WO2023111335A1

WO2023111335A1 - Oligonucleotides capable of increasing glucocerebrosidase expression

Info

Publication number: WO2023111335A1
Application number: PCT/EP2022/086502
Authority: WO
Inventors: Johannes Braun; Ross CORDINER; Lukasz KIELPINSKI
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
Also published as: CN118382697A

Abstract

The present invention relates to oligonucleotides that are complementary to aMTX1P1 mRNA and which lead to increased expression of glucocerebrosidase (GBA) in cells. The present invention further relates to conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with reduced expression of GBA, including Parkinson's disease.

Description

OLIGONUCLEOTIDES CAPABLE OF INCREASING GLUCOCEREBROSIDASE

EXPRESSION

FIELD OF INVENTION

The present invention relates to oligonucleotides that are complementary to a MTX1 P1 transcript and which lead to increased expression of glucocerebrosidase (GBA) in cells. The present invention further relates to conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with reduced expression of GBA, including 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 characterised 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 INVENTION

The invention provides oligonucleotides that are complementary to MTX1 P1 mRNA and are capable of increasing the expression of glucocerebrosidase (GBA) in a cell.

The oligonucleotides of the invention increase GBA expression by binding to MTX1P1 mRNA. MTX1 P1 is a converging transcript downstream of GBA. When MTX1 P1 is transcribed by RNA polymerase II (RNAPII), there is believed to be head-to-head collision with the RNAPII that transcribes GBA, resulting in transcriptional downregulation (Hobson et al., 2012) of GBA. The oligonucleotides of the invention are believed to bind to MTX1 P1 mRNA as it is transcribed, leading to recruitment of RNaseHI , which results in cleavage of MTX1 P1 mRNA. The cleaved MTX1 P1 transcript is believed to be degraded by nuclear exonuclease XRN2, leading to the subsequent release of RNAPII (Lai et al., 2020, Molecular Cell, 77, 1032-1043. Hence there are fewer head-to-head collisions with the RNAPII that transcribes GBA, leading to increased GBA expression.

The oligonucleotides of the invention may therefore be used to restore or to enhance the expression of GBA in cells.

The invention provides oligonucleotides capable of increasing the expression of glucocerebrosidase (GBA) in a cell, wherein the oligonucleotide is 8 to 40 nucleotides in length and comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length, which is complementary to a human MTX1 P1 transcript.

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may comprise a contiguous nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or fully complementary to the human MTX1 P1 transcript.

In some embodiments the oligonucleotide capable of increasing the expression of GBA according to the invention may comprise a contiguous nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or fully complementary to the 5’ region of the human MTX1 P1 transcript.

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may comprise one or more modified nucleoside(s).

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may be capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% in a cell, compared to a control. Herein an increase in expression of GBA may be measured as an increase in GBA mRNA, an increase in expression in GBA protein or an increase in expression of both GBA mRNA and GBA protein.

In some embodiments the control is a cell that has not been exposed to the oligonucleotide. In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may be covalently attached to at least one conjugate moiety.

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may be in the form of a pharmaceutically acceptable salt. The salt may be a sodium salt or a potassium salt

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention may be encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

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

The invention provides for a method for enhancing the expression of GBA in a cell which is expressing GBA, said method comprising exposing an oligonucleotide capable of increasing the expression of GBA of the invention, or the pharmaceutical composition of the invention in an effective amount to said cell.

In some embodiments, the expression of GBA may be increased by 10%, 15% 20%, 30%, 40%, 50% or more than 50% in a cell, compared to a control. In some embodiments the control is a cell that has not been exposed to the oligonucleotide.

The present invention also provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide capable of increasing the expression of GBA 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 oligonucleotide capable of increasing the expression of GBA according to the invention, or a 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 oligonucleotide capable of increasing the expression of GBA according to 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.

In some embodiments, the disease may be associated with reduced expression of GBA.

In some embodiments, 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.

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. GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. SEQ ID NO 38, SEQ ID NO 10, SEQ ID NO 45, SEQ ID NO 3, SEQ ID NO 57, SEQ ID NO 49, SEQ ID NO 34, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 50, SEQ ID NO 4, SEQ ID NO 29, SEQ ID NO 59, SEQ ID NO 13, SEQ ID NO 21 and SEQ ID NO 55 all increase expression of GBA mRNA more than 15% relative to Mock.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified that the expression level of the GBA transcript can be effectively enhanced by targeting the MTX1P1 transcript with oligonucleotides.

Described herein are target sites present on the human MTX1 P1 transcript, which can be targeted by oligonucleotides capable of increasing the expression of GBA according to the invention.

The inventors have surprisingly determined that targeting the 5’ region of human MTX1 P1 transcript, can be particularly effective.

Without wishing to be bound by theory, it is considered that the oligonucleotides of the invention can increase GBA production by binding to MTX1 P1 mRNA as it is transcribed by RNAPII. MTX1 P1 is a converging transcript downstream of GBA and its transcription by RNAPII is believed to result in head-to-head collision with the RNAPII that transcribes GBA, resulting in transcriptional downregulation of GBA (Hobson et al., 2012). Binding of the oligonucleotides of the invention to MTX1 P1 mRNA as it is transcribed leads to the recruitment of RNaseHI to the MTX1 P1 mRNA, resulting in cleavage of MTX1 P1 and subsequent release of RNAPII. Hence there are less head-to-head collisions with the RNAPII that transcribes GBA and increased production of GBA.

Enhanced GBA expression is desirable to treat a range of disorders which are characterised by, or caused by, reduced expression of GBA. These include Gaucher’s disease, Parkinson’s disease dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.

Capable of Increasing the Expression of GBA

The oligonucleotides of the invention are capable of increasing the expression of GBA.

The term “increasing the expression” as used herein is to be understood as an overall term to describe an oligonucleotide's ability to increase the expression of GBA in a cell when compared to a control where the cell is not exposed to the oligonucleotide of the invention.

Without wishing to be bound by theory the increase effected by the oligonucleotide is thought to be related to its ability to reduce, remove, prevent, lessen, lower or terminate the suppression of the GBA transcript, e.g. by degradation or removal of the MTX1 P1 transcript or by blockage or prevention of polymerase activity associated with the MTX1 P1 transcript. The increase can also be viewed as the oligonucleotide's ability to restore or enhance expression of GBA, e.g. by removal or blockage of inhibitory mechanisms affected by the MTX1 P1 transcript.

Herein, the term “increasing the expression of GBA” is understood to mean increasing GBA mRNA levels, increasing GBA protein levels or increasing GBA mRNA and protein levels.

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

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

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

Preferably, the oligonucleotides of the invention induce GBA expression in a cell by degradation or removal of the MTX1 P1 mRNA. In some embodiments the oligonucleotides of the invention are capable of decreasing the level of the MTX1 P1 mRNA by at least 10% compared to a control. More preferably the oligonucleotides capable of increasing the expression of GBA of the present invention may decrease the level of the MTX1 P1 mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more compared to a control.

Control By the term "control", when used in relation to measurements of the effect of an oligonucleotide, it is generally understood that the control is a cell that has not been exposed to the oligonucleotide.

Alternatively the increase in GBA expression may be determined by reference to the amount of GBA mRNA and/or protein expressed before exposure to the oligonucleotide.

In other embodiments, the control may be a cell treated with a non-targeting oligonucleotide.

In some embodiments, the control may be a mock transfection, for example wherein cells are treated with PBS.

Oligonucleotide

The term “oligonucleotide” as used herein is defined as it 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 the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the 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 synthesised, 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.

In some embodiments, the oligonucleotides of the invention are single stranded oligonucleotides.

In some embodiments, the oligonucleotides of the invention are 8 to 40 nucleotides in length.

In some embodiments, the oligonucleotides of the invention are 8 to 40 nucleotides in length and comprise a contiguous nucleotide sequence of 8 to 40 nucleotides. In some embodiments, the oligonucleotides of the invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

In some embodiments the oligonucleotides of the invention are at least 12 nucleotides in length.

In some embodiments the oligonucleotides of the invention are at least 14 nucleotides in length.

In some embodiments the oligonucleotides of the invention are at least 16 nucleotides in length.

In some embodiments the oligonucleotides of the invention are at least 18 nucleotides in length.

Preferably, the oligonucleotides of the invention are 16 to 20 nucleotides in length.

More preferably, the oligonucleotides of the invention are 18 to 20 nucleotides in length.

Contiguous Nucleotide Sequence

The term “contiguous nucleotide sequence” refers to the region of the 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”.

The 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 oligonucleotide cannot be longer than the oligonucleotide as such and that the oligonucleotide cannot be shorter than the contiguous nucleotide sequence. In some embodiments, the entire nucleotide sequence of the oligonucleotide of the invention is the contiguous nucleotide sequence.

The contiguous nucleotide sequence is the sequence of nucleotides in the oligonucleotide 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 to 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 at least 12 nucleotides in length.

In some embodiments the contiguous nucleotide sequence is at least 14 nucleotides in length.

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

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

In a preferred embodiment the contiguous nucleotide sequence is 16 to 20 nucleotides in length.

More preferably, the contiguous nucleotide sequence is 18 to 20 nucleotides in length.

In some embodiments the oligonucleotide of the invention consists of the contiguous nucleotide sequence.

In some embodiments the oligonucleotide of the invention is the contiguous nucleotide sequence

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

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, the oligonucleotide capable of increasing the expression of GBA according to the invention may comprise one or more modified nucleosides.

In some embodiments the contiguous nucleobase sequences (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid. Advantageously, high affinity modified nucleosides are used.

Advantageously, one or more of the modified nucleosides of the oligonucleotide according to 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 oligonucleotide capable of increasing the expression of GBA according to the invention include LNA, 2’-O-MOE, 2’oMe and morpholino nucleoside analogues.

Modified internucleoside linkage

Advantageously, the oligonucleotide capable of increasing the expression of GBA according to the invention comprises one or more modified internucleoside linkages.

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

In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide capable of increasing the expression of GBA according to the invention, or the 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. In some embodiments all of the internucleoside linkages of the oligonucleotide of the invention, or contiguous nucleotide sequence thereof, are phosphorothioate.

In a further embodiment, the oligonucleotide capable of increasing the expression of GBA according to the invention comprises 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 oligonucleotide according to the invention may be phosphorothioate, or all the internucleoside linkages of the oligonucleotide capable of increasing the expression of GBA according to the invention 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 hybridisation. 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 vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

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 oligonucleotide capable of increasing the expression of GBA according to the invention may be a modified oligonucleotide.

The term modified oligonucleotide describes an 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 oligonucleotide capable of increasing the expression of GBA according to the invention to be a chimeric oligonucleotide.

In some embodiments, the oligonucleotide capable of increasing the expression of GBA according to the invention, 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. In an embodiment, the oligonucleotide capable of increasing the expression of GBA according to the invention comprises 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, or at least 19 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 the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (Tm). 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 oligonucleotide capable of increasing the expression of GBA according to 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 02 and 03 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). 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 does not include 2' bridged nucleosides like LNA.

In one embodiment, the oligonucleotide capable of increasing the expression of GBA according to the invention comprises one or more sugar modified nucleosides, such as 2' sugar modified nucleosides. Preferably the oligonucleotide capable of increasing the expression of GBA according to the invention comprises 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 02’ and 04’ 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 al., Bioorganic & Med.Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 , and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

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 oligonucleotide capable of increasing the expression of GBA of the invention comprises or consists 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 capable of increasing the expression of GBA according to the invention may be, for example 8 to 40 morpholino nucleotides in length, such as morpholino 16 to 20 nucleotides in length, such as 18 to 20 nucleotides in length.

Nuclease Mediated Degradation Nuclease mediated degradation refers to an oligonucleotide capable of mediating degradation of a complementary nucleotide sequence when forming a duplex with such a sequence.

In some embodiments, the oligonucleotide may function via nuclease mediated degradation of the target nucleic acid, where the oligonucleotides of the invention are capable of recruiting a nuclease, particularly an endonuclease, preferably endoribonuclease (RNase), such as RNase H. Examples of oligonucleotide designs which operate via nuclease mediated mechanisms are oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing nucleosides, for example gapmers, headmers and tailmers.

RNase H Activity and Recruitment

The RNase H activity of an 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 RNase H. 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 WO 01/23613 (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 RNase H, 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.

Gapmer The term gapmer as used herein refers to an oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap) which is flanked 5' and 3' by one or more affinity enhancing modified nucleosides (flanks). Various gapmer designs are described herein.

Headmers and tailmers are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e. only one of the ends of the oligonucleotide comprises affinity enhancing modified nucleosides. For headmers the 3' flank is missing (i.e. the 5' flank comprises affinity enhancing modified nucleosides) and for tailmers the 5' flank is missing (i.e. the 3' flank comprises affinity enhancing modified nucleosides).

The term LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside.

The term mixed wing gapmer refers to a LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2' substituted modified nucleoside, such as, for example, 2'- O-alkyl-RNA, 2'- O-methyl- RNA, 2'-alkoxy-RNA, 2'- O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, and 2'- F-ANA nucleoside(s). In some embodiments the mixed wing gapmer has one flank which comprises LNA nucleosides (e.g. 5' or 3') and the other flank (3' or 5' respectfully) comprises 2' substituted modified nucleoside(s).

Gapmer Design

In a preferred embodiment the oligonucleotide of the invention has a gapmer design or structure also referred herein merely as "gapmer". In a gapmer structure the oligonucleotide comprises at least three distinct structural regions a 5'-flank, a gap and a 3'-flank, F-G-F' in '5 -> 3' orientation. In this design, flanking regions F and F' (also termed wing regions) comprise a contiguous stretch of modified nucleosides, which are complementary to the MTX1 P1 target nucleic acid, while the gap region, G, comprises a contiguous stretch of nucleotides which are capable of recruiting a nuclease, preferably an endonuclease such as RNase, for example RNase H, when the oligonucleotide is in duplex with the target nucleic acid. Nucleosides which are capable of recruiting a nuclease, in particular RNase H, can be selected from the group consisting of DNA, alpha-L-oxy-LNA, 2'-Flouro-ANA and UNA.

Regions F and F', flanking the 5' and 3' ends of region G, preferably comprise non-nuclease recruiting nucleosides (nucleosides with a 3' endo structure), more preferably one or more affinity enhancing modified nucleosides. In some embodiments, the 3' flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments, the 5' flank comprises at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments both the 5' and 3' flanking regions comprise a LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments all the nucleosides in the flanking regions are LNA nucleosides.

In other embodiments, the flanking regions may comprise both LNA nucleosides and other nucleosides (mixed flanks), such as DNA nucleosides and/or non-LNA modified nucleosides, such as 2' substituted nucleosides. In this case the gap is defined as a contiguous sequence of at least 5 RNase H recruiting nucleosides (nucleosides with a 2' endo structure, preferably DNA) flanked at the 5' and 3' end by an affinity enhancing modified nucleoside, preferably LNA, such as beta-D-oxy-LNA. Consequently, the nucleosides of the 5' flanking region and the 3' flanking region which are adjacent to the gap region are modified nucleosides, preferably non-nuclease recruiting nucleosides. In oligonucleotides with mixed flanks where the flanks comprise DNA the 5' and 3' nucleosides are modified nucleosides.

Region F

Region F (5' flank or 5' wing) attached to the '5 end of region G comprises, contains or consists of at least one modified nucleoside such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides. In an embodiment region F comprises or consists of from 1 to 7 modified nucleosides, such as from 2 to 6 modified nucleosides, such as from 2 to 5 modified nucleosides, such as from 2 to 4 modified nucleosides, such as from 1 to 3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides. In a further embodiment further additional nucleosides may be attached to the '5 end of region F, representing a region D preferably comprising 1, 2 or 3 nucleoside units, such as DNA nucleosides. Region D can take the function of a biocleavable (B) linker described in the definition of "Linkers ".

In some embodiments, the modified nucleosides in region F have a 3' endo structure.

In an embodiment, one or more of the modified nucleosides in region F are 2' modified nucleosides.

In a further embodiment one or more of the 2' modified nucleosides in region F are selected from 2'-0-alkyl-RNA units, 2'-O-methyll-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2'-fluoro-ANA units.

In one embodiment of the invention all the modified nucleosides in region F are LNA nucleosides. In a further embodiment the LNA nucleosides in region F are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and/or ENA, in either the beta-D or alpha-L configurations or combinations thereof. In a preferred embodiment region F has at least one beta-D-oxy LNA unit, at the 5' end of the contiguous sequence.

Region G

Region G (gap region) preferably comprises, contains or consists of at least 4, such as at least 5, such as 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 or at least 16 consecutive nucleosides capable of recruiting the aforementioned nuclease, in particular RNase H. In a further embodiment region G comprises, contains or consists of from 5 to 12, or from 6 to 10, or from 7 to 9, such as 8 consecutive nucleotide units capable of recruiting aforementioned nuclease.

The nucleoside units in region G, which are capable of recruiting nuclease are in an embodiment selected from the group consisting of DNA, alpha-L-LNA, C4' alkylated DNA (as described in PCT/EP2009/050349 and Vester et a/., Bioorg. Med. Chern. Lett. 18 (2008) 2296-2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluter et a/., Mol. Biosyst., 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked "sugar" residue.

In a still further embodiment at least one nucleoside unit in region G is a DNA nucleoside unit, such as from 1 to 16 DNA units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 DNA units, preferably from 2 to 13 DNA units, such as from 4 to 12 DNA units, more preferably from 5 to 11, or from 1 0 to 16, 11 to 15 or 12 to 14 DNA units. In some embodiments, region G consists of 100% DNA units. In a preferred embodiment G consists of, most preferably 10, 11, 12, 13, 14 or 15 DNA units. In further embodiments the region G may consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. Region G may consist of at least 50% DNA, more preferably 60%, 70% or 80 % DNA, and even more preferred 90% or 95% DNA.

In a still further embodiment at least one nucleoside unit in region G is an alpha-L-LNA nucleoside unit, such as at least one alpha-L-LNA unit, such as 2, 3, 4, 5, 6, 7, 8 or 9 alpha- L-LNA units. In a further embodiment, region G comprises the least one alpha-L-LNA is alpha-L-oxy-LNA unit. In a further embodiment region G comprises a combination of DNA and alpha-L-LNA nucleoside units.

In some embodiments the size of the contiguous sequence in region G may be longer, such as 15, 16, 17, 18, 19 or 20 nucleoside units.

In some embodiments, nucleosides in region G have a 2' endo structure.

Region F'

Region F' (3' flank or 3' wing) attached to the '3 end of region G comprises, contains or consists of at least one modified nucleoside such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 modified nucleosides. In an embodiment region F' comprise or consist of from 1 to 7 modified nucleosides, such as from 2 to 6 modified nucleoside, such as from 2 to 4 modified 20 nucleosides, such as from 1 to 3 modified nucleosides, such as 1 , 2, 3 or 4 modified nucleosides. In a further embodiment further additional nucleosides attached to the '3 end of region F', representing a region D', preferably comprising 1, 2 or 3 nucleoside units, such as DNA nucleosides. Region D' can take the function of a biocleavable (B) linker described, in the section "Linkers".

In some embodiments, the modified nucleosides in region F' have a 3' endo structure.

In a preferred embodiment, modified nucleosides in region F' is LNA.

In a further embodiment modified nucleosides in region F' are selected from 2'- O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2'-fluoro-ANA units.

In one embodiment of the invention all the modified nucleosides in region F' are LNA nucleosides. In a further embodiment the LNA nucleosides in region F' are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA, in either the beta-D or alpha-L configurations or combinations thereof. In a preferred embodiment region F' has at least 2 beta-D-oxy LNA unit, at the 3' end of the contiguous sequence.

Region D and D'

The addition of region D’ or D” may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, 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 exonuclease protection or for ease of synthesis or manufacture.

Region D and D' can be attached to the 5' end of region F or the 3' end of region F', respectively. Region D or D' may independently comprise 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. In this respect the oligonucleotide of the invention, may in some embodiments comprise a contiguous nucleotide sequence capable of modulating the target which is flanked at the 5' and/or 3' end by additional nucleotides. Such additional nucleotides 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 may be DNA or RNA. In another embodiment, the additional 5' and/or 3' end nucleotides are modified nucleotides which may for example be included to enhance nuclease stability or for ease of synthesis. In an embodiment of the oligonucleotide of the invention, comprises a region D and/or D' in addition to the contiguous nucleotide sequence.

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 oligonucleotide capable of increasing the expression of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes a gapmer.

In some embodiments the internucleoside linkage positioned between region D’ or D” and the gapmer region is a phosphodiester linkage. The gapmer oligonucleotide of the present invention can be represented by the following formulae:

F-G-F'; in particular Fi-7-G4-i2-F'i-7

D-F-G-F', in particular Di-3-Fi-7-G4-i2-F'i-7

F-G-F'-D', in particular Fi-7-G4-i2-F'i-7-D'i-3

D-F-G-F' -D', in particular Di-3-Fi-7-G4-i2-F'i-7-D'i-3

The preferred number and types of nucleosides in regions F, G and F', D and D' have been described above. The design of the individual oligonucleotide may also have profound impact on the properties of the oligonucleotide in its use for modulating expression of GBA.

In some embodiments the oligonucleotide according to the invention is a gapmer consisting of 14, 15, 16, 17, 18, 19 or 20 nucleotides in length, wherein each of regions F and F' independently consists of 2, 3 or 4 modified nucleoside units and region G consists of 10, 11, 12, 13, 14, 15 or 16 nucleoside units, capable of recruiting nuclease when in duplex with the target nucleic acid.

In a further embodiment, the oligonucleotide is a gapmer wherein each of regions F and F' independently consists of 2, 3 or 4 modified nucleoside units, such as nucleoside units containing a 2'-O-methoxyethyl-ribose sugar (2'-MOE) or nucleoside units containing a 2'- fluorodeoxyribose sugar and/or LNA units, and region G consists of 9, 10, 11, 12, 13, 14, 15 or 16 nucleoside units, such as DNA units or other nuclease recruiting nucleosides such as alpha-L-LNA or a mixture of DNA and nuclease recruiting nucleosides.

In a further specific embodiment, the oligonucleotide is a gapmer wherein each of regions F and F' region consists of two LNA units each, and region G consists of 10, 11, 12, 13, 14, 15 or 16 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include

2-10-2, 2-11-2, 2-12-2, 2-13-2, 2-14-2, 2-15-2 and 2-16-2.

In a further specific embodiment, the oligonucleotide is a gapmer wherein each of regions F and F' independently consists of three LNA units, and region G consists of 10, 11 , 12, 13, 14 or 15 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include

3-10-3, 3-11-3, 3-12-3, 3-13-3, 3-14-3 and 3-15-3.

In a further specific embodiment, the oligonucleotide is a gapmer wherein each of regions F and F' consists of four LNA units each, and region G consists of 10, 11, 12, 13, 14 or 15 nucleoside units, preferably DNA units. Specific gapmer designs of this nature include 4-10- 4, 4-11-4, 4-12-4, 4-1 3-4, 4-14-4 and 4-15-4.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 10 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-10-1 , 2-10-1 , 1-10-2, 1-10-3, 3-10-1 , 1-10-4, 4-10-1 , 2-10-2, 2-10-3, 3-10- 2, 2-10-4, 4-10-2, 3-10-3, 3-10-4, 4-10-3 and 4-10-4 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 11 nucleosides and independently 1 to 4 modified nucleosides in the wings including, 1-11-1 , 2-11-1 , 1-11-2, 1-11-3, 3-11-1 , 1-11-4, 4-11-1 , 2-11-2, 2-11-3, 3-11- 2, 2-11-4, 4-11-2, 3-11-3, 3-11-4, 4-11-3 and 4-11-4 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 12 nucleosides including, 1-12-1 , 2-12-1 , 1-12-2, 1-12-3, 3-12-1 , 1- 12-4, 4-12-1 , 2-12-2, 2-12-3, 3-12-2, 2-12-4, 4-12-2, 3-12-3, 3-12-4, 4-12-3 and 4-12-4 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 13 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-13-1 ,1-13-2, 1-13-3, 3-13-1 , 1-13-4, 4-13-1 , 2-13-1 , 2-13-2, 2-13-3, 3-13-2, 2-13-4, 4-13-2, 3-13-3, 3-13-4, 4-13-3, and 4-13-4 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 14 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-14-1 , 1-14-2, 2-14-1 , 1-14-3, 3-14-1 , 1-14-4, 4-14-1 , 2-14-2, 2-14-3, 3-14- 2, 2-14-4, 4-14-2, 3-14-3, 3-14-4 and 4-14-3 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 15 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-15-1 ,1-15-2, 2-15-1 , 1-15-3, 3-15-1 , 1-15-4, 4-15-1 , 2-15-2, 2-15-3, 3-15-2, 2-15-4, 4-15-2, 3-15-3, 3-15-4 and 4-15-3 gapmers.

Specific gapmer designs of this nature include F-G-F' designs selected from a group consisting of a gap with 16 nucleosides and independently 1 to 4 modified nucleosides in the wings including 1-16-1 ,1-16-2, 2-16-1 , 1-15-3, 3-16-1 , 1-16-4, 4-16-1 , 2-16-2, 2-16-3, 3-16-2 2-16-4, 4-16-2, 3-16-3, 3-16-4 and 4-16-3 gapmers. In some embodiments the F-G-F' design is selected from 2-10-4, 3-10-3 and 4-10-2.

In some embodiments the F-G-F' design is selected from 2-11-4, 3-11-2, 3-11-3 and 4-11-2.

In some embodiments the F-G-F' design is selected from 2-12-2, 2-12-3, 2-12-4, 3-12-2, 3- 12-3, and 4-12-2.

In some embodiments the F-G-F' design is selected from 2-13-2, 2-13-3, 2-13-4, 3-13-3 and

4-13-2.

In some embodiments the F-G-F' design is selected from 2-14-2, 2-14-4, 3-14-3 and 4-14-2.

In some embodiments the F-G-F' design is selected from 2-15-2 and 2-16-2.

In some embodiments the F-G-F' design is selected from the designs indicated in table 2 below.

In all instances the F-G-F' design may further include region D and/or D', which may have 1 , 2 or 3 nucleoside units, such as DNA units. Preferably, the nucleosides in region F and F' are modified nucleosides, while nucleotides in region G are preferably unmodified nucleosides.

In each design, the preferred modified nucleoside is LNA.

In another embodiment all the internucleoside linkages in the gap in a gapmer are phosphorothioate and/or boranophosphate linkages. In another embodiment all the internucleoside linkages in the flanks (F and F' region) in a gapmer are phosphorothioate and/or boranophosphate linkages. In another preferred embodiment all the internucleoside linkages in the D and D' region in a gapmer are phosphodiester linkages.

For specific gapmers as disclosed herein, when the cytosine (C) residues are annotated as

5-methyl-cytosine, in various embodiments, one or more of the C's present in the oligonucleotide may be unmodified C residues.

Further gapmer designs are disclosed in W02004/046160, W02007/146511 and incorporated by reference. 1 The Target

The oligonucleotide of the invention is an antisense oligonucleotide which targets the MTX1 P1 mRNA transcript.

In some embodiments the target sequence is the human MTX1 P1 transcript .

The human MTX1 P1 transcript may be referred to as a target sequence.

In some embodiments the target sequence is human MTX1 P1 transcript, which may be encoded by SEQ ID NO. 1.

An aspect of the present invention relates to an oligonucleotide capable of increasing the expression of GBA, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 1.

In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid shown as SEQ ID NO 1.

In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which may comprise one or two mismatches between the oligonucleotide and the target nucleic acid.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to SEQ ID NO 1.

An aspect of the present invention relates to an oligonucleotide capable of increasing the expression of GBA, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 136.

In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid shown as SEQ ID NO 136.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to SEQ ID NO 136.

An aspect of the present invention relates to an oligonucleotide capable of increasing the expression of GBA, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 137.

In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid shown as SEQ ID NO 137.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to SEQ ID NO 137.

An aspect of the present invention relates to an oligonucleotide capable of increasing the expression of GBA, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 138.

In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid shown as SEQ ID NO 138.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to SEQ ID NO 138.

An aspect of the present invention relates to an oligonucleotide capable of increasing the expression of GBA, which comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length which is complementarity to SEQ ID NO 139. In some embodiments, the oligonucleotide of the invention comprises a contiguous sequence which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid shown as SEQ ID NO 139.

In a preferred embodiment the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is fully complementary (100% complementary) to SEQ ID NO 139.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length with at least 75% complementary, such as at least 80%, at least 85%, at least 90% or at least 95% or 100% complementarity, to a target nucleic acid region selected from the group consisting of 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 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131 , SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134 and SEQ ID NO 135.

Table 1 : Regions of MTX1 P1 which may be targeted using oligonucleotide of the invention and oligonucleotide sequences for targeting such regions. A=adenine; G=guanine; T=thymine; C=cytosine; E=5-methyl cytosine

In some embodiments the contiguous nucleotide sequence is complementary to a 5’ region of a human MTX1P1 transcript . The contiguous nucleotide sequence may be fully complementary to a 5’ region of a human MTX1 P1 transcript. As indicated above, and without wishing to be bound by theory, targeting the 5’ region may increase GBA expression by preventing head-to-head collision of the RNAPII transcribing MTX1P1 with the RNAPII transcribing GBA.

In some embodiments the target sequence is selected from the group consisting of SEQ ID NO 77, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 88, SEQ ID NO 101, SEQ ID NO 105, SEQ ID NO 109, SEQ ID NO 125, SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

In some embodiments the target sequence is selected from the group consisting of SEQ ID NO 88, SEQ ID NO 101, SEQ ID NO 105, SEQ ID NO 109, SEQ ID NO 125, SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

In some embodiments the target sequence is selected from the group consisting of SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

In one embodiment the target sequence is SEQ ID NO 80, or a fragment thereof.

In one embodiment the target sequence is SEQ ID NO 96, or a fragment thereof.

In one embodiment the target sequence is SEQ ID NO 112, or a fragment thereof.

In another embodiment the contiguous nucleotide sequence may be fully complementary to SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

In another embodiment the contiguous nucleotide sequence is fully complementary to SEQ ID NO 80, or a fragment thereof.

In another embodiment the contiguous nucleotide sequence is fully complementary to SEQ ID NO 96, or a fragment thereof.

In another embodiment the contiguous nucleotide sequence is fully complementary to SEQ ID NO 112, or a fragment thereof. 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 vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 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, or at least about 80% complementarity, or at least about 85% complementarity, or at least about 90% complementary, or at least about 95% complementarity to a human MTX1P1 transcript. In some embodiments the contiguous nucleotide sequence may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary to a human MTX1 P1 transcript.

Put another way, for some embodiments, the contiguous nucleotide sequence of the oligonucleotide capable of increasing the expression of GBA according to the invention may include one, two, three, four, five 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.

The oligonucleotide capable of increasing the expression of GBA according to the invention is complementary to the human MTX1 P1 transcript. The human MTX1 P1 transcript sequence is exemplified herein as SEQ ID NO. 1. It will be understood that the target MTX1P1 nucleic acid may be an allelic variant of SEQ ID NO 1 , such as an allelic variant which comprises one or more polymorphism in the human MTX1P1 nucleic acid 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 a contiguous nucleotide sequence within an oligonucleotide of the invention that is complementary to a target sequence also shares a percentage of identity with said complementary sequence.

Hybridisation

The terms “hybridising” or “hybridises” 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 (Tm) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions Tm 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, oligonucleotide of the present invention hybridises 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 of the invention may hybridise 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. In certain embodiments the oligonucleotide of the invention hybridises to a sub-sequence of the target nucleic acid of SEQ ID NO: 1with a AG° below -10 kcal, such as with a AG° between -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.

The GBA increase is triggered by the hybridisation between a contiguous nucleotide sequence of the oligonucleotide according to the invention and the MTX1P1 mRNA. In some embodiments the oligonucleotide of the invention comprises mismatches between the oligonucleotide and the MTX1P1 mRNA. Despite mismatches, hybridisation to the target nucleic acid may still be sufficient to show a desired increase in GBA expression. If required, reduced binding affinity resulting from mismatches may advantageously be compensated by increasing the number of nucleotides in the oligonucleotide and/or an increasing the number of modified nucleosides capable of increasing the binding affinity to MTX1 P1 mRNA, such as 2' modified nucleosides, including LNA, present within the oligonucleotide sequence.

Antisense Oligonucleotides

The term “antisense oligonucleotide” as used herein is defined as an oligonucleotide capable of modulating expression of a target gene by hybridising to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. Antisense oligonucleotides are not generally 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 to 40 nucleotides in length.

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 at least 12 nucleotides in length.

In some embodiments the antisense oligonucleotide is at least 14 nucleotides in length.

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

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

In a preferred embodiment the antisense oligonucleotide is 16 to 20 nucleotides in length.

More preferably, the antisense oligonucleotide is 18 to 20 nucleotides in length.

In some embodiments the oligonucleotide of the invention is the antisense oligonucleotide.

Oligonucleotide design

The oligonucleotide of the invention is an antisense oligonucleotide which is capable of increasing the expression of glucocerebrosidase (GBA) in a cell.

In some embodiments the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 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 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 and SEQ ID NO 68, or a fragment thereof.

In some embodiments the fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 contiguous nucleotides of the contiguous nucleotide sequence preferably at least 10 contiguous nucleotides thereof.

In some embodiments the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO 10, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 21, SEQ ID NO 34, SEQ ID NO 38, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45, or a fragment therefore.

In some embodiments the fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 contiguous nucleotides of the contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

In some embodiments the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 34, SEQ ID NO 38, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45, or a fragment thereof.

In some embodiments the fragment may be at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 contiguous nucleotides of the contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

In some embodiments the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45 or a fragment thereof.

In some embodiments the fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 contiguous nucleotides of the contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof. In one embodiment the contiguous nucleotide sequence is SEQ ID NO 13, or a fragment thereof.

In one embodiment the contiguous nucleotide sequence is a sequence is SEQ ID NO 29, or a fragment thereof.

In one embodiment the contiguous nucleotide sequence is SEQ ID NO 45, or a fragment thereof.

In some embodiments the 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 oligonucleotide cannot be longer than the oligonucleotide as such and that the oligonucleotide cannot be shorter than the contiguous nucleotide sequence.

Conjugate

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

The term “conjugate” as used herein refers to an oligonucleotide capable of increasing the expression of GBA 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 of the invention 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 oligonucleotide capable of increasing the expression of GBA 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 oligonucleotide capable of increasing the expression of GBA conjugate 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 oligonucleotide capable of increasing the expression of GBA 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 oligonucleotide according to the invention, or the conjugate according to the invention.

The invention provides for oligonucleotides according to the invention wherein the oligonucleotides are 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 oligonucleotide according to the invention.

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

Delivery of oligonucleotide

The invention provides for oligonucleotides capable of increasing GBA expression according to the invention wherein the oligonucleotide is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.

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

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

Pharmaceutical Composition

In a further aspect, the invention provides pharmaceutical compositions comprising an oligonucleotide of the invention and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

The invention provides for a pharmaceutical composition according to the invention, wherein the pharmaceutical composition comprises the 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 oligonucleotide of the invention. Suitably the solution, such as phosphate buffered saline solution, of the invention is a sterile solution.

WO 2007/031091 provides suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in W02007/031 091.

Oligonucleotides of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular, with respect to oligonucleotide conjugates, the conjugate moiety of the oligonucleotide is cleaved once the prodrug is delivered to the site of action, e.g. the target cell.

Target Cell

The term “target cell” as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.

Applications The oligonucleotides of the invention may be utilised as, for example, therapeutics and prophylaxis.

Research Reagents

In research, such oligonucleotides may be used to specifically increase the synthesis of GBA mRNA and/or protein in cells (e.g. in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.

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 exposing an 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 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 recognised that treatment as referred to herein may, in some embodiments, be prophylactic.

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

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

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

The invention provides for an oligonucleotide of the invention or a pharmaceutical composition of the invention is typically administered in an effective amount.

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

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

The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of GBA. The disease may in particular be caused by reduced levels and/or activity of GBA protein.

The invention further relates to use of an oligonucleotide of the invention or a pharmaceutical composition of the invention as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of GBA, in particular low levels and/or activity of GBA.

In one embodiment, the invention relates to an oligonucleotide of the invention or a pharmaceutical composition of the invention for use in the treatment of Gaucher’s disease, Parkinson’s disease dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.

The invention provides for the use of an 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.

Administration

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

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

The invention also provides for the use of the oligonucleotide of the invention as described for the manufacture of a medicament wherein the medicament is in a dosage form for intrathecal administration.

The invention also provides for the use of the oligonucleotide of the invention as described for the manufacture 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 oligonucleotide of the invention as described for the manufacture of a medicament wherein the medicament is in a dosage form for intracerebroventricular administration. Combination therapies

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

Method of Manufacture

In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite WO 2017/081223 PCT/EP2016/077383 chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand). In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Compound Table

Helm Annotation Key:

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

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

5 [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,

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

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

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

[mR](G) is a 2’-0-methyl RNA guanine nucleoside, [mR](U) is a 2’-0-methyl RNA DNA uracil nucleoside,

[mR](A) is a 2’-0-methyl RNA DNA adenine nucleoside,

[mR]([C] is a 2’-0-methyl RNA DNA cytosine nucleoside,

15 [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 SK-N-AS neuroblastoma cells were plated to a density of 25000 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 transfected with MTX1P1 targeting gapmers SEQ ID NO 2 to 68 (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 tab. 1. (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 (the error bars represent the standard deviation).

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 38, SEQ ID NO 10, SEQ ID NO 45, SEQ ID NO 3, SEQ ID NO 57, SEQ ID NO 49, SEQ ID NO 34, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 50, SEQ ID NO 4, SEQ ID NO 29, SEQ ID NO 59, SEQ ID NO 13, SEQ ID NO 21 , SEQ ID NO 55 all increase expression of GBA mRNA more than 15% relative to Mock transfected 48h after transfection in SK-N-AS cells.

EMBODIMENTS OF THE INVENTION

1 . An oligonucleotide capable of increasing the expression of glucocerebrosidase (GBA) in a cell, wherein the oligonucleotide is 8 to 40 nucleotides in length and comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length, which is complementary to a human MTX1 P1 transcript.

2. The 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 oligonucleotide of item 2, wherein the contiguous nucleotide sequence is 16 to 20 nucleotides in length.

4. The oligonucleotide of item 3, wherein the contiguous nucleotide sequence is 19 or 20 nucleotides in length.

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

6. The oligonucleotide of any one of items 1 to 5, wherein the contiguous nucleotide sequence is at least 75% complementary to the human MTX1 P1 transcript.

7. The antisense oligonucleotide of item 6, wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary the human MTX1 P1 transcript.

8. The oligonucleotide of item 7, wherein the contiguous nucleotide sequence is fully complementary to the human MTX1 P1 transcript.

9. The oligonucleotide of any one of items 1 to 8, wherein the human MTX1 P1 transcript is SEQ ID NO. 1. 10. The oligonucleotide of any one of items 1 to 9, wherein the contiguous nucleotide sequence is complementary the 5’ region of the human MTX1 P1 transcript.

11. The oligonucleotide of items 10, wherein the 5’ region of the human MTX1 P1 transcript is selected from the group consisting of SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138 and SEQ ID NO 139.

12. The oligonucleotide of any one of items 1 to 9, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 88, SEQ ID NO 101 , SEQ ID NO 105, SEQ ID NO 109, SEQ ID NO 125, SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

13. The oligonucleotide of item 12, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 88, SEQ ID NO 101, SEQ ID NO 105, SEQ ID NO 109, SEQ ID NO 125, SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

14. The oligonucleotide of item 13, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

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

16. The oligonucleotide of any one of items 1 to 15, wherein the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 10, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 21 , SEQ ID NO 34, SEQ ID NO 38, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45, or at least 10 contiguous nucleotides thereof.

17. The oligonucleotide of item 16 wherein the contiguous comprises a sequence selected from the group consisting of SEQ ID NO 21, SEQ ID NO 34, SEQ ID NO 38, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45 or at least 10 contiguous nucleotides thereof. 18. The oligonucleotide of item 17 wherein the contiguous comprises a sequence selected from the group consisting of SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45 or at least 10 contiguous nucleotides thereof.

19. The oligonucleotide of any one of items 1 to 18, comprising one or more modified nucleosides.

20. The oligonucleotide of item 19, wherein the one or more modified nucleoside is independently selected from 2'-O-methyl-RNA and LNA nucleosides.

21. The oligonucleotide of item 18 or item 19, wherein the oligonucleotide comprises 2 or 4 LNAs at the 5’ end and 2 or 4 LNAs at the 3’ end.

22. The oligonucleotide of any one of items 1 to 21 , wherein the oligonucleotide is capable of recruiting RNaseHI .

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

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

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

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

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

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

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

32. The oligonucleotide of any one of items 1 to 31 , wherein the 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 oligonucleotide of any one of items 1 to 32 and a pharmaceutically acceptable diluent, 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 vivo or in vitro method for upregulating or restoring GBA expression in a target cell, the method comprising exposing an oligonucleotide of any one of items 1 to 32 or a 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 either a human cell or a mammalian cell.

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

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

39. A method of treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide of any one of items 1 to 32 or a pharmaceutical composition of item 33 or item 34 to a subject suffering from or susceptible to the disease. 40. The oligonucleotide of any one of items 1 to 32 or a 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 oligonucleotide of any one of items 1 to 32 or a pharmaceutical composition of item 33or 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 oligonucleotide or 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 oligonucleotide or 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 oligonucleotide or pharmaceutical composition for use according to item 40 or the use according to item 41 , wherein the disease is Parkinson’s disease.

Claims

1. An oligonucleotide capable of increasing the expression of glucocerebrosidase (GBA) in a cell, wherein the oligonucleotide is 8 to 40 nucleotides in length and comprises a contiguous nucleotide sequence of 8 to 40 nucleotides in length, which is complementary to a human MTX1 P1 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 the human MTX1 P1 transcript.

3. The oligonucleotide of claim 1 or 2, wherein the human MTX1 P1 transcript is SEQ ID NO. 1.

4. The oligonucleotide of any one of claims 1 to 3, wherein the contiguous nucleotide sequence is complementary the 5’ region of the human MTX1 P1 transcript.

5. The oligonucleotide of claims 4, wherein the 5’ region of the human MTX1 P1 transcript is selected from the group consisting of SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138 and SEQ ID NO 139.

6. The oligonucleotide of any one of claims 1 to 5, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 122, SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 88, SEQ ID NO 101, SEQ ID NO 105, SEQ ID NO 109, SEQ ID NO 125, SEQ ID NO 80, SEQ ID NO 96 and SEQ ID NO 112, or a fragment thereof.

7. The oligonucleotide of any one of claims 1 to 6, wherein the contiguous nucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO 10, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 21 , SEQ ID NO 34, SEQ ID NO 38, SEQ ID NO 42, SEQ ID NO 58, SEQ ID NO 13, SEQ ID NO 29, SEQ ID NO 45, or at least 10 contiguous nucleotides thereof.

8. The oligonucleotide of any one of claims 1 to 7, wherein the oligonucleotide is capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50% in a cell,, compared to a control.

62

9. The oligonucleotide of any one of claims 1 to 8, wherein the oligonucleotide is covalently attached to at least one conjugate moiety.

10. The oligonucleotide of any of claims 1 to 9, wherein the oligonucleotide is in the form of a pharmaceutically acceptable salt.

11. An in vitro method for upregulating or restoring GBA expression in a target cell, the method comprising exposing an oligonucleotide of any one of claims 1 to 10 or a pharmaceutical composition thereof, in an effective amount, to said cell.

12. The oligonucleotide of any one of claims 1 to 10 or a pharmaceutical composition thereof for use as a medicament for the treatment or prevention of a disease in a subject.

13. The method of claim 11 or the oligonucleotide or pharmaceutical composition for use according to claim 12, wherein the disease is associated with reduced expression of GBA.

14. The method of claim 11 or the oligonucleotide or pharmaceutical composition for use according to claim 12, 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 method of claim 11 or the oligonucleotide or pharmaceutical composition for use according to claim 12, wherein the disease is Parkinson’s disease.

63