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WO2023111336A1 - Oligonucleotide gba agonists - Google Patents

Oligonucleotide gba agonists Download PDF

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Publication number
WO2023111336A1
WO2023111336A1 PCT/EP2022/086504 EP2022086504W WO2023111336A1 WO 2023111336 A1 WO2023111336 A1 WO 2023111336A1 EP 2022086504 W EP2022086504 W EP 2022086504W WO 2023111336 A1 WO2023111336 A1 WO 2023111336A1
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Prior art keywords
seq
gba
oligonucleotide
agonist
nucleotide sequence
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PCT/EP2022/086504
Other languages
French (fr)
Inventor
Johannes Braun
Ross CORDINER
Lukasz KIELPINSKI
Original Assignee
F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication date
Application filed by F. Hoffmann-La Roche Ag, Hoffmann-La Roche Inc. filed Critical F. Hoffmann-La Roche Ag
Priority to EP22840080.0A priority Critical patent/EP4448763A1/en
Priority to CN202280083385.5A priority patent/CN118489009A/en
Publication of WO2023111336A1 publication Critical patent/WO2023111336A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to oligonucleotides that upregulate or restore the expression of glucocerebrosidase (GBA) in cells; conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with reduced expression of GBA, including Gaucher’s disease and/or Parkinson’s disease.
  • GBA glucocerebrosidase
  • Glucocerebrosidase 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.
  • GBA Homozygous mutations in the gene encoding GBA cause Gaucher’s disease.
  • macrophages engulf and degrade cell debris.
  • Insufficient GBA activity results in the accumulation of glucocerebroside in the lysosomes of macrophages.
  • Affected macrophages known as ‘Gaucher cells’, build up in areas such as the spleen, liver and bone marrow.
  • Gaucher’s disease is characterized by bruising, fatigue, anaemia, low blood platelet count and enlargement of the liver and spleen.
  • the phenotype is variable, however three clinical forms have been identified: type 1 is the most common and typically causes no neurological damage, whereas types 2 and 3 are characterised by neurological impairment.
  • the condition is inherited in an autosomal recessive pattern. Over 300 variants of the GBA gene have been associated with the disease. Although genetics alone does not determine disease severity, certain mutations are known to cause more severe symptoms. For example, patients with two copies of the L444P mutation usually exhibit neuronopathic forms of the disease, whereas patients with one or two copies of the N370S allele are typically classified as type 1 (Scott et al., 2000, Genet. Med., 2, 65).
  • Parkinson 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.
  • 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.
  • ERT 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.
  • the invention provides oligonucleotide agonists of glucocerebrosidase (GBA) or oligonucleotide GBA agonists, i.e. oligonucleotides that are complementary to a GBA nucleic acid sequence.
  • GBA glucocerebrosidase
  • the invention provides oligonucleotide GBA agonists that target one or more of the GBA promoters. These oligonucleotides are capable of upregulating the expression of GBA.
  • the invention provides oligonucleotide positive modulators i.e. agonists) of GBA.
  • the oligonucleotides of the invention may be used to restore GBA expression in cells, or to enhance expression of GBA in cells.
  • the invention provides oligonucleotide GBA agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary, such as fully complementary, to one or more promoters of the human GBA genomic sequence.
  • the human GBA genomic sequence may be NCBI Reference Sequence: NG_009783.1.
  • the human GBA genomic sequence may be SEQ ID NO 226.
  • the promoter of the human GBA gene may comprise or consist of SEQ ID NO 225.
  • the promoter of the human GBA genomic sequence may be located immediately upstream of exon 1 of the human GBA gene. For example, between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
  • the promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO 224.
  • the promoter of human GBA genomic sequence may be located immediately upstream of exon -2 of the human GBA gene. For example, between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
  • the promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO 223.
  • the invention provides double stranded oligonucleotide GBA agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary, such as fully complementary, to one or more promoters of the human GBA genomic sequence.
  • the double stranded oligonucleotide GBA agonist may be a small activating RNA (saRNA).
  • the oligonucleotide GBA agonist may be or comprise an oligonucleotide mixmer or totalmer.
  • the oligonucleotide GBA agonist may increase the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
  • an increase in expression of GBA may be measured as an increase in GBA mRNA, an increase in expression of GBA protein or an increase in expression of both GBA mRNA and GBA protein.
  • the control may be a cell that has not been exposed to said oligonucleotide GBA agonist.
  • the oligonucleotide GBA agonist may be covalently attached to at least one conjugate moiety.
  • the oligonucleotide GBA agonist may be in the form of a pharmaceutically acceptable salt.
  • the oligonucleotide GBA agonist 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 a pharmaceutical composition
  • a pharmaceutical composition comprising an oligonucleotide GBA agonist and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the invention provides an in vivo or in vitro method for upregulating or restoring GBA expression in a target cell, the method comprising administering the oligonucleotide GBA agonist or pharmaceutical composition of the invention in an effective amount, to said cell.
  • the cell may be a human or mammalian cell.
  • the invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide GBA agonist or the pharmaceutical composition of the invention, to a subject suffering from or susceptible to a disease.
  • the invention provides the oligonucleotide GBA agonist or pharmaceutical composition of the invention, for use as a medicament in the treatment or prevention of a disease in a subject.
  • the invention provides the use of the oligonucleotide GBA agonist or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease in a subject.
  • the disease may be associated with reduced expression of GBA.
  • the disease may be Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.
  • Gaucher’s disease Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.
  • DLB dementia with Lewy bodies
  • REM rapid eye movements
  • Figure 1 shows GBA mRNA expression levels in SK-N-AS neuroblastoma cells at 48 hours post-transfection relative to a mock transfection control.
  • Figure 2 shows GBA mRNA expression levels in H4 neuroglioma cells at 48 hours posttransfection relative to a mock transfection control.
  • the inventors have identified that the expression level of the GBA mRNA transcript, and/or the expression level of encoded protein products, can be effectively enhanced by targeting one or more of the promotor regions of the GBA gene using oligonucleotides, particularly double stranded oligonucleotides such as short activating RNAs (saRNAs).
  • oligonucleotides particularly double stranded oligonucleotides such as short activating RNAs (saRNAs).
  • oligonucleotides of the invention are agonists of GBA, i.e. they increase production of GBA mRNA and/or protein.
  • the invention provides oligonucleotide glucocerebrosidase (GBA) agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary to one or more promoters of the human GBA genomic sequence, NCBI Reference Sequence NG_009783.1 (SEQ ID NO: 226).
  • GBA oligonucleotide glucocerebrosidase
  • the invention relates to oligonucleotide GBA agonists.
  • the oligonucleotides of the present invention are GBA agonists, i.e. they enhance the expression of GBA. This can mean an increase in the expression of GBA nucleic acids, such as GBA mRNA, and/or an increase in the expression of GBA protein. Enhanced GBA expression is desirable to treat, for example, Gaucher’s disease and/or Parkinson’s disease.
  • GBA agonist refers to a compound, in this case an oligonucleotide, which is capable of enhancing the expression of GBA mRNA transcripts and/or GBA protein in a cell, such as a cell which is expressing GBA.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA and protein by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of 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.
  • oligonucleotide as used herein is defined, as is generally understood by the skilled person, as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.
  • Oligonucleotides are commonly made in a laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.
  • the oligonucleotides of the invention are man-made, and are chemically synthesized, and are typically purified or isolated.
  • the oligonucleotides of the invention may comprise one or more modified nucleosides such as 2’ sugar modified nucleosides.
  • the oligonucleotides of the invention may comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.
  • the oligonucleotide GBA agonists of the invention are double stranded oligonucleotides.
  • the oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length. In some embodiments, the oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length and comprise a contiguous nucleotide sequence of 8-40 nucleotides.
  • the oligonucleotide GBA agonists of the invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • the oligonucleotide GBA agonists of the invention are at least 12 nucleotides in length.
  • the oligonucleotide GBA agonists of the invention are at least 14 nucleotides in length.
  • the oligonucleotide GBA agonists of the invention are at least 16 nucleotides in length.
  • the oligonucleotide GBA agonists of the invention are at least 18 nucleotides in length.
  • the oligonucleotide GBA agonists of the invention are 21 nucleotides in length.
  • the length measurement refers to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken as the length of the longest strand.
  • 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.
  • target nucleic acid which may be or may comprise an oligonucleotide motif sequence.
  • contiguous nucleobase 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 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.
  • all the nucleosides of the oligonucleotide constitute 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.
  • the contiguous nucleotide sequence is 8-40 nucleotides in length.
  • 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.
  • the contiguous nucleotide sequence is at least 12 nucleotides in length.
  • the contiguous nucleotide sequence is at least 14 nucleotides in length.
  • the contiguous nucleotide sequence is at least 16 nucleotides in length.
  • the contiguous nucleotide sequence is at least 18 nucleotides in length.
  • the contiguous nucleotide sequence is 19 nucleotides in length.
  • the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.
  • the oligonucleotide consists of the contiguous nucleotide sequence.
  • 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.
  • 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”.
  • the oligonucleotide GBA agonist of the invention may comprise one or more modified nucleosides.
  • 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.
  • one or more of the modified nucleosides of the oligonucleotides of the invention may comprise a modified sugar moiety.
  • 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 GBA agonists of the invention include LNA, 2’-O-MOE, 2’oMe and morpholino nucleoside analogues.
  • the oligonucleotide GBA agonist of the invention comprises one or more modified internucleoside linkage.
  • modified internucleoside linkage is defined as generally understood by the skilled person as linkages, other than phosphodiester (PO) linkages, that covalently couple two nucleosides together.
  • the oligonucleotide GBA agonists of the invention may therefore comprise one or more modified internucleoside linkages such as one or more phosphorothioate internucleoside linkages.
  • At least 50% of the internucleoside linkages in the oligonucleotide GBA agonist, or contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% or more of the internucleoside linkages in the oligonucleotide GBA agonist, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide GBA agonist, or contiguous nucleotide sequence thereof, are phosphorothioate.
  • the oligonucleotide GBA agonist 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.
  • all the internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide GBA agonist may be phosphorothioate, or all the internucleoside linkages of the oligonucleotide GBA agonist may be phosphorothioate linkages.
  • 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.
  • pyrimidine e.g. uracil, thymine and cytosine
  • nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but which are functional during nucleic acid hybridization.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al., 2012, Accounts of Chemical Research, 45, 2055-2065 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1-1.4.32.
  • 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.
  • a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromour
  • 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.
  • the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine.
  • 5-methyl cytosine LNA nucleosides may be used.
  • the oligonucleotide GBA agonist of the invention may be a modified oligonucleotide.
  • modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages.
  • chimeric oligonucleotide is a term that has been used in the literature to describe oligonucleotides comprising sugar modified nucleosides and DNA nucleosides.
  • the oligonucleotide GBA agonist 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.
  • contiguous nucleobase sequences can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid.
  • oligonucleotide design The pattern in which the modified nucleosides (such as high affinity modified nucleosides) are incorporated into the oligonucleotide sequence is generally termed oligonucleotide design.
  • the oligonucleotide GBA agonists of the invention are designed with modified nucleosides and DNA nucleosides.
  • modified nucleosides and DNA nucleosides are used.
  • high affinity modified nucleosides are used.
  • the oligonucleotide GBA agonist 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.
  • a high affinity modified nucleoside is a modified nucleoside which, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T m ).
  • a high affinity modified nucleoside of the present invention preferably results in an increase in melting temperature between +0.5 to +12°C, more preferably between +1.5 to +10°C and most preferably between +3 to +8°C per modified nucleoside.
  • Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann, Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).
  • the oligonucleotide GBA agonist of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
  • 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).
  • HNA hexose ring
  • LNA ribose ring
  • UNA unlinked ribose ring which typically lacks a bond between the 02 and 03 carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nu
  • 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.
  • 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.
  • the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide.
  • 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.
  • substituted sugar modified nucleosides does not include 2’ bridged nucleosides like LNA.
  • the oligonucleotide GBA agonist comprises one or more sugar modified nucleosides, such as 2’ sugar modified nucleosides.
  • the oligonucleotide GBA agonist of 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).
  • LNA locked nucleic acid
  • LNA nucleosides Locked Nucleic Acid Nucleosides
  • 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.
  • BNA bicyclic nucleic acid
  • the locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
  • Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181 , WO 2010/077578,
  • WO 2010/036698 WO 2007/090071 , WO 2009/006478, WO 2011/156202, WO 2008/154401 , WO 2009/067647, WO 2008/150729, Morita et a!., Bioorganic & Med.Chem. Lett., 12, 73-76, Seth et al., J. Org. Chem., 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry, 2016, 59, 9645-9667.
  • 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.
  • the oligonucleotide GBA agonist of the invention comprises or consists of morpholino nucleosides (/.e. is a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO)).
  • 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:
  • morpholino oligonucleotides of the invention may be, for example 8 - 40 morpholino nucleotides in length, such as morpholino 18 - 22 nucleotides in length.
  • the RNase H activity of an oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.
  • WO 01/23613 provides in vitro methods for determining RNase H activity, which may be used to determine the ability to recruit RNase H.
  • 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).
  • 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 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.
  • the oligonucleotide GBA agonists of the invention are not RNase H recruiting gapmer oligonucleotide.
  • RNase H recruitment may be avoided by limiting the number of contiguous DNA nucleotides in the oligonucleotide - therefore mixmer and totalmer designs may be used.
  • the oligonucleotide GBA agonists of the invention, or the contiguous nucleotide sequence thereof do not comprise more than 3 contiguous DNA nucleosides. Further, advantageously the oligonucleotide GBA agonists of the invention, or the contiguous nucleotide sequence thereof, do not comprise more than 4 contiguous DNA nucleosides. Further, advantageously, the oligonucleotide GBA agonists of the invention, or contiguous nucleotide sequence thereof, do not comprise more than 2 contiguous DNA nucleosides.
  • RNase H activity of oligonucleotides may be achieved by designing oligonucleotides which do not comprise a region of more than 3 or more than 4 contiguous DNA nucleosides. This may be achieved by using oligonucleotides or contiguous nucleoside regions thereof with a mixmer design, which comprise sugar modified nucleosides, such as 2’ sugar modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides.
  • nucleosides alternate between 1 LNA and 1 DNA nucleoside, e.g. LDLDLDLDLDLDLDLL, with 5’ and 3’ terminal LNA nucleosides, and every third design, such as LDDLDDLDDLDDLDDL, where every third nucleoside is a LNA nucleoside.
  • a totalmer is an oligonucleotide or a contiguous nucleotide sequence thereof which does not comprise DNA or RNA nucleosides, and may for example comprise only 2’-O-MOE nucleosides, such as a fully MOE phosphorothioate, e.g.
  • the internucleoside nucleosides in mixmers and totalmers may be phosphorothioate, or a majority of nucleoside linkages in mixmers may be phosphorothioate.
  • Mixmers and totalmers may comprise other internucleoside linkages, such as phosphodiester or phosphorodithioate, by way of example.
  • the oligonucleotide GBA agonists are, or comprise, an oligonucleotide mixmer or totalmer.
  • the contiguous nucleotide sequence is a mixmer or a tolalmer.
  • the oligonucleotide GBA agonists of the invention target one or more of the promoters of the human GBA genomic sequence. This may also be referred to herein as the GBA gene.
  • the GNA genomic sequence may be referred to as a target sequence.
  • the target sequence may also be referred to as a target nucleic acid or target site sequence.
  • genomic sequence encompasses both protein coding and non-protein coding sequences. It is understood that such sequences include transcribed and untranscribed sequences, and translated and untranslated sequences. Non-protein coding sequences may comprise regulatory sequences such as enhancers, silencers, promoters, and/or 3’ and 5’ untranslated regions (UTR).
  • GBA nucleic acid sequence may also refer to nucleic acid sequences of the GBA gene in the sense of the definition outlined herein.
  • the target nucleic acid sequences of the GBA gene may refer to sequences as present within the genomic DNA or the same, or antisense sequences, present in a cell in any other form, such as mRNA, or other single or double stranded RNAs, such as miRNAs or siRNAs.
  • genes and their corresponding nucleotide sequences as used herein is not intended to be necessarily limited to either one of the sense or antisense strands thereof. Accordingly, both or either of the sense and antisense sequences may be encompassed.
  • the human GBA gene has the sequence of NCBI Reference NG_009783.1.
  • the human GBA gene comprises a sequence defined by SEQ ID NO 226.
  • SEQ ID NO 226 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 226, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
  • the human GBA genomic sequence consists of SEQ ID NO 226.
  • the oligonucleotide GBA agonists of the invention target one or more promoter regions of the GBA genomic sequence.
  • the promoter of the human GBA gene comprises the intergenic region from FAM189B to GBA translational start codon (SEQ ID NO 225).
  • SEQ ID NO 225 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 225, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
  • the promoter of the human GBA gene comprises or consists of SEQ ID NO 225.
  • SEQ ID NO 225 encompasses Promoters 1 (SEQ ID NO 224) and 2 (SEQ ID NO 223), as discussed below.
  • the oligonucleotide GBA agonist of the invention targets the promoter located immediately upstream of exon 1 of the GBA gene (Promoter 1 , P1). For example, between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
  • the ATG initiation codon corresponds to position 8586 - 8588 of NCBI Reference sequence NG_009783.1 and/or SEQ ID NO: 226.
  • the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 7449 - 8585 of NCBI Reference sequence NG_009783.1 (SEQ ID NO 224). Put another way, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 7449 - 8585 of SEQ ID NO 226 (SEQ ID NO 224).
  • the promoter of the human GBA gene comprises SEQ ID NO 224.
  • the promoter of the human GBA gene consists of SEQ ID NO 224.
  • the target sequence is a sequence of nucleotides within SEQ ID NO 224.
  • SEQ ID NO 224 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 224, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 35 to 55, 36 to 56, 39 to 59, 46 to 66, 68 to 88, 106 to 126, 148 to 168, 152 to 172, 153 to 173, 302 to 322, 313 to 333, 314 to 334, 371 to 391 , 372 to 392, 475 to 495, 517 to 537, 532 to 552, 598 to 618, 656 to 676, 657 to 677, 746 to 766, 747 to 767, 748 to 768, 749 to 769, 750 to 770, 753 to 773, 815 to 835, 816 to 836, 839 to 859, 840 to 860, 884 to 904, 891 to 911 , 904 to 924, 922 to 942, 923 to 943, 996 to 1016
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 39 to 59, 148 to 168, 153 to 173, 302 to 322, 313 to 333, 517 to 537, 532 to 552, 656 to 676, 748 to 768, 750 to 770, 815 to 835, 840 to 860, 904 to 924 and 996 to 1016 of SEQ ID NO 224.
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 748 to 768, 750 to 770, 815 to 835, 904 to 924 and 996 to 1016 of SEQ ID NO 224.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, and SEQ ID NO 40, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 30, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 3, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 7, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 19, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 23, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 25, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 27, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 30, or a fragment thereof. In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 33, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 36, or a fragment thereof.
  • the oligonucleotide GBA agonist of the invention targets the promoter located immediately upstream of exon -2 of the GBA gene (Promoter 2, P2). For example, between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
  • the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 4028 - 5192 of NCBI Reference sequence NG_009783.1 (SEQ ID NO 223). Put another way, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 4028 - 5192 of SEQ ID NO 226 (SEQ ID NO 223).
  • the promoter of the human GBA gene comprises SEQ ID NO 223.
  • the promoter of the human GBA gene consists of SEQ ID NO 223.
  • the target sequence is a sequence of nucleotides within SEQ ID NO 223.
  • SEQ ID NO 223 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 223, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 13 to 33, 34 to 54, 38 to 58, 121 to 141 , 159 to 179, 160 to 180, 173 to 193, 199 to 219, 200 to 220, 313 to 333, 319 to 339, 335 to 355, 336 to 356, 359 to 379, 391 to 411 , 422 to 442, 423 to 443, 454 to 474, 469 to 489, 535 to 555, 548 to 568, 551 to 571 , 589 to 609, 590 to 610, 654 to 674, 669 to 689, 670 to 690, 676 to 696, 685 to 705, 744 to 764, 761 to 781 , 762 to 782, 913 to 933, and 922 to 942 of SEQ ID NO 223.
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121 to 141 , 160 to 180, 173 to 193, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 391 to 411, 423 to 443, 469 to 489, 535 to 555, 548 to 568, 551 to 571, 589 to 609 and 670 to 690 of SEQ ID NO 223.
  • the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121 to 141 , 160 to 180, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 423 to 443, 469 to 489, 548 to 568, 551 to 571 and 670 to 690 of SEQ ID NO 223.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 73, and SEQ ID NO 74, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63 and SEQ ID NO 67, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 62, and SEQ ID NO 67, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 44, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 49, or a fragment thereof. In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 57, or a fragment thereof.
  • the contiguous nucleotide sequence is complementary to SEQ ID NO 67, or a fragment thereof.
  • a fragment of the target sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.
  • the contiguous nucleotide sequence is complementary to at least 8 contiguous nucleotides of any of the target sequences recited herein.
  • the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of any of the target sequences recited herein. In some embodiments the contiguous nucleotide sequence is complementary to at least 12 contiguous nucleotides of any of the target sequences recited herein.
  • the contiguous nucleotide sequence is complementary to at least 14 contiguous nucleotides of any of the target sequences recited herein.
  • the contiguous nucleotide sequence is complementary to at least 16 contiguous nucleotides of any of the target sequences recited herein.
  • the contiguous nucleotide sequence is complementary to at least 18 contiguous nucleotides of any of the target sequences recited herein.
  • the contiguous nucleotide sequence is complementary to at least 19 contiguous nucleotides of any of the target sequences recited herein.
  • Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (II).
  • oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).
  • % complementary 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.
  • nucleobase/nucleotide which does not align 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).
  • the oligonucleotide GBA agonist is a double stranded oligonucleotide, such as an saRNA.
  • the double stranded oligonucleotide may have a nucleotide overhang, such as a 2 nucleotide overhang which may be at the 3’ end of the contiguous nucleotide sequence.
  • complementarity is defined based upon the double stranded sequence without the overhang. For example, if the oligonucleotide is 21 nucleotides in length and includes a 2 nucleotide overhang, complementarity is determined based upon the 19 nucleotides without the two nucleotide overhang.
  • the term “complementary” requires the contiguous nucleotide sequence to be at least about 75% complementary, or at least about 80% complementary, or at least about 85% complementary, or at least about 90% complementary, or at least about 95% complementary, to the target sequence, i.e. one or more promoters of the human GBA gene.
  • the oligonucleotide GBA agonist may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary the target sequence, i.e. one or more promoters of the human GBA gene.
  • the contiguous nucleotide sequence within an oligonucleotide GBA agonist of the invention may include one, two, three or more mis-matches, wherein a mis-match is a nucleotide within the contiguous nucleotide sequence which does not base pair with its target.
  • the contiguous nucleotide sequence is fully complementary to the target sequence.
  • identity 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.
  • 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).
  • hybridizing or “hybridizes” as used herein are to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
  • the affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T m ) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515- 537).
  • 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.
  • ITC isothermal titration calorimetry
  • oligonucleotide GBA agonists of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10- 30 nucleotides in length.
  • the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°.
  • the oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.
  • the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal, or- 16 to -27 kcal such as -18 to -25 kcal.
  • Double stranded oligonucleotide GBA agonists Double stranded oligonucleotide GBA agonists
  • the oligonucleotide GBA agonist is a double stranded oligonucleotide.
  • the double stranded oligonucleotide GBA agonist is a short activating RNA (saRNAs).
  • saRNA short activating RNA
  • saRNAs are capable of inducing gene activation by a process known as RNA activation (RNAa), wherein gene activation is induced by hybridization of the saRNA with a target nucleic acid sequence.
  • RNAa RNA activation
  • Said target nucleic acid sequences typically comprise promoter regions of a gene.
  • a known mechanism of transcriptional upregulation by saRNAs involves Ago2, and is associated with epigenetic modification at target sites, e.g. promotors.
  • Ago2 associates with an saRNA, which guides the complex to a target and facilitates the assembly of an RNA- induced transcriptional activation (RITA) complex.
  • RITA-RNA polymerase II interactions are thought to promote transcription initiation and productive elongation, as well as monoubiquitination of histone 2B (Portnoy et al., Cell Res., 2016, 26(3), 320-335).
  • the oligonucleotide GBA agonist of the invention such as an saRNA, is 19 nucleotides in length.
  • the oligonucleotide GBA agonist of the invention such as an saRNA is 20 nucleotides in length.
  • the oligonucleotide GBA agonist of the invention such as an saRNA, is 21 nucleotides in length.
  • the oligonucleotide GBA agonist of the invention such as an saRNA, is 22 nucleotides in length.
  • the length measurement refers to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken as the length of the longest strand.
  • the oligonucleotide GBA agonist of the invention may have a nucleotide overhang.
  • the nucleotide overhang may be a 2 nucleotide overhang.
  • the overhang may be at the 3’ end of the contiguous nucleotide sequence.
  • the overhang may comprise or consist of two thymine nucleotides (TT).
  • the oligonucleotide GBA agonist of the invention may target a sequence within Promoter 1 of the human GBA genomic sequence.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 100, S
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 77, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 81 , or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 83, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 84, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 85, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 90, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 91, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 93, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 97, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 99, or at least 10 contiguous nucleotides thereof. In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 101 , or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 104, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 107, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 149, S
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 151, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 178, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 151 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 155, or at least 10 contiguous nucleotides thereof. In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 157, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 158, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 159, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 164, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 165, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 167, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 171 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 173, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 175, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 178, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 181 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
  • the oligonucleotide GBA agonist of the invention may target a sequence within Promoter 2 of the human GBA genomic sequence.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 141, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 135, SEQ ID NO 136 and SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 118, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 120, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 121 , or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 122, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 123, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 126, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 127, or at least 10 contiguous nucleotides thereof. In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 128, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 129, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 131 , or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 133, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 134, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 135, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 136, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 137, or at least 10 contiguous nucleotides thereof.
  • the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191 , SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 194, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO 200, SEQ ID NO 201 , SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ ID NO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 , SEQ ID NO 212, SEQ ID NO 213, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219, SEQ ID NO 2
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO 201 , SEQ ID NO 202, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 209, SEQ ID NO 210 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 192, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 194, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 195, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 196, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 197, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 200, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 201 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 202, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 203, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 205, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 207, or at least 10 contiguous nucleotides thereof. In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 208, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 209, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 210, or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 211 , or at least 10 contiguous nucleotides thereof.
  • the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
  • the contiguous nucleotide sequence may be a fragment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of any of the sequences recited herein.
  • double stranded oligonucleotides such as saRNAs
  • oligonucleotide sequences such as oligonucleotide sequences of endogenous RNAs or DNA.
  • a double stranded oligonucleotide of the invention may act through binding to the sense strand, the antisense strand or both strands of the target sequence.
  • the oligonucleotide GBA agonist of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as a mixmer or toalmer region, and further 5’ and/or 3’ nucleosides.
  • the further 5’ and/or 3’ nucleosides may or may not be complementary, such as fully complementary, to the target nucleic acid.
  • Such further 5’ and/or 3’ nucleosides may be referred to as region D’ and D” herein.
  • region D’ or D may be used for the purpose of joining the contiguous nucleotide sequence, such as the mixmer or totoalmer, to a conjugate moiety or another functional group.
  • region D’ or D may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid.
  • the nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these.
  • the D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers).
  • the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA.
  • Nucleotide based biocleavable linkers suitable for use as region D’ or D” are disclosed in WO 2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide.
  • the use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO 2015/113922, where they are used to link multiple antisense constructs within a single oligonucleotide.
  • the oligonucleotide GBA agonist of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes a mixmer or a total mer.
  • the internucleoside linkage positioned between region D’ or D” and the mixmer or totalmer region is a phosphodiester linkage.
  • the invention encompasses an oligonucleotide GBA agonist covalently attached to at least one conjugate moiety. In some embodiments this may be referred to as a conjugate of the invention.
  • the invention provides oligonucleotide GBA agonists covalently attached to at least one conjugate moiety.
  • conjugate refers to an oligonucleotide GBA agonist which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).
  • the conjugate moiety may be covalently linked to the oligonucleotide, optionally via a linker group, such as region D’ or D”.
  • Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.
  • the non-nucleotide 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.
  • 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 GBA agonist 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).
  • the conjugate or oligonucleotide GBA agonist 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).
  • a linker region second region or region B and/or region Y
  • 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 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.
  • the biocleavable linker is susceptible to S1 nuclease cleavage.
  • 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 GBA agonist 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.
  • 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 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 GBA agonists according to the invention, or the conjugate according to the invention.
  • the invention provides for oligonucleotide GBA agonists according to the invention wherein the oligonucleotide GBA agonists are in the form of a pharmaceutically acceptable salt.
  • 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 GBA agonist according to the invention, or the conjugate according to the invention.
  • the invention provides for a pharmaceutically acceptable potassium salt of the oligonucleotide GBA agonist according to the invention, or the conjugate according to the invention.
  • the invention provides for oligonucleotide GBA agonists according to the invention wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.
  • This may be for the purpose of delivering the oligonucleotide GBA agonist of the invention to the targeted cells and/or to improve the pharmacokinetics of the oligonucleotide GBA agonist.
  • lipid-based delivery vehicles examples include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.
  • the invention provides for a pharmaceutical composition comprising the oligonucleotide GBA agonist of the invention, or the conjugate or salt of the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the invention provides for a pharmaceutical composition comprising the oligonucleotide GBA agonists of the invention, or the conjugate of the invention, and a pharmaceutically acceptable salt.
  • the salt may comprise a metal cation, such as a sodium salt or a potassium salt.
  • the invention provides for a pharmaceutical composition according to the invention, wherein the pharmaceutical composition comprises the oligonucleotide GBA agonist of the invention or the conjugate of the invention, or the pharmaceutically acceptable salt 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 GBA agonist of the invention, or the conjugate of the invention, or the pharmaceutically acceptable salt of the invention.
  • a solution such as a phosphate buffered saline solution of the oligonucleotide GBA agonist of the invention, or the conjugate of the invention, or the pharmaceutically acceptable salt of the invention.
  • the solution, such as phosphate buffered saline solution, of the invention is a sterile solution.
  • the invention provides for a method for enhancing, upregulating or restoring the expression of GBA in a cell, such as a cell which is expressing GBA, said method comprising administering an oligonucleotide GBA agonist of the invention or a conjugate of the invention, or a salt of the invention, or the pharmaceutical composition of the invention in an effective amount to said cell.
  • the method is an in vitro method.
  • the method is an in vivo method.
  • the cell is either a human cell or a mammalian cell.
  • the cell is part of, or derived from, a subject suffering from or susceptible to a disease associated with reduced expression of GBA.
  • 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 refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment, as referred to herein may in some embodiments be prophylactic.
  • the invention provides for a method for treating or preventing a disease associated with reduced expression of GBA, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide GBA agonist 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.
  • 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.
  • the disease is Parkinson’s disease.
  • the disease is Gaucher’s disease.
  • 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 GBA agonist of the invention for use as a medicament.
  • the invention provides for an oligonucleotide GBA agonist of the invention for the preparation of a medicament.
  • the invention provides for an oligonucleotide GBA agonist of the invention for use in therapy.
  • the invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for use as a medicament.
  • the invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament.
  • the invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition according to the invention for use in therapy.
  • the invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use as in the treatment of Parkinson’s disease.
  • the invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use as in the treatment of Gaucher’s disease.
  • the invention provides for the use of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Parkinson’s disease.
  • the invention provides for the use of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Gaucher’s disease.
  • the oligonucleotide GBA agonist 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, intra-muscular, intracerebral, intracerebroventricular or intrathecal).
  • the oligonucleotide or pharmaceutical composition of the invention is administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular, administration.
  • a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular, administration.
  • the oligonucleotide is administered intracerebrally or intracerebroventricularly.
  • the oligonucleotide 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.
  • the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA and GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA mRNA and GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
  • the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein compared to a control.
  • the control may be a cell that has not been exposed to the oligonucleotide.
  • the control may be a mock transfection, for example, treatment of cells with PBS. Table 1. saRNA sequences of the invention and their target sequences
  • EXAMPLE 1 EFFECT OF saRNAs ON GBA mRNA
  • SK-N-AS neuroblastoma cells were plated to a density of 25000 per well in 96 well plates in full growth medium (DMEM (Sigma: D6546), 10% FBS, 2mM glutamine, 0.1 mM (1x) NEAA, 25pg/ml Gentamicin).
  • DMEM full growth medium
  • FBS FBS
  • 2mM glutamine 1x
  • NEAA 25pg/ml Gentamicin
  • mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water. 4 pL was used as input for one-step RT-qPCR analysis according to protocol in Table 3. (qScriptTM XLT One-Step RT-qPCR ToughMix®,
  • EXAMPLE 2 EFFECT OF saRNAs ON GBA mRNA
  • H4 neuroglioma cells were plated to a density of 15000 per well in 96 well plates in full growth medium (DMEM Sigma: D0819, 15% FBS, 1 mM Sodium Pyruvate, 25 pg/ml Gentamicin).
  • DMEM Sigma D0819, 15% FBS, 1 mM Sodium Pyruvate, 25 pg/ml Gentamicin.
  • mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water.
  • oligonucleotide glucocerebrosidase (GBA) agonist wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary to one or more promoters of the human GBA genomic sequence.
  • the oligonucleotide GBA agonist of item 1 wherein the contiguous nucleotide sequence is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
  • the oligonucleotide GBA agonist of item 2 wherein the contiguous nucleotide sequence is 19 nucleotides in length.
  • oligonucleotide GBA agonist of any one of items 1 to 3, wherein the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.
  • oligonucleotide GBA agonist of any one of items 1 to 4, wherein the contiguous nucleotide sequence is at least 75% complementary to one or more promoters of the human GBA genomic sequence.
  • oligonucleotide GBA agonist of item 5 wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary to one or more promoters of the human GBA genomic sequence.
  • oligonucleotide GBA agonist of any one of items 1 to 6, wherein the contiguous nucleotide sequence is fully complementary to one or more promoters of the human GBA genomic sequence.
  • oligonucleotide GBA agonist of any one of items 1 to 7, wherein the human GBA genomic sequence is SEQ ID NO 226.
  • oligonucleotide GBA agonist of any one of items 1 to 8, wherein the promoter of the human GBA gene comprises SEQ ID NO 225.
  • oligonucleotide GBA agonist of item 9 wherein the promoter of the human GBA gene consists of SEQ ID NO 225.
  • oligonucleotide GBA agonist of any one of items 1 to 10, wherein the promoter is located immediately upstream of exon 1 of the human GBA gene (P1).
  • oligonucleotide GBA agonist of item 11 wherein the promoter is located between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
  • oligonucleotide GBA agonist of any one of items 17 to 19, wherein the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 151, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 171 , SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 178, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
  • the oligonucleotide GBA agonist of item 27 or item 28 wherein the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 131 , SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
  • oligonucleotide GBA agonist of any one of items 1 to 31 , wherein the oligonucleotide is capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
  • oligonucleotide GBA agonist of any one of items 1 to 33, wherein the oligonucleotide GBA agonist is covalently attached to at least one conjugate moiety.
  • oligonucleotide GBA agonist of any one of items 1 to 34, wherein the oligonucleotide GBA agonist is in the form of a pharmaceutically acceptable salt.
  • oligonucleotide GBA agonist of any one of items 1 to 36, wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.
  • a pharmaceutical composition comprising the oligonucleotide GBA agonist of any one of items 1 to 37 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • composition 39 The pharmaceutical composition of item 38, wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.
  • An in vivo or in vitro method for upregulating or restoring GBA expression in a target cell comprising administering an oligonucleotide GBA agonist of any one of items 1 to 37 or a pharmaceutical composition of item 38 or item 39, in an effective amount, to said cell.
  • control is a cell that has not been exposed to said oligonucleotide GBA agonist.
  • a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide GBA agonist of any of items 1 to 37, or the pharmaceutical composition of item 38 or item 39, to a subject suffering from or susceptible to a disease.
  • oligonucleotide of any one of items 1 to 37 or the pharmaceutical composition of item 38 or item 39 for the preparation of a medicament for treatment or prevention of a disease in a subject.

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Abstract

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

Description

OLIGONUCLEOTIDE GBA AGONISTS
FIELD OF THE INVENTION
The present invention relates to oligonucleotides that upregulate or restore the expression of glucocerebrosidase (GBA) in cells; conjugates, salts and pharmaceutical compositions thereof; and methods for treatment of diseases associated with reduced expression of GBA, including Gaucher’s disease and/or Parkinson’s disease.
BACKGROUND
Glucocerebrosidase (GBA) is a lysosomal enzyme that catalyses the hydrolysis of glucocerebroside (also known as glucosylceramide). Glucocerebroside is a normal component of cell membranes, in particular of red and white blood cells.
Homozygous mutations in the gene encoding GBA cause Gaucher’s disease. During routine cell turnover, macrophages engulf and degrade cell debris. Insufficient GBA activity results in the accumulation of glucocerebroside in the lysosomes of macrophages. Affected macrophages, known as ‘Gaucher cells’, build up in areas such as the spleen, liver and bone marrow.
Gaucher’s disease is characterized by bruising, fatigue, anaemia, low blood platelet count and enlargement of the liver and spleen. The phenotype is variable, however three clinical forms have been identified: type 1 is the most common and typically causes no neurological damage, whereas types 2 and 3 are characterised by neurological impairment.
The condition is inherited in an autosomal recessive pattern. Over 300 variants of the GBA gene have been associated with the disease. Although genetics alone does not determine disease severity, certain mutations are known to cause more severe symptoms. For example, patients with two copies of the L444P mutation usually exhibit neuronopathic forms of the disease, whereas patients with one or two copies of the N370S allele are typically classified as type 1 (Scott et al., 2000, Genet. Med., 2, 65).
Mutations in the GBA gene have also been linked to Parkinson's disease and dementia with Lewy bodies (Riboldi and Di Fonzo, 2019, Cells, 8, 364). Parkinson’s disease is a neurodegenerative disorder of the central nervous system characterised by a wide range of motor and non-motor symptoms. Motor symptoms include bradykinesia (slowness of movement), rigidity, and postural instability. Non-motor symptoms, which may precede motor symptoms by many years, include olfactory loss, rapid eye movement sleep behaviour disorders, dysautonomia, and depression.
Heterozygous mutations of the GBA gene occur in around 8 to 12% of patients with Parkinson’s disease. As with Gaucher’s disease, mutation severity can influence the disease phenotype. For example, the risk for dementia in patients carrying “severe” mutations (such as L444P) is 2- to 3-fold higher than in those carrying “mild” mutations (such as N370S). E326K is the most prevalent GBA mutation in Parkinson’s disease, and patients bearing this mutation show a faster progression of motor symptoms (Avenali et al., 2020, Front. Aging Neurosci.). Current treatments for diseases associated with reduced GBA expression include enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). ERT involves the intravenous administration of recombinant GBA. While most patients respond well to treatment, there is a risk of developing an immune response. Furthermore, GBA is not able to cross the blood-brain barrier and therefore ERT is considered ineffective for patients with Parkinson’s disease or neuronopathic forms of Gaucher’s disease. SRT provides an alternative (or supplementary) treatment for patients who cannot tolerate ERT, or for whom intravenous administration is problematic. SRT works to reduce the build up of glucocerebroside in the lysosome by inhibiting enzymes in the glucocerebroside synthesis pathway. This therapy has a higher incidence of adverse effects than ERT, and long-term reduction of glucocerebroside can affect several different cell functions. Both ERT and SRT are costly and must be continued for life.
There is thus an urgent need for therapeutic agents that can increase or restore the expression of GBA.
SUMMARY OF THE INVENTION
The invention provides oligonucleotide agonists of glucocerebrosidase (GBA) or oligonucleotide GBA agonists, i.e. oligonucleotides that are complementary to a GBA nucleic acid sequence. Specifically, the invention provides oligonucleotide GBA agonists that target one or more of the GBA promoters. These oligonucleotides are capable of upregulating the expression of GBA. Alternatively stated, the invention provides oligonucleotide positive modulators i.e. agonists) of GBA.
The oligonucleotides of the invention may be used to restore GBA expression in cells, or to enhance expression of GBA in cells.
The invention provides oligonucleotide GBA agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary, such as fully complementary, to one or more promoters of the human GBA genomic sequence.
The human GBA genomic sequence may be NCBI Reference Sequence: NG_009783.1.
The human GBA genomic sequence may be SEQ ID NO 226.
The promoter of the human GBA gene may comprise or consist of SEQ ID NO 225.
The promoter of the human GBA genomic sequence may be located immediately upstream of exon 1 of the human GBA gene. For example, between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
The promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO 224.
The promoter of human GBA genomic sequence may be located immediately upstream of exon -2 of the human GBA gene. For example, between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
The promoter of the human GBA genomic sequence may comprise or consist of SEQ ID NO 223.
In some embodiments, the invention provides double stranded oligonucleotide GBA agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary, such as fully complementary, to one or more promoters of the human GBA genomic sequence.
The double stranded oligonucleotide GBA agonist may be a small activating RNA (saRNA).
The oligonucleotide GBA agonist may be or comprise an oligonucleotide mixmer or totalmer.
The oligonucleotide GBA agonist may increase the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control. Herein an increase in expression of GBA may be measured as an increase in GBA mRNA, an increase in expression of GBA protein or an increase in expression of both GBA mRNA and GBA protein.
The control may be a cell that has not been exposed to said oligonucleotide GBA agonist.
The oligonucleotide GBA agonist may be covalently attached to at least one conjugate moiety. The oligonucleotide GBA agonist may be in the form of a pharmaceutically acceptable salt.
The oligonucleotide GBA agonist 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 a pharmaceutical composition comprising an oligonucleotide GBA agonist and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
The invention provides an in vivo or in vitro method for upregulating or restoring GBA expression in a target cell, the method comprising administering the oligonucleotide GBA agonist or pharmaceutical composition of the invention in an effective amount, to said cell.
The cell may be a human or mammalian cell.
The invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide GBA agonist or the pharmaceutical composition of the invention, to a subject suffering from or susceptible to a disease.
The invention provides the oligonucleotide GBA agonist or pharmaceutical composition of the invention, for use as a medicament in the treatment or prevention of a disease in a subject.
The invention provides the use of the oligonucleotide GBA agonist or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease in a subject.
The disease may be associated with reduced expression of GBA.
The disease may be Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.
These and other aspects and embodiments of the invention will be described in further detail below.
BRIEF DESCRIPTION OF FIGURES
Figure 1 shows GBA mRNA expression levels in SK-N-AS neuroblastoma cells at 48 hours post-transfection relative to a mock transfection control.
Figure 2 shows GBA mRNA expression levels in H4 neuroglioma cells at 48 hours posttransfection relative to a mock transfection control. DETAILED DESCRIPTION OF THE INVENTION
The inventors have identified that the expression level of the GBA mRNA transcript, and/or the expression level of encoded protein products, can be effectively enhanced by targeting one or more of the promotor regions of the GBA gene using oligonucleotides, particularly double stranded oligonucleotides such as short activating RNAs (saRNAs).
Described herein are target sites present on the human GBA gene, particularly within the promoter regions, which can be targeted by the oligonucleotides of the invention. The oligonucleotides of the invention are agonists of GBA, i.e. they increase production of GBA mRNA and/or protein.
The invention provides oligonucleotide glucocerebrosidase (GBA) agonists, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary to one or more promoters of the human GBA genomic sequence, NCBI Reference Sequence NG_009783.1 (SEQ ID NO: 226).
GBA agonist
The invention relates to oligonucleotide GBA agonists.
The oligonucleotides of the present invention are GBA agonists, i.e. they enhance the expression of GBA. This can mean an increase in the expression of GBA nucleic acids, such as GBA mRNA, and/or an increase in the expression of GBA protein. Enhanced GBA expression is desirable to treat, for example, Gaucher’s disease and/or Parkinson’s disease.
The term “GBA agonist” as used herein refers to a compound, in this case an oligonucleotide, which is capable of enhancing the expression of GBA mRNA transcripts and/or GBA protein in a cell, such as a cell which is expressing GBA.
In certain embodiments, the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more.
In certain embodiments, the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more.
In certain embodiments, the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA and protein by at least about 10%. In other embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of 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.
Oligonucleotide
The term “oligonucleotide” as used herein is defined, as is generally understood by the skilled person, as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers.
Oligonucleotides are commonly made in a laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotides of the invention are man-made, and are chemically synthesized, and are typically purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides such as 2’ sugar modified nucleosides. The oligonucleotides of the invention may comprise one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.
In some embodiments, the oligonucleotide GBA agonists of the invention are double stranded oligonucleotides.
In some embodiments, the oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length. In some embodiments, the oligonucleotide GBA agonists of the invention are 8-40 nucleotides in length and comprise a contiguous nucleotide sequence of 8-40 nucleotides.
In some embodiments, the oligonucleotide GBA agonists of the invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
In some embodiments the oligonucleotide GBA agonists of the invention are at least 12 nucleotides in length.
In some embodiments the oligonucleotide GBA agonists of the invention are at least 14 nucleotides in length.
In some embodiments the oligonucleotide GBA agonists of the invention are at least 16 nucleotides in length.
In some embodiments the oligonucleotide GBA agonists of the invention are at least 18 nucleotides in length.
In some embodiments, the oligonucleotide GBA agonists of the invention are 21 nucleotides in length.
It will be understood that when discussing embodiments including a double stranded oligonucleotide the length measurement refers to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken as the length of the longest strand.
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”.
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.
In some embodiments all the nucleosides of the oligonucleotide constitute 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 8-40 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 some embodiments, the contiguous nucleotide sequence is 19 nucleotides in length.
In some embodiments, the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.
In some embodiments the oligonucleotide consists of the contiguous nucleotide sequence.
In some embodiments the oligonucleotide 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
Advantageously, the oligonucleotide GBA agonist of the invention may comprise one or more modified nucleosides.
The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.
Advantageously, one or more of the modified nucleosides of the oligonucleotides of the invention may comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing. Exemplary modified nucleosides which may be used in the oligonucleotide GBA agonists of the invention include LNA, 2’-O-MOE, 2’oMe and morpholino nucleoside analogues.
Modified internucleoside linkage
Advantageously, the oligonucleotide GBA agonist of the invention comprises one or more modified internucleoside linkage.
The term “modified internucleoside linkage” is defined as generally understood by the skilled person as linkages, other than phosphodiester (PO) linkages, that covalently couple two nucleosides together. The oligonucleotide GBA agonists 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 GBA agonist, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% or more of the internucleoside linkages in the oligonucleotide GBA agonist, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide GBA agonist, or contiguous nucleotide sequence thereof, are phosphorothioate.
In a further embodiment, the oligonucleotide GBA agonist 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 GBA agonist may be phosphorothioate, or all the internucleoside linkages of the oligonucleotide GBA agonist may be phosphorothioate linkages.
Nucleobase
The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but which are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al., 2012, Accounts of Chemical Research, 45, 2055-2065 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1-1.4.32.
In some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5- thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or II, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used.
Modified oligonucleotide The oligonucleotide GBA agonist of 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 GBA agonist of the invention to be a chimeric oligonucleotide.
In some embodiments, the oligonucleotide GBA agonist or contiguous nucleotide sequence thereof may include modified nucleobases which function as the shown nucleobase in base pairing, for example 5-methyl cytosine may be used in place of methyl cytosine. Inosine may be used as a universal base.
It is understood that the contiguous nucleobase sequences (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity to the target nucleic acid.
The pattern in which the modified nucleosides (such as high affinity modified nucleosides) are incorporated into the oligonucleotide sequence is generally termed oligonucleotide design.
The oligonucleotide GBA agonists of the invention are designed with modified nucleosides and DNA nucleosides. Advantageously, high affinity modified nucleosides are used.
In an embodiment, the oligonucleotide GBA agonist 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 an 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), 293-213).
Sugar modifications
The oligonucleotide GBA agonist of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the 02 and 03 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.
2’ sugar modified nucleosides
A 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradicle capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’- 4’ biradicle bridged) nucleosides.
Indeed, much focus has been spent on developing 2’ sugar substituted nucleosides, and numerous 2’ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2’ substituted modified nucleosides are 2’-O-alkyl-RNA, 2’-O-methyl-RNA (2’oMe). 2’-alkoxy-RNA, 2’-O-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2' substituted modified nucleosides.
Figure imgf000014_0001
In relation to the present invention 2’ substituted sugar modified nucleosides does not include 2’ bridged nucleosides like LNA.
In an embodiment, the oligonucleotide GBA agonist comprises one or more sugar modified nucleosides, such as 2’ sugar modified nucleosides. Preferably the oligonucleotide GBA agonist of the 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 a!., Bioorganic & Med.Chem. Lett., 12, 73-76, Seth et al., J. Org. Chem., 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry, 2016, 59, 9645-9667.
Further non-limiting, exemplary LNA nucleosides are disclosed in Scheme 1.
Scheme 1 :
Figure imgf000016_0001
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 GBA agonist 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:
Figure imgf000017_0001
In some embodiments, morpholino oligonucleotides of the invention may be, for example 8 - 40 morpholino nucleotides in length, such as morpholino 18 - 22 nucleotides in length.
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. WO 01/23613 provides in vitro methods for determining RNase H 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. For effective modulation of splicing, degradation of the pre-mRNA is not desirable, and as such it is preferable to avoid the RNaseH degradation of the target. Therefore, the oligonucleotide GBA agonists of the invention are not RNase H recruiting gapmer oligonucleotide.
RNase H recruitment may be avoided by limiting the number of contiguous DNA nucleotides in the oligonucleotide - therefore mixmer and totalmer designs may be used.
Advantageously the oligonucleotide GBA agonists of the invention, or the contiguous nucleotide sequence thereof, do not comprise more than 3 contiguous DNA nucleosides. Further, advantageously the oligonucleotide GBA agonists of the invention, or the contiguous nucleotide sequence thereof, do not comprise more than 4 contiguous DNA nucleosides. Further, advantageously, the oligonucleotide GBA agonists of the invention, or contiguous nucleotide sequence thereof, do not comprise more than 2 contiguous DNA nucleosides.
Mixmers and Totalmers
For splice modulation it is often advantageous to use oligonucleotides which do not recruit RNAase H. As RNase H activity requires a contiguous sequence of DNA nucleotides, RNase H activity of oligonucleotides may be achieved by designing oligonucleotides which do not comprise a region of more than 3 or more than 4 contiguous DNA nucleosides. This may be achieved by using oligonucleotides or contiguous nucleoside regions thereof with a mixmer design, which comprise sugar modified nucleosides, such as 2’ sugar modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides. Mixmers are exemplified herein by every second design, wherein the nucleosides alternate between 1 LNA and 1 DNA nucleoside, e.g. LDLDLDLDLDLDLDLL, with 5’ and 3’ terminal LNA nucleosides, and every third design, such as LDDLDDLDDLDDLDDL, where every third nucleoside is a LNA nucleoside.
A totalmer is an oligonucleotide or a contiguous nucleotide sequence thereof which does not comprise DNA or RNA nucleosides, and may for example comprise only 2’-O-MOE nucleosides, such as a fully MOE phosphorothioate, e.g.
MMMMMMMMMMMMMMMMMMMM, where M = 2’-O-MOE, or may for example comprise only 2’oMe nucleosides, which are reported to be effective splice modulators for therapeutic use. Alternatively, a mixmer may comprise a mixture of modified nucleosides, such as MLMLMLMLMLMLMLMLMLML, wherein L = LNA and M = 2’-O-MOE nucleosides.
Advantageously, the internucleoside nucleosides in mixmers and totalmers may be phosphorothioate, or a majority of nucleoside linkages in mixmers may be phosphorothioate. Mixmers and totalmers may comprise other internucleoside linkages, such as phosphodiester or phosphorodithioate, by way of example.
In some embodiments, the oligonucleotide GBA agonists are, or comprise, an oligonucleotide mixmer or totalmer. In some embodiments, the contiguous nucleotide sequence is a mixmer or a tolalmer.
Target sequence
The oligonucleotide GBA agonists of the invention target one or more of the promoters of the human GBA genomic sequence. This may also be referred to herein as the GBA gene.
The GNA genomic sequence may be referred to as a target sequence.
The target sequence may also be referred to as a target nucleic acid or target site sequence.
The term “genomic sequence” as used herein, particularly with reference to the GBA genomic sequence, encompasses both protein coding and non-protein coding sequences. It is understood that such sequences include transcribed and untranscribed sequences, and translated and untranslated sequences. Non-protein coding sequences may comprise regulatory sequences such as enhancers, silencers, promoters, and/or 3’ and 5’ untranslated regions (UTR).
The term “GBA nucleic acid sequence” as used herein may also refer to nucleic acid sequences of the GBA gene in the sense of the definition outlined herein. The target nucleic acid sequences of the GBA gene may refer to sequences as present within the genomic DNA or the same, or antisense sequences, present in a cell in any other form, such as mRNA, or other single or double stranded RNAs, such as miRNAs or siRNAs.
Reference to genes and their corresponding nucleotide sequences as used herein is not intended to be necessarily limited to either one of the sense or antisense strands thereof. Accordingly, both or either of the sense and antisense sequences may be encompassed.
In some embodiments, the human GBA gene has the sequence of NCBI Reference NG_009783.1. In some embodiments the human GBA gene comprises a sequence defined by SEQ ID NO 226. SEQ ID NO 226 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 226, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
In some embodiments, the human GBA genomic sequence consists of SEQ ID NO 226.
The oligonucleotide GBA agonists of the invention target one or more promoter regions of the GBA genomic sequence.
In some embodiments, the promoter of the human GBA gene comprises the intergenic region from FAM189B to GBA translational start codon (SEQ ID NO 225). SEQ ID NO 225 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 225, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
In some embodiments, the promoter of the human GBA gene comprises or consists of SEQ ID NO 225.
SEQ ID NO 225 encompasses Promoters 1 (SEQ ID NO 224) and 2 (SEQ ID NO 223), as discussed below.
In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter located immediately upstream of exon 1 of the GBA gene (Promoter 1 , P1). For example, between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
In some embodiments, the ATG initiation codon corresponds to position 8586 - 8588 of NCBI Reference sequence NG_009783.1 and/or SEQ ID NO: 226.
In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 7449 - 8585 of NCBI Reference sequence NG_009783.1 (SEQ ID NO 224). Put another way, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 7449 - 8585 of SEQ ID NO 226 (SEQ ID NO 224).
In some embodiments, the promoter of the human GBA gene comprises SEQ ID NO 224.
In some embodiments, the promoter of the human GBA gene consists of SEQ ID NO 224.
In some embodiments the target sequence is a sequence of nucleotides within SEQ ID NO 224. SEQ ID NO 224 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 224, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 35 to 55, 36 to 56, 39 to 59, 46 to 66, 68 to 88, 106 to 126, 148 to 168, 152 to 172, 153 to 173, 302 to 322, 313 to 333, 314 to 334, 371 to 391 , 372 to 392, 475 to 495, 517 to 537, 532 to 552, 598 to 618, 656 to 676, 657 to 677, 746 to 766, 747 to 767, 748 to 768, 749 to 769, 750 to 770, 753 to 773, 815 to 835, 816 to 836, 839 to 859, 840 to 860, 884 to 904, 891 to 911 , 904 to 924, 922 to 942, 923 to 943, 996 to 1016, 1003 to 1023, 1019 to 1039, 1037 to 1057, and 1098 to 1118 of SEQ ID NO 224.
Unless otherwise stated, all ranges are inclusive of the start and end value, e.g. the target sequence corresponding to positions 4137 to 4157 comprises 21 nucleotides.
In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 39 to 59, 148 to 168, 153 to 173, 302 to 322, 313 to 333, 517 to 537, 532 to 552, 656 to 676, 748 to 768, 750 to 770, 815 to 835, 840 to 860, 904 to 924 and 996 to 1016 of SEQ ID NO 224.
In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 748 to 768, 750 to 770, 815 to 835, 904 to 924 and 996 to 1016 of SEQ ID NO 224.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, and SEQ ID NO 40, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 30, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 3, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 7, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
9, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
10, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
11, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
16, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
17, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 19, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 23, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 25, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 27, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 30, or a fragment thereof. In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 33, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 36, or a fragment thereof.
In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter located immediately upstream of exon -2 of the GBA gene (Promoter 2, P2). For example, between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
In some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 4028 - 5192 of NCBI Reference sequence NG_009783.1 (SEQ ID NO 223). Put another way, in some embodiments, the oligonucleotide GBA agonist of the invention targets the promoter of the human GBA gene within positions 4028 - 5192 of SEQ ID NO 226 (SEQ ID NO 223).
In some embodiments, the promoter of the human GBA gene comprises SEQ ID NO 223.
In some embodiments, the promoter of the human GBA gene consists of SEQ ID NO 223.
In some embodiments the target sequence is a sequence of nucleotides within SEQ ID NO 223.
SEQ ID NO 223 is provided herein as a reference sequence and it will be understood that the target nucleic acid may be an allelic variant of SEQ ID NO 223, such as an allelic variant, which comprises one or more polymorphisms in the human GBA genomic sequence.
In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 13 to 33, 34 to 54, 38 to 58, 121 to 141 , 159 to 179, 160 to 180, 173 to 193, 199 to 219, 200 to 220, 313 to 333, 319 to 339, 335 to 355, 336 to 356, 359 to 379, 391 to 411 , 422 to 442, 423 to 443, 454 to 474, 469 to 489, 535 to 555, 548 to 568, 551 to 571 , 589 to 609, 590 to 610, 654 to 674, 669 to 689, 670 to 690, 676 to 696, 685 to 705, 744 to 764, 761 to 781 , 762 to 782, 913 to 933, and 922 to 942 of SEQ ID NO 223.
In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121 to 141 , 160 to 180, 173 to 193, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 391 to 411, 423 to 443, 469 to 489, 535 to 555, 548 to 568, 551 to 571, 589 to 609 and 670 to 690 of SEQ ID NO 223. In some embodiments, the contiguous nucleotide sequence of the oligonucleotide GBA agonist of the invention is complementary to a sequence selected from the group consisting of nucleotides 121 to 141 , 160 to 180, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 423 to 443, 469 to 489, 548 to 568, 551 to 571 and 670 to 690 of SEQ ID NO 223.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 41 , SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51 , SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71 , SEQ ID NO 72, SEQ ID NO 73, and SEQ ID NO 74, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63 and SEQ ID NO 67, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO 62, and SEQ ID NO 67, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 44, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
46, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
47, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
48, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 49, or a fragment thereof. In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
52, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
53, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
54, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
55, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 57, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
59, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
60, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
61 , or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
62, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO
63, or a fragment thereof.
In some embodiments the contiguous nucleotide sequence is complementary to SEQ ID NO 67, or a fragment thereof.
A fragment of the target sequence may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.
In some embodiments the contiguous nucleotide sequence is complementary to at least 8 contiguous nucleotides of any of the target sequences recited herein.
In some embodiments the contiguous nucleotide sequence is complementary to at least 10 contiguous nucleotides of any of the target sequences recited herein. In some embodiments the contiguous nucleotide sequence is complementary to at least 12 contiguous nucleotides of any of the target sequences recited herein.
In some embodiments the contiguous nucleotide sequence is complementary to at least 14 contiguous nucleotides of any of the target sequences recited herein.
In some embodiments the contiguous nucleotide sequence is complementary to at least 16 contiguous nucleotides of any of the target sequences recited herein.
In some embodiments the contiguous nucleotide sequence is complementary to at least 18 contiguous nucleotides of any of the target sequences recited herein.
In some embodiments the contiguous nucleotide sequence is complementary to at least 19 contiguous nucleotides of any of the target sequences recited herein.
Complementarity
The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (II).
It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).
The term “% complementary” as used herein, refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g. a target sequence or sequence motif). The percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pairs) between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch. Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5’-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
In certain embodiments of the invention the oligonucleotide GBA agonist is a double stranded oligonucleotide, such as an saRNA. In these embodiments the double stranded oligonucleotide may have a nucleotide overhang, such as a 2 nucleotide overhang which may be at the 3’ end of the contiguous nucleotide sequence. In such embodiments complementarity is defined based upon the double stranded sequence without the overhang. For example, if the oligonucleotide is 21 nucleotides in length and includes a 2 nucleotide overhang, complementarity is determined based upon the 19 nucleotides without the two nucleotide overhang.
Within the present invention, the term “complementary” requires the contiguous nucleotide sequence to be at least about 75% complementary, or at least about 80% complementary, or at least about 85% complementary, or at least about 90% complementary, or at least about 95% complementary, to the target sequence, i.e. one or more promoters of the human GBA gene. In some embodiments the oligonucleotide GBA agonist may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary the target sequence, i.e. one or more promoters of the human GBA gene. Put another way, in some embodiments, the contiguous nucleotide sequence within an oligonucleotide GBA agonist of the invention may include one, two, three or more mis-matches, wherein a mis-match is a nucleotide within the contiguous nucleotide sequence which does not base pair with its target.
The term “fully complementary”, refers to 100% complementarity.
In some embodiments the contiguous nucleotide sequence is fully complementary to the target sequence.
Identity
The term “identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif). The percentage of identity is thus calculated by counting the number of aligned nucleobases that are identical (a Match) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
It is therefore to be understood that there is a relationship between identity and complementarity such that a contiguous nucleotide sequence within an oligonucleotide GBA agonist of the invention that is complementary to a target sequence also shares a percentage of identity with said complementary sequence.
Hybridization
The terms “hybridizing” or “hybridizes” as used herein are to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (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 GBA agonists of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10- 30 nucleotides in length.
In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°. The oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length. In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal, or- 16 to -27 kcal such as -18 to -25 kcal.
Double stranded oligonucleotide GBA agonists
In some embodiments, the oligonucleotide GBA agonist is a double stranded oligonucleotide.
In some embodiments the double stranded oligonucleotide GBA agonist is a short activating RNA (saRNAs).
The term “short activating RNA” (saRNA), as used herein, refers to a small double stranded RNA that is typically 21 nucleotides in length and may comprise a 2 nucleotide overhang, optionally at the 3’ end. saRNAs are capable of inducing gene activation by a process known as RNA activation (RNAa), wherein gene activation is induced by hybridization of the saRNA with a target nucleic acid sequence. Said target nucleic acid sequences typically comprise promoter regions of a gene.
A known mechanism of transcriptional upregulation by saRNAs involves Ago2, and is associated with epigenetic modification at target sites, e.g. promotors. Ago2 associates with an saRNA, which guides the complex to a target and facilitates the assembly of an RNA- induced transcriptional activation (RITA) complex. RITA-RNA polymerase II interactions are thought to promote transcription initiation and productive elongation, as well as monoubiquitination of histone 2B (Portnoy et al., Cell Res., 2016, 26(3), 320-335). In some embodiments, the oligonucleotide GBA agonist of the invention, such as an saRNA, is 19 nucleotides in length.
In some embodiments, the oligonucleotide GBA agonist of the invention, such as an saRNA is 20 nucleotides in length.
In some embodiments, the oligonucleotide GBA agonist of the invention, such as an saRNA, is 21 nucleotides in length.
In some embodiments, the oligonucleotide GBA agonist of the invention, such as an saRNA, is 22 nucleotides in length.
It will be understood that when discussing embodiments including a double stranded oligonucleotide the length measurement refers to the length of one of the strands. In embodiments where the two strands may not be the same length, the length is taken as the length of the longest strand.
In some embodiments, the oligonucleotide GBA agonist of the invention, such as an saRNA, may have a nucleotide overhang. The nucleotide overhang may be a 2 nucleotide overhang. In some embodiments the overhang may be at the 3’ end of the contiguous nucleotide sequence. In some embodiments the overhang may comprise or consist of two thymine nucleotides (TT).
Contiguous nucleotide sequence
In one embodiment, the oligonucleotide GBA agonist of the invention may target a sequence within Promoter 1 of the human GBA genomic sequence.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101 , SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 , SEQ ID NO 112, SEQ ID NO 113, and SEQ ID NO 114, or at least 10 contiguous nucleotides thereof.
It will be apparent to the skilled person that a double stranded oligonucleotide can be defined by reference to either the sense strand or the antisense strand. In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 77, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 81 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 83, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 84, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 85, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 90, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 91, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 93, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 97, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 99, or at least 10 contiguous nucleotides thereof. In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 101 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 104, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 107, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151 , SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161 , SEQ ID NO 162, SEQ ID NO 163, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 166, SEQ ID NO 167, SEQ ID NO 168, SEQ ID NO 169, SEQ ID NO 170, SEQ ID NO 171 , SEQ ID NO 172, SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177, SEQ ID NO 178, SEQ ID NO 179, SEQ ID NO 180, SEQ ID NO 181 , SEQ ID NO 182, SEQ ID NO 183, SEQ ID NO 184, SEQ ID NO 185, SEQ ID NO 186, SEQ ID NO 187, and SEQ ID NO 188, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 151, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 178, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 151 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 155, or at least 10 contiguous nucleotides thereof. In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 157, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 158, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 159, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 164, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 165, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 167, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 171 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 173, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 175, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 178, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 181 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
In one embodiment, the oligonucleotide GBA agonist of the invention may target a sequence within Promoter 2 of the human GBA genomic sequence.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141 , SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, and SEQ ID NO 148, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 141, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 135, SEQ ID NO 136 and SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 118, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 120, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 121 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 122, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 123, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 126, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 127, or at least 10 contiguous nucleotides thereof. In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 128, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 129, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 131 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 133, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 134, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 135, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 136, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 137, or at least 10 contiguous nucleotides thereof.
In some embodiments, the sense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191 , SEQ ID NO 192, SEQ ID NO 193, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 194, SEQ ID NO 198, SEQ ID NO 199, SEQ ID NO 200, SEQ ID NO 201 , SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 204, SEQ ID NO 205, SEQ ID NO 206, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 , SEQ ID NO 212, SEQ ID NO 213, SEQ ID NO 214, SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219, SEQ ID NO 220, SEQ ID NO 221 , and SEQ ID NO 222, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO 201, SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO 201 , SEQ ID NO 202, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 209, SEQ ID NO 210 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 192, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 194, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 195, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 196, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 197, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 200, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 201 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 202, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 203, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 205, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 207, or at least 10 contiguous nucleotides thereof. In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 208, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 209, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 210, or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 211 , or at least 10 contiguous nucleotides thereof.
In some embodiments, the antisense strand of the contiguous nucleotide sequence is, or comprises, SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
In certain embodiments wherein the oligonucleotide is a double stranded oligonucleotide the contiguous nucleotide sequence may be a fragment of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of any of the sequences recited herein.
It will be understood that double stranded oligonucleotides, such as saRNAs, comprise two complementary strands, each of which may hybridize with complementary oligonucleotide sequences, such as oligonucleotide sequences of endogenous RNAs or DNA. Without wishing to be bound by theory, it will be appreciated that a double stranded oligonucleotide of the invention may act through binding to the sense strand, the antisense strand or both strands of the target sequence.
Region D’ or D” in an oligonucleotide
The oligonucleotide GBA agonist of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as a mixmer or toalmer region, and further 5’ and/or 3’ nucleosides. The further 5’ and/or 3’ nucleosides may or may not be complementary, such as fully complementary, to the target nucleic acid. Such further 5’ and/or 3’ nucleosides may be referred to as region D’ and D” herein.
The addition of region D’ or D” may be used for the purpose of joining the contiguous nucleotide sequence, such as the mixmer or totoalmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively, it may be used to provide exonucleoase protection or for ease of synthesis or manufacture. Region D’ or D” may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D’ or D” are disclosed in WO 2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO 2015/113922, where they are used to link multiple antisense constructs within a single oligonucleotide.
In one embodiment the oligonucleotide GBA agonist of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes a mixmer or a total mer.
In some embodiments the internucleoside linkage positioned between region D’ or D” and the mixmer or totalmer region is a phosphodiester linkage.
Conjugate
The invention encompasses an oligonucleotide GBA agonist covalently attached to at least one conjugate moiety. In some embodiments this may be referred to as a conjugate of the invention.
In some embodiments, the invention provides oligonucleotide GBA agonists covalently attached to at least one conjugate moiety.
The term “conjugate” as used herein refers to an oligonucleotide GBA agonist which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to the oligonucleotide, optionally via a linker group, such as region D’ or D”.
Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.
In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates (e.g. GalNAc), cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.
Linkers
A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the oligonucleotide GBA agonist 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 GBA agonist 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 GBA agonist 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 GBA agonists according to the invention, or the conjugate according to the invention.
The invention provides for oligonucleotide GBA agonists according to the invention wherein the oligonucleotide GBA agonists 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 GBA agonist according to the invention, or the conjugate according to the invention.
The invention provides for a pharmaceutically acceptable potassium salt of the oligonucleotide GBA agonist according to the invention, or the conjugate according to the invention.
Delivery of oligonucleotide GBA agonist
The invention provides for oligonucleotide GBA agonists according to the invention wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.
This may be for the purpose of delivering the oligonucleotide GBA agonist of the invention to the targeted cells and/or to improve the pharmacokinetics of the oligonucleotide GBA agonist.
Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.
Pharmaceutical compositions
The invention provides for a pharmaceutical composition comprising the oligonucleotide GBA agonist of the invention, or the conjugate or salt of the invention, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant. The invention provides for a pharmaceutical composition comprising the oligonucleotide GBA agonists of the invention, or the conjugate of the invention, and a pharmaceutically acceptable salt. For example, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.
The invention provides for a pharmaceutical composition according to the invention, wherein the pharmaceutical composition comprises the oligonucleotide GBA agonist of the invention or the conjugate of the invention, or the pharmaceutically acceptable salt 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 GBA agonist of the invention, or the conjugate of the invention, or the pharmaceutically acceptable salt of the invention. Suitably the solution, such as phosphate buffered saline solution, of the invention is a sterile solution.
Method for modulating GBA expression
The invention provides for a method for enhancing, upregulating or restoring the expression of GBA in a cell, such as a cell which is expressing GBA, said method comprising administering an oligonucleotide GBA agonist of the invention or a conjugate of the invention, or a salt 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 recognized that treatment, as referred to herein may in some embodiments be prophylactic. The invention provides for a method for treating or preventing a disease associated with reduced expression of GBA, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention to a subject suffering from or susceptible to a disease associated with reduced expression of GBA.
In one embodiment, the disease is selected from the group consisting of Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.
In one embodiment, the disease is Parkinson’s disease.
In one embodiment the disease is Gaucher’s disease.
In some embodiments, the subject is an animal, preferably a mammal such as a mouse, rat, hamster, or monkey, or preferably a human.
The invention provides for an oligonucleotide GBA agonist of the invention for use as a medicament.
The invention provides for an oligonucleotide GBA agonist of the invention for the preparation of a medicament.
The invention provides for an oligonucleotide GBA agonist of the invention for use in therapy.
The invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for use as a medicament.
The invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament.
The invention provides an oligonucleotide GBA agonist of the invention or a pharmaceutical composition according to the invention for use in therapy.
The invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use as in the treatment of Parkinson’s disease.
The invention provides for an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention for use as in the treatment of Gaucher’s disease. The invention provides for the use of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Parkinson’s disease.
The invention provides for the use of an oligonucleotide GBA agonist of the invention or a pharmaceutical composition of the invention, for the preparation of a medicament for the treatment or prevention of Gaucher’s disease.
Administration
The oligonucleotide GBA agonist 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, intra-muscular, intracerebral, intracerebroventricular or intrathecal).
In a preferred embodiment the oligonucleotide or pharmaceutical composition of the invention is administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular, administration. In one embodiment the oligonucleotide is administered intracerebrally or intracerebroventricularly. In another embodiment the oligonucleotide 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, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.
GBA upregulation In certain embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein.
In certain embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
In certain embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
In certain embodiments the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA and GBA protein by at least about 10%. In other embodiments the oligonucleotide GBA agonist of the present invention may enhance the production of GBA mRNA and GBA protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 600% or more.
In some embodiments, the oligonucleotide GBA agonists of the present invention may enhance the production of GBA mRNA, GBA protein, or GBA mRNA and GBA protein compared to a control. The control may be a cell that has not been exposed to the oligonucleotide. In some embodiments, the control may be a mock transfection, for example, treatment of cells with PBS. Table 1. saRNA sequences of the invention and their target sequences
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
EXAMPLES
EXAMPLE 1 : EFFECT OF saRNAs ON GBA mRNA
The day before transfection SK-N-AS neuroblastoma cells were plated to a density of 25000 per well in 96 well plates in full growth medium (DMEM (Sigma: D6546), 10% FBS, 2mM glutamine, 0.1 mM (1x) NEAA, 25pg/ml Gentamicin). The day after plating, the cells were either transfected with saRNA SEQ ID NO 1 to 74 (n=2) or PBS (Mock) using Lipofectamin RNAiMax (Invitrogen) at a final concentration of 10 nM according to Invitrogen’s instructions. 48 h after transfection, mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water. 4 pL was used as input for one-step RT-qPCR analysis according to protocol in Table 3. (qScript™ XLT One-Step RT-qPCR ToughMix®,
Low ROX™, Quanta Bioscience, #95134-500) using custom designed qPCR assay specific to GBA and predesigned assay for TBP (HEX, Hs. PT.58v.39858774 , IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 1. Table 2. GBA qPCR assay: Primers and probe, all sequences 5’-> 3’
Figure imgf000048_0001
Table 3. RT-qPCR protocol
Figure imgf000048_0002
SEQ ID#10, SEQ ID#11, SEQ ID#17, SEQ ID#19, SEQ ID#23, SEQ ID#25, SEQ ID#27, SEQ ID#30, SEQ ID#36, SEQ ID#47, SEQ ID#48, SEQ ID#59, SEQ ID#60, SEQ ID#62, SEQ ID#67 all increase expression of GBA mRNA more than 15% relative to Mock transfected 48 h after transfection in SK-N-AS cells (Figure 1).
EXAMPLE 2: EFFECT OF saRNAs ON GBA mRNA
The day before transfection H4 neuroglioma cells were plated to a density of 15000 per well in 96 well plates in full growth medium (DMEM Sigma: D0819, 15% FBS, 1 mM Sodium Pyruvate, 25 pg/ml Gentamicin). The day after plating, the cells were either transfected with saRNA SEQ ID NO 1 to 74 (n=2) or PBS (Mock) using Lipofectamin RNAiMax (Invitrogen) for final concentration of 10 nM according to Invitrogen’s instructions. 48 h after transfection, mRNA were isolated using the RNeasy® 96 Kit (Qiagen) and extracted in 200 pL RNAse free Water. 4 pL was used as input for one-step RT-qPCR analysis according to protocol in Table 3. (qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™, Quanta Bioscience, #95134-500) using custom designed qPCR assay specifc to GBA and predesigned assay for TBP (HEX, Hs. PT.58v.39858774 , IDT). GBA mRNA concentrations were quantified relative to the housekeeping gene TBP using R Software. See Figure 2.
SEQ ID#3, SEQ ID#7, SEQ ID#9, SEQ ID#16, SEQ ID#23, SEQ ID#25, SEQ ID#27, SEQ ID#33, SEQ ID#36, SEQ ID#44, SEQ ID#46, SEQ ID#49, SEQ ID#52, SEQ ID#53, SEQ ID#54, SEQ ID#55, SEQ ID#57, SEQ ID#59, SEQ ID#61 , SEQ ID#62, SEQ ID# 63, SEQ ID#67 all increase expression of GBA mRNA more than 15% fold relative to Mock transfected 48h after transfection in H4 cells (Figure 2).
EMBODIMENTS
1. An oligonucleotide glucocerebrosidase (GBA) agonist, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary to one or more promoters of the human GBA genomic sequence.
2. The oligonucleotide GBA agonist of item 1 , wherein the contiguous nucleotide sequence is 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. 3. The oligonucleotide GBA agonist of item 2, wherein the contiguous nucleotide sequence is 19 nucleotides in length.
4. The oligonucleotide GBA agonist of any one of items 1 to 3, wherein the contiguous nucleotide sequence is the same length as the oligonucleotide GBA agonist.
5. The oligonucleotide GBA agonist of any one of items 1 to 4, wherein the contiguous nucleotide sequence is at least 75% complementary to one or more promoters of the human GBA genomic sequence.
6. The oligonucleotide GBA agonist of item 5, wherein the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90% or at least 95% complementary to one or more promoters of the human GBA genomic sequence.
7. The oligonucleotide GBA agonist of any one of items 1 to 6, wherein the contiguous nucleotide sequence is fully complementary to one or more promoters of the human GBA genomic sequence.
8. The oligonucleotide GBA agonist of any one of items 1 to 7, wherein the human GBA genomic sequence is SEQ ID NO 226.
9. The oligonucleotide GBA agonist of any one of items 1 to 8, wherein the promoter of the human GBA gene comprises SEQ ID NO 225.
10. The oligonucleotide GBA agonist of item 9, wherein the promoter of the human GBA gene consists of SEQ ID NO 225.
11. The oligonucleotide GBA agonist of any one of items 1 to 10, wherein the promoter is located immediately upstream of exon 1 of the human GBA gene (P1).
12. The oligonucleotide GBA agonist of item 11 , wherein the promoter is located between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
13. The oligonucleotide GBA agonist of item 11 or item 12, wherein the promoter of the human GBA gene comprises SEQ ID NO 224. 14. The oligonucleotide GBA agonist of item 13, wherein the promoter of the human GBA gene consists of SEQ ID NO 224.
15. The oligonucleotide GBA agonist of any one of items 11 to 14, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of nucleotides 39 to 59, 148 to 168, 153 to 173, 302 to 322, 313 to 333, 517 to 537, 532 to 552, 656 to 676, 748 to 768, 750 to 770, 815 to 835, 840 to 860, 904 to 924 and 996 to 1016 of SEQ ID NO 224, or a fragment thereof.
16. The oligonucleotide GBA agonist of any one of items 11 to 14, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 30, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
17. The oligonucleotide GBA agonist of any one of items 1 to 16, wherein the oligonucleotide is a double stranded oligonucleotide.
18. The oligonucleotide GBA agonist of item 17, wherein the oligonucleotide is a saRNA.
19. The oligonucleotide GBA agonist of item 17 or item 18, wherein the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
20. The oligonucleotide GBA agonist of any one of items 17 to 19, wherein the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 151, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 171 , SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 178, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
21. The oligonucleotide GBA agonist of any one of items 1 to 10, wherein the promoter is located immediately upstream of exon -2 of the human GBA gene (P2). 22. The oligonucleotide GBA agonist of item 21 , wherein the promoter is located between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
23. The oligonucleotide GBA agonist of item 21 or item 22, wherein the promoter of the human GBA gene comprises SEQ ID NO 223.
24. The oligonucleotide GBA agonist of item 23, wherein the promoter of the human GBA gene consists of SEQ ID NO 223.
25. The oligonucleotide GBA agonist of any one of items 21 to 24, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of nucleotides 121 to 141, 160 to 180, 173 to 193, 199 to 219, 200 to 220, 335 to 355, 336 to 356, 359 to 379, 391 to 411, 423 to 443, 469 to 489, 535 to 555, 548 to 568, 551 to 571 , 589 to 609 and 670 to 690 of SEQ ID NO 223, or a fragment thereof.
26. The oligonucleotide GBA agonist of any one of items 21 to 24, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63 and SEQ ID NO 67, or a fragment thereof.
27. The oligonucleotide GBA agonist of any one of items 21 to 27, wherein the oligonucleotide is a double stranded oligonucleotide.
28. The oligonucleotide GBA agonist of item 27, wherein the oligonucleotide is a saRNA.
29. The oligonucleotide GBA agonist of item 27 or item 28 wherein the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 131 , SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
30. The oligonucleotide GBA agonist of any one of items 27 to 29, wherein the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO 201 , SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
31. The oligonucleotide GBA agonist of any one of items 1 to 30, wherein the oligonucleotide is or comprises an oligonucleotide mixmer or totalmer.
32. The oligonucleotide GBA agonist of any one of items 1 to 31 , wherein the oligonucleotide is capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
33. The oligonucleotide GBA agonist of item 32, wherein the control is a cell that has not been exposed to the oligonucleotide GBA agonist.
34. The oligonucleotide GBA agonist of any one of items 1 to 33, wherein the oligonucleotide GBA agonist is covalently attached to at least one conjugate moiety.
35. The oligonucleotide GBA agonist of any one of items 1 to 34, wherein the oligonucleotide GBA agonist is in the form of a pharmaceutically acceptable salt.
36. The oligonucleotide GBA agonist of item 35, wherein the salt is a sodium salt or a potassium salt.
37. The oligonucleotide GBA agonist of any one of items 1 to 36, wherein the oligonucleotide GBA agonist is encapsulated in a lipid-based delivery vehicle, covalently linked to or encapsulated in a dendrimer, or conjugated to an aptamer.
38. A pharmaceutical composition comprising the oligonucleotide GBA agonist of any one of items 1 to 37 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
39. The pharmaceutical composition of item 38, wherein the pharmaceutical composition comprises an aqueous diluent or solvent, such as phosphate buffered saline.
40. An in vivo or in vitro method for upregulating or restoring GBA expression in a target cell, the method comprising administering an oligonucleotide GBA agonist of any one of items 1 to 37 or a pharmaceutical composition of item 38 or item 39, in an effective amount, to said cell.
41. The method of item 40, wherein the cell is either a human cell or a mammalian cell.
42. The method of item 40 or item 41 , wherein the expression of GBA mRNA is increased by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
43. The method of any one of items 40 to 42, wherein the expression of GBA protein is increased by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
44. The method of item 42 or item 43, wherein the control is a cell that has not been exposed to said oligonucleotide GBA agonist.
45. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the oligonucleotide GBA agonist of any of items 1 to 37, or the pharmaceutical composition of item 38 or item 39, to a subject suffering from or susceptible to a disease.
46. The oligonucleotide of any one of items 1 to 37 or the pharmaceutical composition of item 38 or item 39 for use as a medicament for the treatment or prevention of a disease in a subject.
47. Use of the oligonucleotide of any one of items 1 to 37 or the pharmaceutical composition of item 38 or item 39, for the preparation of a medicament for treatment or prevention of a disease in a subject.
48. The method of item 45, the oligonucleotide or pharmaceutical composition for use according to item 46 or the use according to item 47, wherein the disease is associated with reduced expression of GBA
49. The method of item 45, the oligonucleotide or pharmaceutical composition for use according to item 46 or the use according to item 47, wherein the disease is selected from the group consisting of Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders. 50. The method of item 45, the oligonucleotide or pharmaceutical composition for use according to item 46 or the use according to item 47, wherein the disease is Parkinson’s disease.
51. The method of item 45, the oligonucleotide or pharmaceutical composition for use according to item 46 or the use according to item 47, wherein the disease is Gaucher’s disease.

Claims

1. An oligonucleotide glucocerebrosidase (GBA) agonist, wherein the oligonucleotide is 8-40 nucleotides in length and comprises a contiguous sequence of 8-40 nucleotides in length, which is complementary to one or more promoters of the human GBA genomic sequence.
2. The oligonucleotide GBA agonist of claim 1 wherein the contiguous nucleotide sequence is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or fully complementary to one or more promoters of the human GBA genomic sequence, wherein the human GBA genomic sequence is SEQ ID NO 226.
3. The oligonucleotide GBA agonist of any one of claims 1 to 3, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO 225.
4. The oligonucleotide GBA agonist of any one of claims 1 to 4, wherein the promoter is located between 1 ,137 and 1 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene (P1).
5. The oligonucleotide GBA agonist of claim 4, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO 224.
6. The oligonucleotide GBA agonist of claim 4 or 5, wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO 30, SEQ ID NO 33 and SEQ ID NO 36, or a fragment thereof.
7. The oligonucleotide GBA agonist of any one of claim 4 to 6, wherein the oligonucleotide GBA agonist is a double stranded oligonucleotide, and wherein the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 77, SEQ ID NO 81 , SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO 104, SEQ ID NO 107 and SEQ ID NO 110, or at least 10 contiguous nucleotides thereof.
8. The oligonucleotide GBA agonist of any one of claims 4 to 7, wherein the oligonucleotide GBA agonist is a double stranded oligonucleotide, and
55 wherein the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 151, SEQ ID NO 155, SEQ ID NO 157, SEQ ID NO 158, SEQ ID NO 159, SEQ ID NO 164, SEQ ID NO 165, SEQ ID NO 167, SEQ ID NO 171, SEQ ID NO 173, SEQ ID NO 175, SEQ ID NO 178, SEQ ID NO 181 and SEQ ID NO 184, or at least 10 contiguous nucleotides thereof.
9. The oligonucleotide GBA agonist of any one of claims 1 to 3, wherein the promoter is located immediately upstream of exon -2 of the human GBA gene (P2).
10. The oligonucleotide GBA agonist of claim 9, wherein the promoter is located between 4,558 and 3,394 nucleotides upstream of the ATG initiation codon in exon 1 of the human GBA gene.
11. The oligonucleotide GBA agonist of claim 9 or claim 10, wherein the promoter of the human GBA gene comprises or consists of SEQ ID NO 223.
12. The oligonucleotide GBA agonist of any one of claims 9 to 11 , wherein the contiguous nucleotide sequence is complementary to a sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63 and SEQ ID NO 67, or a fragment thereof.
13. The oligonucleotide GBA agonist of any one of claims 9 to 12, wherein the oligonucleotide GBA agonist is a double stranded oligonucleotide, and wherein the sense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 118, SEQ ID NO 120, SEQ ID NO 121 , SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 131, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 and SEQ ID NO 141 , or at least 10 contiguous nucleotides thereof.
14. The oligonucleotide GBA agonist of any one of claims 9 to 13, wherein the oligonucleotide GBA agonist is a double stranded oligonucleotide, and wherein the antisense strand of the contiguous nucleotide sequence is, or comprises, a sequence selected from the group consisting of SEQ ID NO 192, SEQ ID NO 194, SEQ ID NO 195, SEQ ID NO 196, SEQ ID NO 197, SEQ ID NO 200, SEQ ID NO
56 201 , SEQ ID NO 202, SEQ ID NO 203, SEQ ID NO 205, SEQ ID NO 207, SEQ ID NO 208, SEQ ID NO 209, SEQ ID NO 210, SEQ ID NO 211 and SEQ ID NO 215, or at least 10 contiguous nucleotides thereof.
15. The oligonucleotide GBA agonist of any one of claims 1 to 14, wherein the oligonucleotide is capable of increasing the expression of GBA by at least 10%, 15%, 20%, 30%, 40%, 50% or more than 50%, compared to a control.
16. The oligonucleotide GBA agonist of any one of claims 1 to 15, wherein the oligonucleotide is a saRNA.
17. The oligonucleotide of any one of claims 1 to 16 for use as a medicament for the treatment or prevention of a disease in a subject.
18. The oligonucleotide for use according to claim 17, wherein the disease is associated with reduced expression of GBA.
19. The oligonucleotide for use according to claim 17 or 18, wherein the disease is selected from the group consisting of Gaucher’s disease, Parkinson’s Disease, dementia, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behaviour disorders.
20. The oligonucleotide for use according to any of claims 17 to 19, wherein the disease is Parkinson’s disease.
57
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