Oligonucleotides that can upregulate glucocerebrosidase expression

Abstract

The present invention provides oligonucleotides, their conjugates, salts, and pharmaceutical compositions that increase the expression of glucocerebrosidase (GBA) in cells, as well as methods for treating diseases associated with reduced GBA expression, including Gaucher disease and / or Parkinson's disease. The oligonucleotides may contain a sequence of bases complementary to the sequence of bases in the 3' untranslated region (UTR) of the GBA mRNA transcript.

Description

Technical Field

[0001] Field of the Invention The present invention relates to oligonucleotides, conjugates, salts, and pharmaceutical compositions thereof that can increase the expression of glucocerebrosidase (GBA) in cells, and their use in methods of treating diseases and disorders associated with reduced expression of GBA, including Gaucher disease and Parkinson's disease.

Background Art

[0002] Background Glucocerebrosidase (GBA) is a lysosomal enzyme that catalyzes the hydrolysis of glucocerebroside (also known as glucosylceramide). Glucocerebroside is a normal component of cell membranes, particularly those of red blood cells and white blood cells.

[0003] Homozygous mutations in the gene encoding GBA cause Gaucher disease. During normal cellular turnover, macrophages phagocytose and degrade cell debris. Insufficient GBA activity results in the accumulation of glucocerebroside in the lysosomes of macrophages. Affected macrophages, known as "Gaucher cells," accumulate in areas such as the spleen, liver, and bone marrow.

[0004] Gaucher disease is characterized by jaundice, fatigue, anemia, low platelet count, and enlargement of the liver and spleen. The phenotype is variable, but three clinical forms have been identified: type 1 is the most common and typically does not cause neurological damage, whereas types 2 and 3 are characterized by neurological disorders.

[0005] This condition is inherited in an autosomal recessive pattern. More than 300 variants of the GBA gene are associated with this disorder. Genetics alone does not determine the severity of the disease, but certain mutations are known to cause more severe symptoms. For example, patients with two copies of the L444P mutation typically exhibit the neuronal phenotype of the disorder, while patients with one or two copies of the N370S allele are typically classified as type 1 (Scott et al., 2000, Genet. Med., 2, 65).

[0006] Mutations in the GBA gene are also associated with Parkinson's disease and Lewy body dementia (Riboldi and Di Fonzo, 2019, Cells, 8, 364). Parkinson's disease is a neurodegenerative disorder of the central nervous system characterized by a wide range of motor and non-motor symptoms. Motor symptoms include bradykinesia (slowness of movement), rigidity, and postural instability. Non-motor symptoms that can precede motor symptoms by many years include loss of smell, rapid eye movement sleep behavior disorder, autonomic dysfunction, and depression.

[0007] Heterozygous mutations in the GBA gene occur in approximately 8-12% of Parkinson's disease patients. Similar to Gaucher disease, the severity of the mutation can affect the disease phenotype. For example, patients with "severe" mutations (such as L444P) have a 2-3 times higher risk of dementia than patients with "mild" mutations (such as N370S). E326K is the most common GBA mutation in Parkinson's disease, and patients with this mutation show a faster progression of motor symptoms (Avenali et al., 2020, Front. Aging Neurosci.).

[0008] Current treatments for diseases associated with reduced GBA expression include enzyme replacement therapy (ERT) and substrate synthesis inhibition therapy (SRT). ERT involves intravenous administration of recombinant GBA. While most patients respond well to the treatment, there is a risk of developing an immune response. Furthermore, GBA cannot cross the blood-brain barrier, and therefore ERT is considered ineffective in patients with Parkinson's disease or neuronal Gaucher disease. SRT provides an alternative (or adjunct) treatment for patients who cannot tolerate ERT or for whom intravenous administration is problematic. SRT works by inhibiting enzymes in the glucocerebroside synthesis pathway, thereby reducing the accumulation of glucocerebroside in lysosomes. This therapy has a higher incidence of adverse effects than ERT, and the long-term reduction in glucocerebroside can affect several different cellular functions. Both ERT and SRT are expensive and must be continued for life.

[0009] There is a need for therapeutic agents that can increase or restore GBA expression. [Overview of the project]

[0010] Summary of the Invention The present invention relates to antisense oligonucleotides that can increase GBA expression, and more particularly to antisense oligonucleotides comprising a sequence of bases complementary to a sequence of bases in the 3' untranslated region (UTR) of a GBA mRNA transcript.

[0011] In a first aspect, the present invention provides an antisense oligonucleotide of 8 to 40 nucleotides in length, comprising a sequence of nucleotides complementary to at least six consecutive bases in the 3' untranslated region (UTR) of an RNA sequence encoding glucocerebrosidase (GBA).

[0012] In some embodiments, antisense oligonucleotides can increase GBA expression.

[0013] In some embodiments, antisense oligonucleotides can reduce the downregulation of microRNA (miR)-mediated GBA expression in target cells, and the miR is selected from its variants containing seed regions that are fully complementary to miR-22-3p and GGCAGCT.

[0014] In some embodiments, antisense oligonucleotides can inhibit the binding of RNA sequences to miRs selected from variants of miR-22-3p and GGCAGCT containing seed regions that are fully complementary to miR-22-3p and GGCAGCT.

[0015] In some embodiments, the continuous nucleotide sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

[0016] In some embodiments, the continuous nucleotide sequence is 16, 18, or 20 nucleotides long.

[0017] In some embodiments, the continuous nucleotide sequence is the same length as the antisense oligonucleotide.

[0018] In some embodiments, the sequence of nucleotides is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the sequence of bases located downstream of the stop codon TAG at positions 1746–1748 of SEQ ID NO: 73.

[0019] In some embodiments, the sequential nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the miR-22-3p binding site located downstream of the stop codon TAG at positions 1746–1748 of SEQ ID NO: 73.

[0020] In some embodiments, the sequence of nucleotides is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the sequence of bases located within the segment at positions 2227–2274 of SEQ ID NO: 73.

[0021] In some embodiments, the RNA sequence encoding GBA is an mRNA sequence that includes a 3'UTR sequence containing consecutive bases at positions 2227–2274 of SEQ ID NO: 73.

[0022] In some embodiments, the RNA sequence encoding GBA is an mRNA sequence containing the sequence of SEQ ID NO: 73 or an allele variant thereof.

[0023] In some embodiments, the continuous nucleotide sequence is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, or to a fragment of at least 10 nucleotides of any of them.

[0024] In some embodiments, the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0025] In some embodiments, the antisense oligonucleotide is a double-stranded oligonucleotide.

[0026] In some embodiments, the continuous nucleotide sequence has, or comprises, a nucleobase sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34, or a fragment of at least 10 nucleotides of any of them.

[0027] In some embodiments, the continuous nucleotide sequence has, or comprises, a nucleobase sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:XX.

[0028] In some embodiments, the antisense oligonucleotide comprises one or more modified nucleoside(s).

[0029] In some embodiments, the antisense oligonucleotide comprises one or more modified nucleoside(s) independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, morpholino, and locked nucleic acid (LNA) nucleosides.

[0030] In some embodiments, the antisense oligonucleotide comprises one or more 2'-MOE RNA nucleosides.

[0031] It should be noted that in the translation of SEQ ID NO:XX in the content of , since the original text is incomplete, it is temporarily replaced with XX. You can adjust it according to the complete information. In some embodiments, the antisense oligonucleotide comprises one or more LNA nucleosides, such as β-D-oxy-LNA nucleosides.

[0032] In some embodiments, the antisense oligonucleotide comprises one or more 2'-O-methylRNA nucleosides.

[0033] In some embodiments, the antisense oligonucleotide is a mixmer or totalmer, and optionally, the mixmer does not contain a DNA or RNA nucleoside.

[0034] In some embodiments, the antisense oligonucleotide is a mixmer of a 2'-MOE and an LNA nucleoside, or a totalmer of a 2'-MOE nucleoside.

[0035] In some embodiments, the antisense oligonucleotide includes at least one modified nucleoside bond.

[0036] In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioate nucleoside interbonds.

[0037] In some embodiments, all nucleoside-to-nucleoside bonds in the antisense oligonucleotide are phosphorothioate nucleoside-to-nucleoside bonds.

[0038] In some embodiments, antisense oligonucleotides can increase GBA expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to a control, optionally.

[0039] In some embodiments, the antisense oligonucleotide is covalently attached to at least one conjugate region.

[0040] In some embodiments, the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt.

[0041] In some embodiments, the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

[0042] In some embodiments, the antisense oligonucleotide may be encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0043] In a second aspect, the present invention provides a pharmaceutical composition comprising an antisense oligonucleotide according to the first aspect and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0044] In some embodiments, the pharmaceutical composition includes an aqueous diluent or solvent, such as phosphate-buffered saline.

[0045] In a third aspect, the present invention provides an antisense oligonucleotide according to the first aspect or a pharmaceutical composition according to the second aspect for use as a pharmaceutical.

[0046] In a fourth aspect, the present invention provides an in vitro or in vivo method for increasing or restoring GBA expression in target cells, comprising administering an effective amount of an antisense oligonucleotide according to the first aspect or a pharmaceutical composition according to the second aspect to the target cells.

[0047] In some embodiments, the cells are mammalian cells, such as human cells.

[0048] In some embodiments, GBA expression is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to a control.

[0049] In some embodiments, the control is a target cell that is not administered with the antisense oligonucleotide.

[0050] In a fifth aspect, the present invention provides a method for treating or preventing a disease or disorder, comprising administering a therapeutically effective amount or a preventively effective amount of an antisense oligonucleotide according to the first aspect, or a pharmaceutical composition according to the second aspect, to a subject suffering from or susceptible to the disease or disorder.

[0051] In a sixth aspect, the present invention provides an antisense oligonucleotide according to the first aspect or a pharmaceutical composition according to the second aspect for use in treating or preventing a disease or disorder.

[0052] In a seventh aspect, the present invention provides the use of an antisense oligonucleotide according to the first aspect or a pharmaceutical composition according to the second aspect for preparing a pharmaceutical for treating or preventing a disease or disorder in a subject.

[0053] In some embodiments of the method according to the fifth aspect, the antisense oligonucleotide or pharmaceutical composition for use according to the sixth aspect, or the use according to the seventh aspect, the disease or disorder is related to reduced expression of GBA.

[0054] In some embodiments, the disease is selected from the group consisting of Gaucher disease, Parkinson's disease, dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder.

[0055] In some embodiments, the disease is Parkinson's disease.

[0056] In some embodiments, the disease is Gaucher disease.

[0057] These and other aspects and embodiments of the present invention will be described in further detail below. [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 shows the GBA mRNA expression levels in H4 glioma cells 48 hours post-transfection compared to a mock transfection control. The gray and black bars represent antisense oligonucleotide concentrations of 5 nM and 25 nM, respectively. Antisense oligonucleotides are indicated herein by sequence numbers having the same numbers as their corresponding compound ID numbers. [Modes for carrying out the invention]

[0059] The inventors have identified that the expression level of the GBA protein product can be effectively increased by targeting GBA mRNA transcripts with antisense oligonucleotides. In particular, the inventors have surprisingly determined that targeting the 3' untranslated region (3'UTR) of the GBA mRNA transcript may be effective.

[0060] Target sites located on human GBA nucleic acid targets, such as GBA mRNA sequences, that can be targeted by the antisense oligonucleotides of the present invention are described herein.

[0061] While we do not wish to be constrained by theory, antisense oligonucleotides are thought to interfere with the microRNA (miR)-mediated degradation of GBA mRNA transcripts, thereby increasing GBA protein expression. In particular, antisense oligonucleotides are thought to interfere with the degradation of GBA mRNA transcripts mediated by miR-22-3p or its variants.

[0062] miR-22-3p (SEQ ID NO: 74) is a non-coding RNA that can act as a single-stranded guide sequence for the miRNA-induced silencing complex (miRISC) to induce GBA mRNA degradation and translational repression. Potential variants of miR-22-3p include those containing a seed region fully complementary to GGCAGCT, and those containing a seed region fully complementary to GGCAGCT with one or two mismatches, e.g., one mismatch. For example, a variant of miR-22-3p may have at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95% % sequence identity with SEQ ID NO: 74, and may contain a seed region that is fully complementary to GGCAGCT, or one or two mismatches. Preferably, a variant of miR-22-3p contains a seed region fully complementary to GGCAGCT.

[0063] Increased expression of GBA The oligonucleotides of the present invention can increase GBA expression.

[0064] Increased GBA expression is desirable to address widespread impairments characterized by or caused by decreased GBA expression. These include Gaucher disease, Parkinson's dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder.

[0065] Unless otherwise indicated or unless inconsistent with the context, terms such as “increase GBA expression,” “upregulate GBA,” “enhance GBA levels,” and “restore GBA expression” as used herein should be understood to refer to or relate to an increase in GBA mRNA transcript, an increase in GBA protein, or an increase in both GBA mRNA and GBA protein in cells.

[0066] Advantageously, the increase in GBA expression induced by an oligonucleotide may be identified in cells exposed to the oligonucleotide compared to a control. The control is typically the GBA expression level in cells not exposed to the oligonucleotide. For example, control cells may be cells treated with a non-targeted oligonucleotide, or cells exposed to a mock transfection in which cells were treated with PBS alone, for example. Alternatively, the control may be a control GBA value that refers to the level of GBA mRNA and / or GBA protein in cells before exposure to the oligonucleotide. Also, when evaluating the ability of an oligonucleotide to restore GBA expression, the cells being tested for increased GBA expression may be cells with lower-than-normal GBA expression, and the control may be cells with a control value that reflects normal GBA expression or normal GBA levels.

[0067] In certain embodiments, oligonucleotides capable of increasing GBA expression may increase GBA mRNA levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression may increase GBA mRNA levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0068] In certain embodiments, oligonucleotides capable of increasing GBA expression may increase GBA protein levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression according to the present invention may increase GBA protein levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0069] In certain embodiments, oligonucleotides capable of increasing GBA expression according to the present invention can increase GBA mRNA and protein levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression according to the present invention can increase GBA mRNA and GBA protein levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0070] In certain embodiments, oligonucleotides capable of increasing GBA expression may increase GBA mRNA levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression may increase GBA mRNA levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0071] In certain embodiments, oligonucleotides capable of increasing GBA expression may increase GBA protein levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression according to the present invention may increase GBA protein levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0072] In certain embodiments, oligonucleotides capable of increasing GBA expression may increase GBA mRNA and GBA protein levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of increasing GBA expression according to the present invention may increase GBA mRNA and GBA protein levels by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0073] In certain embodiments, oligonucleotides capable of restoring GBA expression can restore GBA mRNA levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of restoring GBA expression can restore GBA mRNA levels by at least about 10%, for example, at least about 15%, for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more compared to a control.

[0074] In certain embodiments, oligonucleotides capable of restoring GBA expression can restore GBA protein levels by at least about 5% compared to a control. More preferably, oligonucleotides capable of restoring GBA expression according to the present invention can restore GBA protein levels by at least about 10%, for example, at least about 15%, for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more compared to a control.

[0075] In certain embodiments, oligonucleotides capable of restoring GBA expression according to the present invention can restore GBA mRNA and protein levels to at least about 5% compared to a control. More preferably, oligonucleotides capable of restoring GBA expression according to the present invention can restore GBA mRNA and GBA protein levels to at least about 10%, for example, at least about 15%, for example, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more compared to a control.

[0076] While we do not wish to be bound by theory, the increase brought about by oligonucleotides is thought to be related to their ability to reduce, remove, prevent, mitigate, decrease, or terminate the degradation or translational repression of GBA mRNA transcripts by binding to the 3'-UTR region of GBA mRNA transcripts, thereby reducing, blocking, or preventing the binding of the MiR-22-3p-mediated miRNA-induced silencing complex (miRISC) or its variants to the 3'-UTR region of GBA mRNA transcripts. This increase can also be seen as the ability of oligonucleotides to restore GBA expression to normal levels, for example, by reducing or removing the degradation of GBA mRNA transcripts or by increasing their translational output.

[0077] As a result, the oligonucleotides of the present invention can also, or alternatively, reduce the downregulation of microRNA (miR)-mediated GBA expression in target cells, the miR being selected from miR-22-3p and its variants. Preferably, the miR-22-3p variant contains a seed region that is fully complementary to at least six consecutive nucleotides of GGCAGCT, for example, a seed region fully complementary to GGCAGCT.

[0078] As used herein, downregulation of GBA expression should be understood to refer to or relate to downregulation of GBA mRNA levels, downregulation of GBA protein levels, or downregulation of both GBA mRNA and GBA protein levels, typically in cells, favorably compared to a control. The control is typically the degree of downregulation of GBA levels in cells not exposed to oligonucleotides. For example, control cells may be cells treated with a non-targeted oligonucleotide, or cells exposed to a mock transfection treated with PBS alone, for example. Alternatively, the control may be a control value referring to the degree of downregulation of GBA mRNA and / or GBA protein in cells before exposure to oligonucleotides. As described herein, a decrease in downregulation of GBA expression typically results in an increase in GBA levels.

[0079] Preferably, the oligonucleotides of the present invention can reduce the downregulation of GBA expression mediated by miR-22-3p or its variants by at least about 5%, for example, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%, compared to a control.

[0080] A suitable assay for evaluating the effect of oligonucleotides on GBA mRNA expression is described in Example 1. Other suitable assays for evaluating miR-22-3p-mediated downregulation of GBA mRNA and / or GBA protein levels or GBA expression are known in the art (e.g., Straniero et al., Sci Rep. 2017 Oct 5;7(1):12702).

[0081] The oligonucleotides particularly intended for this invention include antisense oligonucleotides of 8 to 40 nucleotides in length, comprising a sequence of nucleotides complementary to at least six, for example, at least seven, for example, at least eight consecutive bases in the 3' untranslated region (UTR) of the RNA sequence encoding GBA. The RNA sequence encoding GBA is preferably an mRNA sequence comprising the sequence of SEQ ID NO: 73 or an allele variant thereof.

[0082] The oligonucleotides of the present invention may also, or alternatively, inhibit the binding of miR-22-3p and its variants to the RNA sequence encoding GBA. Preferably, the variants contain a seed region that is fully complementary to GGCAGCT.

[0083] Unless otherwise specified or inconsistent with the context, the terms “inhibit,” “block,” or “reduce” the binding of a GBA-encoding RNA sequence, such as miR-22-3p or its variants, to miR should be understood to refer to or relate to the ability of an oligonucleotide to reduce the amount of miR binding to the RNA sequence, preferably compared to a control. A suitable control may be the amount of miR binding to the RNA sequence in the absence of the oligonucleotide, or the level of miR binding to the RNA sequence in the presence of an unrelated control oligonucleotide.

[0084] Preferably, the oligonucleotides of the present invention can inhibit the binding of RNA sequences encoding GBA to miR-22-3p and its variants to miRs selected from these miRs by at least about 5%, for example, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%, compared to a control.

[0085] The present invention also relates to blockmirs. As used herein, “blockmir” is an oligonucleotide containing a sequence of nucleotides complementary to the untranslated region of an mRNA sequence targeted by a miRNA, typically an mRNA sequence. Thus, blockmirs may be able to at least partially inhibit or block the binding of a miRNA to the same site.

[0086] Therefore, in some embodiments, the oligonucleotides of the present invention are block mias, particularly block mias complementary to the mRNA sequence targeted by miRNA-22-3p or its variants. Preferably, the untranslated region of the mRNA targeted by miRNA-22-3p or its variants is the 3'UTR of the mRNA sequence encoding the GBA.

[0087] The block mia particularly intended by the present invention comprises an oligonucleotide of 8 to 40 nucleotides in length, comprising a sequence of nucleotides complementary to at least six, for example, at least seven, for example, at least eight consecutive bases in the 3' untranslated region (UTR) of the RNA sequence encoding GBA. The RNA sequence encoding GBA is preferably an mRNA sequence comprising the sequence of SEQ ID NO: 73 or an allele variant thereof.

[0088] Target sites located in the 3'UTR of human GBA mRNA sequences that can be appropriately targeted by the oligonucleotides of the present invention, such as block mia, are described herein.

[0089] The present invention also relates to GBA agonists. As used herein, the term “GBA agonist” refers to a compound, in this case oligonucleotide or its conjugate, that can increase intracellular GBA, i.e., GBA mRNA transcripts, GBA proteins, or both GBA mRNA transcripts and GBA proteins. Typically, a cell is one that can express several GBA mRNA transcripts and / or GBA proteins. Enhanced GBA expression is desirable, for example, to treat Gaucher disease and / or Parkinson's disease, as described herein.

[0090] Advantageously, GBA agonist activity can be identified in cells exposed to a GBA agonist compared to a control. The control is typically GBA in cells not exposed to an oligonucleotide. For example, control cells could be cells treated with a non-targeted oligonucleotide, or cells exposed to a mock transfection treated with, for example, PBS alone. Alternatively, the control could be a control GBA value referring to GBA mRNA and / or GBA protein in cells before exposure to a GBA agonist. Furthermore, when evaluating GBA agonist activity, cells testing for increased GBA expression may be cells with lower-than-normal GBA expression, and the control could be cells with normal GBA expression or a control value reflecting normal GBA.

[0091] In certain embodiments, a GBA agonist can increase GBA mRNA by at least about 5% compared to a control. More preferably, a GBA agonist can increase GBA mRNA by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0092] In certain embodiments, a GBA agonist can increase GBA protein by at least about 5% compared to a control. More preferably, a GBA agonist can increase GBA protein by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0093] In certain embodiments, a GBA agonist can increase GBA mRNA and GBA protein by at least about 5% compared to a control. More preferably, a GBA agonist can increase GBA mRNA and GBA protein by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

[0094] The GBA agonist particularly intended for this invention is an oligonucleotide or conjugate of 8 to 40 nucleotides in length, comprising a sequence of nucleotides complementary to at least six, for example, at least seven, for example, at least eight consecutive bases in the 3' untranslated region (UTR) of the RNA sequence encoding GBA. The RNA sequence encoding GBA is preferably an mRNA sequence comprising the sequence of SEQ ID NO: 73 or an allele variant thereof.

[0095] oligonucleotides As used herein, the term “oligonucleotide” is defined as a molecule comprising two or more nucleosides linked by covalent bonds, as is commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0096] Oligonucleotides are typically prepared in the laboratory by solid-phase chemical synthesis, followed by purification and isolation. When referring to the sequence of an oligonucleotide, the sequence or order of the nucleic acid base portions of the covalently linked nucleotides or nucleosides, or their modifications, are referred to. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides, such as 2'-saccharide modified nucleosides. The oligonucleotides of the present invention may contain one or more modified nucleoside bonds, such as one or more phosphorothioate nucleoside bonds.

[0097] The oligonucleotides according to the present invention can target GBA mRNA transcripts and may also be referred to herein as “antisense oligonucleotides”.

[0098] The oligonucleotides of the present invention may be single-stranded or double-stranded oligonucleotides. In some preferred embodiments, the oligonucleotides of the present invention are single-stranded oligonucleotides.

[0099] In some embodiments, the oligonucleotides of the present invention are 8 to 40 nucleotides long.

[0100] In some embodiments, the oligonucleotides of the present invention are 8 to 40 nucleotides long and include at least 6 nucleotides, for example, a continuous nucleotide sequence of 6 to 40 nucleotides.

[0101] In some embodiments, the oligonucleotides of the present invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

[0102] In some embodiments, the oligonucleotide of the present invention is at least 12 nucleotides long.

[0103] In some embodiments, the oligonucleotides of the present invention are at least 14 nucleotides long.

[0104] In some embodiments, the oligonucleotide of the present invention is at least 16 nucleotides long.

[0105] In some embodiments, the oligonucleotide of the present invention is at least 18 nucleotides long.

[0106] In some embodiments, the oligonucleotides of the present invention are 16 to 20 nucleotides long, for example, 16, 18, or 20 nucleotides long.

[0107] It is understood that a continuous nucleotide sequence of oligonucleotides cannot be longer than the oligonucleotide itself, and that an oligonucleotide cannot be shorter than a continuous nucleotide sequence.

[0108] In the case of double-stranded oligonucleotides, length measurement refers to the length of the chain containing a sequence of nucleotides complementary to at least six consecutive bases in the 3' untranslated region (UTR) of the RNA sequence encoding glucocerebrosidase (GBA).

[0109] In some embodiments, the oligonucleotide comprises a sequence of nucleotides and optionally includes a nucleotide linker region which can be used to attach further nucleotides, such as functional groups (e.g., conjugate groups), to the sequence of nucleotides. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0110] Sequential nucleotide sequence The term "continuous nucleotide sequence" refers to a region of oligonucleotides that is complementary to the target nucleic acid, which may be or may contain an oligonucleotide motif sequence. This term is used herein interchangeably with the term "continuous nucleic acid base sequence."

[0111] An oligonucleotide comprises a sequence of nucleotides and optionally includes a nucleotide linker region which can be used to attach further nucleotides, such as functional groups (e.g., conjugate groups), to the sequence of nucleotides. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0112] It is understood that a continuous nucleotide sequence of oligonucleotides cannot be longer than the oligonucleotide itself, and that an oligonucleotide cannot be shorter than a continuous nucleotide sequence.

[0113] In some embodiments, the entire nucleotide sequence of the oligonucleotide of the present invention is a continuous nucleotide sequence.

[0114] The continuous nucleotide sequence is a sequence of nucleotides in the oligonucleotide of the present invention that is complementary, and in some cases completely complementary, to the target nucleic acid, target sequence, or target site sequence.

[0115] Target nucleic acids, target sequences, or target site sequences that can be appropriately targeted by the oligonucleotides of the present invention are described elsewhere.

[0116] In some embodiments, the continuous nucleotide sequence includes a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 6 nucleotides from any of them.

[0117] In some embodiments, the continuous nucleotide sequence includes a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 8 nucleotides from any of them.

[0118] In some embodiments, the continuous nucleotide sequence includes a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 10 nucleotides from any of them.

[0119] In some embodiments, the continuous nucleotide sequence has a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 6 nucleotides from any of them.

[0120] In some embodiments, the continuous nucleotide sequence has a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 8 nucleotides from any of them.

[0121] In some embodiments, the continuous nucleotide sequence has a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, or a fragment of at least 10 nucleotides from any of them.

[0122] In some embodiments, the continuous nucleotide sequence includes a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 16, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

[0123] In some embodiments, the continuous nucleotide sequence has a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 16, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

[0124] Sequence IDs 1-34 are RNA nucleic acid base sequences.

[0125] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 1.

[0126] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 2.

[0127] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 3.

[0128] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 4.

[0129] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 5.

[0130] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 6.

[0131] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 7.

[0132] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 8.

[0133] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 9.

[0134] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 10.

[0135] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 11.

[0136] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 12.

[0137] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 13.

[0138] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 14.

[0139] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 15.

[0140] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 16.

[0141] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 17.

[0142] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 18.

[0143] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 19.

[0144] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 20.

[0145] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 21.

[0146] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 22.

[0147] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 23.

[0148] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 24.

[0149] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 25.

[0150] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 26.

[0151] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 27.

[0152] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 28.

[0153] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 29.

[0154] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 30.

[0155] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 31.

[0156] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 32.

[0157] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 33.

[0158] In some embodiments, the continuous nucleotide sequence includes the nucleic acid base sequence of SEQ ID NO: 34.

[0159] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 1 or a fragment thereof.

[0160] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 2 or a fragment thereof.

[0161] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 3 or a fragment thereof.

[0162] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 4 or a fragment thereof.

[0163] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 5 or a fragment thereof.

[0164] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 6 or a fragment thereof.

[0165] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 7 or a fragment thereof.

[0166] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 8 or a fragment thereof.

[0167] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 9 or a fragment thereof.

[0168] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 10 or a fragment thereof.

[0169] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 11 or a fragment thereof.

[0170] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 12 or a fragment thereof.

[0171] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 13 or a fragment thereof.

[0172] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 14 or a fragment thereof.

[0173] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 15 or a fragment thereof.

[0174] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 16 or a fragment thereof.

[0175] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 17 or a fragment thereof.

[0176] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 18 or a fragment thereof.

[0177] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 19 or a fragment thereof.

[0178] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 20 or a fragment thereof.

[0179] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 21 or a fragment thereof.

[0180] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 22 or a fragment thereof.

[0181] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 23 or a fragment thereof.

[0182] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 24 or a fragment thereof.

[0183] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 25 or a fragment thereof.

[0184] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 26 or a fragment thereof.

[0185] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 27 or a fragment thereof.

[0186] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 28 or a fragment thereof.

[0187] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 29 or a fragment thereof.

[0188] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 30 or a fragment thereof.

[0189] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 31 or a fragment thereof.

[0190] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 32 or a fragment thereof.

[0191] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence or a fragment thereof of SEQ ID NO: 33.

[0192] In some embodiments, the continuous nucleotide sequence has the nucleic acid base sequence of SEQ ID NO: 34 or a fragment thereof.

[0193] In some embodiments, the fragment may be at least 6, at least 7, 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, or at least 19 consecutive nucleotides of a consecutive nucleotide sequence. Preferably, the fragment is at least 6 consecutive nucleotides, for example, at least 8 consecutive nucleotides, for example, at least 10 consecutive nucleotides.

[0194] In some embodiments, the continuous nucleotide sequence is 6 to 40 nucleotides long.

[0195] In some embodiments, the continuous nucleotide sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

[0196] In some embodiments, the continuous nucleotide sequence is 6 nucleotides long.

[0197] In some embodiments, the continuous nucleotide sequence is 8 nucleotides long.

[0198] In some embodiments, the continuous nucleotide sequence is 10 nucleotides long.

[0199] In some embodiments, the continuous nucleotide sequence is 12 nucleotides long.

[0200] In some embodiments, the continuous nucleotide sequence is 14 nucleotides long.

[0201] In some embodiments, the continuous nucleotide sequence is 16 nucleotides long.

[0202] In some embodiments, the continuous nucleotide sequence is 18 nucleotides long.

[0203] In a preferred embodiment, the continuous nucleotide sequence is 16 to 20 nucleotides long, for example, 16, 18, or 20 nucleotides long.

[0204] More preferably, the continuous nucleotide sequence is 18 to 20 nucleotides long.

[0205] In some embodiments, the continuous nucleotide sequence is the same length as the antisense oligonucleotide.

[0206] In some embodiments, the oligonucleotides of the present invention consist of a continuous nucleotide sequence.

[0207] In some embodiments, the oligonucleotides of the present invention are a continuous nucleotide sequence.

[0208] In the case of certain sequential nucleotide sequences and oligonucleotides disclosed herein, where a cytosine (C) residue is annotated as 5-methylcytosine (E), in various embodiments, one or more C residues present in the oligonucleotide may be unmodified C residues or modified cytosine residues other than E.

[0209] Nucleotides and nucleosides Nucleotides and nucleosides are the constituent units of oligonucleotides and polynucleotides, and in this invention, this includes both naturally occurring nucleotides and nucleosides, as well as those that do not exist naturally. Nucleotides, such as DNA and RNA nucleotides, naturally consist of a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides may also be interchangeably referred to as “units” or “monomers.”

[0210] Modified nucleoside The terms “modified nucleoside” or “nucleoside modification,” as used herein, refer to a nucleoside modified compared to an equivalent DNA or RNA nucleoside by introducing one or more modifications to the sugar moiety or (nucleic acid) base moiety.

[0211] Advantageously, the oligonucleotide according to the present invention may comprise one or more modified nucleosides.

[0212] In some embodiments, a continuous nucleic acid sequence (motif sequence) may be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid. Advantageously, high-affinity modified nucleosides are used.

[0213] Advantageously, one or more modified oligonucleotide nucleosides according to the present invention may include a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the terms “nucleoside analog” or modified “unit” or modified “monomer.” Nucleosides having an unmodified DNA or RNA sugar moiety are referred herein as DNA or RNA nucleosides. Nucleosides having modifications to the base region of a DNA or RNA nucleoside are still generally referred to as DNA or RNA if they are Watson-Crick base-pairable.

[0214] Examples of modified nucleosides that can be used in oligonucleotides according to the present invention include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, morpholino, and locked nucleic acid (LNA) nucleosides, such as nucleosides from the group consisting of LNA, 2'-O-MOE, and 2'OMe nucleoside analogs.

[0215] In one embodiment, the oligonucleotide comprises one or more 2'-MOE RNA nucleosides.

[0216] In one embodiment, the oligonucleotide comprises one or more LNA nucleosides, such as β-D-oxy-LNA nucleosides.

[0217] In one embodiment, the oligonucleotide comprises one or more 2'-O-methylRNA nucleosides.

[0218] Inter-modified nucleoside bonding Advantageously, the oligonucleotide according to the present invention comprises one or more modified nucleoside interbonds.

[0219] The term "modified nucleoside bond" is defined as is generally understood by those skilled in the art to be a bond other than a phosphodiester (PO) bond that covalently conjugates two nucleosides together. Accordingly, the oligonucleotides of the present invention may contain one or more modified nucleoside bonds, such as nucleoside bonds of one or more phosphorothioates.

[0220] In some embodiments, at least 50%, e.g., at least 60%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, e.g., at least 90%, or more, of the internucleoside bonds in the oligonucleotide or its sequential nucleotide sequence according to the present invention are phosphorothioates. In some embodiments, all of the internucleoside bonds in the oligonucleotide or its sequential nucleotide sequence according to the present invention are phosphorothioates.

[0221] In further embodiments, the oligonucleotide according to the present invention comprises at least one modified internucleoside bond. It is advantageous if at least 75%, for example all, of the internucleoside bonds in the continuous nucleotide sequence are phosphorothioate or boranophosphate internucleoside bonds.

[0222] Advantageously, all nucleoside-to-nucleoside bonds in the continuous nucleotide sequence of the oligonucleotide according to the present invention may be phosphorothioates, or all nucleoside-to-nucleoside bonds in the oligonucleotide according to the present invention may be phosphorothioate bonds.

[0223] Nucleic acid bases The term "nucleic acid base" includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds in nucleic acid hybridization. In the context of this invention, the term "nucleic acid base" may differ from naturally occurring nucleic acid bases, but also includes modified nucleic acid bases that are functional during nucleic acid hybridization. In this context, "nucleic acid base" refers to both naturally occurring nucleic acid bases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

[0224] In some embodiments, the oligonucleotide according to the present invention comprises one or more modified nucleosides, which are modified nucleic acid bases.

[0225] In some embodiments, the nucleic acid base moiety is modified by replacing the purine or pyrimidine with a nucleic acid base selected from modified purines or pyrimidines, such as substituted purines or pyrimidines, for example, isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolocytosine, 5-propynylcytosine, 5-propynyluracil, 5-bromouracil, 5-thiazolouracil, 2-thiouracil, 2'thiothymine, PPG (7-deaza-8-aza-dG-CE phosphoramidite), inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0226] The nucleic acid base portion may be represented by a letter code for each corresponding nucleic acid base, for example, A, T, G, C, or U, where each letter may optionally contain a functionally equivalent modified nucleic acid base. For example, in the illustrated oligonucleotide, the nucleic acid base portion is selected from A, T, G, C, and 5-methylcytosine. Optionally, 5-methylcytosine LNA nucleoside may be used for LNA gapmers. 5-methylcytosine may be denoted as "E".

[0227] Unless otherwise indicated or unless inconsistent with the context, in this disclosure, an oligonucleotide or target sequence that is an RNA sequence may be presented herein together with a thymine (T) nucleic acid base representing a uracil (U) nucleic acid base.

[0228] Modified oligonucleotides The oligonucleotide according to the present invention may be a modified oligonucleotide.

[0229] The term "modified oligonucleotide" describes an oligonucleotide comprising one or more sugar-modified nucleosides and / or modified internucleoside bonds. The term "chimeric oligonucleotide" is a term used in the literature to describe oligonucleotides comprising sugar-modified nucleosides and DNA nucleosides. In some embodiments, it may be advantageous for the oligonucleotide according to the present invention to be a chimeric oligonucleotide.

[0230] In some embodiments, the oligonucleotide or the sequence of nucleotides according to the present invention may include modified nucleic acid bases that function as nucleic acid bases in base pairing, for example, 5-methylcytosine may be used instead of methylcytosine. Inosine may be used as a universal base.

[0231] It is understood that continuous nucleic acid base sequences (motif sequences) can be modified, for example, to increase nuclease resistance and / or binding affinity to target nucleic acids.

[0232] The pattern in which modified nucleosides (such as high-affinity modified nucleosides) are incorporated into oligonucleotide sequences is generally referred to as oligonucleotide design.

[0233] In one embodiment, the oligonucleotide according to the present invention comprises at least one modified nucleoside, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or at least nineteen modified nucleosides.

[0234] Appropriate modifications are described herein under the headings “Modified Nucleosides,” “High-Affinity Modified Nucleosides,” “Sugar Modifications,” “2' Sugar Modifications,” and “Locked Nucleic Acids (LNAs).”

[0235] High affinity modified nucleoside When high affinity modified nucleosides are incorporated into oligonucleotides, for example, their melting temperature (T m The high-affinity modified nucleoside of the present invention is a modified nucleoside that enhances the affinity of the oligonucleotide to its complementary target, as measured by [method / method]. The high-affinity modified nucleoside of the present invention preferably results in an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high-affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0236] sugar modification The oligonucleotide according to the present invention may contain one or more nucleosides having a modified sugar moiety, i.e., a modified sugar moiety compared to the ribose sugar moiety found in DNA and RNA.

[0237] Numerous nucleosides with ribose sugar moieties have been created primarily to improve certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0238] Such modifications include, for example, modifications to the ribose ring structure by replacing it with a hexose ring (HNA), or typically a bicyclic ring (LNA) having a biradicle bridge between the C2 and C4 carbons on the ribose ring, or typically an unconnected ribose ring lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (International Publication No. 2011 / 017521) or tricyclic nucleic acids (International Publication No. 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example, a peptide nucleic acid (PNA) or a morpholino nucleic acid.

[0239] Sugar modifications also include modifications made by changing substituents on the ribose ring to hydrogen or groups other than the naturally occurring 2'-OH groups in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' positions.

[0240] 2' sugar-modified nucleoside 2'-sugar-modified nucleosides are nucleosides having substituents other than H or -OH at the 2' position (2'-substituted nucleosides), or nucleosides containing 2'-linked biradicals that can form a bridge between the 2' carbon and the second carbon of the ribose ring, such as LNA (2'-4' biradical-linked) nucleosides.

[0241] Numerous 2'-substituted nucleosides have been found to possess beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can result in improved binding affinity to oligonucleotides and / or improved nuclease resistance. Examples of 2'-substituted nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for instance, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Examples of several 2' substitution-modified nucleosides are shown in Scheme 1.

[0242] Scheme 1: [ka] [ka]

[0243] In relation to the present invention, the 2'-substituted sugar-modified nucleoside does not contain a 2'-crosslinked nucleoside such as LNA.

[0244] In one embodiment, the oligonucleotide according to the present invention comprises one or more sugar-modified nucleosides, for example, 2'-sugar-modified nucleosides.

[0245] Preferably, the oligonucleotide according to the present invention comprises one or more 2'-saccharide-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), and 2'-fluoro-ANA nucleosides, for example, selected from 2'MOE and 2'oMe.

[0246] Locked nucleic acid nucleosides (LNA nucleosides) LNA nucleosides are modified nucleosides containing a biradical (also called a "2'-4' bridge") that links the C2' and C4' of the ribose sugar ring of the nucleoside, thereby restricting or fixing the conformation of the ribose ring. These nucleosides are also referred to in the literature as cross-linked nucleic acids or bicyclic nucleic acids (BNAs). Fixation of the ribose conformation is associated with improved hybridization affinity (double-strand stabilization) when LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary double-strand.

[0247] Non-restrictive, exemplary LNA nucleosides are described in International Publications 99 / 014226, 00 / 66604, 98 / 039352, 2004 / 046160, 00 / 047599, 2007 / 134181, 2010 / 077578, 2010 / 036698, 2007 / 090071, 2009 / 006478, 2011 / 156202, 2008 / 154401, 2009 / 067647, 2008 / 150729, Morita et al. This is disclosed in Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0248] Further non-limiting and exemplary LNA nucleosides are disclosed in Scheme 2.

[0249] Scheme 2: [ka]

[0250] Specific LNA nucleosides include β-D-oxy-LNA, 6'-methyl-β-D-oxy-LNA, e.g., (S)-6'-methyl-β-D-oxy-LNA(ScET) and ENA.

[0251] The most advantageous LNA is β-D-oxy-LNA.

[0252] Morpholino oligonucleotides In some embodiments, oligonucleotides capable of increasing GBA expression according to the present invention comprise or consist of a morpholino nucleoside (i.e., a morpholino oligomer as a phosphorodiamidate morpholino oligomer (PMO)). For splice-modulated morpholino oligonucleotides, see eteplirsene, a 30nt morpholino oligonucleotide that targets frameshift mutations in DMD and is approved for clinical use, for example, used to treat Duchenne muscular dystrophy. Morpholino oligonucleotides have nucleic acid bases attached to a six-membered morpholino ring rather than a ribose, such as a methylenemorpholine ring linked via a phosphorodiamidate group, as shown in the following diagram of four consecutive morpholino nucleotides. [ka]

[0253] In some embodiments, the morpholino oligonucleotides according to the present invention may have a morpholino nucleotide length of, for example, 8 to 40 morpholino nucleotides, for example, 16 to 20 morpholino nucleotides, or for example, 18 to 20 morpholino nucleotides.

[0254] Activation and recruitment of RNase H RNase H activity of an oligonucleotide refers to its ability to recruit RNase H when in a double helix with a complementary RNA molecule. International Publication No. 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H if, given a complementary target nucleic acid sequence, it is measured in pmol / l / min and has the same base sequence as the modified oligonucleotide being tested, but contains only DNA monomers and has phosphorothioate bonds between all monomers of the oligonucleotide, and if it has at least 5%, e.g., at least 10%, at least 20%, or more than 20% of the initial rate determined using the methodology provided in Examples 91-95 of International Publication No. 01 / 23613 (incorporated herein by reference). Recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland for use in determining RNase H activity.

[0255] DNA oligonucleotides are known to effectively recruit RNase H, such as gapmer oligonucleotides containing regions of DNA nucleosides (typically at least 5 or 6 consecutive DNA nucleosides) where a 2'-glycosylated nucleoside, typically a high-affinity 2'-glycosylated nucleoside, such as a 2-O-MOE and / or LNA-containing region, is adjacent to the 5' and 3' ends.

[0256] To effectively increase GBA expression, degradation of GBA mRNA is undesirable, and therefore, it is preferable to avoid RNase H-mediated degradation of the target. Accordingly, the oligonucleotides of the present invention are not RNase H-mobilizing oligonucleotides, such as gapmer oligonucleotides. RNase H mobilization can be avoided by limiting the number of consecutive DNA nucleotides in the oligonucleotide. Alternatively, oligonucleotide designs that do not mobilize RNase H, such as the mixmers and totalmers described herein, may be used.

[0257] Advantageously, the oligonucleotide or sequential nucleotide sequence of the present invention contains up to four consecutive DNA nucleosides, for example up to three consecutive DNA nucleosides, for example up to two consecutive DNA nucleosides, for example up to one DNA nucleoside, or does not contain any DNA nucleosides.

[0258] Mixmer and Totalmer Mixmers and totalmers are stereoblocking oligonucleotide designs that do not recruit RNase H.

[0259] A mixmer is an oligonucleotide or a sequence of nucleotides thereof containing at least one modified nucleoside. For example, a mixmer may contain a short region of a sugar-modified nucleoside, such as a 2' sugar-modified nucleoside, and a DNA nucleoside, such as one, two, or three DNA nucleosides. Non-limiting examples of mixmer designs include a design where the nucleosides alternate between one LNA nucleoside and one DNA nucleoside, with LNA nucleosides at the 5' and 3' ends, e.g., LDLDLDLDLDLDLDLL, and a design where the nucleosides are LNA nucleosides every three nucleosides, e.g., LDDLDDLDDLDDLDDL. Alternatively, a mixmer may contain a mixture of modified nucleosides such as MLMLMLMLMLMLMLMLML, where L=LNA and M=2'-O-MOE nucleosides. Other mixmer designs include an 18-mer containing 15 2'-O-MOE nucleosides and 3 LNAs at the 3' end.

[0260] A totalmer is an oligonucleotide or a sequence of nucleotides that does not contain DNA or RNA nucleosides, and may contain only 2'-O-MOE nucleosides, such as a complete MOE phosphorothioate, e.g., MMMMMMMMMMMMMMMMMMMM, where M = 2'-O-MOE, or may contain only 2'oMe nucleosides, e.g.

[0261] Advantageously, the internucleosides in the mixmer and totalmer may be phosphorothioates, or the majority of the nucleoside bonds in the mixmer may be phosphorothioates. The mixmer and totalmer may also, or alternatively, contain other internucleoside bonds, such as phosphodiesters or phosphorodithioates, for example.

[0262] In some embodiments, the oligonucleotide of the present invention is a mixer or a totalmer, or comprises a mixer or a totalmer.

[0263] In some embodiments, the oligonucleotide of the present invention is a mixer or comprises a mixer.

[0264] In some embodiments, the oligonucleotide of the present invention is a totalmer or includes a totalmer.

[0265] In some embodiments, the continuous nucleotide sequence of the oligonucleotide of the present invention is a mixmer or a totalmer.

[0266] In some embodiments, the continuous nucleotide sequence of the oligonucleotide of the present invention is a mixmer.

[0267] In some embodiments, the continuous nucleotide sequence of the oligonucleotide of the present invention is a totalmer.

[0268] target sequence The oligonucleotides of the present invention target the 3' untranslated region (UTR) of a GBA mRNA transcript, preferably a human GBA mRNA transcript. This may also be referred to herein as GBA mRNA. The GBA RNA sequence or its segment may also be referred to herein as the target sequence, target nucleic acid, or target site sequence.

[0269] GBA mRNA sequences include any and all naturally occurring mRNA sequences encoding GBA proteins, such as human GBA proteins. GBA mRNA sequences are transcribed from GBA genome sequences, such as human GBA genome sequences. In some embodiments, the human GBA gene has the sequence of NCBI reference NG_009783.1. In some embodiments, the human GBA genome sequence corresponds to ENSG 00000177628(chr1:155234452-155244699, reverse strand GRCh38:CM000663.2) (SEQ ID NO: 72). In some embodiments, the GBA mRNA has the nucleotide sequence shown in SEQ ID NO: 73, which corresponds to ENST00000368373.8. However, it should be understood that GBA mRNA sequences intended as target sequences may include allelic variants of the human GBA genome sequence, such as those transcribed from allelic variants containing one or more polymorphisms.

[0270] In the GBA mRNA sequence, the target sequence is located in the 3'UTR downstream of the stop codon TAG, which is located at positions 1746-1748 in SEQ ID NO: 73. The oligonucleotides of the present invention specifically target a segment of the GBA mRNA 3'UTR that is a binding site for a miR selected from miR-22-3p and its variants. Preferably, the variants contain a seed region that is fully complementary to GGCAGCT except for one or two mismatches, e.g., one mismatch. Preferred target sequences in the 3'UTR of GBA mRNA are located in close proximity to GGCAGCT, which is located as shown at positions 2259-2265 in SEQ ID NO: 73. Target sequences located in the GBA mRNA segment corresponding to the segment defined by positions 2227-2274 in SEQ ID NO: 73 are particularly intended. In some embodiments, the target sequence is selected from a group of GBA mRNA segments defined by the following positions in the GBA genome sequence corresponding to ENSG 00000177628 or SEQ ID NO: 72: 10184-10201, 10184-10201, 10185-10202, 10186-10203, 10187-10204, 10188-10205, 10189-10206, 10190-10207, 10191-10208, 10192-10209, 10193-10210, 10194-10211, 10195-10212, 10196-10213, 10197-10214, 10198-1021 5, 10199~10216, 10200~10217, 10201~10218, 10202~10219, 10203~10220, 10204~10221, 10205~10222, 10205~10222, 10206~10223, 10207~10224, 10208~10225, 10209~10226, 10210~10227, 10211~10228, 10212~10229, 10213~10230, and 10214~10231.

[0271] Unless otherwise specified or unless inconsistent with the context, all scopes herein include both a starting and ending value.

[0272] In some embodiments, the target sequence is or includes an RNA sequence selected from SEQ ID NOs: 36; SEQ ID NOs: 37; SEQ ID NOs: 38; SEQ ID NOs: 39; SEQ ID NOs: 40; SEQ ID NOs: 41; SEQ ID NOs: 42; SEQ ID NOs: 43; SEQ ID NOs: 44; SEQ ID NOs: 45; SEQ ID NOs: 46; SEQ ID NOs: 47; SEQ ID NOs: 48; SEQ ID NOs: 49; SEQ ID NOs: 50; SEQ ID NOs: 51; SEQ ID NOs: 52; SEQ ID NOs: 53; SEQ ID NOs: 54; SEQ ID NOs: 55; SEQ ID NOs: 56; SEQ ID NOs: 57; SEQ ID NOs: 58; SEQ ID NOs: 60; SEQ ID NOs: 61; SEQ ID NOs: 62; SEQ ID NOs: 63; SEQ ID NOs: 64; SEQ ID NOs: 65; SEQ ID NOs: 66; SEQ ID NOs: 67; and SEQ ID NOs: 68 (see Table 1 for nucleic acid base sequences).

[0273] Therefore, in some embodiments, the oligonucleotides of the present invention include at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or a sequence of nucleotides that are completely complementary to the sequence of bases located downstream of the stop codon TAG at positions 1746-1748 of SEQ ID NO: 73.

[0274] In some embodiments, the oligonucleotides of the present invention contain at least 80%, at least 85%, at least 90%, at least 95%, or a fully complementary sequence of nucleotides to the miR-22-3p binding site located downstream of the stop codon TAG at positions 1746-1748 of SEQ ID NO: 73. Preferably, the miR-22-3p binding site is located in close proximity to or includes the nucleotide sequence corresponding to GGCAGCT.

[0275] In some embodiments, the oligonucleotides of the present invention comprise at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a completely complementary sequence of nucleotides to the sequence of bases located in the segment at positions 2227-2274 of SEQ ID NO: 73.

[0276] In one embodiment, the RNA sequence encoding GBA is an mRNA sequence that includes a 3'UTR sequence containing consecutive bases at positions 2227-2274 of sequence number 73.

[0277] In one embodiment, the RNA sequence encoding GBA is an mRNA sequence containing the sequence of SEQ ID NO: 73 or an allele variant thereof.

[0278] In some embodiments, the oligonucleotide of the present invention comprises a target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, or a continuous nucleotide sequence complementary to at least a 6-nucleotide fragment of any of them.

[0279] In some embodiments, the oligonucleotide of the present invention comprises a target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, or a continuous nucleotide sequence complementary to at least 8 nucleotide fragments of any of them.

[0280] In some embodiments, the oligonucleotides of the present invention comprise a contiguous nucleotide sequence that is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, or to at least 10 nucleotide fragments of any of them.

[0281] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence comprising SEQ ID NO: 35, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0282] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence comprising SEQ ID NO: 36, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0283] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence comprising SEQ ID NO: 37, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0284] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence comprising SEQ ID NO: 38, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0285] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 39, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0286] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 40, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0287] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 41, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0288] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 42, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0289] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 43, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0290] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 44, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0291] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 45, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0292] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 46, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0293] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 47, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0294] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 48, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0295] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 49, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0296] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 50, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0297] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 51, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0298] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 52, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0299] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 53, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0300] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 54, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0301] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 55, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0302] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 56, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0303] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 57, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0304] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 58, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0305] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 59, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0306] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 60, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0307] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 61, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0308] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 62, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0309] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 63, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0310] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 64, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0311] In some embodiments, the continuous nucleotide sequence is complementary to the target nucleic acid sequence comprising SEQ ID NO: 65, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0312] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 66, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0313] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 67, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0314] In some embodiments, the sequential nucleotide sequence is complementary to the target nucleic acid sequence containing SEQ ID NO: 68, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or completely complementary.

[0315] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 35 or a fragment thereof.

[0316] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 36 or a fragment thereof.

[0317] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 37 or a fragment thereof.

[0318] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 38 or a fragment thereof.

[0319] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 39 or a fragment thereof.

[0320] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 40 or a fragment thereof.

[0321] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 41 or a fragment thereof.

[0322] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 42 or a fragment thereof.

[0323] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 43 or a fragment thereof.

[0324] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 44 or a fragment thereof.

[0325] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 45 or a fragment thereof.

[0326] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 46 or a fragment thereof.

[0327] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 47 or a fragment thereof.

[0328] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 48 or a fragment thereof.

[0329] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 49 or a fragment thereof.

[0330] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 50 or a fragment thereof.

[0331] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 51 or a fragment thereof.

[0332] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 52 or a fragment thereof.

[0333] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 53 or a fragment thereof.

[0334] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 54 or a fragment thereof.

[0335] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 55 or a fragment thereof.

[0336] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 56 or a fragment thereof.

[0337] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 57 or a fragment thereof.

[0338] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 58 or a fragment thereof.

[0339] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 59 or a fragment thereof.

[0340] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 60 or a fragment thereof.

[0341] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 61 or a fragment thereof.

[0342] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 62 or a fragment thereof.

[0343] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 63 or a fragment thereof.

[0344] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 64 or a fragment thereof.

[0345] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 65 or a fragment thereof.

[0346] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 66 or a fragment thereof.

[0347] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 67 or a fragment thereof.

[0348] In some embodiments, the continuous nucleotide sequence is perfectly complementary to the target nucleic acid sequence containing SEQ ID NO: 68 or a fragment thereof.

[0349] The target sequence fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides long.

[0350] In some embodiments, the sequence of nucleotides is complementary to at least six sequence of nucleotides of any of the target sequences listed herein.

[0351] In some embodiments, the sequence of nucleotides is complementary to at least eight sequence of nucleotides of any of the target sequences listed herein.

[0352] In some embodiments, the sequence of nucleotides is complementary to at least 10 sequence of nucleotides of any of the target sequences listed herein.

[0353] In some embodiments, the sequence of nucleotides is complementary to at least 12 sequence of nucleotides of any of the target sequences listed herein.

[0354] In some embodiments, the sequence of nucleotides is complementary to at least 14 sequence of nucleotides of any of the target sequences listed herein.

[0355] In some embodiments, the sequence of nucleotides is complementary to at least 16 sequence of nucleotides of any of the target sequences listed herein.

[0356] In some embodiments, the sequence of nucleotides is complementary to at least 18 sequence of nucleotides of any of the target sequences listed herein.

[0357] In some embodiments, the sequence of nucleotides is complementary to at least 19 sequence of nucleotides of any of the target sequences listed herein.

[0358] Complementarity The term "complementarity" refers to the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U).

[0359] Oligonucleotides may contain nucleosides with modified nucleic acid bases; for example, 5-methylcytosine is often used in place of cytosine, and therefore the term complementarity will be understood to encompass Watson-Crick base pairing between unmodified and modified nucleic acid bases (see, e.g., Hirao et al. (2012) Accounts of Chemical Research vol. 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0360] The term "% complementary," as used herein, refers to the percentage of nucleotides in a nucleic acid molecule (e.g., oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across a sequence of nucleotides. Therefore, the percentage of complementarity is calculated by counting the number of aligned nucleic acid bases that are complementary (forming Watson-Crick base pairs) between the two sequences (when the oligonucleotide sequences from the target sequence 5'-3' and 3'-5' are aligned), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not permitted in the calculation of the % complementarity of a sequence of nucleotides. It will be understood that, when determining complementarity, chemical modifications of nucleic acid bases are ignored as long as the nucleic acid base retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for the purposes of calculating % identity).

[0361] In the present invention, the term “complementary” means that the sequence nucleotide sequence is at least about 75% complementary to the human GBA mRNA transcript, or at least about 80% complementary, or at least about 85% complementary, or at least about 90% complementary, or at least about 95% complementary. In some embodiments, a sequence of nucleotides may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to the human GBA mRNA transcript.

[0362] In other words, in some embodiments, the sequence of oligonucleotides that can increase the expression of GBA according to the present invention may contain one, two, three, four, five or more mismatches, where the mismatches are nucleotides in the sequence that do not base-pair with their target.

[0363] The term "perfectly complementary" refers to 100% complementarity.

[0364] The oligonucleotides capable of increasing GBA expression according to the present invention are complementary to human GBA mRNA transcripts such as SEQ ID NO: 73. It will be understood that the target GBA nucleic acid may be an allele variant of SEQ ID NO: 73, for example, an allele variant containing one or more polymorphisms in the human GBA nucleic acid sequence.

[0365] identity As used herein, the term "identity" refers to the percentage of nucleotides in a sequence of nucleic acid molecules (e.g., oligonucleotides) that are identical across the sequence to a reference sequence (e.g., a sequence motif).

[0366] Therefore, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleic acid bases between the two sequences (in the sequential nucleotide sequence of the compound of the present invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches × 100) / length of the aligned region (e.g., sequential nucleotide sequence). Insertions and deletions are not permitted in the calculation of the percentage of identity of a sequential nucleotide sequence. When determining identity, it will be understood that chemical modifications of nucleic acid bases are ignored as long as the nucleic acid base retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for the purposes of calculating % identity).

[0367] Therefore, it should be understood that there is a relationship between identity and complementarity, such that a sequence of nucleotides within the oligonucleotide of the present invention that is complementary to the target sequence also shares a percentage of identity with the complementary sequence.

[0368] Hybridization As used herein, the terms “hybridize” or “to hybridize” should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a double helix by forming hydrogen bonds between base pairs on opposing strands. The affinity of the bond between the two nucleic acid strands is the strength of the hybridization. This is often described in terms of the melting temperature (Tm), which is defined as the temperature at which half of the oligonucleotide forms a double helix with the target nucleic acid. Under physiological conditions, Tm is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard-state Gibbs free energy ΔG° more accurately represents the binding affinity and is related to the dissociation constant (Kd) of the reaction by ΔG° = -RTln(Kd) (where R is the gas constant and T is the absolute temperature). Thus, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at an aqueous solution concentration of 1 M, pH 7, and temperature of 37°C. Hybridization of oligonucleotides with target nucleic acids is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by using isothermal titration calorimetry (ITC) methods, as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available instruments are available for measuring ΔG°. ΔG° can be numerically estimated using the nearest neighbor model described in Santa Lucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, and appropriately obtained thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

[0369] In some embodiments, the oligonucleotides of the present invention hybridize to target nucleic acids with an approximate ΔG° value of less than -10 kcal for oligonucleotides of 10 to 30 nucleotides in length.

[0370] In some embodiments, the degree or intensity of hybridization is measured by the Gibbs free energy ΔG° under standard conditions. The oligonucleotides of the present invention may hybridize to a target nucleic acid with estimated ΔG° values ​​less than -10 kcal, e.g., less than -15 kcal, e.g., less than -20 kcal, and e.g., less than -25 kcal. In certain embodiments, the oligonucleotides of the present invention hybridize to a subsequence of the target nucleic acid of SEQ ID NO: 1 with a ΔG° of less than -10 kcal, e.g., -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or -16 to -27 kcal, e.g., -18 to -25 kcal.

[0371] The increase in GBA is made possible by hybridization between the sequential nucleotide sequence of the oligonucleotide according to the present invention and GBA mRNA. In some embodiments, the oligonucleotide of the present invention includes a mismatch between the oligonucleotide and GBA mRNA. Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to show the desired increase in GBA expression. If necessary, the decrease in binding affinity due to the mismatch can be favorably compensated by increasing the number of nucleotides in the oligonucleotide and / or by increasing the number of modified nucleosides, such as 2' modified nucleosides or LNAs, that can increase the binding affinity to GBA mRNA.

[0372] Antisense oligonucleotides As used herein, the term “antisense oligonucleotide” is defined as an oligonucleotide that can hybridize to a target nucleic acid, particularly a continuous sequence on the target nucleic acid. Antisense oligonucleotides are generally not double-stranded and are therefore neither siRNA nor shRNA.

[0373] The antisense oligonucleotides of the present invention may be single-stranded. The single-stranded oligonucleotides of the present invention are understood to be able to form hairpins or intermolecular double structures (double helixes between two molecules of the same oligonucleotide) as long as the degree of intra- or inter-subject complementarity is less than approximately 50% along the entire length of the oligonucleotide.

[0374] In some embodiments, the single-stranded antisense oligonucleotide of the present invention may not contain an RNA nucleoside.

[0375] Advantageously, the antisense oligonucleotides of the present invention comprise one or more modified nucleosides or nucleotides, such as 2'-sugar modified nucleosides. Furthermore, in some antisense oligonucleotides of the present invention, it may be advantageous that the unmodified nucleoside is a DNA nucleoside.

[0376] In some embodiments, the antisense oligonucleotide is 8 to 40 nucleotides long.

[0377] In some embodiments, the antisense oligonucleotide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

[0378] In some embodiments, the antisense oligonucleotide is at least 12 nucleotides long.

[0379] In some embodiments, the antisense oligonucleotide is at least 14 nucleotides long.

[0380] In some embodiments, the antisense oligonucleotide is at least 16 nucleotides long.

[0381] In some embodiments, the antisense oligonucleotide is at least 18 nucleotides long.

[0382] In a preferred embodiment, the antisense oligonucleotide is 16 to 20 nucleotides long.

[0383] More preferably, the antisense oligonucleotide is 18 to 20 nucleotides long.

[0384] In some embodiments, the oligonucleotide of the present invention is an antisense oligonucleotide.

[0385] Region D' or D” within an oligonucleotide In some embodiments, the oligonucleotides of the present invention may comprise, or consist of, a sequence of oligonucleotides complementary to the target nucleic acid, such as a mixmer or totalmer, and further 5' and / or 3' nucleosides. The further 5' and / or 3' nucleosides may be complementary to the target nucleic acid, for example, perfectly complementary, or not. Such further 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0386] The addition of region D' or D'' may be used to link a continuous nucleotide sequence, such as a mixmer or totalmer, to a conjugate moiety or another functional group. When used to link a continuous nucleotide sequence to a conjugate moiety, the addition of region D' or D'' can act as a bio-cleavable linker. Alternatively, the addition of region D' or D'' may be used to provide exonuclease protection or to facilitate synthesis or production.

[0387] Region D' or D'' independently contains or consists of 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid.

[0388] The D' or D'' region can function as a nuclease-sensitive, biocleavable linker (see definition of linker). In some embodiments, additional 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or D'' are disclosed in International Publication 2014 / 076195, which includes, as an example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in International Publication 2015 / 113922, where they are used to link multiple antisense constructs within a single oligonucleotide.

[0389] In one embodiment, the oligonucleotide of the present invention includes a continuous nucleotide sequence constituting a mixmer or totalmer, in addition to regions D' and / or D''.

[0390] In some embodiments, the internucleoside bond located between region D' or D'' and the mixmer or totalmer region is a phosphodiester bond.

[0391] Conjugate The present invention comprises oligonucleotides capable of increasing the expression of GBA covalently attached to at least one conjugate portion. In some embodiments, this may be referred to as the conjugate of the present invention.

[0392] As used herein, the term “conjugate” refers to an oligonucleotide capable of increasing the expression of GBA covalently linked to a non-nucleotide portion (the conjugate portion or region C or a third region). The conjugate portion may optionally be covalently linked to the oligonucleotide of the present invention via a linker group such as region D' or D''.

[0393] Oligonucleotide conjugates and their synthesis have also been reported in comprehensive reviews: Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch.16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0394] In some embodiments, the non-nucleotide portion (conjugate portion) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, active pharmaceutical ingredients, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0395] Linker A conjugate or linker is a connection between two atoms that links one target chemical group or segment to another target chemical group or segment via one or more covalent bonds. The conjugate portion can be attached directly to an oligonucleotide capable of increasing GBA expression, or via a linking portion (e.g., a linker or tether). The linker plays the role of covalently joining a third region, e.g., the conjugate portion (region C), to a first region, e.g., an oligonucleotide or sequence of nucleotides complementary to the target nucleic acid (region A).

[0396] In some embodiments of the present invention, the conjugate or oligonucleotide of the present invention may optionally include a linker region (second region or region B and / or region Y) located between an oligonucleotide or sequential nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate portion (region C or third region).

[0397] Region B refers to biocleavable linkers containing, or comprising, physiologically unstable bonds that can be cleaved under conditions normally encountered or similar to those encountered in the mammalian body. Conditions under which physiologically unstable linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidation or reduction conditions, or drugs, as well as salt concentrations similar to those found in or encountered in mammalian cells. Intracellular mammalian conditions also include the presence of enzyme activity normally present in mammalian cells, such as proteases, hydrolases, or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker contains 1 to 5 nucleosides, such as a DNA nucleoside(s) containing at least two consecutive phosphodiester bonds. Biocleavable linkers containing phosphodiesters are described in detail in International Publication No. 2014 / 076195.

[0398] Region Y refers to a linker that is not necessarily biocleavable but primarily serves to covalently connect the conjugate portion (region C or the third region) to the oligonucleotide (region A or the first region). The linker in region Y may include a chain structure or oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugate of the present invention can be constructed from the following region elements AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, such as a C2-C36 aminoalkyl group including a C6-C12 aminoalkyl group. In some embodiments, the linker (region Y) is a C6 aminoalkyl group.

[0399] salt As used herein, the term “salt” conforms to its commonly known meaning, namely an ionic aggregate of anions and cations.

[0400] The present invention provides pharmaceutically acceptable salts of oligonucleotides according to the present invention, or conjugates according to the present invention.

[0401] The present invention provides oligonucleotides according to the present invention, wherein the oligonucleotide is in the form of a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt may be a sodium salt or a potassium salt.

[0402] The present invention provides pharmaceutically acceptable sodium salts of oligonucleotides according to the present invention.

[0403] The present invention provides pharmaceutically acceptable potassium salts of oligonucleotides according to the present invention.

[0404] Delivery of oligonucleotide GBA agonists The present invention provides oligonucleotides according to the present invention, wherein the oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0405] This may be for the purpose of delivering the oligonucleotide of the present invention to target cells and / or for the purpose of improving the pharmacokinetics of the oligonucleotide of the present invention.

[0406] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0407] Pharmaceutical composition In further embodiments, the present invention provides pharmaceutical compositions comprising the oligonucleotide of the present invention, as well as pharmaceutically acceptable diluents, carriers, salts, and / or adjuvants. Examples of pharmaceutically acceptable diluents include phosphate-buffered saline (PBS), and examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0408] The present invention provides a pharmaceutical composition according to the present invention, comprising the oligonucleotide of the present invention and an aqueous diluent or solvent.

[0409] The present invention provides solutions such as phosphate-buffered saline containing oligonucleotides. Preferably, the solutions such as phosphate-buffered saline of the present invention are sterile solutions.

[0410] International Publication No. 2007 / 031091 provides suitable and preferred examples of pharmaceutically acceptable diluents, carriers, and adjuvants (incorporated herein by reference). Suitable doses, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in International Publication No. 2007 / 031091.

[0411] The oligonucleotides of the present invention can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on several criteria, including, but are not limited to, the route of administration, the severity of the disease, or the dose administered.

[0412] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the present invention is a prodrug. In particular, with respect to oligonucleotide conjugates, when the prodrug is delivered to the site of action, for example, a target cell, the conjugate portion of the oligonucleotide is cleaved.

[0413] target cell As used herein, the term “target cell” refers to a cell expressing a target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell may be a mammalian cell, e.g., a rodent cell, e.g., a mouse cell or a rat cell, or a primate cell, e.g., a monkey cell or a human cell.

[0414] Purpose The oligonucleotides of the present invention can be used, for example, as therapeutic agents including prophylactic agents, as well as research agents.

[0415] Research reagents In research, the oligonucleotides of the present invention may be used to specifically increase the synthesis of GBA mRNA and / or protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating the functional analysis of targets or the evaluation of their usefulness as targets for therapeutic interventions.

[0416] Methods for regulating GBA expression The present invention provides a method for enhancing, upregulating, or restoring GBA expression in cells such as cells expressing GBA, comprising contacting the cells with an effective amount of the oligonucleotide or pharmaceutical composition of the present invention.

[0417] In some embodiments, this method is an in vitro method.

[0418] In some embodiments, the method is an in vivo method.

[0419] In some embodiments, the cells are mammalian cells, such as human cells.

[0420] In some embodiments, the cells are part of a population that is suffering from or susceptible to diseases associated with reduced GBA expression, or originate from a population that is suffering from or susceptible to diseases associated with reduced GBA expression. Such diseases include, but are not limited to, Gaucher disease, Parkinson's disease, dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder.

[0421] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., any disease or disorder referred to herein) and the prevention of a disease, i.e., prevention. Therefore, it will be recognized that, in some embodiments, the treatments referred to herein may be preventative. The present invention relates to a method for treating or preventing a disease, comprising a therapeutically effective amount or a preventively effective amount of oligonucleotide.

[0422] Alternatively, the present invention provides a method comprising administering the pharmaceutical composition to a subject suffering from or susceptible to a disease.

[0423] The present invention provides a method for treating or preventing a disease associated with reduced GBA expression, comprising administering a therapeutically effective or preventively effective amount of the oligonucleotide or pharmaceutical composition of the present invention to a subject suffering from or susceptible to a disease associated with reduced GBA expression.

[0424] In one embodiment, the disease is selected from the group consisting of Gaucher disease, Parkinson's disease, dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder.

[0425] In one embodiment, the disease is Parkinson's disease.

[0426] In one embodiment, the disease is Gaucher disease.

[0427] In some embodiments, the subject is an animal, preferably a mammal, such as a mouse, rat, hamster, or monkey, or most preferably a human.

[0428] The present invention provides oligonucleotides or pharmaceutical compositions of the present invention for use as pharmaceuticals.

[0429] The oligonucleotide or pharmaceutical composition of the present invention is typically administered in an effective amount.

[0430] The present invention provides the use of oligonucleotides or pharmaceutical compositions of the present invention for preparing pharmaceuticals.

[0431] The present invention provides oligonucleotides or pharmaceutical compositions according to the present invention for use in therapeutic applications.

[0432] The method of the present invention is preferably used for the treatment of diseases caused by abnormal levels and / or activity of GBA, such as prophylactic treatment. Diseases may be caused in particular by a decrease in the levels and / or activity of GBA protein.

[0433] The present invention further relates to the use of oligonucleotides or pharmaceutical compositions of the present invention, as defined herein, for producing pharmaceuticals for treating abnormal levels and / or activity of GBA, particularly low levels and / or activity of GBA.

[0434] In one embodiment, the present invention relates to oligonucleotides or pharmaceutical compositions of the present invention for use in the treatment of Gaucher disease, Parkinson's dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder.

[0435] The present invention provides the use of oligonucleotides or pharmaceutical compositions of the present invention for preparing pharmaceuticals for treating or preventing Gaucher disease.

[0436] The present invention provides the use of oligonucleotides or pharmaceutical compositions of the present invention for preparing pharmaceuticals for treating or preventing Parkinson's disease. Administration

[0437] The oligonucleotides or pharmaceutical compositions of the present invention may be administered topically (skin, inhalation, eyes or ears, etc.), enterally (orally or through the gastrointestinal tract, etc.), or parenterally (intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally, etc.).

[0438] In preferred embodiments, the oligonucleotides of the present invention are administered by parenteral routes including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intrathecal or intracranial administration, such as intracerebral or intraventricular. In one embodiment, the oligonucleotides are administered intracerebral or intraventricular. In another embodiment, the oligonucleotides of the present invention are administered intrathecally.

[0439] The present invention also provides the use of oligonucleotides of the present invention, as described, for the manufacture of pharmaceuticals in dosage forms for intrathecal administration.

[0440] The present invention also provides the use of oligonucleotides of the present invention, as described, for the manufacture of pharmaceuticals that are in dosage forms for intracerebral or intraventricular administration.

[0441] The present invention also provides the use of oligonucleotides of the present invention, as described, for the manufacture of pharmaceuticals in dosage forms for intraventricular administration.

[0442] Combination therapy In some embodiments, the oligonucleotide or pharmaceutical composition of the present invention is intended for use in combination with another therapeutic agent.

[0443] Manufacturing method In a further embodiment, the present invention provides a method for producing oligonucleotides, comprising reacting nucleotide units to form a continuous nucleotide unit covalently linked by oligonucleotides. Preferably, this method uses the chemistry of Phosphoramidite International Publication No. 2017 / 081223, PCT / EP2016 / 077383 (see, for example, Caruthers et al, 1987, Methods in Enzymology vol. 154, pp. 287-313).

[0444] In further embodiments, the method further comprises reacting a continuous nucleotide sequence with a conjugate portion (ligand). Further embodiments provide a method for producing a composition of the present invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0445] Table 1 - Base sequences and target site sequences of antisense oligonucleotides The exemplary compounds used in the examples are designed as 18-mer blockmers having a phosphorothioate skeleton, some of which have three LNAs at the 3' end (see Table 2 - Compounds). "E" indicates 5-methylcytosine.

[0446] [Table 1]

[0447] [Table 2]

[0448] Table 2 - Compound table Helm annotation key: [LR](G) is a β-D-oxy-LNA guanine nucleoside, [LR](T) is a β-D-oxy-LNA thymine nucleoside, [LR](A) is a β-D-oxy-LNA adenine nucleoside, [LR]([5meC] is β-D-oxy-LNA5-methylcytosine nucleoside, [dR](G) is a DNA guanine nucleoside, [dR](T) is a DNA thymine nucleoside, [dR](A) is a DNA adenine nucleoside, [dR]([C] is a DNA cytosine nucleoside, [mR](G) is a 2'-O-methylRNA guanine nucleoside, [mR](U) is a 2'-O-methylRNA DNA uracil nucleoside, [mR](A) is a 2'-O-methylRNA DNA adenine nucleoside, [mR]([C] is a 2'-O-methylRNA DNA cytosine nucleoside, [sP] is a phosphorothioate nucleoside bond.

[0449] Further details on how to read HELM sequences are available at www.pistoiaalliance.org / helm-tools / , accessed on December 22, 2022.

[0450] [Table 3] TIFF2026522790000009.tif245170 TIFF2026522790000010.tif245170 TIFF2026522790000011.tif246170

[0451] Examples Example 1: Upcontrol of GBA mRNA The day before transfection, H4 glioblastoma cells were seeded at a density of 10,000 cells per well in a 96-well plate in complete growth medium (DMEM Sigma: D0819, 10% FBS, 1 mM sodium pyruvate). The day after seeding, the cells were transfected with either GBA-targeted antisense oligonucleotide compounds ID numbers 2-34 (n=2), GBA-targeted gapmer (compound ID number 1), or PBS (mock) using Lipofectamin RNAiMax (Invitrogen) at final concentrations of 5 nM and 25 nM according to the manufacturer's instructions. 48 hours after transfection, mRNA was isolated using RNeasy® 96Kit (Qiagen) and extracted with 200 μL of RNAse-free water. 4 μL was used as input for one-step RT-qPCR analysis according to the protocol in Table 3 (qScript® XLT One-Step RT-qPCR ToughMix®, Low ROX®, Quanta Bioscience, #95134-500) using a custom-designed qPCR assay specific to GBA (Table 4) and a pre-designed assay for TBP (HEX, Hs.PT.58v.39858774, IDT). GBA mRNA concentrations were quantified by comparison with the housekeeping gene TBP.

[0452] The results are shown in Figure 1. Compound IDs 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 16, 19, 20, 21, 22, 23, 24, 25, 26, and 27 all resulted in a greater than 10% increase in GBA mRNA expression compared to the mock cells 48 hours after transfection in H4 cells.

[0453] [Table 4]

[0454] In Table 3, ZEN and 3IAbkFQ represent quenchers. The ZEN® internal quencher has a structure owned by Integrated DNA Technologies, Inc. (IDT). 3IAbkFQ is the Iowa Black® quencher manufactured by IDT.

[0455] [Table 5]

[0456] array Sequence ID 72 GBA genome sequence, corresponding to ENSG00000177628 (chr1:155234452-155244699, reverse strand GRCh38:CM000663.2) [ka] TIFF2026522790000015.tif246170 TIFF2026522790000016.tif244170

[0457] Sequence ID 73 Compatible with GBA mRNA and ENST00000368373.8. [ka]

[0458] Sequence ID 74 Mature mir-22-3p sequence, from miRBase database accession number MIMAT0000077 (www.mirbase.org, accessed December 8, 2022). [ka]

[0459] References Straniero et al.(Sci Rep.2017 Oct 5;7(1):12702) International Publication No. 20051398A1

Claims

1. Antisense oligonucleotides 8 to 40 nucleotides long, comprising a sequence of nucleotides complementary to at least six consecutive bases in the 3' untranslated region (UTR) of the RNA sequence encoding glucocerebrosidase (GBA).

2. The antisense oligonucleotide according to claim 1, which can increase GBA expression.

3. The antisense oligonucleotide according to claim 1 or 2, wherein the antisense oligonucleotide can reduce the downregulation of microRNA (miR)-mediated GBA expression in target cells, and the miR is selected from variants thereof containing seed regions that are fully complementary to miR-22-3p and GGCAGCT.

4. The antisense oligonucleotide according to any one of claims 1 to 3, wherein the antisense oligonucleotide can inhibit the binding of the RNA sequence to a variant of the miR selected from those variants containing seed regions completely complementary to miR-22-3p and GGCAGCT.

5. The antisense oligonucleotide according to any one of claims 1 to 4, wherein the continuous nucleotide sequence is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long.

6. The antisense oligonucleotide according to any one of claims 1 to 5, wherein the continuous nucleotide sequence is 16, 18, or 20 nucleotides long.

7. The antisense oligonucleotide according to any one of claims 1 to 6, wherein the continuous nucleotide sequence is the same length as the antisense oligonucleotide.

8. The antisense oligonucleotide according to any one of claims 1 to 7, wherein the sequence of nucleotides is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the sequence of bases located downstream of the stop codon TAG at positions 1746-1748 of SEQ ID NO:

73.

9. The antisense oligonucleotide according to any one of claims 1 to 8, wherein the continuous nucleotide sequence is at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the miR-22-3p binding site located downstream of the stop codon TAG at positions 1746-1748 of SEQ ID NO:

73.

10. The antisense oligonucleotide according to any one of claims 1 to 9, wherein the sequence of nucleotides is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or fully complementary to the sequence of bases located in the segment at positions 2227 to 2274 of SEQ ID NO:

73.

11. The antisense oligonucleotide according to any one of claims 1 to 10, wherein the RNA sequence encoding GBA is an mRNA sequence comprising a 3'UTR sequence containing consecutive bases at positions 2227 to 2274 of SEQ ID NO:

73.

12. The antisense oligonucleotide according to any one of claims 1 to 11, wherein the RNA sequence encoding GBA is an mRNA sequence containing the sequence of SEQ ID NO: 73 or an allele variant thereof.

13. The antisense oligonucleotide according to any one of claims 1 to 12, wherein the continuous nucleotide sequence is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, and 68, or to a fragment of at least 10 nucleotides of any of them.

14. The antisense oligonucleotide according to any one of claims 1 to 13, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

15. The antisense oligonucleotide according to any one of claims 1 to 13, wherein the antisense oligonucleotide is a double-stranded antisense oligonucleotide.

16. The antisense oligonucleotide according to any one of claims 1 to 15, wherein the continuous nucleotide sequence has or comprises a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, SEQ ID NOs: 18, SEQ ID NOs: 19, SEQ ID NOs: 20, SEQ ID NOs: 21, SEQ ID NOs: 22, SEQ ID NOs: 23, SEQ ID NOs: 24, SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, SEQ ID NOs: 33, and SEQ ID NOs: 34, or a fragment of at least 10 nucleotides of any of these.

17. The antisense oligonucleotide according to any one of claims 1 to 16, wherein the continuous nucleotide sequence has or includes a nucleic acid base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 16, 19, 20, 21, 22, 23, 24, 25, 26, and 27.

18. The antisense oligonucleotide according to any one of claims 1 to 17, wherein the antisense oligonucleotide comprises one or more modified nucleosides.

19. The antisense oligonucleotide according to any one of claims 1 to 18, wherein the antisense oligonucleotide comprises one or more modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA, morpholino, and locked nucleic acid (LNA) nucleosides.

20. The antisense oligonucleotide according to any one of claims 1 to 19, wherein the antisense oligonucleotide comprises one or more 2'-MOE RNA nucleosides.

21. The antisense oligonucleotide according to any one of claims 1 to 20, wherein the antisense oligonucleotide comprises one or more LNA nucleosides, such as β-D-oxy-LNA nucleosides.

22. The antisense oligonucleotide according to any one of claims 1 to 21, wherein the antisense oligonucleotide comprises one or more 2'-O-methylRNA nucleosides.

23. The antisense oligonucleotide according to any one of claims 1 to 22, wherein the antisense oligonucleotide is a mixmer or a totalmer, and optionally the mixmer does not contain any DNA or RNA nucleoside.

24. The antisense oligonucleotide according to any one of claims 1 to 23, which is a mixmer of 2'-MOE and LNA nucleoside, or a totalmer of 2'-MOE nucleoside.

25. The antisense oligonucleotide according to any one of claims 1 to 24, wherein the antisense oligonucleotide comprises at least one modified internucleoside bond.

26. The antisense oligonucleotide according to any one of claims 1 to 25, wherein the antisense oligonucleotide comprises one or more phosphorothioate nucleoside interbonding groups.

27. The antisense oligonucleotide according to any one of claims 1 to 26, wherein all nucleoside bonds in the antisense oligonucleotide are phosphorothioate nucleoside bonds.

28. The antisense oligonucleotide according to any one of claims 1 to 27, wherein the antisense oligonucleotide can increase GBA expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to a control.

29. The antisense oligonucleotide according to any one of claims 1 to 28, wherein the antisense oligonucleotide is covalently attached to at least one conjugate portion.

30. The antisense oligonucleotide according to any one of claims 1 to 29, wherein the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt.

31. The antisense oligonucleotide according to any one of claims 1 to 30, wherein the salt is a sodium salt or a potassium salt.

32. The antisense oligonucleotide according to any one of claims 1 to 31, wherein the antisense oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

33. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 32, and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

34. The pharmaceutical composition according to claim 33, wherein the pharmaceutical composition comprises an aqueous diluent or solvent such as phosphate-buffered saline.

35. An antisense oligonucleotide according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33 or 34, for use as a pharmaceutical.

36. An in vitro or in vivo method for increasing or restoring GBA expression in target cells, comprising administering an effective amount of an antisense oligonucleotide according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33 or 34, to the target cells.

37. The method according to claim 36, wherein the cells are mammalian cells such as human cells.

38. The method according to claim 36 or 37, wherein the expression of GBA is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or more than 50% compared to a control.

39. The method according to claim 38, wherein the control is a target cell to which the antisense oligonucleotide is not administered.

40. A method for treating or preventing a disease or disorder, comprising administering to a subject suffering from or susceptible to the disease or disorder a therapeutically effective amount or a preventively effective amount of an antisense oligonucleotide according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33 or 34.

41. An antisense oligonucleotide according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33 or 34, for use in treating or preventing a disease or disorder.

42. Use of an antisense oligonucleotide according to any one of claims 1 to 32, or a pharmaceutical composition according to claim 33 or 34, for preparing a pharmaceutical for treating or preventing a disease or disorder in a subject.

43. The method according to claim 40, the antisense oligonucleotide or pharmaceutical composition for use according to claim 41, or the use according to claim 42, wherein the disease or disorder is related to reduced expression of GBA.

44. The method according to claim 40 or 43, wherein the disease is selected from the group consisting of Gaucher disease, Parkinson's disease, dementia, Lewy body dementia (DLB), and rapid eye movement (REM) sleep behavior disorder, an antisense oligonucleotide or pharmaceutical composition for use according to claim 41 or 43, or the use according to claim 42 or 43.

45. The method according to claim 40, 43, or 44, wherein the disease is Parkinson's disease, an antisense oligonucleotide or pharmaceutical composition for use according to claim 41, 43, or 44, or the use according to claim 42, 43, or 44.

46. The method according to claim 40, 43, or 44, wherein the disease is Gaucher disease, an antisense oligonucleotide or pharmaceutical composition for use according to claim 41, 43, or 44, or the use according to claim 42, 43, or 44.

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