Modified oligomeric compounds comprising tricyclo-DNA nucleosides and uses thereof

Abstract

To provide a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and one or more lipid moieties.SOLUTION: The invention relates to a composition comprising the oligomeric compound to which one or more lipid moieties are covalently linked directly or via a spacer, where, preferably, the oligomeric compound comprises 5-40 monomer subunits, as well as pharmaceutical compositions thereof, and uses thereof in the prevention or treatment of neuromuscular diseases or musculoskeletal diseases such as Duchenne muscular dystrophy or Steinert disease.SELECTED DRAWING: None

Description

[Technical field]

[0001] STATEMENT OF RELATED APPLICATIONS This application claims priority to European Application No. 17167427.8, filed April 20, 2017, and U.S. Provisional Application No. 62 / 562,124, filed September 22, 2017, both of which are incorporated by reference in their entireties. [Background technology]

[0002] Antisense technology is an effective means to reduce the expression of specific gene products and can therefore be useful in therapeutic, diagnostic and research applications. In general, the principle behind antisense technology is that antisense compounds (sequences of nucleotides or their analogs) hybridize to target nucleic acids and modulate the activity or function of gene expression, such as transcription and / or translation. Despite the specific mechanism, due to their sequence specificity, antisense compounds are useful tools for target validation and gene functionalization, as well as for the diagnosis and treatment of diseases. This makes them attractive therapeutic agents that selectively modulate the expression of genes involved in the pathogenesis of

[0003] Antisense compounds typically incorporate chemically modified nucleosides to enhance nucleoside properties such as nuclease resistance, pharmacokinetics or affinity for the target RNA. These chemically modified antisense oligonucleotides (AONs) include structural modifications of natural RNA such as 2'-OH modifications, locked nucleic acids (LNA), peptide nucleic acids (PNA), hexitol nucleic acids (HNA), and tricycloDNA (tcDNA), as well as many others (reviewed in: Bennett CF and Swayze EE, Annu. Rev. Pharmacol. Toxicol., 2010, 50, 259-293; Deleavey GF and Damha M, Chemistry & Biology, 2012, 19, 937-954; Sharma VK et al., Med. Chem. Commun., 2014, 5, 1454-1471; Siva et al., Nucleic Acid Therapeutics, 2014, 24, 69-86; Goyenvalle A. et al., Journal of Neuromuscular Diseases, 2016, 3, 157-167, the disclosures of which are incorporated herein by reference). Also, backbone modifications such as phosphorothioate (PS) linkages, in which one of the non-bridging oxygen atoms of the phosphodiester linkage is replaced with a sulfur atom, are one of the most widely investigated nucleic acid chemical modifications in oligonucleotide therapeutics.

[0004] Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1 in 3500 live male births (Emery. Neuromuscul. Disord., 1991). DMD Dystrophin is caused by mutations in the gene encoding dystrophin, a large protein (427 kDa) found in various tissues, particularly in striated muscle fibers, and in neurons in certain regions of the central nervous system (Kunkel et al., PNAS., 1985; Muntoni F et al., Lancet Neurol., 2003). Dystrophin is located close to the inner surface of the cell membrane and connects the actin cytoskeleton to the extracellular matrix through a membrane dystrophin-associated glycoprotein complex (Culligan et al., 1988). The absence of dystrophin renders muscle fibers particularly vulnerable to mechanical stress and undergoes recurrent cycles of necrosis. As a result, patients present with progressive wasting of skeletal muscles, which are replaced over time by fatty fibrotic tissue, leading to loss of ambulation by the age of 12 and premature death due to either respiratory failure or cardiomyopathy. In addition, approximately one-third of DMD patients also exhibit cognitive impairment suggesting significant disruption of neurons and brain function (Bresolin et al., Neuromuscul. Disord., 1994).

[0005] Full-length dystrophin, translated from a primary 14 kb mRNA transcript made up of 79 exons, is a regulatory protein that can fortunately support multiple exon deletions, provided that the open reading frame is preserved (Koenig et al., Cell, 1987). This phenomenon occurs in the clinically mild disease Becker muscular dystrophy (BMD), in which deletions that maintain the open reading frame result in the synthesis of a truncated, semi-functional form of dystrophin (Monaco et al., Genomics., 1999). 1988). Human DMD is caused by a heterogeneous group of mutations occurring across 79 exons, but the two regions with the highest incidence are observed in the exon 3-7 and exon 45-55 regions. Muntoni, Neuromuscul. Disord., 2009, vol. 20, pp. 355-362. DMD is caused by out-of-frame deletions or caused by inappropriate stop codons in the dystrophin mRNA. This results in out-of-frame truncated mRNA, unstable and incomplete dystrophin, and ultimately the clinical findings of DMD. In-frame truncated mRNA is known to result from different mutations that result in hemifunctional dystrophin, and a much milder form of myopathy known as Becker muscular dystrophy (BMD). Therefore, 15 years ago, it was proposed that disrupting the splicing process of selected exons by using antisense oligonucleotides (AONs) might be a suitable therapeutic approach for DMD (Matsuo M., Brain Dev., 1996). The goal of oligonucleotide therapy against is generally to induce exon skipping (e.g., exon 23 or exon 51, or any other exon), restore dystrophin production, and convert DMD to BMD, thereby reducing mortality and potentially improving cognition. In addition to DMD, many other diseases can also be treated by oligonucleotide-mediated exon skipping approaches, including the diseases described below in this specification.

[0006] Two types of compounds have been extensively tested for antisense-induced exon skipping: 2'-O-methyl modified ribose oligomers with full-length phosphorothioate backbones (2'OMe-PS) and phosphorodiamidate morpholino oligomers (PMOs). Both types of antisense molecules have been shown to rescue dystrophin in skeletal muscle after systemic delivery in animal models of DMD and in clinical trials (van Deutekom et al., New. Engl. J. Med., 2007; Kinali et al., Lancet Neurol., 2009; Goemans et al., New. Engl. J. Med., 2011; Cirak et al., Lancet., 2011). However, Recent studies using 2'OMe-PS and PMO AONs targeting exon 51 in DMD patients have failed to demonstrate significant clinical benefit, likely due to insufficient levels of dystrophin rescue (Lu et al., Mol. Ther. Nucleic Acids, 2 (2014). It is also known from preclinical studies in mice that these two chemicals were not suitable to address cardiac and cognitive abnormalities, which means that it remains a significant challenge to achieve success in many neuromuscular disorders. More recent studies in the mdx mouse model of DMD have demonstrated that tcDNA AONs with a complete PS backbone allow effective skipping of exon 23 to levels 5-6 times higher than achieved with 2'OMe-PS and PMO AONs (Goyenvalle et al., Nat. Med., 2015).

[0007] Importantly, this translated into higher rescue of dystrophin protein levels, especially in the diaphragm and heart, which reached 50% and 40%, respectively, compared to wild-type mice after 12 weeks of treatment. However, while significantly improving biodistribution, oxygen-to-sulfur substitutions in the phosphate backbone have also been shown to be involved in non-specific protein binding and activation of the innate immune system, particularly complement activation, which may result in acute toxicity at worst and long-term toxicity at best (Dirin and Winkler, Expert Opin. Biol. Ther., 2013). This major hurdle, which undermines or at least limits the therapeutic potential of this class of molecules, has prompted us to search for new agents that are more efficient and potentially less toxic. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bennett CF and Swayze EE, Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259-293. Summary of the Invention [Means for solving the problem]

[0009] The present invention relates to a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and one or more lipid moieties covalently linked to said oligomeric compound, either directly or via a spacer.Methods for the treatment of several diseases, including Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), CNS-related symptoms, Pompe disease and myotonic dystrophy type I (DM1), by using said composition of the present invention are also disclosed.

[0010] Surprisingly, it has been found that the compositions of the present invention, comprising an oligomeric compound and one or more lipid moieties covalently linked to said oligomeric compound, are much more efficient and active in penetrating skeletal muscle, heart tissue and CNS after systemic delivery than their tcDNA counterparts that do not comprise said one or more lipid moieties. The latter is the case regardless of the nature of the internucleoside linkages present in the corresponding oligomeric counterparts, and therefore regardless of whether the counterparts have a complete PS backbone or not. The inventors have also found that the compositions of the present invention can exhibit the unexpected property of efficiently penetrating heart tissue and thus crossing the blood-brain barrier, avoiding the need for PS internucleoside linkages, which potentially significantly improves the safety profile and reduces toxicity while maintaining high efficacy. This allows the dosage of the pharmaceutical composition of the present invention to be applied to be increased to meet and satisfy medical needs.

[0011] Furthermore, it has been shown that the composition of the present invention is transported in the bloodstream and taken up by all skeletal muscles, cardiac muscles and CNS after intravenous systemic application.Thus, the composition of the present invention is particularly useful as an antisense oligonucleotide (AON) for obtaining antisense effects in muscle and cardiac cells or in the CNS after systemic delivery.Thus, the present invention also provides a composition for some diseases caused by abnormal gene expression in tissues or cells of a subject.Without being bound by this theory, it is believed that the composition of the present invention forms aggregates with blood albumin, thereby not only ensuring the transport of the composition of the present invention in the bloodstream, but also increasing the circulation half-life and therefore the exposure time of tissues to the composition of the present invention, and also enhancing the resistance to degradation in plasma.

[0012] In addition, the preferred compositions of the present invention, which comprise one or more lipid moieties covalently linked to the oligomeric compound, remain highly effective even when the number of phosphorothioate internucleoside linking groups in the oligomeric compound is reduced in favor of phosphorodiester internucleoside linking groups.Thus, it has been found that the preferred compositions of the present invention, especially those that comprise a fatty acid or fatty diacid moiety such as palmitoyl moiety and contain a large number of phosphorodiester internucleoside linking groups, induce significantly higher levels of exon skipping in multiple tissues compared to their corresponding tc-DNA analogs that do not have the lipid moiety linked thereto.

[0013] Thus, in a first aspect, the present invention provides a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and one or more lipid moieties, preferably exactly one lipid moiety, said one or more lipid moieties being covalently linked to said oligomeric compound, either directly or via a spacer, preferably said oligomeric compound comprising 5 to 40 monomeric subunits.

[0014] In a further aspect, the present invention relates to a pharmaceutical composition comprising the composition of the present invention and further comprising a pharma- ceutically acceptable carrier, preferably said pharmaceutical composition is for use in the prevention, treatment or diagnosis of a neuromuscular disease or a musculoskeletal disease, more preferably said neuromuscular disease or said musculoskeletal disease is selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia-telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2. , macrocephalic leukoencephalopathy type 1 with subcortical cysts, Pelizaeus-Merzbach disease, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), and again more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0015] In another aspect, the present invention provides a pharmaceutical composition of the present invention comprising the composition of the present invention and further comprising a pharma- ceutically acceptable carrier, preferably wherein said pharmaceutical composition is for use in the treatment of a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia-telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, cortical fibrosis, myeloma, myeloma, pulmonary fibrosis ... and again more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0016] In yet a further aspect the present invention relates to a composition of the present invention for use as a medicament in the prevention, treatment or diagnosis of a disease, preferably wherein said disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, large intestine with subcortical cysts, and / or cerebrovascular accidents. Preferably, the neuromuscular disease or the musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), and again more preferably, the neuromuscular disease or the musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0017] In a further aspect the present invention relates to a composition of the present invention for use as a medicament in the treatment of a disease, preferably wherein said disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic white matter encephalopathy with subcortical cysts. In accordance with another aspect of the present invention, there is provided a composition for treating a neuromuscular disease or a musculoskeletal disease, said composition being selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), and again more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0018] In a further aspect the present invention relates to a composition of the present invention for use in a method of treating a disease, preferably wherein said disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic leukoencephalopathy with subcortical cysts. In accordance with another aspect of the present invention, there is provided a composition for treating a neuromuscular or musculoskeletal disease, the composition comprising: a) a neuromuscular disease selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease); and b) a neuromuscular disease selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0019] In a further aspect the present invention relates to a composition of the present invention for use in a method for preventing, treating or diagnosing a disease, preferably wherein said disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephaly with subcortical cysts. Leukoencephalopathy type 1, Pelizaeus-Merzbach disease, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), and again more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0020] In yet a further aspect, the present invention provides a method for treating a neuromuscular or musculoskeletal disease comprising administering to a patient a therapeutically effective dose of a composition of the present invention or a pharmaceutical composition of the present invention, in a preferred embodiment of which said neuromuscular or musculoskeletal disease is selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic leukoencephalopathy with subcortical cysts type 1, Pelizaeus-Merzbach disease, Pompe disease, myotonia, cerebrovascular disease, cerebrovascular accident ... In one embodiment, the neuromuscular or musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), preferably the neuromuscular or musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0021] Further aspects and embodiments of the invention will become apparent as this description continues. In an embodiment of the present invention, for example, the following items are provided: (Item 1) a. an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, preferably comprising 5 to 40 monomeric subunits; and b. one or more lipid moieties A composition comprising: A composition wherein the one or more lipid moieties are covalently linked to the oligomeric compound, either directly or via a spacer. (Item 2) 2. The composition of claim 1, wherein the one or more lipid moieties are independently selected from a fatty acid moiety, a fatty diacid moiety, an alkyl phosphate moiety, and an alkyl phosphonate moiety. (Item 3) The one or more lipid moieties, independently of each other, have the formula (I) AB- * (I) (Wherein, A is C 3~32 Alkyl, C 3~32 Alkenyl, C 3~32 Alkynyl, HOOC-C 3~32 Alkylene, HOOC-C3~32 Alkenylene or HOOC-C 3~32 alkynylene, B is C(O), OP(OH), OP(O)(OH), OP(O)(SH), NH-P(O)(OH), NH-P(O)(SH), NH-C(O), or a pharma- ceutically acceptable salt thereof, and the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer. 3. The composition according to any one of claims 1 or 2, wherein (Item 4) The one or more lipid moieties may each independently be represented by the formula (a)-(u) aC 3~32 Alkyl-C(O)- * 、 bC 3~32 Alkenyl-C(O)- * 、 cC 3~32 Alkynyl-C(O)- * 、 dC 3~32 Alkyl-OP(OH)- * 、 eC 3~32 Alkenyl-OP(OH)- * 、 fC 3~32 Alkynyl-OP(OH)- * 、 gC 3~32 Alkyl-OP(O)(OH)- * 、 hC 3~32 Alkenyl-OP(O)(OH)- * 、 I C 3~32 Alkynyl-OP(O)(OH)- * 、 jC 3~32 Alkyl-OP(O)(SH)- * 、 kC 3~32 Alkenyl-OP(O)(SH)- *、 l.C 3~32 Alkynyl-OP(O)(SH)- * 、 mC 3~32 Alkyl-NH-C(O)- * 、 nC 3~32 Alkenyl-NH-C(O)- * 、 oC 3~32 Alkynyl-NH-C(O)- * 、 p.c. 3~32 Alkyl-NH-P(O)(OH)- * 、 qC 3~32 Alkenyl-NH-P(O)(OH)- * 、 r.C 3~32 Alkynyl-NH-P(O)(OH)- * 、 s.HOOC-C 3~32 Alkylene-C(O)- * 、 t.HOOC-C 3~32 Alkenylene-C(O)- * , and u.HOOC-C 3~32 Alkynylene-C(O)- * 、 (Wherein, the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer. The composition according to any one of items 1 to 3, wherein the composition is selected from any one of the following: (Item 5) The one or more lipid moieties may be independently represented by the formulas (a) and (b): aC 3~32 Alkyl-C(O)- * 、 b.HOOC-C 3~32 Alkylene-C(O)- * (Wherein, the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer, and preferably, 3~32 The alkyl is an unbranched C 3~32 alkyl, more preferably the C 3~32 The alkyl is an unbranched C 3~32 alkyl, preferably 3~32 The alkylene is an unbranched C 3~32 alkylene, more preferably the C 3~32 Alkylene is an unbranched C 3~32 alkylene) 5. The composition according to any one of items 1 to 4, which is a part from any one of the following: (Item 6) The spacer is represented by the formula a.#-NH-C 2~12 Alkylene-§, b. #-NH-C 2~12 Alkylene-OP(OH)-§, c.#-NH-C 2~12 Alkylene-OP(O)(SH)-§, d. #-NH-C 2~12 Alkylene-OP(O)(OH)-§, e.#-NH-C 2~12 Alkylene-NH-C(O)-§, f.#-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and g.#-NH-C 2~12 Alkylene-NH-P(O)(SH)-§, (Wherein, C 2~12 One or more -CH in the alkylene 2 The - moiety is independently -O-, -S-, -NH-, -C(O)-, -C(O)O-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -OP(OH)O-, -OP(O)(SH)O-, -OP(O)(OH)O-, -NHP(O)(OH)O-, -NHP(O)(SH)O-, or -(O-CH 2 -CH 2 ) k - (k is an integer from 1 to 8), 2~12 One or more -CH in the alkylene 2 The - moieties may independently be one or more of -COOH, -NH 2 , -OP(O)(OH)2 or -OH, wherein the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound. and preferably, independently, selected from any one of the formulae: (Item 7) The spacer is represented by the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k-X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently OP(OH), OP(O)(SH), OP(O)(OH), NHP(O)(OH), NHP(O)(SH) or NH-C(O); each k is independently an integer of 1 to 8; each m is independently an integer of 2 to 12; each n is independently an integer of 2 to 4; each p is independently an integer of 1 to 5; each q is independently an integer of 1 to 3, preferably 1 or 2; each r is independently an integer of 1 to 3, preferably 1 or 2; the (#) represents a point of covalent bond with the lipid moiety; and the (§) represents a point of covalent bond with the oligomer compound. and preferably, independently, selected from any one of the formulae: (Item 8) 8. The composition according to any one of items 1 to 7, wherein the one or more lipid moieties are linked, independently of each other, to the oligomeric compound at: (i) a terminal residue of the oligomeric compound; (ii) the 5' end of the oligomeric compound; (iii) the 3' end of the oligomeric compound; (iv) an internal residue of the oligomeric compound. (Item 9) 9. The composition according to any one of items 1 to 8, wherein the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides. (Item 10) 10. The composition according to any one of items 1 to 9, wherein the oligomeric compound comprises 5 to 40 monomeric subunits, the monomeric subunits being nucleosides, one or more of the nucleosides being the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and the monomeric subunits being linked by a plurality of internucleoside linking groups. (Item 11) 11. The composition of claim 10, wherein the internucleoside linking groups are independently selected from phosphorothioate linking groups and phosphorodiester linking groups, and no more than six of the internucleoside linking groups are phosphorothioate linking groups, and preferably no more than three of the internucleoside linking groups are phosphorothioate linking groups. (Item 12) 12. The composition of claim 10 or 11, wherein all of the internucleoside linkage groups are phosphorodiester linkage groups. (Item 13) 13. The composition according to any one of items 1 to 12, wherein the oligomeric compound comprises a sequence selected from any one of the sequences of SEQ ID NOs: 1 to 37. (Item 14) A pharmaceutical composition comprising the composition according to any one of items 1 to 13 and further comprising a pharma- ceutically acceptable carrier, preferably for use in the prevention, treatment or diagnosis of a neuromuscular or musculoskeletal disorder. (Item 15) 15. The composition according to any one of items 1 to 13 or the pharmaceutical composition according to item 14 for use as a medicament in the prevention, treatment or diagnosis of a disease, preferably wherein the disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein the neuromuscular disease or the musculoskeletal disease is selected from Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia-telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic leukoencephalopathy with subcortical cysts type 1, Pelizaeus-Merzbach disease, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease). [Brief description of the drawings]

[0022] [Figure 1]Detection of exon-skipped dystrophin mRNA in mdx muscles and CNS including tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Quad), triceps (Tri), biceps (Bi), diaphragm (Dia), heart, cortex, and cerebellum (Cbl) after 4 weeks of treatment with doses of 200 mg / kg / week for tcDNA-PO M23D (SY-0308), tcDNA-PS M23D (SY-0210), SY-0299, SY-0442, and SY-0455, and 178 mg / kg / week for SY-0343. Exon 23 skipping was quantified by Taqman qPCR and expressed as a percentage of total dystrophin measured by expression levels of exons 4-5 after normalization with endogenous controls. N=4 mice per group; error bars are mean ± SEM.

[0023] [Figure 2-1] Prothrombin time and partial thromboplastin time are two blood tests that measure how long it takes blood to clot in order to predict bleeding problems. 2 mg / ml of the following sequences: tcDNA-PO M23D (SY-0308, SEQ ID NO: 1), or tcDNA-PS M23D (SY-0210, SEQ ID NO: 23), or tcDNA-PS SYN51 (SEQ ID NO: 19, where all nucleotides are tc-DNA and all internucleoside linkages are PS linkages), or tcDNA-PO SYN51 (SEQ ID NO: 19, where all nucleotides are tc-DNA and all internucleoside linkages are PO linkages), or SY-0343, or SY-0442, or SY-0455, were incubated with 50 μl of citrated plasma C at 37° for 30 min, and then prothrombin time (PT) and activated partial thromboplastin time (PTT) assays were performed in a semi-automated START max coagulometer (Stago) according to the manufacturer's instructions (A mouse plasma, B human plasma). PBS (phosphate buffered saline) was used as a negative control and as a positive control, (tcDNA-PS SYN51) with an intact PS backbone, which is highly toxic to mice, was used. [Figure 2-2] Same as above.

[0024] [Diagram 3] One hour after AON injection, blood samples were taken from all mice to measure complement C3. Complement activation in serum samples was determined by Microvue PanSpecific-C3 Converter and SC5b-9 Plus Kit (Quidel Co., San Diego CA, USA). Mouse C3 protein was converted to human SC5b9 using C3 converter reagent (Pan specific C3 reagent kit, Microvue, Quidel) and then detected by SC5b9 Elisa (Quidel). Complement activation was expressed as a percentage of the remaining level of C3 in the samples, considering the level of C3 in PBS condition as 100% of C3 (no activation).

[0025] [Figure 4-1] Serum biochemistry analysis at the end of 4 weeks of treatment. There were no significant changes in serum albumin, creatinine, and urea, suggesting the absence of nephrotoxicity. Only a slight increase in ALP for most palmitoyl-conjugated PO M23D was observed. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above.

[0026] [Diagram 5] Restoration of dystrophin levels quantified by Western blot using the licor Odyssey system in gastrocnemius (Gas), diaphragm (Dia), and triceps (Tri) muscles after 4 weeks of treatment with SY-0299, SY-0455, and SY-0442 at a dose of 200 mg / kg / week.

[0027] [Figure 6-1]Biotinylated tcDNA oligonucleotides (M23D) used for co-precipitation of potentially interacting proteins from mouse and human serum. A: SY-0446; A': SY-0448, an analog of SY-0446, except that the third nucleotide of the tcDNA sequence is changed to 2'OMe-U; B: SY-0445; C: SY-0443, an analog of SY-0445, except that there are two PSs at both ends of the tcDNA oligonucleotide; D: SY-0451. For AONs without palmitoyl residues (SY-0440 and SY-0427), the biotin moiety was attached to the 5' end of the oligonucleotide via a C3 linker (not shown). Characterization and definition of the compositions of the present invention are described in Table 3. [Figure 6-2] Same as above.

[0028] [Figure 7] SDS-PAGE analysis of proteins recovered from mouse (Panel A) and human (Panel B) serum using the oligonucleotides listed and depicted in Figure 6. 20 μg of immobilized AO was incubated in 50 μL of 10-fold diluted serum.

[0029] [Figure 8]Prothrombin times and partial thromboplastin times of the following sequences with variable content of sulfur in the internucleoside linkages: tcDNA-PO M23D (SY-0308, SEQ ID NO: 1), or tcDNA-PS M23D (SY-0210, SEQ ID NO: 23), or tcDNA-PS SYN51 (SEQ ID NO: 19, where all nucleotides are tc-DNA and all internucleoside linkages are PS linkages), or tcDNA-PO SYN51 (SEQ ID NO: 19, where all nucleotides are tc-DNA and all internucleoside linkages are PO linkages), M23D-PS 25sb3 corresponding to (p-CCTCGGCTTA*C*C*T - SEQ ID NO: 42); M23D-PS 33sb3 corresponding to (p-CCTCGGCTT*A*C*C*T - SEQ ID NO: 43); M23D-PS 50sb3, corresponding to (p-CCTCG*G*C*T*T*A*C*C*T - SEQ ID NO: 44); M23D-PS 66sb3, corresponding to (p-CCT*C*G*G*C*T*T*A*C*C*T - SEQ ID NO: 45); and M23D-PS 83sb3, corresponding to (p-CCT*C*G*G*C*T*T*A*C*C*T - SEQ ID NO: 46) (wherein the "*" between the two nucleosides indicates a phosphorothiate internucleoside linkage and p represents the terminal phosphate group).

[0030] [Figure 9] Non-denaturing PAGE of oligonucleotides SY-0357, SY-0299, SY-0442, SY-0455, SY-0343, and SY-0450. Oligonucleotide SY-0221 (CTT TCA TAA TGC TGG - SEQ ID NO: 47), with an intact PS backbone that is toxic in mice, is used to demonstrate migration of multimeric bands on the gel (visualized by StainsAll).

[0031] [Figure 10-1]Exon 23 skipping detected by nested RT-PCR in various tissues from mice treated with SY442 (Figure 10A) or SY450 (Figure 10B) at 200 mg / kg for 72 h, and with SY442 (Figure 10C) or SY450 (Figure 10D) for 2 weeks. The left panel represents RT-PCR between exons 20 and 26, showing the unskipped 901 bp product and the skipped 688 bp product. The right panel represents specific RT-PCR between exon 20 and junctions 22-24, showing only the 401 bp skipped product. The specific PCR shown in the right panel was only used when skipping levels were too low to be detected by conventional RT-PCR. [Figure 10-2] Same as above.

[0032] [Figure 11-1] Exon 23 skipping detected by nested RT-PCR in various tissues from mice treated with 50 mg / kg for 72 h with SY442 (Figure 11A) or SY450 (Figure 11B) with SY0210 as a control (Figure 11C), and for 2 weeks with SY442 (Figure 11D) or SY450 (Figure 11E) with SY0210 as a control (Figure 11F). The left panel represents RT-PCR between exons 20 and 26, showing the non-skipped 901 bp product and the skipped 688 bp product. The right panel represents specific RT-PCR between exon 20 and junctions 22-24, showing only the 401 bp skipped product. The specific PCR shown in the right panel was only used when skipping levels were too low to be detected by conventional RT-PCR. [Figure 11-2] Same as above.

[0033] [Figure 12] Quantification of exon 23 skipping by taqman qPCR in tissues from mice treated with 200 mg / kg tcDNA at 72 hours and 2 weeks post-injection.

[0034] [Figure 13] Quantification of exon 23 skipping by taqman qPCR in tissues from mice treated with 50 mg / kg tcDNA at 72 hours and 2 weeks post-injection.

[0035] [Figure 14] Pooled data comparing efficacy of SY0442 and SY0450 at 72 hours and 2 weeks post-injection. A dose effect can be observed from 50 mg / kg to 200 mg / kg (approximately 4-fold).

[0036] [Figure 15] Quantification of exon 23 skipping at 200 mg / kg comparing intact PO compound with intact PS compound.

[0037] [Figure 16] Quantification of exon 23 skipping at 50 mg / kg comparing intact PO compound with intact PS compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The headings used herein are merely for convenience and should not be construed as limiting the disclosure of any of the aspects and embodiments of the invention. definition

[0039] The term "oligomeric compound," as used herein, refers to a compound comprising preferably eight or more monomeric subunits linked by internucleoside linking groups, at least two of said eight or more monomeric subunits being tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides.

[0040] The term "monomer subunit", as used herein, refers to α-D-ribonucleosides, β-D-ribonucleosides, α-D-2'-deoxyribonucleosides, β-D-2'-deoxyribonucleosides, naturally occurring nucleosides, modified nucleosides, herein, in particular, tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonu ... It is meant to include all manner of monomer units suitable for oligomer synthesis, including, typically and preferably referring to, monomer subunits such as nucleotides, hexitol nucleic acid (HNA) nucleosides; and phosphorodiamidate morpholino (PMO) nucleosides, nucleoside mimetics, naturally occurring nucleotides, modified nucleotides, here in particular tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides, and nucleotide mimetics. Typically and preferably, the term "monomer subunit", as used herein, refers to naturally occurring and modified nucleosides, and herein, in particular, ribonucleosides, deoxyribonucleic acid, tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidates. It refers to morpholino (PMO) nucleosides, as well as naturally occurring and modified nucleotides, and herein specifically refers to ribonucleotides, deoxyribonucleotides, tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides, locked nucleic acid (LNA) nucleotides, peptide nucleic acid (PNA) nucleotides, 2'-deoxy 2'-fluoro-arabinonucleotides, hexitol nucleic acid (HNA) nucleotides, and phosphorodiamidate morpholino (PMO) nucleotides.More preferably, the term "monomer subunit" as used herein refers to modified nucleotides, and herein in particular to tricyclo-deoxyribonucleic acid (tc-DNA) nucleotides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleotides.

[0041] The term "lipid moiety", as used herein, typically and preferably refers to moieties derived from hydrocarbons, oils, fats (such as fatty acids, glycerides), sterols, steroids and derivatives of these compounds. Suitable lipid moieties include fatty acids and their derivatives, hydrocarbons and their derivatives, and moieties derived from sterols such as cholesterol. As used herein, the term lipid moiety also includes amphiphilic compound moieties, which contain both lipid moieties and hydrophilic moieties.

[0042] The term "hydrocarbon" as used herein includes compounds consisting only of hydrogen and carbon connected by covalent bonds. The term includes open-chain (aliphatic) hydrocarbons, including straight (unbranched) chain and branched hydrocarbons, as well as saturated, mono- and polyunsaturated hydrocarbons. The term also includes hydrocarbons containing one or more aromatic rings, and preferably, the term excludes hydrocarbons containing one or more aromatic rings. The terms "straight" and "unbranched" are used interchangeably herein.

[0043] The term "fatty acid" as used herein refers to a hydrocarbon chain terminated in a carboxylic acid group, said hydrocarbon chain being typically and preferably either alkyl or alkenyl, typically 3 to 32 carbons in length, and therefore saturated or unsaturated, and containing one or more, preferably one, carboxylic groups (-COOH). , one or more, preferably one C 1~32alkyl, one or more, preferably one phosphate group (HOP(O)(OH)O-), one or more, preferably one phosphonate group (HOP(O)O-), one or more, preferably one thiophosphate group (HOP(O)(SH)O-), one or more, preferably one dithiophosphate group (HOP(S)(SH)O-), one or more, preferably one diphosphate group (HO-P(O)(OH)-OP(O)(OH)-O-), one or more, preferably one triphosphate group (HO-P(O)(OH)-OP(O)(OH)-OP(O)(OH)-O-), one or more phenyl groups (-C 6 H 5 ), optionally substituted by halogen, preferably iodine, or one or more phenyl groups substituted with a carboxy group. When the fatty acid contains one or more double bonds, and is therefore unsaturated, there is the possibility of either cis or trans geometric isomerism. The term "fatty acid moiety", as used herein, refers to a moiety derived from a fatty acid as defined herein, in which one carboxy group (-COOH) of said fatty acid becomes and is the -C(O)- group of said fatty acid moiety, which is linked to said oligonucleotide according to the invention, either directly or via a spacer. Preferably, the term "fatty acid", as used herein, refers to a hydrocarbon chain terminated by a carboxylic acid group, said hydrocarbon chain being typically and preferably either alkyl or alkenyl, typically 3-32 carbons in length, and therefore saturated or unsaturated, and containing one or more, preferably one carboxy group (-COOH), one or more, preferably one C- 1~32alkyl, one or more, preferably one phosphate group (HOP(O)(OH)O-), one or more, preferably one phosphonate group (HOP(O)O-), one or more, preferably one thiophosphate group (HOP(O)(SH)O-), one or more, preferably one dithiophosphate group (HOP(S)(SH)O-), one or more, preferably one diphosphate group (HO-P(O)(OH)-OP(O)(OH)-O-), one or more, preferably one triphosphate group (HO-P(O)(OH)-OP(O)(OH)-OP(O)(OH)-O-), one or more phenyl groups (-C 6 H 5 ), optionally substituted with halogen, preferably iodine, or one or more phenyl groups substituted with carboxy groups. Preferably, the fatty acid has an even number of carbon atoms, and the carbon atom of the carboxy group (-COOH) of the fatty acid or the -C(O)- group of the fatty acid moiety is included in counting the number of carbon atoms.

[0044] Thus, fatty acids preferably contain an even or odd, preferably an even number of carbon atoms in a linear chain (usually 3 to 32 carbons), can be saturated or unsaturated, and can have a variety of substituents, preferably one or more, preferably one carboxy group (-COOH), one or more, preferably one C 1~32 alkyl, one or more, preferably one phosphate group (HOP(O)(OH)O-), one or more, preferably one phosphonate group (HOP(O)O-), one or more, preferably one thiophosphate group (HOP(O)(SH)O-), one or more, preferably one dithiophosphate group (HOP(S)(SH)O-), one or more, preferably one diphosphate group (HO-P(O)(OH)-OP(O)(OH)-O-), one or more, preferably one triphosphate group (HO-P(O)(OH)-OP(O)(OH)-OP(O)(OH)-O-), one or more phenyl groups (-C 6 H 5), one or more phenyl groups substituted with halogen, preferably iodine, or a carboxy group.

[0045] The term "fatty diacid" refers to a fatty acid as defined herein, but with an additional carboxylic acid group at the omega position. Thus, a fatty diacid is a dicarboxylic acid. The term "fatty diacid moiety" as used herein refers to a moiety derived from a fatty diacid as defined herein, in which one carboxy group (-COOH) of said fatty diacid becomes and is the -C(O)- group of said fatty diacid moiety, which is linked to said oligonucleotide according to the present invention, either directly or via a spacer. A preferred embodiment of a fatty diacid is one or more, preferably one, C(O)- groups. 1~32 alkyl, one or more, preferably one phosphate group (HOP(O)(OH)O-), one or more, preferably one phosphonate group (HOP(O)O-), one or more, preferably one thiophosphate group (HOP(O)(SH)O-), one or more, preferably one dithiophosphate group (HOP(S)(SH)O-), one or more, preferably one diphosphate group (HO-P(O)(OH)-OP(O)(OH)-O-), one or more, preferably one triphosphate group (HO-P(O)(OH)-OP(O)(OH)-OP(O)(OH)-O-), one or more, preferably one phenyl group (-C 6 H 5 ), a halogen, preferably iodine, or a carboxy group, and preferably one phenyl group. A preferred example is a saturated fatty diacid having one C such as 3-pentadecylglutaric acid (PDG). 6~24 Glutaric acid optionally substituted with alkyl is included.

[0046] The term "alkyl phosphate moiety" as used herein means3~32 The alkyl group is represented by the formula: 3~32 Alkyl is independently a C as defined herein. 3~32 is selected from alkyl.

[0047] The term "alkylphosphonate moiety" as used herein means 3~32 The alkyl group is represented by the formula: 3~32 Alkyl is independently a C as defined herein. 3~32 is selected from alkyl.

[0048] The term "alkyl," as used herein, refers to a straight or branched chain hydrocarbon radical containing no unsaturation, having from 1 to 32 carbon atoms, and consisting solely of carbon and hydrogen atoms (e.g., (C 1~32 ) alkyl or C 1~32alkyl), which may be or typically is attached to the remainder of the molecule by a single bond. Whenever it appears herein, a numerical range such as "1-32" refers to each integer within the given range. For example, "1-32 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 32 carbon atoms, although this definition is also intended to cover the occurrence of the term "alkyl" where no numerical range is specifically specified. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (used interchangeably with isopropyl; abbreviated interchangeably herein as iPr or Pri), n-butyl, isobutyl, sec-butyl, isobutyl, tertiary butyl (used interchangeably with 1,1-dimethylethyl or tert-butyl), n-pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. Unless otherwise specifically stated in the specification, alkyl groups are independently selected from the group consisting of alkenyl, alkoxy, carboxy groups (-COOH), heteroalkyl, heteroalkenyl, hydroxyl, phosphate groups (-OP(O)(OH)O-), phosphonate groups (-OP(O)O-), halogen, preferably iodine, or phenyl groups optionally substituted with a carboxy group (-C 6 H 4 Preferably, the term "alkyl" as used herein refers to an unsubstituted alkyl as defined herein.

[0049] The term "alkylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkyl, as defined herein, in which one hydrogen of said alkyl is broken off to generate a second radical of said alkylene. An example of an alkylene is, illustratively, -CH 2 -, -CH 2 -CH 2 -, -CH(CH 3 )-, -CH 2 -CH2 -CH 2 -, -CH(CH 3 )-CH 2 -or-CH(CH 2 CH 3 )-.

[0050] The term "alkenyl," as used herein, refers to a straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond and having 3 to 32 carbon atoms (i.e., (C 3~32 ) alkenyl or C 3~32 alkenyl), which may be or typically is attached to the remainder of the molecule by a single bond. Whenever it appears herein, a numerical range such as "3 to 32" refers to each integer within the given range, for example, "3 to 32 carbon atoms" means that the alkenyl group may consist of 3 carbon atoms, 4 carbon atoms, etc., up to 32 carbon atoms. Exemplary alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, and penta-1,4-dienyl. Each double bond may be in either the (E)- or (Z)-configuration. Thus, alkenyl, where applicable, each of said double bonds may include either its (E)-configuration, its (Z)-configuration, and mixtures thereof in any ratio. Unless otherwise specifically stated in the specification, an alkenyl group is independently an alkenyl, an alkoxy, a carboxy group (-COOH), a heteroalkyl, a heteroalkenyl, a hydroxyl, a phosphate group (-OP(O)(OH)O-), a phosphonate group (-OP(O)O-), a halogen, preferably iodine, or a phenyl group (-C 6 H 4 Preferably, the term "alkenyl" as used herein refers to an unsubstituted alkenyl as defined herein.

[0051] The term "alkenylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkenyl, as defined herein, where one hydrogen of said alkenyl has been cleaved to generate a second radical of said alkenylene.

[0052] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond and having from 2 to 10 carbon atoms (i.e., (C 2~32 ) alkynyl or C 2~32 alkynyl). Whenever it appears in this specification, a numerical range such as "2 to 32" refers to each integer within the given range, for example, "2 to 32 carbon atoms" means that the alkynyl group can consist of 2 carbon atoms, 3 carbon atoms, etc., up to 32 carbon atoms. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Unless otherwise specified in this specification, an alkynyl group is independently selected from the group consisting of alkenyl, carboxy group (-COOH), heteroalkyl, heteroalkenyl, phosphate group (-OP(O)(OH)O-), phosphonate group (-OP(O)O-), halogen, preferably iodine, or phenyl group optionally substituted with a carboxy group (-COOH). 6 H 4 Preferably, the term "alkynyl" as used herein refers to unsubstituted alkynyl as defined herein.

[0053] The term "alkynylene," as used herein, refers to a straight or branched chain hydrocarbon biradical derived from an alkynyl, as defined herein, where one hydrogen of the alkynyl has been cleaved to generate a second radical of the alkynylene.

[0054] The term "alkoxy" refers to groups of -O-alkyl, including linear, branched configurations and combinations thereof, of 1 to 32 carbon atoms attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, and cyclohexyloxy. "Lower alkoxy" refers to an alkoxy group containing 1 to 6 carbons and includes the alkoxy groups (C 1~6 )Alkoxy or OC 1~6 Also called alkyl.

[0055] The term "substituted alkoxy" refers to an alkoxy in which the alkyl component is substituted (i.e., -O-(substituted alkyl)). Unless otherwise specifically stated in the specification, the alkyl portion of the alkoxy group is independently selected from an alkenyl, a carboxy group (-COOH), a heteroalkyl, a heteroalkenyl, a phosphate group (-OP(O)(OH)O-), a phosphonate group (-OP(O)O-), a halogen, preferably iodine, or a phenyl group optionally substituted with a carboxy group (-C 6 H 4 ) is optionally substituted by one or more substituents.

[0056] The term "acyl" refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-, and (heteroalkyl)-C(O)-, which are attached to the parent structure through a carbonyl functionality. Unless otherwise stated specifically in the specification, the alkyl, aryl, or heteroaryl portions of the acyl group are independently selected from alkenyl, carboxy groups (-COOH), heteroalkyl, heteroalkenyl, phosphate groups (-OP(O)(OH)O-), phosphonate groups (-OP(O)O-), halogens, preferably iodine, or phenyl groups optionally substituted with a carboxy group (-COOH). 6 H 4 ) is optionally substituted by one or more substituents.

[0057] Unless otherwise specified in this specification, the term “amino” or “amine” refers to —N(R a ) 2 Refers to the radical group, and each R a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl. a ) 2 Group has 2 R in addition to hydrogen a When substituted, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -N(R a ) 2 is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless otherwise specifically stated in the specification, amino or amine groups are independently selected from alkenyl, carboxy group (-COOH), heteroalkyl, heteroalkenyl, phosphate group (-OP(O)(OH)O-), phosphonate group (-OP(O)O-), halogen, preferably iodine, or phenyl group optionally substituted with a carboxy group (-C 6 H 4 ) is optionally substituted by one or more substituents.

[0058] The term "aromatic" or "aryl" or "Ar" refers to an aromatic radical having 6 to 10 ring atoms that is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl), with at least one ring having a conjugated pi-electron system (e.g., C 6 ~C 10 Aromatic or C 6 ~C 10aryl). Divalent radicals formed from substituted benzene derivatives and having a free valence at a ring atom are named substituted phenylene radicals. Divalent radicals derived from monovalent polycyclic hydrocarbon radicals, named with the suffix "-yl" by the removal of one hydrogen atom from the free valence carbon atom, are named by adding "-idene" to the name of the corresponding monovalent radical, e.g., a naphthyl group having two points of attachment is called naphthylidene. Whenever it appears herein, numerical ranges such as "6-10" refer to respective integers within the given range, e.g., "6-10 ring atoms" means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to a maximum of 10 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups.

[0059] The term "aralkyl" or "arylalkyl" refers to an (aryl)alkyl-radical, where the aryl and alkyl are optionally substituted with one or more substituents disclosed herein and described as suitable substituents for aryl and alkyl, respectively.

[0060] The terms "carboxyl" or "carboxylic" are used interchangeably herein and refer to the -(C=O)OH radical.

[0061] The term "cycloalkyl" refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen and can be saturated or partially unsaturated. Cycloalkyl groups are groups having 3 to 10 ring atoms (i.e., (C 3~10 ) Cycloalkyl or C 3~10Whenever it appears herein, a numerical range such as "3 to 10" refers to each integer within the given range, for example, "3 to 10 carbon atoms" means that the cycloalkyl group consists of 3 carbon atoms, etc., and can contain up to 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, etc.

[0062] The term "fluoroalkyl" refers to an alkyl radical, as defined above, substituted by one or more fluoro radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc. The alkyl portion of the fluoroalkyl radical can be optionally substituted as defined above for an alkyl group.

[0063] The term "halogen" as used herein refers to fluorine, chlorine, bromine, or iodine, preferably iodine. In a preferred embodiment, the halogen substituent is iodine.

[0064] The terms "heteroalkyl" and "heteroalkenyl," as used herein, refer to optionally substituted alkyl and alkenyl radicals having one or more skeletal chain atoms selected from atoms other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, or combinations thereof. Numerical ranges may be given, referring to the total chain length, e.g., C 1 ~C 4 The heteroalkyl, in this example, is four atoms long.

[0065] The term "heteroaryl" or "heteroaromatic" or "HetAr" refers to a 5- to 18-membered aromatic radical containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur, such as C 5 ~C13 Heteroaryl refers to a heteroaryl group, which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system. Whenever it appears herein, a numerical range such as "5 to 18" refers to each integer within the given range, for example, "5 to 18 ring atoms" means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to 18 ring atoms. Divalent radicals derived from monovalent heteroaryl radicals, named with the suffix "-yl" by the removal of one hydrogen atom from the free valence atom, are named by adding "-idene" to the name of the corresponding monovalent radical, for example, a pyridyl group with two points of attachment is a pyridylidene.

[0066] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0067] "Diastereomer" refers to a stereoisomer having two or more centers of chirality where the compound is not a mirror image of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and chemical and biological reactivity. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

[0068] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0069] Stereochemical definitions and conventions used herein generally follow those in McRaw-Hiff Dictionary of Chemical Terms (1984), edited by S.P. Parker, McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0070] In the chemical formula ( * The symbols (), (#) and (§) designate i) points of attachment, ii) radicals, and / or iii) unshared electrons.

[0071] The term "antisense oligonucleotide (AON)" as used herein refers to an oligonucleotide or oligomeric compound capable of altering gene expression by interacting with and / or hybridizing to a pre-mRNA or mRNA having a complementary nucleotide sequence.

[0072] The term "protecting group," as used herein, is intended to mean a group that selectively blocks one or more reactive sites of a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site, and which can then be easily removed or deprotected after the selective reaction is complete. A variety of protecting groups are disclosed, for example, in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, New York, 1999.

[0073] The terms "protecting group for amino", "protecting group for amino group" or "amino protecting group" as used interchangeably herein are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3rd Edition, John Wiley & Sons, New York (1999); Greene's Protective Groups in Organic Synthesis, P.G.M. Wuts, 5th Edition, John Wiley & Sons (2000); 14) as well as those described in detail in Current Protocols in Nucleic Acid Chemistry, edited by S.L. Beaucage et al., June 2012, herein in particular Chapter 2. Suitable "amino protecting groups" for the present invention include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-methylethyl carbamate (Met ... -phenylethyl carbamate (hZ), 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz) and 2,4,6-trimethylbenzyl carbamate, (4-methoxyphenyl)diphenylmethyl (MMTr); as well as formamide, acetamide, benzamide, typically and preferably independently selected from these at each occurrence.

[0074] The terms "protecting group for hydroxyl", "protecting group for a hydroxyl group" or "hydroxyl protecting group", as used interchangeably herein, are well known in the art and are described in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3rd ed., John Wiley & Sons, New York (1999); Greene's Protective Groups in Organic Synthesis, PGM Wuts, 5th ed., John Wiley & Sons (2014) and Current Protocols in Nucleic Acid Chemistry, edited by S.L. Beaucage et al., June 2012, including particularly those detailed herein in Chapter 2. In certain embodiments, the "hydroxyl protecting groups" of the present invention include, typically and preferably independently selected from each occurrence, acetyl, benzoyl, benzyl, β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMTr), methoxymethyl ether (MOM), methoxytrityl[(4-methoxyphenyl)diphenylmethyl] (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl(triphenylmethyl, Tr), t-butyldiphenylsilyl ether (TBDPS), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers, and other silyl ethers; methyl ethers, ethoxyethyl ethers (EE).

[0075] Preferred examples of the "hydroxyl protecting group" of the present invention include acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethylcarbonate, mesylate, tosylate, triflate, 4-monomethoxytrityl (MMTr), 4 ,4'-Dimethoxytrityl (DMTr) and 4,4',4''-Trimethoxytrityl (TMTr), 2-Cyanoethyl (CE or Cne), 2-(Trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl, 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl phenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl). xanthin-9-yl (MOX), and independently at each occurrence are selected from among these.

[0076] The term "nucleobase", as used herein, is abbreviated as Bx, and refers to unmodified or naturally occurring nucleobases, as well as modified or non-naturally occurring nucleobases, and their synthetic mimetics. A nucleobase is any heterocyclic base that contains one or more atoms or groups of atoms that are capable of hydrogen bonding to the heterocyclic base of a nucleic acid.

[0077] Typical and preferred examples of nucleobases are purine bases or pyrimidine bases, preferably, said purine bases are purine or substituted purine, and said pyrimidine bases are pyrimidine or substituted pyrimidine.More preferably, said nucleobases are (i) adenine (A), (ii) cytosine (C), (iii) 5-methylcytosine (MeC), (iv) guanine (G), (v) uracil (U), or (vi) 5-methyluracil (MeU), or derivatives of (i), (ii), (iii), (iv), (v) or (vi).The terms "derivatives of (i), (ii), (iii), (iv), (v) or (vi)" and "nucleobase derivatives" are used interchangeably herein. The derivatives and nucleobase derivatives of (i), (ii), (iii), (iv), (v) or (vi) are known to those skilled in the art, and are described, for example, in Sharma VK et al., Med. Chem. Commun., 2014, vol. 5, pp. 1454-1471, and include, but are not limited to, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, alkyl adenines such as 2-propyladenine, 6-methylguanine, alkylguanines such as 2-propylguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C=C-CH 3 ) Uracil, 5-propynyl (-C=C-CH 3 ) Alkynylpyrimidine bases such as cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil; 8-halo-, 8-amino-, 8-thio 8-substituted purine bases such as 8-thioalkyl-, 8-hydroxyl-adenine or guanine, 5-substituted pyrimidine bases such as 5-halo-, in particular 5-bromo-, 5-trifluoromethyl-uracil or -cytosine; 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, hydrophobic bases, promiscuous bases, size-extended bases, or fluoride salts. In certain embodiments, the nucleobase includes tricyclic pyrimidines, such as, but not limited to, 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one or 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). The term "nucleobase derivative" also includes those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine or 2-pyridone. Additional nucleobases of the present invention include, but are not limited to, those known to those skilled in the art (e.g., U.S. Pat. No. 3,687,808; Swayze et al., The Medicinal Chemistry of Oligonucleotides, in Antisense a Drug Technology, Chapter 6, pp. 143-182 (Crooke, ST, ed., 2008); The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, ed., John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30(6), pp. 613-623; Sanghvi, YS, Antisense Research and Applications, Crooke, ST and Lebleu, B. eds., CRC Press, 1993). (J. Am. Chem. Soc., 2003, pp. 273-302). The term "nucleobase derivative" also includes those in which the purine or pyrimidine base is replaced with a moiety corresponding to the spacer of the present invention, in particular a moiety for linking said one or more lipid moieties within said oligomeric compound, preferably said oligonucleotide. The specific linkage of said moiety corresponding to the spacer is known to those skilled in the art. Preferred nucleobase derivatives include methylated adenine, guanine, uracil and cytosine, and nucleobase derivatives, preferably (i), (ii), (iii) or (iv), wherein each amino group, preferably the exocyclic amino group, is protected by an acyl protecting group or a dialkylformamidino, preferably dimethylformamidino (DMF), and further include nucleobase derivatives such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, and pyrimidine analogs such as pseudoisocytosine and pseudouracil. The preparation of modified nucleobases is known in the art and is described in U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; Nos. 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941.

[0078] The term "internucleoside linking group" as used herein refers to any linking group known in the art that allows for linkage, preferably further linkage of said tricyclo-deoxyribonucleic acid (tc-DNA) nucleoside, either to an additional tc-DNA nucleoside, a nucleoside other than a tc-DNA nucleoside, or a non-nucleoside, including a peptide or a protein. Representative patents which teach such possible linking groups include, but are not limited to, U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,166,315; U.S. Pat. No. 5,185,444; U.S. Pat. No. 5,214,134; U.S. Pat. No. 5,216,141; U.S. Pat. No. 5,235,033; U.S. Pat. No. 5,264,562; U.S. Pat. No. 5,264,564; U.S. Pat. No. 5,405,938; U.S. Pat. No. 5,434,257; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,470,967; U.S. Pat. No. 5,471,967; U.S. Pat. No. 5,472,967; U.S. Pat. No. 5,473,967; U.S. Pat. No. 5,474,967; U.S. Pat. No. 5,475,967; U.S. Pat. No. 5,476,967; U.S. Pat. No. 5,477,967; U.S. Pat. No. 5,478,967; U.S. Pat. No. 5,479,967; U.S. Pat. No. 5,477,967; U.S. Pat. No. 5,479 ... Nos. 5,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,677,439, 5,646,269, and 5,792,608. Thus, the term "internucleoside linking group" includes phosphorus and non-phosphorus linking groups. Non-phosphorus linking groups do not contain a phosphorus atom and examples of non-phosphorus linking groups include, typically and preferably selected from, alkyl, aryl, preferably phenyl, benzyl or benzoyl, cycloalkyl, alkylenearyl, alkylenediaryl, alkoxy, alkoxyalkylene, alkylsulfonyl, alkyne, ether; carboxyl, amide, amine, amino, imine, thiol, sulfide, sulfoxide, sulfone, sulfamate, sulfonate, sulfonamide, siloxane or mixtures thereof, each independently of the other, optionally substituted with cyano, nitro, halogen.Typically and preferably, the internucleoside linking group is a phosphorus linking group, and the phosphorus linking group is P. III or P V It refers to a moiety containing a phosphorus atom in the valence state of 1. More preferably, the internucleoside linking group is a phosphorus linking group. Again more preferably, the internucleoside linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group, a phosphorodithioate linking group, a phosphonate linking group, preferably an H-phosphonate linking group or a methylphosphonate linking group; a phosphonothioate linking group, preferably an H-phosphonothioate linking group, a methylphosphonothioate linking group; a phosphinate linking group, a phosphorthioamidate linking group, a phosphoramidate linking group or a phosphite linking group. In another highly preferred embodiment, the internucleoside linking group is selected from a phosphodiester linking group, a phosphotriester linking group, a phosphorothioate linking group or a phosphonate linking group, the phosphonate being preferably an H-phosphonate linking group or a methylphosphonate linking group.

[0079] As used herein, the term "nucleoside" refers to a compound that comprises a nucleobase and a sugar that is covalently linked to said nucleobase.Furthermore, the term "nucleoside" is meant to include all manner of naturally occurring or modified nucleosides, or nucleoside mimetics that can be incorporated into oligomers using natural or chemical oligomer synthesis.Exemplarily and preferably, the term "nucleoside" as used herein refers to naturally occurring nucleosides, modified nucleosides, or nucleoside mimetics.The term "modified nucleoside" is intended to include modifications made to the sugar and / or nucleobase of the nucleoside described herein, as known to those skilled in the art.The term "nucleoside mimetics" is intended to include those structures that are used to replace sugar and nucleobase. Examples of nucleoside mimetics include nucleosides in which the nucleobase is replaced with a phenoxazine moiety (e.g., a 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one group) and the sugar moiety is replaced with a cyclohexenyl or bicyclo[3.1.0]hexyl moiety. The term "nucleoside" also includes combinations of modifications, such as modifications of two or more nucleobases, modifications of two or more sugars, or modifications of at least one nucleobase and at least one sugar.

[0080] The sugar of the nucleoside includes, but is not limited to, a monocyclic, bicyclic or tricyclic ring system, preferably a tricyclic or bicyclic system, or a monocyclic ribose or de(s)oxyribose. Modifications of the sugar further include, but are not limited to, a modified stereochemical configuration, substitution of at least one group, or deletion of at least one group. The modified sugar is typically and preferably a modified version of a ribosyl moiety (i.e., a furanosyl moiety) naturally occurring in RNA and DNA, such as a bicyclic sugar, a tetrahydropyran, a 2'-modified sugar, a 3'-modified sugar, a 4'-modified sugar, a 5'-modified sugar or a 4'-substituted sugar. Examples of suitable sugar modifications are known to those of skill in the art and include, but are not limited to, 2', 3' and / or 4' substituted nucleosides (e.g., 4'-S-modified nucleosides); 2'-O-alkyl or 2'-O-(substituted)alkyl, e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, and other 2'-O-modified RNA nucleotide residues; 2'-O-(haloalkoxy)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2-thiomethyl)ethyl, and other 2'-O-alkyl, 2'-O-(2-aminoethyl)ethyl, 2'-O-(2-chloroethoxy)ethyl, 2'-O-(2-aminoethyl ... ,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl))ethyl] (DMCE), especially the 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE); or other modified sugar moieties such as morpholino (PMO), cationic morpholino (PMOPlus), or modified morpholino groups such as PMO-X.The term "PMO-X" refers to at least one 3' or 5' end modification such as a 3'-fluorescent tag, a 3' quencher (e.g., 3'-carboxyfluorescein, 3'-Gene Tools Blue, 3'-lissamine, 3'-dabsyl), a 3'-affinity tag and a functional group for chemical linkage (e.g., 3'-biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyldithio), a 5' end modification (5'-primary amine, 5'-dabsyl), a 3'-azide, a 3'-alkyne, a 5'-azide, a 5'-alkyne, or a modified morpholino group, including those disclosed in WO 2011 / 150408 and U.S. Patent Application Publication No. 2012 / 0065169.

[0081] "Bicyclic sugar moiety" includes bicyclic nucleosides having two interconnected ring systems, e.g., the sugar moiety has a 2'-O-CH(alkyl)-4' or 2'-O-CH2-4' group, locked nucleic acid (LNA), xylo-LNA, alpha-L-LNA, beta-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA) or 3'-deoxypyranosyl-DNA (p-DNA).

[0082] In a preferred embodiment, the oligomeric compound is an oligonucleotide.The term "oligonucleotide" as used herein refers to a compound that comprises at least two nucleosides that are linked to each other by internucleoside linking group.Therefore, the term "oligonucleotide" as used herein includes, typically and preferably refers to, an oligomeric compound that comprises at least two nucleosides that are linked by internucleoside linking group, and said at least two nucleosides are independently selected from naturally occurring nucleosides, modified nucleosides or nucleoside mimetics.

[0083] Oligomeric compounds can be single-stranded or double-stranded. In one embodiment, the oligomeric compounds are double-stranded (i.e., duplex). In a preferred embodiment, the oligomeric compounds are single-stranded.

[0084] In preferred embodiments, the one or more lipid moieties are linked to the oligomeric compound, independently of each other, at (i) a terminal residue of the oligomeric compound, (ii) the 5' end of the oligomeric compound, (iii) the 3' end of the oligomeric compound, or (iv) an internal residue of the oligomeric compound.

[0085] The term "'terminal' refers to the end or terminus of an oligomeric compound, and the integer (3', 5, etc.) indicates the carbon atom of the sugar contained in the nucleoside of the oligomeric compound. The terms "5' terminal group" or "3' terminal group", as used herein, refer to the group located at the 5' or 3' end, respectively.

[0086] The terms "natural" or "naturally occurring," as used interchangeably herein, refer to a compound that is of natural origin.

[0087] The term "complementary" refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule, either by traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonds) between complementary nucleosides or nucleotides. "Complementary" (or "specifically hybridizable") is a term that indicates a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an oligomeric compound and a pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule does not require 100% complementarity with a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may not be perfectly complementary. Complementarity may be indicated by the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9 or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90% and 100% complementarity, respectively. "Perfectly" or "completely" complementary nucleic acid molecules mean that all consecutive residues in a first nucleic acid molecule will hydrogen bond with the same number of consecutive residues in a second nucleic acid molecule, whether both nucleic acid molecules have the same number of nucleotides (i.e., the same length) or the two molecules have different lengths.

[0088] The term "exon skipping" refers to the modification of pre-mRNA splicing by targeting splice donor and / or splice acceptor sites in pre-mRNA with one or more complementary antisense oligonucleotides or oligomeric compounds.By blocking the access of spliceosomes to one or more splice donor or splice acceptor sites, or any other sites in exons or introns involved in splicing definition, the oligonucleotides can prevent splicing reaction and cause exon deletion from fully processed mRNA.Exon skipping is achieved in the nucleus during the maturation process of pre-mRNA.Exon skipping involves masking important sequences involved in splicing of targeted exons by using antisense oligonucleotides that are complementary to splice donor sequences in pre-mRNA. For example, the compositions of the invention comprising the oligomeric compounds provided herein can be suitably used for exon skipping by masking splice sites at intron / exon junctions in dystrophin pre-mRNA, thereby facilitating the deletion of mutant exons during processing of the pre-mRNA into mature mRNA.

[0089] The term "exon inclusion" refers to an oligonucleotide-mediated process, such as base pairing of an antisense oligonucleotide to a targeted pre-mRNA, to block exon or intron splicing enhancers, block corresponding splicing repressors, and / or disrupt unfavorable secondary structures, resulting in more efficient recognition of the exon by the spliceosome and restoration of exon expression.

[0090] The term "in vivo" refers to events that take place within a subject's body.

[0091] The term "in vitro" refers to events that take place outside a subject's body. In vitro assays include cell-based assays, in which live or dead cells are used, and can also include cell-free assays, in which no intact cells are used.

[0092] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds described herein sufficient to effect the intended application, including, but not limited to, the treatment of a disease. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or on the human subject and disease state to be treated (e.g., the subject's weight, age, and sex), the severity of the disease state, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term also applies to a dose that induces a particular response in the target cells (e.g., reduced platelet adhesion and / or cell migration). The particular dose will vary depending on the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the bodily delivery system to which the compound is delivered.

[0093] A "therapeutic effect" as the term is used herein encompasses a therapeutic benefit and / or a prophylactic benefit in a human subject. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0094] The term "pharmaceutically acceptable salts" refers to salts derived from various organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. The organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins.Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine.In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium and magnesium salts, and preferably, the pharmaceutically acceptable salt is a sodium salt.

[0095] In the case of a hydroxyl group (OH) or a thiol group (SH) present in said oligomeric compound of the invention, preferably in said oligonucleotide of the invention, typically and preferably bound to P(III) or P(V), typically and preferably present in said lipid moiety or moieties as part of the B group of said lipid moiety or moieties, or present in said spacer, or typically and preferably present as part of said internucleoside linking group selected from phosphorothioate or phosphorodiester, each of said hydroxyl group (OH) or thiol group (SH) can be present, independently of each other, as said OH group or in its ionic state, such as O-anion and a pharma- ceutically acceptable cation, or as said SH group or in its ionic state, such as S-anion and a pharma-ceutically acceptable cation.Furthermore, any combination and any equilibrium state between the above situations in the composition of the invention is included, in particular taking into account oxygen- or sulfur-containing groups on said P(III) or P(V), such as (=O), (=S), another OH group or SH group, which are known to those skilled in the art. For convenience, aspects and embodiments of the invention typically describe only one of the above situations. By way of example, a preferred spacer of the invention is #-NH-C 2~12 As shown, but not limited to, spacers in which the hydrogen is located at the oxygen, thus #-NH-C 2~12 All alkylene-OP(OH)(S)-§ and pharma- ceutically acceptable salts thereof are included herein.

[0096] Thus, a pharma- ceutically acceptable salt in the context of a hydroxyl group (OH) and / or a thiol group (SH) present in said oligomeric compound of the invention, preferably in said oligonucleotide of the invention, typically and preferably linked to P(III) or P(V) and typically and preferably present in said lipid moiety or moieties as part of the B group of said lipid moiety or moieties, or present in said spacer, or present in said oligonucleotide of the invention, typically and preferably selected from phosphorothioate or phosphorodiester, can be a pharma- ceutically acceptable salt in the context of a hydroxyl group (OH) and / or a thiol group (SH) ... refers to a composition of the invention in which one or more of said groups or said SH groups are present, independently of one another, as said OH group or in its ionic state, such as O-anion and its pharma- ceutically acceptable cation, or as said SH group or in its ionic state, such as S-anion and its pharma- ceutically acceptable cation, typically and preferably, said pharma- ceutically acceptable cation is selected from protonated trimethylamine, protonated diethylamine, protonated methylamine, ammonium, sodium or potassium, more preferably said pharma- ceutically acceptable cation is sodium.

[0097] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any solvent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonic and absorption retarding agent, and inactive ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is contemplated for use in the therapeutic composition of the present invention, except where it is incompatible with the active pharmaceutical ingredient. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the compositions and methods described.

[0098] When a range is used herein to describe a physical or chemical property, such as, for example, a molecular weight or a chemical formula, it is intended to include all combinations and subcombinations of the range and specific embodiments therein. The use of the term "about" when referring to a numerical value or numerical range means that the numerical value or numerical range referred to is an approximation within experimental variation (or within statistical experimental error), and thus the numerical value or numerical range may vary. The variation is typically 0% to 15%, 0% to 10%, 0% to 5% of the stated number or numerical range.

[0099] In a first aspect, the present invention provides a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and one or more lipid moieties, preferably exactly one lipid moiety, said one or more lipid moieties being covalently linked to said oligomeric compound, either directly or via a spacer, preferably wherein said oligomeric compound comprises between 5 and 40 monomeric subunits.

[0100] tc-DNA nucleosides In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the composition of the invention is a compound of formula (1): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety); q 1 , q 2 , q 3, q 4 and q 5 are each independently hydrogen (H), halogen, or C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Substituted C 1~6 Alkyl, substituted C 2~6 Alkenyl, substituted C 2~6 Alkynyl, and -(CH 2 ) n -C(O)-R 6’ wherein n is 0 to 6; 6’ OH, NH 2 , O.C. 1~32 Alkyl and NH-C 1~32 selected from the group consisting of alkyl; z 1 and z 2 are each independently H, halogen, or C 1~6 Alkyl, C 1~6 Alkoxyl, OC 2~6 Alkenyl, OC 2~6 Alkynyl, Substituted C 1~6 Alkyl, substituted C 1~6 Alkoxy, substituted OC 2~6 Alkenyl and substituted O-C 2~6 alkynyl), or a pharma- ceutically acceptable salt thereof.

[0101] In an embodiment, the tc-DNA nucleosides of the oligomeric compounds of the present compositions are 5 is H.

[0102] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (1) where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (1) where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (1) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0103] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the composition of the invention is a compound of formula (2): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0104] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (2) where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (2) where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (2) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0105] In embodiments, the tc-DNA nucleoside of the oligomeric compound of the composition of the present invention is a compound of formula (3) (also known as C(6')-functionalized tc-DNA): [ka] (In the formula, Bx is a nucleobase; R 6’ OH, NH 2 , O.C. 1~32 Alkyl and NH-C 1~32 selected from the group consisting of alkyl; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0106] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (3) where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (3) where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (3) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0107] In embodiments, the tc-DNA nucleoside of the oligomeric compound of the composition of the present invention is a compound of formula (4) (also known as 6'-fluoro-tc-DNA): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0108] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (4) where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (4) where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (4) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0109] In embodiments, the tc-DNA nucleoside of the oligomeric compound of the composition of the present invention is a compound of formula (5) (also known as 2'-fluoro-tc-DNA): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0110] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5) where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5) where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0111] Thus, in an embodiment, the tc-DNA nucleoside of said one or more nucleosides of the oligomeric compound of the composition of the invention is a compound of formula (5') (also known as 2'-fluoro-tc-ANA): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0112] In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5') where Bx is selected from the group consisting of thymine, adenine, guanine and cytosine. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5') where Bx is a modified base. In an embodiment, the tc-DNA nucleoside of the oligomeric compound of the present invention comprises a compound of formula (5') where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-bromouracil, inosine and 2,6-diaminopurine.

[0113] General methods for the preparation of compounds of formula (1) and formula (2) for use in oligomeric compounds are known in the art and include those described in U.S. Patent Application Publication Nos. 2015 / 0141637, 2016 / 0002280 and 2014 / 0296323, the disclosures of which are incorporated herein by reference. Standard phosphoramidite building blocks for tc-DNA are known in the art, e.g., Steffens and Leumann, Helv. Chim. Acta, 1997, 80. 2426-2439. Methods for preparing compounds of formula (3) are described, for example, in Lietard and Leumann, J. Org. Chem., 2012, 77, 4566-77. Methods for preparing compounds of formula (4) are described, for example, in Medvecky, Istrate and Leumann, J. Org. Chem., 2006, the disclosure of which is incorporated herein by reference. 15, Vol. 80, pp. 3556-65, the disclosure of which is incorporated herein by reference. Methods for preparing compounds of formula (5) and (5') are described, for example, in Istrate , Medvecky and Leumann, Org. Lett., 2015, vol. 17, pp. 1950-53, the disclosure of which is incorporated herein by reference.

[0114] 2'-Modified RNA Nucleosides and Other Nucleosides In embodiments, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides.

[0115] In embodiments, one or more nucleosides other than tc-DNA nucleosides of the oligomeric compounds of the invention are RNA nucleosides of formula (6): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) It is.

[0116] In an embodiment, the one or more nucleosides other than the tc-DNA nucleosides of the oligomeric compounds of the invention are RNA nucleosides of formula (6) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention include compounds of formula (6) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention include compounds of formula (6) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0117] In an embodiment, the 2'-modified-RNA nucleoside of the preferred oligomeric compound of the composition of the present invention is a compound of formula (7) (2'-O-methyl-RNA nucleoside): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0118] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (7) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (7) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (7) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0119] In embodiments, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention is a compound of formula (8) (2'-O-propargyl-RNA nucleoside): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0120] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (8) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (8) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (8) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0121] In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention is a compound of formula (9) (2'-O-propylamino-RNA nucleoside): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0122] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (9) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (9) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (9) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0123] In embodiments, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention are compounds of formula (10) (2'-amino-RNA nucleosides): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0124] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (10) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (10) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (10) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0125] In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention is a compound of formula (11) (2'-fluoro-RNA nucleoside): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0126] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (11) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (11) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (11) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0127] In embodiments, one or more nucleosides other than tc-DNA nucleosides of the oligomeric compounds of the invention are compounds of formula (11') (2'-deoxy 2'-fluoro-arabinonucleoside (2'-FANA)): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0128] In an embodiment, the one or more nucleosides other than the tc-DNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (11') where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (11') where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (11') where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0129] In embodiments, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention is a compound of formula (12) (2'-O-methoxyethyl-RNA nucleoside, or 2'-MOE nucleoside): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0130] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (12) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (12) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (12) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0131] In embodiments, one or more nucleosides other than tc-DNA nucleosides of the oligomeric compounds of the invention are compounds of formula (13) (morpholino nucleosides): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0132] In an embodiment, the one or more nucleosides other than the tc-DNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (13) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (13) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleoside of the oligomeric compound of the invention comprises a compound of formula (13) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0133] In embodiments, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention are compounds of formula (14) (Locked Nucleic Acid or LNA nucleosides): [ka] (In the formula, Bx is a nucleobase; T 1 and T 2 is an internucleoside linking group, T 1 and T 2 The other is OR 1 , OR 2 , a 5' terminal group, a 3' terminal group or an internucleoside linking group, 1 is H or a hydroxyl protecting group, and R 2 is the phosphorus moiety) Includes.

[0134] In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (14) where Bx is selected from the group consisting of cytosine, adenine, guanine and uracil. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (14) where Bx is a modified base. In an embodiment, the 2'-modified-RNA nucleosides of the oligomeric compounds of the invention comprise a compound of formula (14) where Bx is a modified base selected from the group consisting of 5-methylcytosine, 5-methyluracil, 5-bromouracil, inosine and 2,6-diaminopurine.

[0135] General methods for the preparation of compounds of formula (6)-formula (14) for use in oligomeric compounds are known in the art and include, for example, U.S. Pat. No. 4,981,957; U.S. Pat. No. 5,118,800; U.S. Pat. No. 5,319,080; U.S. Pat. No. 5,359,044; U.S. Pat. No. 5,393,878; U.S. Pat. No. 5,446,137; U.S. Pat. No. 5,466,786; U.S. Pat. No. 5,514,785; U.S. Pat. No. 5,519,134; U.S. Pat. No. 5,567,811; U.S. Pat. No. 5,576,427; U.S. Pat. No. 5,59 No. 1,722; U.S. Patent No. 5,597,909; U.S. Patent No. 5,610,300; U.S. Patent No. 5,627,053; U.S. Patent No. 5,639,873; U.S. Patent No. 5,646,265; U.S. Patent No. 5,670,633; U.S. Patent No. 5,700,920; U.S. Patent No. 5,792,847; and U.S. Patent No. 6,600,032; U.S. Patent Application Publication Nos. 2015 / 0141637, 2016 / 0002280, and 2014 / 0296323; and Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002, the disclosures of which are incorporated herein by reference.

[0136] In embodiments, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other: Ribonucleic acid (RNA) nucleosides; Deoxyribonucleic acid (DNA) nucleosides; 2'-Modified RNA Nucleosides Preferably, 2',4'-BNA with 2'-ONC crosslinking system (2',4'-BNA NC ), bicyclic nucleic acid (2',4'-BNA) nucleosides selected from stereoisomers of LNA-α-L-LNA and ethylene nucleic acid (ENA) nucleosides; Peptide nucleic acid (PNA) nucleosides; 2'-deoxy 2'-fluoro-arabino (FANA) nucleosides; Hexitol nucleic acid (HNA) nucleosides; and Phosphorodiamidate morpholino (PMO) nucleosides is selected from.

[0137] Additional nucleosides useful for the present invention will be known to those of skill in the art, such as other lipophilic 2'-O-alkylRNAs described in Biochemistry, 2005, vol. 44, pp. 9045-9057.

[0138] Non-nucleoside In embodiments, the oligomeric compounds of the invention include non-nucleosides, also known in the art as non-nucleoside linkers, non-nucleotidyl linkers, which are highly flexible substitutes for the sugar carbons of, for example, ribofuranone moieties and can be used to replace nucleosides other than tc-DNA nucleosides and the oligomeric compounds. An example non-nucleoside is the 1,3-propanediol group shown in formula (15), which is shown connecting two example phosphorodiester internucleoside linkages: [ka]

[0139] The wavy lines in formula (15) represent additional oligomeric repeating nucleoside and internucleoside linking units as described herein.

[0140] The non-nucleotides of the invention can be used with any of the internucleoside linkages described herein, including embodiments in which the phosphorodiester internucleoside linkage shown in formula (15) is replaced with one or more phosphorothioate internucleoside linkages.

[0141] In an embodiment, the non-nucleotide is a 1,3-propanediol group. The synthesis and incorporation of 1,3-propanediol groups into oligomeric compounds is known in the art and described, for example, in Seela and Kaiser, Nuc. Acids Res., 1987, vol. 15, pp. 3113-29. In an embodiment, the oligomeric compounds of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 1,3-propanediol groups linked by phosphorothioate internucleoside linkages, phosphorodiester internucleoside linkages or mixtures thereof.

[0142] Alternative non-nucleosides may also be used in the oligomeric compounds of the invention, such as ethylene glycol oligomers of various lengths (i.e., one, two, three or more ethylene glycol units linked to form a single non-nucleoside). A variety of suitable ethylene glycol groups are described, for example, in Pils and Micura, Nuc. Acids Res., 2000, 28, pp. 1859-63. The synthesis and use of non-nucleosides is also described, for example, in U.S. Patent No. 5,573,906, the disclosure of which is incorporated herein by reference.

[0143] Internucleoside Linking Group In embodiments, the internucleoside linking groups of the oligomeric compounds of the invention are independently selected from the group consisting of phosphorothioate, phosphorodithioate, phosphorodiester, phosphotriester, aminoalkylphosphotriester, methylphosphonate, alkylphosphonate, 5'-alkylenephosphonate, phosphonate, phosphinate, phosphoramidate, 3'-aminophosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, and boranophosphate linkages.

[0144] In preferred embodiments, the internucleoside linkages of the oligomeric compounds of the invention are independently selected from the group consisting of phosphorothioate linkages and phosphorodiester linkages. In embodiments, the internucleoside linkages of the oligomeric compounds of the invention comprise only phosphorodiester linkages.

[0145] An exemplary phosphorothioate linkage has the formula (16): [ka] As shown in.

[0146] An example phosphorodiester linkage is represented by formula (17): [ka] As shown in.

[0147] The wavy lines in formula (16) and formula (17) represent additional oligomeric repeating nucleosides and internucleoside linkages described herein.

[0148] General methods for the preparation of internucleoside linkages for use in oligomeric compounds are known in the art and include, for example, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,4 Nos. 5,55233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, the disclosures of which are incorporated herein by reference. Phosphorothioates can be prepared, for example, as described in Krotz et al., Org. Proc. R&D, 2006, 114, 115; Krotz et al., Org. Proc. R&D, 2006, 115 ... Using phenylacetyl disulfide (PADS) chemistry as described in, for example, 2004, vol. 8, pp. 852-58, 2004, they can be prepared from phosphate triesters as part of a solid-phase synthesis using a 3'- to 5'-elongation cycle of four reactions (detritylation, coupling, sulfurization and capping using PADS, followed by the steps of deprotection, cleavage from the support, and purification).

[0149] The term "phosphorus moiety" as used herein means P III or P V This refers to a moiety containing a phosphorus atom in the valence state of formula (18): [ka] (In the formula, W represents O, S, or Se, or W represents an electron pair; R 3 and R 4 are independently H, halogen, OH, OR 5 , N.R. 6 R 7 , S.H., S.R. 8 , C 1 ~C 6 Alkyl, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Haloalkoxy, C 1 ~C 6 R is an aminoalkyl; 5 are each independently cyano, nitro, halogen, -NHC(O)C 1 ~C 3 Alkyl, -NHC(O)C 1 ~C 3 Haloalkyl, C 1 ~C 3 Optionally substituted with alkylsulfonyl, C 1 ~C 9 Alkyl, C 1 ~C 6 Alkoxy; each independently of the others, cyano, nitro, halogen, C 1 ~C 4 Alkoxy, C 1 ~C 4 Haloalkyl, C 1 ~C 4 Haloalkoxy, NHC(O)C 1 ~C 3 Alkyl, NHC(O)C 1 ~C 3 Haloalkyl, C 1 ~C 3 Aryl, optionally substituted with alkylsulfonyl, C 1 ~C 6 Alkylene aryl, C 1 ~C 6 Alkylenediaryl; Acetyl; Hydroxyl protecting group; R 6 and R 7are independently hydrogen, cyano, nitro, halogen, C 2 ~C 6 Alkenyl, C 3 ~C 6 Cycloalkyl, C 1 ~C 3 C optionally substituted with alkoxy 1 ~C 9 Alkyl; Cyano, Nitro, Halogen, C 1 ~C 3 Alkyl, C 1 ~C 3 aryl, optionally substituted with alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, preferably said heterocyclic ring being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being selected from C 1 ~C 3 optionally substituted with alkyl; R 8 is a thiol protecting group; the wavy line represents said OR in any one of formulas (1) to (14), or in a similar manner for nucleosides not expressly represented herein by a formula. 2 When W represents O, S or Se, then the P atom in the phosphorus moiety is represented by the formula: V When W represents an electron pair, the P atom in the phosphorus moiety is then in the valence state III The moiety of formula (18) includes any possible stereoisomers. Additionally, the moiety represented by formula (18) includes salts thereof, typically and preferably, the salts are formed upon treatment with an inorganic base or an amine, typically and preferably, the R 3 and R 4Preferred inorganic bases or amines which effect said salt formation with OH or SH groups are well known in the art and are typically and preferably trimethylamine, diethylamine, methylamine or ammonium hydroxide. These phosphorus moieties included in the present invention are also salts derived from reaction with "O - HB + ” (wherein the above HB + refers to the counter cation that is formed).

[0150] The term "phosphorus moiety," as used herein, includes moieties derived from, typically and preferably, each occurrence independently selected from, phosphonates, phosphite triesters, monophosphates, diphosphates, triphosphates, phosphate triesters, phosphate diesters, thiophosphate esters, dithiophosphate esters, or phosphoramidites. Thus, in embodiments, the ORs in any one of formulas (1)-(14), or in a similar manner for nucleosides not expressly represented herein by a formula, 2 is independently selected at each occurrence from a phosphonate, a phosphite triester, a monophosphate, a diphosphate, a triphosphate, a phosphate triester, a phosphate diester, a thiophosphate ester, a dithiophosphate ester, or a phosphoramidite. Additional phosphorus moieties that can be used in the present invention are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, Vol. 48, pp. 2223-2311), which disclosure is incorporated herein by reference.

[0151] The term "phosphorus moiety" as used herein preferably refers to any one of the R groups defined in any one of formulas (1) to (14), or in an analogous manner for nucleosides not expressly represented herein by any formula. 2 Refers to the group P III or PV This includes a phosphorus atom in a valence state of formula (19), formula (20) or formula (21): [ka] (wherein Y is O, S or Se, Y is preferably O or S, more preferably Y is O; R 5 and R 5’ are independently, at each occurrence, independently of one another, hydrogen; are independently, at each occurrence, independently of one another, cyano, nitro, halogen, -NHC(O)C 1 ~C 3 Alkyl, -NHC(O)C 1 ~C 3 Haloalkyl, C 1 ~C 3 Optionally substituted with alkylsulfonyl, C 1 ~C 9 Alkyl, C 1 ~C 6 Alkoxy; each independently of the others, cyano, nitro, halogen, C 1 ~C 4 Alkoxy, C 1 ~C 4 Haloalkyl, C 1 ~C 4 Haloalkoxy, NHC(O)C 1 ~C 3 Alkyl, -NHC(O)C 1 ~C 3 Haloalkyl, C 1 ~C 3 Aryl, optionally substituted with alkylsulfonyl, C 1 ~C 6 Alkylene aryl, C 1 ~C 6 alkylenediaryl; hydroxyl protecting group; R 6 and R 7 are independently hydrogen, cyano, nitro, halogen, C 2 ~C 6 Alkenyl, C 3 ~C 6 Cycloalkyl, C 1 ~C 3Optionally substituted with alkoxy, C 1 ~C 9 Alkyl; Cyano, Nitro, Halogen, C 1 ~C 3 Alkyl, C 1 ~C 3 aryl, preferably phenyl, optionally substituted with alkoxy; an amino protecting group; or together with the nitrogen atom to which they are attached form a heterocyclic ring, preferably said heterocyclic ring being selected from pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and homopiperazine, said heterocyclic ring being selected from C 1 ~C 3 optionally substituted with alkyl; R 8 is a thiol protecting group; the wavy line represents the OR in any one of formulas (1) to (5). 2 (shows bond to oxygen of the group) Each occurrence of each of the following is expressed independently: composition

[0152] Thus, in a first aspect, the present invention provides a composition comprising an oligomeric compound comprising one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides and one or more lipid moieties, preferably exactly one lipid moiety, said one or more lipid moieties being covalently linked to said oligomeric compound, either directly or via a spacer, preferably said oligomeric compound comprising 5 to 40 monomeric subunits.

[0153] In one embodiment, the one or more lipid moieties are independently selected from a fatty acid moiety, a fatty diacid moiety, a glycerolipid moiety, a glycerophospholipid moiety, a sphingolipid moiety, a phospholipid, an alkyl phosphate moiety, and an alkyl phosphonate moiety.

[0154] In one embodiment, the one or more lipid moieties are independently selected from a fatty acid moiety, a fatty diacid moiety, a phospholipid, an alkyl phosphate moiety, and an alkyl phosphonate moiety.

[0155] In one preferred embodiment, the one or more lipid moieties are, independently of each other, fatty acid moieties. In another preferred embodiment, the one or more lipid moieties are, independently of each other, fatty diacid moieties. In another embodiment, the one or more lipid moieties are, independently of each other, glycerolipid moieties. In another embodiment, the one or more lipid moieties are, independently of each other, glycerophospholipid moieties. In another embodiment, the one or more lipid moieties are, independently of each other, sphingolipid moieties. In another preferred embodiment, the one or more lipid moieties are, independently of each other, alkylphosphate moieties. In another preferred embodiment, the one or more lipid moieties are, independently of each other, alkylphosphonate moieties.

[0156] In one embodiment, the lipid moiety or moieties are negatively charged at pH 7.4, which typically corresponds to physiological pH.

[0157] In a preferred embodiment, the one or more lipid moieties are independently selected from a fatty acid moiety, a fatty diacid moiety, an alkyl phosphate moiety, and an alkyl phosphonate moiety.

[0158] In a preferred embodiment, said one or more lipid moieties, independently of each other, are a fatty acid moiety or a fatty diacid moiety.

[0159] In another preferred embodiment, the one or more lipid moieties, independently of one another, are fatty acid moieties, wherein the fatty acid moieties are saturated fatty acid moieties. In another preferred embodiment, the one or more lipid moieties, independently of one another, are fatty acid moieties, wherein the fatty acid moieties are unsaturated fatty acid moieties.

[0160] In a preferred embodiment, the one or more lipid moieties, independently of one another, are fatty di-acid moieties, and the fatty di-acid moieties are saturated fatty di-acid moieties. In another preferred embodiment, the one or more lipid moieties, independently of one another, are fatty di-acid moieties, and the fatty di-acid moieties are unsaturated fatty di-acid moieties.

[0161] In a highly preferred embodiment, said one or more lipid moieties, independently of each other, are fatty acid moieties, and said fatty acid moieties are saturated, unbranched fatty acid moieties.

[0162] In a preferred embodiment, the one or more lipid moieties, independently of one another, are fatty acid moieties, wherein the fatty acid moieties are derived from saturated, unbranched fatty acids. In a preferred embodiment, the one or more lipid moieties, independently of one another, are fatty di-acid moieties, wherein the fatty di-acid moieties are derived from saturated, unbranched fatty di-acids.

[0163] In a highly preferred embodiment, the one or more lipid moieties are, independently of each other, a fatty acid moiety or a fatty di-acid moiety, wherein the fatty acid moiety is a saturated, unbranched fatty acid moiety and the fatty di-acid moiety is a saturated, unbranched fatty di-acid moiety.

[0164] In a preferred embodiment, the one or more lipid moieties are independently of each other represented by formula (I): AB- * (I) (Wherein, A is C 3~32 Alkyl, C 3~32 Alkenyl, C 3~32 Alkynyl, HOOC-C 3~32 Alkylene, HOOC-C 3~32 Alkenylene or HOOC-C 3~32 alkynylene, B is C(O), OP(OH), OP(O)(OH), OP(O)(SH), NH-C(O), NH-P(O)(OH), NH-P(O)(SH), or a pharma- ceutically acceptable salt thereof, and the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer. This is the part.

[0165] In a further preferred embodiment, the one or more lipid moieties are independently of each other represented by the formulae (a) to (u) aC 3~32 Alkyl-C(O)- * , bC 3~32 Alkenyl-C(O)- * , cC 3~32 Alkynyl-C(O)- * , dC 3~32 Alkyl-OP(OH)- * , eC 3~32 Alkenyl-OP(OH)- * , fC 3~32 Alkynyl-OP(OH)- * , gC 3~32 Alkyl-OP(O)(OH)- * , hC 3~32 Alkenyl-OP(O)(OH)- * , I C 3~32 Alkynyl-OP(O)(OH)- * , jC 3~32 Alkyl-OP(O)(SH)- * , kC 3~32 Alkenyl-OP(O)(SH)- * , lC 3~32 Alkynyl-OP(O)(SH)- * , mC 3~32 Alkyl-NH-C(O)- * , nC 3~32 Alkenyl-NH-C(O)- * , oC 3~32 Alkynyl-NH-C(O)- * , p.c. 3~32 Alkyl-NH-P(O)(OH)- *, qC 3~32 Alkenyl-NH-P(O)(OH)- * , RC 3~32 Alkynyl-NH-P(O)(OH)- * , s.HOOC-C 3~32 Alkylene-C(O)- * , t.HOOC-C 3~32 Alkenylene-C(O)- * , and u.HOOC-C 3~32 Alkynylene-C(O)- * (Wherein, the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer. is selected from one of the following:

[0166] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0167] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0168] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-C(O)- * (Wherein, the asterisk ( *) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0169] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-OP(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0170] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-OP(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0171] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-OP(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0172] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-OP(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0173] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-OP(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0174] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-OP(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0175] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-OP(O)(SH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0176] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-OP(O)(SH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0177] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-OP(O)(SH)- * (Wherein, the asterisk ( *) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0178] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-NH-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0179] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-NH-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0180] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-NH-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0181] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-NH-P(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0182] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-NH-P(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0183] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkynyl-NH-P(O)(OH)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0184] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula HOOC-C 3~32 Alkylene-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0185] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula HOOC-C 3~32 Alkenylene-C(O)- * (Wherein, the asterisk ( * ) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0186] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula HOOC-C 3~32 Alkynylene-C(O)- * (Wherein, the asterisk ( *) is a moiety of ) representing said point of covalent attachment to said oligomeric compound or said spacer, and preferably said composition comprises exactly one lipid moiety.

[0187] In a preferred embodiment, the one or more lipid moieties are independently of each other represented by formula (a) to formula (d): aC 3~32 Alkyl-C(O)- * , b.HOOC-C 3~32 Alkylene-C(O)- * , cC 3~32 Alkyl-OP(O)(OH)- * dC 3~32 Alkyl-OP(O)(SH)- * (Wherein, the asterisk ( * ) represents the point of covalent attachment to the oligomeric compound or the spacer. and preferably, 3~32 The alkyl group is an unbranched C 3~32 alkyl, more preferably the C 3~32 Alkyl is an unbranched C alkyl group having an odd number of carbon atoms. 3~32 alkyl, preferably 3~32 The alkylene is an unbranched C 3~32 alkylene, more preferably the C 3~32 Alkylene is an unbranched C 3~32 It is alkylene.

[0188] In a further preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably 3~32 The alkyl group is an unbranched C 3~32 alkyl, more preferably the C3~32 Alkyl is an unbranched C alkyl group having an odd number of carbon atoms. 3~32 It is an alkyl.

[0189] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably the composition comprises exactly one lipid moiety, and 3~32 The alkyl group is an unbranched C 3~32 It is an alkyl.

[0190] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably the composition comprises exactly one lipid moiety, and 3~32 Alkyl is an unbranched C alkyl group having an odd number of carbon atoms. 3~32 It is an alkyl.

[0191] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably 3~32 Alkenyl is a branched C 3~32 alkenyl, more preferably the C 3~32 Alkenyl is a branched alkyl group having an odd number of carbon atoms. 3~32 It is alkenyl.

[0192] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-C(O)-* (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably the composition comprises exactly one lipid moiety, and 3~32 Alkenyl is a branched C 3~32 It is alkenyl.

[0193] In a preferred embodiment, the one or more lipid moieties, independently of each other, have the formula C 3~32 Alkenyl-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably the composition comprises exactly one lipid moiety, and 3~32 Alkenyl is a branched alkyl group having an odd number of carbon atoms. 3~32 It is alkenyl.

[0194] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, are saturated C 8~26 A fatty acid moiety, preferably the saturated C 8~26 The fatty acid moiety is derived from caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), arachidic acid (C20), lignoceric acid (C22) or cerotic acid (C24).

[0195] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, are saturated fatty acid moieties, preferably said saturated fatty acid moieties are derived from caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), arachidic acid (C20), lignoceric acid (C22) and cerotic acid (C24).

[0196] In a further highly preferred embodiment, the one or more lipid moieties are, independently of one another, saturated fatty acid moieties derived from palmitic acid (C16) or stearic acid (C18), preferably the one or more lipid moieties are saturated fatty acid moieties derived from palmitic acid (C16).

[0197] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, comprise an unsaturated C 14~22 A fatty acid moiety, preferably the unsaturated C 14~22 The fatty acid moieties are derived from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid. In a further highly preferred embodiment, the one or more lipid moieties are saturated fatty acid moieties derived from palmitoleic acid.

[0198] In a further highly preferred embodiment, the one or more lipid moieties, independently of each other, are unsaturated fatty acid moieties derived from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.

[0199] In a further highly preferred embodiment, said one or more lipid moieties are unsaturated fatty acid moieties derived from oleic acid.

[0200] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-C 3~32 Alkylene-C(O)- * (Wherein, the asterisk ( * ) is a moiety representing the point of covalent attachment to the oligomeric compound or the spacer, preferably 3~32 Alkylene is an unbranched C 3~32alkylene, more preferably the C 3~32 Alkylene is an unbranched C alkylene having an odd number of carbon atoms. 3~32 It is alkylene.

[0201] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)(C 5~25 Alkyl)-(CH 2 ) t -C(O)- * (Wherein, the asterisk ( * ) represents the point of covalent bond to the oligomeric compound or the spacer, r's are each independently an integer of 1 to 3, and t's are each independently an integer of 1 to 3.

[0202] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)[(CH 2 ) s CH 3 ]-(CH 2 ) t -C(O)- * (Wherein, the asterisk ( * ) represents the point of connection to the oligomeric compound or the spacer by the covalent bond, each r is independently an integer of 1 to 3, each s is independently an integer of 4 to 24, and each t is independently an integer of 1 to 3.

[0203] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)[(CH 2 ) s CH 3 ]-(CH 2 ) t -C(O)- * (Wherein, the asterisk ( *) represents the point of covalent bond to the oligomeric compound or the spacer, each r is independently an integer of 1 or 2, each s is independently an integer of 5 to 19, and each t is independently an integer of 1 or 2.

[0204] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)[(CH 2 ) s CH 3 ]-(CH 2 ) t -C(O)- * (Wherein, the asterisk ( * ) represents the point of covalent bond to the oligomeric compound or the spacer, r is 1, s is each independently an integer of 4 to 24, preferably 5 to 19, and t is 1.

[0205] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)[(CH 2 ) s CH 3 ]-(CH 2 ) t -C(O)- * (Wherein, the asterisk ( * ) represents the point of covalent bond to the oligomeric compound or the spacer, r is 1, s is each independently an integer of 5 to 19, preferably 11 to 17, and t is 1).

[0206] In a further highly preferred embodiment, said one or more lipid moieties, independently of each other, have the formula (HOOC)-(CH 2 ) r -(CH)[(CH 2 ) s CH 3 ]-(CH 2 )t -C(O)- * (Wherein, the asterisk ( * ) represents said point of covalent attachment to said oligomeric compound or said spacer, r is 1, s is 15 and t is 1.

[0207] Thus, in a further highly preferred embodiment, the one or more lipid moieties is 3-pentadecyl glutaric acid (PDG).

[0208] In a further highly preferred embodiment, the lipid moiety is directly linked to the oligomeric compound.

[0209] In a further highly preferred embodiment, said one or more lipid moieties are linked to said oligomeric compound via a spacer.

[0210] In an embodiment, the spacer has 5 to 30 C atoms, preferably 5 to 25 C atoms, more preferably 5 to 20 C atoms, and most preferably 5 to 17 C atoms. In an additional embodiment, the spacer has 4 to 20 heteroatoms, preferably 4 to 18 heteroatoms, more preferably 4 to 14 heteroatoms, and most preferably 4 to 12 heteroatoms. Particularly preferred examples of heteroatoms are N and O atoms. H atoms are not heteroatoms.

[0211] In a preferred embodiment, the spacer has the formula #-NH-C 2~12 Alkylene-§, #-NH-C 2~12 Alkylene-OP(OH)-§, #-NH-C 2~12 Alkylene-OP(O)(SH)-§, #-NH-C 2~12 Alkylene-OP(O)(OH)-§, #-SH-C 2~12 Alkylene-§, #-NH-C 2~12Alkylene-NH-C(O)-§, #-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and #-NH-C 2~12 Alkylene-NH-P(O)(SH)-§ (Wherein, C 2~12 One or more -CH in the alkylene 2 The - moiety is independently -O-, -S-, -NH-, -C(O)-, -C(O)O-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -OP(OH)O-, OP(O)(SH)O-, OP(O)(OH)O-, NHP(O)(OH)O-, NHP(O)(SH)O-, or -(O-CH 2 -CH 2 ) k - (k is an integer from 1 to 8), 2~12 One or more -CH in the alkylene 2 The - moieties may independently be one or more of -COOH, -NH 2 , -OP(O)(OH) 2 or -OH (hence, 2~12 One or both, preferably one hydrogen atom in one or more -CH2- moieties in an alkylene may be independently substituted with one or more -COOH, -NH 2 , -OP(O)(OH) 2 or -OH), the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0212] In a highly preferred embodiment, the spacer has the formula a.#-NH-C 2~12 Alkylene-§, b. #-NH-C 2~12 Alkylene-OP(OH)-§, c.#-NH-C 2~12Alkylene-OP(O)(SH)-§, d. #-NH-C 2~12 Alkylene-OP(O)(OH)-§, e.#-NH-C 2~12 Alkylene-NH-C(O)-§, f.#-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and g.#-NH-C 2~12 Alkylene-NH-P(O)(SH)-§ (In the formula, the (#) represents a covalent bond point to the lipid moiety, and the (§) represents a covalent bond point to the oligomeric compound.) and preferably, independently selected from any one of the formulas:

[0213] In a highly preferred embodiment, the spacer has the formula a.#-NH-C 2~12 Alkylene-§, b. #-NH-C 2~12 Alkylene-OP(OH)-§, c.#-NH-C 2~12 Alkylene-OP(O)(SH)-§, d. #-NH-C 2~12 Alkylene-OP(O)(OH)-§, e.#-NH-C 2~12 Alkylene-NH-C(O)-§, f.#-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and g.#-NH-C 2~12 Alkylene-NH-P(O)(SH)-§ (Wherein, C 2~12 One or more -CH in the alkylene 2 The - moiety is independently -O-, -S-, -NH-, -C(O)-, -C(O)O-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -OP(OH)O-, OP(O)(SH)O-, OP(O)(OH)O-, NHP(O)(OH)O-, NHP(O)(SH)O-, or -(O-CH 2 -CH2 ) k -(k is an integer from 1 to 8), wherein the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0214] In a highly preferred embodiment, the spacer has the formula a.#-NH-C 2~12 Alkylene-§, b. #-NH-C 2~12 Alkylene-OP(OH)-§, c.#-NH-C 2~12 Alkylene-OP(O)(SH)-§, d. #-NH-C 2~12 Alkylene-OP(O)(OH)-§, e.#-NH-C 2~12 Alkylene-NH-C(O)-§, f.#-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and g.#-NH-C 2~12 Alkylene-NH-P(O)(SH)-§ (Wherein, C 2~12 One or more -CH in the alkylene 2 The - moiety is independently -O-, -S-, -NH-, -C(O)-, -C(O)O-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -OP(OH)O-, OP(O)(SH)O-, OP(O)(OH)O-, NHP(O)(OH)O-, NHP(O)(SH)O-, or -(O-CH 2 -CH 2 ) k - (k is an integer from 1 to 8), 2~12 One or more -CH in the alkylene 2 The - moieties may independently be one or more of -COOH, -NH 2 , -OP(O)(OH) 2or -OH, wherein the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0215] In a highly preferred embodiment, the spacer has the formula a.#-NH-C 2~12 Alkylene-§, b. #-NH-C 2~12 Alkylene-OP(OH)-§, c.#-NH-C 2~12 Alkylene-OP(O)(SH)-§, d. #-NH-C 2~12 Alkylene-OP(O)(OH)-§, e.#-NH-C 2~12 Alkylene-NH-C(O)-§, f.#-NH-C 2~12 Alkylene-NH-P(O)(OH)-§, and g.#-NH-C 2~12 Alkylene-NH-P(O)(SH)-§ (Wherein, C 2~12 One or more -CH in the alkylene 2 The - moiety is independently -O-, -S-, -NH-, -C(O)-, -C(O)O-, phenyl, triazolyl, cyclopentyl, cyclohexyl, succinimidyl, -OP(OH)O-, OP(O)(SH)O-, OP(O)(OH)O-, NHP(O)(OH)O-, NHP(O)(SH)O-, or -(O-CH 2 -CH 2 ) k - (k is an integer from 1 to 8), 2~12 One or more -CH in the alkylene 2 The - moieties may independently be one or more of -COOH, -NH 2 , -OP(O)(OH) 2or -OH, wherein the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0216] In a further highly preferred embodiment, the spacer has the formula a.-NH-(CH 2 ) m -, b. -NH-(CH 2 ) m -X-, c.-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -, d.-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-, e. -NH-CH(COOH)-(CH 2 ) q -, f.-NH-CH(COOH)-(CH 2 ) q -X-, g.-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) m -, h.-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) m -X-, i.-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) n -(O-CH2 -CH 2 ) k -O-(CH 2 ) p -X-, j.-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) r X- (wherein, X's are each independently OP(OH), OP(O)(SH) or OP(O)(OH), k's are each independently an integer of 1 to 8, m's are each independently an integer of 2 to 12, n's are each independently an integer of 2 to 4, p's are each independently an integer of 1 to 5, q's are each independently an integer of 1 to 3, preferably 1 or 2, and r's are each independently an integer of 1 to 3, preferably 1 or 2. and preferably, independently selected from any one of the formulas:

[0217] In a further highly preferred embodiment, the spacer has the formula a. #-NH-(CH 2 ) m -§, b. #-NH-(CH 2 ) m -X-§, c.#-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -§, d. #-NH-(CH 2 ) n -(O-CH 2 -CH2 ) k -O-(CH 2 ) p -X-§、 e.#-NH-CH(COOH)-(CH 2 ) q -§、 f.#-NH-CH(COOH)-(CH 2 ) q -X-§、 g.#-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) m -§、 h.#-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) m -X-§、 i.#-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§、 j.#-NH-CH(COOH)-(CH 2 ) q -C(O)-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) r -X-§ (In the formula, X's are each independently OP(OH), OP(OH)(S) or OP(O)(OH), k's are each independently an integer of 1 to 8, m's are each independently an integer of 2 to 12, n's are each independently an integer of 2 to 4, p's are each independently an integer of 1 to 5, q's are each independently an integer of 1 to 3, preferably an integer of 1 or 2, and r's are each independently an integer of 1 to 3, preferably an integer of 1 or 2, the (#) represents a point of covalent bond to the lipid moiety, and the (§) represents a point of covalent bond to the oligomer compound.) and preferably, independently selected from any one of the formulas:

[0218] In a further highly preferred embodiment, the spacer has the formula a.-Z-NH-(CH 2 ) m -X- b.-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X- c.-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X- d. -Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X- (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 X's are each independently OP(OH), OP(O)(SH), OP(O)(OH), NHP(O)(OH), NHP(O)(SH) or NH-C(O), k's are each independently an integer from 1 to 8, m's are each independently an integer from 2 to 12, n's are each independently an integer from 2 to 4, p's are each independently an integer from 1 to 5, q's are each independently an integer from 1 to 6, preferably 3 or 6, and r's are each independently an integer from 1 to 3, preferably 1 or 2. and preferably, independently selected from any one of the formulas:

[0219] In a further highly preferred embodiment, the spacer has the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r-NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently OP(OH), OP(O)(SH), OP(O)(OH), NHP(O)(OH), NHP(O)(SH) or NH-C(O); each k is independently an integer of 1 to 8; each m is independently an integer of 2 to 12; each n is independently an integer of 2 to 4; each p is independently an integer of 1 to 5; each q is independently an integer of 1 to 6, preferably an integer of 3 or 6; each r is independently an integer of 1 to 3, preferably an integer of 1 or 2; (#) represents a point of covalent bond with the lipid moiety; and (§) represents a point of covalent bond with the oligomer compound. and preferably, independently selected from any one of the formulas:

[0220] In another preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently OP(OH), OP(O)(SH), OP(O)(OH), NHP(O)(OH), NHP(O)(SH) or NH-C(O); each k is independently an integer of 1 or 2; each m is independently an integer of 4 to 8; each n is independently an integer of 2 to 4; each p is independently an integer of 1 or 2; each q is independently an integer of 1 to 6; each r is independently an integer of 1 to 3, preferably 1 or 2; (#) represents a point of covalent bond with the lipid moiety; and (§) represents a point of covalent bond with the oligomer compound. and preferably, independently selected from any one of the formulas:

[0221] In another preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently OP(OH), OP(O)(SH) or OP(O)(OH); each k is independently an integer of 1 or 2; each m is independently an integer of 4 to 8; each n is 2; each p is 1; each q is independently an integer of 1 to 6; each r is independently an integer of 1 to 3, preferably 1 or 2; (#) represents a point of covalent bond with the lipid moiety; and (§) represents a point of covalent bond with the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0222] In another preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 )k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently OP(O)(SH) or OP(O)(OH), each k is independently an integer of 1 or 2, each m is independently an integer of 4 to 8, each n is 2, each p is 1, each q is independently an integer of 1 to 6, each r is independently an integer of 1 to 3, preferably 1 or 2, each (#) represents a point of covalent bond with the lipid moiety, and each (§) represents a point of covalent bond with the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0223] In another highly preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently an integer of 1 or 2; each m is independently an integer of 4 to 8; n is 2; p is 1; each q is independently an integer of 1 to 6; each r is independently an integer of 1 to 3, preferably 1 or 2; (#) represents a point of covalent bond with the lipid moiety; and (§) represents a point of covalent bond with the oligomeric compound), and is preferably independently selected from any one of the formulas.

[0224] In another highly preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-CH[(CH 2 ) 2 each X is independently an integer of 1 or 2; each m is independently an integer of 4 to 8; n is 2; p is 1; each q is independently an integer of 1 to 6; each r is independently an integer of 1 to 3, preferably 1 or 2; (#) represents a point of covalent bond with the lipid moiety; and (§) represents a point of covalent bond with the oligomeric compound), and is preferably independently selected from any one of the formulas.

[0225] In another highly preferred embodiment, the spacer is of the formula a. #-Z-NH-(CH 2 ) m -X-§ b. #-Z-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -X-§ c.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-]-NH-(CH 2 ) q -X-§ d.#-Z[-NH-(CH 2 ) n -(O-CH 2 -CH 2 ) k -O-(CH 2 ) p -C(O)-] r -NH-(CH 2 ) q -(O-CH 2 -CH 2 ) k -X-§ (wherein, -Z- is, independently of each other, a bond or -NH-CH(COOH)-(CH 2 ) 2 -C(O)- or -NH-C[(CH 2 ) 2 X is, independently of each other, OP(O)(SH) or OP(O)(OH), k is 1, m is 6, n is 2, p is 1, q is, independently of each other, an integer of 1 to 6, r is, independently of each other, an integer of 1 to 3, preferably 1 or 2, (#) represents a point of covalent bond to the lipid moiety, and (§) represents a point of covalent bond to the oligomeric compound. and preferably, independently selected from any one of the formulas:

[0226] In another highly preferred embodiment, the spacer comprises #-Z-NH-(CH2)mX-§, preferably #-Z-NH-(CH2)mX-§, where -Z- represents a bond, X is, independently of each other, OP(O)(SH) or OP(O)(OH), m is 6, said (#) represents the point of covalent attachment to said lipid moiety, and said (§) represents the point of covalent attachment to said oligomeric compound.

[0227] In another highly preferred embodiment, the spacer comprises #-Z-NH-(CH2)mX-§, preferably #-Z-NH-(CH2)mX-§, where -Z- represents a bond, X is OP(O)(OH), m is 6, the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound.

[0228] In another highly preferred embodiment, the spacer comprises #-Z-NH-(CH2)mX-§, preferably #-Z-NH-(CH2)mX-§, where -Z- represents a bond, X is OP(O)(SH), m is 6, the (#) represents a point of covalent attachment to the lipid moiety, and the (§) represents a point of covalent attachment to the oligomeric compound.

[0229] In another highly preferred embodiment, said one or more lipid moieties are covalently linked to said oligomeric compound through an -OP(O)(SH)- or -OP(O)(OH)- moiety, either directly or via a spacer, typically and preferably constituted by said one or more lipid moieties or said spacer, said -OP(O)(SH)- or said -OP(O)(OH)- moiety being linked to the 5'- or 3'-terminal OH group of said oligomeric compound.

[0230] In another highly preferred embodiment, the one or more lipid moieties are linked, independently of each other, to the oligomeric compound at: (i) a terminal residue of the oligomeric compound, (ii) the 5' end of the oligomeric compound, (iii) the 3' end of the oligomeric compound, or (iv) an internal residue of the oligomeric compound.

[0231] In another preferred embodiment, said one or more lipid moieties, preferably said exactly one lipid moiety, are independently linked to said oligomeric compound at a terminal residue of said oligomeric compound.

[0232] In another preferred embodiment, said one or more lipid moieties, preferably said exactly one lipid moiety, are linked independently of each other to said oligomeric compound at the 5' end of said oligomeric compound.

[0233] In another preferred embodiment, said one or more lipid moieties, preferably said exactly one lipid moiety, are linked independently of each other to said oligomeric compound at the 3' end of said oligomeric compound.

[0234] In another preferred embodiment, said one or more lipid moieties, preferably said exactly one lipid moiety, are independently linked to said oligomeric compound at an internal residue of said oligomeric compound.

[0235] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, is covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer through an -OP(O)(SH)- or -OP(O)(OH)- or -NHP(O)(OH)- or -NHP(O)(SH)- or -NH-C(O)- moiety, typically and preferably constituted by said one or more lipid moieties or said spacer, said -OP(O)(SH)- or said -OP(O)(OH)- or said -NHP(O)(OH)- or said -NHP(O)(SH)- or said -NH-C(O)- moiety being linked to the 5'- or 3'-terminal OH group of said oligomeric compound.

[0236] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer, through an -OP(O)(SH)- or -OP(O)(OH)- moiety, said -OP(O)(SH)- or said -OP(O)(OH)- moiety being linked to the 5'- or 3'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(SH)- or said -OP(O)(OH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0237] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer through an -OP(O)(SH)- moiety, said -OP(O)(SH)- moiety being linked to the 5'- or 3'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(SH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0238] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer through an -OP(O)(SH)- moiety, said -OP(O)(SH)- moiety being linked to the 5'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(SH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0239] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer through an -OP(O)(SH)- moiety, said -OP(O)(SH)- moiety being linked to the 3'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(SH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0240] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer, through a -OP(O)(OH)- moiety, said -P(O)(OH)- moiety being linked to the 5'- or 3'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(OH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0241] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer, through a -OP(O)(OH)- moiety, said -P(O)(OH)- moiety being linked to the 5'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(OH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0242] In another highly preferred embodiment, said one or more lipid moieties, preferably exactly one lipid moiety, are covalently linked to said oligomeric compound, preferably said oligonucleotide, either directly or via a spacer, through a -OP(O)(OH)- moiety, said -P(O)(OH)- moiety being linked to the 3'-terminal OH group of said oligomeric compound, typically and preferably said -OP(O)(OH)- moiety being constituted by said one or more lipid moieties or said spacer.

[0243] In another preferred embodiment, the composition does not contain any nucleosides other than tc-DNA nucleosides.

[0244] In another preferred embodiment, said oligomeric compound does not contain any nucleosides other than tc-DNA nucleosides.

[0245] In another preferred embodiment, the composition further comprises one or more nucleosides other than tc-DNA nucleosides.

[0246] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides.

[0247] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 50% or more of all nucleosides are tc-DNA nucleosides.

[0248] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 60% or more of all nucleosides are tc-DNA nucleosides.

[0249] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 70% or more of all nucleosides are tc-DNA nucleosides.

[0250] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 75% or more of all nucleosides are tc-DNA nucleosides.

[0251] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 80% or more of all nucleosides are tc-DNA nucleosides.

[0252] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 85% or more of all nucleosides are tc-DNA nucleosides.

[0253] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 90% or more of all nucleosides are tc-DNA nucleosides.

[0254] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides, wherein 95% or more of all nucleosides are tc-DNA nucleosides.

[0255] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other: i. 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides; ii. ribonucleic acid (RNA) nucleosides; iii. Deoxyribonucleic acid (DNA) nucleosides; iv. Locked Nucleic Acid (LNA) nucleosides; v. Peptide nucleic acid (PNA) nucleosides; vi. 2'-deoxy 2'-fluoro-arabinonucleosides; vii. Hexitol nucleic acid (HNA) nucleosides; and viii. Phosphorodiamidate morpholino (PMO) nucleosides is selected from.

[0256] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides.

[0257] In another preferred embodiment, the 2'-modified-RNA nucleosides are incorporated at at least two adjacent positions that form self-complementary Watson-Crick base pairs.

[0258] In another preferred embodiment, the 2'-modified-RNA nucleosides are incorporated at three or more adjacent positions, forming self-complementary Watson-Crick base pairs.

[0259] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, ribonucleic acid (RNA) nucleosides.

[0260] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, deoxyribonucleic acid (DNA) nucleosides.

[0261] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, locked nucleic acid (LNA) nucleosides.

[0262] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, a peptide nucleic acid (PNA) nucleoside.

[0263] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, 2'-deoxy 2'-fluoro-arabinonucleosides.

[0264] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, a hexitol nucleic acid (HNA) nucleoside.

[0265] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, phosphorodiamidate morpholino (PMO) nucleosides.

[0266] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other: i.RNA nucleosides; ii. 2'-O-methyl-RNA nucleosides; iii. 2'-O-propargyl-RNA nucleoside; iv. 2'-O-propylamino-RNA nucleoside; v. 2'-O-amino-RNA nucleosides; vi. 2'-fluoro-RNA nucleosides; vii. 2'-O-methoxyethyl-RNA nucleosides; viii. morpholino nucleosides; and ix. Locked nucleic acid nucleosides is selected from.

[0267] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, RNA nucleosides.

[0268] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, 2'-O-methyl-RNA nucleosides.

[0269] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, 2'-O-propargyl-RNA nucleosides.

[0270] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, 2'-O-propylamino-RNA nucleosides.

[0271] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, 2'-O-amino-RNA nucleosides.

[0272] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, 2'-fluoro-RNA nucleosides.

[0273] In another preferred embodiment, the oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, wherein said one or more nucleosides other than tc-DNA nucleosides are, independently of each other, 2'-O-methoxyethyl-RNA nucleosides.

[0274] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, a morpholino nucleoside.

[0275] In another preferred embodiment, said oligomeric compound further comprises one or more nucleosides other than tc-DNA nucleosides, said one or more nucleosides other than tc-DNA nucleosides being, independently of each other, locked nucleic acid RNA nucleosides.

[0276] In another preferred embodiment, said oligomeric compounds are complementary to a target sequence.

[0277] In another preferred embodiment, the oligomeric compound has a length of up to 40 monomeric subunits, preferably up to 30 monomeric subunits, more preferably up to 30 monomeric subunits, again more preferably up to 20 monomeric subunits or up to 15 monomeric subunits. In a further embodiment, the oligomer comprises from 5 to 40 monomeric subunits, preferably from 8 to 30 monomeric subunits, more preferably from 8 to 25 monomeric subunits, again more preferably from 8 to 20 monomeric subunits.

[0278] In another preferred embodiment, the oligomeric compound comprises 5 to 40 nucleotides, preferably, the oligomeric compound comprises 10 to 30 nucleotides, and more preferably, the oligomeric compound comprises 10 to 25 nucleotides.

[0279] In another preferred embodiment, the oligomeric compound is an oligonucleotide, the oligomeric compound comprises one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, preferably the oligomeric compound comprises 5 to 40 monomeric subunits. In another preferred embodiment, the oligomeric compound is an oligonucleotide, the oligomeric compound comprises one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, the oligomeric compound comprises 5 to 40 monomeric subunits, the monomeric subunits being linked by internucleoside linking groups.

[0280] In another preferred embodiment, the monomeric subunits are independently selected from naturally occurring nucleosides, modified nucleosides and nucleoside mimetics, preferably, the naturally occurring nucleosides, modified nucleosides and nucleoside mimetics are independently selected from tricyclic nucleosides, ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides.

[0281] In another preferred embodiment, the monomeric subunits are independently selected from naturally occurring nucleosides, modified nucleosides and nucleoside mimetics, preferably, the naturally occurring nucleosides, modified nucleosides and nucleoside mimetics are independently selected from tricyclic nucleosides, ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, and the monomeric subunits are linked by multiple internucleoside linking groups.

[0282] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, wherein the monomeric subunits are nucleosides, one or more of the nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and the monomeric subunits are linked by a plurality of internucleoside linking groups, preferably, the oligomeric compound is an oligonucleotide.

[0283] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, wherein the monomeric subunits are nucleosides, one or more of the nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and the monomeric subunits are linked by a plurality of internucleoside linking groups, preferably, the oligomeric compound is an oligonucleotide.

[0284] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, wherein the monomeric subunits are nucleosides, one or more of the nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and the nucleosides are linked by a plurality of internucleoside linking groups, and preferably the oligomeric compound is an oligonucleotide.

[0285] In another preferred embodiment, the composition does not include nucleosides other than tc-DNA nucleosides. In another preferred embodiment, the oligomeric compound does not include nucleosides other than tc-DNA nucleosides. In another preferred embodiment, the composition further includes one or more nucleosides other than tc-DNA nucleosides. In another preferred embodiment, the oligomeric compound further includes one or more nucleosides other than tc-DNA nucleosides.

[0286] In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides. In another preferred embodiment, the oligomeric compound comprises one or more tc-DNA nucleosides and one or more nucleosides other than tc-DNA nucleosides.

[0287] In another preferred embodiment, the one or more tc-DNA nucleosides, or the one or more tc-DNA nucleosides and the one or more nucleosides other than a tc-DNA nucleoside, are linked by multiple internucleoside linking groups.

[0288] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, wherein the monomeric subunits are nucleosides, all of the nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, and the nucleosides are linked by a plurality of internucleoside linking groups, and preferably the oligomeric compound is an oligonucleotide.

[0289] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, one said nucleoside being a nucleoside other than a tc-DNA nucleoside, and all other said nucleosides being said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and preferably said oligomeric compound is an oligonucleotide.

[0290] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, the monomeric subunits being nucleosides, wherein at most 5, preferably at most 4, more preferably at most 3 of the nucleosides are nucleosides other than tc-DNA nucleosides, and all other nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, the nucleosides being linked by a plurality of internucleoside linking groups, and ... Preferably, the at least one, and more preferably at most three, nucleosides other than tc-DNA nucleosides are selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, and preferably, said oligomeric compound is an oligonucleotide.

[0291] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, wherein at most three of said nucleosides are nucleosides other than tc-DNA nucleosides, and all other said nucleosides are said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and wherein said at most three tc-DNA nucleosides are non-nucleoside linking groups. The nucleoside other than nucleoside is selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably said oligomeric compound is an oligonucleotide.

[0292] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, wherein at most two of said nucleosides are nucleosides other than tc-DNA nucleosides, and all other said nucleosides are said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and wherein said at most two tc-DNA nucleosides are non-nucleoside linking groups. The nucleoside other than nucleoside is selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably said oligomeric compound is an oligonucleotide.

[0293] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, at most one of said nucleosides being a nucleoside other than a tc-DNA nucleoside, and all other said nucleosides being said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and said at most one of said tc-DNA nucleosides being a nucleoside other than a tc-DNA nucleoside. The nucleoside other than nucleoside is selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably said oligomeric compound is an oligonucleotide.

[0294] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, one said nucleoside being a nucleoside other than a tc-DNA nucleoside, and all other said nucleosides being said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and said nucleosides other than tc-DNA nucleosides being said one or more tricyclo-deoxyribonucleic acid (tc-DNA) ... Preferably, the nucleoside is selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, and said oligomeric compound is an oligonucleotide.

[0295] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, one said nucleoside being a nucleoside other than a tc-DNA nucleoside, and all other said nucleosides being the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and said nucleosides other than the tc-DNA nucleosides being selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably said oligomeric compound is an oligonucleotide.

[0296] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, the monomeric subunits being nucleosides, one of the nucleosides being a nucleoside other than a tc-DNA nucleoside, and all other of the nucleosides being the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, the nucleosides being linked by a plurality of internucleoside linking groups, the nucleosides other than the tc-DNA nucleosides being selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides and 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, preferably the oligomeric compound is an oligonucleotide.

[0297] In another preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, the monomeric subunits being nucleosides, two of the nucleosides being nucleosides other than tc-DNA nucleosides, and all other of the nucleosides being the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, the nucleosides being linked by a plurality of internucleoside linking groups, and the two nucleosides other than tc-DNA nucleosides being nucleosides other than the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides. The nucleosides are independently selected from ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably said oligomeric compound is an oligonucleotide.

[0298] In another preferred embodiment, the plurality of internucleoside linking groups are independently selected from phosphorothioate, phosphorodithioate, and phosphorodiester, phosphotriester, aminoalkylphosphotriester, methylphosphonate, alkylphosphonate, 5'-alkylenephosphonate, phosphonate, phosphinate, phosphoramidate, 3'-aminophosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate, or boranophosphate linking groups.

[0299] In another preferred embodiment, said internucleoside linkage groups are independently selected from phosphorothioate linkage groups and phosphorodiester linkage groups.

[0300] In another preferred embodiment, no more than 50% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 45% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 40% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 35% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 33% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 30% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 25% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 20% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 20% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 15% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 10% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than 5% of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, none (0%) of the plurality of internucleoside linking groups are phosphorothioate linking groups.

[0301] In another preferred embodiment, no more than eight of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than seven of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than six of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than five of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than four of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than three of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than two of the plurality of internucleoside linking groups are phosphorothioate linking groups. In another preferred embodiment, no more than one of the plurality of internucleoside linking groups is a phosphorothioate linking group. In another preferred embodiment, none of said plurality of internucleoside linkage groups (zero / 0) are phosphorothioate linkage groups.

[0302] In another preferred embodiment, said internucleoside linkage groups are independently selected from phosphorothioate linkage groups and phosphorodiester linkage groups, and no more than 30% of said internucleoside linkage groups are phosphorothioate linkage groups.

[0303] In another preferred embodiment, at least 50% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 55% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 60% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 65% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 66% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 70% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 75% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 80% of the internucleoside linking groups are phosphorodiester linkages. In another preferred embodiment, at least 85% of the internucleoside linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 90% of the internucleoside linkage groups are phosphorodiester linkage groups. In another preferred embodiment, at least 95% of the internucleoside linkage groups are phosphorodiester linkage groups. In another preferred embodiment, all (100%) of the internucleoside linkage groups are phosphorodiester linkage groups.

[0304] In another preferred embodiment, at least 80% of said internucleoside linkage groups are phosphorodiester linkage groups, preferably said internucleoside linkage groups are independently selected from phosphorothioate linkage groups and phosphorodiester linkage groups.

[0305] In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than six of the internucleoside linking groups are phosphorothioate linking groups. In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than five of the internucleoside linking groups are phosphorothioate linking groups. In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than four of the internucleoside linking groups are phosphorothioate linking groups. In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than three of the internucleoside linking groups are phosphorothioate linking groups. In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than two of the internucleoside linking groups are phosphorothioate linking groups. In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorothioate and phosphorodiester linking groups, and no more than one of the internucleoside linking groups is a phosphorothioate linking group.

[0306] In another highly preferred embodiment, the internucleoside linking groups are independently selected from phosphorodiester linking groups. Thus, in another highly preferred embodiment, all of the internucleoside linking groups are phosphorodiester linking groups.

[0307] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, wherein the monomeric subunits are nucleosides, wherein at most three of the nucleosides are nucleosides other than tc-DNA nucleosides, and all other of the nucleosides are the one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, wherein the nucleosides are linked by a plurality of internucleoside linking groups, and wherein the at most three nucleosides other than the tc-DNA nucleosides are selected from the group consisting of ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid nucleosides, (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably the oligomeric compound is an oligonucleotide, and the internucleoside linking groups are independently selected from phosphorothioate linking groups and phosphorodiester linking groups, and no more than 6, preferably no more than 5, more preferably no more than 4, again more preferably no more than 3, more preferably no more than 2, more preferably no more than 1 of the internucleoside linking groups is a phosphorothioate linking group.In a highly preferred embodiment thereof, all of the internucleoside linking groups are independently selected from phosphorodiester linking groups.

[0308] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, wherein at most two of said nucleosides are nucleosides other than tc-DNA nucleosides, and all other said nucleosides are said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and wherein said at most two nucleosides other than tc-DNA nucleosides are selected from the group consisting of ribonucleic acid (RNA) nucleosides, deoxyribonucleic acid (DNA) nucleosides, 2'-modified ribonucleic acid (2'-modified-RNA) nucleosides, locked nucleic acid nucleosides, (LNA) nucleosides, peptide nucleic acid (PNA) nucleosides, 2'-deoxy 2'-fluoro-arabinonucleosides, hexitol nucleic acid (HNA) nucleosides and phosphorodiamidate morpholino (PMO) nucleosides, preferably the oligomeric compound is an oligonucleotide, and the internucleoside linking groups are independently selected from phosphorothioate linking groups and phosphorodiester linking groups, and no more than 6, preferably no more than 5, more preferably no more than 4, again more preferably no more than 3, more preferably no more than 2, more preferably no more than 1 of the internucleoside linking groups is a phosphorothioate linking group.In a highly preferred embodiment thereof, all of the internucleoside linking groups are independently selected from phosphorodiester linking groups.

[0309] In another highly preferred embodiment, the oligomeric compound comprises 5 to 40 monomeric subunits, said monomeric subunits being nucleosides, wherein at most one of said nucleosides is a nucleoside other than a tc-DNA nucleoside, and all other said nucleosides are said one or more tricyclo-deoxyribonucleic acid (tc-DNA) nucleosides, said nucleosides being linked by a plurality of internucleoside linking groups, and wherein said at most one nucleoside other than the tc-DNA nucleoside is a ribonucleic acid (RNA) nucleoside, a deoxyribonucleic acid (DNA) nucleoside, a 2'-modified ribonucleic acid (2'-modified-RNA) nucleoside, a locked nucleoside, a deoxyribonucleic acid (DNA) nucleoside, a 2'-modified ribonucleic acid (2'-modified-RNA) nucleoside, a locked nucleoside, a deoxyribonucleic acid (DNA) nucleoside, a 2'-modified ribonucleic acid (2'-modified-RNA) nucleoside, a locked nucleoside, a deoxyribonucleic acid (DNA) nucleoside, a deoxyribonucleic acid (DNA ... Preferably, the oligomeric compound is an oligonucleotide, and the internucleoside linking groups are independently selected from phosphorothioate linking groups and phosphorodiester linking groups, and no more than 6, preferably no more than 5, more preferably no more than 4, again more preferably no more than 3, more preferably no more than 2, more preferably no more than 1 of the internucleoside linking groups is a phosphorothioate linking group. In a highly preferred embodiment thereof, all of the internucleoside linking groups are independently selected from phosphorodiester linking groups.

[0310] In another preferred embodiment, said oligomeric compounds do not contain direct tc-DNA to tc-DNA phosphorothioate internucleoside linkages.

[0311] The following table provides highly preferred embodiments of the present invention.

[0312] [Table 1-1] [Table 1-2]

[0313] In a preferred embodiment, said oligomeric compound is selected from the oligomeric compounds listed in Table 1, typically and preferably said 2'-modified-RNA is 2'-OMe-RNA.

[0314] In a preferred embodiment, said oligomeric compound, preferably said oligonucleotide, is selected from any one of the sequences SEQ ID NO: 1 to 22, and said 2'-modified-RNA is 2'-OMe-RNA. In a preferred embodiment, said oligomeric compound, preferably said oligonucleotide, is selected from any one of the sequences SEQ ID NO: 1 to 22, and said 2'-modified-RNA is 2'-OMe-RNA, and each of said internucleoside linkages of the sequences SEQ ID NO: 1 to 22 is a phosphorodiester linkage.

[0315] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:1.

[0316] In a further preferred embodiment, said oligomeric compound comprises, and preferably is, the sequence SEQ ID NO:2.

[0317] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO: 3. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO: 3, and said 2'-modified-RNA is 2'-OMe-RNA.

[0318] In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 4, preferably is the sequence SEQ ID NO: 4. In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 4, preferably is the sequence SEQ ID NO: 4, and said 2'-modified-RNA is 2'-OMe-RNA.

[0319] In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 5, preferably the sequence SEQ ID NO: 5. In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 5, preferably the sequence SEQ ID NO: 5, and said 2'-modified-RNA is 2'-OMe-RNA.

[0320] In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 6, preferably the sequence SEQ ID NO: 6. In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 6, preferably the sequence SEQ ID NO: 6, and said 2'-modified-RNA is 2'-OMe-RNA.

[0321] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:7.

[0322] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 8. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 8 and said 2'-modified-RNA is 2'-OMe-RNA.

[0323] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:9.

[0324] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:10.

[0325] In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 11, preferably the sequence SEQ ID NO: 11. In a further preferred embodiment, said oligomeric compound comprises the sequence SEQ ID NO: 11, preferably the sequence SEQ ID NO: 11, and said 2'-modified-RNA is 2'-OMe-RNA.

[0326] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 12. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 12, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0327] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 13. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 13, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0328] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 14. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 14, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0329] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 15. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 15, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0330] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 16. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 16, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0331] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 17. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 17, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0332] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 18. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 18, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0333] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:19.

[0334] In a further preferred embodiment, said oligomeric compound comprises, and is preferably, the sequence of SEQ ID NO: 20. In a further preferred embodiment, said oligomeric compound comprises, and is preferably, the sequence of SEQ ID NO: 20, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0335] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:21.

[0336] In a further preferred embodiment, said oligomeric compound comprises, and preferably is, the sequence SEQ ID NO:22.

[0337] In a further preferred embodiment, said oligomeric compound is selected from the oligomeric compounds listed in Table 2, typically and preferably said 2'-modified-RNA is 2'-OMe-RNA.

[0338] In a highly preferred embodiment, said oligomeric compound, preferably said oligonucleotide, is selected from any one of the sequences SEQ ID NOs: 1-37.

[0339] In a further highly preferred embodiment, said oligomeric compound, preferably said oligonucleotide, is selected from any one of the sequences SEQ ID NO: 1 to 37, and said 2'-modified-RNA is 2'-OMe-RNA.

[0340] [Table 2-1] [Table 2-2] [Table 2-3]

[0341] In a further highly preferred embodiment, said oligomeric compound has a sequence selected from any one of SEQ ID NOs: 1 to 37, each of said internucleoside linking groups of sequences SEQ ID NOs: 1 to 22 is a phosphorodiester linkage, and preferably said 2'-modified-RNA is 2'-OMe-RNA. In a further highly preferred embodiment, said oligomeric compound has a sequence selected from any one of SEQ ID NOs: 1 to 37, each of said internucleoside linking groups of sequences SEQ ID NOs: 1 to 22 is a phosphorodiester linkage, and preferably said 2'-modified-RNA is 2'-OMe-RNA.

[0342] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:23.

[0343] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 24. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 24, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0344] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 25. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 25, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0345] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 26. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 26, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0346] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 27. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 27, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0347] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 28. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 28, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0348] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 29. In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence of SEQ ID NO: 29, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0349] In a further preferred embodiment, said oligomeric compound comprises the sequence of SEQ ID NO: 30, Preferably, the sequence is SEQ ID NO: 30. In a further preferred embodiment, said oligomeric compound comprises, preferably has, the sequence SEQ ID NO: 30, and said 2'-modified-RNA is a 2'-OMe-RNA.

[0350] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:31.

[0351] In a further preferred embodiment, said oligomeric compound comprises, and preferably is, the sequence SEQ ID NO:32.

[0352] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:33.

[0353] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:34.

[0354] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:35.

[0355] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:36.

[0356] In a further preferred embodiment, said oligomeric compound comprises, preferably is, the sequence SEQ ID NO:37.

[0357] In a further highly preferred embodiment, the composition of the present invention is selected from any one of the compositions listed in Table 3.

[0358] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [ka]

[0359] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0299.

[0360] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0343.

[0361] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0357.

[0362] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0427.

[0363] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0440.

[0364] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0442.

[0365] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0443.

[0366] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0444.

[0367] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0445.

[0368] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0446.

[0369] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0448.

[0370] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0450.

[0371] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0451.

[0372] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0455.

[0373] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0457.

[0374] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0458.

[0375] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0459.

[0376] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0460.

[0377] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0487.

[0378] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0488.

[0379] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0489.

[0380] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0490.

[0381] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0491.

[0382] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0492.

[0383] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0493.

[0384] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0494.

[0385] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0495.

[0386] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0496.

[0387] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0497.

[0388] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0498.

[0389] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0499.

[0390] In a further highly preferred embodiment, the compositions of the invention comprise, preferably are, SY-0487.

[0391] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0500.

[0392] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0501.

[0393] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0502.

[0394] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0503.

[0395] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0504.

[0396] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0505.

[0397] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0506.

[0398] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0507.

[0399] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0508.

[0400] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0509.

[0401] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0510.

[0402] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0511.

[0403] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0512.

[0404] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0513.

[0405] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0514.

[0406] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0515.

[0407] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0516.

[0408] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0517.

[0409] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0518.

[0410] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0519.

[0411] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0520.

[0412] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0521.

[0413] In a further highly preferred embodiment, the compositions of the invention comprise, and preferably are, SY-0522.

[0414] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0523.

[0415] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0524.

[0416] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0525.

[0417] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0526.

[0418] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0527.

[0419] In a further highly preferred embodiment, the composition of the invention comprises, preferably is, SY-0543.

[0420] Methods of Treating Disease The compositions described herein can be used in methods for treating diseases. In some embodiments, the disease is a disease that can be treated using exon skipping oligomeric compounds. In some embodiments, the disease is a disease that can be treated using antisense-mediated exon inclusion oligomeric compounds. In embodiments of the invention, the compositions described herein cross the blood-brain barrier and are therefore useful in treating diseases of the central nervous system, behavioral disorders, psychiatric disorders and / or behavioral symptoms of diseases. In some embodiments, the disease is a disease of the central nervous system (CNS). In some embodiments, the disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), epilepsy, Creutzfeldt-Jakob disease (CJ), Menkes disease or Huntington's disease (HD). In some embodiments, the disease is a disease that affects the function of the cerebellum, including but not limited to ataxia. In some embodiments, the disease is a disease affecting the function of the amygdala, including but not limited to Urbach-Wiete disease. In some embodiments, the disease is a disease affecting the function of the hippocampus, including but not limited to memory loss. In some embodiments, the disease to be treated is a mental or behavioral disorder, including but not limited to mood disorders, dementia, anxiety, bipolar disorder, schizophrenia, sleep disorders, post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), and depressive disorders.

[0421] In some embodiments of the present invention, the oligonucleotide of the present invention is used to treat sleep disorders and / or cognitive disorders and their associated symptoms. In some embodiments, the sleep disorder is insomnia or slow wave sleep disturbance. In some embodiments, the cognitive disorder is schizophrenia. When the disease to be treated is schizophrenia, both the positive and negative symptoms of schizophrenia can be treated. In some embodiments, the positive symptoms of schizophrenia are hallucinations, delusions, or disturbances in logical thinking processes. In some embodiments, the negative symptoms of schizophrenia include lack of motivation, lack of spontaneity, inability to think abstractly, lack of mood expression, lack of cognition, lack of ability to feel pleasure, flattened emotions, allopathic, amotivational, dysphoric moods including anger, anxiety and depression, disturbed sleep patterns, poor impulse control, lack of judgment, abnormal psychomotor activity such as pacing or locking, and movement disorders such as tardive dyskinesia. In some embodiments, the domains of cognition such as verbal memory, verbal fluency, memory consolidation and executive function are improved by administration of one or more oligonucleotide compounds of the present invention. In some embodiments, administration of one or more oligonucleotide compounds of the present invention increases slow wave sleep, thereby improving cognition. In some embodiments, the disease is selected from the group consisting of Duchenne muscular dystrophy (DMD), familial dysautonomia, spinal muscular atrophy (SMA), ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia (FTD), Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic leukoencephalopathy with subcortical cysts type 1, Pelizaeus-Merzbach disease, Pompe disease, and myotonic dystrophy type 1.Methods of using tc-DNA oligonucleotides for the treatment of Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), spinocerebellar ataxia type 3 (SCA3), and other diseases are known in the art and are described, for example, in U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,577,428; 5,579,520; 5,578,521; 5,579,522; 5,579,523; 5,579,524; 5,579,525; 5,579,526; 5,579,527; 5,579,528; 5,579,529 ... Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847; and 6,600,032; as well as U.S. Patent Application Publication Nos. 2015 / 0141637, 2016 / 0002280, 2014 / 0296323, and 2012 / 0149756, the disclosures of which are incorporated herein by reference.

[0422] Table A provides a list of certain neurodegenerative diseases and their targets for which the compositions of the present invention are useful. [Table A]

[0423] The efficacy of the compositions described herein in treating, preventing and / or managing the indicated disease or disorder can be tested using a variety of models known in the art, which provide guidance for the treatment of human disease. Models for diseases that can be treated using exon-skipping oligomeric compounds are described, for example, in Siva et al., Nucleic Acids and Proteins, 1999, 113:1311-1323, 19 ... Acid Therapeutics, 2014, vol. 24, pp. 69-86. Models for diseases that can be treated using antisense-mediated exon inclusion oligomeric compounds are described, for example, in Hua and Krainer, Methods Mol. Biol., 2012, vol. 867, pp. 307-323.

[0424] Genetic animal models for DMD are known in the art. The mdx mouse carries a nonsense mutation in exon 23 of the dystrophin gene that precludes the synthesis of full-length wild-type dystrophin protein. Grounds et al., Neurobiol. Dis., 2008, vol. 31. , pp. 1-19. The GRMD (Golden Retriever Muscular Dystrophy) canine model lacks functional dystrophin due to a splice site mutation in intron 6 that disrupts the reading frame. In the GRMD model, similar to human DMD, progressive fiber degradation leads to disintegration of the skeletal musculature with significant endomysial and perimysial fibrosis. Other models for DMD include dystrophin / utrophin double knockout mice, humanized DMD mice, mdx52 mice (with a deletion of exon 52 in mouse DMD) and 4CV mice (with a nonsense mutation in exon 53). Goyenvalle et al., Mol. Ther., 2010, vol. 18, pp. 198-05; Bremmer-Bout et al. , Mol. Ther., 2004, vol. 10, pp. 232-240; Aoki et al., Mol. Ther., 2010, vol. 18, pp. 1995-2005; Mitrpant et al., J. Gene. Med., 2009, 1 Volume 1, pages 46-56.

[0425] Spinal muscular atrophy (SMA) is a class of genetic disorders resulting from the deletion of the survival motor neuron gene (SMN1), which maps to chromosome 5q11.2-13.3. Overall, SMA is characterized by the loss of motor neurons in the spinal cord and brainstem, resulting in muscle atrophy from the loss of nerve contact. Each type of SMA has an incidence of approximately 1 in 6,000. Type I SMA, also known as Werdnig-Hoffmann disease or severe childhood SMA, affects infants within the first year of life and is generally fatal. Type II SMA, also known as moderate SMA, affects children and causes muscle weakness that results in patients being unable to stand or walk, although they may be able to sit, with increasing weakness with age. Patients with type III SMA are able to walk at some point in their development.

[0426] SMA is caused by loss of a functional SMN1 gene and a mutation in exon 7 of the SMN2 paralogue that causes substantial skipping of exon 7 and production of only low levels of functional protein, such that the SMN2 protein cannot compensate for the loss of SMN1. Cartegni et al., Am. J. Hum. Genet., 2006, 78, 63-78. Page 77. Oligonucleotide-mediated exon inclusion methods for the treatment of SMA have been explored, including methods to compensate for deleterious mutations in SMN2 by masking intron silencing sequences and / or terminal stem-loop sequences in the SMN2 gene to obtain a modified functional SMN2 protein comprising an amino acid sequence encoded by exon 7, which is capable of at least partially compensating for non-functional SMN1 protein. See, for example, International Publication No. WO 2010 / 115993 A1, the disclosure of which is incorporated herein by reference. In addition to SMA, numerous other diseases, including those diseases described herein, can also potentially be treated by the exon inclusion approach provided by the compositions of the present invention.

[0427] Accordingly, the present invention relates to a pharmaceutical composition comprising the composition of the present invention and further comprising a pharma- ceutical acceptable carrier, preferably said pharmaceutical composition being for use in the prevention, treatment or diagnosis of a neuromuscular disease or a musculoskeletal disease, more preferably said neuromuscular disease or said musculoskeletal disease being selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia-telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, dermatomyositis, rheumatoid arthr ... and even more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0428] The present invention further relates to a pharmaceutical composition of the present invention comprising the composition of the present invention, further comprising a pharma- ceutical acceptable carrier, preferably said pharmaceutical composition being for use in the treatment of a neuromuscular disease or a musculoskeletal disease, more preferably said neuromuscular disease or said musculoskeletal disease being selected from the group consisting of Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia-telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, subcortical capsule and preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease), and again more preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease).

[0429] The present invention further relates to a composition of the present invention for use as a medicament in the prevention, treatment or diagnosis of a disease, preferably wherein said disease is a neuromuscular disease or a musculoskeletal disease, more preferably wherein said neuromuscular disease or said musculoskeletal disease is Duchenne muscular dystrophy, familial dysautonomia, spinal muscular atrophy, ataxia telangiectasia, congenital disorders of glycosylation, frontotemporal dementia, Parkinsonism linked to chromosome 17, Niemann-Pick disease type C, neurofibromatosis type 1, neurofibromatosis type 2, macrocephalic white matter with subcortical cysts, and preferably, said neuromuscular disease or said musculoskeletal disease is selected from Duchenne muscular dystrophy, spinal muscular atrophy, Pompe disease, myotonic dystrophy type 2 (DM2, or proximal myotonic myopathy) and myotonic dystrophy type 1 (DM1, or Steinert disease). EXAMPLES

[0430] The embodiments encompassed herein will now be described with reference to the following examples, which are provided for illustrative purposes only, and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein. material and method

[0431] Animals: The mdx mouse (Bulfield et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 1189-1192; Ryder-Cook et al., EMBO J., 1988, 7, 3017-3021) has a single base substitution in exon 23 of the dystrophin gene that causes premature termination of the polypeptide chain (Sicinski et al., Science, 1989, 244, 1578-1581). -1580), so that the full-length 427 kDa muscle isoform of dystrophin is not produced. However, the other isoform, produced from a different promoter (located 3' to the point mutation), is unaffected. Mutants are viable and fertile. They have no obvious symptoms and remain mechanically unimpaired throughout their lives unless muscle damage is caused by mechanical or chemical insults (Reimann et al., Neuromusc. Disord., 2000, 10, 276-282; Connolly et al., Neuromusc. Disord., 2001, 10, 276-282). 11, pp. 703-712).

[0432] At the histological level, mdx shows classical features of dystrophic muscle, characterized by numerous necrotic fibers with subsequent infiltration of scavenger cells (Coulton et al., Neuropathol. Appl. Neurobiol., 1988, vol. 14, pp. 299-314). However, unlike DMD, efficient unknown compensatory mechanisms counteract the degeneration and thus maintain the regenerative process, restoring the constant mechanical damage. The number of revertant fibers is low, usually present in about 1% of all fibers, but their number increases with the age of the mice (Lu et al., J. Cell. Biol, 2000, vol. 148, pp. 985-995). Exon phasing (ph According to Asing, skipping of exon 23 during mRNA splicing allows translation of a truncated dystrophin.

[0433] Oligonucleotide synthesis: Oligonucleotides for in vivo experiments were synthesized on a 260 μmol scale using an Aekta Oligo Pilot 10 synthesizer and a NittoPhase® UnyLinker™ 200, universal solid support. In the case of compositions of the invention containing one or more lipid moieties and / or said spacers at the 3'-end, the corresponding modified, typically and preferably Fmoc-protected phosphoramidites were introduced in the first cycle of the synthesis. A modified synthesis cycle was used, typically using 2.2 equivalents of phosphoramidite and a coupling time of 4 minutes. In the case of compositions of the invention containing one or more lipid moieties and / or spacers at the 5'-end, the corresponding modified phosphoramidites were used in the last cycle. To remove the Fmoc and cyanoethyl protecting groups, the solid support was treated with 20% diethylamine in MeCN.

[0434] Oligonucleotides for in vitro experiments were synthesized on a 1 μmol scale using the Expedite Nucleic Acids Synthesis System and the universal solid support High Load Glen UnySupport™. In the case of compositions of the invention containing said lipid moieties and / or spacers at the 3'-end, the corresponding modified phosphoramidite was introduced in the first cycle of synthesis. In the case of compositions of the invention containing said biotin moieties and / or spacers at the 3'-end, the corresponding biotin phosphoramidite was introduced in the first cycle of synthesis, followed by a C4 spacer phosphoramidite in the second cycle of synthesis, and a corresponding, typically and preferably Fmoc-protected spacer phosphoramidite in the third cycle of synthesis. A modified synthesis cycle was used, typically using 9 equivalents of phosphoramidite and a coupling time of 3.5 minutes. In the case of compositions of the invention containing said one or more lipid moieties and / or spacers at the 5'-end, the corresponding modified phosphoramidite was used in the last cycle. To remove the Fmoc and cyanoethyl protecting groups, the solid support was treated with 20% diethylamine in MeCN.

[0435] Deprotection of solid supported oligonucleotides with saturated NH 4 This was carried out by heating with EtOH at 65° C. for 3 h. The mixture was then cooled to ambient temperature and 4M NaOH and MeOH were added so that the final composition of the mixture was 0.4M NaOH and 60% MeOH, and treatment was continued for an additional 2.5 h at ambient temperature. The solid was filtered off and diluted with EtOH / H 2 Wash with 1:1 H2O and 2M NaHCO 2 PO 4 The filtrate was neutralized by the addition of 100 mM NaCl. The resulting solution of crude oligonucleotide was desalted against water using TFF and lyophilized. For compositions of the invention containing one or more -COOH functional groups, previously described conditions (Surzhikov et al., Nucleic Acids Res., 2000, vol. 28, p. e29) were used.

[0436] Oligonucleotides for in vivo experiments were purified by isocratic RP HPLC (Waters x Bridge prep C18, 5 μm, 10 x 150 mm) using 42% MeCN in ammonium acetate buffer (pH 7) at a flow rate of 4 mL / min at 75°C. Fractions containing sufficiently pure product (>70% FLP) were combined and partially lyophilized. Approximately 20 equivalents of NaCl were added to the solution, which was subsequently desalted against water using TFF.

[0437] Oligonucleotides for in vitro experiments were purified by isocratic RP HPLC (Waters x Bridge prep C18, 5 μm, 4.6 x 150 mm) using approximately 30% iPrOH in ammonium bicarbonate buffer (pH 7) at a flow rate of 1 mL / min at 60° C. Fractions containing products of sufficient purity were combined and subsequently desalted using SEC (GE Healthcare, HiPrep 26 / 10) with water as eluent.

[0438] Linking lipid moieties to oligomeric compounds: According to the present invention, there are several approaches to link said lipid moiety or moieties to oligonucleotides, either directly on a solid support or in solution. These approaches follow classical conjugation chemistry well known to those skilled in the art and widely described in the literature (Bioconjugate Techniques, Greg T. Hermanson, Pierce Biotechnology; Singh, Chem. Soc. Rev., 2013). 10, 39, 2054-2070; H. Rosemeyer, Chem. Biodiversity, 2005, 2, 977-1062). As indicated, in the case of the compositions of the invention comprising said one or more lipid moieties and / or spacers at the 3'- or 5'-terminus, the corresponding modified, typically and preferably Fmoc- or MMTr-protected phosphoramidites were introduced in the first or last cycle of the synthesis.

[0439] Formula (I)AB- * (Wherein, the asterisk ( * Typical and preferred linking procedures for linking said one or more lipid moieties of (wherein, represents said point of covalent attachment to said oligomeric compound or said spacer according to the present invention) include, but are not limited to, the following: i. When B is preferably OP(O)(OH), OP(O)(SH), more preferably OP(O)(SH), the 5' end of the oligomeric compound is linked to the oligonucleotide, respectively, according to the following reaction scheme: 5'-HO-OLIGO-3'+AOP(O-PG)-X → 5'-AOP(O-PG)-O-OLIGO-3'+HX P(III) is subsequently oxidized to P(V), where X is a suitable leaving group and PG is a suitable protecting group; ii. When B is preferably NH-P(O)(OH), the 5' end of the oligomeric compound is linked to the oligonucleotide via phosphoramidate chemistry, respectively, according to the following reaction scheme: 5'-HO-OLIGO-3' → 5'-H-(O-PG)(O)PO-OLIGO-3' 5'-H-(O-PG)(O)PO-OLIGO-3' + H 2 NA → 5'-A-NH-(O-PG)(O)PO-OLIGO-3' (where PG is a suitable protecting group); iii. When B is preferably NH-P(S)(OH), the 5' end of the oligomeric compound is linked to the oligonucleotide via phosphoramidate chemistry, respectively, according to the following reaction scheme: 5'-HO-OLIGO-3' → 5'-XP(O-PG)-O-OLIGO-3' 5'-XP(O-PG)-O-OLIGO-3' + H 2 NA → 5'-A-NH-P(O)(O-PG)-O-OLIGO-3' P(III) is then oxidized to P(V) using a suitable sulfur transfer reagent, where PG is a suitable protecting group; iv. When B is preferably NH-C(O), the 5' end of the oligomeric compound is linked to the oligonucleotide via carbamate chemistry, respectively, according to the following reaction scheme: 5'-HO-OLIGO-3' → 5'-XC(O)-O-OLIGO-3' 5'-XC(O)-O-OLIGO-3' + H 2 NA → 5'-A-NH-C(O)-O-OLIGO-3' + HX (wherein X is a suitable leaving group); or 5'-HO-OLIGO-3' + OCN-A → 5'-A-NH-C(O)-O-OLIGO-3'.

[0440] The ligation procedures described above are also applicable for ligation to the 3' ends of oligomeric compounds and oligonucleotides, typically during reverse 5' to 3' oligomeric compound and oligonucleotide synthesis, respectively.

[0441] As an example: for the synthesis of preferred compositions of the invention such as SY-0457, SY-0458, SY-0459, and SY-0460, synthesis was performed on solid support utilizing 10 equivalents of NHS-ester in DMSO in the presence of DIPEA (15 equivalents) or in 1:1 DMSO / buffer (0.1M NaHCO) at 37° C. 3 The lipid moieties were conjugated by reaction of the amino-modified oligonucleotides with the corresponding NHS-ester in solution utilizing 3×10 equivalents of NHS-ester in 10 mL of 10% HO (pH 8.5).

[0442] As a further example: an oligomeric compound of SEQ ID NO: 1 (having a complete PO internucleoside linkage group) may be modified by adding -NH-C 6 Alkylene-OP(O)(SH)- or -NH-C 6 For the synthesis of preferred compositions of the invention such as SY-0299, SY-0343, SY-0442, and SY-0455, which contain a palmitoyl residue as one lipid moiety linked by either an alkylene-OP(O)(OH)-spacer, 5'-Palmitate-CE-Phosphoramidite (Link, P / N 2199) was used as the phosphoramidite in the last cycle. The moiety attached to the 3' end of SY-0343 and SY-0299, i.e. -P(O)(SH)O-CH 2 CH-(CH 2 OH)-(CH 2 ) 4 NH 2 The moiety was introduced in the first cycle of the synthesis by coupling of the corresponding Fmoc-protected phosphoramidite to the 3'-terminal OH group of the first nucleotide.

[0443] Certain preferred compositions of the present invention and compositions used in the experimental section are specifically characterized in Table 3 with further reference to their respective names and abbreviations typically used throughout the specification.

[0444] Analysis of oligonucleotides by RP-HPLC-DAD-MS: The following parameters were used for analytical HPLC. A C18 column with 1.7 μm particle size was used. The column temperature was set at 75 °C. Mobile phase A was 400 mM hexafluoroisopropanol (HFIP) and 15 mM triethylamine + 10% methanol. Mobile phase B was methanol. A gradient of 32-52% mobile phase B was applied. The flow rate was set at 0.25 mL / min. Oligonucleotides were detected using a UV photometer at 260 nm and a time-of-flight mass spectrometer.

[0445] Detection of multimers: Polyacrylamide gel electrophoresis (PAGE) experiments were performed to detect self-multimers. The following chemicals were used: Tris(hydroxymethyl)aminomethane (Tris), (TCI A0321); acetic acid (Merck 1.00063); acrylamide / Bis solution, 29:1 (40%, Serva 10680. 01); tetramethylethylenediamine (TEMED, Sigma-Aldrich T9281); ammonium persulfate (Sigma-Aldrich 248614); and glycerol (Sigma-Aldrich G9012). Buffer A was prepared by dissolving 60 g of Tris in 200 mL of water. Adjust the pH to 7.4 with glacial acetic acid (approximately 25-30 mL). Dilute the solution to 500 mL with water and store at +4 °C. Buffer B was prepared by diluting 16 mL of buffer A to 800 mL with water. Ammonium persulfate 10% (w / v) was prepared by dissolving 100 mg of ammonium persulfate in 0.9 mL of water. Preparation of the gel was performed by mixing the following solutions in a glass beaker: 9.4 mL of acrylamide / bis solution; 15 mL of water; 500 μL of buffer A; 125 μL of 10% (w / v) ammonium persulfate, and 38 μL of TEMED. Test solutions were 1 mg / mL in 10% glycerol and 10 μL was applied (corresponding to 10 μg of oligonucleotide). Pre-migration settings were 40 min / 90 V in Buffer B. Migration settings were 45-60 min / 120 V in Buffer B in addition to 90 min / 90 V or 15 min / 90 V. 5-7 μL of 6× DNA loading dye was also transferred. Detection was performed by placing the gel on a TLC plate and examining under UV light at 254 nm. Staining with Stains-All (Sigma-Aldrich, 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide, 3,3′-diethyl-9-methyl-4,5,4′,5′-dibenzothiacarbocyanine) was then performed according to the manufacturer's protocol.

[0446] Isolation of proteins interacting with oligonucleotides: Isolation of blood proteins was performed using streptavidin beads (High Capacity Streptavidin Agarose). Biotinylated oligonucleotides immobilized on Streptavidin Agarose (Pierce) The assay was carried out using a ELISA kit. Biotinylated oligonucleotides were immobilized on the beads (10 μl of beads for 20 μg of oligonucleotide) for 30 min, and excess oligonucleotides were removed by washing three times with phosphate buffered saline (PBS). The beads thus prepared were incubated with 5–50 μl of mouse or human serum for 1 h, and then unbound proteins were removed by four successive washes in PBS. After the last wash, precipitated proteins were either dissolved in Laemmli sample buffer for further analysis by SDS-PAGE or directly digested with trypsin for analysis by mass spectrometry on an ORBI-TRAP instrument.

[0447] Commercially available human serum (SIGMA, St. Louis, MO) or serum samples from healthy human adults obtained according to regulatory guidelines were used in the experiments. Blood samples from C57BL / 6 mice were collected from the tail vein or retro-orbital plexus. Animals were All procedures, including those described herein, were performed in accordance with the guidelines of the Animal Ethics Committee of our institution.

[0448] Identification of captured proteins by mass spectrometry using OrbiTrap technology: For analysis by mass spectrometry, precipitated proteins were directly digested with trypsin in a buffer containing 100 mM ammonium carbonate pH 8.0 and 500 ng trypsin (Sequence Grade Trypsin, Promega) for 16 h at 37 °C and stored at -20 °C until use. The peptide mixture was desalted using ZipTip μ-C18 pipette tips (Millipore) and separated on an Easy nano-LC Proxeon system (Thermo Fisher Scientific) equipped with a reversed-phase C18 column (Easy-Column Proxeon C18, L 15 cm, ID 75 μm). The eluate was monitored on an LTQ VelosOrbitrap mass spectrometer (Thermo Fisher Scientific) and tandem MS (MS / MS) data were processed by Proteome Discoverer 1.4 software (Thermo Fisher Scientific) coupled with an in-house Mascot search server (Matrix Science, version 2.3.2) using the SwissProt database as previously described (Rouillon, 2015). The relative abundance of each identified protein was estimated by label-free quantification using Progenesis LC MS software (Nonlinear Dynamics, version 4.0). The average normalized abundance (ANA), which reflects the relative amount of the protein in the Progenesis analysis, was used to compare the amount of protein bound to the vector.

[0449] Protein identification after SDS-PAGE separation: Protein identification was performed as previously described (Denard, Proteomics, 2009, vol. 9, pp. 3666-3676). SDS-PAGE (4-12% gradient, NuPAGE Novex Bis-Tris Gel After separation of isolated proteins with 1.0 mm gel (Life Technologies), the gel was visualized by staining with Coomassie Blue (InstantBlue Protein Stain, Expedion) and bands of interest were sliced ​​for further analysis. Gel slices were washed with 1 ml of 96% CH 2 After washing in CHOH for 10 min, the alcohol was carefully removed and replaced with 20 μl of a solution containing 100 mM ammonium carbonate pH 8.0 and 100 ng trypsin (Promega). Samples were digested for 16 h at 37° C. and stored at −20° C. until use. Peptide mixtures were desalted using ZipTip μ-C18 pipette tips (Millipore) and deposited on MALDI plaques with α-cyano-4-hydroxycinnamic acid (HCCA) (5 μg) matrix in 80% acetonitrile, 0.1% formic acid.

[0450] MALDI-TOF MS analysis was performed on a MALDI TOF / TOF ABI 4800+ (AB Sciex). All spectra were acquired in positive reflector mode. The resulting mass list was searched using an in-house Mascot search server (Matrix Science, version 2.3.2 213) and the SwissProt database human / mouse, one remaining cleavage site considering partial oxidation of methionine, and a mass tolerance setting of 50 ppm.

[0451] Systemic administration of AONs: Animal procedures were carried out in accordance with national and European legislation and approved by the French government (Ministère de l'Enseignement Superieur The study was approved by the FASB, the National Institute of Health and Human Services (NHI), and the Autorisation APAFiS #6518 (FHSS). Mdx (C57BL / 10ScSc-Dmdmdx / J) and C57BL / 10 mice were bred in our animal facility at the Platform 2Care, UFR des Sciences de la sante, Universite de Versailles Saint Quentin, and maintained on a standard 12-h light / dark cycle with free access to food and water. Mice were weaned at 4–5 weeks postnatal age and housed 2–5 individuals per cage.

[0452] Six- to eight-week-old mdx mice were intravenously injected with different AONs (15mer or 13mer, PO or PS, conjugated or unconjugated) into the retroorbital sinus under general anesthesia using 1.5–2% isoflurane once a week for periods ranging from 4 to 12 weeks. Age-matched C57 / BL10 (WT) and mdx groups that received equivalent volumes of sterile saline were included as controls. One hour after the first injection, blood samples were collected from all mice to measure complement C and cytokine / chemokine levels. Additional blood samples were collected one week after the sixth injection (middle of treatment) and one week after the end of treatment. Mice were then placed in metabolic cages for urine collection over a 24-h period. Animals were sacrificed two weeks after the last injection. Muscles and tissues were harvested, flash frozen in liquid nitrogen-cooled isopentane, and stored at -80 °C until further analysis. Sample sizes and n values ​​are indicated in the legends of each figure. Investigators were blinded to the RNA and protein analyses.

[0453] Serum and urine analysis: Blood samples were collected from tail bleeding under general anesthesia. Analysis of serum creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, creatinine, urea, and albumin levels was performed by the pathology laboratory at the Mary Lyon Centre, Medical Research Council, Harwell, Oxfordshire, UK. Cytokine and chemokine levels in serum were analyzed by multiplex assays using Luminex® technology. Bio-Plex Pro Mouse Cytokine 10-Plex Immunoassay Panel (Bio-Rad, Hercules, CA) was used to detect levels of IL-1β, IL-6, IL-10, IL-12p70, IL-13, IL-17, IFN-γ, MCP-1, RANTES, and TNF-α, according to the manufacturer's instructions. Immunoassays were read using a Bio-Plex MAGPIX Multiplex reader, and results were analyzed with Bio-Plex manager 6.1 software (Bio-Rad, France).

[0454] Urine was collected directly in refrigerated tubes (4°C) using metabolic cages over a 24-h period. After collection, urine was centrifuged at 10,000 × g for 10 min, and the supernatant was aliquoted and frozen at -80°C for further analysis. Urinary creatinine was measured using a creatinine assay kit (R&D Systems, Inc, Minneapolis, MN) according to the manufacturer's instructions. Total protein in urine samples was measured as previously described (Swayze et al., Nucleic Acids Res., 2007, 35, 687-700). Briefly, protein was determined by centrifugation of 40 μL of dHO. 2 Urine samples were precipitated by adding 200 μL of dHO and 200 μL of pre-chilled acetone to 10 μL of urine. The samples were then incubated at -20°C for 30 min and then centrifuged at 14,000 × g for 15 min at 4°C. The pellet was resuspended in 40 μl of dHO and washed with 100 μL of acetone. 2Resuspend in 0 and run on Pierce BCA Assay (Thermo Protein concentrations were measured using a ELISA kit (Bethy Laboratories, Rockford, IL). Albumin from urine samples was measured using an Albumin ELISA kit (Bethy Laboratories, Montgomery, TX) according to the manufacturer's instructions. Levels of acute kidney injury (AKI) biomarkers were analyzed by multiplex assays using Luminex® technology. Levels of beta-2-microglobulin (B2M), renin, kidney injury molecule 1 (KIM-1), interferon gamma-inducible protein 10 (IP-10), vascular endothelial growth factor (VEGF), cystatin C, epidermal growth factor (EGF), lipocalin-2-NGAL, clusterin, and osteopontin (OPN) were measured using a multiplex kidney injury panel (MKI1MAG-94K, MKI2MAG-94K, Merck-Millipore) according to the manufacturer's instructions. Results were read using a Bio-Plex MAGPIX Multiplex reader and analyzed with Bio-Plex manager 6.1 software (Bio-Rad, France).

[0455] Assay of complement activation: Complement activation in mouse serum samples was measured by Microvue PanSpecific-C3 Converter and SC5b-9 Plus Kit (Quidel Co., San Diego CA, USA). Briefly, mouse C3 protein was converted to human SC5b9 using C3 converter reagent (Pan specific C3 reagent kit, Microvue, Quidel), and then detected by SC5b9 Elisa (Quidel).

[0456] For in vitro complement activation studies, AON molecules were incubated with pooled normal human serum (1:10) (Seralab, UK) for 45 min at 37°C. Complement activation determination was performed using the Human SC5b-9 Plus kit (Quidel Co., San Jose, CA). Complement activation was assessed using 5 mg / ml zymosan (Complement Technology, Inc., San Diego CA, USA). 5 mg / ml zymosan (Complement Technology, Inc., Texas, USA) was used as a positive control. Complement activation was expressed as a percentage of the remaining level of C3 in the sample, considering the level of C3 in PBS condition as 100% of C3 (no activation).

[0457] Clotting assays: Mouse blood samples were collected into tubes containing 3.2% sodium citrate. Whole blood samples were centrifuged at 2,500 × g for 15 min, after which plasma was immediately separated and stored at -80 °C until use. After incubating 2 mg / mL tcDNA-AON with 50 μL of citrated plasma C for 30 min at 37 °C, prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays were performed on a semi-automated STARTmax coagulometer (Stago) according to the manufacturer's instructions.

[0458] RNA analysis: Total RNA was isolated from interstitial muscle sections collected during cryosectioning using TRIzol reagent according to the manufacturer's instructions (ThermoFisher Scientific, USA). An aliquot of 500 ng of total RNA was used for RT-PCR analysis using the Access RT-PCR System (Promega, USA) in a 50 μL reaction using the following external primers: Ex 20Fo (5'-CAGAATTCTGCCAATTGCTGAG-3' - SEQ ID NO: 38) and Ex 26Ro (5'-TTCTTCAGCTTGTGTCATCC-3' - SEQ ID NO: 39).

[0459] cDNA synthesis was carried out at 45° C. for 45 min, followed directly by primary PCR with 30 cycles of 95° C. (30 s), 55° C. (1 min), and 72° C. (2 min). 2 μL of these reactions were then reamplified in a nested PCR with 22 cycles of 95° C. (30 s), 55° C. (1 min), and 72° C. (2 min) using the following internal primers: Ex 20Fi (5'-CCCAGTCTACCACCCTATCAGAGC-3' - SEQ ID NO: 40) and Ex 26Ri (5'-CCTGCCTTTAAGGCTTCCTT-3' - SEQ ID NO: 41).

[0460] PCR products were analyzed on 2% agarose gels. Exon 23 skipping was also measured by Taqman quantitative RT-PCR as previously described (Goyenvalle et al., Hum. Mol. Genet. 2012, 21:2559-2571; Straub et al., Lancet Neurol. 2016, 15:882-890).

[0461] We also isolated total RNA from renal cortex samples as previously described (Frazier et al., Toxicol. Pathol., 2014, 42, 923-935) and performed quantitative RT-PCR for the following kidney injury biomarker (KIB) genes: interferon gamma (IFNg), interleukin 6 (IL6), granzyme B (Gzmb), interferon gamma-inducible protein 10 (IP-10), tumor necrosis factor (TNF), chemokine ligand 2 (Ccl2), chemokine ligand 3 (Ccl3), beta-2-microglobulin (B2M), kidney injury molecule 1 (KIM-1), renin 1 (Ren1), and epidermal growth factor (EGF).

[0462] Statistical analysis: Data were analyzed with GraphPad Prism5 software (San Diego, California, USA) and are presented as mean ± SEM. "n" refers to the number of mice per group. Comparisons of statistical significance were assessed by the non-parametric Mann-Whitney U test. Significance levels were: * P < 0.05, ** P < 0.01, *** P was set at <0.001.

[0463] As described herein, the oligomeric compound contained in the composition of the present invention is, in a preferred embodiment, an antisense oligonucleotide (AON) designed to be complementary to specific mRNA or pre-mRNA.This preferred class of compounds of the present invention can be used to treat many diseases.The exemplary diseases provided below do not limit the present invention, and the compositions of the present invention provided can be used to treat any disease that can be treated by administering AON.

[0464] Example 1 Compositions of the Invention for the Treatment of Duchenne Muscular Dystrophy Efficacy assessment Adult mdx mice were treated weekly for 4 weeks with intravenous injections of various 13-mer AONs targeting the donor splice site (M23D:+2-11) of exon 23 of the dystrophin pre-mRNA, namely SY-0308, SY-0210, and SY-0299, SY-0343, SY-0442, and SY-0455 of the present invention. SY-0308 (also referred to interchangeably herein as "tcDNA-PO M23D") corresponds to p-CCTCGGCTTACCT-OH of SEQ ID NO: 1, where all nucleotides are tc-DNA, all internucleoside linkages are phosphorodiester linkages, and p is the phosphate moiety at the 5' terminus. SY-0210 (also referred to herein interchangeably as "tcDNA-PS M23D") corresponds to p-CCTCGGCTTACCT-OH of SEQ ID NO:1, where all nucleotides are tc-DNA, all internucleoside linkages are phosphorothioate linkages, and p is the 5'-terminal phosphate moiety. Composition SY-0343 of the present invention is referred to herein interchangeably as "Palm-2PS-tcDNA-PO M23D" and is shown below: [ka]

[0465] Composition SY-0442 of the present invention is referred to interchangeably herein as "Palm-1PS-tcDNA-PO M23D" and is shown below: [ka]

[0466] Composition SY-0299 of the present invention is referred to interchangeably herein as "Palm-2PO-tcDNA-PO M23D" and is shown below: [ka]

[0467] Composition SY-0455 of the present invention is referred to interchangeably herein as "Palm-1PO-tcDNA / 2OMe-PO M23D" and is shown below: [ka]

[0468] SY-0308, SY-0210, SY-0299, SY-0442 and SY-0455 were used at a dose of 200 mg / kg body weight / week, and SY-0343 was used at a dose of 178 mg / kg body weight / week. Two weeks after the last injection, muscles were harvested and RNA samples were analyzed by quantitative RT-PCR to determine the level of exon 23 skipping of the dystrophin gene (Figure 1), and the level of restored dystrophin was quantified by Western blot using the licor Odyssey system (Figure 5). Figure 1 shows that the level of skipping was always consistently higher with palmitoyl-conjugated AONs than with tcDNA-PO M23D. The level of exon 23 skipping was higher in most tissues from mice treated with palmitoyl-conjugated AONs compared to those treated with tcDNA-PS M23D AONs, with the most notable differences in the heart, cortex, and cerebellum. The percentage of dystrophin mRNA with exon 23 skipping exceeded the value of 15% in some skeletal muscles and in the heart. For the heart, the fold changes of SY-0299, SY-0343, SY-0442, and SY-0455 relative to SY-0308 or SY-0210 ranged from 4.4 to 9.2 and 4.0 to 8.5, respectively. Importantly, skipping was clearly detected in the cortex and cerebellum immediately after 4 weeks of treatment with palmitoyl-conjugated AONs, but not yet with the other two compounds.

[0469] Thus, palmitoyl-conjugated tcDNA AONs, in which the internucleoside linkage group is PO, are more efficient than the naked tcDNA equivalent AONs, with or without PS bonds.

[0470] This example shows that systemic delivery of a composition of the invention comprising an antisense oligomer linked to the 5'-palmitoyl-C6-amino lipid moiety and a spacer and having an intact PO backbone (palmitoyl-conjugated PO M23D) allowed greater rescue of dystrophin compared to an equivalent sequence made of tcDNA having either a PO or PS backbone.

[0471] Toxicity assessment No significant clinical signs were observed during the course of experiments with three AONs targeting the (M23D:+2-11) region of dystrophin in mice. However, acute adverse effects have been observed with other sequences of interest, such as (H51:+67+81 - AAGATGGCATTTCTA - SEQ ID NO: 19), targeting exon 51 of human dystrophin, herein referred to as SYN51. Thus, SY-0206, also referred to interchangeably herein as "tcDNA-PS SYN51" and corresponding to p-AAGATGGCATTTCT-OH of SEQ ID NO: 19, in which all nucleotides are tc-DNA, all internucleoside linkages are phosphorothioate linkages, and p is the phosphate moiety at the 5' end, threatens further clinical development. Historically, PO internucleoside linkages were replaced with PS internucleoside linkages to stabilize oligomers and prevent their degradation. Although modification and replacement of PO with PS modifications is not a priori necessary because tcDNA oligonucleotides are highly stable, nevertheless, such modifications significantly increased the tcDNA-PS biodistribution after systemic delivery and allowed a better and more extensive skipping effect compared to tcDNA-PO, as shown in Figure 1. This phenomenon was likely due to the increased interaction of PS residues with serum proteins.

[0472] One of the major acute effects of toxic tcDNA-PS AONs was that they caused blood coagulation, thrombosis, and complement activation.

[0473] Here, we used two complementary blood clotting tests, prothrombin time (PT) and partial thromboplastin time (aPTT), to check for potential bleeding problems caused by various tcDNA AONs, namely, SY-0308 (tcDNA-PO M23D), SY-0210 (tcDNA-PS M23D), SY-0343 (Palm-2PS-tcDNA-PO M23D), SY-0442 (Palm-1PS-tcDNA-PO M23D), SY-0455 (Palm-1PO-tcDNA / 2OMe-PO M23D), SY-0206 (tcDNA-PS SYN51), and SY-0252. SY-0252, also referred to interchangeably herein as "tcDNA-PO SYN51," corresponds to p-AGATGGCATTTCT-OH of SEQ ID NO: 19, where all nucleotides are tc-DNA, all internucleoside linkages are phosphorodiester linkages, and p is the 5'-terminal phosphate moiety. SYN51 is known to be a highly toxic sequence to mice in the presence of PS linkages.

[0474] Figure 2 shows that the PT and aPTT profiles were very similar in mouse and human plasma. In both cases, tcDNAs containing PO bonds (tcDNA-PO M23D as well as tcDNA-PO SYN51 and palmitoyl-conjugated AONs) did not cause significant changes in the prothrombin time (PT), whereas tcDNAs containing PS bonds, especially tcDNA-PS SYN51, increased the PT and always caused acute toxicity in mice after intravenous injection. Moreover, tcDNA-PS AONs also strongly increased the aPTT, especially in human plasma, as did tcDNA-POs with the SYN51 sequence (Figure 2D). Considering the clinical signs caused by the various AONs, it became clear that the PT test reflected the magnitude of acute adverse effects, at least in mice. All tcDNA-PO AONs were safe in mice and did not increase the PT. On the other hand, tcDNA-PS M23D caused only a slight increase in PT and toxicity was never mentioned, whereas tcDNA-PS SYN51 caused a strong increase in PT and was highly toxic.

[0475] Figure 8 shows that PT and aPTT decrease proportionally with the reduction of the PS linkages in the backbone of the oligonucleotide. Here, we used tc-DNA M23D sequences with various amounts of internucleoside PS linkages, and thus M23D-PS 25sb3 is (p-CCTCGGCTTA * C * C * T - corresponding to sequence number 42) and M23D-PS 33sb3 is (p-CCTCGGCTT * A * C * C * T - SEQ ID NO: 43), and M23D-PS 50sb3 corresponds to (p-CCTCGGC * T * T * A * C * C * T - SEQ ID NO: 44), and M23D-PS 66sb3 corresponds to (p-CCTCG * G * C * T * T * A * C * C * T - SEQ ID NO: 45), and M23D-PS 83sb3 corresponds to (p-CCT * C * G * G * C * T * T * A * C * C * T - SEQ ID NO: 46), where " * " is phosphorothioate indicates the internucleoside linkage, and p represents the terminal phosphate group. As shown so far, the aPTT test is more sensitive to the PS content than to the PT, reaching its maximum measurement time with six internucleoside PS (compound M23D-PS 50sb3). A lower number of PS linkages induces only a very limited increase in PT and aPTT.

[0476] Complement activation was also examined in mice following systemic injection of AONs at a high dose of 200 mg / kg.

[0477] Figure 3 shows that safe AONs containing phosphorodiester linkages as internucleoside linkages, specifically tcDNA-PO M23D, tc-DNA-PS M23D, tcDNA-PO SYN51, and palmitoyl-conjugated AONs of the present invention (SY-0299, SY-0343, SY-0442, and SY-0455), did not significantly activate the complement cascade. Only toxic tcDNA-PS SYN51 clearly activated the cleavage of C3 (50%). Note that tcDNA-PS M23D, which was considered safe, still slightly activated the consumption of C3.

[0478] Serum was collected at the end of 4 weeks of treatment with compounds SY-0299, SY-0343, SY-0442, and SY-0455, and serum biochemistry was evaluated (Figure 4). There were no significant changes in serum albumin, creatinine, and urea, suggesting a lack of nephrotoxicity. Only a slight increase in ALP for most palmitoyl-conjugated PO M23D was observed (Figure 4E).

[0479] Thus, as shown, systemic delivery of the inventive composition (palmitoyl-conjugated PO M23D, SY-0343) comprising an antisense oligomer linked to the 5'-palmitoyl-C6-amino lipid moiety and spacer and having a complete PO backbone allowed greater rescue of dystrophin compared to the equivalent sequence made of tcDNA with either a PO or PS backbone. Furthermore, the inventive composition (SY-0299, SY-0343, SY-0442, and SY-0455) and AON, respectively, do not cause adverse consequences in the blood compartment after systemic delivery. Furthermore, the lack of sulfur in the phosphate backbone of AONs significantly reduced non-specific binding of serum proteins, thus significantly reducing the risk of adverse effects of complement activation and / or coagulation. The compositions of the present invention, including at least one lipid moiety, such as palmitoyl-conjugated tcDNA AONs with an internucleoside linking group that is PO, have conferred new properties to PO-tcDNA containing molecules that are now believed to preferentially bind to serum proteins such as albumin and lipoproteins, which are natural carriers of poorly water-soluble molecules that contain fatty acids. Furthermore, and in summary, the above results also indicate that tcDNA AONs that modify coagulation properties also activate the complement cascade systemically in vivo. A significant increase in PTT values ​​(>180 seconds) is a sign of potential risk, which becomes reality when PT values ​​are also dramatically increased (>100 seconds). Finally, tcDNA-PO and the compositions of the present invention Palm-tcDNA-PO are much safer than their related tcDNA-PS. Example 2 Coprecipitation experiments and proteomics

[0480] The oligonucleotides of interest were synthesized as biotinylated conjugates for subsequent co-precipitation of potentially interacting serum proteins by using streptavidin beads. The biotinylated derivatives used in this study were SY-0440, SY-0427, SY-0446, SY-0448, SY-0445, SY-0443, and SY-0451, which are defined and characterized in Table 3 and shown in Figure 6.

[0481] Biotinylated oligonucleotides immobilized on streptavidin beads were incubated in the presence of serum for 1 h. Beads were collected by low-speed centrifugation, washed, and then solubilized in appropriate buffers for further protein analysis by SDS-PAGE, Orbitrap LC-MS / MS, and SDS-PAGE / MALDI-TOF.

[0482] Figure 7 illustrates SDS-PAGE analysis of protein recovery from mouse and human serum for the oligonucleotides used in this study. SY-0440, the tcDNA M23D with an intact PS, appeared to retain much more serum protein than its tcDNA-PO counterpart, SY-0427. This result is consistent with previous studies (not shown) using a variety of sequences (M23D, SYN51, and Poly T) and backbone chemistries (DNA, tcDNA, 2'OMe-PS, and PMO), which showed that oligomers lacking PS linkages did not bind significantly to serum proteins, suggesting that protein adsorption occurs preferentially at the level of the thiophosphate backbone and does not involve consensus sequence motifs.

[0483] In general, we found that tcDNA-PS oligonucleotides bind to many proteins related to the complement pathway and coagulation. Orbitrap analysis revealed that tcDNA-PS oligonucleotides retain mannose-binding protein (MBL), which is beneficial for innate immunity via the lectin pathway. MBL binds to MASPs (MBL-associated serine proteases - MASP-1;-2;-3), which also form complexes with ficolins. When MBL binds to its target (e.g., mannose on the surface), the MASP proteins function to cleave the blood protein C4 into C4a and C4b. The C4b fragment can then bind to surfaces and initiate the formation of the C3 convertase. The MBL / MASP-1 complex also has thrombin-like activity (thrombin clots fibrin to initiate blood clotting). All of these proteins were found in tcDNA-PS samples, but not in complexes with oligonucleotides lacking the PS internucleotide linkage.

[0484] As shown in Figure 7, the tcDNA-palmitoyl conjugates of the present invention showed comparable patterns of protein recovery, which were very different from those obtained with naked tcDNA-PS and tcDNA-PO. Considering the fact that tcDNA-PO was not consistently and significantly bound to serum proteins, the patterns obtained for A, B, C, A', and D molecules could be largely contributed by their palmitoyl and alkyl phosphate moieties. SDS-PAGE MALDI-ToF MS analysis of the major proteins recovered from mouse and human serum showed that the main proteins were albumin and lipoprotein components of LDL (low density lipoprotein) and HDL (high density lipoprotein) complexes, all involved in lipid transport (Tables 4 and 5).

[0485] [Table 4]

[0486] [Table 5]

[0487] At first glance, the C7 part at the 3' end of the tcDNA sequence does not contribute significantly to serum protein binding (compare A(SY-0446) with B(SY-0445)), but B retained more dimeric albumin in mice. Comparison of B with C(SY-0443) showed that the presence of thiophosphates at both ends of the tcDNA sequence did not enhance protein binding, quite the opposite, showing a slight decrease in some proteins in human serum (fragments of factor H and complement C3). The A'(SY-0448) molecule was similar to A, except that in A' the third nucleotide of the sequence was replaced by 2'OMe-U. The A / A' profile was almost identical in mouse conditions, but with some differences in human serum, in particular A' retained less factor H and complement C3, possibly suggesting a better safety profile. Finally, the D molecule (SY-0451), lacking both C7 and C6, retained slightly less albumin and HDL components compared to A and A', leaving room for additional proteins as indicated by the increased intensity of previously marginal bands.

[0488] The C7 motif was introduced first to allow the grafting of ligands for further tissue-specific targeting. Although our studies showed that the C7 moiety does not affect serum protein retention, it may still be advantageous to keep it at the 3' end of the AON to expand the size of the compound and thereby improve steric hindrance at the level of its pre-mRNA target. On the other side of the compound, moving the palmitoyl moiety away from the 5' end of the tcDNA sequence by C6 seems to increase preferential binding to albumin and lipoprotein complexes involved in fatty acid transport. Example 3 Stability of the compositions of the present invention in human serum

[0489] The stability of preferred compositions of the invention in human serum was studied with oligonucleotides SY-0343, SY-0442, SY-0299, SY-0450, SY-0455, and SY-0357. The compositions of the invention are defined and characterized in Table 3.

[0490] A mixture (80 μL total volume) consisting of 40 μL human serum, 20 μL oligonucleotide stock solution (2x in PBS), and milliQ water was prepared in a 0.2 mL PCR vial to a final oligonucleotide concentration of 2 μM and incubated at 37° C. Aliquots were removed after 4, 24, and 120 h. The mixture was digested in a PCR vial with proteinase K (80 μL of the mixture, 100 μL of proteinase K buffer 2x (200 mM Tris-HCl, pH 8.5, 400 mM NaCl, 10 mM EDTA, 0.4% SDS), 20 μL of proteinase K (20 mg / mL in 100 mM Tris-HCl pH 8.5, 10 mM CaCl2) for 2 h at 55 °C. The mixture was then cooled to room temperature, centrifuged (13400 rpm, 10 min), and the supernatant was collected. An aliquot of 50 μL was then taken for SPE and diluted with 50 μL of 0.2 M TEAB buffer. After 30 min, 50 μL of Condi solution (15 mM Et3N, 100 mM HFIP) was added. An SPE column (Oasis HLB 1 cc (10 mg) extraction cartridge, Waters 186000383) was conditioned by successively passing 1 mL MeOH and 2 × 500 μL Condi solution. The above test solution was quantitatively applied to the SPE column, and the column was successively washed by passing 2 × 250 μL Condi solution and 300 μL TEAB (0.2 M) solution. The oligonucleotide was washed by applying 2 × 200 μL eluent (Condi solution / MeOH 4:6) and 150 μL MeOH, and the eluate was collected. The eluate was lyophilized to dryness in a speed-vac and then diluted with 100 μL milliQ water. The mixture was then analyzed by HPLC-DAD-MS (Waters Acquity OST C18, 100 × 2.1 mm, 1.7 μm, mobile phase A: 400 mM HFIP, 15 mM TEA + The analysis was performed using 10% methanol, mobile phase B: methanol, flow rate 0.2 mL / min, 70°C.

[0491] As noted in Tables 6 and 7, the predominant degradation products following incubation in human serum were the parent oligonucleotides for all sequences tested, as shown below. [ka]

[0492] No other degradation products were detected in significant amounts. In particular, after 120 h, no cleavage was observed between tc-nucleosides and 2'-OMe nucleosides (sequence SY-0455) and no cleavage of the C7-amino group at the 3' end (sequences SY-0343, SY-0299, SY-0357). Sequences with PO linkages between the oligonucleotide and the spacer / lipid moiety were significantly less stable in human serum compared to their PS counterparts. After 24 h, a significant amount of the spacer / lipid moiety was cleaved, and after 120 h, almost no original oligonucleotide was present in sequences linked via PO, whereas in the case of fatty acids connecting via PS linkers, only about 1 and 8% of the spacer / lipid moiety was cleaved after 24 and 120 h, respectively.

[0493] [Table 6]

[0494] [Table 7] Example 4 Identification of multimers

[0495] Polyacrylamide gel electrophoresis (PAGE) experiments were performed to detect self-multimerization. The following chemicals were used: tris(hydroxymethyl)aminomethane (Tris), (TCI A0321); acetic acid (Merck 1.00063); acrylamide / bis solution, 29:1 (40%, Serva 10680.01); tetramethylethylenediamine (TEMED, Sigma-Aldrich T9281); ammonium persulfate (Sigma-Aldrich 248614); and glycerol (Sigma-Aldrich G9012). Buffer A was prepared by dissolving 60 g of Tris in 200 mL of water. Adjust the pH to 7.4 with glacial acetic acid (approximately 25-30 mL). Dilute the solution to 500 mL with water and store at +4 °C. Buffer B was prepared by diluting 16 mL of buffer A to 800 mL with water. Ammonium persulfate 10% (w / v) was prepared by dissolving 100 mg of ammonium persulfate in 0.9 mL of water. Preparation of the gel was performed by mixing the following solutions in a glass beaker: 9.4 mL of acrylamide / bis solution; 15 mL of water; 500 μL of buffer A; 125 μL of 10% (w / v) ammonium persulfate, and 38 μL of TEMED. The test solution was 2 mg / mL in 50% glycerol, and 10 μL was applied (corresponding to 10 μg of oligonucleotide). Pre-migration settings were 40 min / 90 V in buffer B. Migration settings were 45–60 min / 120 V in buffer B in addition to 90 min / 90 V or 15 min / 90 V. 5–7 μL of 6× DNA loading dye was also transferred. Detection was performed by placing the gel on a TLC plate and examining under UV light at 254 nm. Staining with Stains-All (Sigma-Aldrich, 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide, 3,3′-diethyl-9-methyl-4,5,4′,5′-dibenzothiacarbocyanine) was then performed according to the manufacturer's protocol.

[0496] As mentioned above, tc-DNA oligonucleotides with acute toxicity were observed to show an additional band migrating at a level of 40-60 BP in polyacrylamide gel electrophoresis experiments. This rather sharp band was proposed to be a multimer of the oligonucleotide (e.g., duplex, trimer, or larger aggregates). The results of a gel electrophoresis experiment without any obvious appearance of a multimer band for any of the studied oligonucleotides are shown in Figure 9. As an example of a multimer band, the oligonucleotide SY-0221, which showed toxicity in vivo, is also shown. Example 5 Exon 23 skipping studies performed on mouse M23D surrogate sequences

[0497] SY0442 and SY0450 were selected for further investigation into the efficacy of exon 23 skipping. SY0210 (intact PS) and SY0308 (intact PO) were used as controls for some of the experiments detailed herein. The purpose of these experiments was to test efficacy and tcDNA content in tissues and to determine potential toxicity of the test compounds.

[0498] Mdx23 mice were injected with the compounds at 50 mg / kg / week or 200 mg / kg / week for 4 weeks. Group 1 was analyzed 72 hours after the last injection and Group 2 was analyzed 2 weeks after the last injection. The in vitro and mouse model studies discussed in Examples 1 and 2 were performed and the results were consistent with those Examples.

[0499] Exon 23 skipping levels were assessed using nested RT-PCR. Total RNA was extracted from specific tissues in the mouse model and reverse transcribed using the SSIII cDNA synthesis kit. RT-PCR was performed between exons 20 and 26, as well as specific RT-PCR to detect only the skipped product. Figure 10A-D demonstrates the RT-PCR results for 200 mg / kg / week at 72 hours (Figures 10A and 10B) and 2 weeks (Figures 10C and 10D). Clear bands corresponding to exon 23 skipping products in all muscles treated with the compound can be observed. The skipping levels are particularly high in the heart in all cases. Using specific nested PCR, exon 23 skipping was also detected in the CNS, smooth muscle of the gastrointestinal tract, and retina. Similar results were obtained in the 50 mg / kg / week cohort (Figure 11).

[0500] Exon 23 skipping was further quantified more precisely using the more sensitive taqman qPCR protocol. Figures 12A and 12B demonstrate similar results across the 200 mg / kg (Figure 12A) and 50 mg / kg (Figure 12B) cohorts. The ratios calculated between the 72 hour and 2 week cohorts were in most cases close to 1 for SY0442 and SY0450, respectively, at each dose: [Table 8]

[0501] The ratio of exon skipping was also calculated between SY0442 and SY0450, demonstrating that SY0442 generally had a slightly higher overall level of skipping than SY0450: [Table 9]

[0502] The data was further manipulated in Figure 14, showing pooled data comparing the efficacy of SY0442 and SY0450 at 72 hours and 2 weeks post-injection. A dose effect can be observed between 50 mg / kg and 200 mg / kg (approximately 4-fold). Quantitation of exon 23 skipping at 200 mg / kg (Figure 15) and 50 mg / kg (Figure 16) comparing the full PO compound with the full PS compound was also determined.

[0503] To identify the most potent compounds, the potency / amount ratio of compounds found in tissues was calculated for 200 mg / kg: [Table 10]

[0504] Also calculated for 50mg / kg: [Table 11]

Claims

[Claim 1] An object, method or system as described in this specification and drawings.

Images