemically reactive functional groups, one at RNA 5' end and another at the 3' end. The functional group at the 5' end is incorporated co-transcriptionally, whereas the reactive group at the 3' end is incorporated post-transcriptionally. The invention encompasses RNAs of different lengths (from 2 nt to 20000 nt), including messenger RNAs (RNAs encoding a protein). The circRNAs encoding a protein according to this invention undergo translation with an efficiency higher than chemically unmodified circRNAs of the same sequence. One of the embodiments of the invention discloses circRNA analogs that are obtained from pre-circRNAs containing a functional group which can be used for coupling desirable tags or markers, such as nucleotide and oligonucleotide, including mRNA 5' cap analog, nucleic acid, fluorophore, hapten, chelator, (radio)isotopic tag, lipid, amino acid, oligopeptide, protein, saccharide, oligosaccharide, polymer, polymer composite, metal-organic framework, or a solid support. This approach offers high versability regarding the desired functionalization and overcomes many technical problems that have been associated with prior art methods of preparing functionalized circRNA, where the functionalization has generally been via one of the nucleoside residues which had to be introduced before cyclisation.

[0052] In other words, the present invention provides analogs of circRNAs that can be prepared from pre-circRNAs via a chemical reaction (chemical circularization of RNA), regardless of the circRNA sequence, length or size. Due to chemical nature of the circularization process, it allows the attachment of tags, markers, etc. after the circularization at a position in close proximity to the position where the circRNA was circularized.

[0053] In addition, the presence of such tags, markers etc. at the circRNA analog molecule has been found to increase its translational activity, in comparison to chemically unmodified circRNAs of the same sequence. Therefore, the present disclosure gives unprecedented access to circRNAs that undergo translation via a mechanism which is aided by the presence of tags, markers, etc.

[0054] The present disclosure provides a circRNA analog prepared by in vitro transcription reaction followed by a subsequent chemical circularization reaction, wherein said circularization is obtained in the absence of protein enzymes or catalytic nucleic acid sequences. Functional groups present at positions other than the nucleosides, typically in proximity to the site at which chemical circularization occurred (e.g., within about 1 to 25 (preferably 1 to 15) bonds from the morpholine ring formed by the circularization method), then allow the attachment of desired tags, markers, etc.

[0055] Description of the Figures

[0056] The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, embodiments are shown in the drawings, which are presently exemplified. It should be understood, however, that the invention is not limited to the precise arrangement and instrumentalities of the embodiments shown in the drawings.

[0057] Fig. 1 shows the general concept of the preparation of circRNA analogs. A) First, the precursor of circRNA analog (FG-linker-RNA) is synthesized during an in vitro transcription (IVT) reaction. Due to the presence of a transcription initiator (FG-linker-AG), the transcribed RNA contains a reactive functional group (FG) at the 5' end. Next, the 3' end reactive functional group is incorporated into RNA post-synthetically, by either enzymatic reaction or preferably chemical reaction. The 5' and 3' reactive functional groups are reactive towards each other either spontaneously or in the presence of a specific trigger. B) As an example, a precursor of circRNA analog (EDA-linker-RNA) is synthesized during IVT. Due to the presence of a transcription initiator EDA-linker-AG, the transcribed RNA contains an ethylenediamine (EDA) moiety at the 5' end. The precursor is first chemically modified at the 3' end by treatment with periodate. This causes selective oxidation of the 3' ribose, which is prone to nucleophilic attack by the EDA moiety. Finally, the addition of a reductive agent (i.e. NaBH₃CN) leads to an irreversible covalent linkage of RNA's 5' and 3' ends.

[0058] Fig. 2 shows that chemical circularization results in higher yields and product homogeneity than enzymatic circularization. A) The 5'-modified RNA (pre-circRNA2) was used as a precursor for chemical circularization. As a reference, 5'-monophosphorylated RNA of the same sequence (pre-circRNAl) was used as a substrate in enzymatic circularization catalyzed by T4 RNA ligase I. The chemical circularization results in higheryield of the desired circularized product; B) The 5'-modified RNA (pre-circRNA21) was used as a precursor for chemical circularization. As a reference, 5'-monophosphorylated RNA of the same sequence (pre-circRNA20) was used as a substrate in enzymatic circularization catalyzed by T4 RNA ligase I. The chemical circularization results in higher yield of the desired circularized product and notably lower content of side-products (linear dimer, circular dimer). The substrates and products of chemical and enzymatic reactions were analyzed by PAGE (6% AA, AA: MBAA = 19:1, 7 M Urea, lx TBE). The yields of the reactions were determined by densitometry. M refers to linear RNA length marker. HMW refers to linear RNA of length ranging from 1500 nt to 6000 nt.

[0059] Fig. 3 shows chemical circularization of long RNA molecules results in higher yield and less side-products than ribozymatic cricularization (permuted intron-exon (PIE) splicing reaction). The splicing reaction was conducted using a self-splicing RNA precursor (pre-circRNA22), while the recircularization reaction was performed at increasing concentrations of mRNA. The precursor for chemical circularization included 5'-modified long RNAs (pre-circRNA19, pre-circRNAlO, pre-circ RNA18). The substrates and products of chemical and selfsplicing reactions were analyzed by PAGE (6% AA, AA: MBAA = 37.5:0.5, 7 M Urea, lx TBE). The yields of the reaction were determined by densitometry. M refers to linear RNA length marker. HMW refers to linear RNA of length ranging from 1500 nt to 6000 nt.

[0060] Fig.4 shows an RNase R-mediated removal of linear RNA in the presence of circular RNA. Samples of a linear precursor of circRNA analog (pre-circRNA2) and products of chemical circularization (a mixture of pre-circRNA2 and circRNA2) were treated with a processive 3' exonuclease (RNase R) that selectively hydrolyses linear RNA species. After RNase R treatment the samples were analyzed by PAGE (6% AA, AA: MBAA = 38:1, 7 M Urea, lx TBE). M refers to linear RNA length marker. HMW refers to linear RNA of length ranging from 2000 nt to 6000 nt.

[0061] Fig. 5 shows a RNase H digestion assay to confirm the circular topology of chemically circularized RNAs. Samples of a circRNA analog (circRNA2) and its linear precursor (pre-circRNA2) underwent DNA-dependent endonucleolytic cleavage catalyzed by RNase H and subsequent PAGE. In presence of targeting DNA oligonucleotide, the circRNA analog is nicked. As a result, nicked circRNA exhibits electrophoretic mobility of a linear precursor. On the other hand, cleavage of the linear precursor leads to two RNA molecules of lower size. PAGE conditions: 6% AA, AA: MBAA = 38:1, 7 M Urea, 1x TBE. M refers to linear RNA length marker. HMW refers to linear RNA of length ranging from 2000 nt to 6000 nt.

[0062] Fig. 6 shows a polyacrylamide gel electrophoresis of circRNA - influence of gel crosslinking degree. Samples of a RNA length marker, a linear precursor of circRNA analog (pre-circRNA2), and products of chemical circularization (a mixture pre-circRNA2 and circRNA2) were analyzed by PAGE. Two types of 6% polyacrylamide gels were used in parallel: one with higher crosslinking degree (AA: MBAA = 19:1, 7 M Urea, 1x TBE) and one with lower crosslinking degree (AA: MBAA = 38:1, 7 M Urea, 1x TBE). The migration of circRNA is slower than that of linear RNA. Electrophoretic mobility differences are pronounced in gel with a higher crosslinking degree. HMW refers to linear RNA of length ranging from 2000 nt to 6000 nt.

[0063] Fig. 7 shows an optimization of RNA conformation using complementary DNA oligonucleotides. A) Prediction of minimum free energy structure of RNA5 sequence generated with RNAfold web server. B) Prediction of tertiary structure of an RNA sequence comprising flanking regions of RNA5 sequence generated with RNAComposer web server. The predictions indicate, that 3' poly-A sequence may distance 5' and 3' ends and hinder any reaction between them. Fluorescence spectra of Cy5-RNA5-Cy3 probe recorded at 4°C (C) or 20°C (D) before (w / o ON) or after hybridization with of one of the complementary DNA oligonucleotides (ON2-ON5). Emergence of 660 nm emission band indicates the occurrence of the FRET phenomenon facilitated by decreased distance between 5' and 3' ends.

[0064] Fig. 8 shows the influence of complementary DNA oligonucleotides on chemical circularization. Pre-circRNA10 was used as a precursor for chemical circularization. Before the oxidation step, the precursor was annealed with one of the complementary DNA oligonucleotides (ON2-ON5). After the reaction, the samples were analyzed by PAGE (6% AA, AA: MBAA = 38:1, 7 M Urea, 1x TBE). As a reference, samples of precursor before reaction (NR) and products of reaction without complementary DNA (w / o ON) were included. M refers to linear RNA length marker. HMW refers to linear RNA of length ranging from 3000 nt to 6000 nt. The relative amounts of circular product (P), linear dimer (D), and unreacted substrate (S) were determined by densitometry.

[0065] Fig. 9 shows an attempted separation of linear and circular RNA using IE-HPLC (A), SEC-HPLC (B) and RP-HPLC (C). A sample containing a mixture of linear and circular RNA (pre-circRNA2 + circRNA2) was chromatographed using three different modes and columns. As a reference, linear RNA (pre-circRNA2) was resolved in same conditions. IE-HPLC conditions: CIMac PrimaS™ 0.1 mL Analytical Column (2 μm); A - 50 mM HEPES pH 7.0; B - 200 mM sodium pyrophosphate, 50 mM HEPES pH 8.5; 0% B for 2 min, 0-100% B in 6 min, 100% B for 2 min, 100-0% B in 2 min, 0% B for 1 min; flow 1.0 ml / min at 25°C. SEC-HPLC conditions: Yarra 3 μm SEC-4000 300x7.8 mm; A - 100 mM Na2HPO4, 0.025% NaN3pH 6.8; 100% A for 50 min; flow 1.2 ml / min at 25°C; RP-HPLC conditions: RNASept Prep C18 50x7.8 mm 2 μm; A - 100 mM HAA, 10% MeCN, pH 7.0; B - 100 mM HAA, 75% MeCN, pH 7.0; 20-70% B in 25 min; flow: 1.0 ml / min at 60 °C.

[0066] Fig. 10 shows the purification of circRNA using reversed-phase high-pressure liquid chromatography. Products of chemical circularization of pre-circRNA4 were chromatographed under denaturing conditions (60°C, hexylammonium acetate buffer and acetonitrile mixture as a mobile phase). The UV-absorbing eluate was collected and separated into fractions that corresponded to different peaks of absorbance. Next, the fractions were concentrated and analyzed by PAGE, along with EP5-GLuc before and after circularization. NR - precursor (pre-circRNA4), Cr - crude products of circularization, F1-F3 - concentrated HPLC fractions, M -linear RNA length marker. HMW refers to linear RNA of length ranging from 3000 nt to 6000 nt. PAGE conditions: 6% AA, AA: MBAA = 38:1, 7 M Urea, 1x TBE. Purified circRNA is in fraction 2.

[0067] Fig. 11 shows the influence of mobile phase composition on RP-HPLC of circRNA. A mixture of linear (pre-circRNA14) and circular (circRNA14) RNAs was resolved with RP-HPLC. Each chromatography was performed in presence of different ion-pairing agent: A) hexylammonium acetate; B) triethylammonium acetate; C) 2-ethylhexylammonium acetate; D) dibutylammonium acetate; E) / V, / V-dimethylhexylammonium acetate; F) N, N-diisopropylammonium acetate.

[0068] Fig. 12 shows a PAGE analysis of selected circRNA precursors and circRNA after HPLC and RNase R treatment. M - linear RNA length marker. HMW refers to linear RNA of length ranging from 3000 nt to 6000 nt. PAGE conditions: 6% AA, AA: MBAA = 38:1, 7 M Urea, lx TBE.

[0069] Fig. 13 shows the translational activity of circRNA analogs in HEK293 cells. Cells were transfected with unmodified, i.e. enzymatically obtained circRNA (circRNA11) or modified, i.e. chemically obtained circRNA (circRNA12 with unnatural linkage, and circRNA13 with a cap structure) encoding Gaussia luciferase, a reporter protein secreted by the cells to the medium. The circRNA precursors (pre-circRNA11-13) were used as references. The cell media were harvested at timepoints (4, 24, 48, 72, 96, 120, 144 and 168 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay. The detected luminescence from all timepoints was summarized to give the total luminescence. The data presented in the graph derives from three independent replicates, error bars represent a standard error of the mean (SEM).

[0070] Fig. 14 shows the translational activity of circRNA analogs in HEK 293 cells (continued). Cells were transfected with unmodified (circRNA11) or modified circRNA (circRNA12, 13) encoding Gaussia luciferase, a reporter protein secreted by the cells to the medium. The circRNA precursors (pre-circRNA11-13) were used as a reference. The cell media were harvested at timepoints (4, 24, 48, 72, 96, 120, 144 and 168 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay. The data presented in the graph derives from three independent replicates, error bars represent a standard error of the mean (SEM).

[0071] Fig. 15 shows the translational activity of circRNA analog in vitro compared to its linear precursor. HEK 293T, Hep G2, A549, HeLa were transfected with modified circRNA (circRNA12) or its linear precursor (pre-circRNA12) encoding Gaussia luciferase, a reporter protein secreted by the cells to the medium. Mock transfection (water instead of RNA) was used as a reference. Cell media were harvested at timepoints (4, 24, 48, 72, 96, 120, 144 and 168 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay. The detected luminescence from all timepoints was summarized to give the total luminescence, that was subsequently normalizes to the average luminescence obtained for linRNA23. The data presented in the graph derives from three independent repetitions, error bars represent a standard error of the mean (SEM).

[0072] Fig. 16 shows the translational activity of circRNA analog in A549 cells. Cells were transfected with chemically obtained circRNA (circRNA27) and its linear RNA precursor (pre-circRNA27) with azide group, both encoding Gaussia luciferase, a reporter protein secreted by the cells to the medium. Cell culture media were collected at timepoints (4, 24, 48, 72, 96, 120 and 144 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay. The detected luminescence from all timepoints was summarized to give the total luminescence. The data presented in the graph derives from three independent replicates, error bars represent a standard error of the mean (SEM).

[0073] Fig. 17 shows the efficiency of SPAAC reactions performed on circular RNA (circRNA27) with different DBCO-labelled 7-methylguanosine cap analogs. Mixtures comprising uncapped circular RNA (circRNA27) and capped circular RNA species (circRNA29-34) were analyzed by RP-HPLC. The separation was carried out using a PolymerX RP-1 column (3 μm 100 Å, 4.1 x 150 mm) at a flow rate of 1.0 mL / min and a temperature of 60°C. Solvent A: 100 mM TEAA pH 7.0; solvent B: 200 mM TEAA pH 7.0 / acetonitrile 1:1 (v / v); elution: from 20% to 32.5% B in 25 min, then from 32.5% to 100% B in 2 min, then 100% B for 2 min. As a reference, unmodified circular RNA (circRNA27) was analyzed under the same chromatographic conditions.

[0074] Fig. 18 shows the translational activity of modified circRNA analogs in vitro compared to their chemically obtained unmodified circRNA. A549 were transfected with modified circRNAs (circRNA35-40) or their circular unmodified azide precursor (circRNA28), all encoding Gaussia luciferase, a reporter protein secreted by the cells to the medium. Mock transfection (water instead of RNA) was used as a reference. Cell media were harvested at timepoints (4, 24, 48, 72, 96, 120 and 144 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay. The data presented in the graph derives from three independent repetitions, error bars represent a standard error of the mean (SEM).

[0075] Fig. 19 shows the efficiency of SPAAC reactions performed on circular RNA (circRNA28) with different DBCO-labelled tags. A mixture comprising uncapped circular RNA (circRNA28) and capped circular RNA species (circRNA35-40) was analyzed by RP-HPLC. The separation was carried out using a PolymerX RP-1 column (3 μm 100 Å, 4.1 x 150 mm) at a flow rate of 1.0 mL / min and a temperature of 60°C. Solvent A: 100 mM TEAA pH 7.0; solvent B: 200 mM TEAA pH 7.0 / acetonitrile 1:1 (v / v); elution: from 20% to 32.5% B in 25 min, then from 32.5% to 100% B in 2 min, then 100% B for 2 min. As a reference, unmodified circular RNA (circRNA28) was analyzed under the same chromatographic conditions.

[0076] Fig. 20 shows the efficiency of SPAAC reaction performed on circular RNA under different conditions (excess of DBCO-cap analog, reaction time, temperature). A mixture comprising uncapped circular RNA (circRNA27) and capped circular RNA (circRNA31) was analyzed by A) PAGE electrophoresis: 6% AA, AA: MBAA = 38:1, 7 M Urea, 1x TBE, and B) RP-HPLC. The separation was carried out using a PolymerX RP-1 column (3 μm 100 Å, 4.1 x 150 mm) at a flow rate of 1.0 mL / min and a temperature of 60°C. Solvent A: 100 mM TEAA pH 7.0; solvent B: 200 mM TEAA pH 7.0 / acetonitrile 1:1 (v / v); elution: from 20% to 32.5% B in 25 min, then from 32.5% to 100% B in 2 min, then 100% B for 2 min.

[0077] Fig. 21 shows the efficiency of SPAAC reactions between circular RNA molecule (circRNA27) and different DBCO-tags, i.e. AcPFVYLI (CPP), CLS-PEG4, or TriGalNAc. A mixture comprising nonconjugated circular RNA (circRNA27) and its conjugates (circRNA41-43) was analyzed by A) PAGE electrophoresis: 6% AA, AA: MBAA = 38:1, 7 M Urea, 1x TBE and, B) RP-HPLC. The separation was carried out using a PolymerX RP-1 column (3 μm 100 Å, 4.1 x 150 mm) at a flow rate of 1.0 mL / min and a temperature of 60°C. Solvent A: 100 mM TEAA pH 7.0; solvent B: 200 mM TEAA pH 7.0 / acetonitrile 1:1 (v / v); elution: from 20% to 32.5% B in 55 min, then from 32.5% to 100% B in 2 min, then 100% B for 2 min. As a reference, unmodified circular RNA (circRNA27) was analyzed under the same chromatographic conditions.

[0078] Fig. 22 shows electrophoretic mobility shift assay of circRNA samples (circRNA27 and circRNA44) after incubation with or without streptavidin for 30 and 60 minutes. An agarose gel (0.5%) illustrates the migration of biotinylated circRNA44 incubated with streptavidin for 30 minutes (upper panel) and 60 minutes (lower panel). Distinct bands correspond to the main reaction products. As a reference for the conjugation reaction efficiency, circRNA27 was used and subsequently incubated with or without streptavidin.

[0079] Fig. 23 shows the translational activity of circRNA analogs in K562 or HepG2 cells. The cells were transfected with chemically synthesized circRNAs (circRNA41-44) carrying different biologically active DBCO-tags, such as AcPFVYLI, CLS-PEG4, and TriGalNAc as well as with their circular RNA precursor (circRNA27) containing an azide group. All circRNA constructs encoded Gaussia luciferase, a reporter protein secreted by the cells to the medium. Cell culture media were collected at timepoints (4, 24, 48, 72, 96, 120 and 144 hours post transfection) and used for quantification of the reporter protein levels with luciferase bioluminescence assay (A). The detected luminescence from all timepoints was summarized to give the total luminescence (B). The data presented in the graph derives from three independent replicates, error bars represent a standard error of the mean (SEM). Detailed description of the invention

[0080] The present invention relates to circular RNA molecules, methods of producing circular RNA molecules, circular and non-circular intermediates thereof, and methods of treating or preventing a disease.

[0081] The compound of Formula I

[0082] The present invention relates to a compound of Formula

[0083]

[0084] A preferred form of Formula I is the following Formula la:

[0085]

[0086] Any statements herein regarding Formula I are also applicable to Formula la.

[0087] In Formula I, n is any integer with a value of from 1 to 20000. Typically, n is any integer with a value of from 90 to 20000, preferably from 100 to 10000, more preferably from 500 to 8 000, even more preferably from 800 to 8000. Each X is independently selected from O’, BHs’, NH’, S’, and Se’. Typically, each X is independently selected from O’, S’ and Se’; preferably each X is independently selected from O’ and S’; more preferably each X is O’.

[0088] Each G is independently selected from O and S. Preferably, each G is O.

[0089] Each A is independently selected from CH2, O, S, NH and a bond. Typically, each A is independently selected from O, S, NH and a bond; preferably each A is independently selected from O, S and a bond; more preferably each A is independently selected from O and a bond; even more preferably each A is O.

[0090] Each E is independently selected from CH2, O, S and NH. Preferably, each E is independently selected from O and S. More preferably each E is O.

[0091] Each D is independently selected from CH2, O, S and NH. Preferably each D is independently selected from O and S. More preferably each D is O.

[0092] Each R1is independently selected from a natural or modified nucleoside base. Typically, each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O (RR, -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0093] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH, preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine;

[0094] More preferably, each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0095] Each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C O) RR, - C(O)NH2, -C(O) H(RR), -C(O (RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR)), - NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)N H(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl. Typically, each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N (a lkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN. Preferably each R2is independently selected from H, alkyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl is optionally substituted with one or more selected from F, Cl, Br and CN.

[0096] More preferably each R2is independently selected from H, Me, OH, OMe, O-CH2-aryl and F. Even more preferably each R2is independently selected from H, OH, OMe and F. Still more preferably each R2is independently selected from OH and OMe. Still even more preferably each R2is OH.

[0097] R3is selected from a natural or modified nucleoside base. Typically, R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), - C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)N H(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0098] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH. Preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine. More preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0099] L is a trivalent group having from 1 to 50 non-H atoms. Typically, L is a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. L is preferably a trivalent group having from 1 to 40 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms, More preferably, L is a trivalent group having from 1 to 30 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. Even more preferably, L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 30 hydrogen atoms, such as a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S and P, and up to 30 hydrogen atoms.

[0100] L preferably fulfils one or more, preferably all of:

[0101] - L preferably contains at least one -P(=O)(O-)O- group;

[0102] - L preferably contains at least one branch selected from ternary carbon and an aryl group having at least 3 non-H-substituents; and / or

[0103] - L contains at least one secondary amine group and / or ether group.

[0104] It is preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom. It is more preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2group). It is even more preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2group) which is bound to another carbon atom bearing two hydrogens (a CH2group), thus representing a -CH2-CH2- group. Still even more preferably the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2 group) which is bound to another carbon atom bearing two hydrogens (a CH2 group), which in turn is bound to a nitrogen, thus representing a -CH2- CH2-N< group. It is to be understood that the nitrogen in this group may be bound to any further groups present within L.

[0105] L is preferably represented by the following Formula L-l:

[0106]

[0107] wherein the P is preferably linked to A, XL2is preferably linked to Q and XL3is preferably linked to the nitrogen of the morpholine group, as seen in Formula I.

[0108] ZLis selected from CH, C(Ci-6 alkyl), a trivalent mono- or bicyclic cycloalkyl group, a trivalent mono- or bicyclic cycloheteroalkyl group, a trivalent mono- or bicyclic aryl group and a trivalent mono- or bicyclic heteroaryl group. ZLpreferably has 1 to 15 (more preferably 1 to 10, such as 1 to 6) non-H atoms. More preferably, ZLis selected from CH, C(Ci-6 alkyl), a trivalent benzene ring and a six-membered trivalent heterocyclic ring. Even more preferably, ZLis selected from CH, a trivalent benzene ring and a trivalent triazine ring. The term "trivalent" in this context reflects the fact that ZLis bound to each of XL1, XL2and XL3.

[0109] Each of XL1, XL2and XL3are preferably independently selected from a bond, an alkylene group and a heteroalkylene group. For example, XL1may be a bond and each of XL2and XL3are preferably independently selected from an alkylene group and a heteroalkylene group.

[0110] Each alkylene group preferably has from 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms, and is preferably linear. Each alkylene group may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0111] Each heteroalkylene more preferably corresponds to a (preferably linear) C1-15alkylene wherein one to six CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may preferably be substituted with one or more selected from F, OH, CN, O(Me), NH2, NH(Me), and N(Me)2. More preferably, the heteroalkylene corresponds to a (preferably linear) C1-10alkylene wherein one to five CH2groups are independently replaced by S, O, NH, N(alkyl), P(=O)(O-) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0112] L is more preferably represented by the following Formula L-2:

[0113]

[0114] wherein each of ZL, XL1, XL2and XL3are as defined above. Preferably, the alkylene or heteroalkylene group in XL3of Formula L-2 is shorter by two CH2units than defined for XL3of Formula L-1.

[0115] More preferably, L is selected from

[0116]

[0117] It is preferred that the moiety -A-L(Q)- does not represent any of the following formulae:

[0118]

[0119] Q is a monovalent group comprising one or more groups selected from nucleotide and oligonucleotide, including mRNA 5' cap analog, nucleic acid, fluorophore, hapten, chelator, (radio)isotopic tag, lipid, amino acid, oligopeptide, protein, saccharide, oligosaccharide, polymer, polymer composite, metal-organic framework, or a solid support such as nanoparticle, nanomaterial, glass, metal, ceramics or metal-organic framework.

[0120] Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, a nucleic acid, a fluorophore, a chelator, a radioisotope, a wax, a sterol, a vitamin selected from Vitamins A, D, E and K, a monoglyceride, a diglyceride, a phospholipid, an amino acid, an oligopeptide, a protein, a monosaccharide, an oligosaccharide or a polysaccharide. Preferably Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, a nucleic acid, a fluorophore, chelating ligand and a radioisotope. More preferably Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, and a fluorophore.

[0121] Even more preferably Q is any of the above and contains a triazole group.

[0122] In any of the definitions of Q, it is preferred that Q comprises 10 to 500 non-hydrogen-atoms, more preferably 10 to 300 non-hydrogen-atoms.

[0123] Still more preferably, Q is selected from the following formulae:

[0124] O A HN-p V l!

[0125] O o o', — / \

[0126] O-P-O-P O ~Pzp"-N / h'-N

[0127] I I I IZ

[0128] o o 6 o' A, Ax A

[0129] OH OH n N

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] and

[0137]

[0138] Most preferably, Q is selected from the following formulae:

[0139] CH

[0140] Nr.-N+H5. N" %r. N To*i OH OH H HN

[0141] "bT '□ H H N

[0142]

[0143] N

[0144]

[0145]

[0146]

[0147]

[0148] The compound of Formula II

[0149] In addition, the present invention also relates to compounds which can be used to prepare the compound of Formula I. For example, a compound of Formula I can be obtained by coupling a compound of Formula II as described in the following with a compound containing a desired tag such as a nucleotide, oligonucleotide (e.g. including an mRNA 5' cap analog), nucleic acid, fluorophore, hapten, chelator, (radio)isotopic tag, lipid, amino acid, oligopeptide, protein, saccharide, oligosaccharide, polymer, polymer composite, metal-organic framework, or a solid support such as nanoparticle, nanomaterial, glass, metal or ceramics.

[0150] Thus, the present invention furthermore relates to a compound of Formula II

[0151]

[0152] A preferred form of Formula II is the following Formula Ila:

[0153]

[0154] Any statements herein regarding Formula II are also applicable to Formula Ila.

[0155] In Formula II, n is any integer with a value of from 1 to 20000. Typically, n is any integer with a value of from 90 to 20000, preferably from 100 to 10000, more preferably from 500 to 8 000, even more preferably from 800 to 8000.

[0156] Each X is independently selected from O’, BHs’, NH’, S’, and Se’. Typically, each X is independently selected from O’, S’ and Se-; preferably each X is independently selected from O’ and S’; more preferably each X is O’. Each G is independently selected from O and S. Preferably, each G is O.

[0157] Each A is independently selected from CH2, O, S, NH and a bond. Typically, each A is independently selected from O, S, NH and a bond; preferably each A is independently selected from O, S and a bond; more preferably each A is independently selected from O and a bond; even more preferably each A is O.

[0158] Each E is independently selected from CH2, O, S and NH. Preferably, each E is independently selected from O and S. More preferably each E is O.

[0159] Each D is independently selected from CH2, O, S and NH. Preferably each D is independently selected from O and S. More preferably each D is O.

[0160] Each R1is independently selected from a natural or modified nucleoside base. Typically, each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O) RR), -C(O)NH2, -C(O)NH(RR), -C(O N(RR?, -C(O)OH, -C(O O(RR, - NH?., -NH RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O) H(RR, -NHC O) (RR?, - NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)(RR)2, -OC(O) H(RR), -OC(O)NH2, ■ OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0161] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH, preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine.

[0162] More preferably, each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0163] Each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), - C(O)NH2, -C(O)NH(RR), -C(O)N(RRk, -C(O)OH, -C(O)O(RR), -NH?, -NH(RR, -N RR)2, -NHC(O)(RR)), ■ NHC(O)H, -NHC(O)NH2, --NHC(O)NH(RR), NHC(O N(RR)2, NHC(O O(RR), -NO2, -OH, -O-RR, - OC(O (RR), -OC(O)N(RRk, -OC(O)NH(RR), OC(O)NH2, OC(O O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl. Typically, each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N (a lkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN. Preferably each R2is independently selected from H, alkyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl is optionally substituted with one or more selected from F, Cl, Br and CN.

[0164] More preferably each R2is independently selected from H, Me, OH, OMe, O-CH2-aryl and F. Even more preferably each R2is independently selected from H, OH, OMe and F. Still more preferably each R2is independently selected from OH and OMe. Still even more preferably each R2is OH.

[0165] R3is selected from a natural or modified nucleoside base. Typically, R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), - C(O)NH2, -C(O)NH(RR), -C(O N(RR2, -C(O)OH, -C(O)O(RR), -NH2, - H RR, -N(RR)2, -NHC(O)(RR), - NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), - HC(O) (RR)2, - HC(O)O(RR), -NO2, -OH, -O-RR, - OC(O)(RR), -OC(O)N(RR)2, OC O H(RR, -OC(O)NH2, -OC(O O(RR, -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0166] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH. Preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine.

[0167] More preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0168] L is a trivalent group having from 1 to 50 non-H atoms. Typically, L is a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 50 hydrogen atoms. L is preferably a trivalent group having from 1 to 40 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. More preferably, L is a trivalent group having from 1 to 30 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. Even more preferably, L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 30 hydrogen atoms, such as a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S and P, and up to 30 hydrogen atoms. L preferably fulfils one or more, preferably all of:

[0169] - L preferably contains at least one -P(=O)(O-)O- group;

[0170] - L preferably contains at least one branch selected from ternary carbon and an aryl group having at least 3 non-H-substituents; and / or

[0171] - L contains at least one secondary amine group and / or ether group.

[0172] It is preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom. It is more preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2group). It is even more preferred that the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2group) which is bound to another carbon atom bearing two hydrogens (a CH2group), thus representing a -CH2-CH2- group. Still even more preferably the atom of L being directly bound to the N of the morpholine ring shown in the above formula be a carbon atom bearing two hydrogens (a CH2 group) which is bound to another carbon atom bearing two hydrogens (a CH2 group), which in turn is bound to a nitrogen, thus representing a -CH2-CH2-N< group. It is to be understood that the nitrogen in this group may be bound to any further groups present within L.

[0173] L is preferably represented by the following Formula L-l:

[0174]

[0175] wherein the P is preferably linked to A, XL2is preferably linked to J and XL3is preferably linked to the nitrogen of the morpholine group, as seen in Formula II.

[0176] ZLis selected from CH, C(Ci-6 alkyl), a trivalent mono- or bicyclic cycloalkyl group, a trivalent mono- or bicyclic cycloheteroalkyl group, a trivalent mono- or bicyclic aryl group and a trivalent mono- or bicyclic heteroaryl group. ZLpreferably has 1 to 15 (more preferably 1 to 10, such as 1 to 6) non-H atoms. More preferably, ZLis selected from CH, C(Ci-6 alkyl), a trivalent benzene ring and a six-membered trivalent heterocyclic ring. Even more preferably, ZLis selected from CH, a trivalent benzene ring and a trivalent triazine ring. The term "trivalent" in this context reflects the fact that ZLis bound to each of XL1, XL2and XL3.

[0177] Each of XL1, XL2and XL3are preferably independently selected from a bond, an alkylene group and a heteroalkylene group. For example, XL1may be a bond and each of XL2and XL3are preferably independently selected from an alkylene group and a heteroalkylene group.

[0178] Each alkylene group preferably has from 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms, and is preferably linear. Each alkylene group may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0179] Each heteroalkylene more preferably corresponds to a (preferably linear) C1-15alkylene wherein one to six CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may preferably be substituted with one or more selected from F, OH, CN, O(Me), NH2, NH(Me), and N(Me)2. More preferably, the heteroalkylene corresponds to a (preferably linear) C1-10alkylene wherein one to five CH2groups are independently replaced by S, O, NH, N(alkyl), P(=O)(O-) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0180] L is more preferably represented by the following Formula L-2:

[0181]

[0182] wherein each of ZL, XL1, XL2and XL3are as defined above. Preferably, the alkylene or heteroalkylene group in XL3of Formula L-2 is shorter by two CH2units than defined for XL3of Formula L-1.

[0183] More preferably, L is selected from

[0184]

[0185] J is a monovalent group having from 1 to 70 non-H atoms (typically having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S), wherein J preferably comprises at least one group selected from CEC bond, -N3, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F. Preferably, J is a monovalent group having 0 to 20 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein J comprises at least one group selected from CEC bond, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

[0186] The maleimide is preferably represented by

[0187]

[0188] The diazirine is preferably represented by

[0189]

[0190] including any versions thereof where the carbon of the diazirine is optionally substituted with an optional substituent defined herein.

[0191] The epoxide is preferably represented by

[0192]

[0193] including any versions thereof where the carbon at the lower right corner of the epoxide ring is optionally substituted with an optional substituent defined herein.

[0194] The tetrazine is preferably represented by

[0195] N

[0196]

[0197] including any versions thereof where the carbon at the lower right corner of the tetrazine is optionally substituted with an optional substituent defined herein.

[0198] More preferably, J is selected from the following formulae:

[0199] NH2

[0200]

[0201]

[0202] and

[0203] Still more preferably, J is selected from the following formulae:

[0204] 0 H

[0205] |~O“P“O' V 'i

[0206]

[0207] and ’ ° °

[0208] It is preferred that J does not comprise -CH2-N3. More preferably J is not -CH2-N3. Even more preferably J is not N3. Still more preferably J does not comprise N3. It is furthermore preferred that the moiety — A-L(J)— does not represent any of the following formulae:

[0209]

[0210] The compound of Formula III

[0211] The present invention also relates to primers that can be used to prepare compounds of formulae I and II, such as to a compound of Formula III

[0212]

[0213] A preferred form of Formula III is the following Formula Illa: H0_ _0H

[0214]

[0215] Any statements herein regarding Formula III are also applicable to Formula Illa.

[0216] In Formula III, X is selected from O’, BHs’, NH’, S’, and Se’. X is preferably selected from O’, S’ and Se-. More preferably X is selected from O’ and S’. Even more preferably X is O’.

[0217] G is selected from O and S. G is preferably O.

[0218] A is selected from CH2, O, S, NH and a bond. A is typically selected from O, S, NH and a bond. Preferably A is selected from O, S and a bond. More preferably A is selected from O and a bond. Even more preferably A is O.

[0219] E is selected from CH2, O, S and NH. E is preferably selected from O and S. More preferably E is O.

[0220] Dbis selected from CH2, O, S and NH. Dbis preferably selected from O and S. More preferably Dbis O.

[0221] Each of R1and Rlbare independently selected from a natural or modified nucleoside base. Preferably, R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O N(RR)2, - C(O)OH, -C(O)O(RR), -NH2, - H(RR, -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(RR), -NHC(O N(RR)Z, -NHC(0)0 RR), -NO2, -OH, -O-RR, -OC(O) RR), -OC(O)N(RR)?, - OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0222] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH.

[0223] More preferably R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6- thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine.

[0224] Even more preferably R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0225] R2is selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein Each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)( RR), -C(O)NH?, -C(O)NH(RR), -C O)N RR)2, -C(O)OH, -C(O)O(RR), -NHZ, -N H(RR), -N(RR)2, - HC(O (RR, -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR?, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC O) RR), -OC(O)N(RR)Z, - OC(O NH(RR), -OC(O NH2, OC(O)O(RR, -SH, and -S-RR, wherein Each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein Each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl. It is preferred that R2be selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN. More preferably R2is selected from H, alkyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN. Even more preferably R2is selected from H, Me, OH, OMe, O-CH2-aryl and F. Still even more preferably R2is selected from H, OH, OMe and F, such as OH and OMe. Most preferably R2is OH.

[0226] L is a trivalent group having from 1 to 50 non-H atoms. Typically, L is a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 50 hydrogen atoms. Typically, L is a trivalent group having from 1 to 40 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. More preferably, L is a trivalent group having from 1 to 30 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms. Even more preferably, L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 30 hydrogen atoms, such as a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S and P, and up to 30 hydrogen atoms.

[0227] L preferably fulfils one or more, preferably all of:

[0228] - L preferably contains at least one -P(=O)(O-)O- group;

[0229] - L preferably contains at least one branch selected from ternary carbon and an aryl group having at least 3 non-H-substituents; and / or

[0230] - L contains at least one secondary amine group and / or ether group.

[0231] It is preferred that the atom of L being directly bound to the N of the NH2 group shown in the above formula be a carbon atom. It is more preferred that the atom of L being directly bound to the N of the NH2 group shown in the above formula be a carbon atom bearing two hydrogens (a CH2group). It is even more preferred that the atom of L being directly bound to the N of the NH2 group shown in the above formula be a carbon atom bearing two hydrogens (a CH2group) which is bound to another carbon atom bearing two hydrogens (a CH2group), thus representing a -CH2-CH2- group. Still even more preferably the atom of L being directly bound to the N of the NH2 group shown in the above formula be a carbon atom bearing two hydrogens (a CH2 group) which is bound to another carbon atom bearing two hydrogens (a CH2 group), which in turn is bound to a nitrogen, thus representing a -CH2-CH2-N< group. It is to be understood that the nitrogen in this group may be bound to any further groups present within L.

[0232] L is preferably represented by the following Formula L-l:

[0233]

[0234] wherein the P is preferably linked to A, XL2is preferably linked to J and XL3is preferably linked to the nitrogen of the NH2 group, as seen in Formula III.

[0235] ZLis selected from CH, C(Ci-6 alkyl), a trivalent mono- or bicyclic cycloalkyl group, a trivalent mono- or bicyclic cycloheteroalkyl group, a trivalent mono- or bicyclic aryl group and a trivalent mono- or bicyclic heteroaryl group. ZLpreferably has 1 to 15 (more preferably 1 to 10, such as 1 to 6) non-H atoms. More preferably, ZLis selected from CH, C(Ci-6 alkyl), a trivalent benzene ring and a six-membered trivalent heterocyclic ring. Even more preferably, ZLis selected from CH, a trivalent benzene ring and a trivalent triazine ring. The term "trivalent" in this context reflects the fact that ZLis bound to each of XL1, XL2and XL3.

[0236] Each of XL1, XL2and XL3are preferably independently selected from a bond, an alkylene group and a heteroalkylene group. For example, XL1may be a bond and each of XL2and XL3are preferably independently selected from an alkylene group and a heteroalkylene group.

[0237] Each alkylene group preferably has from 1 to 15 carbon atoms, more preferably 1 to 10 carbon atoms, and is preferably linear. Each alkylene group may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0238] Each heteroalkylene more preferably corresponds to a (preferably linear) C1-15alkylene wherein one to six CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may preferably be substituted with one or more selected from F, OH, CN, O(Me), NH2, NH(Me), and N(Me)2. More preferably, the heteroalkylene corresponds to a (preferably linear) C1-10alkylene wherein one to five CH2groups are independently replaced by S, O, NH, N(alkyl), P(=O)(O-) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0239] L is more preferably represented by the following Formula L-2:

[0240]

[0241] wherein each of ZL, XL1, XL2and XL3are as defined above. Preferably, the alkylene or heteroalkylene group in XL3of Formula L-2 is shorter by two CH2units than defined for XL3of Formula L-1.

[0242] More preferably, L is selected from

[0243]

[0244] L preferably does not comprise a natural, modified or unnatural 7-methylguanosine cap structure.

[0245] J is a monovalent group having from 1 to 70 non-H atoms (typically having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S), wherein J preferably comprises at least one group selected from C=C bond, -N3, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F. Typically, J is a monovalent group having 0 to 20 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein J comprises at least one group selected from C=C bond, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F. Preferably J is selected from the following formulae:

[0246] NH2

[0247]

[0248] Still more preferably, J is selected from the following formulae: o~

[0249] O-p- 0 - 6

[0250] 0 H

[0251] < 1.-x „hk

[0252] v S -o-p:l-o' " " ■'-" Y I: i

[0253] 0 O

[0254]

[0255] and

[0256] J preferably does not comprise -CH2-N3. More preferably J is not -CH2-N3. Even more preferably J is not N3. Still more preferably J does not comprise N3. In addition, the moiety -A-L(J )— preferably does not represent any of the following formulae:

[0257]

[0258] The compound of Formula IV

[0259] The present invention furthermore relates to compounds that can be reacted with compounds of Formula II to form compounds of Formula I. These include compounds of Formula IV as follows:

[0260]

[0261] A preferred form of Formula IV is the following Formula IVa:

[0262]

[0263] Any statements herein regarding Formula IV are also applicable to Formula IVa.

[0264] In Formula IV, RC1and RC2are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl, and (hetero)aryl, wherein the alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl are optionally substituted with one or more selected from F, -Br, - Cl, -RR, -CN, -C(O)H, -C(O)(RR, -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), - C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, - NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, - OC(O)O(alkyl), -SH, and -S- alkyl. RC1is preferably selected from H and alkyl, more preferably alkyl, and even more preferably methyl. RC2is preferably selected from H and alkyl. More preferably, RC2is H.

[0265] RC3and RC4are independently selected from the group consisting of OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, NH2, NH(alkyl), F, Cl, Br, alkyl, alkenyl, and alkynyl, or RC3and RC4may be taken together to form -O-alkylidene-O-.

[0266] It is preferred that RC3be selected from OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, F, Cl and Br. More preferably RC3is selected from OH and OBn. Still more preferably RC3is OH.

[0267] RC4is preferably selected from OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, F, Cl and Br. RC4is more preferably from OH and OBn. RC4is even more preferably OH.

[0268] W1and W2are independently selected from the group consisting of O, S, Se, NH, CH2, alkylene, alkenylyne, alkynylene and a bond. Preferably, W1is selected from O, S, Se, NH, methylene, CH2O and a bond. More preferably W1is selected from O and S, even more preferably O. W2is preferably selected from O, S, Se, NH, methylene, CH2O and a bond. More preferably W2is O, S and methylene, even more preferably O and methylene.

[0269] m and n are independently an integer selected from the group consisting of 0, 1 and 2. Preferably, m is 0 or 1 and n is 1 or 2. More preferably m is 0 and n is 1.

[0270] XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are independently selected from the group consisting of O, S, Se, BH3 and NH. Preferably one, two, three, four, five, six or seven of XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are selected from O and S. More preferably, one, two, three, four, five, six or seven of XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4is O.

[0271] Still more preferably each of XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are selected from O and S. even more preferably, each of XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4is O. ZC1, ZC2, ZC3are independently selected from the group consisting of O, NH, CH2, CH(alkyl), CF(alkyl), CH(alkyl)₂, CHF, CF2, CFCI, CHCI and CCI2, wherein alkyl is optionally haloalkyl. Preferably, ZC1, ZC2and ZC3are independently selected from O and NH. More preferably, ZC1, ZC2and ZC3are O.

[0272] L2comprises one or more groups selected from optionally substituted heteroalkylene, optionally substituted alkylene, peptides, oligonucleotides and their block co-polymers; chemically modified peptides, chemically modified oligonucleotides and their block copolymers; polyethylene glycol), poly(lactic acid) and their block co-polymers, wherein the wherein the one or more optional substituents of the heteroalkylene and the alkylene are selected from F, -Br, -Cl, -RR, -CN, C(O)H, C(O)(RR), -C(O)NH2, C(O)NH(RR), -C(O)N(RR)2, •• C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, - OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl.

[0273] L2preferably comprises optionally substituted alkylene or optionally substituted heteroalkylene, wherein the wherein the one or more optional substituents of the heteroalkylene and the alkylene are selected from OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl, Br and CN. More preferably, L2consists of optionally substituted heteroalkylene, at least one nucleoside and optionally 1 to 15 further atoms selected from halogen, C, N, P, O, S. Even more preferably L2is of formula (C-l):

[0274]

[0275] wherein the OH can optionally be replaced by O-alkyl;

[0276] RCL1is selected from optionally substituted heteroalkylene, and

[0277] RCS1and RCS2are each independently selected from a natural or modified nucleoside base. Preferably, RCS1and RCS2are each independently a nucleobase optionally having a substituent selected from alkyl and alkylene-aryl at an exocyclic nitrogen.

[0278] It is to be understood that in the partial formula

[0279]

[0280] OH OH the RCS1is selected from a natural or modified nucleoside base which is further linked to the remainder of the molecule.

[0281] Even still more preferably L2is of formula (C-l):

[0282]

[0283] wherein the OH can optionally be replaced by O-alkyl; RCL1is selected from optionally substituted heteroalkylene, and

[0284] RCS1and RCS2are each independently selected from a natural or modified nucleoside base. Preferably, RCS1and RCS2are each independently a nucleobase optionally having a substituent selected from alkyl and alkylene-aryl at an exocyclic nitrogen.

[0285] Still even more preferable L2is selected from

[0286]

[0287]  Among these, the following are particularly preferred:

[0288]

[0289] Q2is a group having from 0 to 70 non-H atoms (typically having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S), wherein the Q2preferably comprises at least one group selected from C=C bond, -N3, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

[0290] Q2is preferably a group having from 1 to 70 non-H atoms (typically having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S), wherein Q2comprises at least one group selected from C≡C bond. More preferably Q2is a cyclic group having 2 to 25 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein Q2 comprises at least one C≡C bond. Even more preferably Q2comprises at least one eightmembered ring having a C=C bond in the ring. Still even more preferably, Q2is selected from

[0291]

[0292] The optionally substituted heteroalkylene typically corresponds to a (preferably linear) C1-30 alkylene wherein one to ten CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2. Preferably the optionally substituted heteroalkylene preferably corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2. More preferably the optionally substituted heteroalkylene preferably corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0293] Preferred examples of Formula IV are represented by the following formulae: I o T

[0294] o

[0295]

[0296] i

[0297]

[0298]

[0299] Among these, the following are particularly preferred: OH OH

[0300]

[0301]

[0302] Intermediates and uses of the compounds of the present invention

[0303] The present invention furthermore relates to the use of the compound of Formula II for the manufacture of a compound of Formula I.

[0304] In addition, the present invention relates to the use of the compound of Formula III for the manufacture of a compound of Formula I. The manufacture of the compound of Formula I preferably involves reacting a compound of the following Formula V

[0305]

[0306] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II, 5 to form a compound of the following Formula VI:

[0307]

[0308] preferably

[0309]

[0310] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II.

[0311] Reacting the compound of Formula V typically involves oxidation of the compound of Formula V. This oxidation is preferably conducted in the presence of a periodate, such as NaIO4. The compound of Formula V is preferably at a concentration below 10 mM. Reacting the compound of Formula V with the periodate is preferably conducted at a temperature below 60°C.

[0312] The manufacture of the compound of Formula I preferably involves reacting a compound of the following Formula VI

[0313]

[0314] preferably

[0315]

[0316] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II,

[0317] to form a compound of Formula II.

[0318] The compound of Formula VI is typically reacted with a borohydride, preferably Na[BH3(CN)]. The pH during the reaction is preferably in the range of from 5 to 8. The compound of Formula VI is typically at a concentration of less than 100 mM.

[0319] The present invention also relates to the use of the compound of Formula IV for the manufacture of a compound of Formula I. The compound of Formula IV is preferably reacted with the compound of Formula II to form the compound of Formula I.

[0320] In addition, the present invention concerns a compound which is obtainable by reacting the compound of Formula II with the compound of Formula IV.

[0321] It is preferred that J in the compound of Formula II contains an azide group and Q2in the compound of Formula IV contains an alkyne group (preferably a cycloalkyne group). Alternatively, J in the compound of Formula II preferably contains an alkyne group (preferably a cycloalkyne group) and Q2in the compound of Formula IV contains an azide group.

[0322] The present invention also relates to further intermediates which may be used in the preparation of compounds of formulae I, II and III. For example, the present invention relates to a compound the following Formula VII:

[0323]

[0324] wherein A, L, Q, E, G, X, D, R1, R2, R3and n are as defined for Formula I.

[0325] In addition, the present invention relates to a compound the following Formula VIII:

[0326]

[0327] preferably

[0328]

[0329] wherein A, L, Q, E, G, X, D, R1, R2, R3and n are as defined for Formula I.

[0330] The compound of Formula VII or the compound Formula VII may be used for the manufacture of a compound of Formula I. Preferably, the compound of Formula VII is oxidized to form the compound of Formula VIII, preferably in the presence of a periodate, such as NaIO4, wherein the compound of Formula VII is preferably at a concentration below 10 mM and / or wherein reacting the compound of Formula VII with the periodate is preferably conducted at a temperature below 60°C.

[0331] The compound of Formula VIII can be reacted with a borohydride, preferably Na[BH3(CN)], preferably at a pH in the range of from 5 to 8, preferably while the compound of Formula VIII is at a concentration of less than 100 mM, to form the compound of Formula I.

[0332] The kit of the present invention

[0333] The present invention furthermore relates to a kit of parts comprising one or more of the compounds disclosed herein. The kit typically comprises a compound of any of formulae II, III, IV, V, VI, VII and VIII, as well as instructions for the preparation of a compound of Formula I.

[0334] The medical use of the compounds of the present invention

[0335] The present invention additionally provides the compounds described herein for medial use. For example, the compounds described herein may be part of a pharmaceutical composition. The pharmaceutical composition may comprise one or more the compounds described herein and optionally one or more pharmaceutically acceptable excipients.

[0336] The compounds, kits or pharmaceutical compositions are preferably for use in the treatment of a cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, or a rare disease.

[0337] In addition, the compounds, kits or pharmaceutical compositions are preferably for the prevention of a cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, or a rare disease. In particular, the prevention is accomplished by using the compounds, kits or pharmaceutical compositions in vaccination against any of these diseases.

[0338] In other words, the present invention also relates to a method for treating or preventing a disease selected from of a cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, or a rare disease. The method may be a method of vaccination against any of these diseases. The method typically involves the administration of an effective dose of a compound, kit or pharmaceutical composition of the present invention to a person in need thereof. The person in need thereof is typically a person having one or more of these diseases. In the case of the method of preventing, the person in need thereof is typically a person at a risk of contracting one or more of these diseases.

[0339] Circular RNAs (circRNAs) have emerged as promising drug candidates due to several unique characteristics that make them advantageous over traditional linear RNAs. The advantages include increased stability due to lack of free 5' and 3' ends and consequent resistance to degradation by exonucleases. This results in significantly longer half-lives compared to linear RNAs, enabling prolonged activity within cells. This entails that fewer doses may be needed to achieve satisfactory outcome or, in case of chronic diseases (addressed e.g. by protein or gene replacement therapies), less frequent administration of the drug may be required, which could improve the safety and lower costs. Furthermore, circRNAs can potentially evoke a lower immune response than linear mRNAs because of their structural properties, reducing the risk of inflammation or immune-related side effects. This is particularly significant for applications requiring repeated or high-dose administrations (cf. the review article on: " Circular RNAs as Therapeutic Agents and Drug Targets" by Z. Li, M. Ruan, C. Wang, and Y. Yang, Frontiers in Molecular Biosciences, 2021, DOI: 10.3389 / fmolb.2021.629590).

[0340] Furthermore, using the circRNA design according to the present invention may enable further advancement in the circRNA design. For example, the circRNA may be conjugated with a small molecule, tag, hapten, peptide, protein or other chemical individuum that enhances cellular uptake of the circRNA or offers tissue- or cell-specific targeting. Moreover, combination with molecules enabling detection, such as chelators of specific radioisotopes or fluorescent dyes, may enable in cel I u Io and in vivo visualization or tracking of the circ RNAs. Combination with biological tags such as biotin may enable isolation of the circRNA from biological material postadministration.

[0341] The cancer is preferably selected from breast cancer, lung cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, esophageal cancer, melanoma, lymphoma, leukemia.

[0342] Cardiovascular diseases are preferably selected from myocardial infarction, heart failure, atherosclerosis, hypertension, cardiac hypertrophy, and arrhythmias.

[0343] Neurological disorders are preferably selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, multiple sclerosis, Huntington's disease, epilepsy, and traumatic brain injury.

[0344] The metabolic disorders are preferably selected from diabetes mellitus (type 1 and type 2), obesity, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Infectious diseases are preferably selected from viral infections (e.g., hepatitis B and C, HIV, influenza, SARS-COV-2), bacterial infections (e.g., tuberculosis, staphylococcus aureus), fungal infections (e.g., candida), and parasitic infections (e.g., malaria).

[0345] The genetic disorders is preferably selected from Duchenne muscular dystrophy, cystic fibrosis, spinal muscular atrophy, hemophilia, sickle cell disease, and thalassemia.

[0346] Inflammatory and autoimmune diseases are preferably selected from rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, and asthma.

[0347] The ophthalmic diseases are preferably selected from age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma.

[0348] pulmonary diseases are preferably selected from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, asthma, and cystic fibrosis.

[0349] Renal diseases are preferably selected from chronic kidney disease (CKD), acute kidney injury, and polycystic kidney disease.

[0350] Gastrointestinal diseases are preferably selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), gastric ulcers, and liver cirrhosis.

[0351] dermatological diseases are preferably selected from psoriasis, atopic dermatitis, vitiligo, and melanoma.

[0352] The musculoskeletal diseases are preferably selected from osteoarthritis, rheumatoid arthritis, osteoporosis, and muscular dystrophies.

[0353] Endocrine disorders are preferably selected from thyroid diseases (e.g., hypothyroidism, hyperthyroidism), adrenal insufficiency, and polycystic ovary syndrome (PCOS). hematological diseases are preferably selected from anemia, leukemia, lymphoma, and hemophilia.

[0354] The rare diseases are preferably selected from Huntington's disease, Gaucher disease, Fabry disease, and Pompe disease.

[0355] In addition, the compounds, kits or pharmaceutical compositions may be used for imaging. For this use, the compounds of the present invention preferably include a fluorophore or radioisotope such as18F,14C,99mTc,123l,125l,131l,68Ga,67Ga,150,13N,82Rb,62Cu,32P,89Sr,153Sm,186Re. Preferred isotopes include those used for PET such as18F and68Ga. It is preferred in the present invention not include a circRNA analog according to formula 1

[0356]

[0357] Formula 1

[0358] wherein:

[0359] n is any integer with a value from 1 to 20000;

[0360] X and each of Xnis independently OH, BH3, NH2, or SH;

[0361] Y and each of Ynis independently O or S;

[0362] Z and each of Znis independently O, S or NH;

[0363] W and each of Wnis independently O, S or NH;

[0364] R and each of Rnis independently H, OH, alkyl, O-alkyl or halogen;

[0365] B, B' and each of Bnis independently a natural, modified or unnatural nucleoside base; L is selected from the following:

[0366] O

[0367]

[0368] Formula 5

[0369]

[0370]

[0371] The present invention may be summarized by the following items:

[0372] Item 1. A compound of Formula I

[0373]

[0374] wherein:

[0375] n is any integer with a value of from 1 to 20000;

[0376] each X is independently selected from O’, BHs’, NH’, S’, and Se’;

[0377] each G is independently selected from O and S;

[0378] each A is independently selected from CH2, O, S, NH and a bond;

[0379] each E is independently selected from CH2, O, S and NH;

[0380] each D is independently selected from CH2, O, S and NH;

[0381] each R1is independently selected from a natural or modified nucleoside base;

[0382] each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)( RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;

[0383] R3is selected from a natural or modified nucleoside base, L is a trivalent group having from 1 to 50 non-H atoms, wherein L is preferably a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms,

[0384] Q is a monovalent group comprising one or more groups selected from nucleotide and oligonucleotide, including mRNA 5' cap analog, nucleic acid, fluorophore, hapten, chelator, (radio)isotopic tag, lipid, amino acid, oligopeptide, protein, saccharide, oligosaccharide, polymer, polymer composite, metal-organic framework, or a solid support such as nanoparticle, nanomaterial, glass, metal, ceramics or metal-organic framework.

[0385] Item 2. The compound according to item 1, wherein

[0386] Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, a nucleic acid, a fluorophore, a chelator, a radioisotope, a wax, a sterol, a vitamin selected from Vitamins A, D, E and K, a monoglyceride, a diglyceride, a phospholipid, an amino acid, an oligopeptide, a protein, a monosaccharide, an oligosaccharide or a polysaccharide;

[0387] preferably Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, a nucleic acid, a fluorophore, chelating ligand and a radioisotope;

[0388] more preferably Q is a monovalent group comprising one or more groups selected from a nucleotide, an oligonucleotide, and a fluorophore;

[0389] even more preferably Q is any of the above and contains a triazole group;

[0390] Q preferably comprises 10 to 500 non-hydrogen-atoms, more preferably 10 to 300 non-hydrogen-atoms;

[0391] most preferably Q is selected from the following formulae:

[0392] O CH3

[0393] N." X,r A X HN - X IL J0 0o7z

[0394] N" 7 r-O-p-O-P-O-P— ''

[0395] o o

[0396] OH OH

[0397]

[0398]

[0399]

[0400] 

[0401]

[0402]

[0403] Item 3. A compound of Formula II

[0404]

[0405] wherein:

[0406] n is any integer with a value of from 1 to 20000;

[0407] each X is independently selected from O’, BHs’, NH’, S’, and Se_;

[0408] each G is independently selected from O and S;

[0409] each A is independently selected from CH2, O, S, NH and a bond;

[0410] each E is independently selected from CH2, O, S and NH;

[0411] each D is independently selected from CH2, O, S and NH;

[0412] each R1is independently selected from a natural or modified nucleoside base;

[0413] each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;

[0414] R3is selected from a natural or modified nucleoside base, L is a trivalent group having from 1 to 50 non-H atoms, wherein L is preferably a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 50 hydrogen atoms,

[0415] J is a monovalent group having from 1 to 70 non-H atoms, wherein J is preferably a monovalent group having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S, wherein J preferably comprises at least one group selected from C=C bond, -N3, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

[0416] Item 4. The compound according to item 3, wherein

[0417] J is a monovalent group having 0 to 20 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein J comprises at least one group selected from C=C bond, -NH2, -SH, -NHC(O)CH₂I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F; preferably J is selected from the following formulae:

[0418] O~

[0419] 6

[0420]

[0421] still more preferably, J is selected from the following formulae: o~

[0422] O-p-0 - 6

[0423] 0 H

[0424] < 1.-x „hk

[0425] v-o-p-o' " " ■'-" Y i

[0426] S:l I:

[0427] . 0 O

[0428]

[0429] and

[0430] Item 5. The compound according to any one of the preceding items, wherein

[0431] L is a trivalent group having from 1 to 40 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms,

[0432] L is a trivalent group having from 1 to 30 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms,

[0433] L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 30 hydrogen atoms,

[0434] preferably L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S and P, and up to 30 hydrogen atoms,

[0435] L preferably contains at least one -P(=O)(O-)O- group; and / or L preferably contains at least one branch selected from ternary carbon and an aryl group having at least 3 non-H-substituents; and / or L contains at least one secondary amine group and / or ether group; more preferably, L is selected from

[0436]

[0437] Item 6. The compound according to any one of the preceding items, wherein n is any integer with a value of from 90 to 20000, preferably from 100 to 10000, more preferably from 500 to 8000, even more preferably from 800 to 8000.

[0438] Item 7. The compound according to any one of the preceding items, wherein each X is independently selected from O’, S’ and Se’; preferably each X is independently selected from O’ and S’; more preferably each X is O’.

[0439] Item 8. The compound according to any one of the preceding items, wherein each G is O. Item 9. The compound according to any one of the preceding items, wherein each A is independently selected from O, S, NH and a bond; preferably each A is independently selected from O, S and a bond; more preferably each A is independently selected from O and a bond; even more preferably each A is O.

[0440] Item 10. The compound according to any one of the preceding items, wherein each E is independently selected from O and S; preferably each E is O.

[0441] Item 11. The compound according to any one of the preceding items, wherein each D is independently selected from O and S; preferably each D is O. Item 12. The compound according to any one of the preceding items, wherein each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0442] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH, preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5- hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine;

[0443] more preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0444] Item 13. The compound according to any one of the preceding items, wherein

[0445] each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN;

[0446] preferably each R2is independently selected from H, alkyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl is optionally substituted with one or more selected from F, Cl, Br and CN;

[0447] more preferably each R2is independently selected from H, Me, OH, OMe, O-CH2-aryl and F; even more preferably each R2is independently selected from H, OH, OMe and F;

[0448] still more preferably each R2is independently selected from OH and OMe;

[0449] still even more preferably each R2is OH;

[0450] Item 14. The compound according to any one of the preceding items, wherein R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O RR2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, - HC(O RR, -NHC(O)H, -NHC(O)NH?, - HC(O) H(RR, -NHC(O N(RR?, -NHC(O)O(RR), -NO2, -OH, -O-RR, - OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0451] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH, preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine;

[0452] more preferably R3is selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0453] Item 15. The compound according to any one of the preceding items, wherein J does not comprise -CH2-N3, preferably J is not -CH2-N3, more preferably J does not comprise N3. Item 16. The compound according to any one of the preceding items, wherein

[0454] the moiety –A-L(Q)– or –A-L(J)– does not represent any of the following formulae:

[0455]

[0456] wherein

[0457] X is selected from O’, BHs’, NH’, S’, and Se’;

[0458] G is selected from O and S;

[0459] A is selected from CH2, O, S, NH and a bond;

[0460] E is selected from CH2, O, S and NH;

[0461] Dbis selected from CH2, O, S and NH;

[0462] R1and Rlbare independently selected from a natural or modified nucleoside base;

[0463] R2is selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH₂, -C(O)NH(RR), -C(O)N(RR)?, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O) HZ, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR, -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;

[0464] L is a trivalent group having from 1 to 50 non-H atoms, wherein L is preferably a trivalent group having from 1 to 50 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 50 hydrogen atoms,

[0465] J is a monovalent group having from 1 to 70 non-H atoms, wherein J is preferably a monovalent group having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S, wherein J preferably comprises at least one group selected from C=C bond, -N3, -NH2, - SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

[0466] Item 17. The compound according to item 16, wherein

[0467] J is a monovalent group having 0 to 20 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein J comprises at least one group selected from C=C bond, -NH2, -SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F; preferably J is selected from the following formulae:

[0468] O - P - Oxo - p - o

[0469] II > I I

[0470] 0 0

[0471] o _ Q

[0472] J.-O-P-O "S- z t-O-P-O

[0473] T il ’ Ji

[0474] SH Z I „|

[0475] =■ n > s' 'H'-

[0476]

[0477] 0

[0478] ?>-O-P-O"'

[0479] N3

[0480]

[0481] and

[0482] still more preferably, J is selected from the following formulae:

[0483] 0

[0484] i-o-p-o

[0485] 11

[0486] 0

[0487]

[0488] Item 18. The compound according to item 16 or 17, wherein

[0489] L is a trivalent group having from 1 to 40 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms,

[0490] L is a trivalent group having from 1 to 30 atoms selected from halogen, C, N, O, S, Se, P and B and additionally up to 50 hydrogen atoms,

[0491] L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S, Se, P and B, and up to 30 hydrogen atoms,

[0492] preferably L is a trivalent group having from 3 to 30 atoms selected from halogen, C, N, O, S and P, and up to 30 hydrogen atoms, L preferably contains at least one -P(=O)(O O- group; and / or L preferably contains at least one branch selected from ternary carbon and an aryl group; and / or L contains at least one secondary amine group and / or ether group;

[0493] more preferably, L is selected from

[0494]

[0495] and / or

[0496] wherein L preferably does not comprise a natural, modified or unnatural 7-methylguanosine cap structure.

[0497] Item 19. The compound according to any one of items 16 to 18, wherein X is selected from O’, S’ and Se_; preferably X is selected from O’ and S’; more preferably X is O’.

[0498] Item 20. The compound according to any one of items 16 to 19, wherein G is O.

[0499] Item 21. The compound according to any one of items 16 to 20, wherein A is selected from O, S, NH and a bond; preferably A is selected from O, S and a bond; more preferably A is selected from O and a bond; even more preferably A is O.

[0500] Item 22. The compound according to any one of items 16 to 21, wherein E is selected from O and S; preferably E is O. Item 23. The compound according to any one of items 16 to 22, wherein Dbis selected from O and S; preferably Dbis O.

[0501] Item 24. The compound according to any one of items 16 to 23, wherein R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, - NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, - OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl,

[0502] and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH, preferably R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2- methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine;

[0503] more preferably R1and Rlbare independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0504] Item 25. The compound according to any one of items 16 to 24, wherein

[0505] R2is selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH₂-aryl, SH, S-alkyl, NH₂, NH(alkyl), N(alkyl)₂, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN;

[0506] preferably R2is selected from H, alkyl, OH, O-alkyl, O-CH₂-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN; more preferably R2is selected from H, Me, OH, OMe, O-CH2-aryl and F;

[0507] even more preferably R2is selected from H, OH, OMe and F;

[0508] still more preferably R2is selected from OH and OMe;

[0509] still even more preferably R2is OH;

[0510] Item 26. The compound according to any one of items 16 to 25, wherein J does not comprise -CH2-N3, preferably J is not -CH2-N3, more preferably J does not comprise N3.

[0511] Item 27. The compound according to any one of items 16 to 26, wherein

[0512] the moiety — A-L(J)— does not represent any of the following formulae:

[0513]

[0514] Item 28. A compound of Formula IV

[0515]

[0516] RC1and RC2are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl, and (hetero)aryl, wherein the alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl are optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), - NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;

[0517] RC3and RC4are independently selected from the group consisting of OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, NH2, NH(alkyl), F, Cl, Br, alkyl, alkenyl, and alkynyl, or RC3and RC4may be taken together to form -O-alkylidene-O-;

[0518] W1and W2are independently selected from the group consisting of O, S, Se, NH, CH2, alkylene, alkenylyne, alkynylene and a bond;

[0519] m and n are independently an integer selected from the group consisting of 0, 1 and 2.

[0520] XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are independently selected from the group consisting of O, S, Se, BH3and NH;

[0521] ZC1, ZC2, ZC3are independently selected from the group consisting of O, NH, CH2, CH(alkyl), CF(alkyl), CH(a lkyl)2, CHF, CF2, CFCI, CHCI and CCI2, wherein alkyl is optionally haloalkyl; L2comprises one or more groups selected from optionally substituted heteroalkylene, optionally substituted alkylene, peptides, oligonucleotides and their block co-polymers; chemically modified peptides, chemically modified oligonucleotides and their block copolymers; polyethylene glycol), poly(lactic acid) and their block co-polymers, wherein the wherein the one or more optional substituents of the heteroalkylene and the alkylene are selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), - HC(O) (RR2, -NHC O O(RR, -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, - OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;

[0522] Q2is a group having from 0 to 70 non-H atoms (such as 1 to 70 non-H atoms), wherein Q2is preferably a group having 0 to 50 carbon atoms and up to 20 heteroatoms selected from N, O, P and S, wherein the Q2preferably comprises at least one group selected from C=C bond, -N3, -SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F. Item 29. The compound of Formula IV of item 28, wherein

[0523] L2comprises optionally substituted alkylene or optionally substituted heteroalkylene, wherein the wherein the one or more optional substituents of the heteroalkylene and the alkylene are selected from OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(a I kyl)2, F, Cl, Br and CN; preferably L2consists of optionally substituted heteroalkylene, at least one nucleoside and optionally 1 to 15 further atoms selected from halogen, C, N, P, O, S;

[0524] more preferably L2is of formula (C-l):

[0525]

[0526] wherein the OH can optionally be replaced by O-alkyl;

[0527] RCL1is selected from optionally substituted heteroalkylene, and

[0528] RCS1and RCS2are each independently selected from a natural or modified nucleoside base; preferably, RCS1and RCS2are each independently a nucleobase optionally having a substituent selected from alkyl and alkylene-aryl at an exocyclic nitrogen;

[0529] even more preferable L2is selected from

[0530]

[0531] Item 30. The compound of Formula IV of item 28 or 29, wherein

[0532] Q2is a group having 0 to 50 carbon atoms and up to 20 heteroatoms selected from halogen, N, O, P and S, wherein Q2comprises at least one group selected from C=C bond, preferably Q2is a cyclic group having 2 to 25 carbon atoms and up to 10 heteroatoms selected from halogen, N, O, P and S, wherein Q2comprises at least one C≡C bond;

[0533] more preferably Q2comprises at least one eight-membered ring having a C=C bond in the ring; even more preferably, Q2is selected from

[0534]

[0535] Item 31. The compound of Formula IV of any one of items 28 to 30, wherein RC1is selected from H and alkyl, preferably alkyl, more preferably methyl.

[0536] Item 32. The compound of Formula IV of any one of items 28 to 31, wherein RC2is selected from H and alkyl, preferably H.

[0537] Item 33. The compound of Formula IV of any one of items 28 to 32, wherein RC3is selected from OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, F, Cl and Br, preferably from OH and OBn, more preferably OH.

[0538] Item 34. The compound of Formula IV of any one of items 28 to 33, wherein RC4is selected from OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, F, Cl and Br, preferably from OH and OBn, more preferably OH.

[0539] Item 35. The compound of Formula IV of any one of items 28 to 34, wherein W1is selected from O, S, Se, NH, methylene, CH2O and a bond, preferably O and S, more preferably O and / or W2is selected from O, S, Se, NH, methylene, CH2O and a bond, preferably O, S and methylene, more preferably O and methylene.

[0540] Item 36. The compound of Formula IV of any one of items 28 to 35, wherein XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are selected from O and S, preferably O.

[0541] Item 37. The compound of Formula IV of any one of items 28 to 36, wherein ZC1, ZC2, ZC3are independently selected from O and NH, preferably O.

[0542] Item 38. The compound of Formula IV of any one of items 28 to 37, wherein m is 0 or 1 and n is 1 or 2, preferably wherein m is 0 and n is 1.

[0543] Item 39. The compound of Formula IV of any one of items 28 to 38, wherein

[0544] the optionally substituted heteroalkylene corresponds to a (preferably linear) C1-30 alkylene wherein one to ten CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and which may be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2;

[0545] preferably the optionally substituted heteroalkylene preferably corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and which may be substituted with one or more selected from F, OH, CN, O(Me), NH2, NH(Me), and N(Me)2,

[0546] more preferably the optionally substituted heteroalkylene preferably corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2groups are independently replaced by S, O, NH, N(alkyl) or C=O and which may be substituted with one or more selected from F, OH, CN, O(Me), NH2, NH(Me), and N(Me)2.

[0547] Item 40. Use of the compound of Formula II of any one of items 3 to 15 for the manufacture of a compound of Formula I of any of items 1, 2 and 5 to 15.

[0548] Item 41. Use of the compound of Formula III of any one of items 16 to 27 for the manufacture of a compound of Formula I of any of items 1, 2 and 5 to 15.

[0549] Item 42. The use according to item 41, wherein the manufacture of the compound of Formula I involves reacting a compound of the following Formula V

[0550]

[0551] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II in any of items 3 to 15,

[0552] to form a compound of the following Formula VI:

[0553]

[0554] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II in any of items 3 to 15.

[0555] Item 43. The use according to item 42, wherein reacting the compound of Formula V involves oxidation of the compound of Formula V, preferably in the presence of a periodate, such as NaIO4, wherein the compound of Formula V is preferably at a concentration below 10 mM and / or wherein reacting the compound of Formula V with the periodate is preferably conducted at a temperature below 60°C.

[0556] Item 44. The use according to any one of items 41 to 43, wherein the manufacture of the compound of Formula I involves reacting a compound of the following Formula VI

[0557]

[0558] wherein each of A, L, J, E, G, X, D, R1, R2, R3and n are as defined for Formula II,

[0559] to form a compound of Formula II.

[0560] Item 45. The use according to item 44, wherein the compound of Formula VI is reacted with a borohydride, preferably Na[BH3(CN)], preferably at a pH in the range of from 5 to 8, preferably while the compound of Formula VI is at a concentration of less than 100 mM. Item 46. Use of the compound of Formula IV of any one of items 28 to 39 for the manufacture of a compound of Formula I of any of items 1, 2 and 5 to 15.

[0561] Item 47. The use of item 46, wherein the compound of Formula IV is reacted with the compound of Formula II of any one of items 3 to 15 to form the compound of Formula I of any of items 1, 2 and 5 to 15.

[0562] Item 48. A compound which is obtainable by reacting the compound of Formula II of any one of items 3 to 15 with the compound of Formula IV of any of items 28 to 39.

[0563] Item 49. The use of item 47 or the compound of item 48, wherein

[0564] J in the compound of Formula II contains an azide group and Q2in the compound of Formula IV contains an alkyne group (preferably a cycloalkyne group), or J in the compound of Formula II contains an alkyne group (preferably a cycloalkyne group) and Q2in the compound of Formula IV contains an azide group.

[0565] Item 50. A compound the following Formula VII:

[0566]

[0567] wherein A, L, Q, E, G, X, D, R1, R2, R3and n are as defined for Formula I in any of items 1, 2 and 5 to 15.

[0568]

[0569] 5 to 15.

[0570] Item 52. Use of the compound of Formula VII of item 50 or compound Formula VII of item 51 for the manufacture of a compound of Formula I of any of items 1, 2 and 5 to 15.

[0571] Item 53. The use according to item 52, wherein the compound of Formula VII is oxidized to form the compound of Formula VIII, preferably in the presence of a periodate, such as NaIO4, wherein the compound of Formula VII is preferably at a concentration below 10 mM and / or wherein reacting the compound of Formula VII with the periodate is preferably conducted at a temperature below 60°C. Item 54. The use according to item 52 or 53, wherein the compound of Formula VIII is reacted with a borohydride, preferably Na[BH3(CN)], preferably at a pH in the range of from 5 to 8, preferably while the compound of Formula VIII is at a concentration of less than 100 mM, to form the compound of Formula I.

[0572] Item 55. A kit of parts, the kit comprising a compound of any of formulae II, III, IV, V, VI, VII and VIII, as defined in any one of items 3 to 51, and instructions for the preparation of a compound of Formula I as defined in any of items 1, 2 and 5 to 15.

[0573] Item 56. A compound according to any of items 1 to 39, and 48 to 51, or the kit according to item 55, wherein the compound or kit is for the treatment of a cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, ora rare disease; wherein the cancer is preferably selected from breast cancer, lung cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, esophageal cancer, melanoma, lymphoma, leukemia; wherein cardiovascular diseases are preferably selected from myocardial infarction, heart failure, atherosclerosis, hypertension, cardiac hypertrophy, and arrhythmias;

[0574] wherein neurological disorders are preferably selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, multiple sclerosis, Huntington's disease, epilepsy, and traumatic brain injury;

[0575] wherein metabolic disorders are preferably selected from diabetes mellitus (type 1 and type 2), obesity, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome; wherein infectious diseases are preferably selected from viral infections (e.g., hepatitis B and C, HIV, influenza, SARS-COV-2), bacterial infections (e.g., tuberculosis, staphylococcus aureus), fungal infections (e.g., candida), and parasitic infections (e.g., malaria);

[0576] wherein genetic disorders is preferably selected from Duchenne muscular dystrophy, cystic fibrosis, spinal muscular atrophy, hemophilia, sickle cell disease, and thalassemia; wherein inflammatory and autoimmune diseases are preferably selected from rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, and asthma; wherein ophthalmic diseases are preferably selected from age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma;

[0577] wherein pulmonary diseases are preferably selected from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, asthma, and cystic fibrosis;

[0578] wherein renal diseases are preferably selected from chronic kidney disease (CKD), acute kidney injury, and polycystic kidney disease;

[0579] wherein gastrointestinal diseases are preferably selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), gastric ulcers, and liver cirrhosis;

[0580] wherein dermatological diseases are preferably selected from psoriasis, atopic dermatitis, vitiligo, and melanoma;

[0581] wherein musculoskeletal diseases are preferably selected from osteoarthritis, rheumatoid arthritis, osteoporosis, and muscular dystrophies;

[0582] wherein endocrine disorders are preferably selected from thyroid diseases (e.g., hypothyroidism, hyperthyroidism), adrenal insufficiency, and polycystic ovary syndrome (PCOS);

[0583] wherein hematological diseases are preferably selected from anemia, leukemia, lymphoma, and hemophilia.;

[0584] wherein rare diseases are preferably selected from Huntington's disease, Gaucher disease, Fabry disease, and Pompe disease.

[0585] Item 57. A compound according to any of items 1 to 39 and 48 to 51, or the kit according to item 55, wherein the compound or kit is for the prevention (preferably by vaccination) of a cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, ora rare disease; wherein the cancer is preferably selected from breast cancer, lung cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, esophageal cancer, melanoma, lymphoma, leukemia; wherein cardiovascular diseases are preferably selected from myocardial infarction, heart failure, atherosclerosis, hypertension, cardiac hypertrophy, and arrhythmias; wherein neurological disorders are preferably selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), stroke, multiple sclerosis, Huntington's disease, epilepsy, and traumatic brain injury;

[0586] wherein metabolic disorders are preferably selected from diabetes mellitus (type 1 and type 2), obesity, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome; wherein infectious diseases are preferably selected from viral infections (e.g., hepatitis B and C, HIV, influenza, SARS-COV-2), bacterial infections (e.g., tuberculosis, staphylococcus aureus), fungal infections (e.g., candida), and parasitic infections (e.g., malaria);

[0587] wherein genetic disorders is preferably selected from Duchenne muscular dystrophy, cystic fibrosis, spinal muscular atrophy, hemophilia, sickle cell disease, and thalassemia; wherein inflammatory and autoimmune diseases are preferably selected from rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis, and asthma;

[0588] wherein ophthalmic diseases are preferably selected from age-related macular degeneration (AMD), retinitis pigmentosa, diabetic retinopathy, and glaucoma;

[0589] wherein pulmonary diseases are preferably selected from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, asthma, and cystic fibrosis;

[0590] wherein renal diseases are preferably selected from chronic kidney disease (CKD), acute kidney injury, and polycystic kidney disease;

[0591] wherein gastrointestinal diseases are preferably selected from inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), gastric ulcers, and liver cirrhosis;

[0592] wherein dermatological diseases are preferably selected from psoriasis, atopic dermatitis, vitiligo, and melanoma;

[0593] wherein musculoskeletal diseases are preferably selected from osteoarthritis, rheumatoid arthritis, osteoporosis, and muscular dystrophies;

[0594] wherein endocrine disorders are preferably selected from thyroid diseases (e.g., hypothyroidism, hyperthyroidism), adrenal insufficiency, and polycystic ovary syndrome (PCOS);

[0595] wherein hematological diseases are preferably selected from anemia, leukemia, lymphoma, and hemophilia.;

[0596] wherein rare diseases are preferably selected from Huntington's disease, Gaucher disease, Fabry disease, and Pompe disease. Description of further aspects relevant to the present invention:

[0597] The present invention relates to circular RNA (circRNA) analogs obtained by circularization of a circRNA precursor containing two reactive functional groups, one at the 5'-end and a second at the 3' end. [Fig.1A] The reactive functional group at the 5'-end is incorporated into RNA during enzymatic synthesis (i.e. in vitro transcription reaction). The 3'-end reactive functional group is incorporated into RNA post-synthetically, by either enzymatic reaction or, preferably, by chemical reaction. The 5' and 3' reactive groups are reactive towards each other either spontaneously or in the presence of a specific trigger, such as the addition of a chemical reagent, addition of a catalysts, a change of temperature, presence of light, or a change in the pH of the solution, etc. Representative pairs of 5' and 3' reactive groups reactive towards each other include, but are not limited to: EDA and dialdehyde, hydrazide and dialdehyde, azide and alkyne, azide and cyclooctyne derivative, amino and carboxylic group, amino and fluorosulfonylgroup, azide and phosphine, etc..

[0598] A representative example of a functional group at the 5'-end is the EDA motif (EDA-linker-RNA). [Fig.1B] The precursor is a modified nucleic acid that can be prepared by in vitro transcription. The 5'-EDA motif can be readily installed during the in vitro transcription (IVT) reaction if the transcription is carried out in the presence of an EDA-modified transcription initiator [see Table 1 for examples].

[0599] This is beneficial, as unlike other methods of chemical circularization, the precursor's length is not limited by the efficiency of RNA solid-phase synthesis.

[0600] The 5'-EDA motif and the nucleic acid chain of the EDA-linker-RNA are joined by a linker. The linker itself may be a linear or branched combination of simple chemical moieties (such as carbohydrate or PEG linkers), or it may contain more complex elements, such as detectable labels. The circRNA precursor can bear additional modifications, such as detectable labels, as long as they do not interfere with the circularization reaction.

[0601] This is beneficial, as in contrast to enzymatic circularization, the user is not restricted by the activity and substrate-specificity of a particular enzyme.

[0602] The chemical circularization reaction is a two-step process. First, the circRNA precursor is selectively oxidized with oxidizing agents such as periodate at the 3' end to introduce a reactive functional group (a dialdehyde) at the 3' end of RNA. This occurs via ring-opening oxidation and the formation of a reactive acyclic dialdehyde derivative of the terminal 3'-end ribose. During the second step, the 3'-end dialdehyde derivative spontaneously reacts with the 5' EDA motif. This step requires reductive environment for optimal performance. If a reductive environment is ensured during this reaction, the reduction of the formed intermediate occurs, making the whole process irreversible. Except for the presence of the 5' EDA motif (5' reactive group) and the 3' dialdehyde motif (3' reactive group), chemical circularization has no requirements concerning chemical structure or the sequence of the circRNA precursor.

[0603] This is beneficial, as in contrast to circularization techniques based on activity of catalytic nucleic acids (PIE and Tornado systems), the invented chemical circularization does not require sequence engineering and construct cloning for preparation of the circRNA precursor. CircRNA precursors containing EDA motif were prepared by means of IVT and subsequently circularized [Table 1]. As a comparison, precursors bearing a 5' monophosphate moiety and an identical sequence were subjected to enzymatic circularization catalyzed by T4 RNA ligase I or T4 RNA ligase II. Unexpectedly, the yield of chemical circularization, in comparison to enzymatic circularization, is less dependent on the precursor sequence and is often more efficient [Fig.2A], In some of the investigated enzymatic circularization processes, the predominant outcome involves the generation of linear and circular dimers, while the chemical circularization predominantly results in the formation of the desired circular RNA product [Fig. 2B],

[0604] The method for chemical circularization of mRNAs as disclosed herein is applicable for circularization of long RNA molecules. Crude products of circularization reaction are a noncomplex mixture composed of the desired circRNA product and unreacted linear pre-circRNA. The yield of circularization reaction is high and independent of pre-circRNA length. The circularization procedure requires no additional steps, in contrast to PIE methodology that often requires a recircularization step. The above-mentioned recircularization step, due to prolonged incubation of RNA in presence of magnesium salts, can lead to degradation (nicking) of the desired circRNA product, as well as the formation of a complex mixture of byproducts, which can interfere with the subsequent purification steps [Fig. 3], The circular topology of the circRNA analogs obtained by chemical circularization can be verified with exonucleolytic digestion assays, endonucleolytic assays, as well as electrophoretic mobility assays. Exonucleolytic digestion assays allow one to distinguish linear RNAs from circular RNAs, as exonucleases can hydrolyze only RNA molecules that contain free 5' or 3' end. On the other hand, circular RNAs are resistant to exonucleolytic hydrolysis as they lack free 5' and 3' ends. Rnase R is a processive 3' exonuclease, that is most commonly used for such assay (NPTL3) The products of chemical circularization were treated with Rnase R [Fig. 4], In contrary to the precursors, the circRNA analogs were resistant to Rnase R hydrolysis.

[0605] Endonucleolytic assays are based on a phenomenon of nicking and linearization of circRNA. Site-selective and sequence-dependent cleavage of a circular RNA molecule leads to a single linear RNA molecule of equal size. On the other hand, cleavage of a linear RNA molecule leads to two linear RNA molecules of smaller sizes. Such assay can be performed with Rnase H, an endonuclease, that nicks RNA in RNA-DNA duplexes (NPTL25). Rnase H and targeting DNA oligonucleotides were used for nicking of the products of chemical circularization [Fig. 5], The assay confirmed that, in contrast to the precursors, nicking of the circRNA analogs leads to their linearization.

[0606] Electrophoretic mobility of nucleic acids is affected by their size, charge, conformation, and topology (NPTL26 and 27). Thanks to that, circRNA and their precursors can be separated during polyacrylamide gel electrophoresis in denaturing conditions [Fig. 6], The crosslinking degree of the polyacrylamide gel matrix is a crucial factor for the separation of RNA based on their topology. Electrophoretic separation of linear and circular RNA using a cross-linked polyacrylamide gel is significantly better than with regular or commercially available agarose gels (NPTL27).

[0607] The circularization efficiency is affected by the sequence of the circRNA precursor. [Tab. 2] In some cases, such as sequences containing a 3' polyA sequence, the circularization yield can be increased by the addition of a complementary nucleic acid oligomer. [Tab. 3] During the reaction, the oligomer and precursor hybridize and affect the conformation of the precursor. Using an oligomer with an optimal design, the conformational change of the precursor decreases the distance between the 5' and 3' ends [Fig. 7]. In consequence, the optimal distance and orientation of the 5' and 3' ends increase the circularization yield [Fig. 8].

[0608] The present invention also relates to methods of preparation and isolation of circRNA analogs. This includes development of chromatographic methods of linear and circular RNA resolution. Previously reported methods of chromatography of circularization products were based on size-exclusion chromatography in an HPLC setup (SEC-HPLC) (NPTL11, 12 and 14). This method Ill

[0609] allows one to separate sample components based on hydrodynamic radius and is considered to provide one of the mildest chromatographic conditions. SEC-HPLC was used to resolve the products of PIE-based circularization (NPTL11, 12 and 14). In these cases, the precursor and circRNA significantly differ in molecular size between each other, as well as form side products (such as free introns). In case of synthetic polymers (such as polystyrene or poly(ethyneneoxide)) the hydrodynamic radius (Stokes radius) is decreased upon circularization (NPTL28). This property was used to separate linear and cyclic polymers using gel permeation chromatography (GPC). However, the decrease in the Stokes radius of an RNA molecule caused by its circularization has not been documented. One could speculate, that intramolecular interactions, that contribute to the existence of a landscape of RNA conformers, may diminish the influence of topological change on the differentiation of Stokes radii of circular and linear RNA (NPTL29). This could also explain why SEC-HPLC under mild conditions leads to the coelution of intact and nicked circRNA (NPTL12 and 14).

[0610] During the development of the invention, we found that both size-exclusion chromatography (SEC-HPLC) and ion-exchange HPLC (IE-HPLC) are insufficiently effective for separation of linear and circular RNA of the same molar mass [Fig. 9], Reversed-phase HPLC (RP-HPLC) in ion-pair mode was found to be the most suitable method for the separation of circularization products [Fig. 9, 10], Chromatography was performed using polystyrene-divinylbenzene (PSDVB) resins, elevated temperature (50-80 °C), and a mixture of an aqueous solution of ion pair reagent with acetonitrile as a mobile phase. The separation of linear and circular RNA is affected by composition of the mobile phase, namely the chosen ion pair reagent. The most effective resolution was obtained with 100 mM n-hexylammonium acetate (HAA), with a linear gradient of acetonitrile (45-58,5% in 25 min, flowrate 1.00 ml / min at 60 °C) [Fig. 11], The present invention also relates to therapeutic application of circRNA analogs. To demonstrate the applicability of protein-encoding circRNA analogs in gene delivery, circRNA analogs encoding the reporter protein, Gaussia luciferase were synthesized and expressed in cell lines HEK 293T, A549, HeLa and Hep G2. The levels of the reporter protein were higher in case of expression of chemically modified circRNA analogs (circRNA12, 13) than unmodified circRNA (circRNAll) [Fig. 13], Both modified and unmodified circRNAs showed increased duration of protein expression in comparison to their linear precursors (pre-circRNAll-12) [Fig. 14], The chemically circularized mRNAs had higher translation efficiency compared to their liner precursors [Fig. 15], The result shows that chemical modification within the circRNA analogs is beneficial in the context of therapeutic protein expression.

[0611] The following tables brief descriptions are provided for an improved understanding of the present invention:

[0612] Table 1: List of DNA templates (linearized plasmids) and corresponding RNA sequences with description and properties. Column description: # plasmid - designation of a plasmid; Restrictase - name of the enzyme used for linearization of the plasmid; # RNA sequence -designation of a RNA sequence encoded in the DNA template for IVT; RNA sequence elements - 5' and 3' untranslated regions (5' UTR and 3' UTR), protein-coding sequence (CDS), sequence downstream of the 3' UTR (3' tail); RNA length - total length of the transcript; RNA E260 -molar extinction coefficient calculated for particular RNA sequence (NPTL30). HBB: human betaglobin UTR (5' or 3') sequence, HBB (30nt): 30 consecutive nucleotides of human betaglobin sequence, Kozak sequence, EMCV: Encephalomyocarditis virus IRES, RSV: respiratory syncytial virus IRES, OligolRES: synthetic IRES, eGFP: enhanced green fluorescent protein sequence, Glue: Gaussia-Dura luciferase sequence, A30: 30 nucleotide-long polyadenine sequence. # Plasmid Restrictase # RNA sequence RNA sequence elements RNA length RNA E260[nt] [μM-1cm-15' UTR CDS 3' UTR 3' tail

[0613] PL1 Bam HI RNA1 - eGFP - - 740 8.93 PL2 Bam HI RNA2 - Glue - - 578 6.99 PL3 Bam HI RNA3 HBB - - 626 7.58 Pme\ RNA4 HBBx2 - 918 11.0 Aar\ RNA5 HBBx2 A30 952 11.5 PL4 Bam HI RNA6 EMCV - - 1152 13.9 PL5 RNA7 RSV - - 598 7.25 PL6 RNA8 OligolRES - - 711 9.35 PL7 Esp3\ RNA9 EMCV HBBx2 A30 1446 17.86 PL8 Aar\ RNA10 Kozak HBBx2 A30 908 10.99 PL9 Esp3\ RNA11 EMCV eGFP - pAC 1492 13.51 PL10 EcoS2\ RNA12 EMCV Glue - pAC 1612 19.61 PL11 Esp3\ RNA13 EMCV - A30 1207 14.39 PL12 PCR RNA14 EMCV eGFP - A30 1493 16.31 PL13 Xba\ RNA15 EMCV - - 1458 15.82 PL14 RNA16 HBB - 1494 16.19 (30nt)

[0614] PL15 RNA17 - A30 1494 16.34 PL16 RNA18 HBB - 1597 17.34 PL17 RNA19 HBB - - 925 10.05 PL18 RNA20 HBB(30nt) - 961 10.41 PL19 RNA21 - A30 968 10.65 PL2O RNA22 HBB - 1064 11.56 PL21 Xba\ RNA23 EMCV hEpo HBB(30nt) - 1221

[0615] PL22 Xba\ RNA24 HBB HBB(30nt) - 688

[0616]

[0617] PL23 Xba\ RNA25 EMCV GLuc - A30 1200 13.18

[0618]

[0619] PL24 Xba\ RNA26 HBB - A30 677 7.41 Table 2: List of circRNA and yields of their chemical or enzymatic circularization. Column description: # circRNA - designation of a circRNA and corresponding pre-circRNA; # RNA sequence -designation of an RNA sequence encoded in the DNA template for IVT; IVT initiator - compound used as transcription initiator in IVT reaction (GMP: guanosine 5'-monophosphate, EDA-PEG5-Cap1: trinucleotide cap analog with EDA motif (see Wasinska-Kalwa, et al. 2025, Nat. Commun. https: / / doi.org / 10.1038 / s41467-025-61775-1), Cap1: m7GpppAmpG (m7G(5')ppp(5')(2'OMeA)pG) trinucleotide cap structure, EDA-PEG5-AG: Example Al, N3-glE-pApG: Example B-l-5, NA: Not applicable); DBCO-Tag - compounds used in conjugation reaction (SPAAC) with (SPAAC) respective circRNAs (NA: Not applicable, MGC-1: Example B-2-3, MGC-14B: Example B-2-35, MGC-7B: Example B-2-19, MGC-5B: Example B-2-12, MGC-11: Example B-2-32, MGC-3B: Example B-2-7, AcPFVYLI: commercially available compound (see https: / / lipopharm.pl / index.php / en / peptides-and-lipopeptides-synthesis), CLS-PEG4: commercially available compound (see: https: / / www.biochempeg.com / product / CLS-PEG4-DBCO.html (CatalogID: 12584)), Tri-GalNAc: commercially available compound (see: https: / / www.medchemexpress.com / tri-galnac-dbco.html (Catalog No.: HY-148476), Biotin-PEG4: commercially available compound (see: https: / / broadpharm.com / product / bp-22295 (Catalog No.: BP22295)); Circularization type - type of reaction (chemical (Chem.) or enzymatic catalyzed by T4RNA ligase I or T4 RNA ligase II) used for RNA circularization; DNA oligonucleotide - information on whether a complementary DNA oligonucleotide was used during particular circularization reaction; Circularization yield - fraction of the circRNA precursor converted to particular circRNA product during circularization reaction, determined by PAGE of crude circularization products and densitometric analysis; DBCO-Tag conversion - fraction of the circRNAs with azide group converted to particular conjugated circRNAs product during SPAAC reaction, determined by HPLC analysis. # circRNA # RNA IVT initiator DBCO-Tag Circularization type DNA Circularization yield DBCO-Tag sequence oligonucleotide [%] conversion [%] circRNAl RNA1 GMP NA Enz. (T4 Lig. 1) w / o ON 5-10 NA circRNA2 EDA-PEG5-AG NA Chem. 30-45 NA circRNA3 RNA2 GMP NA Enz. (T4 Lig. 1) 36 NA circRNA4 EDA-PEG5-AG NA Chem. 60 NA circRNA5 EDA-PEG5-Cap1 53 NA circRNA6 RNA3 EDA-PEG5-Cap1 NA Chem. 46 NA circRNA? RNA4 GMP NA Enz. (T4 Lig. 1) w / o ON 13 NA Enz. (T4 Lig. 2) w / o ON 31 NA ONI 1-45 NA circRNA8 EDA-PEG5-Cap1 NA Chem. w / o ON 10 NA ONI 50 NA circRNA9 RNA5 GMP NA Enz. (T4 Lig. 1) w / o ON 9 NA Enz. (T4 Lig. 2) w / o ON 32 NA ON4 61 NA circRNAlO EDA-PEG5-Cap1 NA Chem w / o ON 2 NA ON4 40 NA circRNAll RNA6 GMP NA Enz. (T4 Lig. 1) w / o ON 10 NA Enz. (T4 Lig. 2) ON6 46 NA circRNA12 EDA-PEG5-AG NA Chem w / o ON 44 NA ON6 44 NA circRNA13 EDA-PEG5-Cap1 ON6 41 NA circRNA14 RNA7 GMP NA Enz. (T4 Lig. 1) w / o ON 40 NA Enz. (T4 Lig. 2) 84 NA circRNA15 EDA-PEG5-AG NA Chem 52 NA circRNA16 RNA8 GMP NA Enz. (T4 Lig. 1) 5-10 NA

[0620]

[0621] circRNA17 EDA-PEG5-AG Chem 53 NA circRNA18 RNA9 EDA-PEG5-Cap1 NA Chem ON7 57 NA

[0622] circRNA19 RNA10 EDA-PEG5-Cap1 NA Chem ON8 58 NA circRNA20 RNA11 GMP NA Enz. (T4 Lig. 1) ON9 1-3 NA circRNA21 EDA-PEG5-AG Chem 37-51 NA circRNA22 RNA12 NA PIE NA 34-54 NA linRNA23 RNAO5 Capl NA NA NA NA circRNA24 RNA13 N3-glE-pApG NA Chem ON7 55 NA circRNA25 RNA14 30

[0623] circRNA26 RNA25 EDA-PEG5-AG NA Chem ONIO 64 NA circRNA27 N3-glE-pApG 54

[0624] circRNA28 RNA26 N3-glE-pApG NA Chem ON11 54 NA circRNA29 RNA25 N3-glE-pApG MGC-1 Chem NA NA

[0625] circRNA30 MGC-14B 91 circRNA31 MGC-7B 82 circRNA32 MGC-5B 97 circRNA33 MGC-11

[0626] circRNA34 MGC-3B 94 circRNA35 RNA26 N3-glE-pApG MGC-1 Chem NA NA

[0627] circRNA36 MGC-14B 87 circRNA37 MGC-7B 86 circRNA38 MGC-5B 96 circRNA39 MGC-11

[0628] circRNA40 MGC-3B 97 circRNA41 RNA25 N3-glE-pApG AcPFVYLI Chem NA NA

[0629] circRNA42 CLS-PEG4 <90 circRNA43 Tri-GalNAc <90

[0630]

[0631] circRNA44 Biotin-PEG4 <90 Table 3: List of complementary DNA oligonucleotides used for RNA circularization and FRET experiments. Column descriptions: WON - designation of a DNA oligonucleotide sequence; W RNA sequence - designation of an RNA sequence; ON sequence - sequence of the designated oligonucleotide.

[0632] W ON W RNA sequence ON sequence

[0633] ONI RNA4

[0634] ON2

[0635] ON3

[0636] ON4 RNA5

[0637] ON5

[0638] ON6 RNA6

[0639] RNA9

[0640] ON7

[0641] RNA13

[0642] ON8 RNA10

[0643] ON9 RNA11

[0644] ONIO RNA25

[0645]

[0646] ON11 RNA26 Table 4: List of plasmids and inserted sequences, used for preparation of IVT templates. Underlined is the coding sequence from the T7 RNA polymerase transcription start site to the site of linearization. The recognition sequence for BamHI (GGATCC), Pme\ (GTTTAAAC) and Aarl (AAAAAAAAAGCAGGTG (SEQ ID NO: 10)A Esp3\ (CGTCTC / V), Eco52\ (CGGCCG) and Xbal (TCTAGA) are in bold with the cut site between nucleotides in italic.

[0647] GCCGAATTATATTAI 1 1 1 IGCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCA 1 1 1 AAATTCTTAGATGATACTTCATC TGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGTAATACGACTCACTATAGGGGGTACGCCACCATGGTGAGCAAGGG CGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCC GGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT GGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCT TCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCC GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGG PL1 TGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGATCCTAGG GCCGCAGCTCGCTTTCTTGCTGTCCAAI 1 1 C 1 A 1 FAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATT ATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTA 1 1 1 1 CA TTGCG 1 1 1 AGCTCGC 1 1 1 C 1 1 GCTGTCCAAT TTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTG CCTAATAAAAAACATTTATTTTCATTGCATTTAAATGTTTAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAAC (SEQ ID NO: 11) GCCCCTGCAGCCGAATTATATTAI 1 1 1 I GCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGTAATACGACTCACTATAGGGGGTACGCCACCATGGG AGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCG TGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGA GGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGT GCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCAT AGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCG PL2 ATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTG CCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGGA TCCTAGGGCCGCAGCTCGC 1 1 1 C 1 1 GCTGTCCAA 1 1 1 C 1 A 1 1 AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGG GGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACA 1 1 1 ATTTTCATTGCG 1 1 1 AGCTCGC 1 1 1 C 1 1 GC TGTCCAAI 1 ICIAI 1 AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTT G G ATTCTG CCTAATAAAAAAC ATTTATTTTCATTG CATTTAAATGTTTAAACAAAAAAAAAAAA AAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAAC (SEQ ID NO: 12) CTGCAGCCGAATTATATTAI 1 1 1 1 GCCAAATAA 1 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCA 1 1 1 AAATTCTTAGATGAT AC TTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGATAATCTAGACATTTGCTT CTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGT GGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTC GATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAA GCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACG CTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCT PL3 GGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAA AGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCC AGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGGATCCTAGGGCCGCAGCTCGC 1 1 I CI 1 GCTGTCCAAT TTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTG CCTAATAAAAAACATTTAT 1 1 1 CA ITGCGTTT AGCTCGC 1 1 1 C 1 1 GCTGTCCAA 1 1 1 C 1 ATTAAAGGTTCCTTTGTTCCCTAAG TCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCA TTTAAATGTTTAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAAC AAACGGCCGC (SEQ ID NO: 13)

[0648] CCTGCAGCCGAATTATATTA 1 1 1 1 IGCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCA 1 1 1 AAATTCTTAGATGAT A CTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGTACCCCTCTCCCTCCC PL4 CCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG 1 1 1 G 1 C 1 A 1 ATGTTA I l l i CCACCATATTGCCGTC

[0649]

[0650] TTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGG AATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCC TTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAG GCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAA GGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTT AGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCC ACACATATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGA CTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGC TGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTC CCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCAC AGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATC GCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAA GAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGG TGACTAAGGATCCTAGGGCCGCAGCTCGC I 1 I C I I GCTGTCCAA I 1 I C I A l 1 AAAGGTTCCTTTGTTCCCTAAGTCCAACTA CTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGTTTAGCTC GC 1 1 1 C 1 1 GCTGTCCAA H I L I ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCC TTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCATTTAAATGTTTAAACAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAACGGCCGCT (SEQ ID NO: 14) CCTGCAGCCGAATTATATTA 1 1 1 1 I GCCAAATAA I 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCA 1 1 1 AAATTCTTAGATGATA CTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGTAATGAGTCCTGGAC TGAAACGGACTCATATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAAC AACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCG GCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGAT CTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAG AGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGA PL5 G CAGTTCATCGCAC AG GTCG ATCTGTGTGTG G ACTG CACAACTG GCTG CCTCAAAG G GCTTG CCAACGTGCAGTGTTCTG ACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGG GGCCGGTGGTGACTAAGGATCCTAGGGCCGCAGCTCGC 1 1 1 C 1 1 GCTGTCCAA H I L I ATTAAAGGTTCC 1 1 1 GTTCCCTAA GTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC GTTTAGCTCGC 1 1 1 C 1 1 GCTGTCCAA H I L I ATTAAAGGTTCC 1 1 1 GTTCCCTAAGTCCAACTACTAAACTGGGGGATATTAT GAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCATTTAAATGTTTAAACAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAACGGCCGCT (SEQ ID NO: 15) GCCCCTGCAGCCGAATTATATTA I 1 1 1 I GCCAAATAA I 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGTAGGCGCGCCA GTCCTTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTA TAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTC CTCAAGCGTATTCAACAAG CG ACTCG G ACATATG G G AGTCAAAGTTCTGTTTG CCCTG ATCTG CATCG CTGTGG CCG AG G CCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGAC CGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGC ACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACAC PL6 CTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAG GACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGC CAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGG TGGACAAGATCAAGGGGGCCGGTGGTGACTAAGGATCCTAGGGCCGCAGCTCGC 1 1 1 C 1 1 GCTGTCCAA 1 1 1 C 1 A 1 1 AAA GGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAA AAACATTTA I l l i CATTGCG 1 1 1 AGCTCGC 1 1 1 C 1 1 GCTGTCCAA 1 1 1 C 1 A 1 1 AAAGGTTCCTTTGTTCCCTAAGTCCAACTAC TAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCATTTAAATGT TTAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAACGGCCG CT (SEQ ID NO: 16)

[0651] GCCCCTGCAGCCGAATTATATTA I 1 1 1 I GCCAAATAA I 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGTACCCCTCTCCCT CCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCC GTC 1 1 1 1 GGCAA 1 G 1 GAGGGCCCGGAAACC 1 GGCCC 1 G 1 C 1 1 C 1 1 GACGAGCA 1 1 CC 1 AGGGG 1 C 1 1 1 CCCCTCTCGCCAA AGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGA CCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCA AAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAA CAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTG PL7 TTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATG GCCACACATATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGA AGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAG AAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCC TGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCC GCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGT TCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGC

[0652]

[0653] TCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCG GTGGTGACTAAGGATCCTAGGGCCGCAGCTCGC 1 1 1 C 1 1 GCTGTCCAA H I LI ATTAAAGGTTCC 1 1 1 G 1 ICCCTAAGTCCA ACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGTTTA GCTCGC 1 1 1 C 1 1 GC 1 G 1 CCAA H I LI ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAG GGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCATTTAAATGTTTAAACAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA4GAGACGGGGTACGTTCGTGATACGGCCGCTTCGAGCAGA (SEQ ID NO: 17) GCCCCTGCAGCCGAATTATATTAI 1 1 1 I GCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATCACGCTGTGTAATACGACTCACTATAGGGGGTACGCCACCATGGG AGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCG TGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGA GGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGT GCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCAT AGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCG PL8 ATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTG CCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGGA TCCTAGGGCCGCAGCTCGC 1 1 1 C 1 1 GG 1 G 1 CCAA H ILI ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGG GGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGTTTAGCTCGCTTTCTTGC TGTCCAA 1 1 1 C 1 ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTT G G ATTCTG CCTAATAAAAAAC ATTTATTTTCATTG CATTTAAATGTTTAAACAAAAAAAAAAAA AAAAAAAAAAAAAAAAA AAAAAGCAGGTGGGAGGGTACGTTCGTGATAAACAAACGGCCGCT (SEQ ID NO: 18) GCCCCTGCAGCCGAATTATATTAI 1 1 1 I GCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGTAAAACAAAACAC ACCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTC CACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTC CCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAA CGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAA GATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTC AAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACA TGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACAC GATGATAATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGG PL9 ACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGA AGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTC AGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGGTACGTGCAGGAGCGGACCA TCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGGACACCCTGGTGAACCGCATCGA GCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTG TACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCAGCGTGC AGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTCCTGCCCGACAACCACTACCTGTCCACCC AGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGCATCAC CCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTCCCGCCGTGGCCGCCCAGGACGACG GCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGACCGGCACCCGGCCGCCTGCGCCAGCGCCCGGATCAACGT GTGATAGGGATCCAACAAAAAAAACAAAAAAAAAACCAAAGAGACGCGGCCGCTTCGAGCAGACATGATAAGATACATT

[0654] (SEQ ID NO: 19)

[0655] GCCCCTGCAGCCGAATTATATTAI 1 1 1 I GCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGAGACCCTCGACC GTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACGGGAGCTACCCTAACGT CAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTT GACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCACGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAA ACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTT TCCACCATATTGCCGTC 1 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTT TCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAAC AACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTAT AAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCC TCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCAC PL10 ATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACA CGATGATAATATGGCCACACATATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCA CCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAA GTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGG CTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAG GCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGA GCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGC AGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAA GATCAAGGGGGCCGGTGGTGACTAAAAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGAGACGCTACGGACT TAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAG GCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGACCAGTGGACAATCGACGGATAACAGCATATCTAGCGGCCGCTT

[0656]

[0657] CGAGCAGACATGATAAGATACATTGATGATC (SEQ ID NO: 20) GCCCCTGCAGCCGAATTATATTAI 1 1 1 I GCCAAATAAI 1 1 1 1 AACAAAAGCTCTGAAGTCTTCTTCATTTAAATTCTTAGATG ATACTTCATCTGGAAAATTGTCCCAATTAGTAGCATGCCACGCTGTGTAATACGACTCACTATAGGGGGTACCCCTCTCCCT CCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCC GTCI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGG 1 Cl 1 1 CCCCTCTCGCCAA AGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGA CCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCA AAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAA CAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTG TTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATG PL11 GCCACACATATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGA AGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAG AAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCC TGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCC GCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGT TCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGC TCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCG GTG GTG ACTAAG G ATCCTAG GG CATTACG CGTTTAAACAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAGAGACGG G GTACGTTCGTGATACGGCCGCTTCGAGCAGACATGATA (SEQ ID NO: 21) TAATACGACTCACTATAAGGGGTACCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTG TGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTG ACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTG GAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTG CGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTG GAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATC TGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGA CGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTG GTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCT PL12 GACCTACGGCGTGCAGTGCTTCAGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGG GGTACGTGCAGGAGCGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGG ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTA CAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCAC AACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCCTGC CCGACAACCACTACCTGTCCACCCAGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGA GTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTC CCGCCGTGGCAGCCCAGGACGATGGCACACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCCGC CTGTGCAAGTGCCAGGATCAACGTGTGATAGGGATCCTAGGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0658] (SEQ ID NO: 22) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTACCCC TCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCA TATTGCCG I CI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACA CCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGAT AATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCG ACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA PL13 TCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGG TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGGTACGTGCAGGAGCGGACCATCTTCTT CAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCAGCGTGCAGCTG GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCCTGCCCGACAACCACTACCTGTCCACCCAGAG CGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTG GGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTCCCGCCGTGGCAGCCCAGGACGATGGCA CACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCCGCCTGTGCAAGTGCCAGGATCAACGTGTG ATAGTCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTA TCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA (SEQ ID NO: 23) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG PL14 TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTACCCC

[0659]

[0660] TCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCA TATTGCCG I CI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACA CCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGAT AATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCG ACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA TCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGG TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGGTACGTGCAGGAGCGGACCATCTTCTT CAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCAGCGTGCAGCTG GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCCTGCCCGACAACCACTACCTGTCCACCCAGAG CGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTG GGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTCCCGCCGTGGCAGCCCAGGACGATGGCA CACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCCGCCTGTGCAAGTGCCAGGATCAACGTGTG ATAGGGATCCAGCTCGC 1 1 IC I IGCTGTCCAAI 1 ILIAI 1 / CTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATA (SEQ ID NO: 24) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTACCCC TCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCA TATTGCCG I CI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACA CCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGAT AATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCG ACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA PL15 TCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGG TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGGTACGTGCAGGAGCGGACCATCTTCTT CAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCAGCGTGCAGCTG GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCCTGCCCGACAACCACTACCTGTCCACCCAGAG CGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTG GGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTCCCGCCGTGGCAGCCCAGGACGATGGCA CACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCCGCCTGTGCAAGTGCCAGGATCAACGTGTG ATAGGGATCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCG TAATCATG GTCATAG CTGTTTCCTGTGTG AAATTGTTATCCG CTCAC AATTCC ACACAACATACG AG CCG G AAG CATAAAG

[0661] (SEQ ID NO: 25) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTACCCC TCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCA TATTGCCG I CI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACA CCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGAT AATATGGCCACACATATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCG PL16 ACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA TCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGG TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGGTACGTGCAGGAGCGGACCATCTTCTT CAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGGGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTGTACATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCAGCGTGCAGCTG GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCCTGCCCGACAACCACTACCTGTCCACCCAGAG CGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTG GGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCCTCCCGCCGTGGCAGCCCAGGACGATGGCA CACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCCGCCTGTGCAAGTGCCAGGATCAACGTGTG

[0662]

[0663] ATAGGGATCCAGCTCGC 1 1 1 C 1 1 GCTGTCCAA 1 1 1 L 1 A 1 1 AAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGG AT ATT AT G AAG G G CCTTG AG CATTTGG ATTCTGCCTAATAAAAAACATTTATTTTC ATTG C TCTAGAGTCG ACTGCAG AG G CCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTC (SEQ ID NO: 26) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTAACAT TTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCG TGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGA GGGGTACGTGCAGGAGCGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG GGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA PL17 GTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGG CACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCC TGCCCGACAACCACTACCTGTCCACCCAGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCC TCCCGCCGTGGCAGCCCAGGACGATGGCACACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCC GCCTGTGCAAGTGCCAGGATCAACGTGTGATAGTCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCAT GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAA

[0664] (SEQ ID NO: 27) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTAACAT TTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCG TGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGA GGGGTACGTGCAGGAGCGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG GGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA PL18 GTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGG CACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCC TGCCCGACAACCACTACCTGTCCACCCAGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCC TCCCGCCGTGGCAGCCCAGGACGATGGCACACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCC GCCTGTGCAAGTGCCAGGATCAACGTGTGATAGGGATCCAGCTCGCTTTCTTGCTGTCCAAI 1 1 Cl Al 1 TCTAGAGTCG AC TGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT (SEQ ID NO: 28) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTAACAT TTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCG TGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGA GGGGTACGTGCAGGAGCGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG GGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA PL19 GTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGG CACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCC TGCCCGACAACCACTACCTGTCCACCCAGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCC TCCCGCCGTGGCAGCCCAGGACGATGGCACACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCC GCCTGTGCAAGTGCCAGGATCAACGTGTGATAGGGATCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAGCAG GTGGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT

[0665] (SEQ ID NO: 29) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTAACAT TTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGTGTCTAAGGGGGAGGAGCTGTTCACCGGCG TGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGGGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACAACGGGGAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACC CTGACCTACGGCGTGCAGTGCTTCAGCCGGTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGA GGGGTACGTGCAGGAGCGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG PL20 GGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGA GTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGG CACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCAGTGCTCC TGCCCGACAACCACTACCTGTCCACCCAGAGCGCCCTGAGCAAGGACCCAAACGAGAAGCGGGACCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAGCGCTCCCGCGACATCAGCCACGGCTTCCC TCCCGCCGTGGCAGCCCAGGACGATGGCACACTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGGCATCCGGCC

[0666]

[0667] GCCTGTGCAAGTGCCAGGATCAACGTGTGATAGGGATCCAGCTCGCTTTCTTGCTGTCCAAI 1 1 Cl Al 1 AAAGGTTCC 1 1 1 G TTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATTTGGATTCTGCCTAATAAAAAACATTTATT TTCATTGCTCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG (SEQ ID NO: 30) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTACCCC TCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCA TATTGCCG I CI 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCT CGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTG TAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACA CCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCG AG GTTAAAAAACGTCTAG G CCCCCCG AACCACG G GG ACGTG GTTTTCCTTTG AAAAACACG ATG AT PL21 AATATGGCCACACATATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCTGCTGCTGAGCCTGCTGAGCCTGCCCCTGGG CCTGCCCGTGCTGGGCGCCCCCCCCCGGCTGATCTGCGACAGCCGGGTGCTGGAGCGGTACCTGCTGGAGGCCAAGGAG GCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCAGCCTGAACGAGAACATCACCGTGCCCGACACCAAGGTGAACTT CTACGCCTGGAAGCGGATGGAGGTGGGCCAGCAGGCCGTGGAGGTGTGGCAGGGCCTGGCCCTGCTGAGCGAGGCCGT GCTGCGGGGCCAGGCCCTGCTGGTGAACAGCAGCCAGCCCTGGGAGCCCCTGCAGCTGCACGTGGACAAGGCCGTGAG CGGCCTGCGGAGCCTGACCACCCTGCTGCGGGCCCTGGGCGCCCAGAAGGAGGCCATCAGCCCCCCCGACGCCGCCAGC GCCGCCCCCCTGCGGACCATCACCGCCGACACCTTCCGGAAGCTGTTCCGGGTGTACAGCAACTTCCTGCGGGGCAAGCT GAAGCTGTACACCGGCGAGGCCTGCCGGACCGGCGACCGGTGATAGGGATCCAGCTCGCI 1 1 C 1 1 GC 1 GTCCAA 1 1 ILIA TTTCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC CGCTCACAATTCCACACAACATACGAGCCGG (SEQ ID NO: 31) GGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCTAATACGACTCACTATAAGGATAATGGTAACAT TTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCT GCTGCTGAGCCTGCTGAGCCTGCCCCTGGGCCTGCCCGTGCTGGGCGCCCCCCCCCGGCTGATCTGCGACAGCCGGGTGC TGGAGCGGTACCTGCTGGAGGCCAAGGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCAGCCTGAACGAGA ACATCACCGTGCCCGACACCAAGGTGAACTTCTACGCCTGGAAGCGGATGGAGGTGGGCCAGCAGGCCGTGGAGGTGTG PL22 GCAGGGCCTGGCCCTGCTGAGCGAGGCCGTGCTGCGGGGCCAGGCCCTGCTGGTGAACAGCAGCCAGCCCTGGGAGCC CCTGCAGCTGCACGTGGACAAGGCCGTGAGCGGCCTGCGGAGCCTGACCACCCTGCTGCGGGCCCTGGGCGCCCAGAAG GAGGCCATCAGCCCCCCCGACGCCGCCAGCGCCGCCCCCCTGCGGACCATCACCGCCGACACCTTCCGGAAGCTGTTCCG GGTGTACAGCAACTTCCTGCGGGGCAAGCTGAAGCTGTACACCGGCGAGGCCTGCCGGACCGGCGACCGGTGATAGGG ATCCAGCTCGCTTTCTTGCTGTCCAAI 1 ICIAI 1 TCTAGAGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGG TCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT (SEQ ID NO: 32) GCTGTGTAATACGACTCACTATAAGGATAATGGTACCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA TAAGGCCGGTGTGCGTTTGTCTATATGTTAT 1 1 1 CCACCATATTGCCG 1 C 1 1 1 1 GGCAATGTGAGGGCCCGGAAACCTGGC CCTGTCTTCTTGACGAGCATTCCTAGGGG 1 C 1 1 1 CCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAA GCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGA CAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTT GGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCA TTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCG AACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACACATGCCACCATGGGAGTCAAAGTTCTGT PL23 TTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGC AACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGT TGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATG AAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATC GTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGA CTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTG CGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAGGATCCAAAAAAAAAA AAAAAAAAAAAAAAAAAAAATCTAGA (SEQ ID NO: 34) GCTGTGTAATACGACTCACTATAAGGATAATGGTAACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGA CACCGCCACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACG AAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAA GAAGCTGCCGCTGGAGGTGCTCAAAGAGTTGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGC PL24 CTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTC CGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCTTGGAGCAG TTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTG CTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCG

[0668]

[0669] GTGGTGACTAAGGATCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAGAGCAGGTG (SEQ ID NO: 35) Definitions

[0670] The following definitions apply throughout the present specification and the claims, unless specifically indicated otherwise.

[0671] Various groups are referred to as being "optionally substituted" in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the "optionally substituted" groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.

[0672] Where a group is indicated as having a certain number of carbon atoms, a certain number of non-H atoms, or a certain number of other atoms, it is to be understood that the group furthermore contains hydrogen atoms, even if this is not explicitly mentioned. A skilled person is well aware of the fact that a group (in particular an organic group) containing a certain number of non-H atoms usually has to also contain an adequate number of hydrogen atoms in order to saturate the valencies of the non-H atoms in as far as these are not saturated by bonds between the respective non-H atoms. For example, the term "group having from 1 to 50 non-H atoms" etc. as used herein, preferably indicates that the group consists of 1 to 50 non-H atoms and an adequate number of hydrogen atoms.

[0673] The term "non-H" as used herein preferably encompasses any atom selected from Li, B, C, N, O, F, Na, Mg, Al, Si, P, S, Cl, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, I, Cs, Ba, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, TI, Pb, Bi, Po, At, Fr, Ra, Ac, Th, Pa, and U. The term "non-H" as used herein more preferably encompasses any atom selected from Li, B, C, N, O, F, Na, Mg, Si, P, S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, I, Cs, Ba, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt and Au. The term "non-H" as used herein even more preferably encompasses any atom selected from Li, B, C, N, O, F, Na, Mg, Si, P, S, Cl, K, Ca, Se, Br, Rb, Sr, I. The term "non-H" as used herein still even more preferably encompasses any atom selected from B, C, N, O, F, Si, P, S, Cl, Se, Br, I. The term "non-H" as used herein most preferably encompasses any atom selected from C, N, O, F, P, S, Cl, Br and I.

[0674] As is set out below, the present invention also encompasses salts of the compounds of the present invention. Thus, even if the definition of a certain group does not explicitly mention it, the group may include an anion or cation and it is evident that such anion or cation may be accompanied by a counterion which may include atoms other than specified in the definition of the group. For example, a group specified as containing 1 to 10 non-H atoms selected from C, N, O, S, P, F and Cl may include a carboxylic group, a phosphate group, an ammonium group etc., each for which may have a suitable counterion, such as Na+, K+, PFe’, etc.

[0675] As used herein, the expression "group having" is used synonymously with the expression "groups comprising".

[0676] Any substituent or optional substituent not otherwise defined in the context is preferably selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O (RR), -C(O)NH2, -C(O)NH(RR, -C(O N(RR)2, - C(O)OH, -C(O)O(RR), - H?, -NH(RR), -N(RR)?, - HC(O)(RR, -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(RR), - HC(O)N(RRh, -NHC(O O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, - OC(O NH(RR), -OC(O NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(0)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(0)N(alkyl)2, -C(O)OH, -C(0)0(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(0)0(alkyl), -NO2, -OH, -O- alkyl, -0C(0)(alkyl), -0C(0)N(alkyl)2, -0C(0)NH(alkyl), -OC(O)NH2, -0C(0)0(alkyl), -SH, and -S- alkyl. In cases of substituents or optional substituents of alkyl groups, the substituent or optional substituent is preferably not an alkyl group.

[0677] More preferably, the substituent or optional substituent is selected form -OH, 0-alkyl, OBn, SH, S-alkyl, NH2, NH(alkyl), N(alkyl)2, F, Cl, Br and CN. Any group being modified or chemically modified is preferably a group having one or more (preferably 1 to 5, more preferably 1 or 2) substituents selected from -F, -Br, -Cl, -RR, -CN, - C(O)H, -C(O)(RR), -C(O)NH2, -C(O NH RR, -C O N RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), - RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), - HC O RR)2, -NHC(O)O(RR), - NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC O O RR, -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl.

[0678] As used herein, the term "heteroatom" refers to any atom other than carbon and hydrogen. Preferably, the term "heteroatom" refers to N, O, S, P, F, Cl, Br, I, Si, B and Se, more preferably N, O, S, P, F, Cl, Br and I, even more preferably N, O, S, P, F and Cl.

[0679] As used herein, the term "alkyl" refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an "alkyl" group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A " Ci-5 alkyl" denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term "alkyl" preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.

[0680] As used herein, the term "heteroalkyl" indicates an alkyl group in which one or more CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and optionally one or more tertiary CH groups are replaced by N, and which may preferably be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2. Preferably, the term "heteroalkyl" indicates a (preferably linear) C1-30 alkyl wherein one to ten CH2groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and which may be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2. The heteroalkyl more preferably corresponds to a (preferably linear) C1-30 alkyl wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and which may preferably be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2. Even more preferably, the heteroalkyl corresponds to a (preferably linear) C1-30 alkyl wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2.

[0681] As used herein, the term "alkenyl" refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term " C2-5 alkenyl" denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-l-en-l-yl, prop-l-en-2-yl, or prop-2-en-l-yl), butenyl, butadienyl (e.g., buta-l,3-dien-l-yl or buta-l,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term "alkenyl" preferably refers to C2-4 alkenyl.

[0682] As used herein, the term "alkynyl" refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term " C2-5 alkynyl" denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term "alkynyl" preferably refers to C2-4 alkynyl.

[0683] As used herein, the term "alkylene" refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A " C1-5 alkylene" denotes an alkylene group having 1 to 5 carbon atoms; the term " C0-5 alkylene" indicates that a covalent bond (corresponding to the option " Co alkylene") or a C1-5 alkylene is present. Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2-CH2-CH2-CH2-). Unless defined otherwise, the term "alkylene" preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene. As used herein, the term "heteroalkylene" indicates an alkylene group in which one or more CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene or C=O and optionally one or more tertiary CH groups are replaced by N, and which may preferably be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2.Preferably, the term "heteroalkylene" indicates a (preferably linear) C1-30 alkylene wherein one to ten CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may be substituted with one or more selected from F, Cl, Br, OH, CN, O(alkyl), NH2, NH(alkyl), and N(alkyl)2. The heteroalkylene more preferably corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl), triazolene, P(=O)(O-) or C=O and which may preferably be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2. Even more preferably, the heteroalkylene corresponds to a (preferably linear) C1-30 alkylene wherein one to eight CH2 groups are independently replaced by S, O, NH, N(alkyl) or C=O and which may be substituted with one or more selected from F, OH, CN, 0(Me), NH2, NH(Me), and N(Me)2. The optional substituents of the "heteroalkylene" are preferably only present on the carbon atoms of the "heteroalkylene".

[0684] In any "heteroalkylene", it is preferred that (except for carbon), like atoms are not bound to like atoms. For example an O is preferably not bound to O. Furthermore, N is preferably not directly bound to N. N is preferably not directly bound to F. Preferably no more than one triazolene is present in each "heteroalkylene".

[0685] As used herein the term "-O-alkylidene-O-" preferably refers to "-O-C(Ci-6alkyl)2-O-", more preferably "-O-C(Ci-3alkyl)2-O-", even more preferably "-O-CfCHsh-O-".

[0686] As used herein, the term "alkenylene" refers to an alkenediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. A " C2-5 alkenylene" denotes an alkenylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term "alkenylene" preferably refers to C2-4 alkenylene (including, in particular, linear C2-4 alkenylene).

[0687] As used herein, the term "alkynylene" refers to an alkynediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. A " C2-5alkynylene" denotes an alkynylene group having 2 to 5 carbon atoms. Unless defined otherwise, the term "alkynylene" preferably refers to C2-4alkynylene (including, in particular, linear C2-4alkynylene).

[0688] As used herein, the term "carbocyclyl" refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, "carbocyclyl" preferably refers to aryl, cycloalkyl or cycloalkenyl.

[0689] As used herein, the term "heterocyclyl" refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two O atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, "heterocyclyl" preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.

[0690] As used herein, the term "aryl" refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). If the aryl is a bridged and / or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group). " Aryl" may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1, 2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1H-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl. Unless defined otherwise, an "aryl" preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.

[0691] As used herein, the term "heteroaryl" refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two O atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heteroaryl" may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-l-benzopyranyl or 4H-l-benzopyranyl), isochromenyl (e.g., 1H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [l,10]phenanthrolinyl, [l,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[l,5-a]pyrimidinyl (e.g., pyrazolo[l,5-a]pyrimidin-3-yl), l,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl, lH-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl, 1,2,4-triazinyl, or 1,3,5-triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or l,3-dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g., imidazo[l,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or 1,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term "heteroaryl" preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroaryl" refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a "heteroaryl" include pyridinyl (e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), imidazolyl, thiazolyl, lH-tetrazolyl, 2H-tetrazolyl, thienyl (i.e., thiophenyl), or pyrimidinyl.

[0692] As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). " Cycloalkyl" may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, "cycloalkyl" preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred "cycloalkyl" is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Moreover, unless defined otherwise, particularly preferred examples of a "cycloalkyl" include cyclohexyl or cyclopropyl, particularly cyclohexyl.

[0693] As used herein, the term "cycloalkylene" refers to a cycloalkyl group, as defined herein above, but having two points of attachment, i.e. a divalent saturated hydrocarbon ring group. " Cycloalkylene" may, e.g., refer to cyclopropylene (e.g., cyclopropan-l,l-diyl or cyclopropan-1,2-diyl), cyclobutylene (e.g., cyclobutan-l,l-diyl, cyclobutan-l,2-diyl, or cyclobutan-l,3-diyl), cyclopentylene (e.g., cyclopentan-1, 1-diyl, cyclopentan-1, 2-diyl, or cyclopentan-1, 3-diyl), or cyclohexylene (e.g., cyclohexan-1, 1-diyl, cyclohexan-1, 2-diyl, cyclohexan-1, 3-diyl, or cyclohexan-l,4-diyl). Unless defined otherwise, "cycloalkylene" preferably refers to a C3-7 cycloalkylene, and more preferably refers to a C3-5 cycloalkylene. Moreover, unless defined otherwise, a particularly preferred example of a "cycloalkylene" is cyclopropylene.

[0694] As used herein, the term "heterocycloalkyl" refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two O atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. " Heterocycloalkyl" may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g., 1,4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1,3-dioxolanyl, tetrahydropyranyl, 1,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1,3-dithiolanyl, thianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl. Unless defined otherwise, "heterocycloalkyl" preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl" refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized. Moreover, unless defined otherwise, particularly preferred examples of a "heterocycloalkyl" include tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or tetrahydrofuranyl.

[0695] As used herein, the term "heterocycloalkylene" refers to a heterocycloalkyl group, as defined herein above, but having two points of attachment. " Heterocycloalkylene" may, e.g., refer to aziridinylene, azetidinylene, pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene, piperazinylene, azepanylene, diazepanylene (e.g., 1,4-diazepanylene), oxazolidinylene, isoxazolidinylene, thiazolidinylene, isothiazolidinylene, morpholinylene, thiomorpholinylene, oxazepanylene, oxiranylene, oxetanylene, tetrahydrofuranylene, 1,3-dioxolanylene, tetrahydropyranylene, 1,4-dioxanylene, oxepanylene, thiiranylene, thietanylene, tetrahydrothiophenylene (i.e., thiolanylene), 1,3-dithiolanylene, thianylene, or thiepanylene. Unless defined otherwise, "heterocycloalkylene" preferably refers to a divalent 3 to 7 membered saturated monocyclic ring group, wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkylene" refers to a divalent 3 to 5 membered saturated monocyclic ring group containing one or two (preferably one) ring heteroatoms independently selected from O, S and N, wherein the remaining ring atoms are carbon atoms. Moreover, unless defined otherwise, particularly preferred examples of a "heterocycloalkylene" include aziridinylene, oxiranylene, thiiranylene, azetidinylene (e.g., azetidi n-3,3-diyl), oxetanylene (e.g., oxetan-3,3-diyl), thietanylene (e.g., thietan-3,3-diyl), pyrrolidinylene, tetrahydrofuranylene, or tetrahydrothiophenylene.

[0696] As used herein, the term "cycloalkenyl" refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. " Cycloalkenyl" may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, "cycloalkenyl" preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl. A particularly preferred "cycloalkenyl" is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.

[0697] As used herein, the term "heterocycloalkenyl" refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent ring atomsand does not comprise any triple bond between adjacent ring atoms. For example, each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two O atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. " Heterocycloalkenyl" may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5-dihydro-1-imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1,2-dihydropyridinyl or 2,3-di hyd ropy ridi ny I), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), di hydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, octahydroquinolinyl (e.g., 1, 2, 3, 4, 4a, 5,6,7-octahydroquinolinyl), or octahydroisoquinolinyl (e.g., 1,2,3,4,5,6,7,8-octahydroisoquinolinyl). Unless defined otherwise, "heterocycloalkenyl" preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenyl" refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from O, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms.

[0698] As used herein, the term "halogen" refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-1).

[0699] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. " Haloalkyl" may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A particularly preferred "haloalkyl" group is -CF3.

[0700] The terms "bond" and "covalent bond" are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.

[0701] The term "oligo" as used herein indicates that 2 to 100 units (preferably 2 to 50, more preferably 2 to 20, even more preferably 2 to 8 units) of the respective entity are linked to each other. For example, the term "oligonucleotide" typically comprises 2 to 100 nucleotides.

[0702] As used herein, the term "mRNA 5' cap analog" preferably refers to a modified guanine nucleotide (7-methylguanosine) linked to another nucleotide via a 5'-5' triphosphate bridge. As used herein, the term "fluorophore" preferably refers to a fluorescent chemical compound that can re-emit light upon light excitation. When a fluorophore absorbs photons (light energy) at a specific wavelength, it becomes excited to a higher energy state. As the fluorophore returns to its ground state, it emits photons at a longer wavelength, which is observed as fluorescence. Examples of fluorophores include fluorescein and its derivatives: Fluorescein isothiocyanate (FITC), Carboxyfluorescein (FAM), Oregon Green, Alexa Fluor 488; Rhodamine and its derivatives: Rhodamine B, Tetramethylrhodamine (TAMRA), Texas Red, Alexa Fluor 546, Alexa Fluor 594; Cyanine Dyes: Cy3, Cy5, Cy7, Alexa Fluor 647; BODIPY Dyes: BODIPY FL, BODIPY TMR, BODIPY TR; Alexa Fluor Dyes: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 700, Alexa Fluor 750; Atto Dyes: Atto 390, Atto 425, Atto 488, Atto 550, Atto 565, Atto 647N, Atto 655; DyLight Dyes: DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755; Quantum Dots: Qdot 525, Qdot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot 800; Green Fluorescent Protein (GFP) and its variants: Enhanced GFP (EGFP), Yellow Fluorescent Protein (YFP), Cyan Fluorescent Protein (CFP), Blue Fluorescent Protein (BFP), mCherry, mOrange, mKate; Phycoerythrin (PE) and its derivatives: R-Phycoerythrin (R-PE), B-Phycoerythrin (B-PE), Allophycocyanin (APC); Peridinin-Chlorophyll-Protein Complex (PerCP); DAPI (4',6-diamidino-2-phenylindole); Hoechst Dyes: Hoechst 33258, Hoechst 33342; SYBR Green (nucleic acid staining), PicoGreen (dsDNA quantification), TOTO and YOYO dyes (nucleic acid staining). Further examples are EDANS, Xanthene derivatives in general (e.g. fluorescein, Rhodamine, Oregon green, eosin), Cyanine derivatives (e.g., indocarbocyanine, oxacarbocyanine, merocyanine), Squaraine derivatives (e.g., Seta, Se Tau, Square dyes), Naphthalene derivatives (e.g., dansyl or prodan derivatives), Coumarin derivatives (e.g. 7-Amino-4-methylcoumarin (AMC), Coumarin 343), Oxadiazole derivatives, Anthracene derivatives (e.g., Anthraquinones such as DRAQ5, DRAQ7, CyTRAK Orange), Pyrene derivatives (e.g., cascade blue), Oxazine derivatives (e.g., Nile red, Nile blue, Cresyl violet), Acridine derivatives (e.g., Proflavin, Acridine Orange, Acridine Yellow), Arylmethine derivatives (e.g., Auramine, Crystal Violet, Malachite Green), or Tetrapyrrole derivatives (e.g., Parphin, Phthal ocyanine, Bilirubin). As used herein, the term "hapten" preferably refers to a molecule (usually having not more than 100 non-H atoms) that, by itself, is not capable of eliciting an immune response. However, when a hapten is covalently attached to a larger carrier molecule, typically a protein, it can become immunogenic. Examples of haptens include Penicillin, Aspirin (acetylsalicylic acid), Ibuprofen, Sulfonamides (e.g., sulfamethoxazole), Methotrexate, Chloramphenicol, Hydralazine, Procainamide, Quinidine, Phenytoin, Carbamazepine, Estradiol, Testosterone, Progesterone, Cortisol, Thyroxine (T4), Triiodothyronine (T3), Dinitrophenol (DNP), Trinitrophenol (TNP), Benzene, Toluene, Formaldehyde, Parathion (an organophosphate pesticide), Dioxins, Polychlorinated biphenyls (PCBs), Benzoic acid, Sulfites, Tartrazine (a synthetic lemon yellow azo dye), Monosodium glutamate (MSG), Aspartame, Bilirubin, Uric acid, Creatinine, Histamine, Serotonin, Dopamine, Biotin, Folic acid, Nicotinic acid (niacin), Riboflavin (vitamin B2), Thiamine (vitamin Bl), Pyridoxal phosphate (vitamin B6), Amoxicillin, Cephalosporins, Tetracycline, Erythromycin, Vancomycin, 2,4-Dinitrophenylhydrazine (DNPH), 4-Hydroxy-3-nitrophenylacetyl (NP), 2,4,6-Trinitrobenzenesulfonic acid (TNBS), 2,4,6-Trinitrophenyl (TNP), 2,4-Dinitrofluorobenzene (DNFB), Fluorescein isothiocyanate (FITC), Dinitrobenzene (DNB), Dinitrophenyl (DNP), 4-Hydroxy-3-nitrophenylacetic acid (NIP), 2,4-Dinitrophenylacetyl (DNP-A).

[0703] As used herein, the term "chelator" preferably refers to chemical compound that can form multiple bonds with a single metal ion. It can effectively "sequester" the metal ion by surrounding it with a stable, ring-like structure known as a chelate complex. Examples of commonly used chelators include Ethylenediaminetetraacetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), Deferoxamine, Dimercaprol (BAL), Dimercaptosuccinic acid (DMSA), Dimercaptopropanesulfonic acid (DMPS), Penicillamine, Desferrioxamine B, Deferiprone, Deferasirox, Triethylenetetramine (TETA), Nitrilotriacetic acid (NTA), Ethylenediamine-N, N'-bis(2-hydroxyphenylacetic acid) (EDDHA), 1,10-Phenanthroline, Bipyridine, Tetracycline, Oxalate, Citrate, Phytate, Phytic acid, Salicylate, Hydroxamic acids, Aminopolycarboxylic acids, Cyclam, Crown ethers, Cryptands, Porphyrins, Phthalocyanines, Siderophores, Enterobactin, Ferrichrome, Pyoverdine, Aurothioglucose, Thiomalate, Trientine, Succimer, Glutathione, Histidine, Cysteine, Methionine, Phosphonates, Polyphosphates, Zeolites, Humic acids, Fulvic acids, Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), Diethylenetriamine (DETA), Triethylenetetramine (TETA), Tetraethylenepentamine (TEPA), Hexadentate ligands, Polyaminocarboxylates, Polyaminophosphonates, Polyaminopolycarboxylates, Polyaminopolyphosphonates, Iminodiacetic acid (IDA), Hydroxyethylethylenediaminetriacetic acid (HEDTA), Ethylenediaminedisuccinic acid (EDDS), N, N'-Bis(carboxymethyl)-L-lysine (BCML), N, N'-Bis(2-hydroxybenzyl)ethylenediamine-N, N'-diacetic acid (HBED), N, N'-Bis(2-hydroxy-5-sulfobenzyl)ethylenediamine-N, N'-diacetic acid (HBED-SS), N, N'-Bis(2-hydroxybenzyl)ethylenediamine-N, N'-diacetic acid (HBED), N, N'-Bis(2-hydroxy-5-sulfobenzyl)ethylenediamine-N, N'-diacetic acid (HBED-SS), and N, N'-Bis(2-hydroxybenzyl)ethylenediamine-N, N'-diacetic acid (HBED).

[0704] As used herein, the term "(radio)isotopic tag" preferably refers to an isotope of an element having a half-time of decay of less than 109years. Examples of radioisotopes include iodine-131, iodine-125, technetium-99m, cobalt-60, cesium-137, radium-226, phosphorus-32, phosphorus-33, sulfur-35, calcium-45, chromium-51, iron-59, zinc-65, selenium-75, yttrium-90, molybdenum-99, ruthenium-106, palladium-103, silver-111, indium-111, xenon-133, samarium-153, gadolinium-153, holmium-166, erbium-169, thulium-170, ytterbium-169, lutetium-177, rhenium-186, iridium-192, gold-198, mercury-203, thallium-201, radium-223, actinium-225, thorium-227, radon-222, fluorine-18, carbon-11, nitrogen-13, oxygen-15, gallium-67, gallium-68, strontium-89, iodine-123, iodine-124, copper-64, copper-67, scandium-44, zirconium-89, terbium-161, terbium-149, terbium-152, terbium-155, lead-212, bismuth-213, bismuth-212 and astatine-211.

[0705] As used herein, the term "lipid" preferably refers to C18-50 carboxylic acids, triglycerides, phospholipids (typically consisting of glycerol, two fatty acids, and a phosphate group), steroids, glycolipids and waxes. Examples of lipids include cholesterol, sphingolipids, ceramides, sphingomyelins, glycosphingolipids, gangliosides, cerebrosides, eicosanoids, prostaglandins, thromboxanes, leukotrienes, lipoxins, isoprenoids, terpenes, carotenoids, retinoids, tocopherols, tocotrienols, ubiquinones, plasmalogens, cardiolipins, lysophospholipids, diacylglycerols, monoacylglycerols, waxes, arachidonic acid, linoleic acid, alpha-linolenic acid, palmitic acid, stearic acid, oleic acid, myristic acid, lauric acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, cardiolipin, lipoproteins, chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), apolipoproteins, lipopolysaccharides, lipoteichoic acids, sulfolipids, ether lipids, alkylglycerols, alkenylglycerols, polyprenols, dolichols, prenylated proteins, farnesylated proteins, geranylgeranylated proteins, phosphonolipids, aminolipids, lipoglycans. As used herein, the term "amino acid" preferably refers to a-amino acids such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine. The term "amino acid" preferably may also refer to β-amino acids such as β-alanine, β-leucine, β-lysine, β-arginine, β-glutamate, β-glutamine, β-phenylalanine and β-tyrosine.

[0706] As used herein, the term "oligopeptide" preferably refers to a peptide having from 2 to 100 amino acids.

[0707] As used herein, a "protein" preferably contains more than 100 amino acids.

[0708] As used herein, the term "saccharide" preferably refers to mono-saccharides, such as glucose, fructose, galactose, mannose, ribose, xylose, arabinose, lyxose, allose, altrose, gulose, idose, talose, erythrose, threose, ribulose, xylulose, psicose, sorbose, tagatose, fucose, rhamnose, deoxyribose, sedoheptulose, glucoheptose, galactoheptose, mannoheptose, idoheptose, alloheptose, altroheptose, guloheptose, taloheptose, arabinoheptose, lyxoheptose, erythroheptose, threoseheptose, riboheptose, xyloheptose, arabinohexose, lyxohexose, erythro hexose, threosehexose, ribohexose, xylohexose, arabinoheptose, lyxoheptose, erythroheptose, threoseheptose, riboheptose or xyloheptose. However, the term "saccharide" also encompasses derivatives of these compounds wherein a OH group is replaced by an NH2group (such as glucosamine), and desoxysaccharides, wherein at least one OH group is replaced by H (e.g. desoxiribose).

[0709] As used herein, an "oligosaccharide" preferably from 2 to 100 saccharides. A preferred type of oligosaccharide is a dissacharide. Exemplary dissacharides are sucrose, lactose, maltose, trehalose, cellobiose, isomaltose, gentiobiose, turanose, melibiose, sophorose, laminaribiose, nigerose, kojibiose, maltulose, leucrose, palatinose, lactulose, mannobiose, xylobiose, rutinose, primeverose, sambubiose, vicianose, isomaltulose, neotrehalose, neohesperidose, thevetose, amygdalose, gentianose, diglucose, digalactose, difructose, glucosylfructose, galactosylglucose, fructosylglucose, fructosylga lactose, glucosylgalactose, galactosylfructose. As used herein, the term "polymer" preferably refers a molecule containing repeating structural units called monomers, which are covalently bonded together in a two-dimensional or three-dimensional structure. This term preferably does not include polysaccharides, oligonucleotides or peptides. Preferred examples of polymers are polyethylenglycole (PEG), polyoxyethylene (PEG) or polyglycerol (e.g. polyglycerol-polyricinoleate (PGPR).

[0710] As used herein, the term "polymer composite" preferably refers to a composite of a polymer with one or more strengthening fillers such as fibers (e.g. glass fibers, carbon fibers, aramid fibers, jute, hemp or flax), particles (such as silica, alumina, calcium carbonate, metal particles) or nanomaterials (such as carbon nanotubes, graphene, nanoclays).

[0711] As used herein, the term "metal-organic framework" preferably refers to structures containing metal ions or metal clusters coordinated to organic ligands to form one-, two-, or three-dimensional structures.

[0712] As used herein, the term "solid support" preferably refers to a material that is solid at 25°C, 1 atm, and can serve as a substrate that provides a surface or matrix for the immobilization, attachment, or linkage of molecules.

[0713] As used herein, the term "nanoparticle" preferably refers to particles with at least one dimension less than 100 nm, preferably with a volume median particle diameter D50 of less than 100 nm, preferably measured by dynamic light scattering (DLS).

[0714] As used herein, the term "nanomaterial" preferably refers to materials consisting of particles with at least one dimension less than 100 nm, preferably with a volume median particle diameter D50 of less than 100 nm, preferably measured by dynamic light scattering (DLS).

[0715] As used herein, the term "glass" preferably refers to amorphous solid (25°C, 1 atm) material that is typically transparent (preferably at least 90% transparent at 550 nm). The "glass" is preferably selected from borosilicate glass, aluminosilicate glass, soda-lime glass and quartz glass. The compounds of the present invention may be attached to glass by using a silane coupling agent such as APTES ((3-Aminopropyl)triethoxysilane). The terms "nucleic acid" and "nucleic acid polymer" refer to a polymeric substance composed of ribo or deoxyribo derivatives and of naturally occurring or synthetic nucleoside monomers, including noncanonical linkages and any degree of polymerization. These phrases include, but are not limited to natural or synthetic RNA and DNA, as well as nucleic acids composed of phosphorothioate, boranophosphate, selenophosphate, and phosphonate moieties, morpholino nucleic acids (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), and threose nucleic acid (TNA).

[0716] The terms "modification", "modifying group", and "modified" in the context of nucleic acids refer to any chemical moiety that may be attached to a nucleic acid molecule. This includes, but is not limited to attachment of the modifying group to the sugar, nucleobase, internucleotide phosphate, or 5'-5'-oligophosphate of a nucleic acid molecule.

[0717] The term "internucleotide linkage" refers to a covalent or non-covalent bond or bonds that connect two monomers of a nucleic acid polymer.

[0718] The terms "nucleic acid sequence", "nucleotide sequence", "nucleoside sequence", "nucleobase sequence" and "sequence" refer to the order and composition of a series of nucleobases within a nucleic acid molecule. The terms "nucleoside base" and "nucleobase" are used interchangeably.

[0719] The terms "nucleobase pair" and "base pair" refer to a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. The term "base pairing" refers to specific hydrogen bonding between nucleobases, that can be canonical (Watson-Crick base paring) or non-canonical (e.g. wobble base paring, Hoogsten base paring).

[0720] The terms "hybridization" and "hybridize" refer to interaction of two or more nucleic acid strands. It is characterized by that, that under appropriate conditions distinct nucleic acid strands maintain a sequence-dependent structure (e.g. a double helix, a triple helix, an l-motif, a G-quadruplex).

[0721] The term "complementary" refers to relation between sequences of two or more strands of a nucleic acid or nucleic acids. The complementary sequences or strands are characterised by that they undergo hybridization and form a sequence-dependent structure. The complementarity can be full or partial.

[0722] The terms "linear" and "circular" in context of nucleic acids refer to the topology of a nucleic acid molecule. Linear nucleic acids are linear polymers with no linkage between 5' and 3' ends. Circular nucleic acids are polymers with natural or modified linkage between 5' and 3' ends.

[0723] The terms "circularization" and "cyclization" refer to a process of transformation of a linear nucleic acid polymer to a circular nucleic acid polymer.

[0724] The terms "circRNA precursor" and "pre-circRNA" refer to a linear nucleic acid polymer that can undergo circularization leading to a circular nucleic acid polymer.

[0725] The term "linker" in the context of circular nucleic acids refers to any natural or synthetic combination of chemical moieties that covalently join 5' and 3' ends of the polymer. The term "linker" includes, but is not limited to any linear or branched combination of alkyl, alkenyl, alkynyl, ether, ester, amide, amine, carbamate, triazol, phosphoester, amidophosphate, or phosphonate moieties. In particular it can be selected among of following structures:

[0726]

[0727] wherein m is a natural number from 1 to 100.

[0728] The term "cap analog" refers to any compound that contains natural or modified 5'-5'- oligophosphate linking a natural or modified nucleosides, nucleotides, or nucleic acids. The term " Cap analog" refers to canonical, as well as non-canonical cap structure and includes, but is not limited to CapO, Capl, Cap2, TMG-cap, NAD-cap, and FAD-cap, as well as cap structures modified within oligophosphate bridge, ribose, or nucleobases. In particular, it can be selected among of structures described in any document cited in subject description, esspecially: WO 2009 / 149253, WO 2008 / 157688, WO 2011 / 015347, WO 2022 / 191725, WO 2018 / 015845, WO 2014 / 093627, WO 2021 / 162566, WO 2021 / 162567 and WO 2017 / 130151. The terms "internal ribosomal entry site" and " IRES" refer to RNA sequences that are characterised by their ability facilitate cap-independent initiation of translation by interaction or binding cellular proteins involved in formation of ribosomes and translation. The term " IRES sequence" includes, but is not limited to Encephalomyocarditis virus (EMCV) IRES, Coxsackievirus B3 (CVB3) IRES, respiratory syncytial virus (RSV) m6A methylation sites, viral IRES sequences, human IRES sequences, m6A methylation sites, sequences containing m6A modification, elF4G aptamers, and artificial IRES sequences (NPTL 11, 12, 22 and 24).

[0729] The terms "transcription" or "transcription reaction" refer to methods for enzymatic synthesis of RNA that is complementary to a DNA template. Transcription can occur in cells, in cellular systems or in artificial setups (as in in vitro transcription reaction). Transcription is carried out in presence of a DNA-dependent RNA polymerase, ribonucleoside triphosphates (NTPs), and a DNA template. During the reaction, the sequence of the DNA template is copied and amplified in form of RNA molecules. Optionally, the transcription can be carried out in the presence of additional reagents, such as transcription initiators, transcription terminators, or modified nucleoside triphosphates.

[0730] The terms "label" and "detectable label" refer to any compound, particle, or any combination of compounds and / or particles, that may be directly or indirectly detected. The label can be associated with a target molecule by covalent and / or non-covalent interactions. The label can be a radioisotope, NMR label, spin label, a chromophore, a fluorophore, a hapten. The preferred label includes but is not limited to2H,13C,15C,32P,19F, cyanines, fluoresceines, rhodamines, coumarines, perylenes, naphthalimides, nitro dyes, azo dyes, BODIPY, biotin, desthiobiotin, folic acid and N-acetylgalactosamine.

[0731] The terms "periodate oxidation", "cis-diol oxidation" and "ribose oxidation" refer to a process of cleavage of carbon-carbon bonds of vicinal cis-diols present in the structure of a natural or synthetic nucleic acids leading to a dialdehyde derivative of a nucleic acid.

[0732] The terms "reductive amination" and "amination" in the context of nucleic acid modification or circularization refer to amination reaction involving a dialdehyde derivative of a nucleic acid. The term "reductive environment" refers to conditions under which chemical reduction reactions occur. The referred reductive environment includes but is not limited to the presence of sodium cyanoborohydride, sodium borohydride, pyridine borane, or sodium ascorbate.

[0733] The terms "ethylenediamine motif", " EDA motif", " EDA group", and " EDA" refer to a chemical motif of R-CH2-NH-(CH2)2-NH2, where R is any substituent. Structure of the EDA motif may as well be recognized in an ionic form or in a form of a salt.

[0734] The term "reactive functional group" refers to any functional group that is incorporated at the RNA 5' or 3' end that can react with another (same or different) reactive group in a selective manner in presence of other (natural or unnatural) chemical moieties contained within structure of the circRNA precursor. The representative pairs of 5' and 3' reactive groups which are reactive towards each other, include but are not limited to EDA and dialdehyde, hydrazide and dialdehyde, azide and alkyne, azide and cyclooctyne derivative, amino and carboxylic group, amino and fluorosulfonylgroup, azide and phosphine, etc.

[0735] The terms "nucleic acids alcohol precipitation", "nucleic acids precipitation", and "alcohol precipitation" refer to the process of nucleic acid isolation. It is a technique to de-salt and concentrate nucleic acids from aqueous solutions. Addition of polar organic solvents (e. g. ethanol, isopropanol, acetone) and salts (e. g. sodium acetate, ammonium acetate, lithium chloride, sodium perchlorate, lithium perchlorate) to nucleic acid solution causes the precipitation of nucleic acids.

[0736] The terms "polyacrylamide gel electrophoresis" and " PAGE" refer to a technique used for separation of biological macromolecules according to their electrophoretic mobility. Electrophoretic mobility is a function of the mass, conformation, topology, and charge of the molecule. When PAGE is performed in denaturing conditions, it provides information on the composition of the nucleic acid species in the sample.

[0737] The terms "reversed-phase high pressure chromatography", " RP-HPLC, and " HPLC" refer to a technique for separation, identification, quantification, and isolation of components of a sample mixture. It relies on pumps constituting a flow of pressurized liquid mobile phase (a mixture of solvents and solutions) through a solid matrix of the stationary phase. Components of the sample interact differently with mobile and stationary phases and are in equilibrium between adsorbed and solubilized states. This is facilitated by a combination of hydrophobic, dipole-dipole and ionic interactions. As chromatography progresses, the analytes are resolved based on their elution velocity, can be detected by their molecular properties, and collected into separate fractions (peaks). The elution velocity of individual components of the sample is parameterized by the retention time of the fraction and the peak shape. The active component of the stationary phase is a monolith or particle solid resin of organic and / or inorganic composition, such as silica, poli(styren-co-divinylbenzen), or other polymers. The mobile phase is an aqueous or organic solution or a mixture of aqueous and organic solutions. The chromatography may be performed in ion-pair mode, in elevated temperatures, and for analytical as well as preparative purposes.

[0738] The terms "translation in vitro", "protein synthesis in vitro", and "protein expression in vitro" refer to a process characterised as an enzymatic production of polypeptides based on sequence of RNA or RNA analog in an artificial system. An in vitro system includes but is not limited to artificial reaction mixture, cellular lysates, nuclear lysates, cell cultures, and tissue cultures.

[0739] The terms "translation in vivo", "protein synthesis in vivo", and "protein expression in vivo" refer to a process characterised as an enzymatic production of polypeptides based on sequence of RNA or RNA analog in a natural system. An in vivo system includes but is not limited to whole organisms, organs, cellular and tissue transplants of human, animal, plant, fungal, or bacterial origin.

[0740] The terms "therapeutic protein" or "therapeutic proteins" refer to a polypeptide molecule that may be utilized in the course of therapy or medical studies. The classes of therapeutic proteins include but are not limited to antigens, antibodies, immunogenic proteins, fluorescent proteins, reporter proteins, protein hormones, membrane proteins, transcription factors, regulatory proteins, systems for gene editing and systems for gene regulation.

[0741] The terms "therapeutic gene" or "therapeutic genes" refer to a sequence of a nucleic acid containing an open-reading frame encoding the amino acid sequence of the therapeutic protein. Therapeutic genes may consist of but are not limited to sequences encoded in natural or synthetic viral DNA or RNA, plasmid vectors, RNA, mRNA or circRNA. The term "pharmaceutical composition" refers to a composition of substances comprising of active substances and helper substances, such as pharmaceutically acceptable solvents and / or carriers. Pharmaceutical composition may be formulated as a solid, liquid, aqueous solution, aqueous emulsion, suspension, lipid nanoparticles, liposomes, or liposomal suspension. The term pharmaceutical composition includes but is not limited to compositions designed for expression of a therapeutic genes and proteins in in vitro and in vivo setup.

[0742] As used herein, the term "metal" preferably refers to any element selected from lithium, beryllium, sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, thorium, protactinium, uranium, neptunium and plutonium. Preferred metals are scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, cesium, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum and gold. The compounds of the present invention may be attached to a metal such as gold by lipoic acid modification.

[0743] As used herein, the term "ceramics" preferably refers to oxides, nitrides, carbides, or silicates of metallic and non-metallic elements. Preferred ceramics may contain or consist of one or more selected from alumina, zirconia, silicon carbide, silicon nitride, boron carbide, boron nitride, titanium carbide, titanium nitride, tungsten carbide, magnesium oxide, calcium oxide, barium titanate, lead zirconate titanate (PZT), lithium niobate, lithium tantalate, yttria-stabilized zirconia (YSZ), mullite, cordierite, steatite, forsterite, spinel, aluminum nitride, cerium oxide, hafnium oxide, lanthanum oxide, neodymium oxide, praseodymium oxide, samarium oxide, gadolinium oxide, terbium oxide, dysprosium oxide, holmium oxide, erbium oxide, thulium oxide, ytterbium oxide, lutetium oxide, scandium oxide, indium oxide, tin oxide, zinc oxide, iron oxide, chromium oxide, manganese oxide, nickel oxide, cobalt oxide, copper oxide, vanadium oxide, molybdenum oxide, tungsten oxide, tantalum oxide, niobium oxide, bismuth oxide, antimony oxide, arsenic oxide, cadmium oxide, gallium oxide, germanium oxide, tellurium oxide, thallium oxide, uranium oxide, thorium oxide, zirconium silicate, aluminum silicate, magnesium silicate, calcium silicate, barium silicate, strontium silicate, sodium silicate, potassium silicate, lithium silicate, borosilicate and aluminosilicate.

[0744] The terms "nucleoside" and "nucleoside moiety" as use herein reference a nucleic acid subunit including a sugar group and a heterocyclic base, as well as analogs of such subunits, such as a modified or naturally occurring deoxyribonucleoside or ribonucleoside or any chemical modifications thereof. Other groups (e.g., protecting groups) can be attached to any component(s) of a nucleoside. Modifications of the nucleosides include, but are not limited to, 2'-, 3'- and 5'-position sugar modifications, 5- and 6-position pyrimidine modifications, 2-, 6-and 8-position purine modifications, modifications at exocyclic amines, substitution of 5-bromo-uracil, and the like. Nucleosides can be suitably protected and derivatized to enable oligonucleotide synthesis by methods known in the field, such as solid phase automated synthesis using nucleoside phosphoramidite monomers, H-phosphonate coupling or phosphate triester coupling.

[0745] As used herein, the term "nucleotide" or "nucleotide moiety" refers to a sub-unit of a nucleic acid which includes a phosphate group, a sugar group and a heterocyclic base, as well as analogs of such subunits. Other groups (e.g., protecting groups) can be attached to any component(s) of a nucleotide.

[0746] As used herein, the term "natural nucleoside base" preferably refers to adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

[0747] As used herein, the term "modified nucleoside base" preferably refers to 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5-fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1-methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 5- (methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7- methylguanine, isoguanine, Nl-methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2-phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8-methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl-methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6-isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6-isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5-ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4-benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6-thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5-nitroimidazole and 6-mercaptopurine. Furthermore, the term "modified nucleoside base" includes any nucleoside bases selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), C(O)NH2, C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl. In addition, one or two of the C=O groups of the nucleoside bases selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and one or two of C-OH groups of the nucleoside bases selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH. Modifications also include protecting groups such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, dibutylformamidine, dimethylacetamidine, N, N-diphenyl carbamate, etc. The present invention often mentions that one compound comprises another compound. For example, " Q is a monovalent group comprising [...] fluorophore", which may be represented by the expression " A comprises B". As a skilled person will understand, in cases where B is described in terms of fully defined compounds, such as " Rhodamine", this is to be understood in the sense that the " Rhodamine" in this group is no longer a stand-alone " Rhodamine" but the " Rhodamine" has been attached via one of its non-H atoms. In other words, when the present invention indicates that a compound, which is usually considered to be a stand-alone compound, is part of a certain chemical group or substituent, it is evident that this compound has been attached via a suitable means. A skilled person is well aware of such means which usually include chemical functionalization. Furthermore, many commonly used compounds are also commercially available with suitable functionalization, e.g. fluorophores which have been functionalized in that a maleimide has been attached which provides a position of attachment to another compound.

[0748] Terms such as "typically", "preferable", "preferably", etc. as used indicate features or embodiments which are desirable but not mandatory in the present invention. Thus, a feature or embodiment described in such terms is not limiting on the present invention, but certain further improvements may be achieved if such feature or embodiment is fulfilled.

[0749] As used herein, the terms "optional", "optionally" and "may" denote that the indicated feature may be present but can also be absent. Whenever the term "optional", "optionally" or "may" is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression " X is optionally substituted with Y" (or " X may be substituted with Y") means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional", the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

[0750] A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, preferred attachment positions for the various specific substituent groups are as illustrated in the examples. As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a", "an" and "the" are used interchangeably with "one or more" and "at least one". Thus, for example, a composition comprising "a" compound of formula (I) can be interpreted as referring to a composition comprising "one or more" compounds of formula (I).

[0751] It is to be understood that wherever numerical ranges are provided / disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.

[0752] As used herein, the term "comprising" (or "comprise", "comprises", "contain", "contains", or "containing"), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia", i.e., "containing, among further optional elements, In addition thereto, this term also includes the narrower meanings of "consisting essentially of" and "consisting of". For example, the term " A comprising B and C" has the meaning of " A containing, inter alia, B and C", wherein A may contain further optional elements (e.g., " A containing B, C and D" would also be encompassed), but this term also includes the meaning of " A consisting essentially of B and C" and the meaning of " A consisting of B and C" (i.e., no other components than B and C are comprised in A).

[0753] The scope of the present invention embraces all pharmaceutically acceptable salt forms of the compounds of the present invention which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N, N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. A pharmaceutically acceptable salt of the compounds of the present invention is preferably not a hydroiodide salt. Preferred pharmaceutically acceptable salts of the compounds of the present invention include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, an oxalate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compounds of the present invention is a hydrochloride salt. Accordingly, if a compounds of the present invention, including any one of the specific compounds of the present invention described herein, is provided in the form of a pharmaceutically acceptable salt, it is preferred that the respective compound is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, an oxalate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that it is in the form of a hydrochloride salt.

[0754] The present invention also specifically relates to the compounds of the present invention, including anyone of the specific compounds of the present invention described herein, in nonsalt form.

[0755] Moreover, the scope of the invention embraces the compounds of the present invention in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds of the present invention are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds of the present invention are likewise embraced by the invention.

[0756] Furthermore, the compounds of the present invention may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis / trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto / enol tautomers or thione / thiol tautomers). All such isomers of the compounds of the present invention are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures / racemates). The racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present invention further encompasses any tautomers of the compounds of the present invention. It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms. The formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.

[0757] The scope of the invention also embraces compounds of the present invention, in which one or more atoms are replaced by a specific isotope of the corresponding atom. For example, the invention encompasses compounds of the present invention, in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e.,2H; also referred to as " D"). Accordingly, the invention also embraces compounds of the present invention which are enriched in deuterium. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 (1H) and about 0.0156 mol-% deuterium (2H or D). The content of deuterium in one or more hydrogen positions in the compounds of the present invention can be increased using deuteration techniques known in the art. For example, a compound of the present invention or a reactant or precursor to be used in the synthesis of the compounds of the present invention can be subjected to an H / D exchange reaction using, e.g., heavy water (D2O). Further suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy. Unless specifically indicated otherwise, it is preferred that the compounds of the present invention are not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or1H hydrogen atoms in the compounds of the present invention is preferred.

[0758] The present invention also embraces compounds of the present invention, in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g.,18F,11C,13N,15O,76Br,77Br,120I and / or124I. Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET). The invention thus includes (i) compounds of the present invention, in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by18F atoms, (ii) compounds of the present invention, in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by11C atoms, (iii) compounds of the present invention, in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by13N atoms, (iv) compounds of the present invention, in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by15O atoms, (v) compounds of the present invention, in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by76Br atoms, (vi) compounds of the present invention, in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by77Br atoms, (vii) compounds of the present invention, in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by120I atoms, and (viii) compounds of the present invention, in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by124I atoms. In general, it is preferred that none of the atoms in the compounds of the present invention are replaced by specific isotopes.

[0759] The compounds of the present invention may be administered as compounds per se or may be formulated as medicaments. The medicaments / pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and / or solubility enhancers.

[0760] The pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including polyethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, a-cyclodextrin, P-cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin, hydroxypropyl-fj-cyclodextrin, hydroxyethyl-y-cyclodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl-fj-cyclodextrin, sulfobutylether- -cyclodextrin, sulfobutylether-y-cyclodextrin, glucosyl-a-cyclodextrin, glucosyl-P-cyclodextrin, diglucosyl-P-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-p-cyclodextrin, maltosyl-y-cyclodextrin, maltotriosyl-P-cyclodextrin, maltotriosyl-y-cyclodextrin, dimaltosyl-p-cyclodextrin, methyl-P-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.

[0761] The pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.

[0762] The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in " Remington: The Science and Practice of Pharmacy", Pharmaceutical Press, 22ndedition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.

[0763] The compounds of the present invention orthe above described pharmaceutical compositions comprising a compounds of the present invention may be administered to a subject by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.

[0764] If said compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternal ly, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and / or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

[0765] The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and / or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0766] For oral administration, the compounds or pharmaceutical compositions are preferably administered by oral ingestion, particularly by swallowing. The compounds or pharmaceutical compositions can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as "oral-gastrointestinal" administration.

[0767] Alternatively, said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.

[0768] Said compounds or pharmaceutical compositions may also be administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid. Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing a compound of the invention.

[0769] Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

[0770] It is also envisaged to prepare dry powder formulations of the compounds of the present invention for pulmonary administration, particularly inhalation. Such dry powders may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification / spray drying process.

[0771] For topical application to the skin, said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.

[0772] The present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Particularly preferred routes of administration are by injection, preferably subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly.

[0773] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.

[0774] A proposed, yet non-limiting dose of the compounds according to the invention for administration to a human (of approximately 70 kg body weight) may be 0.05 to 2000 mg, preferably 0.1 mg to 1000 mg, of the active ingredient per unit dose. The unit dose may be administered, e.g., 1 to 3 times per day. The unit dose may also be administered 1 to 7 times per week, e.g., with not more than one administration per day. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient / subject as well as the severity of the condition to be treated. The precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.

[0775] The compounds of the present invention or a pharmaceutical composition comprising the compounds of the present invention can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated or prevented with the compounds of the present invention). However, the compounds of the present invention or a pharmaceutical composition comprising the compounds of the present invention can also be administered in combination with one or more further therapeutic agents. If the compounds of the present invention is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used. The combination of the compounds of the present invention with one or more further therapeutic agents may comprise the simultaneous / concomitant administration of the compounds of the present invention and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential / separate administration of the compounds of the present invention and the further therapeutic agent(s). If administration is sequential, either the compounds of the present invention according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compounds of the present invention, or they may be administered in two or more different (separate) pharmaceutical formulations.

[0776] The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, the subject / patient to be treated in accordance with the invention is a human.

[0777] The term "treatment" of a disorder or disease, as used herein, is well-known in the art. " Treatment" of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient / subject. A patient / subject suspected of suffering from a disorder or disease typically shows specific clinical and / or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).

[0778] The "treatment" of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment" of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject / patient suffering from the disorder or disease. Accordingly, the "treatment" of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject / patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).

[0779] The term "prevention" of a disorder or disease, as used herein, is also well-known in the art. In the context of the present invention, in particular the use of the compounds of the present invention for vaccination is contemplated. For example, a patient / subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject / patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient / subject (for example, the patient / subject does not show any clinical or pathological symptoms). Thus, the term "prevention" comprises the use of a compound of the present invention before any clinical and / or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.

[0780] It is to be understood that the present invention specifically relates to each and every combination of features described herein, including any combination of general and / or preferred features. In particular, the invention specifically relates to each combination of meanings (including general and / or preferred meanings) for the various groups and variables comprised in the formulae of the compounds of the present invention.

[0781] In this specification, a number of documents including patent applications, scientific literature and manufacturers' manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The reference in this specification to any prior publication (or information derived therefrom) is not and should not be taken as an acknowledgment or admission or any form of suggestion that the corresponding prior publication (or the information derived therefrom) forms part of the common general knowledge in the technical field to which the present specification relates.

[0782] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. EXAMPLES

[0783] The compounds / examples described in this section are defined by their chemical formulae and their corresponding chemical names. In case of conflict between any chemical formula and the corresponding chemical name indicated herein, the present invention relates to both the compound / example defined by the chemical formula and the compound / example defined by the chemical name, and particularly relates to the compound / example defined by the chemical formula.

[0784] Part A:

[0785] Examples Al to A3 demonstrate the synthesis of representative transcription initiators containing EDA group suitable for the synthesis of pre-circRNAs - starting material for the circularization according to this invention. Example A4 describes the generation of DNA templates for the synthesis of RNAs by transcription in vitro. Examples A5 to All describes the preparation of circRNAs according to the invention as well as reference circRNAs obtained by state-of-the-art methods used in comparative studies. Examples A12 to A14 describe the procedures for circRNA purification and structure confirmation. Examples A15 and A16 described the optimization of RNA conformation for optimal chemical circularization. Example A17 described the evaluation of...

Claims

New PCT-Patent Application(based on Priority Application: EP 24461634.8)Siec Badawcza Lukasiewicz -PORT Polski Osrodek Rozwoju TechnologiiVossius Ref.: AJ3700 PCT S3CLAIMS1. A compound of Formula Iwherein:n is any integer with a value of from 1 to 20000;each X is independently selected from O’, BHs’, NH’, S’, and Se_;each G is independently selected from O and S;each A is independently selected from CH2, O, S, NH and a bond;each E is independently selected from CH2, O, S and NH;each D is independently selected from CH2, O, S and NH;each R1is independently selected from a natural or modified nucleoside base; each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N (a lkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, --NHC(O)NH(RR), -NHC(O)N(RR)2, - NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, - OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, - haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, - C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;R3is selected from a natural or modified nucleoside base,L is a trivalent group having from 1 to 50 non-H atoms,Q is a monovalent group comprising one or more groups selected from nucleotide and oligonucleotide, including mRNA 5' cap analog, nucleic acid, fluorophore, hapten, chelator, (radio)isotopic tag, lipid, amino acid, oligopeptide, protein, saccharide, oligosaccharide, polymer, polymer composite, metal-organic framework, or a solid support.

2. A compound of Formula IIwherein:n is any integer with a value of from 1 to 20000;each X is independently selected from O’, BHs’, NH’, S’, and Se_;each G is independently selected from O and S;each A is independently selected from CH2, O, S, NH and a bond;each E is independently selected from CH2, O, S and NH;each D is independently selected from CH2, O, S and NH;each R1is independently selected from a natural or modified nucleoside base;each R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N (a lkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C O)(RR, -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, - OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, - haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, - C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, - NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;R3is selected from a natural or modified nucleoside base,L is a trivalent group having from 1 to 50 non-H atoms,J is a monovalent group having from 1 to 70 non-H atoms, wherein J preferably comprises at least one group selected from C=C bond, -N3, -NH2, -SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

3. The compound according to claim 1 or 2, wherein each X is independently selected from O’, S’ and Se_; preferably each X is independently selected from O’ and S’; more preferably each X is O’.

4. The compound according to any one of the preceding claims, wherein each G is O.

5. The compound according to any one of the preceding claims, wherein each A is independently selected from O, S, NH and a bond; preferably each A is independently selected from O, S and a bond; more preferably each A is independently selected from O and a bond; even more preferably each A is O.

6. The compound according to any one of the preceding claims, wherein each E is independently selected from O and S; preferably each E is O.

7. The compound according to any one of the preceding claims, wherein each D is independently selected from O and S; preferably each D is O.

8. The compound according to any one of the preceding claims, wherein each R1and R3is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), each adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) optionally having one, two or three (preferably one or two, more preferably one) substituents selected from -F, -Br, -Cl, -RR, -CN, -C(O)H, C(O)(RR), -C(O)NH2, -C(O)NH(RR), C 0 N(RR2, -C(O)OH, C(O)O(RR), -NHZ, - H(RR), -N(RR)2, -NHC(O) RR, -NHC(O)H, NHC(O)NH2, •• NHC(O)NH(RR), - HC(O N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), - OC(O (RR2, -OC(O) H(RR), -OC(O)NH2, -OC(O)O RR, -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cycloalkyl, -(CH2)o- 3-heterocycloalkyl, -(CH2)o-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(a Ikyl), -C(O)NH2, -C(O)N H(a Ikyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), - NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), - SH, and -S- alkyl,and wherein one or two of the C=O groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C=S and / or one or two of C-OH groups of the adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) may be replaced by C-SH,preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), 2-thiouracil, 5-bromouracil, 5-carboxyuracil, 5-ethynyluracil, 5- fluorouracil, 5-formyluracil, 5-hydroxyuracil, 5-iodouracil, 5-methoxycarbonylmethyl-2- thiouracil, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 5-propynyluracil, 1- methylpseudouracil, 5-(2-bromovinyl)uracil, 5-(carboxyhydroxymethyl)uracil, 5- (carboxymethylaminomethyl)-uracil, 5-(methylaminomethyl)uracil, 5-chlorouracil, 5-hydroxymethyluracil, 5-methyl-2-thiouracil, 5-propyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 6-thioguanine, 7-methylguanine, isoguanine, Nl- methylguanine, N2-benzoylguanine, N2-isobutyrylguanine, N2-methylguanine, N2- phenylacetylguanine, 8-aminoguanine, 8-bromoguanine, 8-chloroguanine, 8- methylguanine, 8-thioguanine, 2,2-dimethylguanine, 2-methylguanine, Nl- methyladenine, N6-benzoyladenine, N6-methyladenine, N6-phenylacetyladenine, 6- isopentyladenine, 8-bromoadenine, 2-methyladenine, 2-methylthio-N6- isopentyladenine, N, N-dimethyladenine, 2-thiocytosine, 5-carboxycytosine, 5- ethylcytosine, 5-formylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5- methoxycytosine, 5-methylcytosine, 5-propynylcytosine, N4-acetylcytosine, N4- benzoylcytosine, N4-isobutyrylcytosine, N4-methylcytosine, 3-methylcytosine, isocytosine, inosine-base, 1-methylinosine-base, pseudouracil, queuosine-base, wyosine-base, 2-aminopurine, xanthine, hypoxanthine, 2,6-diaminopurine, 6- thiopurine, 6-hydroxyaminopurine, 5-nitroindole, 5-nitrobenzimidazole, 5- nitroimidazole and 6-mercaptopurine;more preferably each R1is independently selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).

9. The compound according to any one of the preceding claims, whereineach R2is independently selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N (a lkyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, Cl, Br and CN; preferably each R2is independently selected from H, alkyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, F, Cl and Br, wherein each alkyl is optionally substituted with one or more selected from F, Cl, Br and CN;more preferably each R2is independently selected from H, Me, OH, OMe, O-CH2-aryl and F;even more preferably each R2is independently selected from H, OH, OMe and F; still more preferably each R2is independently selected from OH and OMe;still even more preferably each R2is OH;10. The compound according to any one of the preceding claims, whereinthe moiety –A-L(Q)– or –A-L(J)– does not represent any of the following formulae:

11. A compound of Formula IIIwhereinX is selected from O’, BHs’, NH’, S’, and Se’;G is selected from O and S;A is selected from CH2, O, S, NH and a bond;E is selected from CH2, O, S and NH;Dbis selected from CH2, O, S and NH;R1and Rlbare independently selected from a natural or modified nucleoside base; R2is selected from H, alkyl, alkenyl, alkynyl, OH, O-alkyl, O-CH2-aryl, SH, S-alkyl, NH2, NH(alkyl), N(a I kyl)2, F, Cl and Br, wherein each alkyl, alkenyl, alkynyl and aryl is optionally substituted with one or more selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -HNO2, -OH, -O-RR, -OC(O)(RR, -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)o-3-cydoalkyl, -(CH2)o-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)0-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(a I kyl), - C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), - N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, - NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), - OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;L is a trivalent group having from 1 to 50 non-H atoms,J is a monovalent group having from 1 to 70 non-H atoms, wherein J preferably comprises at least one group selected from C=C bond, -N3, -NH2, -SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

12. A compound of Formula IVwhereinRC1and RC2are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl, and (hetero)aryl, wherein the alkyl, alkenyl, alkynyl, aryl, alkylene(hetero)aryl are optionally substituted with one or more selected from F, - Br, -Cl, -RR, -CN, -C(O)H, -C(O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O)N(RR)2, -C(O)OH, -C(O)O(RR), -NH2, -NH(RR), -N(RR)2, -NHC(O)(RR), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(RR), -NHC(O)N(RR)2, -NHC(O)O(RR), -NO2, -OH, -O-RR, -OC(O)(RR), -OC(O)N(RR)2, -OC(O)NH(RR), -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), -OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;RC3and RC4are independently selected from the group consisting of OH, O-CH2-aryl, O-alkyl, SH, S-alkyl, NH2, NH(alkyl), F, Cl, Br, alkyl, alkenyl, and alkynyl, or RC3and RC4may be taken together to form -O-alkylidene-O-;W1and W2are independently selected from the group consisting of O, S, Se, NH, CH2, alkylene, alkenylyne, alkynylene and a bond;m and n are independently an integer selected from the group consisting of 0, 1 and 2. XC1, YC1, XC2, YC2, XC3, YC3, XC4, YC4are independently selected from the group consisting of O, S, Se, BH3and NH;ZC1, ZC2, ZC3are independently selected from the group consisting of O, NH, CH2, CH(alkyl), CF(alkyl), CH(alkyl)2, CHF, CF2, CFCI, CHCI and CCI2, wherein alkyl is optionally haloalkyl;L2comprises one or more groups selected from optionally substituted heteroalkylene, optionally substituted alkylene, peptides, oligonucleotides and their block co-polymers; chemically modified peptides, chemically modified oligonucleotides and their block copolymers; polyethylene glycol), poly(lactic acid) and their block co-polymers, wherein the wherein the one or more optional substituents of the heteroalkylene and the alkylene are selected from F, -Br, -Cl, -RR, -CN, -C(O)H, -C O)(RR), -C(O)NH2, -C(O)NH(RR), -C(O (RR2, -C(O)OH, -C(O)O(RR), -NH2, - H(RR, -N(RR)2, -NHC(O) RR), -NHC(O)H, -NHC(O)NH2, --NHC(O)NH(RR), -NHC(O) (RR2, -NHC(O)O(RR), -NO2, -OH, -O-RR, - OC(O)(RR), -OC O)N(RR)2, -OC(O NH(RR, -OC(O)NH2, -OC(O)O(RR), -SH, and -S-RR, wherein each RRis independently selected from -alkyl, -haloalkyl, -heteroalkyl, -(CH2)0-3-cycloalkyl, -(CH2)0-3-heterocycloalkyl, -(CH2)0-3-aryl, -(CH2)o-3-heteroaryl, wherein each heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one or more selected from -F, -Br, -Cl, -alkyl, -CN, -C(O)H, -C(O)(alkyl), -C(O)NH2, -C(O)NH(alkyl), -C(O)N(alkyl)2, -C(O)OH, -C(O)O(alkyl), -NH2, -NH(alkyl), -N(alkyl)2, -NHC(O)(alkyl), -NHC(O)H, -NHC(O)NH2, -NHC(O)NH(alkyl), -NHC(O)N(alkyl)2, -NHC(O)O(alkyl), -NO2, -OH, -O- alkyl, -OC(O)(alkyl), -OC(O)N(alkyl)2, -OC(O)NH(alkyl), - OC(O)NH2, -OC(O)O(alkyl), -SH, and -S- alkyl;Q2is a group having 0 to 50 carbon atoms and up to 20 heteroatoms selected from N, O, P and S, wherein the Q2preferably comprises at least one group selected from C≡C bond, -N3, -SH, -NHC(O)CH2I, maleimide, diazirine, epoxide, tetrazine, -SO2F, -OSO2F, and -NHSO2F.

13. Use of the compound of Formula II of any one of claims 2 to 10 for the manufacture of a compound of Formula I of any of claims 1 and 3 to 10; oruse of the compound of Formula III of claim 11 for the manufacture of a compound of Formula I of any of claims 1 and 3 to 10; oruse of the compound of Formula IV of claim 12 for the manufacture of a compound of Formula I of any of claims 1 and 3 to 10, preferably by reacting the compound of Formula IV of claim 12 with the compound of Formula II of any one of claims 2 to 10.

14. A kit of parts, the kit comprising a compound of any of formulae II, III, and IV, as defined in any one of claims 2 to 12, and instructions for the preparation of a compound of Formula I as defined in any of claims 1 and 3 to 10.

15. A compound according to any of claims 1 to 12, or the kit according to claim 14, wherein the compound or kit is for the treatment or prevention of cancer, a cardiovascular disease, a neurological disorder, a metabolic disorder, an infectious disease, a genetic disorder, an inflammatory and autoimmune disease, an ophthalmic disease, a pulmonary disease, a renal disease, a gastrointestinal disease, a dermatological disease, a musculoskeletal disease, an endocrine disorder, a hematological disease, Huntington's disease, Gaucher disease, Fabry disease, and / or Pompe disease.