Antisense oligonucleotides

Abstract

The present invention relates to antisense oligonucleotides, conjugates, salts, and pharmaceutical compositions thereof that upregulate or restore expression of GBA in cells, and their use in the treatment of Parkinson's disease and Gaucher disease.

Description

[Technical field]

[0001] The present invention relates to antisense oligonucleotides, conjugates, salts and pharmaceutical compositions thereof that upregulate or restore expression of glucocerebrosidase (GBA) in cells; methods of treating diseases associated with decreased expression of GBA, including Gaucher disease and / or Parkinson's disease. [Background technology]

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

[0003] Homozygous mutations in the gene encoding GBA cause Gaucher disease. During routine cell turnover, macrophages phagocytose and degrade cellular debris. Insufficient GBA activity leads to 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 bruising, fatigue, anemia, low platelet counts, and enlarged liver and spleen. Although the phenotype is variable, 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 impairment.

[0005] The condition is inherited in an autosomal recessive pattern. Over 300 variants of the GBA gene have been associated with the disease. 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 usually exhibit a neuropathic form of the disease, whereas 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 dementia with Lewy bodies (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 may precede motor symptoms by many years include anosmia, 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. As with Gaucher disease, the severity of the mutation can affect the disease phenotype. For example, the risk of dementia in patients carrying "severe" mutations (such as L444P) is 2-3 times higher than in patients carrying "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 reduction therapy (SRT). ERT involves intravenous administration of recombinant GBA. Although most patients respond well to the treatment, there is a risk of developing an immune response. Furthermore, ERT is thought to be ineffective in patients with Parkinson's disease or neuropathic Gaucher disease because GBA cannot cross the blood-brain barrier. SRT offers an alternative (or adjunct) treatment for patients who cannot tolerate ERT or for whom intravenous administration is problematic. SRT works to reduce the accumulation of glucocerebroside in lysosomes by inhibiting enzymes in the glucocerebroside synthesis pathway. This treatment has a higher incidence of adverse effects than ERT, and long-term reduction of glucocerebroside may affect several different cellular functions. Both ERT and SRT are expensive and must be continued lifelong.

[0009] Therefore, there is an urgent need for therapeutic agents that can increase or restore the expression of GBA. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Scott et al.,2000,Genet.Med.,2,65 [Non-Patent Document 2] Riboldi and Di Fonzo,2019,Cells,8,364 [Non-Patent Document 3] Avenali et al.,2020,Front.Aging Neurosci. Summary of the Invention

[0011] The present invention provides an antisense oligonucleotide of 8 to 40 nucleotides in length, comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length, wherein the contiguous nucleotide sequence is complementary to a sequence within the 5' untranslated region (5'UTR) located upstream of the canonical AUG start codon of a glucocerebrosidase (GBA) mRNA transcript.

[0012] The GBA mRNA transcript is the mRNA sequence encoded by the glucocerebrosidase (GBA) gene.

[0013] Without wishing to be bound by theory, it is believed that the antisense oligonucleotides of the invention can reduce and / or prevent translation initiation from an upstream non-canonical AUG (uAUG) site, which can result in increased translation from a downstream canonical AUG initiation site, thereby increasing GBA protein production.

[0014] The contiguous nucleotide sequence can be complementary to an upstream open reading frame (uORF) within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0015] The start of a uORF is defined by a uAUG.

[0016] The contiguous nucleotide sequence can be 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 in length.

[0017] The contiguous nucleotide sequence can be 16 or 18 nucleotides in length.

[0018] The contiguous nucleotide sequence may be as long as the antisense oligonucleotide.

[0019] In some embodiments, the contiguous nucleotide sequence can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or completely complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0020] The contiguous nucleotide sequence can be perfectly complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0021] In some embodiments, the GBA gene can comprise a nucleic acid sequence according to SEQ ID NO: 107. The canonical AUG start codon can correspond to positions 8586-8588 of SEQ ID NO:107.

[0022] In some embodiments, the GBA mRNA transcript can comprise a nucleic acid sequence according to SEQ ID NO: 108. The canonical AUG start codon can correspond to positions 138-140 of SEQ ID NO:108.

[0023] In some embodiments, the 5'UTR may comprise a uAUG.

[0024] In some embodiments, the uAUG may be located 14 nt upstream of the canonical AUG. The uAUG may be at a position corresponding to positions 8572-8574 of SEQ ID NO: 107. The uAUG may be at a position corresponding to positions 124-126 of SEQ ID NO: 108.

[0025] In some embodiments, the contiguous nucleotide sequence may be complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:109.

[0026] The antisense oligonucleotide may be a single-stranded antisense oligonucleotide.

[0027] In some embodiments, the contiguous nucleotide sequence may be or may include a sequence selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:38, or at least 10 contiguous nucleotides thereof. In one embodiment, the contiguous nucleotide sequence may be SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0028] In some embodiments, the antisense oligonucleotides may contain one or more modified nucleosides.

[0029] The one or more modified nucleosides may be 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, and locked nucleic acid (LNA) nucleosides.

[0030] The one or more modified nucleosides may be an LNA nucleoside. The LNA nucleoside may be beta-D-oxy-LNA.

[0031] The one or more modified nucleosides can be 2'-O-methyl-RNA nucleosides.

[0032] The oligonucleotide may be a mixmer.

[0033] The antisense oligonucleotides may contain at least one modified internucleoside linkage.

[0034] One or more or all of the modified internucleoside linkages may comprise phosphorothioate linkages. All internucleoside linkages present in the antisense oligonucleotide may be phosphorothioate internucleoside linkages.

[0035] In some embodiments, the contiguous nucleotide sequence can increase expression of GBA by 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control, which can be cells not exposed to the oligonucleotide.

[0036] In some embodiments, the antisense oligonucleotide may be covalently attached to at least one conjugate moiety.

[0037] In some embodiments, the antisense oligonucleotides may be in the form of a pharma- ceutically acceptable salt. The salt may be a sodium or potassium salt.

[0038] The antisense oligonucleotides may be encapsulated in a lipid-based delivery vehicle, covalently linked or encapsulated in a dendrimer, or conjugated to an aptamer.

[0039] The present invention also provides a pharmaceutical composition comprising an antisense oligonucleotide of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0040] The pharmaceutical composition may include an aqueous diluent or solvent, such as phosphate buffered saline.

[0041] The present invention also provides an in vitro or in vivo method for upregulating or restoring GBA expression in a target cell, comprising administering to the cell an effective amount of an antisense oligonucleotide of the present invention or a pharmaceutical composition of the present invention.

[0042] The cell can be a human cell or a mammalian cell.

[0043] Expression of GBA may be increased by 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control, which may be cells not exposed to the oligonucleotide.

[0044] The present invention also provides a method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention to a subject suffering from or susceptible to a disease.

[0045] The present invention also provides the antisense oligonucleotide of the present invention or the pharmaceutical composition of the present invention for use as a medicament for treating or preventing a disease in a subject.

[0046] The present invention also provides the use of an antisense oligonucleotide of the present invention or a pharmaceutical composition of the present invention for the preparation of a medicament for treating or preventing a disease in a subject.

[0047] The disease may be associated with decreased expression of GBA.

[0048] The disease may be selected from the group consisting of Gaucher's disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0049] In some embodiments, the disease can be Parkinson's disease.

[0050] In some embodiments, the disease can be Gaucher's disease. [Brief description of the drawings]

[0051] [Figure 1] Figure 1 shows upregulation of GBA mRNA in SK-N-AS neuroblastoma cells 48 hours after transfection compared to mock-transfected controls. Cells were seeded at a density of 25,000 cells per well. GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. [Diagram 2] Figure 1 shows upregulation of GBA mRNA in SK-N-As neuroblastoma cells 72 hours after transfection compared to mock-transfected controls. Cells were seeded at a density of 10,000 cells per well. GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. [Diagram 3] Figure 1 shows the upregulation of GBA protein in SK-N-As neuroblastoma cells 72 hours after transfection compared to mock transfection control. Cells were seeded at a density of 60000 cells per well. GBA protein abundance was quantified relative to HPRT1 protein using "Compass for SW" software and Microsoft Excel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The inventors have identified that expression levels of GBA protein products can be effectively enhanced by targeting GBA mRNA transcripts with antisense oligonucleotides, in particular antisense oligonucleotides containing high affinity sugar-modified nucleosides, such as high affinity 2' sugar-modified nucleosides, such as LNA nucleosides or 2'-O-methoxyethyl (MOE) nucleosides.

[0053] Described herein are target sites present on human GBA nucleic acid targets, such as the GBA mRNA sequence, which can be targeted by the antisense oligonucleotides of the invention.

[0054] The inventors have surprisingly determined that targeting the 5' untranslated region (5'UTR) of the GBA mRNA transcript can be particularly effective.

[0055] The 5'UTR may contain one or more upstream initiation codon sites (uAUGs), which define the start of the upstream open reading frame (uORF). Without wishing to be bound by theory, it is believed that the antisense oligonucleotides of the present invention can increase GBA production by binding to these 5' regions and affecting (e.g., decreasing) the initiation of translation from these uAUG sites. This ensures that protein translation is initiated from the downstream canonical initiation site (ORF), increasing GBA production.

[0056] Thus, the inventors have identified that the antisense oligonucleotides of the invention can be used to effectively enhance expression levels of the encoded protein product by targeting GBA mRNA transcripts with antisense oligonucleotides that block one or more upstream open reading frames (uORFs) located upstream of the canonical AUG start codon and / or prevent translation from the uAUG sites.

[0057] In some embodiments, the antisense oligonucleotide of the present invention comprises a continuous nucleotide sequence that is complementary to the sequence in the 5'UTR that comprises uORF located upstream of the canonical AUG start codon of GBA mRNA transcript.Therefore, antisense oligonucleotide can directly bind to uORF, which, without wishing to be bound by theory, can cause steric blocking of uORF and prevent protein translation starting from uAUG site.

[0058] In some embodiments, the antisense oligonucleotide of the present invention comprises the continuous nucleotide sequence complementary to the sequence in 5'UTR that is located upstream of the canonical AUG start codon of GBA mRNA transcript and does not directly include uAUG site.Thus, antisense oligonucleotide can bind to the sequence in 5'UTR that does not directly include uAUG, but does not want to be bound by theory, can still cause steric blockage and prevent protein translation from uAUG site.

[0059] Oligonucleotides, such as RNase H recruiting single stranded antisense oligonucleotides or siRNAs, have been widely used in the art to inhibit target RNA, ie, as antagonists of their complementary nucleic acid targets.

[0060] The antisense oligonucleotides of the invention are agonists, ie, they enhance expression of their complementary target GBA nucleic acid, thereby enhancing expression of GBA protein.

[0061] Thus, the present invention relates to antisense oligonucleotides that enhance the expression of GBA.

[0062] Enhancement of GBA expression is desirable for treating diseases associated with decreased GBA expression, such as Gaucher disease and / or Parkinson's disease. Oligonucleotides

[0063] As used herein, the term "oligonucleotide" is defined as a molecule that contains two or more covalently linked nucleosides as commonly understood by those of skill in the art. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer.

[0064] Oligonucleotides are usually made in the laboratory by solid phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. The oligonucleotides of the invention are artificial, chemically synthesized, and usually purified or isolated. The oligonucleotides of the invention may contain one or more modified nucleosides, such as, for example, 2' sugar modified nucleosides. The oligonucleotides of the invention may contain one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages. Antisense oligonucleotides

[0065] The oligonucleotide is an antisense oligonucleotide.

[0066] In some embodiments, the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0067] The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid.Antisense oligonucleotides are not double-stranded in nature, and therefore are not siRNA or shRNA.Antisense oligonucleotides of the present invention can be single-stranded.It is understood that the single-stranded oligonucleotides of the present invention can form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide), as long as the degree of complementarity within or between themselves is less than about 50% over the entire length of the oligonucleotide.

[0068] In some embodiments, the single-stranded antisense oligonucleotides of the invention may not contain RNA nucleosides.

[0069] Advantageously, the oligonucleotides of the invention contain one or more modified nucleosides or nucleotides, such as, for example, 2' sugar modified nucleosides. Furthermore, in some antisense oligonucleotides of the invention, the unmodified nucleosides may advantageously be DNA nucleosides.

[0070] In some embodiments, the antisense oligonucleotides are 8-40 nucleotides in length.

[0071] In some embodiments, the antisense oligonucleotides are 8-40 nucleotides in length and comprise a contiguous nucleotide sequence of 8-40 nucleotides.

[0072] 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 in length.

[0073] In some embodiments, the antisense oligonucleotide is 16 nucleotides in length.

[0074] In some embodiments, the antisense oligonucleotide is 18 nucleotides in length.

[0075] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an antisense oligonucleotide that is complementary to a target nucleic acid, which may be or may include an oligonucleotide motif sequence. This term is used interchangeably herein with the term "contiguous nucleobase sequence."

[0076] In some embodiments, antisense oligonucleotides comprise a contiguous nucleotide sequence, and may optionally comprise a nucleotide linker region that can be used to attach additional nucleotides, such as functional groups (e.g., conjugate groups), to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0077] It is understood that the contiguous nucleotide sequence of an antisense oligonucleotide cannot be longer than the antisense oligonucleotide itself, and that the antisense oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

[0078] In some embodiments, all of the nucleotides of the antisense oligonucleotide may constitute a contiguous nucleotide sequence.

[0079] A contiguous nucleotide sequence is a sequence of nucleotides in the antisense oligonucleotide of the invention that is complementary, and in some cases completely complementary, to a target nucleic acid, target sequence, or target site sequence.

[0080] In some embodiments, the contiguous nucleotide sequence is between 8 and 40 nucleotides in length.

[0081] In some embodiments the contiguous nucleotide sequence 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 in length.

[0082] In some embodiments, the contiguous nucleotide sequence is 16 nucleotides in length.

[0083] In some embodiments, the contiguous nucleotide sequence is 18 nucleotides in length.

[0084] In some embodiments, the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.

[0085] In some embodiments, the antisense oligonucleotide consists of a contiguous nucleotide sequence.

[0086] In some embodiments, the antisense oligonucleotide is a contiguous nucleotide sequence.

[0087] 5'UTR and uORF of GBA The antisense oligonucleotides of the invention may comprise a contiguous nucleotide sequence complementary to an upstream open reading frame (uORF) located within the 5'UTR upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript.

[0088] The GBA mRNA transcript is the mRNA sequence encoded by the glucocerebrosidase (GBA) gene.

[0089] Thus, the antisense oligonucleotides of the present invention can provide steric blocking of uAUG and prevent protein translation from the uAUG site, which can result in increased translation from the canonical AUG start codon and increased expression of the GBA protein.

[0090] The antisense oligonucleotides of the present invention may contain a contiguous nucleotide sequence complementary to a sequence in the 5'UTR located upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript, and although the sequence in the 5'UTR may not encompass the uAUG, upon binding to the sequence in the 5'UTR, may provide steric blocking and prevent protein translation from the uAUG site.

[0091] The term "gene" as used herein, particularly with respect to the GBA gene, encompasses both protein-coding and non-protein-coding sequences. It is understood that such sequences include transcribed and non-transcribed sequences, as well as translated and non-translated sequences. Non-protein-coding sequences may include regulatory sequences, such as enhancers, silencers, promoters, and / or 3' and 5' untranslated regions (UTRs).

[0092] A uORF is a gene expression regulatory element that is an open reading frame (ORF) that begins with an upstream start AUG (uAUG) codon located within the 5' untranslated region (5'UTR) of an mRNA. The uAUG is located upstream of the canonical AUG start codon of the primary coding region of a gene.

[0093] In some embodiments, the contiguous nucleotide sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e., completely) complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0094] Without wishing to be bound by theory, targeting the 5'UTR can increase GBA expression by binding to upstream initiation sites (AUG sites) and affecting, e.g., decreasing, protein translation from these upstream AUG sites, ensuring that protein translation is initiated from downstream canonical initiation sites (ORFs) and increasing GBA production.

[0095] In some embodiments, the contiguous nucleotide sequence is at least 75% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0096] In some embodiments, the contiguous nucleotide sequence is at least 80% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0097] In some embodiments, the contiguous nucleotide sequence is at least 85% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0098] In some embodiments, the contiguous nucleotide sequence is at least 90% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0099] In some embodiments, the contiguous nucleotide sequence is at least 95% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0100] In some embodiments, the contiguous nucleotide sequence is at least 96% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0101] In some embodiments, the contiguous nucleotide sequence is at least 97% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0102] In some embodiments, the contiguous nucleotide sequence is at least 98% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0103] In some embodiments, the contiguous nucleotide sequence is at least 99% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0104] In some embodiments, the contiguous nucleotide sequence is fully complementary (ie, 100% complementary) to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0105] In some embodiments, the contiguous nucleotide sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e., completely) complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0106] In some embodiments, the contiguous nucleotide sequence is at least 75% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0107] In some embodiments, the contiguous nucleotide sequence is at least 80% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0108] In some embodiments, the contiguous nucleotide sequence is at least 85% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0109] In some embodiments, the contiguous nucleotide sequence is at least 90% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0110] In some embodiments, the contiguous nucleotide sequence is at least 95% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0111] In some embodiments, the contiguous nucleotide sequence is at least 96% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0112] In some embodiments, the contiguous nucleotide sequence is at least 97% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0113] In some embodiments, the contiguous nucleotide sequence is at least 98% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0114] In some embodiments, the contiguous nucleotide sequence is at least 99% complementary to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0115] In some embodiments, the contiguous nucleotide sequence is fully complementary (ie, 100% complementary) to a uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0116] In some embodiments, the GBA gene may consist of the human GBA genome sequence according to NCBI reference sequence: NG_009783.

[0117] In some embodiments, the GBA gene may comprise a nucleic acid sequence according to SEQ ID NO:107.

[0118] SEQ ID NO: 107 is provided herein as a reference sequence, and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO: 107, for example an allelic variant that includes one or more polymorphisms in the human GBA nucleic acid sequence.

[0119] In some embodiments, the GBA gene may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e., complete) sequence identity to a sequence according to SEQ ID NO:107.

[0120] In some embodiments, the GBA gene may consist of the nucleic acid sequence according to SEQ ID NO:107.

[0121] It will be understood that the sequence according to SEQ ID NO: 107 is the GBA genomic sequence. Any reference to the GBA genomic sequence includes a reference to the GBA mRNA transcript sequence corresponding to the GBA genomic sequence. Thus, the GBA mRNA transcript may be the mRNA sequence encoded by SEQ ID NO: 107. Thus, a reference to an "ATG" codon will be understood to refer to the start codon of the GBA genomic sequence corresponding to "AUG" at the position corresponding to the position of the GBA mRNA transcript.

[0122] In some embodiments, the canonical AUG start codon of the GBA mRNA transcript can correspond to positions 8586-8588 of the GBA genomic sequence of SEQ ID NO: 107. Thus, the "A" of the canonical AUG start codon can be at a position corresponding to position 8586 of SEQ ID NO: 107, the "U" of the canonical AUG start codon can be at a position corresponding to position 8587 of SEQ ID NO: 107, and the "G" of the canonical AUG start codon can be at a position corresponding to position 8588 of SEQ ID NO: 107.

[0123] In some embodiments, the GBA mRNA transcript may comprise a nucleic acid sequence according to SEQ ID NO:108.

[0124] SEQ ID NO: 108 is provided herein as a reference sequence, and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO: 108, for example an allelic variant that contains one or more polymorphisms in the human GBA mRNA sequence.

[0125] In some embodiments, the GBA mRNA transcript may comprise a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e., complete) sequence identity to a sequence according to SEQ ID NO:108.

[0126] In some embodiments, the GBA mRNA transcript may consist of a nucleic acid sequence according to SEQ ID NO:108.

[0127] In some embodiments, the GBA mRNA sequence according to SEQ ID NO:108 may correspond to the GBA genomic sequence according to SEQ ID NO:107.

[0128] In some embodiments, the canonical AUG start codon of the GBA mRNA transcript corresponds to positions 138-140 of SEQ ID NO: 108. Thus, the "A" of the canonical AUG start codon can be at a position corresponding to position 138 of SEQ ID NO: 108, the "U" of the canonical AUG start codon can be at a position corresponding to position 139 of SEQ ID NO: 108, and the "G" of the canonical AUG start codon can be at a position corresponding to position 140 of SEQ ID NO:108.

[0129] The uAUG is located upstream of the canonical AUG start codon position, where upstream is understood to mean the 5' position relative to the canonical AUG codon.

[0130] In some embodiments, the uORF may be located 1-100 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-100 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-50 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-50 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-40 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-40 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-30 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-30 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-20 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-20 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-15 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-15 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript.

[0131] In some embodiments, the uAUG of the uORF may be located 14 nucleotides (nt) upstream of the canonical AUG start codon position. In other words, there may be 11 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript, i.e., there are 11 nucleotides between the G of the uAUG and the A of the canonical AUG. Thus, the "A" of the uAUG may be located 14 nucleotides upstream of the canonical AUG codon, the "U" of the uAUG may be located 13 nucleotides upstream of the canonical AUG codon, and the "G" of the uAUG may be located 12 nucleotides upstream of the canonical AUG codon.

[0132] In some embodiments, the uAUG of the uORF is at a position corresponding to positions 8572-8574 of the GBA genomic sequence of SEQ ID NO: 107. Thus, the "A" of the uAUG can be at a position corresponding to position 8572 of SEQ ID NO: 107, the "U" of the uAUG can be at a position corresponding to position 8573 of SEQ ID NO: 107, and the "G" of the uAUG can be at a position corresponding to position 8574 of SEQ ID NO: 107.

[0133] In some embodiments, the uAUG of the uORF is at a position corresponding to positions 124-126 of the GBA mRNA sequence of SEQ ID NO: 108. Thus, the "A" of the uAUG can be at a position corresponding to position 124 of SEQ ID NO: 108, the "U" of the uAUG can be at a position corresponding to position 125 of SEQ ID NO: 108, and the "G" of the uAUG can be at a position corresponding to position 126 of SEQ ID NO: 108.

[0134] Unless otherwise stated, all ranges are inclusive of the starting and ending values.

[0135] Complementarity to GBA target sequence The contiguous nucleotide sequence of the antisense oligonucleotide of the present invention can be complementary to the GBA target nucleic acid sequence. The contiguous nucleotide sequence can be completely complementary to the GBA target nucleic acid sequence. The contiguous nucleotide sequence can be partially complementary to the GBA target nucleic acid sequence, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e. completely) complementary to the GBA target nucleic acid sequence.

[0136] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence, which may be selected from the group consisting of 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, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, and SEQ ID NO:106, or a fragment thereof.

[0137] In some embodiments, the contiguous nucleotide sequence is 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, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 Complementary to a target nucleic acid sequence which may be selected from the group consisting of SEQ ID NO: 106 and SEQ ID NO: 109, or a fragment thereof.

[0138] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:90.

[0139] In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:109.

[0140] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:60, or a fragment thereof.

[0141] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:61, or a fragment thereof.

[0142] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:62, or a fragment thereof.

[0143] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:63, or a fragment thereof.

[0144] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:64, or a fragment thereof.

[0145] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:65, or a fragment thereof.

[0146] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:66, or a fragment thereof.

[0147] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:67, or a fragment thereof.

[0148] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:81, or a fragment thereof.

[0149] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:82, or a fragment thereof.

[0150] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:87, or a fragment thereof.

[0151] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:89, or a fragment thereof.

[0152] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:90, or a fragment thereof.

[0153] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 109, or a fragment thereof.

[0154] Fragments of the target sequence can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0155] In some embodiments, the fragment may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the corresponding sequence.

[0156] In some embodiments, the fragment may differ from the corresponding sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0157] Targeting the GBA target sequence The contiguous nucleotide sequence of the antisense oligonucleotide of the present invention may comprise a sequence that targets a GBA target nucleic acid sequence.

[0158] In some embodiments, the contiguous nucleotide sequence is or comprises a sequence that may be 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, SEQ ID NO:34, 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, and SEQ ID NO:105, or at least 10 contiguous nucleotides thereof.

[0159] In some embodiments, the contiguous nucleotide sequence is or comprises a sequence that may be 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, SEQ ID NO:34, 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:105, and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0160] In some embodiments, the contiguous nucleotide sequence is or comprises a sequence that may be selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:38, or at least 10 contiguous nucleotides thereof.

[0161] In some embodiments, the contiguous nucleotide sequence is or comprises a sequence that may be selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0162] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:8 or at least 10 contiguous nucleotides thereof.

[0163] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:9 or at least 10 contiguous nucleotides thereof.

[0164] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:10 or at least 10 contiguous nucleotides thereof.

[0165] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:11 or at least 10 contiguous nucleotides thereof.

[0166] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO: 12 or at least 10 contiguous nucleotides thereof.

[0167] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:13 or at least 10 contiguous nucleotides thereof.

[0168] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:14 or at least 10 contiguous nucleotides thereof.

[0169] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:15 or at least 10 contiguous nucleotides thereof.

[0170] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:29 or at least 10 contiguous nucleotides thereof.

[0171] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:30 or at least 10 contiguous nucleotides thereof.

[0172] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:35 or at least 10 contiguous nucleotides thereof.

[0173] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:37 or at least 10 contiguous nucleotides thereof.

[0174] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:38 or at least 10 contiguous nucleotides thereof.

[0175] In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO:110 or at least 10 contiguous nucleotides thereof.

[0176] In certain embodiments, the contiguous nucleotide sequence may be or may comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides of the recited sequence.

[0177] It is particularly understood that the sequence of SEQ ID NO:110 is longer than a typical oligonucleotide sequence. In certain embodiments, the contiguous nucleotide sequence may be or may include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides of SEQ ID NO:110.

[0178] Nucleotides and Nucleosides Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides and nucleosides.Naturally, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0179] Modified Nucleosides Advantageously, the antisense oligonucleotides of the invention may contain one or more modified nucleosides.

[0180] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified compared to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or (nucleo) base moieties.

[0181] Advantageously, one or more of the modified nucleosides of the antisense oligonucleotides of the present invention may contain modified sugar moieties. The term modified nucleoside may also be used interchangeably with the terms "nucleoside analogue" or modified "unit" or modified "monomer". Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing. Exemplary modified nucleosides that may be used in the antisense oligonucleotides of the present invention include LNA, 2'-O-MOE, 2'oMe and morpholino nucleoside analogues.

[0182] Modified Internucleoside Linkages Advantageously, the antisense oligonucleotides of the invention may contain one or more modified internucleoside linkages.

[0183] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides together as commonly understood by those skilled in the art. Thus, the antisense oligonucleotides of the present invention can include one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

[0184] In some embodiments, at least 50% of the internucleoside linkages of the antisense oligonucleotide or its contiguous nucleotide sequence are phosphorothioate, for example at least 60%, such as at least 70%, for example at least 75%, for example at least 80%, for example at least 90% or more of the internucleoside linkages of the antisense oligonucleotide or its contiguous nucleotide sequence are phosphorothioate. In some embodiments, all of the internucleoside linkages of the antisense oligonucleotide or its contiguous nucleotide sequence are phosphorothioate.

[0185] In a further embodiment, the antisense oligonucleotide may contain at least one modified internucleoside linkage. Advantageously, at least 75%, for example all, of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate linkages.

[0186] Conveniently, all of the internucleoside linkages of the contiguous nucleotide sequence of the antisense oligonucleotide may be phosphorothioate, or all of the internucleoside linkages of the antisense oligonucleotide may be phosphorothioate linkages.

[0187] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al., 2012, Accounts of Chemical Research, 45, 2055-2065 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1-1.4.32.

[0188] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0189] The nucleobase moiety may be represented by the letter code for each corresponding nucleobase, for example, A, T, G, C, or U, where each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides may be used. 5-methylcytosine may be represented as "E".

[0190] Modified Oligonucleotides The antisense oligonucleotides of the present invention may be modified antisense oligonucleotides.

[0191] The term "modified antisense oligonucleotide" refers to an antisense oligonucleotide that comprises one or more sugar-modified nucleosides and / or modified internucleoside linkages.The term "chimeric oligonucleotide" is a term used in the literature to describe oligonucleotides that comprise sugar-modified nucleosides and DNA nucleosides.In some embodiments, it may be advantageous for the antisense oligonucleotide of the present invention to be a chimeric oligonucleotide.

[0192] In some embodiments, the antisense oligonucleotide or its contiguous nucleotide sequence may contain modified nucleobases that function as the indicated nucleobases in base pairing, for example, 5-methylcytosine may be used instead of methylcytosine. Inosine may be used as a universal base.

[0193] It is understood that the consecutive nucleobase sequence (motif sequence) may be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0194] The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into an oligonucleotide sequence is commonly referred to as the oligonucleotide design.

[0195] The antisense oligonucleotides of the invention are designed with modified nucleosides and DNA nucleosides. Advantageously, high affinity modified nucleosides are used.

[0196] In one embodiment, the antisense oligonucleotide comprises at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, 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, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 modified nucleosides.

[0197] Suitable modifications are described herein under the headings "modified nucleosides," "high affinity modified nucleosides," "sugar modifications," "2' sugar modifications," and "Locked Nucleic Acids (LNA)."

[0198] High-affinity modified nucleosides High affinity modified nucleosides, when incorporated into oligonucleotides, can, for example, increase the melting temperature (T m ) of the present invention. The high affinity modified nucleosides of the present invention preferably provide 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 many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

[0199] sugar modification The antisense oligonucleotides of the invention may contain one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety compared to the ribose sugar moiety found in DNA and RNA.

[0200] A number of nucleosides with modifications of the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0201] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA) or bicyclic ring (typically having a biradical bridge between the C2 and C4 carbons of the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0202] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0203] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or a nucleoside that contains a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as an LNA (2'-4' biradical bridged) nucleoside.

[0204] Indeed, much attention has been paid to the development of 2' sugar-substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2' modified sugars can confer improved binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2' substituted modified nucleosides are 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, e.g., 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. Below are illustrative examples of some 2'-substituted modified nucleosides. [ka]

[0205] In the context of the present invention, 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides such as LNA.

[0206] In one embodiment, the antisense oligonucleotide comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides.

[0207] In one embodiment, the antisense oligonucleotide comprises 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, 34, 35, 36, 37, 38, 39 or 40 sugar modified nucleosides.

[0208] Preferably, the antisense oligonucleotide of the present invention comprises one or more 2' sugar 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, arabinonucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. It is advantageous if one or more of the modified nucleosides is a locked nucleic acid (LNA).

[0209] Locked Nucleoside (LNA) Nucleoside "LNA nucleosides" are 2' modified nucleosides that contain a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, restricting or fixing the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixing the conformation of the ribose is associated with improved hybridization affinity (stabilization of the duplex) when LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0210] Non-limiting exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett., 12, 73-76, Seth et al., J. Org. Chem., 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry, 2016, 59, 9645-9667.

[0211] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: [ka]

[0212] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0213] A particularly advantageous LNA is beta-D-oxy-LNA.

[0214] Morpholino Oligonucleotides In some embodiments, the antisense oligonucleotide of the present invention comprises or consists of morpholino nucleosides (i.e., morpholino oligomers, as phosphorodiamidate morpholino oligomers (PMOs)). Splice-modulating morpholino oligonucleotides have been approved for clinical use, see, for example, eteplirsen, a 30 nt morpholino oligonucleotide that targets frameshift mutations in DMD and is used to treat Duchenne muscular dystrophy. Morpholino oligonucleotides have a nuclease linked to a six-membered morpholine ring rather than a ribose, such as, for example, a methylene morpholine ring linked via a phosphorodiamidate group, as shown in the following diagram of four consecutive morpholino nucleotides: [ka]

[0215] In some embodiments, the morpholino oligonucleotides of the invention can be, for example, 8 to 40 morpholino nucleotides in length.

[0216] RNase H activity and recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate measured in pmol / l / min that is at least 5%, e.g., at least 10%, at least 20%, or more than 20% of the initial rate determined when using an oligonucleotide that has the same base sequence as the modified oligonucleotide being tested but contains only DNA monomers with phosphorothioate bonds between all monomers in the oligonucleotide and using the methodology provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference). Recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland for use in determining RNase H activity.

[0217] DNA oligonucleotides are known to effectively recruit RNaseH, as are gapmer oligonucleotides that contain regions of DNA nucleosides (typically at least 5 or 6 consecutive DNA nucleosides) flanked at 5' and 3' by regions containing 2' sugar-modified nucleosides, typically high affinity 2' sugar-modified nucleosides, such as 2-O-MOE and / or LNA. To effectively promote steric blocking, degradation of pre-mRNA is undesirable, and thus it is preferable to avoid RNaseH degradation of the target. Thus, the antisense oligonucleotides of the present invention are not RNaseH recruiting gapmer oligonucleotides.

[0218] RNaseH recruitment can be avoided by limiting the number of consecutive DNA nucleotides in oligonucleotides, so that mixmer and totalmer designs can be used.Advantageously, in some embodiments, the antisense oligonucleotide of the present invention or its consecutive nucleotide sequence does not contain more than 3 consecutive DNA nucleosides.Furthermore, advantageously, in some embodiments, the antisense oligonucleotide of the present invention or its consecutive nucleotide sequence does not contain more than 4 consecutive DNA nucleosides.Furthermore, advantageously, in some embodiments, the antisense oligonucleotide of the present invention or its consecutive nucleotide sequence does not contain more than 2 consecutive DNA nucleosides.

[0219] Mixmar and Totalmar Due to steric blocking, it is often advantageous to use antisense oligonucleotides that do not recruit RNAase H and cause destruction of the target RNA. Because RNase H activity requires a consecutive sequence of DNA nucleotides, recruitment of RNase H can be prevented by designing oligonucleotides that do not contain a region of more than three or more than four consecutive DNA nucleosides. This can be achieved by using oligonucleotides or consecutive nucleoside regions thereof in mixmer designs that contain a sugar-modified nucleoside, such as a 2' sugar-modified nucleoside, and a short region of DNA nucleosides, such as one, two or three DNA nucleosides. Mixmers are exemplified herein by each second design in which the nucleosides alternate between one LNA nucleoside and one DNA nucleoside, for example LDLDLDLDLDLDLDLL with 5' and 3' terminal LNA nucleosides, and each third design, such as LDDLDDLDDLDDLDDL, in which each third nucleoside is an LNA nucleoside.

[0220] In one embodiment, a mixmer may comprise or consist of alternating nucleosides of 1, 2 or 3 consecutive DNA nucleosides followed by 1 or 2 consecutive LNA nucleosides.

[0221] A totalmer is an oligonucleotide or contiguous nucleotide sequence thereof that does not contain any DNA or RNA nucleosides and may, for example, contain only complete 2'-MOE phosphorothioates, e.g., MMMMMMMMMMMMMMMMMMMM, where M=2'-O-MOE, or, for example, contain only 2'oMe nucleosides, which have been reported to be effective steric blockers for therapeutic use.

[0222] Alternatively, the mixmer may comprise a mixture of modified nucleosides such as MLMLMLMLMLMLMLMLML, where L=LNA and M=2'-O-MOE nucleoside.

[0223] Advantageously, the internucleosides in the mixmers and totalmers may be phosphorothioates or the majority of the nucleoside linkages in the mixmers may be phosphorothioates. Mixmers and totalmers may contain other internucleoside linkages such as, for example, phosphodiester or phosphorodithioate.

[0224] In some embodiments, the antisense oligonucleotide is or comprises an oligonucleotide mixmer or totalmer. In some embodiments, the contiguous nucleotide sequence is a mixmer or totalmer.

[0225] Complementarity The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U).

[0226] Oligonucleotides may contain nucleosides having modified nucleobases, for example, 5-methylcytosine is often substituted for cytosine, and thus it will be understood that the term complementarity encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).

[0227] The term "% complementary" as used herein refers to the percentage of nucleotides of a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned nucleobases (from Watson-Crick base pairs) that are complementary between two sequences (when aligned with the target sequence 5'-3' and the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in the calculation of the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0228] Within the present invention, the term "complementary" requires that a contiguous nucleotide sequence is at least about 75% complementary, at least about 80% complementary, at least about 85% complementary, at least about 90% complementary, or at least about 95% complementary to a target sequence, e.g., a sequence of a 5'UTR or uORF located upstream of a canonical AUG start codon. In some embodiments, the antisense oligonucleotide or a contiguous sequence thereof may be at least about 75%, 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 or 100% complementary to a target sequence, e.g., a sequence of a 5'UTR or uORF located upstream of a canonical AUG start codon.

[0229] In some embodiments, a contiguous nucleotide sequence in an antisense oligonucleotide of the invention may contain one, two, three or more mismatches, where a mismatch is a nucleotide in the contiguous nucleotide sequence that does not base pair with its target.

[0230] The term "fully complementary" refers to 100% complementarity.

[0231] In some embodiments, the contiguous nucleotide sequence is perfectly complementary to the target sequence.

[0232] identity As used herein, the term "identity" refers to the proportion of nucleotides (expressed as a percentage) of a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence.

[0233] Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleobases between the two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

[0234] It is therefore understood that a relationship exists between identity and complementarity, such that a contiguous nucleotide sequence within an antisense oligonucleotide of the invention which is complementary to a target sequence also shares a percentage of identity with the complementary sequence.

[0235] Hybridization The term "hybridize" or "hybridizing" as used herein should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is measured by the melting temperature (T), defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often explained by T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The Gibbs free energy ΔG° at standard conditions more accurately represents the binding affinity, ΔG°=-RTln(K d ) to calculate the dissociation constant (K d), 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 the reaction at an aqueous concentration of 1M, pH of 7, and temperature of 37°C. Hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and ΔG° in the case of a spontaneous reaction is less than zero. ΔG° can be experimentally measured, for example, by isothermal titration calorimetry (ITC), 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 equipment is available for measuring ΔG°. ΔG° can also be estimated numerically using appropriately derived thermodynamic parameters described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

[0236] In some embodiments, the antisense oligonucleotides of the invention hybridize to a target nucleic acid with an estimated ΔG° value of less than −10 kcal for oligonucleotides 10-30 nucleotides in length.

[0237] In some embodiments, the degree or strength of hybridization is measured by the Gibbs free energy ΔG° under standard conditions. The oligonucleotide may hybridize to the target nucleic acid with an estimated ΔG° value in the range of less than -10 kcal, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal for an oligonucleotide of 8 to 30 nucleotides in length. In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal, or -16 to -27 kcal, such as -18 to -25 kcal.

[0238] Region D' or D" in the oligonucleotide The antisense oligonucleotides of the invention may, in some embodiments, comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, e.g., a mixmer or totalmer region, as well as further 5' and / or 3' nucleosides. The further 5' and / or 3' nucleosides may be complementary, e.g., fully complementary, to the target nucleic acid, or may not be complementary, e.g., fully complementary. Such further 5' and / or 3' nucleosides may be referred to herein as regions D' and D".

[0239] The addition of region D' or D" can be used for the purpose of linking a contiguous nucleotide sequence, such as a mixmer or totalmer, to a conjugate moiety or another functional group. When used for linking, the contiguous nucleotide sequence with the conjugate moiety can serve as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0240] Region D' or D" independently comprises or consists of 1, 2, 3, 4 or 5 additional nucleotides and may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides such as DNA or RNA or base-modified versions thereof. The D' or D' region may serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the 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 WO 2014 / 076195, including by way of example phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, where they have been used to link multiple antisense constructs within a single oligonucleotide.

[0241] In one embodiment, the antisense oligonucleotide of the invention comprises, in addition to the contiguous nucleotide sequence constituting the mixmer or totalmer, regions D' and / or D".

[0242] In some embodiments, the internucleoside linkage located between region D' or D" and the mixmer or totalmer region is a phosphodiester bond.

[0243] Conjugates The present invention comprises an antisense oligonucleotide covalently attached to at least one conjugate moiety, which in some embodiments may be referred to as a conjugate of the present invention.

[0244] In some embodiments, the present invention provides an antisense oligonucleotide covalently linked to at least one conjugate moiety.

[0245] The term "conjugate" as used herein refers to an antisense oligonucleotide covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or a third region). The conjugate moiety may be covalently attached to the oligonucleotide, optionally via a linker group such as region D' or D".

[0246] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by 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.

[0247] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate (e.g., GalNAc), a cell surface receptor ligand, a drug substance, a hormone, a lipophile, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0248] Linker Bond or linker is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.The conjugate moiety can be directly or through a linking moiety (e.g., linker or tether) to the antisense oligonucleotide.The linker serves to covalently connect the third region, e.g., the conjugate moiety (region C), to the first region, e.g., the oligonucleotide or continuous nucleotide sequence (region A) that is complementary to the target nucleic acid.

[0249] In some embodiments of the invention, the conjugates or antisense oligonucleotides of the invention may optionally comprise a linker region (second region or region B and / or region Y) located between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

[0250] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally encountered or similar to those encountered in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions, or drugs, as well as salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker comprises 1-5 nucleosides, such as DNA nucleosides that contain at least two consecutive phosphodiester bonds. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195.

[0251] Region Y refers to a linker that is not necessarily biocleavable but serves primarily to covalently attach the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units or aminoalkyl groups. Antisense oligonucleotide conjugates of the 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, for example, a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In some embodiments, the linker (region Y) is a C6 aminoalkyl group.

[0252] salt The term "salt" as used herein corresponds to its commonly understood meaning, i.e., an ionic collection of an anion and a cation.

[0253] The present invention provides pharma- ceutically acceptable salts of the antisense oligonucleotides of the present invention.

[0254] The present invention provides the antisense oligonucleotides of the present invention in the form of a pharma- ceutically acceptable salt. In some embodiments, the pharma-ceutically acceptable salt may be a sodium salt or a potassium salt.

[0255] The present invention provides pharma- ceutically acceptable sodium salts of the antisense oligonucleotides of the invention.

[0256] The present invention provides pharma- ceutically acceptable potassium salts of the antisense oligonucleotides of the invention.

[0257] Delivery of antisense oligonucleotides The present invention provides antisense oligonucleotides of the present invention, wherein the antisense oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked or encapsulated in a dendrimer, or conjugated to an aptamer.

[0258] This may be for the purpose of delivering the antisense oligonucleotides of the invention to targeted cells and / or improving the pharmacokinetics of the antisense oligonucleotides.

[0259] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0260] Pharmaceutical Compositions The invention provides pharmaceutical compositions comprising an antisense oligonucleotide of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0261] The present invention provides a pharmaceutical composition comprising an antisense oligonucleotide of the present invention and a pharma- ceutically acceptable salt. For example, the salt may include a metal cation, such as a sodium salt or a potassium salt.

[0262] The invention provides a pharmaceutical composition of the invention, comprising an antisense oligonucleotide of the invention and an aqueous diluent or solvent.

[0263] The invention provides solutions, such as phosphate buffered saline solutions, of the antisense oligonucleotides of the invention. In some embodiments, the solutions, such as phosphate buffered saline solutions, of the invention are sterile solutions.

[0264] Methods for modulating GBA expression The present invention provides a method for enhancing, upregulating or restoring expression of GBA in a cell, such as a cell expressing GBA, comprising administering to the cell an effective amount of an antisense oligonucleotide of the present invention or a pharmaceutical composition of the present invention.

[0265] In some embodiments, the method is an in vitro method.

[0266] In some embodiments, the method is an in vivo method.

[0267] In some embodiments, the cell is an animal cell, preferably a mammalian cell, such as a mouse cell, a rat cell, a hamster cell, or a monkey cell, or preferably a human cell.

[0268] In some embodiments, the cells are either human or mammalian cells.

[0269] In some embodiments, the cell is part of or derived from a subject suffering from or susceptible to a disease associated with reduced expression of GBA, including, but not limited to, Gaucher disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0270] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Thus, it will be recognized that the treatments referred to herein may, in some embodiments, be prophylactic.

[0271] The invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

[0272] The disease may be associated with decreased expression of GBA.

[0273] In some embodiments, the present invention provides a method for treating or preventing a disease associated with decreased expression of GBA, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the present invention or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to a disease associated with decreased expression of GBA.

[0274] In one embodiment, the disease is selected from the group consisting of Gaucher's disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0275] In one embodiment, the disease is Parkinson's disease.

[0276] In one embodiment, the disease is Gaucher's disease.

[0277] In some embodiments, the subject is an animal, preferably a mammal such as a mouse, rat, hamster, or monkey, or preferably a human. In some embodiments, the subject is a human.

[0278] The present invention provides the antisense oligonucleotide of the present invention for use as a medicament.

[0279] The present invention provides an antisense oligonucleotide of the present invention for the preparation of a medicament.

[0280] The present invention provides an antisense oligonucleotide of the invention for use in therapy.

[0281] The present invention provides the antisense oligonucleotide of the present invention or the pharmaceutical composition of the present invention for use as a medicament.

[0282] The present invention provides an antisense oligonucleotide of the present invention or a pharmaceutical composition of the present invention for preparing a medicament.

[0283] The present invention provides an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention for use in therapy.

[0284] The present invention provides an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention for use as a medicament in the treatment of Parkinson's disease.

[0285] The present invention provides an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention for use as a medicament in the treatment of Gaucher disease.

[0286] The present invention provides the use of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment or prevention of Parkinson's disease.

[0287] The present invention provides the use of an antisense oligonucleotide of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment or prevention of Gaucher disease.

[0288] Administration The antisense oligonucleotides of the invention or pharmaceutical compositions of the invention may be administered topically (such as to the skin, inhalation, eye or ear) or enterally (such as orally or through the digestive tract) or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0289] In a preferred embodiment, the antisense oligonucleotide of the present invention or the pharmaceutical composition of the present invention is administered by parenteral routes, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular administration. In one embodiment, the antisense oligonucleotide of the present invention is administered intracerebrally or intraventricularly. In another embodiment, the antisense oligonucleotide of the present invention is administered intrathecally.

[0290] The present invention also provides the use of the described antisense oligonucleotide of the invention or the pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intrathecal administration.

[0291] The present invention also provides the use of the described antisense oligonucleotide of the invention or the pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intracerebral or intraventricular administration.

[0292] The present invention also provides the use of the described antisense oligonucleotide of the invention or the pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intracerebroventricular administration.

[0293] Combination therapy In some embodiments, the antisense oligonucleotide of the invention or the pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.

[0294] GBA upregulation In certain embodiments, the antisense oligonucleotides of the present invention can enhance or increase the expression of GBA by at least about 10%. In other embodiments, the antisense oligonucleotides of the present invention can enhance or increase the expression of GBA by 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%, or at least about 600% or more.

[0295] In certain embodiments, the antisense oligonucleotides of the present invention can enhance or increase the expression of GBA protein by at least about 10%. In other embodiments, the antisense oligonucleotides of the present invention can enhance or increase the expression of GBA protein by 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%, or at least about 600% or more.

[0296] It is understood that the above enhancement or increase in expression of GBA protein is the increase in % compared to a control, and the control is a cell that is not exposed to the oligonucleotide. In some embodiments, the control can be mock transfection, for example, treatment of cells with PBS. Here, the increase in expression of GBA can be measured as an increase in GBA protein.

[0297] [Table 1] TIFF2025501498000005.tif248170 TIFF2025501498000006.tif248170

[0298] [Table 2] TIFF2025501498000008.tif255170 TIFF2025501498000009.tif255170 TIFF2025501498000010.tif255170 TIFF2025501498000011.tif255170 EXAMPLES

[0299] Example 1: Upregulation of GBA mRNA The day before the transfection procedure, SK-N-AS neuroblastoma cells were seeded at a density of 25000 / well in 96-well plates in complete growth medium (DMEM (Sigma:D6546), 10% FBS, 2 mM glutamine, 0.1 mM (1x) NEAA, 25 μg / ml gentamicin). The day after plating, cells were transfected with SEQ ID NOs: 1-40 (n=2) or PBS (mock) using Lipofectamin RNAiMax (Invitrogen) at a final concentration of 10 nM according to the Invitrogen instructions. 48 hours after transfection, mRNA was isolated using the RNeasy® 96 Kit (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 4 (qScript™ XLT One-Step RT-qPCR ToughMix™, Low ROX™, Quanta Bioscience, #95134-500) using a custom designed qPCR assay specific for GBA and a pre-designed assay for TBP (HEX, Hs.PT.58v.39858774, IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 1. [Table 3]

[0300] [Table 4]

[0301] As shown in Figure 1, SEQ ID NO:9, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38 all increase GBA mRNA expression by more than 10% fold compared to mock transfection 48 hours after transfection in SK-N-AS cells.

[0302] Example 2: Upregulation of GBA mRNA SK-N-As neuroblastoma cells were seeded at a density of 10000 cells / well in 96-well plates in complete growth medium (DMEM (Sigma:D6546), 10% FBS, 2 mM glutamine, 0.1 mM (1x) NEAA, 25 μg / ml gentamicin). The day after plating, cells were treated (10 μM final concentration) with either SEQ ID NOs: 1-40 (n=2) or PBS (mock). After 72 hours of treatment, mRNA was isolated using the RNeasy® 96 Kit (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 4 (qScript™ XLT One-Step RT-qPCR ToughMix™, Low ROX™, Quanta Bioscience, #95134-500) using a custom-designed qPCR assay specific for GBA and a pre-designed assay for TBP (HEX, Hs.PT.58v.39858774, IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 2.

[0303] As shown in Figure 2, 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 and SEQ ID NO:30 all increase GBA mRNA expression by more than 10% compared to mock after 72 hours of gymnosis in SK-N-AS cells.

[0304] Example 3: Upregulation of GBA protein SK-N-As neuroblastoma cells were seeded at a density of 60000 cells / well in 24-well plates in complete growth medium (DMEM (Sigma:D6546), 10% FBS, 2 mM glutamine, 0.1 mM (1x) NEAA, 25 μg / ml gentamicin). The day after plating, cells were treated with either SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:41 (n=2) or PBS (mock) (50 μM final concentration). After 72 hours of treatment, proteins were isolated using 75 μl RIPA (ThermoFisher Scientific, #89900) supplemented with 1 mM DTT buffer. Proteins were detected using Wes analysis (ProteinSimple) using a 12-230 kDa Wes separation module (ProteinSimple, #SM-W004) and anti-glucocerebrosidase (Sigma Aldrich, #G4046) and HPRT (Santa Cruz Biotechnology, #FL-218) antibodies according to the manufacturer's recommendations. GBA protein abundance was quantified relative to HPRT1 protein using "Compass for SW" software and Microsoft Excel. See Figure 3.

[0305] As shown in Figure 3, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:35 and SEQ ID NO:38 all increase GBA protein expression by more than 30% compared to mock after 72 hours of gymnosis in SK-N-AS cells. SEQ ID NO:105 reduces GBA protein by more than 70%.

[0306] Embodiment 1. An antisense oligonucleotide of 8 to 40 nucleotides in length, comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length, wherein the contiguous nucleotide sequence is complementary to a sequence within the 5' untranslated region (5'UTR) located upstream of the canonical AUG start codon of a glucocerebrosidase (GBA) mRNA transcript.

[0307] 2. The antisense oligonucleotide of item 1, wherein the contiguous nucleotide sequence 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 in length.

[0308] 3. The antisense oligonucleotide according to item 2, wherein the contiguous nucleotide sequence is 16 or 18 nucleotides in length.

[0309] 4. The antisense oligonucleotide according to any one of items 1 to 3, wherein the contiguous nucleotide sequence is the same length as the antisense oligonucleotide.

[0310] 5. The antisense oligonucleotide according to any one of items 1 to 4, wherein the contiguous nucleotide sequence is at least 75% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0311] 6. The antisense oligonucleotide according to any one of items 1 to 5, wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0312] 7. The antisense oligonucleotide according to any one of items 1 to 6, wherein the contiguous nucleotide sequence is perfectly complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

[0313] 8. The antisense oligonucleotide according to any one of items 1 to 7, wherein the GBA mRNA transcript comprises a nucleic acid sequence according to SEQ ID NO: 108.

[0314] 9. The antisense oligonucleotide according to any one of items 1 to 8, wherein the canonical AUG start codon corresponds to positions 138 to 140 of SEQ ID NO: 108.

[0315] 10. The antisense oligonucleotide according to any one of items 1 to 9, wherein the 5'UTR comprises an upstream AUG (uAUG).

[0316] 11. The antisense oligonucleotide according to item 10, wherein the uAUG is located 14 nt upstream of the canonical AUG.

[0317] 12. The antisense oligonucleotide according to item 10 or 11, wherein uAUG is at a position corresponding to positions 124 to 126 of SEQ ID NO: 108.

[0318] 13. The antisense oligonucleotide according to any one of items 1 to 12, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90.

[0319] 14. The antisense oligonucleotide according to any one of items 1 to 13, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:109.

[0320] 15. The antisense oligonucleotide according to any one of items 1 to 14, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

[0321] 16. The antisense oligonucleotide according to any one of items 1 to 15, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37 and SEQ ID NO:38 or at least 10 contiguous nucleotides thereof.

[0322] 17. The antisense oligonucleotide according to any one of items 1 to 16, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:110 or at least 10 contiguous nucleotides thereof.

[0323] 18. The antisense oligonucleotide according to any one of items 1 to 17, comprising one or more modified nucleosides.

[0324] 19. The antisense oligonucleotide of item 18, wherein the one or more modified nucleosides are 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, and locked nucleic acid (LNA) nucleosides.

[0325] 20. The antisense oligonucleotide according to item 18 or 19, wherein one or more modified nucleosides are LNA nucleosides.

[0326] 21. The antisense oligonucleotide according to item 19 or 20, wherein the LNA nucleoside is beta-D-oxy-LNA.

[0327] 22. The antisense oligonucleotide according to any one of items 18 to 21, wherein the one or more modified nucleosides are 2'-O-methyl-RNA nucleosides.

[0328] 23. The antisense oligonucleotide according to any one of items 1 to 22, wherein the oligonucleotide is a mixmer.

[0329] 24. The antisense oligonucleotide according to any one of items 1 to 23, comprising at least one modified internucleoside linkage.

[0330] 25. The antisense oligonucleotide according to any one of items 1 to 24, wherein one or more or all of the modified internucleoside linkages comprise a phosphorothioate linkage.

[0331] 26. The antisense oligonucleotide according to item 25, wherein all of the internucleoside linkages present in the antisense oligonucleotide are phosphorothioate internucleoside linkages.

[0332] 27. The antisense oligonucleotide according to any one of items 1 to 26, wherein the contiguous nucleotide sequence is capable of increasing expression of GBA by 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

[0333] 28. The oligonucleotide according to item 27, wherein the control is a cell not exposed to the oligonucleotide.

[0334] 29. The antisense oligonucleotide according to any one of items 1 to 28, wherein the antisense oligonucleotide is covalently linked to at least one conjugate moiety.

[0335] 30. The antisense oligonucleotide according to any one of items 1 to 29, wherein the antisense oligonucleotide is in the form of a pharma- ceutically acceptable salt.

[0336] 31. The antisense oligonucleotide according to item 30, wherein the salt is a sodium salt or a potassium salt.

[0337] 32. The antisense oligonucleotide according to any one of items 1 to 31, wherein the antisense oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked or encapsulated in a dendrimer, or conjugated to an aptamer.

[0338] 33. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of items 1 to 32 and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0339] 34. The pharmaceutical composition according to item 33, comprising an aqueous diluent or solvent such as phosphate buffered saline.

[0340] 35. An in vitro or in vivo method for upregulating or restoring GBA expression in a target cell, comprising administering to said cell an effective amount of an antisense oligonucleotide according to any one of items 1 to 32 or a pharmaceutical composition according to item 33 or item 34.

[0341] 36. The method according to item 35, wherein the cell is a human cell or a mammalian cell.

[0342] 37. The method according to item 35 or item 36, wherein expression of GBA is increased by 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

[0343] 38. The oligonucleotide according to item 37, wherein the control is a cell not exposed to the oligonucleotide.

[0344] 39. A method for treating or preventing a disease, comprising administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of an antisense oligonucleotide according to any one of items 1 to 32, or a pharmaceutical composition according to item 33 or item 34.

[0345] 40. The antisense oligonucleotide according to any one of items 1 to 32 or the pharmaceutical composition according to item 33 or item 34 for use as a medicament for treating or preventing a disease in a subject.

[0346] 41. Use of an antisense oligonucleotide according to any one of items 1 to 32 or a pharmaceutical composition according to item 33 or item 34 for the preparation of a medicament for treating or preventing a disease in a subject.

[0347] 42. The method according to item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41, wherein the disease is associated with a decrease in the expression of GBA.

[0348] 43. The method according to item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41, wherein the disease is selected from the group consisting of Gaucher disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0349] 44. The method according to item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41, wherein the disease is Parkinson's disease.

[0350] 45. The method according to item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41, wherein the disease is Gaucher's disease.

Claims

1. An antisense oligonucleotide of 8 to 40 nucleotides in length, comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length, said contiguous nucleotide sequence being complementary to a sequence within the 5' untranslated region (5'UTR) located upstream of the canonical AUG start codon of a glucocerebrosidase (GBA) mRNA transcript.

2. The antisense oligonucleotide of claim 1, wherein the contiguous nucleotide sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or completely complementary to a sequence within the 5'UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

3. The antisense oligonucleotide of claim 1 or 2, wherein the GBA mRNA transcript comprises a nucleic acid sequence according to SEQ ID NO:

108.

4. The antisense oligonucleotide of any one of claims 1 to 3, wherein the canonical AUG start codon corresponds to positions 138 to 140 of SEQ ID NO:

108.

5. The antisense oligonucleotide of any one of claims 1 to 4, wherein the 5'UTR comprises an upstream AUG (uAUG).

6. The antisense oligonucleotide of claim 5 , wherein the uAUG is located 14 nt upstream of the canonical AUG.

7. The antisense oligonucleotide of claim 5 or 6, wherein the uAUG is at a position corresponding to positions 124 to 126 of SEQ ID NO:

108.

8. The antisense oligonucleotide of any one of claims 1 to 7, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence or a fragment thereof selected from the group consisting of 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:81, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:

109.

9. The antisense oligonucleotide according to any one of claims 1 to 8, wherein the antisense oligonucleotide is a single-stranded antisense oligonucleotide.

10. The antisense oligonucleotide of any one of claims 1 to 9, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of 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:29, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

11. The antisense oligonucleotide of any one of claims 1 to 10, wherein the contiguous nucleotide sequence is capable of increasing expression of GBA by 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

12. 12. The antisense oligonucleotide of any one of claims 1 to 11 for use as a medicament for treating or preventing a disease in a subject.

13. The antisense oligonucleotide for use according to claim 12, wherein the disease is associated with a decreased expression of GBA.

14. 14. The antisense oligonucleotide for use according to claim 12 or 13, wherein the disease is selected from the group consisting of Gaucher's disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB), and rapid eye movement (REM) sleep behavior disorder.

15. The antisense oligonucleotide for use according to any one of claims 12 to 14, wherein the disease is Parkinson's disease.

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