Compounds and methods for modulating gene splicing

Abstract

The present invention relates to compounds and methods for modulating gene splicing, in particular to compounds, compositions and methods for modulating gene splicing. In some embodiments, modulating gene splicing increases expression of a target protein or target functional RNA.

Description

[0001] This divisional application of the present invention is based on the patent application for invention titled "Compounds and Methods for Modulating Gene Splicing" with the application number 202080080092.2 (International Application Number PCT / US2020 / 023598) and the filing date of March 19, 2020.

[0002] Cross - reference to related applications

[0003] This application claims the priority of U.S. Provisional Application No. 62 / 902,603 filed on September 19, 2019 and U.S. Provisional Application No. 62 / 943,539 filed on December 4, 2019. The entire contents of the above - mentioned applications are hereby incorporated herein by reference. Background of the Invention

[0004] The potential of antisense oligonucleotide therapies was first proposed in an article published in 1978 (Zamecnik & Stephenson, Proc. Natl. Acad. Sci. U.S.A. 75:280 - 284 and 285 - 288 (1978)); it disclosed that a 13 - mer synthetic oligonucleotide complementary to a portion of the Rous sarcoma virus (RSV) genome inhibited the replication of RSV in infected chicken fibroblasts and inhibited the transformation of primary chicken fibroblasts mediated by RSV into malignant sarcoma cells.

[0005] Antisense oligonucleotide methods utilize the sequence - specific binding of DNA - and / or RNA - based oligonucleotides to selected mRNA, microRNA, precursor RNA, or mitochondrial RNA targets and the resulting inhibition of translation. This oligonucleotide - based inhibition of translation and ultimately gene expression is the result of one or more cellular mechanisms, which may include, but are not limited to, (i) direct (steric) blockage of translation, (ii) ribonuclease H - mediated inhibition, and (iii) RNA interference - mediated inhibition (e.g., small interfering RNA (siRNA), microRNA (miRNA), splicing modulation, inhibition of non - coding RNA and single - strand RNA interference (ssRNAi)).

[0006] The history of antisense technology shows that while it is relatively simple to identify antisense oligonucleotides that bind to mRNA, it is not simple to optimize antisense oligonucleotides that have the true potential to inhibit gene expression and thus become good clinical candidates. Oligonucleotide - based antisense technology has the inherent problems of being unstable in living organisms and having the potential to produce off - target effects, such as unexpected immune stimulation (Agrawal & Kandimalla (2004) Nature Biotech. 22:1533 - 1537).

[0007] Methods for optimizing these technologies have focused on trying to address biostability, affinity for RNA targets, cellular permeability, and activity in vivo. Generally, these represent competing factors. For example, traditional antisense oligonucleotides utilize phosphodiester internucleotide linkages, which have proven to be too biologically unstable to be effective. Accordingly, methods for optimizing these technologies have focused on modifying antisense oligonucleotides to make them more biologically stable. Early methods focused on modifying the internucleotide linkages to make them more resistant to degradation by cellular nucleases. However, these modifications can cause the molecule to reduce its target specificity and generate unwanted biological activities.

[0008] In addition, throughout oligonucleotide research, it has been recognized that these molecules are susceptible to degradation by exonucleases in vivo, with the major degradation occurring at the 3'-end of the molecule (Temsamani et al., (1993) Analytical Biochem. 215:54-58). Accordingly, methods to avoid this exonuclease activity have been exploited.

[0009] Despite extensive research, efforts to improve stability and maintain recognition of RNA targets without off-target effects have not generally produced oligonucleotides with a higher probability of clinical success. Accordingly, if methods for downregulating gene expression based on oligonucleotides are to succeed, optimized antisense oligonucleotides that can most effectively achieve this result are still needed. There are two key mechanisms of antisense activity. The first mechanism involves an antisense oligonucleotide hybridizing to the target RNA and the formation of an activated ribonuclease H, which cleaves the target RNA and inhibits its expression. The second mechanism is that the antisense oligonucleotide blocks the processing of the target RNA when hybridizing to the target, and processing includes splicing, thereby inhibiting or enhancing gene expression. The combination of these antisense mechanisms can also lead to nonsense-mediated decay, thereby inhibiting or enhancing gene expression. When using these two methods, off-target effects have been observed, and new antisense designs are needed to mitigate off-target activity and improve efficacy.

[0010] To modulate splicing, antisense oligonucleotides are designed to bind to the target RNA with high affinity and selectivity. To date, antisense candidates applied to this mechanism include modified RNA oligonucleotides, such as 2'-O-methyloligoribonucleosides, which were used to modulate splicing in cells in the first study. (Sierakowska et al., (1996) Proc Natl Acad Sci USA, v93(23):12840-4; Wilton et al., Neuromuscul Disord (1999) v9(5):330-8). Since then, several other modified oligonucleotides have been evaluated, such as oligonucleotides with 2'-methoxyethoxy, LNA, HNA, CeNa, ANA, or mixtures of these modifications.

[0011] However, other new designs are also needed. SUMMARY OF THE INVENTION

[0012] The present invention provides a method for modulating RNA processing, comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, said linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein said antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0013] The present invention also provides a method for selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, said method comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, said linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target pre-mRNA; wherein said antisense oligonucleotide targets the splicing site of the pre-mRNA of a second mRNA transcript, thereby blocking the splicing site of said second mRNA transcript and directing the splicing of said pre-mRNA to said first mRNA transcript; and wherein said antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0014] The present invention also provides a method for treating a disease or disorder in a subject, wherein modulating RNA processing will be beneficial for treating the subject, said method comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, said linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein said antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0015] The present invention also provides a method for inducing nonsense-mediated decay of a target RNA, comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, said linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein said antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0016] The present invention also provides a method for increasing the level of protein encoded by mRNA or the level of functional mRNA and increasing the expression of protein or functional mRNA, which comprises administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein the antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or a combination thereof.

[0017] The present invention also provides an antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target precursor RNA comprising a retained intron, wherein the antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or a combination thereof.

[0018] Specifically, the present invention includes but is not limited to the following items:

[0019] 1. A method for regulating RNA processing, which comprises administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein the antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or a combination thereof.

[0020] 2. A method for selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target precursor mRNA; wherein the antisense oligonucleotide targets a splicing site of the precursor mRNA of a second mRNA transcript, thereby blocking the splicing site of the second mRNA transcript and directing splicing of the precursor mRNA to the first mRNA transcript; and wherein the antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or a combination thereof.

[0021] 3. A method of treating a disease or disorder in a subject, wherein modulating the RNA processing process will be beneficial for treating the subject, the method comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein the antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0022] 4. A method of inducing nonsense-mediated decay of a target RNA, comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein the antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0023] 5. A method of increasing the level of a protein encoded by an mRNA or the level of a functional mRNA and increasing the expression of a protein or a functional mRNA, comprising administering an antisense oligonucleotide comprising 14-30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target RNA, wherein the antisense oligonucleotide comprises 1-3 regions, each region independently comprising 2-5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0024] 6. The method according to any one of items 1-5, wherein the target RNA comprises a retained intron.

[0025] 7. The method according to any one of items 1-6, wherein the 2'-substituted nucleotides are selected from 2'O-methyl ribonucleotides or 2'-MOE.

[0026] 8. The method according to any one of items 1-7, wherein the antisense oligonucleotide comprises 1 region, the region comprising 2-5 consecutive deoxyribonucleotides.

[0027] 9. The method according to item 8, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide, at the 3' end of the antisense oligonucleotide, flanked by 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0028] 10. The method according to item 9, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide.

[0029] 11. The method according to item 9, wherein the consecutive deoxyribonucleotides are located at the 3' end of the antisense oligonucleotide.

[0030] 12. The method according to any one of items 1-11, wherein the length of the consecutive deoxyribonucleotides is 2-4 nucleotides.

[0031] 13. The method according to item 12, wherein the length of the consecutive deoxyribonucleotides is 4 nucleotides.

[0032] 14. The method according to any one of items 1-13, wherein the 5' splice site of the retained intron is flanked by exons.

[0033] 15. The method according to any one of items 1-13, wherein the 3' splice site of the retained intron is flanked by exons.

[0034] 16. The method according to any one of items 1-13, wherein the 5' splice site of the retained intron is flanked by exons, and the 3' splice site of the retained intron is flanked by exons.

[0035] 17. The method according to item 2 and any one of items 6-16 when item 2 is cited, wherein the 5' side of the splice site of the second mRNA transcript is an exon.

[0036] 18. The method according to item 2 and any one of items 6-16 when item 2 is cited, wherein the 3' side of the splice site of the second mRNA transcript is an exon.

[0037] 19. The method according to item 2 and any one of items 6-16 when item 2 is cited, wherein the 5' side of the splice site of the second mRNA transcript is an exon, and the 3' side of the splice site of the second mRNA transcript is an exon.

[0038] 20. The method according to any one of items 1-19, wherein the method can be used to treat a subject suffering from a disorder caused by insufficient amount or activity of a protein or insufficient amount or activity of the functional mRNA expressed from the precursor mRNA.

[0039] 21. The method according to item 20, wherein the insufficient amount or activity of the target protein or functional mRNA is caused by haploinsufficiency of the protein or functional RNA.

[0040] 22. The method according to any one of items 1 - 21, wherein the antisense oligonucleotide is part of a composition comprising a pharmaceutically acceptable carrier.

[0041] 23. The method according to any one of items 1 - 22, wherein the antisense oligonucleotide is administered topically.

[0042] 24. The method according to any one of items 1 - 23, wherein the antisense oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

[0043] 25. The method according to item 24, wherein at least half of the internucleotide linkages are phosphorothioates.

[0044] 26. The method according to item 24, wherein all of the internucleotide linkages are phosphorothioates.

[0045] 27. The method according to any one of items 1 - 26, wherein the antisense oligonucleotide is single - stranded.

[0046] 28. The method according to any one of items 1 - 27, wherein the antisense oligonucleotide is at least 90% complementary to a portion of the target mRNA over its entire length.

[0047] 29. The method according to any one of items 1 - 27, wherein the RNA is selected from precursor mRNA, mRNA, non - coding RNA.

[0048] 30. An antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal - length portion of a target precursor RNA comprising a retained intron, wherein the antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0049] 31. The oligonucleotide according to item 30, wherein the 2'-substituted nucleotides are selected from 2'O - methyl ribonucleotides or 2'-MOE.

[0050] 32. The oligonucleotide according to item 30 or 31, wherein the antisense oligonucleotide comprises 1 region, the region comprising 2 - 5 consecutive deoxyribonucleotides.

[0051] 33. The oligonucleotide according to item 32, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide, at the 3' end of the antisense oligonucleotide, flanked by 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0052] 34. The oligonucleotide according to item 33, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide.

[0053] 35. The oligonucleotide according to item 33, wherein the consecutive deoxyribonucleotides are located at the 3' end of the antisense oligonucleotide.

[0054] 36. The oligonucleotide according to any one of items 30 - 35, wherein the length of the consecutive deoxyribonucleotides is 2 - 4 nucleotides.

[0055] 37. The oligonucleotide according to item 36, wherein the length of the consecutive deoxyribonucleotides is 4 nucleotides.

[0056] 38. The oligonucleotide according to any one of items 30 - 37, wherein the 5' splice site of the retained intron is flanked by exons.

[0057] 39. The oligonucleotide according to any one of items 30 - 37, wherein the 3' splice site of the retained intron is flanked by exons.

[0058] 40. The oligonucleotide according to any one of items 30 - 37, wherein the 5' splice site of the retained intron is flanked by exons, and the 3' splice site of the retained intron is flanked by exons.

[0059] 41. The oligonucleotide according to any one of items 30 - 40, wherein the antisense oligonucleotide is administered locally.

[0060] 42. The oligonucleotide according to any one of items 30 - 41, wherein the antisense oligonucleotide comprises at least one phosphorothioate internucleotide linkage.

[0061] 43. The oligonucleotide according to item 42, wherein at least half of the internucleotide linkages are phosphorothioate.

[0062] 44. The oligonucleotide according to item 42, wherein all of the internucleotide linkages are phosphorothioate.

[0063] 45. The oligonucleotide according to any one of items 30 - 44, wherein the antisense oligonucleotide is single - stranded.

[0064] 46. The oligonucleotide according to any one of items 30 - 45, wherein the antisense oligonucleotide is at least 90% complementary to a portion of the target mRNA over its entire length.

[0065] 47. The oligonucleotide according to any one of items 30 - 46, wherein the RNA is selected from precursor mRNA, mRNA, non - coding RNA.

[0066] 48. A pharmaceutical composition comprising the oligonucleotide according to any one of items 30 - 47 and a pharmaceutically acceptable carrier.

[0067] 49. The method according to item 1, wherein the RNA processing process includes splicing.

[0068] 50. The method according to item 3, wherein the RNA processing process includes splicing. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1A and Figure 1B are schematic diagrams of embodiments of the present invention. DETAILED DESCRIPTION

[0070] The present invention relates to compounds, compositions and methods for regulating gene splicing. In some embodiments, regulating gene splicing increases the expression of a target protein and inhibits the expression of an unwanted protein or target functional RNA.

[0071] By convention, unless otherwise stated, the sequences discussed in the present invention are listed from 5' to 3'. In addition, unless otherwise stated, the strand containing the sequence numbered SEQ ID NO is the sequence from 5' to 3'.

[0072] When the term "3'" is used directionally, it generally refers to a region or position 3' (close to the 3' end of the nucleotide) in a polynucleotide or oligonucleotide from another region or position in the same polynucleotide or oligonucleotide. The term "3' end" generally refers to the 3' - terminal nucleotide of the oligonucleotide in composition.

[0073] When the term "5'" is used directionally, it generally refers to a region or position 5' (close to the 5' end of the nucleotide) in a polynucleotide or oligonucleotide from another region or position in the same polynucleotide or oligonucleotide. As used in the present invention, the term "5' end" generally refers to the 5' - terminal nucleotide of the oligonucleotide in composition.

[0074] The term "about" generally means that the exact number is not important. Thus, oligonucleotides with one or two fewer nucleoside residues, or one to several additional nucleoside residues are considered equivalents of each of the above - mentioned embodiments.

[0075] "Antisense activity" means any detectable or measurable activity caused by hybridization of an antisense oligonucleotide compound with its target nucleic acid. In some embodiments, the antisense activity is a reduction in the amount or expression of the target nucleic acid or the protein encoded by such target nucleic acid. In some embodiments, the antisense activity is the modulation of splicing, thereby inhibiting or increasing the expression of the protein encoded by such target nucleic acid.

[0076] "Antisense inhibition" means a reduction in the level of a target nucleic acid or a target protein in the presence of an antisense oligonucleotide complementary to the target nucleic acid as compared to the level of the target nucleic acid or the target protein in the absence of the antisense oligonucleotide.

[0077] "Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleic acid base sequence that permits hybridization with a corresponding region or segment of a target nucleic acid.

[0078] The term "co-administration" (or "co-administered") generally means the administration of at least two different substances. Co-administration means simultaneous administration, and also means sequential administration with a time interval of several days, with at least two different substances being administered in any order, in a single dose or in different doses.

[0079] The term "combination" generally means the administration of an oligonucleotide-based compound according to the present invention and another agent useful for treating a disease or disorder, and the other agent does not eliminate the activity of the present compound during the treatment of a patient. Such administration can be carried out in any order, including simultaneous administration, and sequential administration with a time interval from a few seconds to several days. Such combination therapy may also include more than a single administration of the compound according to the present invention and / or separate administration of the other agent. The compound according to the present invention and the other agent can be administered by the same or different routes.

[0080] The term "individual" or "subject" or "patient" generally means a mammal, such as a human. The term "mammal" specifically includes warm-blooded vertebrates, including but not limited to humans, non-human primates, rats, mice, cats, dogs, horses, cattle (cattle and cows), pigs, sheep, and rabbits. As used in the present invention, "an individual in need thereof" means a human or non-human animal selected for treatment or therapy who requires such treatment or therapy.

[0081] As used in the present invention, "inhibit expression or activity" means to reduce or block the expression or activity of RNA or protein, and does not necessarily mean complete elimination of expression or activity.

[0082] The term "nucleoside" generally refers to a compound composed of a sugar and a purine or pyrimidine base, where the sugar generally refers to ribose, deoxyribose, pentose, arabinose or hexose. For the purposes of the present invention, if the base is not guanine, cytosine, adenine, thymine or uracil, the base is considered non-natural, and if the sugar is not β-ribofuranoside or 2'-deoxyribofuranoside, the sugar is considered non-natural.

[0083] The term "nucleotide" generally refers to a nucleoside containing a phosphorus group attached to the sugar. As used herein, "linked nucleotides" may or may not be linked by a phosphodiester bond, and thus nucleotides include, but are not limited to, "linked nucleotides". As used herein, "linked nucleotides" are nucleotides linked in a continuous sequence (i.e., there are no additional nucleotides between the linked nucleotides).

[0084] The term "nucleic acid" includes genomic regions or RNA molecules transcribed from genomic regions. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is microRNA. In some embodiments, the nucleic acid is non-coding RNA.

[0085] As used herein, "nucleic acid base" refers to a moiety that can be attached to a sugar moiety to produce a nucleoside capable of being incorporated into an oligonucleotide, and wherein said moiety is capable of bonding to a complementary nucleic acid base naturally occurring in another oligonucleotide or nucleic acid. Nucleic acid bases can be naturally occurring or can be modified. As used herein, "nucleic acid base sequence" refers to the order of consecutive nucleic acid bases independent of any sugar, bond or nucleic acid base modification.

[0086] As used herein, the term "unmodified nucleic acid base" or "naturally occurring nucleic acid base" refers to the heterocyclic nucleic acid bases naturally occurring in RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U).

[0087] As used herein, "modified nucleic acid base" refers to any nucleic acid base that is not naturally occurring.

[0088] As used herein, "modified nucleoside" refers to a nucleoside that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. Modified nucleosides contain a modified sugar moiety and / or a modified nucleic acid base.

[0089] As used herein, "oligonucleotide" refers to a compound comprising multiple linked nucleosides. In some embodiments, the oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA). In some embodiments, the oligonucleotide comprises only unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA). In some embodiments, the oligonucleotide comprises one or more modified nucleosides.

[0090] As used herein, "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and / or at least one modified sugar.

[0091] As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, "naturally occurring internucleoside linkage" refers to a 3' to 5' phosphodiester bond. As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than a naturally occurring internucleoside linkage.

[0092] Phrases such as "oligonucleotide complementary to a single-stranded RNA sequence" refer to an oligonucleotide that, under physiological conditions, forms a sufficient number of hydrogen bonds through Watson-Crick interactions of its nucleic acid bases with the nucleic acid bases of a single-stranded RNA sequence to form a double helix with the single-stranded RNA sequence. This is in contrast to oligonucleotides that form triple helices with double-stranded DNA or RNA through Hoogsteen hydrogen bonding.

[0093] As used herein, "chemical modification" refers to a chemical difference in a compound when compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleic acid base modifications) and internucleoside linkage modifications. With respect to oligonucleotides, chemical modification does not solely include differences in the nucleic acid base sequence.

[0094] The term "complementary" means an oligonucleotide that binds to a nucleic acid sequence under physiological conditions, for example, through Watson-Crick base pairing (interaction between an oligonucleotide and a single-stranded nucleic acid) or through Hoogsteen base pairing (interaction between an oligonucleotide and a double-stranded nucleic acid) or by any other means, including in cases where the oligonucleotide binds to RNA and results in pseudoknot formation. Binding through Watson-Crick or Hoogsteen base pairing under physiological conditions is measured by observing interference with the function of the nucleic acid sequence.

[0095] "Fully complementary" or "100% complementary" means that each nucleic acid base of a first nucleic acid has a complementary nucleic acid base in a second nucleic acid. In some embodiments, the first nucleic acid is an antisense compound and the target nucleic acid is the second nucleic acid.

[0096] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In some embodiments, the complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

[0097] "Nonsense-mediated decay" refers to any number of cellular mechanisms that do not rely on ribonuclease H or RISC to degrade mRNA or precursor mRNA. In some embodiments, nonsense-mediated decay eliminates and / or degrades mRNA transcripts containing premature termination codons. In some embodiments, nonsense-mediated decay eliminates and / or degrades any form of abnormal mRNA and / or precursor mRNA transcripts.

[0098] The term "pharmaceutically acceptable" refers to non-toxic materials that do not interfere with the effects of the compounds according to the present invention or the biological activity of the compounds according to the present invention.

[0099] "Segment" refers to a defined number of consecutive (i.e., linked) nucleic acid bases of a nucleic acid. In some embodiments, the segment is a defined number of consecutive nucleic acid bases of a target nucleic acid. In some embodiments, the segment is a defined number of consecutive nucleic acid bases of an antisense compound.

[0100] The term "prophylactically effective amount" generally refers to an amount sufficient to prevent or reduce the development of an unwanted biological effect.

[0101] As used herein, "sugar moiety" refers to the naturally occurring sugar moiety of a nucleoside or a modified sugar moiety. As used herein, "naturally occurring sugar moiety" refers to the furanosyl group found in naturally occurring RNA or the deoxyfuranosyl group found in naturally occurring DNA. As used herein, "modified sugar moiety" refers to a substituted sugar moiety or sugar substitute, such as but not limited to a 2'-modified sugar or a restricted sugar.

[0102] The term "therapeutically effective amount" or "pharmacologically effective amount" generally refers to an amount sufficient to effect a desired biological effect, such as a beneficial result, including but not limited to preventing, alleviating, improving or eliminating the signs or symptoms of a disease or disorder. Thus, the total amount of each active ingredient of a pharmaceutical composition or method is sufficient to demonstrate a meaningful patient benefit, such as but not limited to the recovery from a chronic disease characterized by immune stimulation. Thus, a "pharmacologically effective amount" will depend on the circumstances of administration. A pharmacologically effective amount may be administered in one or more prophylactic or therapeutic administrations. When an active ingredient is administered to an individual, alone, the term refers to the administration of that single ingredient. When applied to a combination, whether administered in combination, sequentially or simultaneously, the term refers to the combined amount of the active ingredients that produces a therapeutic effect.

[0103] The term "treatment" generally refers to a method intended to obtain a beneficial or desired result, which may include alleviating symptoms, or delaying or improving disease progression.

[0104] The term "gene expression" generally refers to the process by which information from a gene is used to synthesize a functional gene product, which can be a protein. The process may involve transcription of the protein, RNA splicing, translation, and post-translational modification, and may include mRNA, precursor mRNA, non-coding RNA, small nucleolar RNA, ribosomal RNA, and other protein synthesis templates.

[0105] "Targeting" (or "targeted") refers to the process of designing and selecting an antisense oligonucleotide that specifically hybridizes to a target nucleic acid and induces a desired effect. "Target gene", "target allele", "target nucleic acid", "target RNA", "target mRNA", and "target RNA transcript" all refer to the nucleic acid and antisense oligonucleotide that specifically hybridize. "Target allele" is an allele whose expression is selectively targeted. "Target segment", "target region", and "target site" all refer to the nucleotide sequence of the target nucleic acid targeted by the antisense oligonucleotide.

[0106] The target region is the region defined by the target nucleic acid structure. For example, the target region may include 3'UTR, 5'UTR, exon, intron, exon / intron junction, coding region, translation initiation region, translation termination region, or other defined nucleic acid regions.

[0107] Some embodiments provide compositions and methods that include administering to an animal an antisense compound or composition disclosed herein. In some embodiments, the antisense compound is administered to prevent, treat, ameliorate, or slow the progression of a disease or disorder associated with gene expression or protein activity. In some embodiments, the animal is a human.

[0108] The present invention provides a new design for an antisense oligonucleotide for regulating splicing. In this design, the antisense oligonucleotide has two domains (see Figure 1). The first domain consists of ribonucleotides (RNA), modified RNA, or a combination thereof, which provide affinity for the target RNA. The second domain consists of phosphodiester or phosphorothioate oligodeoxynucleotides (DNA), which allow recruitment of ribonuclease H but do not allow ribonuclease H to cleave the antisense oligonucleotide-target RNA duplex. The recruitment of ribonuclease H and its binding to the oligonucleotide-target RNA duplex provide steric hindrance at the duplex site and promote splicing. As used herein, modified RNA includes, but is not limited to, 2'-substituted nucleotides, nonionic nucleotides, or restricted sugar nucleotides.

[0109] Any method disclosed herein includes administering an antisense oligonucleotide disclosed herein.

[0110] In some embodiments, the present invention provides a method for regulating splicing. In some embodiments, the present invention provides a method for regulating RNA splicing. In an embodiment, the RNA includes, but is not limited to, precursor mRNA, mRNA, non-coding RNA. In an embodiment, the RNA is precursor mRNA. In an embodiment, the RNA is mRNA. In an embodiment, the RNA is non-coding RNA. In some embodiments, the target RNA contains a retained intron.

[0111] In some embodiments, the target precursor mRNA contains a retained intron. In some embodiments, one or both sides of the retained intron are exons. In some embodiments, the exon flanks the 5' splice site of the retained intron. In some embodiments, the exon flanks the 3' splice site of the retained intron. In some embodiments, the exon flanks the 5' splice site of the retained intron and the exon flanks the 3' splice site of the retained intron.

[0112] In some embodiments, the retained intron is constitutively spliced from the target RNA; thereby increasing the mRNA encoding protein level or the functional mRNA level and increasing the protein or functional mRNA expression. In some embodiments, the present invention provides a method for increasing the mRNA encoding protein level or the functional mRNA level and increasing the protein or functional mRNA expression.

[0113] In some embodiments, the method for regulating splicing can be used to treat a subject suffering from a disorder caused by insufficient amount or activity of a protein or insufficient amount or activity of a functional mRNA; and wherein the insufficient amount or activity of the protein or functional mRNA is caused by haploinsufficiency of the target protein or target functional RNA.

[0114] In some embodiments, the present invention provides a method for treating a disease or disorder of a subject, wherein regulating splicing will be beneficial for treating the subject. In an embodiment, the disease or disorder is caused by insufficient amount or activity of a protein or insufficient amount or activity of a functional mRNA. In an embodiment, the insufficient amount or activity of the protein or functional mRNA is caused by haploinsufficiency of the target protein or target functional RNA.

[0115] In some embodiments, the antisense oligonucleotide compound contains a sequence complementary to the target RNA region. In some embodiments, the antisense oligonucleotide compound contains a sequence complementary to the region of the target RNA containing the retained intron.

[0116] In one embodiment, the present invention provides a method for selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to a co - length portion of a target pre - mRNA; wherein the antisense oligonucleotide targets a splice site of the pre - mRNA of a second mRNA transcript, thereby blocking the splice site of the second mRNA transcript and directing the pre - mRNA to be spliced to the first mRNA transcript; and wherein the antisense oligonucleotide comprises 1 to 3 nucleotide regions, the nucleotide regions comprising 2 to 5 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0117] In any embodiment of the present invention, the retained intron is constitutively spliced from the target RNA; thereby increasing the mRNA - encoded protein level or the functional mRNA level and increasing protein or functional mRNA expression. In some embodiments, the present invention provides a method for increasing the mRNA - encoded protein level or the functional mRNA level and increasing protein or functional mRNA expression.

[0118] In an embodiment, the antisense oligonucleotide comprises 1 nucleotide region, the nucleotide region comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0119] In an embodiment, the antisense oligonucleotide comprises 2 nucleotide regions, the nucleotide regions comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In some embodiments, the 2 nucleotide regions are not adjacent.

[0120] In an embodiment, the antisense oligonucleotide comprises 3 nucleotide regions, the nucleotide regions comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In some embodiments, the deoxyribonucleotide regions are not adjacent.

[0121] In one embodiment, the present invention provides a method of selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides, the linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target pre-mRNA; wherein the antisense oligonucleotide targets a splice site of a pre-mRNA of a second mRNA transcript, thereby blocking the splice site of the second mRNA transcript and directing splicing of the pre-mRNA to the first mRNA transcript; and wherein the antisense oligonucleotide comprises 1 - 3 nucleotide regions, the nucleotide regions comprising 2 - 4 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0122] In an embodiment, the antisense oligonucleotide comprises 1 nucleotide region, the nucleotide region comprising 2 - 4 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0123] In an embodiment, the antisense oligonucleotide comprises 2 nucleotide regions, the nucleotide regions comprising 2 - 4 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In some embodiments, the 2 nucleotide regions are not adjacent.

[0124] In an embodiment, the antisense oligonucleotide comprises 3 nucleotide regions, the nucleotide regions comprising 2 - 4 consecutive deoxyribonucleotides and the remaining nucleotides being 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In some embodiments, the deoxyribonucleotide regions are not adjacent.

[0125] In one embodiment, the present invention provides a method of selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target pre-mRNA; wherein the antisense oligonucleotide targets a splice site of a pre-mRNA of a second mRNA transcript, thereby blocking the splice site of the second mRNA transcript and directing splicing of the pre-mRNA to the first mRNA transcript; and wherein the antisense oligonucleotide comprises a deoxyribonucleotide region that comprises 2 - 5 consecutive deoxyribonucleotides at the 3'-end of the antisense oligonucleotide and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In an embodiment, the deoxyribonucleotide region comprises 4 consecutive deoxyribonucleotides at the 5'-end of the antisense oligonucleotide and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

[0126] In one embodiment, the present invention provides a method of selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target pre-mRNA; wherein the antisense oligonucleotide targets a splice site of a pre-mRNA of a second mRNA transcript, thereby blocking the splice site of the second mRNA transcript and directing splicing of the pre-mRNA to the first mRNA transcript; and wherein the antisense oligonucleotide comprises a deoxyribonucleotide region that comprises 2 - 5 consecutive deoxyribonucleotides at the 5'-end of the antisense oligonucleotide and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or combinations thereof. In an embodiment, the deoxyribonucleotide region comprises 4 consecutive deoxyribonucleotides at the 5'-end of the antisense oligonucleotide and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides.

[0127] In some embodiments, the present invention provides a method for modulating the processing of a target RNA, the method comprising contacting a cell with an antisense oligonucleotide as described in the present invention, wherein the processing of a target precursor transcript is modulated. In some embodiments, the processing of the target RNA includes, but is not limited to, splicing, cleavage, transport, translation, and degradation of coding and non-coding RNAs. In some embodiments, the RNA processing includes inhibiting an RNA binding protein. In some embodiments, the RNA processing includes splicing of coding and non-coding RNAs. In some embodiments, the RNA processing includes cleavage of coding and non-coding RNAs. In some embodiments, the RNA processing includes transport of coding and non-coding RNAs. In some embodiments, the RNA processing includes translation of coding and non-coding RNAs. In some embodiments, the RNA processing includes degradation of coding and non-coding RNAs.

[0128] In some embodiments, a method for treating a disease or disorder by modulating the processing of a target precursor transcript comprises administering an antisense oligonucleotide as described in the present invention.

[0129] In some embodiments, the present invention provides a method for inducing nonsense-mediated decay of a target RNA, comprising administering an antisense oligonucleotide as described in the present invention.

[0130] In some embodiments, the antisense oligonucleotide as described in the present invention modulates the splicing of one or more target nucleic acids, and such modulation causes degradation and / or reduction of the target nucleic acid via nonsense-mediated decay.

[0131] In some embodiments, the antisense oligonucleotide complementary to the target nucleic acid as described in the present invention can increase the inclusion of an exon, and the inclusion of the exon causes the nonsense-mediated decay pathway to recognize and degrade the mRNA containing the exon.

[0132] In some embodiments, the antisense oligonucleotide complementary to the target nucleic acid as described in the present invention can enhance the exclusion of an exon, and the exclusion of the exon causes the nonsense-mediated decay pathway to recognize and degrade the mRNA in the absence of the exon.

[0133] Nonsense-mediated decay is a surveillance pathway that functions to reduce errors in aberrant gene expression by eliminating and / or degrading aberrant mRNA transcripts. In some embodiments, the mechanism of nonsense-mediated decay selectively degrades mRNAs that result from errors in the processing of precursor mRNAs. For example, many precursor mRNA transcripts contain multiple exons and introns that can be alternatively spliced to yield any number of mRNA transcripts containing various combinations of exons. The mRNA transcripts are then translated into any number of protein isoforms. In some embodiments, the precursor mRNA is processed to include one or more exons, the inclusion of which results in an mRNA that encodes or would encode a non-functional or misfolded protein. In some embodiments, the precursor mRNA is processed to include one or more exons, the inclusion of which results in an mRNA that contains a premature stop codon. In some such embodiments, the mechanism of nonsense-mediated decay recognizes an mRNA transcript that contains an additional exon and degrades the mRNA transcript prior to translation. In some such embodiments, the mechanism of nonsense-mediated decay recognizes an mRNA transcript that contains a premature stop codon and degrades the mRNA transcript prior to translation.

[0134] In some embodiments, the precursor mRNA is processed to exclude one or more exons, the exclusion of which results in an mRNA that encodes a non-functional protein. In some embodiments, the precursor mRNA is processed to exclude one or more exons, the exclusion of which results in an mRNA that contains a premature stop codon. In some such embodiments, the mechanism of nonsense-mediated decay recognizes an mRNA transcript that lacks an exon and degrades the mRNA transcript prior to translation. In some such embodiments, the mechanism of nonsense-mediated decay recognizes an mRNA transcript that lacks an exon and contains a premature stop codon and degrades the mRNA transcript prior to translation.

[0135] Without wishing to be bound by any particular theory, the antisense oligonucleotides of the invention permit the antisense oligonucleotide to bind to the target RNA and complex with ribonuclease H; however, the antisense oligonucleotide inactivates ribonuclease H. In other words, the antisense oligonucleotide / target RNA-ribonuclease H complex is not cleaved by ribonuclease H. In some embodiments, the antisense oligonucleotide is administered locally.

[0136] In some embodiments, the antisense compound comprises or consists of an oligonucleotide that comprises a region that is complementary to the target nucleic acid. In some embodiments, the target nucleic acid is an endogenous RNA molecule. In some embodiments, the target nucleic acid is a precursor mRNA. In some embodiments, the antisense oligonucleotide modulates the splicing of the precursor mRNA.

[0137] In some embodiments, the antisense oligonucleotide is complementary to the nucleotide sequence of a target pre-mRNA, wherein the antisense oligonucleotide comprises 14-30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target pre-mRNA, wherein the antisense oligonucleotide comprises 1-3 nucleotide regions that each comprise 2-5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0138] In some embodiments, the antisense oligonucleotide is complementary to the nucleotide sequence of a target pre-mRNA, wherein the antisense oligonucleotide comprises 14-30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target pre-mRNA, wherein the antisense oligonucleotide comprises 1-3 nucleotide regions that each comprise 2-4 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0139] In some embodiments, the antisense oligonucleotide is complementary to the nucleotide sequence of a target pre-mRNA, wherein the antisense oligonucleotide comprises 14-30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target pre-mRNA, wherein the antisense oligonucleotide comprises a deoxyribonucleotide region that comprises 2-5 consecutive deoxyribonucleotides at the 3'-end of the antisense oligonucleotide, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0140] In some embodiments, the antisense oligonucleotide is complementary to the nucleotide sequence of a target pre-mRNA, wherein the antisense oligonucleotide comprises 14-30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target pre-mRNA, wherein the antisense oligonucleotide comprises a deoxyribonucleotide region that comprises 2-5 consecutive deoxyribonucleotides at the 5'-end of the antisense oligonucleotide, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0141] In some embodiments, the antisense oligonucleotide comprises 1 region that comprises 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof. In some embodiments, the antisense oligonucleotide comprises 2 deoxyribonucleotide regions, each region independently comprises 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof. In some embodiments, the antisense oligonucleotide comprises 3 deoxyribonucleotide regions, each region independently comprises 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0142] In some embodiments, the deoxyribonucleotide region comprises 2 to 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof. In some embodiments, the deoxyribonucleotide region comprises 2 to 4 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof. In some embodiments, the deoxyribonucleotide region comprises 4 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides, or restricted sugar nucleotides, or combinations thereof.

[0143] In some embodiments, the 2'-substituted nucleotides are selected from, but not limited to, 2'-O-methyl ribonucleotides, 2'-O-methoxy-ethyl (2'-MOE) ribonucleotides, 2'-halogen (e.g., fluorine) nucleotides, and morpholino-modified nucleic acids. In some embodiments, the restricted sugar nucleotides include bicyclic nucleosides. In some embodiments, the bicyclic nucleosides include locked nucleic acids and bridged nucleic acids. In some embodiments, the restricted sugar nucleotides are selected from, but not limited to, locked nucleic acids (LNA), peptide nucleic acids (PNA), hexitol nucleic acids (HNA), cyclohexenyl nucleic acids (CeNA), sorbitol nucleic acids (ANA), restricted MOE (cMOE), restricted ethyl (cEt), ethylene-bridged nucleic acids (ENA), serine nucleic acids (SNA), and twist-embedded nucleic acids (TINA). In some embodiments, the non-ionic includes, but is not limited to, methylphosphonates, phosphotriesters, and morpholino (PMO). In some embodiments, the nucleotide can be 2'-substituted and have a restricted sugar.

[0144] In some embodiments, the antisense oligonucleotide comprises 1 deoxyribonucleotide region that comprises 2, 3, 4, or 5 consecutive deoxyribonucleotides.

[0145] In some embodiments, the antisense oligonucleotide comprises one deoxyribonucleotide region that contains 2, 3, or 4 consecutive deoxyribonucleotides. In some embodiments, the antisense oligonucleotide comprises one deoxyribonucleotide region that contains 2 consecutive deoxyribonucleotides. In some embodiments, the antisense oligonucleotide comprises one deoxyribonucleotide region that contains 3 consecutive deoxyribonucleotides. In some embodiments, the antisense oligonucleotide comprises one deoxyribonucleotide region that contains 4 consecutive deoxyribonucleotides. In some embodiments, the consecutive deoxyribonucleotides are located at the 3'-end of the antisense oligonucleotide.

[0146] In some embodiments, the consecutive deoxyribonucleotides are located at the 5'-end of the antisense oligonucleotide.

[0147] In some embodiments, the antisense oligonucleotide comprises two deoxyribonucleotide regions, each independently containing 2, 3, or 4 consecutive deoxyribonucleotides. In some embodiments, the antisense oligonucleotide comprises three deoxyribonucleotide regions, each independently containing 2, 3, or 4 consecutive deoxyribonucleotides.

[0148] In some embodiments, the consecutive deoxyribonucleotides are located at the 5'-end of the antisense oligonucleotide, at the 3'-end of the antisense oligonucleotide, flanked by 2'-substituted oligonucleotides, non-ionic oligonucleotides, or restricted sugar oligonucleotides, or combinations thereof. In some embodiments, the consecutive deoxyribonucleotides are located at the 5'-end of the antisense oligonucleotide. In some embodiments, the consecutive deoxyribonucleotides are located at the 3'-end of the antisense oligonucleotide. In some embodiments, the consecutive deoxyribonucleotides are flanked by 2'-substituted oligoribonucleotides.

[0149] In some embodiments, the consecutive deoxyribonucleotides are naturally occurring nucleotides. In some embodiments, the consecutive deoxyribonucleotides are unmodified nucleotides. In some embodiments, one or more of the consecutive deoxyribonucleotides are modified.

[0150] The antisense oligonucleotides of the present invention are pharmaceutically acceptable. The antisense oligonucleotides of the present invention are injectable. In some embodiments, the target RNA can be mRNA. Some embodiments provide an antisense oligonucleotide, wherein the antisense oligonucleotide is single-stranded.

[0151] In some embodiments, the present invention provides an antisense oligonucleotide compound that is 17 nucleotides in length and comprises at least 12 complementary consecutive nucleic acid bases of an equal-length portion of the target sequence.

[0152] In some embodiments, the present invention provides an antisense oligonucleotide compound having a length of 18-25 nucleotides, and the antisense oligonucleotide compound comprises at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target sequence. In some embodiments, the length of the antisense oligonucleotide compound is 18 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 19 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 20 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 21 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 22 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 23 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 24 nucleotides. In some embodiments, the length of the antisense oligonucleotide compound is 25 nucleotides.

[0153] In some embodiments, the present invention provides an antisense oligonucleotide compound having a length of 20 nucleotides, and the antisense oligonucleotide compound comprises at least 12 consecutive nucleic acid bases complementary to an equal-length portion of a target sequence. In some embodiments, the antisense oligonucleotide comprises a nucleotide region comprising 2-4 consecutive deoxyribonucleotides at the 3'-end of the antisense oligonucleotide, and the remaining nucleotides are 2'-substituted nucleotides, non-ionic nucleotides or restricted sugar nucleotides, or a combination thereof.

[0154] In some embodiments, the length of the antisense oligonucleotide of the present invention can be at least 14 nucleotides, for example, the length is between 14-30 nucleotides. Thus, the length of the antisense oligonucleotide of the present invention can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, the length of the antisense oligonucleotide of the present invention can be between 14-25 nucleotides. In some embodiments, the length of the antisense oligonucleotide of the present invention can be between 17-22 nucleotides. In some embodiments, the length of the antisense oligonucleotide of the present invention can be between 19-28 nucleotides.

[0155] The length of the antisense oligonucleotides of the present invention can be 17, 18, 19, 20, 21 or 22 nucleotides. In some embodiments, the length of the antisense oligonucleotides of the present invention can be 17 nucleotides. The length of the antisense oligonucleotides of the present invention can be 18 nucleotides. The length of the antisense oligonucleotides of the present invention can be 19 nucleotides. The length of the antisense oligonucleotides of the present invention can be 20 nucleotides. The length of the antisense oligonucleotides of the present invention can be 21 nucleotides. The length of the antisense oligonucleotides of the present invention can be 22 nucleotides. The length of the antisense oligonucleotides of the present invention can be 23 nucleotides. The length of the antisense oligonucleotides of the present invention can be 24 nucleotides. The length of the antisense oligonucleotides of the present invention can be 25 nucleotides. The length of the antisense oligonucleotides of the present invention can be 26 nucleotides. The length of the antisense oligonucleotides of the present invention can be 27 nucleotides. The length of the antisense oligonucleotides of the present invention can be 28 nucleotides. The length of the antisense oligonucleotides of the present invention can be 29 nucleotides. The length of the antisense oligonucleotides of the present invention can be 30 nucleotides.

[0156] The natural or unmodified bases in RNA are adenine (A) and guanine (G), and the pyrimidine bases are cytosine (C) and uracil (U) (thymine (T) in DNA). In contrast, modified bases, also known as heterocyclic base moieties, include other nucleic acid bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine and guanine and other alkyl derivatives of adenine and guanine, 2-propyladenine and guanine and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7-methyladenine, 2-fluoro-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deaza-azaadenine and 3-deaza-aza-guanine and 3-deaza-azaadenine.

[0157] In some embodiments, the modified nucleic acid bases are selected from: universal bases, hydrophobic bases, mixed bases, size extended bases, and fluorinated bases as defined in the present invention. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, and guanine, and other alkyl derivatives of adenine and guanine, 2-propyladenine and guanine, and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-fluoro-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deaza-adenine, 3-deazaguanine, and 3-deaza-adenine. Further modified nucleic acid bases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H))-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytosine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include bases in which the purine or pyrimidine base is replaced by other heterocyclic compounds such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In some embodiments, the modified nucleic acid base is 5-methylcytosine.

[0158] Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituents at one or more of their 2', 3', or 4' positions, and sugars having substituents that replace one or more hydrogen atoms of the sugar. In some embodiments, the sugar is modified by having a substituent at the 2' position. In additional embodiments, the sugar is modified by having a substituent at the 3' position. In other embodiments, the sugar is modified by having a substituent at the 4' position. It is also contemplated that the sugar may have modifications at more than one of these positions, or that the antisense oligonucleotide may have one or more nucleotides having a sugar modification at one position and one or more nucleotides having a sugar modification at a different position.

[0159] Sugar modifications in antisense oligonucleotides are contemplated to include, but are not limited to, sugar substituents selected from the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. In some embodiments, these groups may be selected from: O(CH2) x OCH3, O((CH2) x O) y CH3, O(CH2) x NH2, O(CH2) x CH3, O(CH2) x ONH2, and O(CH2) x ON((CH2) x CH3)2, where x and y are each from 1 to 10.

[0160] In some embodiments, the modified sugar contains a substituent selected from the following: C1-C 10Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, Cl, Br, CN, OCN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, target group, intercalator, a group for improving the pharmacokinetic properties of an antisense oligonucleotide or a group for improving the pharmacodynamic properties of an antisense oligonucleotide, and other substituents having similar properties. In one embodiment, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also referred to as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995), i.e., an alkoxyalkoxy. Another modification includes 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also referred to as 2'-DMAOE and 2'-dimethylaminoethoxyethoxy (also referred to in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2.

[0161] Additional sugar substituents include allyl (-CH2-CH═CH2), -O-allyl (-CH2-CH═CH2), methoxy (-O-CH3), aminopropoxy (-OCH2CH2NH2), and fluorine (F). The sugar substituent at the 2'-position (2'-) can be in the arabinose (up) position or the ribose (down) position. One 2'-arabinose modification is 2'-F. Other similar modifications can also be made at other positions of the oligomeric compound, particularly at the 3'-terminal nucleoside or at the 3'-position of the sugar in a 2'-5'-linked oligonucleotide and at the 5'-position of the 5'-terminal nucleotide. The oligomeric compound can also have a sugar analogue, such as a cyclobutyl moiety, in place of the furanose. U.S. patent examples that disclose methods for preparing modified sugar structures include, but are not limited to, 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; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, the entire contents of which are hereby incorporated by reference.

[0162] Representative sugar substituents include the groups described in U.S. Patent Application Publication 2005 / 0261218, which is hereby incorporated by reference. In certain embodiments, the sugar modification is a 2'-O-Me modification, 2'F modification, 2'H modification, 2'-amino modification, 4'-thioribose modification, or a thiophosphate modification on a carboxyl group attached to the 6'-position carbon, or a combination thereof.

[0163] In some embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from: F, OCH3, and OCH2CH2OCH3.

[0164] Some modified sugar moieties contain substituents that bridge two atoms of the furanose ring to form a second ring, thereby producing a bicyclic sugar moiety (also referred to as a restricted sugar). In some such embodiments, the bicyclic sugar moiety contains a bridge between the 4'-furanose ring atom and the 2'-furanose ring atom. Examples of such 4'-2'-bridged sugar substituents include, but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-O-2' ("ENA"), 4'-CH(CH3)-O-2' (referred to as "constrained ethyl" or "cEt"), 4'-CH2-O-CH2-2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-O-2' ("constrained MOE" or "cMOE") and their analogs (see, e.g., Seth et al., US 7,399,845; Bhat et al., US 7,569,686; Swayze et al., US 7,741,457 and Swayze et al., US 8,022,193), 4'-C(CH3)(CH3)-O-2' and its analogs (see, e.g., Seth et al., US8,278,283), 4'-CH2-N(OCH3)-2' and its analogs (see, e.g., Prakash et al., US 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., Allerson et al., US 7,696,345 and Allerson et al., US 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and its analogs (see, e.g., Seth et al., US 8,278,426), 4'-C(R a R b )-N(R)-O-2', 4,-C(R a R b)-ON(R)-2’,4’-CH2-ON(R)-2’ and 4’-CH2-N(R)-O-2’, where R, Ra and Rb are each independently H, a protecting group or a C1-C 12 alkyl (see, for example, Imanishi et al., U.S. 7,427,672).

[0165] In some embodiments, such 4’-2’ bridges independently comprise 1-4 members independently selected from: -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a );

[0166] where:

[0167] x is 0, 1 or 2;

[0168] n is 1, 2, 3 or 4;

[0169] R a and R b are each independently H, a protecting group, a hydroxyl group, a C1-C 12 alkyl, a substituted C1-C 12 alkyl, a C2-C 12 alkenyl, a substituted C2-C 12 alkenyl, a C2-C 12 alkynyl, a substituted C2-C 12 alkynyl, a C5-C 20 aryl, a substituted C5-C 20 aryl, a heterocyclic group, a substituted heterocyclic group, a heteroaryl, a substituted heteroaryl, a C5-C7 cycloalkyl, a substituted C5-C7 cycloalkyl, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(=O)-H), a substituted acyl group, CN, a sulfonyl group (S(=O)2-J1) or a sulfinyl group (S(=O)-J1); and J1 and J2 are each independently H, a C1-C 12 alkyl, a substituted C1-C 12 alkyl, a C2-C 12 alkenyl, a substituted C2-C 12Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl or protecting group.

[0170] Additional bicyclic sugar moieties are well known in the art, see for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al J Am. Chem. Soc, 20017, 129, 8362-8379; Wengel et al., U.S. 7,053,207; Imanishi et al., U.S. 6,268,490; Imanishi et al., U.S. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. 6,794,499; Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133; Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582; and Ramasamy et al., U.S. 6,525,191; Torsten et al., WO 2004 / 106356; Wengel et al., WO1999 / 014226; Seth et al WO 2007 / 134181; Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S. 8,088,746; Seth et al., U.S. 7,750,131; Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S. 8,546,556; Seth et al., U.S. 8,530,640; Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; and U.S. Patent Publication: Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0171] In some embodiments, the isomeric configuration further defines the bicyclic sugar moiety and the nucleoside incorporated into such bicyclic sugar moiety. For example, an LNA nucleoside (as described in the present invention) can be in the α-L configuration or the β-D configuration.

[0172]

[0173] α-L-methoxy (4’-CH2-O-2’) or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). In the present invention, the general description of bicyclic nucleosides includes two isomeric configurations. Unless otherwise specified, the position of a particular bicyclic nucleoside (e.g., LNA or cEt) in the exemplary embodiments of the present invention is in the β-D configuration.

[0174] In some embodiments, the modified sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5’-substituted sugar and 4’-2’-bridged sugar).

[0175] In some embodiments, the modified sugar moiety is a sugar replacement. In some such embodiments, the oxygen atom of the sugar moiety is replaced with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moiety further comprises bridging and / or non-bridging substituents as described in the present invention. For example, some sugar replacements comprise a 4’-sulfur atom and a substituent at the 2’-position (see, e.g., Bhat et al., US 7,875,733 and Bhat et al., US 7,939,677) and / or at the 5’-position.

[0176] In some embodiments, the sugar replacement includes a ring other than a five-membered ring. For example, in some embodiments, the sugar replacement includes a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (“HNA”), anisol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), F-HNA:

[0177]

[0178] ("F-HNA", see Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906; F-HNA may also be referred to as F-THP or 3'-fluorotetrahydropyran) and a nucleoside comprising an additionally modified THP compound, the nucleoside having the following formula:

[0179]

[0180] Wherein the modified THP nucleosides are each independently:

[0181] Bx is a base moiety;

[0182] T3 and T4 are each independently a internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide, or one of T3 and T4 is a internucleoside linking group that links the modified THP nucleoside to the remainder of the oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;

[0183] q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; R1, R2 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, where X is O, S or NJ1, and J1, J2, and J3 are each H or C1-C6 alkyl.

[0184] In some embodiments, modified THP nucleosides are provided, wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is not H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In some embodiments, modified THP nucleosides are provided, wherein one of R1 and R2 is F. In some embodiments R1 is F and R2 is H, in some embodiments, R1 is methoxy and R2 is H, and in some embodiments, R1 is methoxyethoxy and R2 is H.

[0185] In some embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than one heteroatom. For example, nucleosides containing a morpholino sugar moiety and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506). As used herein, the term "morpholino" refers to a sugar substitute having the following structure:

[0186]

[0187] In some embodiments, the morpholino can be modified, for example, by adding or changing different substituents from the above morpholino structure. Such sugar substitutes are referred to herein as "modified morpholinos".

[0188] In some embodiments, the sugar substitute comprises an acyclic moiety. Examples of nucleosides and oligonucleotides containing such acyclic sugar substitutes include, but are not limited to: peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and the nucleosides and oligonucleotides described in Manoharan et al., WO2011 / 133876.

[0189] Many other bicyclic and tricyclic sugars and sugar substitute ring systems are known in the art and can be used in modified nucleosides.

[0190] The nucleoside residues of antisense oligonucleotides can be coupled to each other through any of a number of known internucleoside linkages. The two main types of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, those containing a phosphodiester bond ("P=O") (also referred to as an unmodified or naturally occurring bond), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate ("P=S") and dithiophosphonate ("HS-P=S") phosphates. Representative phosphorus-free internucleoside linking groups include, but are not limited to, methyleneimino (-CH2-N(CH3)-O-CH2-), thiodiester, thiocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.

[0191] Such internucleoside linkages include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylthiophosphonate, triester phosphate, phosphoramidate, siloxane, carbonate, alkoxycarbonyl, acetamidate, carbamate, morpholino, borane, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. In some embodiments, the synthetic antisense oligonucleotides of the invention may comprise a combination of internucleotide linkages. In some embodiments, the synthetic antisense oligonucleotides of the invention may comprise a combination of phosphorothioate and phosphodiester internucleotide linkages. In some embodiments, more than half but less than all of the internucleotide linkages are phosphorothioate internucleotide linkages. In some embodiments, all of the internucleotide linkages are phosphorothioate internucleotide linkages.

[0192] Modified oligonucleotides comprising internucleoside linkages having chiral centers can be prepared as a population of modified oligonucleotides comprising stereorandom internucleoside linkages or as a population of modified oligonucleotides comprising phosphorothioate bonds of a specific stereochemical configuration. In some embodiments, the population of modified oligonucleotides comprises phosphorothioate internucleoside linkages, wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in a random selection of the stereochemical configuration of each phosphorothioate bond. However, as is well known to those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In some embodiments, the population of modified oligonucleotides is enriched in modified oligonucleotides that comprise one or more phosphorothioate internucleoside linkages in a specific, independently selected stereochemical configuration.

[0193] In some embodiments, the phosphorothioate bonds can be a mixture of Rp and Sp enantiomers, or they can be made stereoregular or substantially stereoregular in the Rp or Sp configuration. In embodiments where the bonds are a mixture of Rp and Sp enantiomers, the Rp and Sp configurations can be located at defined positions within the antisense oligonucleotide or randomly placed throughout the oligonucleotide.

[0194] In some embodiments, the present invention provides antisense oligonucleotides as described herein and optionally one or more conjugate groups and / or terminal groups. A conjugate group consists of one or more conjugate moieties and a conjugate bond that links the conjugate moiety to the oligonucleotide. The conjugate group can be linked to one or both ends of the oligonucleotide and / or any internal position. In some embodiments, the conjugate group is linked to the 2'-position of the nucleoside of the modified oligonucleotide. In some embodiments, a conjugate group linked to one or both ends of the oligonucleotide is a terminal group. In some such embodiments, the conjugate group or terminal group is linked at the 3' and / or 5'-end of the oligonucleotide. In some such embodiments, the conjugate group (or terminal group) is linked at the 3'-end of the oligonucleotide. In some such embodiments, the conjugate group is linked near the 3'-end of the oligonucleotide. In some such embodiments, the conjugate group (or terminal group) is linked at the 5'-end of the oligonucleotide. In some such embodiments, the conjugate group is linked near the 5'-end of the oligonucleotide.

[0195] Examples of terminal groups include, but are not limited to, conjugate groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

[0196] Some conjugate bases and conjugate moieties have been described previously in the literature, such as: cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett, 1993, 3, 2765-2770), cholesteryl thioether (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecyl diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid, hexadecyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexanamino-carbonyl-hydroxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), tocopherol groups (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or GalNAc clusters (e.g., WO2014 / 179620).

[0197] The synthetic antisense compounds of the present invention can be prepared by methods well known in the art. For example, phosphoramidite or H-phosphonate chemical reactions can be carried out manually or by an automated synthesizer. The synthetic antisense compounds of the present invention can also be modified in a variety of ways that do not impair their ability to hybridize to mRNA.

[0198] In some embodiments, the oligonucleotide-based compounds of the present invention are synthesized by a linear synthesis method.

[0199] At the end of the synthesis according to a linear or parallel synthesis protocol, if modified nucleosides are incorporated, the oligonucleotide-based compounds of the present invention can be conveniently protected with concentrated ammonia solution or as recommended by the phosphoramidite supplier. The product oligonucleotide-based compounds are preferably purified by reverse-phase HPLC, detritylated, desalted, and dialyzed.

[0200] A non-limiting list of the antisense oligonucleotides of the present invention is shown in Table 1. The antisense oligonucleotides in Table 1 are designed to induce exon 23 skipping in the mouse dystrophin gene transcript. Unless otherwise indicated, the antisense oligonucleotides have phosphorothioate (PS) backbone linkages. However, those skilled in the art will recognize that other linkages based on phosphodiester or non-phosphodiester moieties can be included.

[0201] Table 1

[0202] Compound# Sequence SEQ ID NO: 1 <![CDATA[5’- GGC CAAACCUCGGCUUACCU-3’]]> 1 2 <![CDATA[5’-GGCC AAA CCUCGGCUUACCU-3’]]> 2 3 <![CDATA[5’-GGCCAAAC CTC GGCUUACCU-3’]]> 3 4 <![CDATA[5’-GGCCAAACCUCG GCT UACCU-3’]]> 4 5 <![CDATA[5’- GGC CAAACCUCGGCUU AC CU-3’]]> 5 6 <![CDATA[5’-GGCCAAACCUCGGCUUA CCT -3’]]> 6 7 <![CDATA[5’- GGCC AAACCUCGGCUUACCU-3’]]> 7 8 <![CDATA[5’-GGCC AAAC CUCGGCUUACCU-3’]]> 8 9 <![CDATA[5’-GGCCAAAC CTCG GCUUACCU-3’]]> 9 10 <![CDATA[5’-GGCCAAACCUCG GCTT ACCU-3’]]> 10 11 <![CDATA[5’-GGCCAAACCUCGGCUU ACCT -3’]]> 11 12 <![CDATA[5’-GGCCAAACCUCGG CTTACCT -3’]]> 12 13 <![CDATA[5’-GGCCAAACCUCGGC TTA CCT -3’]]> 13 14 <![CDATA[5’-GGCCAAACCUCGGCU TA CCT -3’]]> 14 15 <![CDATA[5’-GGCCAAACCUCGGCUUA CCT -3’]]> 15 16 <![CDATA[5’-GGCCAAACCUCGGCUUAC CT -3’]]> 16

[0203] Underlined = deoxynucleotide; non-underlined = 2'-O-methyl nucleotide

[0204] In some embodiments, the target nucleic acid is the mouse sequence of the target. In some embodiments, the target nucleic acid is the human sequence of the target.

[0205] The present invention provides a pharmaceutical composition comprising the antisense oligonucleotides described herein and a pharmaceutically acceptable carrier. The term "carrier" generally includes any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulation, or other materials for pharmaceutical formulations. It should be understood that the properties of the carrier, excipient, or diluent depend on the route of administration for a particular use. The preparation of pharmaceutically acceptable formulations containing these materials is described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

[0206] The composition may also comprise one or more other agents. Such agents may include, but are not limited to, vaccines, antigens, antibodies, cytotoxins, chemotherapeutic agents (conventional chemotherapy and modern targeted therapies), kinase inhibitors, allergens, antibiotics, agonists, antagonists, antisense oligonucleotides, ribozymes, RNA interference molecules, small interfering RNA molecules, microRNA molecules, oligonucleotide aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, costimulatory molecules, or combinations thereof.

[0207] The nucleic acid sequence complementary to the oligonucleotide according to the present invention will vary depending on the agent to be inhibited. For example, an antisense oligonucleotide according to the present invention may have an oligonucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state. The nucleic acid sequences of several such cellular genes have been described in the art. An antisense oligonucleotide according to the present invention may have any oligonucleotide sequence, provided that the sequence is partially or fully complementary to the target RNA nucleotide sequence.

[0208] In some embodiments, the antisense oligonucleotide may be at least 90% complementary to a portion of the target mRNA over its entire length. In some embodiments, the antisense oligonucleotide may be at least 93% complementary to a portion of the target RNA over its entire length. In some embodiments, the antisense oligonucleotide may be at least 95% complementary to a portion of the target RNA over its entire length. In some embodiments, the antisense oligonucleotide may be at least 98% complementary to a portion of the target RNA over its entire length. In some embodiments, the antisense oligonucleotide may be at least 99% complementary to a portion of the target RNA over its entire length. In some embodiments, the antisense oligonucleotide may be at least 100% complementary to a portion of the target RNA over its entire length.

[0209] Some embodiments provide compounds that target a gene, wherein the compound comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or 22 consecutive nucleic acid bases that are complementary to an equi-length portion of any target RNA. In some embodiments, the antisense oligonucleotide may comprise at least 12 consecutive nucleic acid bases that are complementary to an equi-length portion of the target RNA.

[0210] The antisense oligonucleotides of the present invention can be administered alone or in combination with any other agent or therapy. The agent or therapy can be co-administered or administered simultaneously. Such agents or therapies can be used to treat or prevent a disease or disorder without reducing the gene expression regulatory effect of the antisense oligonucleotides according to the present invention. Agents that can be used to treat or prevent a disease or disorder include, but are not limited to, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxin agents, kinase inhibitors, allergens, antibiotics, small interfering RNA molecules, antisense oligonucleotides, TLR antagonists (such as TLR3 and / or TLR7 antagonists and / or TLR8 antagonists and / or TLR9 antagonists), chemotherapeutic agents (conventional chemotherapy and modern targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants and co-stimulatory molecules (such as cytokines, chemokines, protein ligands, transactivators, peptides or peptides containing modified amino acids) or combinations thereof. Alternatively, the antisense oligonucleotides according to the present invention can be administered in combination with other compounds (such as lipids or liposomes) to enhance the specificity or magnitude of the gene expression regulation of the antisense oligonucleotides according to the present invention.

[0211] The antisense oligonucleotides of the present invention can be administered by any suitable route, including but not limited to, parenteral, mucosal delivery, oral, sublingual, transdermal, topical, inhalation, intratumoral, intravenous, subcutaneous, intrathecal, intranasal, spray, intraocular, intratracheal, rectal, intravaginal, by gene gun, in the form of a skin patch or eye drops or mouthwash. In any method according to the present invention, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, can be directly administered to a tissue or organ, such as but not limited to the bladder, liver, lung, kidney or lung. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered intramuscularly. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered mucosally. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered orally. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered rectally. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered intrathecally. In some embodiments, the antisense oligonucleotides according to the present invention, alone or in combination with any other agent, are administered intratumorally.

[0212] Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, normal saline, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and a reagent for adjusting tonicity, such as sodium chloride or glucose. The pH value can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0213] Administration of the antisense oligonucleotides according to the present invention can be carried out using known procedures, using an effective amount, and the administration time is such that it can effectively relieve the symptoms of the disease or replace the biomarker. For example, the effective amount of the antisense oligonucleotides according to the present invention for treating a disease and / or disorder can be an amount necessary to relieve or reduce symptoms, or delay or mitigate tumors, cancers, or bacterial, viral, or fungal infections. In the case of administering a composition for regulating gene expression, the effective amount of the antisense oligonucleotides according to the present invention is an amount sufficient to achieve the desired regulation compared to the gene expression in the absence of the antisense oligonucleotides according to the present invention. The effective amount for any particular application can vary depending on factors such as the disease or disorder being treated, the particular oligonucleotide being administered, the size of the subject, or the severity of the disease or disorder. Those of ordinary skill in the art can determine the effective amount of a particular antisense oligonucleotide empirically without undue experimentation.

[0214] When administered systemically, the therapeutic composition is preferably administered in a sufficient dose to bring the blood level of the compound of the present invention to about 0.0001 micromoles to about 10 micromoles. For topical administration, concentrations lower than this may be effective, and concentrations higher than this may be tolerated. Preferably, the total dose of the compound according to the present invention ranges from about 0.001 mg per patient per day to about 200 mg per kg body weight per day. In some embodiments, the total dose can be 0.08, 0.16, 0.32, 0.48, 0.32, 0.64, 1, 10, or 30 mg / kg body weight administered daily, twice a week, or once a week. It may be desirable to administer one or more therapeutically effective amounts to an individual as a single treatment event simultaneously or sequentially. As a single treatment phase for an individual, it may be desirable to administer one or more therapeutically effective amounts of the therapeutic composition of the present invention simultaneously or sequentially.

[0215] The method according to this aspect of the present invention can be used for model studies of gene expression. The method can also be used for preventing or treating human or animal diseases. For example, the method can be used for inhibition in pediatric and veterinary applications of gene expression.

[0216] Some embodiments provide a kit for treating, preventing or ameliorating a disease, disorder or condition as described herein, wherein the kit comprises: (i) an antisense oligonucleotide as described herein; and optionally (ii) a second agent or therapy as described herein. The kits of the invention may also include instructions for using the kit to treat, prevent or ameliorate a disease, disorder or condition as described herein.

[0217] Cell culture and antisense compound treatment

[0218] The effect of an antisense compound on the level, activity or expression of a target nucleic acid can be tested in vitro in a variety of cell types. Cell types for such assays are available from commercial suppliers (e.g., American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the supplier's instructions using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, Calif.). Exemplary cell types include, but are not limited to, HepG2 cells, Hep3B cells and primary hepatocytes.

[0219] In vitro testing of antisense oligonucleotides

[0220] The invention describes methods of treating cells with antisense oligonucleotides, which can be appropriately modified for treatment with other antisense compounds.

[0221] When cells reach approximately 60 - 80% confluency in culture, the cells can be treated with an antisense oligonucleotide.

[0222] One reagent commonly used to introduce antisense oligonucleotides into cultured cells is the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). The antisense oligonucleotide can be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of the antisense oligonucleotide and a concentration of LIPOFECTIN ranging from 2 to 12 μg / mL per 100 nM antisense oligonucleotide.

[0223] Another reagent for introducing antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, California). The antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 Reduced Serum Medium (Invitrogen, Carlsbad, California) to achieve the desired concentration of the antisense oligonucleotide and a concentration of LIPOFECTAMINE ranging from 2 to 12 μg / mL per 100 nM of the antisense oligonucleotide.

[0224] Another technique for introducing antisense oligonucleotides into cultured cells involves electroporation.

[0225] Cells are treated with the antisense oligonucleotide by conventional methods. Cells can be harvested 16 - 24 hours after antisense oligonucleotide treatment, at which time the RNA or protein levels of the target nucleic acid are measured by methods known in the art and the methods described in the present invention. Typically, when the treatment is repeated multiple times, the data are presented as the average of the repeated treatments.

[0226] The concentration of the antisense oligonucleotide used varies depending on the cell line. Methods for determining the optimal concentration of the antisense oligonucleotide for a particular cell line are well known in the art. When transfected with LIPOFECTAMINE, the antisense oligonucleotide is typically used at a concentration of 1 nM to 300 nM. When transfected by electroporation, the concentration range of the antisense oligonucleotide used is from 625 to 20,000 nM.

[0227] RNA Isolation

[0228] RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using TRIZOL reagent (Invitrogen, Carlsbad, California) according to the manufacturer's recommended protocol.

[0229] Analysis of Inhibition of Target Levels or Expression

[0230] Inhibition of the level or expression of a target nucleic acid can be analyzed by a variety of methods known in the art. For example, the level of a target nucleic acid can be quantified by, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using commercially available ABI PRISM 7600, 7700, or 7900 sequence detection systems, available from PE-Applied Biosystems, Foster City, Calif., and used according to the manufacturer's instructions.

[0231] Quantitative real-time PCR analysis of target RNA levels

[0232] Quantification of RNA levels can be accomplished by quantitative real-time PCR using an ABI PRISM 7600, 7700, or 7900 sequence detection system (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Methods for quantitative real-time PCR are well known in the art.

[0233] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction which generates complementary DNA (cDNA), and the complementary DNA is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are carried out sequentially in the same sample well. RT and real-time PCR reagents are available from Invitrogen Corporation (Carlsbad, Calif.). The RT and real-time PCR reactions are performed by methods well known to those skilled in the art.

[0234] The amount of gene (or RNA) target obtained by real-time PCR is normalized either using the expression level of a gene with a constant expression (e.g., cyclophilin A), or by quantifying total RNA using RIBOGREEN (Invitrogen Corporation, Carlsbad, Calif.). Cyclophilin A expression is quantified by real-time PCR, run simultaneously with the target, multiplexed, or run separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invitrogen, Eugene, Oreg.). The method for RNA quantification by RIBOGREEN is taught in Jones, L.J. et al. (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

[0235] Design probes and primers for hybridization to target nucleic acids. Methods for designing real-time PCR probes and primers are well known in the art and may include using software such as PRIMER EXPRESS software (Applied Biosystems, Foster City, California).

[0236] Protein level analysis

[0237] Protein levels can be evaluated or quantified by a variety of methods well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemical reactions, immunocytochemical reactions, or fluorescence-activated cell sorting (FACS). Antibodies directed to the target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Michigan), or can be prepared by conventional monoclonal or polyclonal antibody production methods well known in the art.

[0238] In vivo testing of antisense compounds

[0239] Testing can be performed in normal animals or experimental disease models. For administration to animals, the antisense oligonucleotide is formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration such as intraperitoneal, intravenous, and subcutaneous. Calculation of the antisense oligonucleotide dose and dosing frequency is within the capabilities of those skilled in the art and depends on factors such as the route of administration and the animal body weight. After treatment with the antisense oligonucleotide for a period of time, RNA is isolated and changes in nucleic acid expression are measured.

[0240] Some indications

[0241] In some embodiments, the present invention provides methods of treating an individual, including administering one or more pharmaceutical compositions of the present invention. Some embodiments include treating a subject in need thereof by administering a therapeutically effective amount of an antisense compound of the present invention to the individual.

[0242] In one embodiment, administration of a therapeutically effective amount of an antisense compound targeting a nucleic acid is accompanied by monitoring the corresponding target level in the individual to determine the individual's response to the administration of the antisense compound. The response of the individual to the administration of the antisense compound can be used by a physician to determine the amount and duration of therapeutic intervention.

[0243] Examples

[0244] Synthesis of antisense oligonucleotides

[0245] The antisense oligonucleotides according to the present invention can be synthesized by procedures well known in the art, such as phosphoramidite or H-phosphonate chemical reactions that can be carried out manually or by an automated synthesizer. For example, the antisense oligonucleotides of the present invention can be synthesized by a linear synthesis method.

[0246] The ARNA compounds used in the study were synthesized using phosphoramidite chemistry. These protocols are described in detail, for example, in https: / / pubs.rsc.org / en / content / chapter / bk9781788012096-00453 / 978-1-78801-209-6, which is incorporated herein by reference.

[0247] Cell culture and transfection

[0248] H-2Kb-tsA58 mdx myoblasts 42,43 (H2K mdx cells) can be cultured and differentiated as previously described in the art. Briefly, 24-well plates were pretreated with 50 μg / mL poly-D-lysine (Merck Millipore), followed by treatment with 100 μg / ml Matrigel (CellLens, provided by In Vitro Technologies). Myoblast cultures at 60%-80% confluence were treated with trypsin (Thermo Fisher Scientific) and seeded onto the above-pretreated 24-well plates at a seeding density of 2×10 4 cells / well. Incubated at 37 °C in 5% CO2 in DMEM (Thermo Fisher Scientific) containing 5% horse serum for 24 hours, the cells can differentiate into myotubes. AO can be complexed with liposomes (Thermo Fisher Scientific) at a ratio of 2:1 (w / w) (liposome / AO) and used at a final transfection volume of 500 μL / well in 24-well plates according to the manufacturer's instructions.

[0249] RNA extraction and RT-PCR

[0250] RNA can be extracted from transfected cells using the Direct-zol RNAMiniPrep Plus and TRI reagent (kit, provided by Integrated Science) according to the manufacturer's instructions. Then, dystrophin transcripts can be analyzed by RT-PCR using SuperScriptIII reverse transcriptase (Thermo Fisher Scientific) spanning exons 20 - 26. PCR products can be separated on a 2% agarose gel in acetate-EDTA buffer and images can be captured on a Fusion Fx gel documentation system (gel imager, Marne-la-Vallée, France). Densitometry can be performed using ImageJ software. The actual exon skipping efficiency can be determined by expressing the amount of RT-PCR product with exon 23 skipped as a percentage of the total dystrophin transcript. The results are shown in the table below.

[0251] SEQ ID NO: Sequence Exon 23 Skipping Percentage 7 <![CDATA[5’- GGCC AAACCUCGGCUUACCU-3’]]> 34 8 <![CDATA[5’-GGCC AAAC CUCGGCUUACCU-3’]]> 30 9 <![CDATA[5’-GGCCAAAC CTCG GCUUACCU-3’]]> 0 10 <![CDATA[5’-GGCCAAACCUCG GCTT ACCU-3’]]> 32 11 <![CDATA[5’-GGCCAAACCUCGGCUU ACCT -3’]]> 42 12 <![CDATA[5’-GGCCAAACCUCGG CTTACCT -3’]]> 25 13 <![CDATA[5’-GGCCAAACCUCGGC TTA CCT -3’]]> 25 14 <![CDATA[5’-GGCCAAACCUCGGCU TA CCT -3’]]> 29 15 <![CDATA[5’-GGCCAAACCUCGGCUUA CCT -3’]]> 34 16 <![CDATA[5’-GGCCAAACCUCGGCUUAC CT -3’]]> 34

[0252] While the invention has been specifically shown and described in terms of its preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as contained in the appended claims.

Claims

1. A method of modulating an RNA processing event, comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to a co - linear portion of a target RNA, wherein the antisense oligonucleotide comprises 1 - 3 regions each independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

2. A method of selecting a first mRNA transcript in a gene comprising at least two mRNA transcripts, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to a co - linear portion of a target pre - mRNA; wherein the antisense oligonucleotide targets a splicing site of the pre - mRNA of a second mRNA transcript, thereby blocking the splicing site of the second mRNA transcript and directing splicing of the pre - mRNA to the first mRNA transcript; and wherein the antisense oligonucleotide comprises 1 - 3 regions each independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

3. A method of treating a disease or disorder in a subject, wherein modulating an RNA processing event would be beneficial to the treatment of the subject, the method comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to a co - linear portion of a target RNA, wherein the antisense oligonucleotide comprises 1 - 3 regions each independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

4. A method of inducing nonsense - mediated decay of a target RNA, comprising administering an antisense oligonucleotide comprising 14 - 30 linked nucleotides having at least 12 consecutive nucleic acid bases complementary to a co - linear portion of a target RNA, wherein the antisense oligonucleotide comprises 1 - 3 regions each independently comprising 2 - 5 consecutive deoxyribonucleotides and the remaining nucleotides are 2'-substituted nucleotides, non - ionic nucleotides or restricted sugar nucleotides, or combinations thereof.

5. A method for increasing the level of protein encoded by mRNA or the level of functional mRNA and increasing the expression of protein or functional mRNA, comprising administering an antisense oligonucleotide, said antisense oligonucleotide comprising 14 - 30 linked nucleotides, said linked nucleotides having at least 12 consecutive nucleic acid bases complementary to an equal-length portion of the target RNA, wherein said antisense oligonucleotide comprises 1 - 3 regions, each region independently comprising 2 - 5 consecutive deoxyribonucleotides, and the remaining nucleotides are 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

6. The method according to any one of claims 1 - 5, wherein the target RNA comprises a retained intron.

7. The method according to any one of claims 1 - 6, wherein the 2'-substituted nucleotides are selected from 2'O-methyl ribonucleotides or 2'-MOE.

8. The method according to any one of claims 1 - 7, wherein the antisense oligonucleotide comprises 1 region, said region comprising 2 - 5 consecutive deoxyribonucleotides.

9. The method according to claim 8, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide, at the 3' end of the antisense oligonucleotide, flanked by 2'-substituted nucleotides, nonionic nucleotides or restricted sugar nucleotides, or combinations thereof.

10. The method according to claim 9, wherein the consecutive deoxyribonucleotides are located at the 5' end of the antisense oligonucleotide.

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