WO2023111337A1

WO2023111337A1 - Antisense oligonucleotide

Info

Publication number: WO2023111337A1
Application number: PCT/EP2022/086505
Authority: WO
Inventors: Johannes Braun; Ross CORDINER; Lukasz KIELPINSKI; Soren V RASMUSSEN; Disa Elisabet TEHLER
Original assignee: F. Hoffmann-La Roche Ag; Hoffmann-La Roche Inc.
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2023-06-22

Abstract

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

Description

ANTISENSE OLIGONUCLEOTIDE

FIELD OF THE INVENTION

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

BACKGROUND

Glucocerebrosidase (GBA) is a lysosomal enzyme that catalyses the hydrolysis of glucocerebroside (also known as glucosylceramide). Glucocerebroside is a normal component of cell membranes, in particular of red and white blood cells.

Homozygous mutations in the gene encoding GBA cause Gaucher’s disease. During routine cell turnover, macrophages engulf and degrade cell debris. Insufficient GBA activity results in the accumulation of glucocerebroside in the lysosomes of macrophages. Affected macrophages, known as ‘Gaucher cells’, build up in areas such as the spleen, liver and bone marrow.

Gaucher’s disease is characterized by bruising, fatigue, anaemia, low blood platelet count and enlargement of the liver and spleen. The phenotype is variable, however three clinical forms have been identified: type 1 is the most common and typically causes no neurological damage, whereas types 2 and 3 are characterised by neurological impairment.

The condition is inherited in an autosomal recessive pattern. Over 300 variants of the GBA gene have been associated with the disease. Although genetics alone does not determine disease severity, certain mutations are known to cause more severe symptoms. For example, patients with two copies of the L444P mutation usually exhibit neuronopathic forms 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).

Mutations in the GBA gene have also been linked to 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 characterised 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 many years, include olfactory loss, rapid eye movement sleep behaviour disorders, dysautonomia, and depression. Heterozygous mutations of the GBA gene occur in around 8 to 12% of patients with Parkinson’s disease. As with Gaucher’s disease, mutation severity can influence the disease phenotype. For example, the risk for dementia in patients carrying “severe” mutations (such as L444P) is 2- to 3-fold higher than in those carrying “mild” mutations (such as N370S). E326K is the most prevalent GBA mutation in Parkinson’s disease, and patients bearing 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 reduction therapy (SRT). ERT involves the intravenous administration of recombinant GBA. While most patients respond well to treatment, there is a risk of developing an immune response. Furthermore, GBA is not able to cross the blood-brain barrier and therefore ERT is considered ineffective for patients with Parkinson’s disease or neuronopathic forms of Gaucher’s disease. SRT provides an alternative (or supplementary) treatment for patients who cannot tolerate ERT, or for whom intravenous administration is problematic. SRT works to reduce the build up of glucocerebroside in the lysosome by inhibiting enzymes in the glucocerebroside synthesis pathway. This therapy has a higher incidence of adverse effects than ERT, and long-term reduction of glucocerebroside can affect several different cell functions. Both ERT and SRT are costly and must be continued for life.

There is thus an urgent need for therapeutic agents that can increase or restore the expression of GBA.

SUMMARY OF THE INVENTION

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

A GBA mRNA transcript is the mRNA sequence encoded by a glucocerebrosidase (GBA) gene.

Without wishing to be bound by theory, it is considered that the antisense oligonucleotides of the invention can reduce and/or prevent translation from being initiated from upstream, non- canonical AUG (uAUG) sites, which may lead to increased translation from the downstream canonical AUG start site, increasing GBA protein production. The contiguous nucleotide sequence may be complementary to an upstream open reading frame (uORF) in the 5’ UTR, which is located upstream of the canonical AUG start codon of the GBA mRNA transcript.

The beginning of the uORF is defined by an uAUG.

The contiguous nucleotide sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

The contiguous nucleotide sequence may be 16 or 18 nucleotides in length.

The contiguous nucleotide sequence may be the same length as the antisense oligonucleotide.

In some embodiments, the contiguous nucleotide sequence may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or fully complementary to a sequence in a 5’ UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

The contiguous nucleotide sequence may be fully complementary to a sequence in a 5’ UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

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

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

In some embodiments, the 5’UTR may comprise an uAUG.

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

In some embodiments, the contiguous nucleotide sequence may be complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 90 and SEQ ID NO 109, or a fragment thereof. The antisense oligonucleotide may be a single stranded antisense oligonucleotide.

In some embodiments, the contiguous nucleotide sequence may be or may comprise a sequence selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37 and SEQ ID NO 38, or at least 10 contiguous nucleotides thereof. In an embodiment, the contiguous nucleotide sequence may be or may SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.

In some embodiments, the antisense oligonucleotide may comprise one or more modified nucleoside(s).

The one or more modified nucleosides may be independently selected from the group consisting of 2’-O-alkyl-RNA, 2’-O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA, 2’- amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA, and locked nucleic acid (LNA) nucleosides.

The one or more modified nucleosides may be a LNA nucleoside. The LNA nucleoside may be a beta- D-oxy- LNA.

The one or more modified nucleosides may be a 2’-O-methyl-RNA nucleoside.

The oligonucleotide may be a mixmer.

The antisense oligonucleotide may comprise at least one modified internucleoside linkage.

One or more, or all, of the modified internucleoside linkages may comprise a phosphorothioate linkage. All of the internucleoside linkages present within the antisense oligonucleotide may be phosphorothioate internucleoside linkages.

In some embodiments, the contiguous nucleotide sequence may be capable of increasing the expression of GBA by 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control, wherein the control may be a cell that has not been exposed to said oligonucleotide.

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

In some embodiments, the antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt. The salt may be a sodium salt or a potassium salt The antisense oligonucleotide may be encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

The present invention also provides a pharmaceutical composition comprising the antisense oligonucleotide of the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

The pharmaceutical composition may comprise an aqueous diluent or solvent, such as phosphate buffered saline.

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

The cell may be a human cell or a mammalian cell.

The expression of GBA may be increased by 10%, 15% 20%, 30%, 40%, 50% or more than 50%, compared to a control, wherein the control may be a cell that has not been exposed to said oligonucleotide.

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

The present invention also provides an antisense oligonucleotide of the invention or the pharmaceutical composition of the invention, for use as a medicament for the treatment or prevention of a disease in a subject.

The present invention also provides the use of the antisense oligonucleotide of the invention or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease in a subject.

The disease may be associated with reduced expression of GBA.

The disease may be selected from the group consisting of Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.

In some embodiments, the disease may be Parkinson’s disease. In some embodiments, the disease may be Gaucher’s disease.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows upregulation of GBA mRNA in SK-N-AS neuroblastoma cells 48 hours after transfection relative to a mock transfection control. Cells were plated to a density of 25000 per well. GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software.

Figure 2 shows upregulation of GBA mRNA in SK-N-As neuroblastoma cells 72 hours after transfection relative to a mock transfection control. Cells were plated to a density of 10000 cells per well. GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software.

Figure 3 shows upregulation of GBA protein in SK-N-As neuroblastoma cells 72 hours after transfection relative to a mock transfection control. Cells were plated to a density of 60000 cells per well. GBA protein abundances were quantified relative to HPRT1 protein using “Compass for SW” software and Microsoft Excel.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified that the expression level of GBA protein products can be effectively enhanced by targeting the GBA mRNA transcript with antisense oligonucleotides, particularly antisense oligonucleotides which comprise high affinity sugar modified nucleosides, such as high affinity 2’ sugar modified nucleosides, such as LNA nucleosides or 2’-O-methoxyethyl (MOE) nucleosides.

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

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

The 5' UTR may contain one or more upstream start codon sites (uAUGs), wherein an uAUG defines the beginning of an upstream open reading frame (uORF). Without wishing to be bound by theory, it is considered that the antisense oligonucleotides of the invention can increase GBA production by binding to these 5’ regions and affecting, such as reducing, the initiation of translation from these uAUG sites. This ensures that protein translation is initiated from the downstream canonical start site (ORF), increasing GBA production. Thus, the inventors have identified that the expression level of the encoded protein products, can be effectively enhanced by targeting the GBA mRNA transcript with antisense oligonucleotides, which either block one or more upstream open reading frames (uORFs) located upstream of the canonical AUG start codon, and/or prevent translation from the uAUG sites, using antisense oligonucleotides of the invention.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide sequence which is complementary to a sequence in a 5’UTR comprising an uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript. Thus, the antisense oligonucleotide may bind directly to an uORF, which without wishing to be bound by theory, may lead to steric blocking of the uORF and prevent protein translation starting from the uAUG site.

In some embodiments, the antisense oligonucleotides of the invention comprise a contiguous nucleotide sequence which is complementary to a sequence in a 5’UTR, which does not directly encompass an uAUG site, located upstream of the canonical AUG start codon of the GBA mRNA transcript. Thus, the antisense oligonucleotide may bind to a sequence in a 5’UTR which does not directly encompass an uAUG, but which, without wishing to be bound by theory, may still lead to steric blocking and prevent protein translation from the uAUG site.

Oligonucleotides, such as RNaseH recruiting single stranded antisense oligonucleotides or siRNAs are used extensively in the art to inhibit target RNAs - i.e. are used as antagonists of their complementary nucleic acid target.

The antisense oligonucleotides of the present invention are agonists, i.e. they enhance the expression of their complementary target, GBA nucleic acids, and thereby enhance the expression of GBA protein.

Thus, the invention relates to antisense oligonucleotides that enhance the expression of GBA.

Enhanced GBA expression is desirable to treat diseases associated with reduced expression of GBA, for example, Gaucher’s disease and/or Parkinson’s disease.

Oligonucleotide The term “oligonucleotide” as used herein is defined, as is generally understood by the skilled person, as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.

Oligonucleotides are commonly made in a laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotides of the invention are man-made, and are chemically synthesized, and are typically purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides such as 2’ sugar modified nucleosides. The oligonucleotides of the invention may comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

Antisense Oligonucleotide

The oligonucleotide is an antisense oligonucleotide.

In some embodiments, the antisense oligonucleotide is a single stranded antisense oligonucleotide.

The term “antisense oligonucleotide” as used herein is defined as an oligonucleotide capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. The antisense oligonucleotides of the present invention may be single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than approximately 50% across of the full length of the oligonucleotide.

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

Advantageously, the antisense oligonucleotides of the invention comprise one or more modified nucleosides or nucleotides, such as 2’ sugar modified nucleosides. Furthermore, in some antisense oligonucleotides of the invention, it may be advantageous that the nucleosides which are not modified are DNA nucleosides. In some embodiments, the antisense oligonucleotide is 8-40 nucleotides in length.

In some embodiments, antisense oligonucleotide is 8-40 nucleotides in length and comprises a contiguous nucleotide sequence of 8-40 nucleotides.

In some embodiments, the antisense oligonucleotide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

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

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

Contiguous Nucleotide Sequence

The term “contiguous nucleotide sequence” refers to the region of the antisense oligonucleotide which is complementary to a target nucleic acid, which may be or may comprise an oligonucleotide motif sequence. The term is used interchangeably herein with the term “contiguous nucleobase sequence”.

In some embodiments, the antisense oligonucleotide comprises the contiguous nucleotide sequence, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group (e.g. a conjugate group) to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

It is understood that the contiguous nucleotide sequence of the antisense oligonucleotide cannot be longer than the antisense oligonucleotide as such and that the antisense oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

In some embodiments, all of the nucleosides of the antisense oligonucleotide may constitute the contiguous nucleotide sequence.

The contiguous nucleotide sequence is the sequence of nucleotides in the antisense oligonucleotides of the invention which are complementary to, and in some instances fully complementary to, the target nucleic acid, target sequence, or target site sequence.

In some embodiments, the contiguous nucleotide sequence is 8-40 nucleotides in length. 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.

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

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

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

In some embodiments, the antisense oligonucleotide consists of the contiguous nucleotide sequence.

In some embodiments, the antisense oligonucleotide is the contiguous nucleotide sequence.

5’UTR and uORF of GBA

The antisense oligonucleotides of the invention may comprise a contiguous nucleotide sequence which is complementary to an upstream open reading frame (uORF) located in the 5’ UTR upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript.

Thus, the antisense oligonucleotides of the invention may lead to steric blocking of the uAUG, preventing protein translation from the uAUG site. This may lead to increased translation from the canonical AUG start codon, and increased expression of the GBA protein.

The antisense oligonucleotides of the invention may comprise a contiguous nucleotide sequence which is complementary to a sequence in a 5’UTR located upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript, wherein the sequence in the 5’UTR may not encompass an uAUG, but binding to the sequence in the 5’UTR may lead to steric blocking and prevent protein translation from the uAUG site.

The term “gene” as used herein, particularly with reference to the GBA gene, encompasses both protein coding and non-protein coding sequences. It is understood that such sequences include transcribed and untranscribed sequences, and translated and untranslated sequences. Non-protein coding sequences may comprise regulatory sequences such as enhancers, silencers, promoters, and/or 3’ and 5’ untranslated regions (UTR). An uORF is a gene expression regulatory element which is an open reading frame (ORF) beginning with an upstream initiation AUG (uAUG) codon which is located within the 5' untranslated region (5'UTR) of an mRNA. The uAUG is located upstream of the canonical AUG initiation codon of the main coding region of a gene.

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

Without wishing to be bound by theory, targeting the 5’UTR may increase GBA expression by binding to an upstream start site (AUG site) and affecting, such as reducing, protein translation from these upstream AUG sites. This ensures that protein translation is initiated from the downstream canonical start site (ORF), increasing GBA production.

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

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

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

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

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

In some embodiments, the contiguous nucleotide sequence is at least 96% complementary to a sequence in a 5’UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript. In some embodiments, the contiguous nucleotide sequence is at least 97% complementary to a sequence in a 5’UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

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

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

In some embodiments, the contiguous nucleotide sequence is fully complementary (i.e. 100% complementary) to a sequence in a 5’UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

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

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

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

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

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

In some embodiments, the contiguous nucleotide sequence is at least 95% complementary to an uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript. In some embodiments, the contiguous nucleotide sequence is at least 96% complementary to an uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

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

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

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

In some embodiments, the contiguous nucleotide sequence is fully complementary (i.e. 100% complementary) to an uORF located upstream of the canonical AUG start codon of the GBA mRNA transcript.

In some embodiments, the GBA gene may comprise the human GBA genomic sequence according to NCBI Reference Sequence: NG_009783.

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

SEQ ID NO 107 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 107, such as an allelic variant, which comprises one or more polymorphisms in the human GBA nucleic acid sequence.

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

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

It will be understood that the sequence according to SEQ ID NO 107 is the GBA genomic sequence. Any reference to the GBA genomic sequence also encompasses reference to the GBA mRNA transcript sequence corresponding to the GBA genomic sequence. Accordingly, the GBA mRNA transcript may be an mRNA sequence which is encoded by SEQ ID NO 107. Accordingly, reference to an “ATG” codon will be understood as referring to a start codon of the GBA genomic sequence, which corresponds to an “AUG” at a position corresponding to a positon of the GBA mRNA transcript.

In some embodiments, the canonical AUG start codon of the GBA mRNA transcript may correspond to positions 8586-8588 of the GBA genomic sequence of SEQ ID NO 107. Thus, the “A” of the canonical AUG start codon may be located at a position corresponding to position 8586 of SEQ ID NO 107; the “U” of the canonical AUG start codon may be located at a position corresponding to position 8587 of SEQ ID NO 107; and the “G” of the canonical AUG start codon may be located at a position corresponding to position 8588 of SEQ ID NO 107.

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

SEQ ID NO 108 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 108, such as an allelic variant, which comprises one or more polymorphisms in the human GBA mRNA sequence.

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

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

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

In some embodiments, the canonical AUG start codon of the GBA mRNA transcript corresponds to positions 138-140 of SEQ ID NO 108. Thus, the “A” of the canonical AUG start codon may be located at a position corresponding to position 138 of SEQ ID NO 108; the “U” of the canonical AUG start codon may be located at a position corresponding to position 139 of SEQ ID NO 108; and the “G” of the canonical AUG start codon may be located at a position corresponding to position 140 of SEQ ID NO 108.

An uAUG is located upstream of the canonical AUG start codon position. Here it will be understood that upstream means in a 5’ position relative to the canonical AUG codon. In some embodiments, the uORF may be located 1-100 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-100 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-50 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-50 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-40 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-40 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-30 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-30 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-20 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-20 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript. In some embodiments, the uORF may be located 1-15 nucleotides upstream of the canonical AUG start codon position. In other words, there may be 1-15 nucleotides between the uAUG codon of the uORF and the canonical AUG codon of the GBA mRNA transcript.

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

In some embodiments, the uAUG of the uORF is located at a position corresponding to positions 8572-8574 of the GBA genomic sequence of SEQ ID NO 107. Thus, the “A” of the uAUG may be located at a position corresponding to positions 8572 of SEQ ID NO 107; the “U” of the uAUG may be located at a position corresponding to positions 8573 of SEQ ID NO 107; and the “G” of the uAUG may be located at a position corresponding to positions 8574 of SEQ ID NO 107.

In some embodiments, the uAUG of the uORF is located at a position corresponding to positions 124-126 of the GBA mRNA sequence of SEQ ID NO 108. Thus, the “A” of the uAUG may be located at a position corresponding to positions 124 of SEQ ID NO 108; the “II” of the uAUG may be located at a position corresponding to positions 125 of SEQ ID NO 108; and the “G” of the uAUG may be located at a position corresponding to positions 126 of SEQ ID NO 108.

Unless otherwise stated, all ranges are inclusive of the start and end value.

Complementarity to the GBA target sequence

The contiguous nucleotide sequence of the antisense oligonucleotides of the invention may be complementary to a GBA target nucleic acid sequence. The contiguous nucleotide sequence may be fully complementary to a GBA target nucleic acid sequence. The contiguous nucleotide sequence may be partially complementary to a GBA target nucleic acid sequence, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (i.e. fully) complementary to a GBA target nucleic acid sequence.

In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence which may be selected from the group consisting of SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, and SEQ ID NO 106, or a fragment thereof.

In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence which may be selected from the group consisting of SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91 , SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104 SEQ ID NO 106 and SEQ ID NO 109, or a fragment thereof.

In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence which may be selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89, and SEQ ID NO 90, or a fragment thereof.

In some embodiments, the contiguous nucleotide sequence is complementary to a target nucleic acid sequence which may be selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 90 and SEQ ID NO 109, or a fragment thereof.

In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO

60 or a fragment thereof.

61 or a fragment thereof.

62 or a fragment thereof.

63 or a fragment thereof.

64 or a fragment thereof.

65 or a fragment thereof.

66 or a fragment thereof.

67 or a fragment thereof.

In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO 81 or a fragment thereof. In some embodiments, the contiguous nucleotide sequence is complementary to SEQ ID NO 82 or a fragment thereof.

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

89 or a fragment thereof.

90 or a fragment thereof.

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

A fragment of the target sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

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

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

Targeting the GBA target sequence

The contiguous nucleotide sequence of the antisense oligonucleotides of the invention may comprise a sequence which targets a GBA target nucleic acid sequence.

In some embodiments, the contiguous nucleotide sequence is or comprises a sequence which may be selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52 and SEQ ID NO 105, or at least 10 contiguous nucleotides thereof.

In some embodiments, the contiguous nucleotide sequence is or comprises a sequence which may be selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 105 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.

In some embodiments, the contiguous nucleotide sequence is or comprises a sequence which may be selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37 and SEQ ID NO 38, or at least 10 contiguous nucleotides thereof.

In some embodiments, the contiguous nucleotide sequence is or comprises a sequence which may be selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 38, and SEQ ID NO 110 or at least 10 contiguous nucleotides thereof.

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

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

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

In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO 11 , or at least 10 contiguous nucleotides thereof. In some embodiments, the contiguous nucleotide sequence is or comprises SEQ ID NO 12, or at least 10 contiguous nucleotides thereof.

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

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

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

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

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

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

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

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

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

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

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

Nucleotides and nucleosides Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non- naturally occurring nucleotides and nucleosides. In nature, nucleotides, such as DNA and RNA nucleotides, comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.

Modified nucleoside

Advantageously, the antisense oligonucleotides of the invention may comprise one or more modified nucleoside(s).

The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.

Advantageously, one or more of the modified nucleosides of the antisense oligonucleotides of the invention may comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing. Exemplary modified nucleosides which may be used in the antisense oligonucleotides of the invention include LNA, 2’-O-MOE, 2’oMe and morpholino nucleoside analogues.

Modified internucleoside linkage

Advantageously, the antisense oligonucleotides of the invention may comprise one or more modified internucleoside linkage.

The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages, other than phosphodiester (PO) linkages, that covalently couple two nucleosides together. The antisense oligonucleotides of the invention may therefore comprise one or more modified internucleoside linkages such as one or more phosphorothioate internucleoside linkage.

In some embodiments at least 50% of the internucleoside linkages in the antisense oligonucleotide, or contiguous nucleotide sequence thereof, 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 antisense oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the antisense oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

In a further embodiment, the antisense oligonucleotide may comprise at least one modified internucleoside linkage. It is advantageous if at least 75%, such as all, of the internucleoside linkages within the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.

Advantageously, all the internucleoside linkages of the contiguous nucleotide sequence of the antisense oligonucleotide may be phosphorothioate, or all the internucleoside linkages of the antisense oligonucleotide may be phosphorothioate linkages.

Nucleobase

The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but which 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, as well as non-naturally occurring variants. Such variants are for example described 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.

In some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 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.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or II, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used. 5-methyl cytosine may be denoted as “E”.

Modified oligonucleotide

The antisense oligonucleotides of the invention may be modified antisense oligonucleotides.

The term “modified antisense oligonucleotide” describes an antisense oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term “chimeric oligonucleotide” is a term that has been used in the literature to describe oligonucleotides comprising sugar modified nucleosides and DNA nucleosides. In some embodiments, it may be advantageous for the antisense oligonucleotides of the invention to be chimeric oligonucleotide.

In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence thereof may include modified nucleobases which function as the shown nucleobase in base pairing, for example 5-methyl cytosine may be used in place of methyl cytosine. Inosine may be used as a universal base.

It is understood that the contiguous nucleobase sequences (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid.

The pattern in which the modified nucleosides (such as high affinity modified nucleosides) are incorporated into the oligonucleotide sequence is generally termed oligonucleotide design.

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

In an embodiment, the antisense oligonucleotides comprise at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39 or at least 40 modified nucleosides.

Suitable modifications are described herein under the headings “modified nucleoside”, “high affinity modified nucleosides”, “sugar modifications”, “2’ sugar modifications” and Locked nucleic acids (LNA)”. High affinity modified nucleosides

A high affinity modified nucleoside is a modified nucleoside which, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T^m). A high affinity modified nucleoside of the present invention preferably results in an increase in melting temperature between +0.5 to +12°C, more preferably between +1.5 to +10°C and most preferably between +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann, Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

Sugar modifications

The antisense oligonucleotides of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks 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 where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2', 3', 4' or 5' positions.

2' sugar modified nucleosides

A 2' sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle capable of forming a bridge between the 2' carbon and a second carbon in the ribose ring, such as LNA (2'- 4' biradicle bridged) nucleosides.

Indeed, much focus has been spent on developing 2' sugar substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2' modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. 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 nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development 2000, 3(2), 203-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2' substituted modified nucleosides.

In relation to the present invention 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides like LNA.

In an embodiment, the antisense oligonucleotide comprises one or more sugar modified nucleosides, such as 2' sugar modified nucleosides.

In an embodiment, the antisense oligonucleotide comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 sugar modified nucleosides.

Preferably the antisense oligonucleotides of the invention comprise one or more 2' sugar modified nucleoside 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 nucleoside(s) is a locked nucleic acid (LNA).

Locked Nucleic Acid Nucleosides (LNA nucleoside)

A "LNA nucleoside" is a 2'- modified nucleoside which comprises a biradical linking the C2' and C4' of the ribose sugar ring of said nucleoside (also referred to as a "2' - 4' bridge"), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.

Non limiting, exemplary LNA nucleosides are disclosed 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 a!., 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.

Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 1.

Scheme 1 :

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.

A particularly advantageous LNA is beta-D-oxy-LNA. Morpholino oligonucleotides

In some embodiments, the antisense oligonucleotides of the invention comprise or consist of morpholino nucleosides (/.e. is a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO)). Splice modulating morpholino oligonucleotides have been approved for clinical use - see for example eteplirsen, a 30nt morpholino oligonucleotide targeting a frame shift mutation in DMD, used to treat Duchenne muscular dystrophy.

Morpholino oligonucleotides have nucleases attached to six membered morpholino rings rather ribose, such as methylenemorpholine rings linked through phosphorodiamidate groups, for example as illustrated by the following illustration of 4 consecutive morpholino nucleotides:

In some embodiments, morpholino oligonucleotides of the invention may be, for example 8 - 40 morpholino nucleotides in length.

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10%, at least 20% or more than 20%, of the initial rate determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Examples 91 - 95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland. DNA oligonucleotides are known to effectively recruit RNaseH, as are gapmer oligonucleotides which comprise a region of DNA nucleosides (typically at least 5 or 6 contiguous DNA nucleosides), flanked 5’ and 3’ by regions comprising 2’ sugar modified nucleosides, typically high affinity 2’ sugar modified nucleosides, such as 2-O-MOE and/or LNA. For effective facilitation of steric blocking, degradation of the pre-mRNA is not desirable, and as such it is preferable to avoid the RNaseH degradation of the target. Therefore, the antisense oligonucleotides of the invention are not RNaseH recruiting gapmer oligonucleotides.

RNaseH recruitment may be avoided by limiting the number of contiguous DNA nucleotides in the oligonucleotide - therefore mixmer and totalmer designs may be used. Advantageously, in some embodiments, the antisense oligonucleotides of the invention, or the contiguous nucleotide sequences thereof, does not comprise more than 3 contiguous DNA nucleosides. Further, advantageously, in some embodiments, the antisense oligonucleotides of the invention, or the contiguous nucleotide sequences thereof, do not comprise more than 4 contiguous DNA nucleosides. Further advantageously, in some embodiments, the antisense oligonucleotides of the invention, or contiguous nucleotide sequences thereof, do not comprise more than 2 contiguous DNA nucleosides.

Mixmers and Totalmers

For steric blocking it is often advantageous to use antisense oligonucleotides which do not recruit RNAase H and do not cause destruction of target RNA. As RNase H activity requires a contiguous sequence of DNA nucleotides, RNase H recruitment may be prevented by designing oligonucleotides which do not comprise a region of more than 3 or more than 4 contiguous DNA nucleosides. This may be achieved by using oligonucleotides or contiguous nucleoside regions thereof with a mixmer design, which comprise sugar modified nucleosides, such as 2’ sugar modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides. Mixmers are exemplified herein by every second design, wherein the nucleosides alternate between 1 LNA and 1 DNA nucleoside, e.g. LDLDLDLDLDLDLDLL, with 5’ and 3’ terminal LNA nucleosides, and every third design, such as LDDLDDLDDLDDLDDL, where every third nucleoside is a LNA nucleoside.

In one embodiment, the mixmer may comprise or consist of nucleosides that alternate between 1 , 2 or 3 sequential DNA nucleosides, followed by 1 or 2 sequential LNA nucleosides.

A totalmer is an oligonucleotide or a contiguous nucleotide sequence thereof which does not comprise DNA or RNA nucleosides, and may for example comprise only 2’-O-MOE nucleosides, such as a fully MOE phosphorothioate, e.g.

MMMMMMMMMMMMMMMMMMMM, where M = 2’-O-MOE, or may for example comprise only 2’oMe nucleosides, which are reported to be effective in steric blockers for therapeutic use.

Alternatively, a mixmer may comprise a mixture of modified nucleosides, such as MLMLMLMLMLMLMLMLMLML, wherein L = LNA and M = 2’-O-MOE nucleosides.

Advantageously, the internucleoside nucleosides in mixmers and totalmers may be phosphorothioate, or a majority of nucleoside linkages in mixmers may be phosphorothioate. Mixmers and totalmers may comprise other internucleoside linkages, such as phosphodiester or phosphorodithioate, by way of example.

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

Complementarity

The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (II).

It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).

The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g. a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pairs) between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5’-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

Within the present invention, the term “complementary” requires the contiguous nucleotide sequence to be at least about 75% complementary, at least about 80% complementary, at least about 85% complementary, at least about 90% complementary, or at least about 95% complementary to the target sequence, e.g. the sequence of the 5’UTR or the uORF located upstream of the canonical AUG start codon. In some embodiments the antisense oligonucleotide, or contiguous sequence thereof, may be at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary, or 100% complementary to the target sequence, e.g. the sequence of the 5’UTR or the uORF located upstream of the canonical AUG start codon.

In some embodiments, the contiguous nucleotide sequences within the antisense oligonucleotides of the invention may include one, two, three or more mis-matches, wherein a mis-match is a nucleotide within the contiguous nucleotide sequence which does not base pair with its target.

The term “fully complementary”, refers to 100% complementarity.

In some embodiments the contiguous nucleotide sequence is fully complementary to the target sequence.

Identity

The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif).

The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) 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. Therefore, Percentage of Identity = (Matches x 100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

It is therefore to be understood that there is a relationship between identity and complementarity such that contiguous nucleotide sequences within the antisense oligonucleotides of the invention that are complementary to a target sequence also share a percentage of identity with said complementary sequence.

Hybridization

The terms “hybridizing” or “hybridizes” as used herein are to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T_m) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T_m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515- 537). The standard state Gibbs free energy AG° is a more accurate representation of binding affinity and is related to the dissociation constant (Kd) of the reaction by AG°=- RTIn(Kd), where R is the gas constant and T is the absolute temperature. Therefore, a very low AG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong hybridization between the oligonucleotide and target nucleic acid. AG° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37°C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements. AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43: 5388- 5405. In some embodiments, antisense oligonucleotides of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10- 30 nucleotides in length.

In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°. The oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length. In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal, or- 16 to -27 kcal such as -18 to -25 kcal.

Region D’ or D” in an oligonucleotide

The antisense oligonucleotides of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequences of the oligonucleotides which are complementary to the 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 or may not be complementary, such as fully complementary, to the target nucleic acid. Such further 5’ and/or 3’ nucleosides may be referred to as region D’ and D” herein.

The addition of region D’ or D” may be used for the purpose of joining the contiguous nucleotide sequence, such as the mixmer or totalmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively, it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.

Region D’ or D” may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D’ or D” are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to link multiple antisense constructs within a single oligonucleotide. In one embodiment the antisense oligonucleotides of the invention comprise a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes a mixmer or a total mer.

In some embodiments the internucleoside linkage positioned between region D’ or D” and the mixmer or totalmer region is a phosphodiester linkage.

Conjugate

The invention encompasses an antisense oligonucleotide covalently attached to at least one conjugate moiety. In some embodiments this may be referred to as a conjugate of the invention.

In some embodiments, the invention provides antisense oligonucleotides covalently attached to at least one conjugate moiety.

The term “conjugate” as used herein refers to an antisense oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to the oligonucleotide, optionally via a linker group, such as region D’ or D”.

Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

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, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the antisense oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A). In some embodiments of the invention, the conjugate or antisense oligonucleotide of the invention may optionally comprise a linker region (second region or region B and/or region Y) which is positioned 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).

Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments the nuclease susceptible linker comprises between 1 and 5 nucleosides, such as DNA nucleoside(s) comprising at least two consecutive phosphodiester linkages. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195.

Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The antisense oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In some embodiments the linker (region Y) is a C6 amino alkyl group.

Salts

The term “salts” as used herein conforms to its generally known meaning, i.e. an ionic assembly of anions and cations.

The invention provides for pharmaceutically acceptable salts of the antisense oligonucleotide of the invention.

The invention provides for an antisense oligonucleotide of the invention wherein the antisense oligonucleotide is in the form of a pharmaceutically acceptable salt. In some embodiments the pharmaceutically acceptable salt may be a sodium salt or a potassium salt.

The invention provides for a pharmaceutically acceptable sodium salt of the antisense oligonucleotide of the invention.

The invention provides for a pharmaceutically acceptable potassium salt of the antisense oligonucleotide of the invention.

Delivery of antisense oligonucleotides

The invention provides for antisense oligonucleotides of the invention wherein the antisense oligonucleotides are encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

This may be for the purpose of delivering the antisense oligonucleotides of the invention to the targeted cells and/or to improve the pharmacokinetics of the antisense oligonucleotide.

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

Pharmaceutical compositions

The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide of the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

The invention provides for a pharmaceutical composition comprising the antisense oligonucleotide of the invention, and a pharmaceutically acceptable salt. For example, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

The invention provides for a pharmaceutical composition of the invention, wherein the pharmaceutical composition comprises the antisense oligonucleotide of the invention, and an aqueous diluent or solvent.

The invention provides for a solution, such as a phosphate buffered saline solution of the antisense oligonucleotide of the invention. In some embodiments, the solution, such as phosphate buffered saline solution, of the invention is a sterile solution.

Method for modulating GBA expression

The invention provides for a method for enhancing, upregulating or restoring the expression of GBA in a cell, such as a cell which is expressing GBA, said method comprising administering an antisense oligonucleotide of the invention, or the pharmaceutical composition of the invention in an effective amount to said cell.

In some embodiments the method is an in vitro method.

In some embodiments the method is an in vivo method.

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

In some embodiments, the cell is either a human cell or a mammalian cell.

In some embodiments, the cell is part of, or derived from, a subject suffering from or susceptible to a disease associated with reduced expression of GBA. Such diseases include but are not limited Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.

Treatment

The term “treatment” as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment, as referred to herein may in some embodiments be prophylactic.

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

The disease may be associated with reduced expression of GBA.

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

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 movements (REM) sleep behaviour disorders.

In one embodiment, the disease is Parkinson’s disease. In one embodiment the disease is Gaucher’s disease.

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

The invention provides for an antisense oligonucleotide of the invention for use as a medicament.

The invention provides for an antisense oligonucleotide of the invention for the preparation of a medicament.

The invention provides for an antisense oligonucleotide of the invention for use in therapy.

The invention provides for an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention, for use as a medicament.

The invention provides for an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention, for the preparation of a medicament.

The invention provides for an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention for use in therapy.

The invention provides for an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention for use as a medicament in the treatment of Parkinson’s disease.

The invention provides for an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention for use as a medicament in the treatment of Gaucher’s disease.

The invention provides for the use of an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Parkinson’s disease.

The invention provides for the use of an antisense oligonucleotide of the invention, or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Gaucher’s disease.

Administration

The antisense oligonucleotide of the invention or the pharmaceutical composition of the invention may be administered topically (such as, to the skin, inhalation, ophthalmic or otic) or enteral (such as, orally or through the gastrointestinal tract) or parenterally (such as, intravenous, subcutaneous, intra-muscular, intracerebral, intracerebroventricular or intrathecal).

In a preferred embodiment the antisense oligonucleotide of the invention or pharmaceutical composition of the 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 antisense oligonucleotide of the invention is administered intracerebrally or intracerebroventricularly. In another embodiment the antisense oligonucleotide of the invention is administered intrathecally.

The invention also provides for the use of the antisense oligonucleotide of the invention or pharmaceutical composition of the invention as described for the preparation of a medicament wherein the medicament is in a dosage form for intrathecal administration.

The invention also provides for the use of the antisense oligonucleotide of the invention or pharmaceutical composition of the invention as described for the preparation of a medicament wherein the medicament is in a dosage form for intracerebral or intraventricular administration.

The invention also provides for the use of the antisense oligonucleotide of the invention or pharmaceutical composition of the invention as described for the preparation of a medicament wherein the medicament is in a dosage form for intracerebroventricular administration.

Combination therapies

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

GBA upregulation

In certain embodiments the antisense oligonucleotides of the present invention may enhance or increase the expression of GBA by at least about 10%. In other embodiments the antisense oligonucleotides of the present invention may enhance or increase the expression of GBA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.

In certain embodiments the antisense oligonucleotides of the present invention may enhance or increase the expression of GBA protein by at least about 10%. In other embodiments the antisense oligonucleotides of the present invention may enhance or increase the expression of GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.

It will be understood that the % increase in the enhancement or increase in the expression of GBA protein referred to above will be a % increase relative to a control, wherein the control is a cell that has not been exposed to said oligonucleotide. In some embodiments, the control may be a mock transfection, for example, treatment of cells with PBS. Herein, an increase in expression of GBA may be measured as an increase in GBA protein.

Table 1 : Antisense oligonucleotides sequences and corresponding target site sequences. Letter code for each corresponding nucleobase:

A=adenine; G=guanine; T=thymine; C=cytosine; E=5-methyl cytosine.

Table 2: Compound ID

Helm Annotation Key:

5 [LR](G) is a beta-D-oxy-LNA guanine nucleoside,

[LR](T) is a beta-D-oxy-LNA thymine nucleoside,

[LR](A) is a beta-D-oxy-LNA adenine nucleoside,

[LR]([5meC] is a beta-D-oxy-LNA 5-methyl cytosine nucleoside,

[dR](G) is a DNA guanine nucleoside,

10 [dR](T) is a DNA thymine nucleoside,

[dR](A) is a DNA adenine nucleoside,

[dR]([C] is a DNA cytosine nucleoside,

[mR](G) is a 2’-O-methyl RNA guanine nucleoside,

[mR](U) is a 2’-O-methyl RNA DNA uracil nucleoside,

15 [mR](A) is a 2’-O-methyl RNA DNA adenine nucleoside,

[mR]([C] is a 2’-O-methyl RNA DNA cytosine nucleoside,

[sP] is a phosphorothioate internucleoside linkage.

Further details regarding how to read a HELM sequence are provided at www.pistoiaalliance.org/helm-tools/.

EXAMPLES

Example 1 : UPREGULATION OF GBA mRNA

The day before transfection treatment SK-N-AS neuroblastoma cells were plated to a density of 25000 per well in 96 well plates in full growth medium (DMEM (Sigma: D6546), 10% FBS, 2mM glutamine, 0.1 mM (1x) NEAA, 25pg/ml Gentamicin). The day after plating, the cells were either transfected with SEQ ID NO 1 to 40 (n=2) or PBS (Mock) using Lipofectamin RNAiMax (Invitrogen) at a final concentration of 10 nM according to Invitrogen’s instructions. 48 h after transfection, mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water. 4 pL was used as input for one-step RT-qPCR analysis according to protocol in Table 4 (qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™, Quanta Bioscience, #95134-500) using custom designed qPCR assay specifc to GBA and predesigned assay for TBP (HEX, Hs. PT.58v.39858774 , IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 1.

Table 3. GBA gPCR assay: Primers and probe, all sequences 5’-> 3’

Table 4. RT-qPCR protocol

As shown in Figure 1 , SEQ ID NO 9, SEQ ID NO 29, SEQ ID NO 35, SEQ ID NO 37 and SEQ ID NO 38 all increase expression of GBA mRNA more than 10% fold relative to Mock transfected 48h after transfection in SK-N-AS cells.

Example 2: UPREGULATION OF GBA mRNA

SK-N-As neuroblastoma cells were plated to a density of 10000 cells per well in 96 well plates in full growth medium (DMEM (Sigma: D6546), 10% FBS, 2mM glutamine, 0.1 mM (1x) NEAA, 25pg/ml Gentamicin). The day after plating, the cells were either treated with SEQ ID NO 1 to 40 (n=2) or PBS (Mock) a final concentration of 10 pM. 72 h after treatment, mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water. 4 pL was used as input for one-step RT-qPCR analysis according to protocol in Table 4 (qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™, Quanta Bioscience, #95134-500) using custom designed qPCR assay specifc to GBA and predesigned assay for TBP (HEX, Hs. PT.58v.39858774 , IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 2.

As shown in Figure 2, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 30 all increase expression of GBA mRNA more than 10 % relative to Mock after 72 h of gymnosis in SK-N- AS cells.

Example 3: UPREGULATION OF GBA protein

SK-N-As neuroblastoma cells were plated to a density of 60000 cells per well in 24 well plates in full growth medium (DMEM (Sigma: D6546), 10% FBS, 2mM glutamine, 0.1 mM (1x) NEAA, 25pg/ml Gentamicin). The day after plating, the cells were either treated with SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 21 , SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 37 and SEQ ID NO 41 (n=2) or PBS (Mock) a final concentration of 50 pM. 72 h after treatment, proteins were isolated using 75 pl RIPA (ThermoFisher Scientific, # 89900) supplemented with 1 mM DTT buffer. Proteins were detected using Wes analysis (ProteinSimple) using the 12-230 kDa Wes separation module (ProteinSimple, # SM-W004) according to the manufacturers recommendations and Anti-Glucocerebrosidase antibody (Sigma Aldrich, # G4046) and HPRT Antibody (Santa Cruz Biotechnoligy, # FL-218). GBA protein abundances were quantified relative to HPRT1 protein using “Compass for SW’ software and Microsoft Excel. See Figure 3. As shown in Figure s, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 , SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35 and SEQ ID NO 38 all increase expression of GBA protein more than 30 % relative to Mock after 72 h of gymnosis in SK-N- AS cells. SEQ ID NO 105 reduces GBA protein by more than 70 %.

EMBODIMENTS

1. An antisense oligonucleotide of 8 to 40 nucleotides in length, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length; wherein the contiguous nucleotide sequence is complementary to a sequence in a 5’ untranslated region (5'IITR) located upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript.

2. The antisense oligonucleotide of item 1, wherein the contiguous nucleotide sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

3. The antisense oligonucleotide of item 2, wherein the contiguous nucleotide sequence is 16 or 18 nucleotides in length.

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

5. The antisense oligonucleotide of any one of items 1 to 4, wherein the contiguous nucleotide sequence is at least 75% complementary to a sequence in a 5’UTRIocated upstream of the canonical AUG start codon of the GBA mRNA transcript.

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

7. The antisense oligonucleotide of any one of items 1 to 6, wherein the contiguous nucleotide sequence is fully complementary to a sequence in a 5’UTR located upstream of the canonical AUG start codon of the GBA mRNA transcript.

8. The antisense oligonucleotide of any one of items 1 to 7, wherein the GBA mRNA transcript comprises the nucleic acid sequence according to SEQ ID NO 108. 9. The antisense oligonucleotide of any one of items 1 to 8, wherein the canonical AUG start codon corresponds to positions 138-140 of SEQ ID NO 108.

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

11. The antisense oligonucleotide of item 10, wherein the uAUG is located 14nt upstream of the canonical AUG.

12. The antisense oligonucleotide of item 10 or item 11 , wherein the uAUG is located at a position corresponding to positions 124-126 of SEQ ID NO 108.

13. The antisense oligonucleotide of any one of items 1 to 12, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89 and SEQ ID NO 90, or a fragment thereof.

14. The antisense oligonucleotide of any one of items 1 to 13, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 90 and SEQ ID NO 109, or a fragment thereof.

15. The antisense oligonucleotide of any one of items 1 to 14, where the antisense oligonucleotide is a single stranded antisense oligonucleotide.

16. The antisense oligonucleotide of any one of items 1 to 15, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37 and SEQ ID NO 38, or at least 10 contiguous nucleotides thereof.

17. The antisense oligonucleotide of any one of items 1 to 16, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 38 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.

18. The antisense oligonucleotide of any one of items 1 to 17, wherein the antisense oligonucleotide comprises one or more modified nucleoside(s).

19. The antisense oligonucleotide of item 18, wherein the one or more modified nucleoside is independently selected from the group consisting of 2’-O-alkyl-RNA, 2’- O-methyl-RNA, 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro- DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA, and locked nucleic acid (LNA) nucleosides.

20. The antisense oligonucleotide of item 18 or 19, wherein the one or more modified nucleoside is a LNA nucleoside.

21. The antisense oligonucleotide of item 19 or 20, wherein the LNA nucleoside is beta- D-oxy-LNA.

22. The antisense oligonucleotide of any one of items 18 to 21, wherein the one or more modified nucleoside is a 2’-O-methyl-RNA nucleoside.

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

24. The antisense oligonucleotide of any one of items 1 to 23, wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.

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

26. The antisense oligonucleotide of item 25, wherein all of the internucleoside linkages present within the antisense oligonucleotide are phosphorothioate internucleoside linkages.

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

28. The oligonucleotide of item 27, wherein the control is a cell that has not been exposed to said oligonucleotide. 29. The antisense oligonucleotide of any one of items 1 to 28, wherein the antisense oligonucleotide is covalently attached to at least one conjugate moiety.

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

31. The antisense oligonucleotide of item 30, wherein the salt is a sodium salt or a potassium salt

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

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

34. The pharmaceutical composition of item 33 wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.

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

36. The method of item 35, wherein the cell is a human cell or a mammalian cell.

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

38. The oligonucleotide of item 37, wherein the control is a cell that has not been exposed to said oligonucleotide.

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

40. The antisense oligonucleotide of any one of items 1 to 32 or the pharmaceutical composition of item 33 or item 34, for use as a medicament for the treatment or prevention of a disease in a subject. 41. Use of the antisense oligonucleotide of any one of items 1 to 32 or the pharmaceutical composition of item 33 or item 34, for the preparation of a medicament for treatment or prevention of a disease in a subject.

42. The method of item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41 , wherein the disease is associated with reduced expression of GBA.

43. The method of item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41 , wherein the disease is selected from the group consisting of Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements

(REM) sleep behaviour disorders.

44. The method of item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41 , wherein the disease is Parkinson’s disease. 45. The method of item 39, the antisense oligonucleotide or the pharmaceutical composition for use according to item 40, or the use according to item 41 , wherein the disease is Gaucher’s disease.

Claims

1 . An antisense oligonucleotide of 8 to 40 nucleotides in length, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length; wherein the contiguous nucleotide sequence is complementary to a sequence in a 5’ untranslated region (5'UTR) located upstream of the canonical AUG start codon of the glucocerebrosidase (GBA) mRNA transcript.

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

3. The antisense oligonucleotide of claim 1 or 2, wherein the GBA mRNA transcript comprises the nucleic acid sequence according to SEQ ID NO 108.

4. The antisense oligonucleotide of any one of claims 1 to 3, wherein the canonical AUG start codon corresponds to positions 138-140 of SEQ ID NO 108.

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

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

7. The antisense oligonucleotide of claim 5 or claim 6, wherein the uAUG is located at a position corresponding to positions 124-126 of SEQ ID NO 108.

8. The antisense oligonucleotide of any one of claims 1 to 7, wherein the contiguous nucleotide sequence is complementary to a target nucleic acid sequence selected from the group consisting of SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 81 , SEQ ID NO 82, SEQ ID NO 87, SEQ ID NO 89 SEQ ID NO 90 and SEQ ID NO 109, or a fragment thereof.

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

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10. The antisense oligonucleotide of any one of claims 1 to 9, wherein the contiguous nucleotide sequence is or comprises a sequence selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 38 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.

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

12. The antisense oligonucleotide of any one of claims 1 to 11 , for use as a medicament for the treatment or prevention of a disease in a subject.

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

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

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

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