Oligonucleotide GBA agonists

Abstract

The present invention relates to oligonucleotides that upregulate or restore expression of glucocerebrosidase (GBA) in cells; conjugates, salts and pharmaceutical compositions thereof; and methods for the treatment of diseases associated with reduced expression of GBA, including Gaucher disease and / or Parkinson's disease.

Description

[Technical field]

[0001] The present invention relates to oligonucleotides that upregulate or restore expression of glucocerebrosidase (GBA) in cells; conjugates, salts and pharmaceutical compositions thereof; and methods for the treatment of diseases associated with reduced 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 subcutaneous bleeding, fatigue, anemia, low platelet count, and enlarged liver and spleen. Although the phenotype is heterogeneous, 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] This condition is inherited in an autosomal recessive pattern. More than 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 the 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, which may precede motor symptoms by years, include anosmia, rapid eye movement sleep behavior disorder, autonomic neuropathy 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 those 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.). Current treatments for diseases associated with reduced GBA expression include enzyme replacement therapy (ERT) and substrate synthesis suppression therapy (SRT). ERT involves the intravenous administration of recombinant GBA. Although most patients respond well to the treatment, there is a risk of developing an immune response. Furthermore, GBA cannot cross the blood-brain barrier, and therefore ERT is considered ineffective in patients with Parkinson's disease or the neuropathic form of Gaucher disease. 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 therapy has a higher incidence of adverse effects than ERT, and the long-term reduction in glucocerebroside may affect several different cellular functions. Both ERT and SRT are expensive and must be continued lifelong.

[0008] Therefore, there is an urgent need for therapeutic agents that can increase or restore the expression of GBA. Summary of the Invention

[0009] The present invention provides oligonucleotide agonists of glucocerebrosidase (GBA) or oligonucleotide GBA agonists, i.e., oligonucleotides complementary to GBA nucleic acid sequences. Specifically, the present invention provides oligonucleotide GBA agonists that target one or more of the GBA promoters. These oligonucleotides can upregulate the expression of GBA. Alternatively, the present invention provides oligonucleotide positive modulators (i.e., agonists) of GBA.

[0010] The oligonucleotides of the invention can be used to restore GBA expression in a cell or to enhance the expression of GBA in a cell.

[0011] The present invention provides oligonucleotide GBA agonists, the oligonucleotides being 8-40 nucleotides in length and comprising a contiguous sequence of 8-40 nucleotides in length that is complementary, e.g., fully complementary, to one or more promoters of the human GBA genomic sequence.

[0012] The human GBA genomic sequence can be the NCBI reference sequence: NG_009783.1.

[0013] The human GBA genomic sequence can be SEQ ID NO:226.

[0014] The promoter of the human GBA gene may comprise or consist of SEQ ID NO:225.

[0015] The promoter of the human GBA genomic sequence can be located immediately upstream of exon 1 of the human GBA gene, for example, between nucleotides 1,137 and 1 upstream of the ATG start codon in exon 1 of the human GBA gene.

[0016] The promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO:224.

[0017] The promoter of the human GBA genomic sequence can be located immediately upstream of exon-2 of the human GBA gene, for example, between nucleotides 4,558 and 3,394 upstream of the ATG start codon in exon 1 of the human GBA gene.

[0018] The promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO:223.

[0019] In some embodiments, the invention provides double-stranded oligonucleotide GBA agonists, the oligonucleotides being 8-40 nucleotides in length and comprising a contiguous sequence of 8-40 nucleotides in length that is complementary, e.g., fully complementary, to one or more promoters of the human GBA genomic sequence.

[0020] The double-stranded oligonucleotide GBA agonist can be a small activating RNA (saRNA).

[0021] An oligonucleotide GBA agonist may be or may include an oligonucleotide mixmer or totalmer.

[0022] An oligonucleotide GBA agonist may increase expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control, where increased expression of GBA may be measured as an increase in GBA mRNA, an increase in GBA protein expression, or an increase in both GBA mRNA and GBA protein expression.

[0023] Controls can be cells that have never been exposed to the oligonucleotide GBA agonist.

[0024] The oligonucleotide GBA agonist may be covalently attached to at least one conjugate moiety.

[0025] The oligonucleotide GBA agonists may be in the form of a pharma- ceutically acceptable salt.

[0026] Oligonucleotide GBA agonists may be encapsulated in a lipid-based delivery vehicle, covalently attached to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0027] The present invention provides pharmaceutical compositions comprising an oligonucleotide GBA agonist and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0028] The present invention provides an in vivo or in vitro method for upregulating or restoring GBA expression in a target cell, comprising administering to said cell an effective amount of an oligonucleotide GBA agonist or pharmaceutical composition of the present invention.

[0029] The cells can be human or mammalian cells.

[0030] The present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide GBA agonist or pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease.

[0031] The invention provides an oligonucleotide GBA agonist or a pharmaceutical composition of the invention for use as a medicament in the treatment or prevention of a disease in a subject.

[0032] The invention provides the use of an oligonucleotide GBA agonist or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment or prevention of a disease in a subject.

[0033] The disease may be associated with reduced expression of GBA.

[0034] The disease may be Gaucher's disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0035] These and other aspects and embodiments of the invention are described in further detail below. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 shows GBA mRNA expression levels in SK-N-AS neuroblastoma cells 48 hours after transfection compared to mock-transfected controls. [Diagram 2] FIG. 2 shows GBA mRNA expression levels in H4 glioma cells 48 hours after transfection compared to mock-transfected controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The inventors have identified that by targeting one or more of the promoter regions of the GBA gene using oligonucleotides, particularly double-stranded oligonucleotides such as small activating RNAs (saRNAs), the expression levels of GBA mRNA transcripts and / or the expression levels of the encoded protein products can be effectively enhanced.

[0038] Described herein are target sites present on the human GBA gene, particularly within the promoter region, that can be targeted by the oligonucleotides of the invention. The oligonucleotides of the invention are agonists of GBA, i.e., they increase the production of GBA mRNA and / or protein.

[0039] The present invention provides oligonucleotide glucocerebrosidase (GBA) agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length that is complementary to one or more promoters of the human GBA genomic sequence, NCBI Reference Sequence NG_009783.1 (SEQ ID NO: 226).

[0040] GBA agonist The present invention relates to oligonucleotide GBA agonists.

[0041] The oligonucleotide of the present invention is a GBA agonist, i.e. enhances the expression of GBA. This can mean increasing the expression of GBA nucleic acid, such as GBA mRNA, and / or increasing the expression of GBA protein. Enhanced GBA expression is desirable, for example, to treat Gaucher disease and / or Parkinson's disease.

[0042] As used herein, the term "GBA agonist" refers to a compound, in this case an oligonucleotide, that is capable of enhancing expression of GBA mRNA transcripts and / or GBA protein in a cell, such as a cell expressing GBA.

[0043] In some embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA mRNA by at least about 10%. In other embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA mRNA 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%, at least about 600%, or more.

[0044] In some embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA protein by at least about 10%. In other embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production 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%, at least about 600%, or more.

[0045] In some embodiments, the oligonucleotide GBA agonists of the invention can enhance GBA mRNA and protein production by at least about 10%. In other embodiments, the oligonucleotide GBA agonists of the invention can enhance GBA mRNA and protein production 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%, at least about 600%, or more.

[0046] Oligonucleotides As used herein, the term "oligonucleotide" is defined as a molecule that contains two or more covalently linked nucleosides, as is commonly understood by those of skill in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0047] 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 covalently linked nucleotides or nucleosides, or modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides, such as 2' sugar modified nucleosides. The oligonucleotides of the present invention may contain one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

[0048] In some embodiments, the oligonucleotide GBA agonists of the invention are double-stranded oligonucleotides.

[0049] In some embodiments, oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length.

[0050] In some embodiments, oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length and comprise a contiguous nucleotide sequence of 8-40 nucleotides.

[0051] In some embodiments, oligonucleotide GBA agonists of the invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0052] In some embodiments, oligonucleotide GBA agonists of the invention are at least 12 nucleotides in length.

[0053] In some embodiments, oligonucleotide GBA agonists of the invention are at least 14 nucleotides in length.

[0054] In some embodiments, oligonucleotide GBA agonists of the invention are at least 16 nucleotides in length.

[0055] In some embodiments, oligonucleotide GBA agonists of the invention are at least 18 nucleotides in length.

[0056] In some embodiments, the oligonucleotide GBA agonists of the invention are 21 nucleotides in length.

[0057] When discussing embodiments involving double stranded oligonucleotides, it will be understood that the length measurements refer to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken to be the length of the longest strand.

[0058] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an 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."

[0059] In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence and may optionally comprise a nucleotide linker region that may 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.

[0060] It is understood that the contiguous nucleotide sequence of an oligonucleotide cannot be longer than the oligonucleotide itself, and that the oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

[0061] In some embodiments, all nucleosides of the oligonucleotide constitute a contiguous nucleotide sequence.

[0062] A contiguous nucleotide sequence is a sequence of nucleotides in an oligonucleotide of the invention that is complementary, and optionally completely complementary, to a target nucleic acid, target sequence, or target site sequence.

[0063] In some embodiments, the contiguous nucleotide sequence is between 8 and 40 nucleotides in length.

[0064] 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.

[0065] In some embodiments, the contiguous nucleotide sequence is at least 12 nucleotides in length.

[0066] In some embodiments, the contiguous nucleotide sequence is at least 14 nucleotides in length.

[0067] In some embodiments, the contiguous nucleotide sequence is at least 16 nucleotides in length.

[0068] In some embodiments, the contiguous nucleotide sequence is at least 18 nucleotides in length.

[0069] In some embodiments, the contiguous nucleotide sequence is 19 nucleotides in length.

[0070] In some embodiments, the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.

[0071] In some embodiments, the oligonucleotide consists of a contiguous nucleotide sequence.

[0072] In some embodiments, the oligonucleotide is a contiguous nucleotide sequence.

[0073] Nucleotides and Nucleosides Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and in 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 nucleic acid base moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers".

[0074] Modified Nucleosides Advantageously, the oligonucleotide GBA agonists of the present invention may contain one or more modified nucleosides.

[0075] 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.

[0076] Advantageously, one or more of the modified nucleosides of the oligonucleotide of the present invention may include a modified sugar moiety. The term modified nucleoside may also be used herein 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 oligonucleotide GBA agonists of the present invention include LNA, 2'-O-MOE, 2'oMe and morpholino nucleoside analogues.

[0077] Modified Internucleoside Linkages Advantageously, the oligonucleotide GBA agonists of the invention contain one or more modified internucleoside linkages.

[0078] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides to each other as commonly understood by those skilled in the art. The oligonucleotide GBA agonists of the present invention may therefore contain one or more modified internucleoside linkages, for example, one or more phosphorothioate internucleoside linkages.

[0079] In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide GBA agonist or its contiguous nucleotide sequence are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% or more of the internucleoside linkages in the oligonucleotide GBA agonist or its contiguous nucleotide sequence are phosphorothioate. In some embodiments, all of the internucleoside linkages in the oligonucleotide GBA agonist or its contiguous nucleotide sequence are phosphorothioate.

[0080] In a further embodiment, the oligonucleotide GBA agonist comprises at least one modified internucleoside linkage. Advantageously, at least 75%, for example all, of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.

[0081] Conveniently, all internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide GBA agonist may be phosphorothioate linkages, or all internucleoside linkages of the oligonucleotide GBA agonist may be phosphorothioate linkages.

[0082] 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.

[0083] 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.

[0084] The nucleobase moieties may be represented by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplary oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides may be used.

[0085] Modified Oligonucleotides The oligonucleotide GBA agonists of the present invention may be modified oligonucleotides.

[0086] The term modified oligonucleotide refers to the 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 the oligonucleotide that comprises sugar-modified nucleosides and DNA nucleosides.In some embodiments, the oligonucleotide GBA agonist of the present invention may advantageously be a chimeric oligonucleotide.

[0087] In some embodiments, the oligonucleotide GBA agonist 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.

[0088] 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.

[0089] 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.

[0090] The oligonucleotide GBA agonists of the present invention are designed using modified nucleosides and DNA nucleosides. Advantageously, high affinity modified nucleosides are used.

[0091] In one embodiment, the oligonucleotide GBA agonist 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, or at least 19 modified nucleosides.

[0092] Suitable modifications are described herein under the headings "modified nucleosides," "high affinity modified nucleosides," "sugar modifications," "2' sugar modifications," and "Locked Nucleic Acids (LNA)."

[0093] High-affinity modified nucleosides High affinity modified nucleosides, when incorporated into oligonucleotides, can, for example, increase the melting temperature (T m ) of the high affinity modified nucleosides of the present invention provide an increase in melting temperature of preferably +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), 293-213).

[0094] sugar modification Oligonucleotide GBA agonists of the present invention may contain one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0095] A number of nucleosides with modifications in the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0096] Such modifications include, for example, those in which the ribose ring structure has been modified by replacing it with a hexose ring (HNA), or a bicyclic ring, typically having a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring, typically lacking a bond between the C2 and C3 carbons (e.g., UNA). 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.

[0097] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or by altering the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0098] 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 biradicle that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as an LNA (2'-4' biradicle bridged) nucleoside.

[0099] 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, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrative examples of some 2'-substituted modified nucleosides. [ka]

[0100] In the context of the present invention, 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides such as LNA.

[0101] In one embodiment, the oligonucleotide GBA agonist comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides.Preferably, the oligonucleotide GBA agonist 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, arabino nucleic 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).

[0102] 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 said 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 (duplex stabilization) when LNA is incorporated into an oligonucleotide to a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0103] 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.

[0104] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: [ka]

[0105] 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.

[0106] A particularly advantageous LNA is beta-D-oxy-LNA.

[0107] Morpholino Oligonucleotides In some embodiments, the oligonucleotide GBA agonist of the present invention comprises or consists of a morpholino nucleoside (i.e., a morpholino oligomer, as a phosphorodiamidate morpholino oligomer (PMO)). Splice-modulating morpholino oligonucleotides have been approved for clinical use, see, for example, eteplirsen, a 30 nt morpholino oligonucleotide that targets a frameshift mutation in DMD and is used to treat Duchenne muscular dystrophy. Morpholino oligonucleotides have nucleases attached to a 6-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]

[0108] In some embodiments, the morpholino oligonucleotides of the invention can be, for example, 8 to 40 morpholino nucleotides in length, for example, 18 to 22 morpholino nucleotides in length.

[0109] RNase H activity and recruitment RNaseH activity of an oligonucleotide refers to its ability to recruit RNaseH when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNaseH activity that can be used to determine the ability to recruit RNaseH. Typically, an oligonucleotide is considered capable of recruiting RNaseH 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 linkages between all monomers in the oligonucleotide and using the methodology provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference). Recombinant RNaseH1 is available from Lubio Science GmbH, Lucerne, Switzerland for use in determining RNaseH activity.

[0110] DNA oligonucleotides are known to effectively recruit RNaseH, such as gapmer oligonucleotides that contain regions of 2' sugar-modified nucleosides, typically high affinity 2' sugar-modified nucleosides, such as 2-O-MOE and / or LNA-containing regions of DNA nucleosides (typically at least 5 or 6 consecutive DNA nucleosides) flanked at 5' and 3'. For effective regulation of splicing, degradation of pre-mRNA is undesirable, and thus it is preferable to avoid RNaseH degradation of the target. Thus, the oligonucleotide GBA agonist of the present invention is not a gapmer oligonucleotide that recruits RNaseH.

[0111] RNaseH recruitment can be avoided by limiting the number of consecutive DNA nucleotides in oligonucleotide, so that mixmer and totalmer designs can be used.Advantageously, the oligonucleotide GBA agonist of the present invention or its consecutive nucleotide sequence does not contain more than 3 consecutive DNA nucleosides.More advantageously, the oligonucleotide GBA agonist of the present invention or its consecutive nucleotide sequence does not contain more than 4 consecutive DNA nucleosides.More advantageously, the oligonucleotide GBA agonist of the present invention or its consecutive nucleotide sequence does not contain more than 2 consecutive DNA nucleosides.

[0112] Mixmar and Totalmar For splice regulation, it is often advantageous to use oligonucleotides that do not recruit RNAaseH. Because RNaseH activity requires a consecutive sequence of DNA nucleotides, RNaseH activity of an oligonucleotide can be achieved by designing an oligonucleotide that does not contain a region of more than three or more than four consecutive DNA nucleosides. This can be achieved by using an oligonucleotide or a region of consecutive nucleosides thereof in a mixmer design that contains 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 every other design with 5' and 3' terminal LNA nucleosides, where the nucleosides alternate between one LNA nucleoside and one DNA nucleoside, such as LDLDLDLDLDLDLDLL, and every third design, such as LDDLDDLDDLDDLDDL, where every third nucleoside is an LNA nucleoside.

[0113] A totalmer is an oligonucleotide or contiguous nucleotide sequence thereof that does not contain DNA or RNA nucleosides and may, for example, contain only 2'-O-MOE nucleosides, such as a full MOE phosphorothioate, e.g., MMMMMMMMMMMMMMMMMMMM, where M=2'-O-MOE, or may, for example, contain only 2'oMe nucleosides, which have been reported to be effective splice modulators for therapeutic use.

[0114] Alternatively, the mixmer may comprise a mixture of modified nucleosides such as MLMLMLMLMLMLMLMLML, where L=LNA and M=2'-O-MOE nucleoside.

[0115] Conveniently, 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.

[0116] In some embodiments, the oligonucleotide GBA agonist is or comprises an oligonucleotide mixmer or totalmer. In some embodiments, the contiguous nucleotide sequence is a mixmer or totalmer.

[0117] Target sequence The oligonucleotide GBA agonists of the present invention target one or more of the promoters of the human GBA genomic sequence, which may also be referred to herein as the GBA gene.

[0118] The GNA genomic sequence may be referred to as the target sequence.

[0119] The target sequence may also be referred to as a target nucleic acid or a target site sequence.

[0120] The term "genomic sequence" as used herein, particularly with respect to the GBA genomic sequence, 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).

[0121] The term "GBA nucleic acid sequence" as used herein may also refer to the nucleic acid sequence of the GBA gene in the sense of the definition outlined herein. The target nucleic acid sequence of the GBA gene may refer to the sequence present in genomic DNA, or the same or antisense sequence present in the cell in any other form, such as mRNA, or other single-stranded or double-stranded RNA, such as miRNA or siRNA.

[0122] References to genes and their corresponding nucleotide sequences as used herein are not necessarily intended to be limited to either the sense or antisense strands thereof, and thus may include both or either the sense and antisense sequences.

[0123] In some embodiments, the human GBA gene has the sequence of NCBI reference NG_009783.1.

[0124] In some embodiments, the human GBA gene comprises a sequence defined by SEQ ID NO: 226. SEQ ID NO: 226 is provided herein as a reference sequence, and it will be understood that the target nucleic acid can be an allelic variant of SEQ ID NO: 226, for example, an allelic variant that includes one or more polymorphisms in the human GBA genomic sequence.

[0125] In some embodiments, the human GBA genomic sequence consists of SEQ ID NO:226.

[0126] The oligonucleotide GBA agonists of the present invention are targeted to one or more promoter regions of the GBA genomic sequence.

[0127] In some embodiments, the promoter of the human GBA gene comprises the intergenic region from FAM189B to the GBA translation start codon (SEQ ID NO: 225). SEQ ID NO: 225 is provided herein as a reference sequence, and it will be understood that the target nucleic acid can be an allelic variant of SEQ ID NO: 225, for example an allelic variant that includes one or more polymorphisms in the human GBA genomic sequence.

[0128] In some embodiments, the promoter of the human GBA gene comprises or consists of SEQ ID NO:225.

[0129] SEQ ID NO:225 encompasses promoters 1 (SEQ ID NO:224) and 2 (SEQ ID NO:223), as discussed below.

[0130] In some embodiments, the oligonucleotide GBA agonists of the invention target the promoter (promoter 1, P1) located immediately upstream of exon 1 of the GBA gene, for example, between nucleotides 1,137 and 1 upstream of the ATG start codon in exon 1 of the human GBA gene.

[0131] In some embodiments, the ATG start codon corresponds to positions 8586-8588 of the NCBI reference sequence NG_009783.1 and / or SEQ ID NO:226.

[0132] In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene (SEQ ID NO: 224) within positions 7449-8585 of the NCBI reference sequence NG_009783.1. In other words, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene (SEQ ID NO: 224) within positions 7449-8585 of SEQ ID NO: 226.

[0133] In some embodiments, the promoter of the human GBA gene comprises SEQ ID NO:224.

[0134] In some embodiments, the promoter of the human GBA gene consists of SEQ ID NO:224.

[0135] In some embodiments, the target sequence is a sequence of nucleotides within SEQ ID NO:224.

[0136] SEQ ID NO:224 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:224, for example an allelic variant that includes one or more polymorphisms in the human GBA genomic sequence.

[0137] In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is selected from the group consisting of nucleotides 35-55, 36-56, 39-59, 46-66, 68-88, 106-126, 148-168, 152-172, 153-173, 302-322, 313-333, 314-334, 371-391, 372-392, 475-495, 517-537, 532-552, 598-618, 656-662, 657-663, 659-671, 660-672, 661-673, 662-674, 663-675, 664-676, 665-678, 666-679, 667-679, 671-679, 672-679, 673-674, 675-675, 676-679, 677-679, 678-679, 679-679, 680-680, 681-681, 682-682, 683-683, 684-684, 685-685, 686-686, 687-687, 688-688, 689-700, 690-700, 70 ... 76, 657-677, 746-766, 747-767, 748-768, 749-769, 750-770, 753-773, 815-835, 816-836, 839-859, 840-860, 884-904, 891-911, 904-924, 922-942, 923-943, 996-1016, 1003-1023, 1019-1039, 1037-1057, and 1098-1118.

[0138] Unless otherwise stated, all ranges are inclusive of the starting and ending values, for example, the target sequence corresponding to positions 4137-4157 contains 21 nucleotides.

[0139] In some embodiments, the contiguous nucleotide sequence of an oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 39-59, 148-168, 153-173, 302-322, 313-333, 517-537, 532-552, 656-676, 748-768, 750-770, 815-835, 840-860, 904-924, and 996-1016 of SEQ ID NO:224.

[0140] In some embodiments, the contiguous nucleotide sequence of an oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 748-768, 750-770, 815-835, 904-924, and 996-1016 of SEQ ID NO:224.

[0141] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, or a fragment thereof.

[0142] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:36, or a fragment thereof.

[0143] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33 and SEQ ID NO:36, or a fragment thereof.

[0144] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:3, or a fragment thereof.

[0145] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:7, or a fragment thereof.

[0146] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:9, or a fragment thereof.

[0147] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 10, or a fragment thereof.

[0148] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:11, or a fragment thereof.

[0149] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 16, or a fragment thereof.

[0150] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 17, or a fragment thereof.

[0151] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 19, or a fragment thereof.

[0152] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 23, or a fragment thereof.

[0153] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 25, or a fragment thereof.

[0154] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:27, or a fragment thereof.

[0155] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 30, or a fragment thereof.

[0156] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 33, or a fragment thereof.

[0157] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 36, or a fragment thereof.

[0158] In some embodiments, the oligonucleotide GBA agonists of the invention target the promoter (promoter 2, P2) located immediately upstream of exon-2 of the GBA gene, for example, between nucleotides 4,558 and 3,394 upstream of the ATG start codon in exon 1 of the human GBA gene.

[0159] In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene (SEQ ID NO: 223) within positions 4028-5192 of the NCBI reference sequence NG_009783.1. In other words, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene (SEQ ID NO: 223) within positions 4028-5192 of SEQ ID NO: 226.

[0160] In some embodiments, the promoter of the human GBA gene comprises SEQ ID NO:223.

[0161] In some embodiments, the promoter of the human GBA gene consists of SEQ ID NO:223.

[0162] In some embodiments, the target sequence is a sequence of nucleotides within SEQ ID NO:223.

[0163] SEQ ID NO:223 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:223, for example an allelic variant that includes one or more polymorphisms in the human GBA genomic sequence.

[0164] In some embodiments, the contiguous nucleotide sequence of an oligonucleotide GBA agonist of the invention is selected from the group consisting of nucleotides 13-33, 34-54, 38-58, 121-141, 159-179, 160-180, 173-193, 199-219, 200-220, 313-333, 319-339, 335-355, 336-356, 359-379, 391-411 of SEQ ID NO:223. , 422 to 442, 423 to 443, 454 to 474, 469 to 489, 535 to 555, 548 to 568, 551 to 571, 589 to 609, 590 to 610, 654 to 674, 669 to 689, 670 to 690, 676 to 696, 685 to 705, 744 to 764, 761 to 781, 762 to 782, 913 to 933 and 922 to 942.

[0165] In some embodiments, the contiguous nucleotide sequence of an oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121-141, 160-180, 173-193, 199-219, 200-220, 335-355, 336-356, 359-379, 391-411, 423-443, 469-489, 535-555, 548-568, 551-571, 589-609, and 670-690 of SEQ ID NO:223.

[0166] In some embodiments, the contiguous nucleotide sequence of an oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121-141, 160-180, 199-219, 200-220, 335-355, 336-356, 359-379, 423-443, 469-489, 548-568, 551-571, and 670-690 of SEQ ID NO:223.

[0167] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74, or a fragment thereof.

[0168] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:67, or a fragment thereof.

[0169] In some embodiments, the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:67, or a fragment thereof.

[0170] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 44, or a fragment thereof.

[0171] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 46, or a fragment thereof.

[0172] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 47, or a fragment thereof.

[0173] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 48, or a fragment thereof.

[0174] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 49, or a fragment thereof.

[0175] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:52, or a fragment thereof.

[0176] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:53, or a fragment thereof.

[0177] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:54, or a fragment thereof.

[0178] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:55, or a fragment thereof.

[0179] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:57, or a fragment thereof.

[0180] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:59, or a fragment thereof.

[0181] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO: 60, or a fragment thereof.

[0182] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:61, or a fragment thereof.

[0183] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:62, or a fragment thereof.

[0184] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:63, or a fragment thereof.

[0185] In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO:67, or a fragment thereof.

[0186] Fragments of the target sequence can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0187] In some embodiments, the contiguous nucleotide sequence is complementary to at least 8 contiguous nucleotides of any of the target sequences listed herein.

[0188] In some embodiments, the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of any of the target sequences listed herein.

[0189] In some embodiments, the contiguous nucleotide sequence is complementary to at least 12 contiguous nucleotides of any of the target sequences listed herein.

[0190] In some embodiments, the contiguous nucleotide sequence is complementary to at least 14 contiguous nucleotides of any of the target sequences listed herein.

[0191] In some embodiments, the contiguous nucleotide sequence is complementary to at least 16 contiguous nucleotides of any of the target sequences listed herein.

[0192] In some embodiments, the contiguous nucleotide sequence is complementary to at least 18 contiguous nucleotides of any of the target sequences listed herein.

[0193] In some embodiments, the contiguous nucleotide sequence is complementary to at least 19 contiguous nucleotides of any of the target sequences listed herein.

[0194] 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).

[0195] 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).

[0196] The term "% complementary" as used herein refers to the proportion (in percent) of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) over a 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 the two sequences (when aligning the target sequence 5'-3' with 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 calculating the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded as long as the nucleobases retain the functional ability to form Watson-Crick base pairs (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0197] In some embodiments of the present invention, oligonucleotide GBA agonist is a double-stranded oligonucleotide, such as saRNA.In these embodiments, double-stranded oligonucleotide can have nucleotide overhang, such as 2-nucleotide overhang that can be at the 3' end of the continuous nucleotide sequence.In such embodiments, complementarity is defined based on the double-stranded sequence without overhang.For example, if the oligonucleotide is 21 nucleotides long and contains 2-nucleotide overhang, complementarity is determined based on the 19 nucleotides without 2-nucleotide overhang.

[0198] In the present invention, the term "complementary" requires that the contiguous nucleotide sequence is at least about 75% complementary, or at least about 80% complementary, or at least about 85% complementary, or at least about 90% complementary, or at least about 95% complementary to the target sequence, i.e., one or more promoters of the human GBA gene. In some embodiments, the oligonucleotide GBA agonist may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to the target sequence, i.e., one or more promoters of the human GBA gene. In other words, in some embodiments, the contiguous nucleotide sequence in the oligonucleotide GBA agonist of the present invention may contain one, two, three or more mismatches, which are nucleotides in the contiguous nucleotide sequence that do not base pair with its target.

[0199] The term "fully complementary" refers to 100% complementarity.

[0200] In some embodiments, the contiguous nucleotide sequence is perfectly complementary to the target sequence.

[0201] 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.

[0202] Thus, the percentage of identity is calculated by counting the number of aligned nucleobases that are identical (matching) between 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, the 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 of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of nucleobases are disregarded as long as the nucleobase retains the functional ability to form Watson-Crick base pairs (e.g., 5'-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

[0203] It is therefore understood that a relationship exists between identity and complementarity, such that contiguous nucleotide sequences within the oligonucleotide GBA agonists of the present invention that are complementary to a target sequence also share a percentage of identity with said complementary sequence.

[0204] 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 often 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 ) in physiological conditions. m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents the binding affinity, ΔG°=-RTln(K d ) to calculate the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Thus, the very low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the 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. The hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, and the ΔG° in the case of a spontaneous reaction is less than zero. ΔG° can be experimentally measured, for example, by using an isothermal titration calorimetry (ITC) method 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 be estimated numerically by using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using appropriately derived thermodynamic parameters as described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

[0205] In some embodiments, oligonucleotide GBA agonists of the invention hybridize to target nucleic acids with estimated ΔG° values ​​of less than −10 kcal for oligonucleotides that are 10-30 nucleotides in length.

[0206] 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.

[0207] Double-stranded oligonucleotide GBA agonists In some embodiments, the oligonucleotide GBA agonist is a double-stranded oligonucleotide.

[0208] In some embodiments, the double-stranded oligonucleotide GBA agonist is a small activating RNA (saRNA).

[0209] The term "small activating RNA" (saRNA) as used herein refers to small double-stranded RNA that is typically 21 nucleotides long and may optionally contain a 2-nucleotide overhang at the 3' end. saRNA can induce gene activation by a process known as RNA activation (RNAa), where gene activation is induced by hybridization of saRNA with a target nucleic acid sequence. The target nucleic acid sequence typically includes the promoter region of a gene.

[0210] Known mechanisms of transcriptional upregulation by saRNA involve Ago2 and involve epigenetic modifications at target sites, e.g., promoters. Ago2 associates with saRNA, which guides the complex to its target and promotes assembly of the RNA-induced transcriptional activation (RITA) complex. RITA-RNA polymerase II interaction is thought to promote transcription initiation and productive elongation, as well as monoubiquitination of histone 2B (Portnoy et al., Cell Res., 2016, 26(3), 320-335).

[0211] In some embodiments, the oligonucleotide GBA agonists of the invention, such as saRNA, are 19 nucleotides in length.

[0212] In some embodiments, the oligonucleotide GBA agonists of the invention, such as saRNA, are 20 nucleotides in length.

[0213] In some embodiments, the oligonucleotide GBA agonists of the invention, such as saRNA, are 21 nucleotides in length.

[0214] In some embodiments, the oligonucleotide GBA agonists of the invention, such as saRNA, are 22 nucleotides in length.

[0215] When discussing embodiments involving double stranded oligonucleotides, it will be understood that the length measurements refer to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken to be the length of the longest strand.

[0216] In some embodiments, the oligonucleotide GBA agonist of the present invention, such as saRNA, can have a nucleotide overhang. The nucleotide overhang can be a 2-nucleotide overhang. In some embodiments, the overhang can be at the 3' end of the contiguous nucleotide sequence. In some embodiments, the overhang can comprise or consist of two thymine nucleotides (TT).

[0217] Contiguous nucleotide sequence In one embodiment, oligonucleotide GBA agonists of the invention may target sequences within promoter 1 of the human GBA genomic sequence.

[0218] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of 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, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114, or at least 10 contiguous nucleotides thereof.

[0219] It will be apparent to one of skill in the art that a double-stranded oligonucleotide can be defined by reference to either the sense strand or the antisense strand.

[0220] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107 and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0221] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:107 and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0222] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:77 or at least 10 contiguous nucleotides thereof.

[0223] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:81 or at least 10 contiguous nucleotides thereof.

[0224] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:83 or at least 10 contiguous nucleotides thereof.

[0225] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:84 or at least 10 contiguous nucleotides thereof.

[0226] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:85 or at least 10 contiguous nucleotides thereof.

[0227] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:90 or at least 10 contiguous nucleotides thereof.

[0228] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:91 or at least 10 contiguous nucleotides thereof.

[0229] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:93 or at least 10 contiguous nucleotides thereof.

[0230] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:97 or at least 10 contiguous nucleotides thereof.

[0231] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:99 or at least 10 contiguous nucleotides thereof.

[0232] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:101 or at least 10 contiguous nucleotides thereof.

[0233] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:104 or at least 10 contiguous nucleotides thereof.

[0234] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 107 or at least 10 contiguous nucleotides thereof.

[0235] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:110 or at least 10 contiguous nucleotides thereof.

[0236] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188, or at least 10 contiguous nucleotides thereof.

[0237] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:178, SEQ ID NO:181, and SEQ ID NO:184, or at least 10 contiguous nucleotides thereof.

[0238] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:181 and SEQ ID NO:184, or at least 10 contiguous nucleotides thereof.

[0239] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 151 or at least 10 contiguous nucleotides thereof.

[0240] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 155 or at least 10 contiguous nucleotides thereof.

[0241] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 157 or at least 10 contiguous nucleotides thereof.

[0242] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 158 or at least 10 contiguous nucleotides thereof.

[0243] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 159 or at least 10 contiguous nucleotides thereof.

[0244] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 164 or at least 10 contiguous nucleotides thereof.

[0245] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 165 or at least 10 contiguous nucleotides thereof.

[0246] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 167 or at least 10 contiguous nucleotides thereof.

[0247] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 171 or at least 10 contiguous nucleotides thereof.

[0248] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 173 or at least 10 contiguous nucleotides thereof.

[0249] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 175 or at least 10 contiguous nucleotides thereof.

[0250] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 178 or at least 10 contiguous nucleotides thereof.

[0251] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 181 or at least 10 contiguous nucleotides thereof.

[0252] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 184 or at least 10 contiguous nucleotides thereof.

[0253] In one embodiment, oligonucleotide GBA agonists of the invention may target sequences within promoter 2 of the human GBA genomic sequence.

[0254] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, and SEQ ID NO:148, or at least 10 contiguous nucleotides thereof.

[0255] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:141, or at least 10 contiguous nucleotides thereof.

[0256] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:136 and SEQ ID NO:141, or at least 10 contiguous nucleotides thereof.

[0257] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:118 or at least 10 contiguous nucleotides thereof.

[0258] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:120 or at least 10 contiguous nucleotides thereof.

[0259] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:121 or at least 10 contiguous nucleotides thereof.

[0260] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 122 or at least 10 contiguous nucleotides thereof.

[0261] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 123 or at least 10 contiguous nucleotides thereof.

[0262] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 126 or at least 10 contiguous nucleotides thereof.

[0263] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 127 or at least 10 contiguous nucleotides thereof.

[0264] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 128 or at least 10 contiguous nucleotides thereof.

[0265] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 129 or at least 10 contiguous nucleotides thereof.

[0266] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:131 or at least 10 contiguous nucleotides thereof.

[0267] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 133 or at least 10 contiguous nucleotides thereof.

[0268] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 134 or at least 10 contiguous nucleotides thereof.

[0269] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 135 or at least 10 contiguous nucleotides thereof.

[0270] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 136 or at least 10 contiguous nucleotides thereof.

[0271] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 137 or at least 10 contiguous nucleotides thereof.

[0272] In some embodiments, the sense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:141 or at least 10 contiguous nucleotides thereof.

[0273] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, and SEQ ID NO:222, or at least 10 contiguous nucleotides thereof.

[0274] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:215, or at least 10 contiguous nucleotides thereof.

[0275] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:210 and SEQ ID NO:215, or at least 10 contiguous nucleotides thereof.

[0276] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 192 or at least 10 contiguous nucleotides thereof.

[0277] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 194 or at least 10 contiguous nucleotides thereof.

[0278] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:195 or at least 10 contiguous nucleotides thereof.

[0279] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 196 or at least 10 contiguous nucleotides thereof.

[0280] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 197 or at least 10 contiguous nucleotides thereof.

[0281] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 200 or at least 10 contiguous nucleotides thereof.

[0282] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 201 or at least 10 contiguous nucleotides thereof.

[0283] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 202 or at least 10 contiguous nucleotides thereof.

[0284] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 203 or at least 10 contiguous nucleotides thereof.

[0285] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 205 or at least 10 contiguous nucleotides thereof.

[0286] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 207 or at least 10 contiguous nucleotides thereof.

[0287] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 208 or at least 10 contiguous nucleotides thereof.

[0288] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 209 or at least 10 contiguous nucleotides thereof.

[0289] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 210 or at least 10 contiguous nucleotides thereof.

[0290] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO:211 or at least 10 contiguous nucleotides thereof.

[0291] In some embodiments, the antisense strand of the contiguous nucleotide sequence is or comprises SEQ ID NO: 215 or at least 10 contiguous nucleotides thereof.

[0292] In certain embodiments where the oligonucleotide is a double-stranded oligonucleotide, the contiguous nucleotide sequence can be a 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide fragment of any of the sequences listed herein.

[0293] It will be understood that a double-stranded oligonucleotide, such as saRNA, comprises two complementary strands, each of which can hybridize with a complementary oligonucleotide sequence, such as an oligonucleotide sequence of an endogenous RNA or DNA. Without wishing to be bound by theory, it will be understood that the double-stranded oligonucleotide of the present invention can act by binding to the sense strand, the antisense strand, or both strands of a target sequence.

[0294] Region D' or D'' in the oligonucleotide The oligonucleotide GBA agonist of the present invention may, in some embodiments, comprise or consist of a contiguous nucleotide sequence of an oligonucleotide that is complementary to a target nucleic acid, such as a mixmer or totalmer region, and 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''.

[0295] The addition of a 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 to link a conjugate moiety to a conjugate moiety, the addition of a region D' or D'' can serve as a biocleavable linker. Alternatively, the addition of a region D' or D'' can be used to provide exonuclease protection or to facilitate synthesis or manufacture.

[0296] Region D' or D'' may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which 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 are used to link multiple antisense constructs within a single oligonucleotide.

[0297] In one embodiment, the oligonucleotide GBA agonist of the invention comprises regions D' and / or D'' in addition to the contiguous nucleotide sequence constituting the mixmer or totalmer.

[0298] In some embodiments, the internucleoside linkage located between region D' or D'' and the mixmer or totalmer region is a phosphodiester bond.

[0299] Conjugates The present invention encompasses an oligonucleotide GBA agonist covalently linked to at least one conjugate moiety, which in some embodiments may be referred to as a conjugate of the present invention.

[0300] In some embodiments, the present invention provides an oligonucleotide GBA agonist covalently attached to at least one conjugate moiety.

[0301] The term "conjugate" as used herein refers to an oligonucleotide GBA agonist covalently attached to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently attached to the oligonucleotide, optionally via a linker group such as region D' or D''.

[0302] 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.

[0303] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0304] Linker Bond or linker is the 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 oligonucleotide GBA agonist.The linker serves to covalently connect the third region, for example, the conjugate moiety (region C), to the first region, for example, the oligonucleotide or continuous nucleotide sequence (region A) that is complementary to the target nucleic acid.

[0305] In some embodiments of the invention, the conjugates or oligonucleotide GBA agonist conjugates 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).

[0306] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally 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, oxidizing or reducing conditions or oxidizing or reducing agents, and 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.

[0307] 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. The oligonucleotide GBA agonist conjugates of the invention may 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.

[0308] salt The term "salt" as used herein conforms to its commonly understood meaning, namely, an ionic assembly of anions and cations.

[0309] The invention provides a pharma- ceutically acceptable salt of an oligonucleotide GBA agonist according to the invention, or a conjugate according to the invention.

[0310] The present invention provides oligonucleotide GBA agonists according to 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.

[0311] The invention provides a pharma- ceutically acceptable sodium salt of an oligonucleotide GBA agonist according to the invention or a conjugate according to the invention.

[0312] The present invention provides a pharma- ceutically acceptable potassium salt of an oligonucleotide GBA agonist according to the invention or a conjugate according to the invention.

[0313] Delivery of oligonucleotide GBA agonists The present invention provides an oligonucleotide GBA agonist according to the present invention, wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0314] This may be for the purpose of delivering the oligonucleotide GBA agonists of the invention to targeted cells and / or improving the pharmacokinetics of the oligonucleotide GBA agonists.

[0315] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0316] Pharmaceutical Compositions The invention provides a pharmaceutical composition comprising an oligonucleotide GBA agonist of the invention or a conjugate or salt of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0317] The present invention provides a pharmaceutical composition comprising the oligonucleotide GBA agonist of the present invention or the conjugate 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.

[0318] The invention provides a pharmaceutical composition according to the invention comprising an oligonucleotide GBA agonist of the invention or a conjugate of the invention, or a pharma- ceutically acceptable salt of the invention, and an aqueous diluent or solvent.

[0319] The present invention provides a solution of the oligonucleotide GBA agonist of the present invention or the conjugate of the present invention, or a pharma- ceutically acceptable salt of the present invention, such as a phosphate buffered saline solution.Suitably, the solution of the present invention, such as a phosphate buffered saline solution, is a sterile solution.

[0320] Methods for regulating 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 said cell an effective amount of an oligonucleotide GBA agonist of the present invention, or a conjugate of the present invention, or a salt of the present invention, or a pharmaceutical composition of the present invention.

[0321] In some embodiments, the method is an in vitro method.

[0322] In some embodiments, the method is an in vivo method.

[0323] In some embodiments, the cells are either human or mammalian cells.

[0324] In some embodiments, the cell is part of a subject suffering from or susceptible to a disease associated with reduced expression of GBA, or is derived from a subject suffering from or susceptible to a disease associated with reduced expression of GBA.Such diseases include, but are not limited to, Gaucher disease, Parkinson's disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movement (REM) sleep behavior disorder.

[0325] 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.

[0326] The present invention provides a method for treating or preventing a disease associated with reduced expression of GBA, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide GBA agonist of the present invention or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to a disease associated with reduced expression of GBA.

[0327] 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.

[0328] In one embodiment, the disease is Parkinson's disease.

[0329] In one embodiment, the disease is Gaucher's disease.

[0330] In some embodiments, the subject is an animal, preferably a mammal such as a mouse, rat, hamster or monkey, or preferably a human.

[0331] The present invention provides an oligonucleotide GBA agonist of the invention for use as a pharmaceutical.

[0332] The invention provides an oligonucleotide GBA agonist of the invention for the preparation of a medicament.

[0333] The present invention provides oligonucleotide GBA agonists of the invention for use in therapy.

[0334] The present invention provides an oligonucleotide GBA agonist or a pharmaceutical composition of the present invention for use as a medicament.

[0335] The invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for the preparation of a medicament.

[0336] The present invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition according to the invention for use in therapy.

[0337] The present invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use in the treatment of Parkinson's disease.

[0338] The present invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use in the treatment of Gaucher disease.

[0339] The present invention provides the use of an oligonucleotide GBA agonist 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.

[0340] The present invention provides the use of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment or prevention of Gaucher disease.

[0341] Administration The oligonucleotide GBA agonists or pharmaceutical compositions of the invention may be administered topically (such as to the skin, inhalation, eye or ear), enterally (such as orally or through the digestive tract), or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0342] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the present invention is administered by a parenteral route, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular administration. In one embodiment, the oligonucleotide is administered intracerebrally or intraventricularly. In another embodiment, the oligonucleotide is administered intrathecally.

[0343] The invention also provides the use of an oligonucleotide of the invention as described for the manufacture of a medicament in a dosage form for intrathecal administration.

[0344] The present invention also provides the use of an oligonucleotide of the invention as described for the manufacture of a medicament in a dosage form for intracerebral or intraventricular administration.

[0345] The invention also provides the use of an oligonucleotide of the invention as described for the manufacture of a medicament in a dosage form for intraventricular administration.

[0346] Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is used in a combination treatment with another therapeutic agent.

[0347] GBA upregulation In certain embodiments, oligonucleotide GBA agonists of the invention may enhance production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein.

[0348] In some embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA mRNA by at least about 10%. In other embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA mRNA 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.

[0349] In some embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA protein by at least about 10%. In other embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production 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.

[0350] In some embodiments, the oligonucleotide GBA agonists of the invention can enhance the production of GBA mRNA and GBA protein by at least about 10%. In other embodiments, the oligonucleotide GBA agonists of the invention can enhance the production of GBA mRNA and 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.

[0351] In some embodiments, the oligonucleotide GBA agonist of the present invention can enhance the production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein compared to a control.The control can be cells that have never been exposed to the oligonucleotide.In some embodiments, the control can be mock transfection, for example, treatment of cells with PBS. [Table 1] TIFF2024546887000005.tif250170 TIFF2024546887000006.tif250170 EXAMPLES

[0352] Example 1: Effect of saRNA on GBA mRNA The day before transfection, 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 seeding, cells were transfected with either saRNA of SEQ ID NO: 1-74 (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 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 3. A custom designed qPCR assay specific for GBA and a pre-designed assay for TBP (HEX, Hs.PT.58v.39858774, IDT) were used (qScript™ XLT One-Step RT-qPCR ToughMix™, Low ROX™, Quanta Bioscience, #95134-500). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 1. [Table 2] [Table 3]

[0353] SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:67 all increase GBA mRNA expression by more than 15% compared to mock transfection 48 hours post-transfection in SK-N-AS cells (Figure 1).

[0354] Example 2: Effect of saRNA on GBA mRNA The day before transfection, H4 glioma cells were seeded at a density of 15000 / well in 96-well plates in complete growth medium (DMEM Sigma:D0819, 15% FBS, 1 mM sodium pyruvate, 25 μg / ml gentamicin). The day after seeding, cells were transfected with either saRNA of SEQ ID NO: 1-74 (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 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 3. A custom designed qPCR assay specific for GBA and a pre-designed assay for TBP (HEX, Hs.PT.58v.39858774, IDT) are used (qScript™ XLT One-Step RT-qPCR ToughMix™, Low ROX™, Quanta Bioscience, #95134-500). GBA mRNA concentrations were quantified relative to the housekeeping gene HPRT1 using R Software. See Figure 2.

[0355] SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:67 all increase GBA mRNA expression by more than 15% fold compared to mock transfection in H4 cells 48 hours post-transfection (Figure 2).

[0356] Embodiment 1. An oligonucleotide glucocerebrosidase (GBA) agonist, the oligonucleotide being 8-40 nucleotides in length and comprising a contiguous sequence of 8-40 nucleotides in length that is complementary to one or more promoters of the human GBA genomic sequence.

[0357] 2. The oligonucleotide GBA agonist 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.

[0358] 3. The oligonucleotide GBA agonist according to item 2, wherein the contiguous nucleotide sequence is 19 nucleotides in length.

[0359] 4. The oligonucleotide GBA agonist according to any one of items 1 to 3, wherein the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.

[0360] 5. The oligonucleotide GBA agonist according to any one of items 1 to 4, wherein the contiguous nucleotide sequence is at least 75% complementary to one or more promoters of the human GBA genomic sequence.

[0361] 6. The oligonucleotide GBA agonist of item 5, wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary to one or more promoters of the human GBA genomic sequence.

[0362] 7. The oligonucleotide GBA agonist according to any one of items 1 to 6, wherein the contiguous nucleotide sequence is fully complementary to one or more promoters of the human GBA genomic sequence.

[0363] 8. The oligonucleotide GBA agonist according to any one of items 1 to 7, wherein the human GBA genomic sequence is SEQ ID NO: 226.

[0364] 9. The oligonucleotide GBA agonist according to any one of items 1 to 8, wherein the promoter of the human GBA gene comprises SEQ ID NO: 225.

[0365] 10. The oligonucleotide GBA agonist according to item 9, wherein the promoter of the human GBA gene consists of SEQ ID NO: 225.

[0366] 11. The oligonucleotide GBA agonist according to any one of items 1 to 10, wherein the promoter is located immediately upstream of exon 1 of the human GBA gene (P1).

[0367] 12. The oligonucleotide GBA agonist of item 11, wherein the promoter is located between nucleotides 1,137 and 1 upstream of the ATG start codon in exon 1 of the human GBA gene.

[0368] 13. The oligonucleotide GBA agonist of item 11 or item 12, wherein the promoter of the human GBA gene comprises SEQ ID NO: 224.

[0369] 14. The oligonucleotide GBA agonist according to item 13, wherein the promoter of the human GBA gene consists of SEQ ID NO: 224.

[0370] 15. The oligonucleotide GBA agonist according to any one of items 11 to 14, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of nucleotides 39 to 59, 148 to 168, 153 to 173, 302 to 322, 313 to 333, 517 to 537, 532 to 552, 656 to 676, 748 to 768, 750 to 770, 815 to 835, 840 to 860, 904 to 924 and 996 to 1016 of SEQ ID NO: 224 or a fragment thereof.

[0371] 16. The oligonucleotide GBA agonist according to any one of items 11 to 14, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:36, or a fragment thereof.

[0372] 17. The oligonucleotide GBA agonist according to any one of items 1 to 16, wherein the oligonucleotide is a double-stranded oligonucleotide.

[0373] 18. The oligonucleotide GBA agonist of item 17, wherein the oligonucleotide is a saRNA.

[0374] 19. The oligonucleotide GBA agonist of item 17 or item 18, wherein the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107 and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

[0375] 20. The oligonucleotide GBA agonist according to any one of items 17 to 19, wherein the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 178, SEQ ID NO: 181 and SEQ ID NO: 184 or at least 10 contiguous nucleotides thereof.

[0376] 21. The oligonucleotide GBA agonist according to any one of items 1 to 10, wherein the promoter is located immediately upstream of exon-2 of the human GBA gene (P2).

[0377] 22. The oligonucleotide GBA agonist of item 21, wherein the promoter is located between 4,558 and 3,394 nucleotides upstream of the ATG start codon in exon 1 of the human GBA gene.

[0378] 23. The oligonucleotide GBA agonist of item 21 or item 22, wherein the promoter of the human GBA gene comprises SEQ ID NO: 223.

[0379] 24. The oligonucleotide GBA agonist according to item 23, wherein the promoter of the human GBA gene consists of SEQ ID NO: 223.

[0380] 25. The oligonucleotide GBA agonist according to any one of items 21 to 24, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of nucleotides 121 to 141, 160 to 180, 173 to 193, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 391 to 411, 423 to 443, 469 to 489, 535 to 555, 548 to 568, 551 to 571, 589 to 609 and 670 to 690 of SEQ ID NO: 223, or a fragment thereof.

[0381] 26. The oligonucleotide GBA agonist according to any one of items 21 to 24, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:67 or a fragment thereof.

[0382] 27. The oligonucleotide GBA agonist according to any one of items 21 to 27, wherein the oligonucleotide is a double-stranded oligonucleotide.

[0383] 28. The oligonucleotide GBA agonist of item 27, wherein the oligonucleotide is a saRNA.

[0384] 29. The oligonucleotide GBA agonist according to item 27 or item 28, wherein the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137 and SEQ ID NO:141 or at least 10 contiguous nucleotides thereof.

[0385] 30. The oligonucleotide GBA agonist according to any one of items 27 to 29, wherein the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211 and SEQ ID NO: 215 or at least 10 contiguous nucleotides thereof.

[0386] 31. The oligonucleotide GBA agonist according to any one of items 1 to 30, wherein said oligonucleotide is an oligonucleotide mixmer or totalmer or comprises an oligonucleotide mixmer or totalmer.

[0387] 32. The oligonucleotide GBA agonist according to any one of items 1 to 31, wherein the oligonucleotide is capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

[0388] 33. The oligonucleotide GBA agonist according to item 32, wherein the control is a cell that has not been exposed to the oligonucleotide GBA agonist.

[0389] 34. The oligonucleotide GBA agonist according to any one of items 1 to 33, wherein the oligonucleotide GBA agonist is covalently linked to at least one conjugate moiety.

[0390] 35. The oligonucleotide GBA agonist according to any one of items 1 to 34, wherein said oligonucleotide GBA agonist is in the form of a pharma- ceutically acceptable salt.

[0391] 36. The oligonucleotide GBA agonist according to item 35, wherein the salt is a sodium salt or a potassium salt.

[0392] 37. The oligonucleotide GBA agonist according to any one of items 1 to 36, wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

[0393] 38. A pharmaceutical composition comprising an oligonucleotide GBA agonist according to any one of items 1 to 37 and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0394] 39. The pharmaceutical composition according to item 38, wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.

[0395] 40. An in vivo or in vitro method for upregulating or restoring GBA expression in a target cell, comprising administering to said cell an effective amount of an oligonucleotide GBA agonist described in any one of items 1 to 37 or a pharmaceutical composition described in item 38 or item 39.

[0396] 41. The method according to item 40, wherein the cell is either a human cell or a mammalian cell.

[0397] 42. The method according to item 40 or item 41, wherein expression of GBA mRNA is increased by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

[0398] 43. The method according to any one of items 40 to 42, wherein expression of GBA protein is increased by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

[0399] 44. The method of claim 42 or 43, wherein the control is a cell that has never been exposed to the oligonucleotide GBA agonist.

[0400] 45. 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 oligonucleotide GBA agonist according to any one of items 1 to 37, or a pharmaceutical composition according to item 38 or item 39.

[0401] 46. ​​The oligonucleotide according to any one of items 1 to 37 or the pharmaceutical composition according to item 38 or item 39 for use as a medicament for the treatment or prevention of a disease in a subject.

[0402] 47. Use of an oligonucleotide according to any one of items 1 to 37 or a pharmaceutical composition according to item 38 or item 39 for the preparation of a medicament for the treatment or prevention of a disease in a subject.

[0403] 48. The method according to item 45, the oligonucleotide for use or the pharmaceutical composition according to item 46 or the use according to item 47, wherein the disease is associated with reduced expression of GBA.

[0404] 49. The method according to item 45, the oligonucleotide or pharmaceutical composition for use according to item 46, or the use according to item 47, 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.

[0405] 50. The method according to item 45, the oligonucleotide or pharmaceutical composition for use according to item 46, or the use according to item 47, wherein the disease is Parkinson's disease.

[0406] 51. The method according to item 45, the oligonucleotide or pharmaceutical composition for use according to item 46, or the use according to item 47, wherein the disease is Gaucher's disease.

Claims

1. An oligonucleotide glucocerebrosidase (GBA) agonist, said oligonucleotide being 8-40 nucleotides in length and comprising a contiguous sequence of 8-40 nucleotides in length that is complementary to one or more promoters of a human GBA genomic sequence.

2. The oligonucleotide GBA agonist 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 one or more promoters of the human GBA genomic sequence, and the human GBA genomic sequence is SEQ ID NO:

226.

3. The oligonucleotide GBA agonist of any one of claims 1 to 3, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO:

225.

4. The oligonucleotide GBA agonist of any one of claims 1 to 4, wherein the promoter is located between nucleotides 1,137 and 1 upstream of the ATG start codon in exon 1 of the human GBA gene (P1).

5. The oligonucleotide GBA agonist of claim 4, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO:

224.

6. The oligonucleotide GBA agonist of claim 4 or 5, wherein the contiguous nucleotide sequence is complementary to a sequence or a fragment thereof selected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33 and SEQ ID NO:

36.

7. the oligonucleotide GBA agonist is a double-stranded oligonucleotide; The oligonucleotide GBA agonist of any one of claims 4 to 6, wherein the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107 and SEQ ID NO:110, or at least 10 contiguous nucleotides thereof.

8. the oligonucleotide GBA agonist is a double-stranded oligonucleotide; The oligonucleotide GBA agonist of any one of claims 4 to 7, wherein the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:178, SEQ ID NO:181 and SEQ ID NO:184 or at least 10 contiguous nucleotides thereof.

9. The oligonucleotide GBA agonist of any one of claims 1 to 3, wherein the promoter is located immediately upstream of exon-2 of the human GBA gene (P2).

10. 10. The oligonucleotide GBA agonist of claim 9, wherein the promoter is located between 4,558 and 3,394 nucleotides upstream of the ATG start codon in exon 1 of the human GBA gene.

11. 11. The oligonucleotide GBA agonist of claim 9 or claim 10, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO:

223.

12. The oligonucleotide GBA agonist of any one of claims 9 to 11, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and SEQ ID NO:67 or a fragment thereof.

13. the oligonucleotide GBA agonist is a double-stranded oligonucleotide; The oligonucleotide GBA agonist of any one of claims 9 to 12, wherein the sense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137 and SEQ ID NO:141 or at least 10 contiguous nucleotides thereof.

14. the oligonucleotide GBA agonist is a double-stranded oligonucleotide; The oligonucleotide GBA agonist of any one of claims 9 to 13, wherein the antisense strand of the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211 and SEQ ID NO:215 or at least 10 contiguous nucleotides thereof.

15. The oligonucleotide GBA agonist of any one of claims 1 to 14, wherein the oligonucleotide is capable of increasing expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% compared to a control.

16. The oligonucleotide GBA agonist of any one of claims 1 to 15, wherein the oligonucleotide is saRNA.

17. An oligonucleotide according to any one of claims 1 to 16 for use as a medicament for the treatment or prevention of a disease in a subject.

18. 18. The oligonucleotide for use according to claim 17, wherein the disease is associated with reduced expression of GBA.

19. 19. The oligonucleotide for use according to claim 17 or 18, 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.

20. The oligonucleotide for use according to any one of claims 17 to 19, wherein the disease is Parkinson's disease.

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