WO2020011744A2

WO2020011744A2 - Antisense oligonucleotides targeting cers5

Info

Publication number: WO2020011744A2
Application number: PCT/EP2019/068322
Authority: WO
Inventors: Marie Wirkström LINDHOLM; Steffen Schmidt
Original assignee: Roche Innovation Center Copenhagen AS
Current assignee: Roche Innovation Center Copenhagen AS
Priority date: 2018-07-11
Filing date: 2019-07-09
Publication date: 2020-01-16
Anticipated expiration: 2021-01-11
Also published as: WO2020011744A3

Abstract

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to CERS5 pre-mRNA exon and junction sequences, which are capable of inhibiting the expression of CERS5 protein. Inhibition of CERS5 expression is beneficial for a range of medical disorders including obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING CERS5

FIELD OF INVENTION

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to CERS5 pre-mRNA exon and junction sequences, which are capable of inhibiting the expression of CERS5. Inhibition of CERS5 expression is beneficial for a range of medical disorders including obesity, insulin resistance, diabetes such as type 2 diabetes,

atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

BACKGROUND

Ceramide synthases (EC 2.3.1.24) such as CERS5 (Ceramide synthase 5) are highly conserved among eukaryotes and are involved in de novo ceramide synthesis (Mizutani et al., Biochem. J.,2005, 390: 263-271 ).They form a group of six enzymes (CERS1 to CERS6) that catalyse the formation of dihydroceramide or ceramide through N-acylation of dihydrosphingosine or sphingosine. Different ceramide synthases use a restricted subset of acyl-CoAs, producing ceramides with specific fatty acid chain lengths. CERS5 synthesizes mostly C16-Ceramides (Cingolani et al., Chemistry and Physics of Lipids, 2016, 197: 25-32). Various ceramide synthase isoforms such as CERS5 are implicated as drivers of obesity, insulin resistance, and diabetes. It has been shown that pharmacological and genetic interventions that prevent de novo ceramide synthesis ameliorate many critical features of obesity-related diseases such as insulin resistance, atherosclerosis, and cardiomyopathy (Turpin et al., Cell Metabolism, 2014, (20)4: 678-686).

Gosejacob et al. reported that CERS5 Is essential to maintain C16:0-ceramide pools and ontributes to the development of diet-induced obesity. It was shown that the loss of CerS5 is associated with reduced weight gain and improved systemic health, including maintenance of glucose homeostasis and reduced white adipose tissue inflammation after high fat diet challenge. It was concluded that reduction of endogenous C16:0-ceramide by genetic inhibition of CerS5 would ameliorate obesity and its comorbidities (Gosejakob et al., The Journal of Biological Chemistry, 2016, 291 , 6989-7003).

It is known that ceramide levels decrease in various cancers (see e.g. Cingolani et al., Chemistry and Physics of Lipids, 2016, 197: 25-32). However, high CERS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer (Fitzgerald et al., J Pathol Clin Res. 2015 Jan; 1 (1 ): 54-65). US2012/328602 discloses that inhibitors of ceramide biosynthesis can be used for preventing or delaying physical dependence on opioid drug, e.g. CERS5 inhibitors (such as antisense molecules which target the CERS5 transcript).

Mullen et al. describes the use of small interfering RNAs against the six CerS family members in order to specifically reduce the expression of each isoform in MCF-7 breast adenocarcinoma cells (Mullen et al., J Lipid Res. 2011 Jan; 52(1 ): 68-77).

Various small molecule inhibitors of ceramide synthases have been identified so far. E.g., FB1 is a well characterized inhibitor and belongs to a family of toxins of fungal origin which are food contaminants and cause liver and kidney toxicity, neurotoxicity, immunological disorders and cancer. The toxic effect of FB1 is directly related to its role as a Ceramide synthase inhibitor, with the subsequent accumulation of sphingoid long chain bases. Other well characterized ceramide synthase inhibitors are aminopentol, australifungin, 1- deoxyphingosin and FTY720. However, there are currently no isoform-specific ceramide synthase inhibitors (Cingolani et al., Chemistry and Physics of Lipids, 2016, 197: 25-32).

We have screened a large number of LNA gapmers targeting mouse and human CERS5 and identified sequences and compounds which are particularly potent and effective to specifically target for CERS5 antisense in vitro (human and mouse cells).

There is a need for therapeutic agents which can inhibit CERS5 specifically.

OBJECTIVE OF THE INVENTION

The inventors have identified particularly effective regions of the CERS5 transcript ( CERS5 ) for antisense inhibition in vitro or in vivo, and provides for antisense oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the CERS5 premRNA or mature mRNA. The present invention identifies oligonucleotides which inhibit human CERS5 which are useful in the treatment of a range of medical disorders including obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

STATEMENT OF THE INVENTION The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human CERS5 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human CERS5 target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully

complementary, to a sequence selected from the group consisting of SEQ ID NO 15, 16, 17, 18, 19, 20, 21 and 22.

complementary to SEQ ID NO 15.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90%

complementary, such as fully complementary, to SEQ ID NO 15 wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90%

complementary, such as fully complementary, to SEQ ID NO 15, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 15, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 15, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a sequence selected from the group consisting of SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5. The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1 1 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 13, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 14, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript. The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 23, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 24, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human CERS5 transcript in a cell which is expressing human CERS5 transcript.

The oligonucleotide of the invention as referred to or claimed herein may be in the form of a pharmaceutically acceptable salt.

The invention provides for a conjugate comprising the oligonucleotide according to the invention, and at least one conjugate moiety covalently attached to said oligonucleotide.

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

The invention provides for an in vivo or in vitro method for modulating CERS5 expression in a target cell which is expressing CERS5, said method comprising administering an oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount to said cell.

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

In some embodiments, the disease is selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in medicine.

The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

The invention provides for the use of the oligonucleotide, conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : Testing in vitro efficacy of various antisense oligonucleotides targeting human and mouse CERS5 mRNA in A549 and HeLa cell lines at single concentration.

Figure 2: Comparison of in vitro efficacy for antisense oligonucleotides targeting human CERS5 mRNA in A549 and HeLa cell lines at single concentration shows good correlation. Two motifs with very efficient targeting are highlighted.

Figure 3: Testing selected oligonucleotides targeting human (and mouse) CERS5 mRNA in vitro for concentration dependent potency and efficacy in HeLa cell line.

Figure 4: Testing selected oligonucleotides targeting human (and mouse) CERS5 mRNA in vitro for concentration dependent potency and efficacy in A549 cell line.

Figure 5: Testing selected oligonucleotides targeting (human and) mouse Cers5 mRNA in vitro for concentration dependent potency and efficacy in RAW264.7 cell line.

Figure 6: Mouse in vivo efficacy: remaining Cers5 mRNA transcript in mouse tissues after 16 days of treatment, Intravenous IV (tail vein).

Figure 7: Trivalent GalNAc conjugates. The wavy line represents the covalent bond to the phosphodiester linkage at the 5’ end of the oligonucleotide. DEFINITIONS

Oligonucleotide

The term“oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated. The oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.

Antisense oligonucleotides

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

Contiguous Nucleotide Sequence

The term“contiguous nucleotide sequence” refers to the region of the oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term“contiguous nucleobase sequence” and the term“oligonucleotide motif sequence”.

In some embodiments all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence. In some embodiments the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. Adventurously, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid. Nucleotides

Nucleotides 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. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as“units” or“monomers”.

Modified nucleoside

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

Modified internucleoside linkages

The term“modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. The oligonucleotides of the invention may therefore comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F’.

In an embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester.

A preferred modified internucleoside linkage is phosphorothioate.

Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

Nuclease resistant linkages, such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F’ for gapmers. Gapmer oligonucleotides may, in some embodiments comprise one or more phosphodiester linkages in region F or F’, or both region F and F’, which the internucleoside linkage in region G may be fully phosphorothioate.

Advantageously, all the internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.

It is recognized that, as disclosed in EP2 742 135, antisense oligonucleotide may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate), for example alkyl phosphonate / methyl phosphonate internucleosides, which according to EP2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate gap region.

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 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 vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid

Chemistry Suppl. 37 1.4.1.

In a 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 nucleobased 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 U, 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 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 with modified nucleosides.

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

Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1 ).

The term“% complementary” as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are complementary to (/^'.e. form Watson Crick base pairs with) a contiguous sequence of nucleotides, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid or target sequence). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing 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.

Preferably, insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.

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

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 bases that are identical (a match) between two sequences (e.g. 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 aligned region 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).

Hybridization

The term“hybridizing” or“hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T_m) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T_m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy AG° is a more accurate representation of binding affinity and is related to the dissociation constant (K_d) of the reaction by AG°=-RTIn(K_d), 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 1 M, 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 a!., 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 34:11211-1 1216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°. The oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below - 20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.

In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or- 16 to -27 kcal such as -18 to -25 kcal.

Target nucleic acid

According to the present invention, the target nucleic acid is a nucleic acid which encodes mammalian CERS5 and may for example be a gene, a CERS5 RNA, a mRNA, a pre-mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an CERS5 target nucleic acid.

Suitably, the target nucleic acid encodes an CERS5 protein, in particular mammalian CERS5, such as the human CERS5 gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 15, 16, 17, 18, 19, 20, 21 or 22. In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO 15, 16, 17, 18, 19, 20, 21 or 22, or naturally occurring variants thereof (e.g. CERS5 sequences encoding a mammalian CERS5 protein).

If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro application, the oligonucleotide of the invention is typically capable of inhibiting the expression of the CERS5 target nucleic acid in a cell which is expressing the CERS5 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the CERS5 target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other non- complementary terminal nucleotides (e.g. region D’ or D”). The target nucleic acid is a messenger RNA, such as a mature mRNA or a pre-mRNA which encodes mammalian CERS5 protein, such as human CERS5, e.g. the human CERS5 pre-mRNA sequence, such as that disclosed as SEQ ID NO 15, or CERS5 mature mRNA, such as that disclosed as SEQ ID NO 16, 17, 18, 19, 20, 21 or 22. SEQ ID NO 15 to 22 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 16.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 17.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 18.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 19.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 20.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 21.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 22.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15 and at least one of, such as two, three, four, five, six or seven of SEQ ID NO 16, 17, 18, 19, 20, 21 and 22.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15 16, 17, 18, 19, 20, 21 and 22.

Target Sequence

The term“target sequence” as used herein refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention. In some embodiments, the target sequence consists of a region on the target nucleic acid which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.

Herein are provided numerous target sequence regions, as defined by regions of the human CERS5 pre-mRNA (using SEQ ID NO 15 as a reference) which may be targeted by the oligonucleotides of the invention. In some embodiments the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a sub-sequence of the target nucleic acid, such as a target sequence described herein.

The oligonucleotide comprises a contiguous nucleotide sequence which are complementary to a target sequence present in the target nucleic acid molecule. The contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 10 contiguous nucleotides, such as 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.

Target Sequence Regions

The inventors have identified particularly effective sequences of the CERS5 target nucleic acid which may be targeted by the oligonucleotide of the invention.

In some embodiments the target sequence is SEQ ID NO 1 1.

In some embodiments the target sequence is SEQ ID NO 12.

In some embodiments the target sequence is SEQ ID NO 13.

In some embodiments the target sequence is SEQ ID NO 14.

In some embodiments the target sequence is SEQ ID NO 23.

In some embodiments the target sequence is SEQ ID NO 24.

In some embodiments the target sequence is SEQ ID NO 25.

SEQ ID NO 1 1 : CTT ACT AAATT CTGT GAAAG CAT GT AAGT A (15)

SEQ ID NO 12: TGGAATT AGATTT CTCTGGTCGGT GAGT CT (15)

SEQ ID NO 13: AAATTCTGTGAAAGCATGT (15, 16, 17, 18, 19, 20, 21 , 22)

SEQ ID NO 14: AGATTT CTCTGGTCGG (15)

SEQ ID NO 23: CTT ACT AAATT CTGT GAAAG CAT GT (15, 16, 17, 18, 19, 20, 21 , 22) SEQ ID NO 24: AGATTTCTCTGGTCG (15, 16, 17, 18, 19, 20, 21 , 22) SEQ ID NO 25: T G GAATT AG ATTT CTCTGGTCG (15, 16, 17, 18, 19, 20, 21 , 22)

(numbers in brackets refer to the SEQ ID of CERS5 premRNA or mRNA transcript in which the target sequence is found). In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an exon region of SEQ ID NO 15, selected from the group consisting if Ex_1 - Ex_10.

In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 15, selected from the group consisting of 1 12 - 433; 23477 - 23582; 24330 - 24460; 25424 - 25481 ; 28917 - 28967; 29714 - 29806; 31465 - 31593; 31696 - 31802; 32832 - 32988; 36840 - 37736; In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an intron region of SEQ ID NO 15, selected from the group consisting of lnt_1 - lnt_9.

In a further aspect, the invention provides for an antisense oligonucleotide, 10-30

nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 15, selected from the group consisting 433 - 23477; 23582 - 24330; 24460 - 25424; 25481 - 28917; 28967 - 29714; 29806 - 31465; 31593 - 31696; 31802 - 32832; 32988 -

36840;

nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 15, selected from the group consisting of 230 - 244; 279 - 293; 310 - 325; 381 - 397; 501 - 515; 1018 - 1032; 1190 - 1213; 1204 - 1223; 1324 - 1339; 1441 - 1455; 2976 - 2992; 3101 - 3115; 3245 - 3260; 3250 - 3268; 3545 - 3562; 3556 - 3574; 3766 - 3787; 3877 - 3891 4213 - 4228; 4808 - 4823; 4924 - 4938; 5327 - 5349; 5360 - 5374; 5537 - 5552; 5632 - 5647

5641 - 5664; 5656 - 5673; 5733 - 5749; 6808 - 6826; 7215 - 7232; 7223 - 7239; 7478 - 7494

7818 - 7833; 8204 - 8221 ; 8344 - 8360; 8461 - 8477; 8882 - 8897; 9448 - 9475; 1 1096 -

1 11 13; 1 11 14 - 11 130; 11 195 - 11209; 11413 - 11437; 11428 - 11442; 11770 - 11800;

12645 - 12662; 12745 - 12760; 12773 - 12787; 12845 - 12859; 13164 - 13179; 13560 - 13582; 13645 - 13659; 13846 - 13860; 14132 - 14147; 14238 - 14266; 14714 - 14732;

15042 - 15056; 17034 - 17056; 17783 - 17797; 18492 - 18507; 18564 - 18589; 18587 - 18606; 18605 - 18632; 18921 - 18937; 19188 - 19206; 19229 - 19243; 19576 - 19590;

19627 - 19643; 20106 - 20128; 20380 - 20398; 20668 - 20690; 22376 - 22399; 23471 - 23488; 24040 - 24056; 24055 - 24069; 24384 - 24430; 24444 - 24463; 24967 - 24988;

251 16 - 25131 ; 25228 - 25244; 25417 - 25435; 25467 - 25489; 25575 - 25597; 26677 - 26696; 27224 - 27250; 27721 - 27739; 28023 - 28050; 28964 - 28979; 29701 - 29715;

29771 - 29789; 31046 - 31064; 3131 1 - 31329; 31460 - 31475; 31706 - 31720; 31740 - 31757; 32183 - 32225; 32826 - 32843; 32934 - 32953; 33441 - 33462; 34269 - 34285;

34403 - 34418; 34423 - 34440; 35127 - 35143; 35247 - 35261 ; 35956 - 35978; 36089 - 36103; 36105 - 36122; 36142 - 36156; 36867 - 36891 ; 37088 - 37107; 37257 - 37271 ;

37331 - 37348; and 38008 - 38033.

nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 15, selected from the group consisting of 45 - 77; 85 - 106; 1 13 - 139; 144 - 220; 221 - 308; 320 - 355; 363 - 430; 478 - 499; 501 - 524; 526 - 547; 557 - 582; 584 - 600; 606 - 643; 645 - 664: 666 - 691 : 700 - 733: 735 - 762: 764 - 799: 814 - 840: 849 - 881 : 903 - 921 : 932 -

1015; 1017 - 1038; 1054 - 11 15; 1 147 - 1174; 1 179 1220; 1221 - 1243; 1248 - 1266; 1293 - 1328; 1319 - 1337; 1341 - 1359; 1360 - 1390; 1383 1402; 1391 - 1412; 1414 - 1529; 1531 - 1547; 1550 - 1579; 1579 - 1593; 1594 - 1642; 1644 1670; 1672 - 1690; 1698 - 1724; 1737 - 1793; 1821 - 1866; 1906 - 1928; 1932 - 1954; 1960 2003; 2001 - 2058; 2079 - 2098; 2087 - 2102; 2094 - 2108; 2100 - 21 15; 2106 - 2128; 2120 2159; 2159 - 2178; 2168 - 2188; 2180 - 2197; 2197 - 2211 ; 2218 - 2232; 2234 - 2252; 2257 2291 ; 2313 - 2334; 2341 - 2355; 2344 - 2380; 2438 - 2465; 2467 - 2515; 2517 - 2534; 2524 2552; 2567 - 2585; 2587 - 2617; 2619 - 2647; 2649 - 2686; 2689 - 2719; 2735 - 2774; 2776 2852; 2854 - 2876; 2881 - 2903; 2923 - 2957; 2959 - 2978; 2993 - 3015; 3066 - 3080; 3095 31 12; 3122 - 3147; 3174 - 3192; 3189 - 3205; 3194 - 3214; 3221 - 3235; 3245 - 3260; 3251 3271 ; 3276 - 3330; 3329 - 3401 ; 3403 - 3419; 3413 - 3429; 3427 - 3478; 3480 - 3500; 3490 3513; 3518 - 3535; 3538 - 3582; 3585 - 3599; 3591 - 3632; 3673 - 3689; 3691 - 3706; 3710 3731 ; 3729 - 3746; 3758 - 3781 ; 3787 - 3801 ; 3853 - 3871 ; 3953 - 3968; 3963 - 3977; 4049 4098; 4095 - 4121 ; 4132 - 4172; 4189 - 4255; 4246 - 4265; 4257 - 4278; 4283 - 4298; 4306 4328; 4324 - 4341 ; 4342 - 4378; 4377 - 441 1 ; 4433 - 4447; 4451 - 4520; 4522 - 4536; 4535 4552; 4544 - 4563; 4571 - 4638; 4642 -

12686 - 12702; 12728 - 12746; 12764 - 12847; 12871 - 12931 ; 12933 - 13019; 13028 - 13046; 13046 - 13062; 13068 - 13097; 13099 - 13124; 13126 - 13164; 13162 - 13177; 13184 - 13199; 13208 - 13229; 13232 - 13246; 13251 - 13266; 13258 - 13296; 13294 - 13308; 13297 - 1331 1 ; 13313 - 13327; 13332 - 13375; 13365 - 13382; 13398 - 13413; 13406 - 13420; 13409 - 13444; 13450 - 13484; 13490 - 13519; 13521 - 13535; 13537 - 13554; 13556 - 13601 ; 13597 - 13615; 13624 - 13663; 13665 - 13687; 13686 - 13702; 13722 - 13744; 13746 - 13772; 13765 - 13807; 13805 - 13836; 13838 - 13852; 13843 - 13862; 13852 - 13870; 13872 - 13925; 13927 - 13949; 13976 - 14008; 14016 - 14033; 14043 - 14065; 14055 - 14097; 14099 - 14113; 14110 - 14129; 14130 - 14180; 14170 - 14192; 14202 - 14226; 14233 - 14266; 14268 - 14320; 14322 - 14342; 14344 - 14370; 14372 - 14403; 14405 - 14422; 14447 - 14481 ; 14478 - 14531 ; 14541 - 14592; 14594 - 14614; 14609 - 14642; 14648 - 14693; 14683 - 14698; 14700 - 14739; 14747 - 14766; 14768 - 14782; 14802 - 14822; 14824 - 14842; 14867 - 14890; 14892 - 14937; 14939 - 14972; 14965 - 14994; 14988 - 15031 ; 15035 - 15063; 15065 - 15093; 15095 - 15145; 15147 - 15171 ; 15173 - 15191 ; 15193 - 15248; 15279 - 15390; 15406 - 15421 ; 15463 - 15501 ; 15504 - 15576; 15578 - 15592; 15608 - 15632; 15621 - 15640; 15656 - 15682; 15703 - 15720; 15747 - 15763; 15764 - 15778; 15790 - 15817; 15821 - 15852; 15855 - 15877; 15879 - 15905; 15932 - 15950; 15960 - 16003; 16005 - 16062; 16092 - 16126; 16145 - 16193; 16195 - 16246; 16248 - 16280; 16282 - 16316; 16318 - 16349; 16356 - 16388; 16392 - 16420; 16422 - 16476; 16479 - 16508; 1651 1 - 16530; 16532 - 16552; 16564 - 16613; 16647 - 16663; 16664 - 16689; 16693 - 16719; 16736 - 16755; 16757 - 16778; 16791 - 16809; 16822 - 16890; 16896 - 16933; 16950 - 16969; 17025 - 17063; 17084 - 17099; 17114 - 17133; 17149 - 17178; 17182 - 17240; 17235 - 17250; 17239 - 17279; 17294 - 17313; 17315 - 17331 ; 17328 - 17349; 17347 - 17397; 17399 - 17423; 17425 - 17444; 17461 - 17477; 17479 - 17500; 17512 - 17539; 17539 - 17553; 17550 - 17568; 17557 - 17577; 17570 - 17584; 17573 - 17588; 17592 - 17645; 17647 - 17673; 17664 - 17684; 17675 - 17690; 17692 - 17708; 17707 - 17726; 17749 - 17766; 17764 - 17779; 17769 - 17791 ; 17799 - 17874; 17901 - 17940; 17948 - 18030; 18039 - 18069; 18071 - 18094; 18096 - 18115; 18117 - 18165; 18167 - 18202; 18213 - 18240; 18242 - 18264; 18269 - 18286; 18288 - 18310; 18341 - 18363; 18382 - 18401 ; 18403 - 18506; 18509 - 18529; 18542 - 18556; 18587 - 18606; 18596 - 18612; 18605 - 18642; 18631 - 18646; 18656 - 18679; 18679 - 18695; 18706 - 18732; 18768 - 18815; 18813 - 18828; 18817 - 18838; 18832 - 18861 ; 18917 - 18935; 18939 - 18953; 18957 - 18981 ; 18983 - 19006; 19015 - 19043; 19032 - 19047; 19049 - 19063; 19065 - 19180; 19182 - 19212; 19222 - 19249; 19251 - 19279; 19287 - 19305; 19315 - 19395; 19397 - 19412; 19414 - 19442; 19435 - 19461 ; 19469 - 19523; 19521 - 19581 ; 19585 - 19662; 19669 - 19718; 19720 - 19768; 19770 - 19786; 19788 - 19807; 19808 - 19849; 19851 - 19904; 19906 - 19938; 19941 - 19966; 19968 - 20013; 20028 - 20045; 20047 - 20063; 20069 - 20093; 20106 - 20137; 20129 - 20147; 20145 - 20159; 20178 - 20199; 20205 - 20227; 20229 - 20258; 20281 - 20348; 20338 - 20360; 20349 - 20364; 20357 - 20407; 20423 - 20442; 20448 - 20469; 20474 - 20488; 20488 - 20518; 20520 - 20538; 20539 - 20582; 20609 - 20628; 20647 - 20664; 20666 - 20703; 20703 - 20731 ; 20731 - 20780; 20790 - 20817; 20808 - 20824; 20818 - 20873; 20872 - 20903; 20893 - 20945; 20972 - 21036; 21038 - 21064; 21085 - 21 124; 21 126 - 21 144; 21 162 - 21 194; 21 198 - 21237; 21239 - 21259; 21296 - 21310; 21313 - 21358; 21374 - 21398; 21430 - 21448; 21450 - 21509; 21521 - 21536; 21537 - 21556; 21565 - 21588; 21590 - 21620; 21647 - 21670; 21674 - 21705; 21725 - 21745; 21747 - 21766; 21774 - 21819; 21831 - 21861 ; 21875 - 21907; 21909 - 21931 ; 21933 - 21983; 21996 - 22017; 22032 - 22055; 22062 - 22165; 22166 - 22181 ; 22221 - 22270; 22272 - 22318; 22331 - 22347; 22347 - 22369; 22374 - 22409; 22423 - 22447; 22441 - 22470; 22473 - 22526; 22516 - 22541 ; 22547 - 22588; 22604 - 22684; 22686 - 22708; 22710 - 22765; 22767 - 22814; 22816 - 22833; 22847 - 22876; 22878 - 22892; 22912 - 22937; 22939 - 23022; 23029 - 23054; 23056 - 23077; 23085 - 23099; 23101 - 23126; 23128 - 23151 ; 23171 - 2321 1 ; 23213 - 23241 ; 23245 - 23261 ; 23253 - 23270; 23263 - 23278; 23297 - 23335; 23337 - 23356; 23358 - 23387; 23389 - 23422; 23458 - 23476; 23478 - 23501 ; 23504 - 23566; 23577 - 23609; 23619 - 23640; 23664 - 23688; 23711 - 23777; 23789 - 23815; 23817 - 23833; 23851 - 23879; 23881 - 23896; 23913 - 23933; 23944 - 23990; 23992 - 2401 1 ; 24000 - 24022; 24029 - 24106; 24108 - 24123; 24135 - 24204; 24205 - 24236; 24237 - 24253; 24326 - 24410; 24412 - 24439; 24441 - 24467; 24478 - 24497; 24499 - 24524; 24536 - 24558; 24560 - 24576; 24578 - 24594; 24596 - 24642; 24682 - 24833; 24835 - 24856; 24871 - 24907; 24909 - 24926; 24966 - 24997; 24994 - 25009; 25000 - 25020; 25013 - 25030; 25037 - 25059; 25065 - 251 10; 251 15 - 25144; 25145 - 25177; 25179 - 25202; 25206 - 25220; 25209 - 25241 ; 25238 - 25253; 25255 - 25295; 25307 - 25333; 25343 - 25361 ; 25385 - 25404; 25416 - 25516; 25518 - 25540; 25543 - 25569; 25563 - 25606; 25603 - 25621 ; 25622 - 25636; 25636 - 25662; 25670 - 25687; 25689 - 25721 ; 25729 - 25777; 25767 - 25807; 25796 - 25822; 25814 - 25828; 25823 - 25844; 25853 - 25868; 25878 - 25892; 25884 - 25918; 25914 - 25932; 25934 - 25986; 26008 - 26022; 26031 - 26105; 26107 - 26128; 26144 - 26162; 26164 - 26186; 26189 - 2621 1 ; 26205 - 26243; 26252 - 26293; 26283 - 26301 ; 26292 - 26306; 26338 - 26359; 26372 - 26386; 26384 - 26402; 26400 - 26415; 26438 - 26482; 26490 - 26505; 26507 - 26524; 26558 - 26585; 26607 - 26629; 26640 - 26682; 26678 - 26696; 26713 - 26740; 26745 - 26782; 26797 - 26853; 26855 - 26886; 26909 - 26937; 26960 - 27008; 27020 - 27046; 27061 - 27089; 27104 - 27118; 2711 1 - 27147; 27141 - 27156; 27159 - 27181 ; 27197 - 2721 1 ; 27217 - 27251 ; 27254 - 27280; 27282 - 27309; 27308 - 27329; 27342 - 27376; 27378 - 27405; 27437 - 27481 ; 27483 - 27528; 27564 - 27585; 27591 - 27609; 27622 - 27651 ; 27667 - 27682; 27698 - 27731 ; 27721 - 27739; 27731 - 27784; 27786 - 27830; 27832 - 27857; 27850 - 27867; 27870 - 27921 ; 27944 - 27974; 27977 - 28017; 28007 - 28021 ; 28035 - 28050; 28055 - 28082; 28076 - 28091 ; 28096 - 28136; 28138 - 28159; 28167 - 28185; 28197 - 28218; 28210 - 28225; 28218 - 28232; 28249 - 28285; 28290 - 2831 1 ; 28306 - 28334; 28336 - 28380; 28397 - 28413; 28437 - 28479; 28486 - 28530; 28535 - 28575; 28582 - 28596; 28612 - 28635; 28637 - 28662; 28698 - 28720; 28722 - 28739; 28759 - 28780; 28792 - 28810; 28817 - 28849; 28855 - 28888; 28890 - 28993; 29030 - 29058; 29060 - 29075; 29104 - 29125; 29133 - 29170; 29172 - 29192; 29194 - 29221 ; 29256 - 29289; 29291 - 29317; 29328 - 29345; 29347 - 29364; 29368 - 29390; 29388 - 29406; 29417 - 29465; 29467 - 29494; 29496 - 29514; 29531 - 29589; 29591 - 29607; 29609 - 29659; 29682 - 29709; 2971 1 - 29734; 29736 - 29820; 29844 - 29879; 29881 - 29902; 29904 - 29942; 29944 - 30013; 30015 - 30083; 30093 - 30125; 30138 - 30159; 30159 - 30183; 30200 - 30217; 30219 - 30252; 30254 - 30272; 30274 - 30299; 30301 - 30345; 30363 - 30379; 30393 - 30414; 30416 - 30459; 30461 - 30505; 30507 - 30599; 30601 - 30659; 30671 - 30699; 30701 - 30721 ; 30726 - 30771 ; 30781 - 30815; 30817 - 30838; 30843 - 30860; 30915 - 30948; 30976 - 31020; 31023 - 31074; 31077 - 31 105; 31 105 - 31 129; 31 144 - 31 189; 31205 - 31236; 31242 - 31287; 31289 - 31333; 31340 - 31356; 31363 - 31377; 31382 - 31398; 31400 - 31420; 31422 - 31508; 31510 - 31543; 31546 - 31604; 31610 - 31628; 31638 - 31654; 31657 - 31703; 31705 - 31721 ; 31723 - 31751 ; 31753 - 31781 ; 31783 - 31855; 31853 - 31904; 31910 - 31950; 31952 - 31966; 31970 - 31998; 32000 - 32041 ; 32063 - 32087; 32089 - 32113; 32126 - 32144; 32146 - 32185; 32472 - 32509; 32531 - 32548; 32550 - 32564; 32587 - 32630; 32637 - 32673; 32675 - 32700; 32702 - 32761 ; 32771 - 32785; 32813 - 32922; 32924 - 33016; 33024 - 33047; 33049 - 33124; 33138 - 33155; 33157 - 33195; 33186 - 33210; 33244 - 33293; 33288 - 33318; 33323 - 33350; 33360 - 33374; 33367 - 33382; 33403 - 33418; 33412 - 33466; 33464 - 33482; 33477 - 33503; 33505 - 33529; 33532 - 33546; 33537 - 33582; 33571 - 33591 ; 33602 - 33630; 33632 - 33671 ; 33672 - 33689; 33704 - 33737; 33744 - 33774; 33793 - 33826; 33828 - 33877; 33879 - 33914; 33910 - 33929; 33933 - 33956; 33962 - 33993; 3401 1 - 34031 ; 34035 - 34054; 34060 - 34080; 34083 - 34097; 34099 - 34136; 34138 - 34165; 34167 - 34196; 34198 - 34234; 34243 - 34304; 34324 - 34358; 34361 - 34402; 34404 - 34454; 34455 - 34491 _; 34493 . 34604; 34606 - 34630; 34632 - 34664; 34666 - 34707; 34709 - 34732; 34756 - 34777; 34803 - 34835; 34837 - 34855; 34877 - 34944; 34946 - 34962; 34964 - 35003; 35012 - 35065; 35097 - 35116; 35115 - 35129; 35122 - 35138; 35127 - 35141 ; 35145 - 35176; 35182 - 35204; 35221 - 35262; 35263 - 35287; 35282 - 35313; 35327 - 35357; 35361 - 35405; 35428 - 35444; 35449 - 35517; 35519 - 35544; 35578 - 35608; 35618 - 35657; 35668 - 35696; 35703 - 35718; 35734 - 35759; 35772 - 35791 ; 35793 - 35810; 35832 - 35894; 35896 - 35928; 35930 - 35944; 35941 - 35955; 35945 - 35996; 36003 - 36025; 36018 - 36049; 36051 - 36102; 36114 - 36129; 36119 - 36163; 36166 - 36201 ; 36200 - 36214; 36203 - 36255; 36258 - 36273; 36275 - 36334; 36355 - 36422; 36424 - 36443; 36445 - 36463; 36490 - 36527; 36535 - 36560; 36563 - 36599; 36601 - 36619; 36628 - 36647; 36649 - 36672; 36675 - 36690; 36692 - 36714; 36715 - 36775; 36777 - 36791 ; 36788 - 36802; 36805 - 36835; 36846 - 36916; 36918 - 36958; 36960 - 36999; 37014 - 37031 ; 37033 - 37071 ; 37093 - 37119; 37121 - 37144; 37150 - 37170; 37172 - 37199; 37201 - 37218; 37227 - 37253; 37255 - 37271 ; 37282 - 37298; 37308 - 37349; 37373 - 37392; 37410 - 37441 ; 37451 - 37470; 37482 - 37529; 37533 - 37562; 37563 - 37599; 37601 - 37628; 37630 - 37664; 37666 - 37697; 37919 - 37953; 37967 - 37989; 38010 - 38036.

Target Cell

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

In preferred embodiments the target cell expresses CERS5 mRNA, such as the CERS5 pre- mRNA, e.g. SEQ ID NO 15, or CERS5 mature mRNA (SEQ ID NO 16, 17, 18, 19, 20, 21 and/or 22). The poly A tail of CERS5 mRNA is typically disregarded for antisense oligonucleotide targeting.

Naturally occurring variant

The term“naturally occurring variant” refers to variants of CERS5 gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof.

The homo sapiens CERS5 gene is located at chromosome 12, 50129306..50167533, complement (NC_000012.12, Gene ID 91012).

In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian CERS5 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 15, 16, 17, 18, 19, 20, 21 and 22. In some embodiments the naturally occurring variants have at least 99% homology to the human CERS5 target nucleic acid of SEQ ID NO 15.

Modulation of expression

The term“modulation of expression” as used herein is to be understood as an overall term for an oligonucleotide’s ability to alter the amount of CERS5 protein or CERS5 mRNA when compared to the amount of CERS5 or CERS5 mRNA prior to administration of the oligonucleotide. Alternatively modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).

One type of modulation is an oligonucleotide’s ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of CERS5, e.g. by degradation of CERS5 mRNA.

High affinity modified nucleosides

A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the 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 result 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 oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA. Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO201 1/017521 ) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

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

2’ sugar modified nucleosides.

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

Indeed, much focus has been spent on developing 2’ 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’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’- alkoxy-RNA, 2’-0-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.

In relation to the present invention 2’ substituted does not include 2’ bridged molecules like

LNA.

Locked Nucleic Acids (LNA)

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

Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO

00/66604, WO 98/039352 , WO 2004/046160, WO 00/047599, WO 2007/134181 , WO

2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 2011/156202, WO 2008/154401 , WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic &

Med.Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 , and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

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

Scheme 1 :

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

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

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a 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 Example 91 - 95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland. Gapmer

The antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof may be a gapmer. The antisense gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions a 5’-flank, a gap and a 3’-flank, F-G-F’ in the‘5 -> 3’ orientation. The“gap” region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5’ flanking region (F) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and by a 3’ flanking region (F’) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides. The one or more sugar modified nucleosides in region F and F’ enhance the affinity of the oligonucleotide for the target nucleic acid ( i.e . are affinity enhancing sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in region F and F’ are 2’ sugar modified nucleosides, such as high affinity 2’ sugar modifications, such as independently selected from LNA and 2’-MOE.

In a gapmer design, the 5’ and 3’ most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5’ (F) or 3’ (F’) region respectively. The flanks may further defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5’ end of the 5’ flank and at the 3’ end of the 3’ flank.

Regions F-G-F’ form a contiguous nucleotide sequence. Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F-G-F’.

The overall length of the gapmer design F-G-F’ may be, for example 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to17, such as 16 to18 nucleosides.

By way of example, the gapmer oligonucleotide of the present invention can be represented by the following formulae:

FI-8-G₅-I6-F’I-₈, such as

F1-8-G7-16-F 2-8

with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length.

Regions F, G and F’ are further defined below and can be incorporated into the F-G-F’ formula. Gapmer - Region G

Region G (gap region) of the gapmer is a region of nucleosides which enables the oligonucleotide to recruit RNaseH, such as human RNase H1 , typically DNA nucleosides. RNaseH is a cellular enzyme which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule. Suitably gapmers may have a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5 - 16 contiguous DNA nucleosides, such as 6 - 15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8 - 12 contiguous DNA nucleotides, such as 8 - 12 contiguous DNA nucleotides in length. The gap region G may, in some embodiments consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous DNA nucleosides. One or more cytosine (C) DNA in the gap region may in some instances be methylated (e.g. when a DNA c is followed by a DNA g) such residues are either annotated as 5-methyl-cytosine (^meC). In some embodiments the gap region G may consist of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 contiguous phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages.

Whilst traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides which allow for RNaseH recruitment when they are used within the gap region. Modified nucleosides which have been reported as being capable of recruiting RNaseH when included within a gap region include, for example, alpha-L-LNA, C4’ alkylated DNA (as described in PCT/EP2009/050349 and Vester et a!., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661 ), UNA

(unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst, 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked“sugar” residue. The modified nucleosides used in such gapmers may be nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region, i.e. modifications which allow for RNaseH recruitment). In some embodiments the DNA Gap region (G) described herein may optionally contain 1 to 3 sugar modified nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region.

Region G -“Gap-breaker”

Alternatively, there are numerous reports of the insertion of a modified nucleoside which confers a 3’ endo conformation into the gap region of gapmers, whilst retaining some RNaseH activity. Such gapmers with a gap region comprising one or more 3’endo modified nucleosides are referred to as“gap-breaker” or“gap-disrupted” gapmers, see for example WO2013/022984. Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RNaseH recruitment. The ability of gapbreaker

oligonucleotide design to recruit RNaseH is typically sequence or even compound specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses“gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA. Modified nucleosides used within the gap region of gap- breaker oligonucleotides may for example be modified nucleosides which confer a 3’endo confirmation, such 2’ -O-methyl (OMe) or 2’-0-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2’ and C4’ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

As with gapmers containing region G described above, the gap region of gap-breaker or gap-disrupted gapmers, have a DNA nucleosides at the 5’ end of the gap (adjacent to the 3’ nucleoside of region F), and a DNA nucleoside at the 3’ end of the gap (adjacent to the 5’ nucleoside of region F’). Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5’ end or 3’ end of the gap region. Exemplary designs for gap-breaker oligonucleotides include

wherein region G is within the brackets [D_n-E_r- D_m], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (the gap-breaker or gap-disrupting nucleoside), and F and F’ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length. In some embodiments, region G of a gap disrupted gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNaseH recruitment.

Gapmer - flanking regions, F and F’

Region F is positioned immediately adjacent to the 5’ DNA nucleoside of region G. The 3’ most nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F’ is positioned immediately adjacent to the 3’ DNA nucleoside of region G. The 5’ most nucleoside of region F’ is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F is 1 - 8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length. Advantageously the 5’ most nucleoside of region F is a sugar modified nucleoside. In some embodiments the two 5’ most nucleoside of region F are sugar modified nucleoside. In some embodiments the 5’ most nucleoside of region F is an LNA nucleoside. In some embodiments the two 5’ most nucleoside of region F are LNA nucleosides. In some embodiments the two 5’ most nucleoside of region F are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 5’ most nucleoside of region F is a 2’ substituted nucleoside, such as a MOE nucleoside.

Region F’ is 2 - 8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length. Advantageously, embodiments the 3’ most nucleoside of region F’ is a sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are LNA nucleosides. In some embodiments the 3’ most nucleoside of region F’ is an LNA nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 3’ most nucleoside of region F’ is a 2’ substituted nucleoside, such as a MOE nucleoside. It should be noted that when the length of region F or F’ is one, it is advantageously an LNA nucleoside.

In some embodiments, region F and F’ independently consists of or comprises a contiguous sequence of sugar modified nucleosides. In some embodiments, the sugar modified nucleosides of region F may be independently selected from 2’-0-alkyl-RNA units, 2’-0- methyl-RNA, 2’-amino-DNA units, 2’-fluoro-DNA units, 2’-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2’-fluoro-ANA units.

In some embodiments, region F and F’ independently comprises both LNA and a 2’ substituted modified nucleosides (mixed wing design).

In some embodiments, region F and F’ consists of only one type of sugar modified nucleosides, such as only MOE or only beta-D-oxy LNA or only ScET. Such designs are also termed uniform flanks or uniform gapmer design.

In some embodiments, all the nucleosides of region F or F’, or F and F’ are LNA

nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET

nucleosides. In some embodiments region F consists of 1-5, such as 2-4, such as 3-4 such as 1 , 2, 3, 4 or 5 contiguous LNA nucleosides. In some embodiments, all the nucleosides of region F and F’ are beta-D-oxy LNA nucleosides. In some embodiments, all the nucleosides of region F or F’, or F and F’ are 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments region F consists of 1 , 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides. In some embodiments only one of the flanking regions can consist of 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments it is the 5’ (F) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 3’ (F’) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides. In some embodiments it is the 3’ (F’) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 5’ (F) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.

In some embodiments, all the modified nucleosides of region F and F’ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details). In some embodiments, all the modified nucleosides of region F and F’ are beta-D-oxy LNA nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).

In some embodiments the 5’ most and the 3’ most nucleosides of region F and F’ are LNA nucleosides, such as beta-D-oxy LNA nucleosides or ScET nucleosides.

In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F’ and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkages between the nucleosides of region F or F’, F and F’ are phosphorothioate internucleoside linkages.

LNA Gapmer

An LNA gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of LNA nucleosides. A beta-D-oxy gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of beta-D-oxy LNA nucleosides.

In some embodiments the LNA gapmer is of formula: [LNA]i_₅-[region G] -[LNA]_I-5, wherein region G is as defined in the Gapmer region G definition.

MOE Gapmers

A MOE gapmers is a gapmer wherein regions F and F’ consist of MOE nucleosides. In some embodiments the MOE gapmer is of design [MOE]i-e-[Region G]-[MOE] 1-8, such as [MOE]2-7-[Region G]s-i₆-[MOE] 2-7, such as [MOE]3-6-[Region G]-[MOE] 3-6, wherein region G is as defined in the Gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

Mixed Wing Gapmer

A mixed wing gapmer is an LNA gapmer wherein one or both of region F and F’ comprise a 2’ substituted nucleoside, such as a 2’ substituted nucleoside independently selected from the group consisting of 2’-0-alkyl-RNA units, 2’-0-methyl-RNA, 2’-amino-DNA units, 2’- fluoro-DNA units, 2’-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2’-fluoro- ANA units, such as a MOE nucleosides. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least one LNA nucleoside, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least two LNA nucleosides, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of region F and F’ may further comprise one or more DNA nucleosides.

Mixed wing gapmer designs are disclosed in W02008/049085 and WO2012/109395, both of which are hereby incorporated by reference.

Alternating Flank Gapmers

Oligonucleotides with alternating flanks are LNA gapmer oligonucleotides where at least one of the flanks (F or F’) comprises DNA in addition to the LNA nucleoside(s). In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F and/or F’ region are LNA nucleosides.

In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F or F’ region are LNA nucleosides, and there is at least one DNA nucleoside positioned between the 5’ and 3’ most LNA nucleosides of region F or F’ (or both region F and F’).

Region D’ or D” in an oligonucleotide

The oligonucleotide 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 the gapmer F-G-F’, and further 5’ and/or 3’ nucleosides. The further 5’ and/or 3’ nucleosides may or may not be 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 gapmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.

Region D’ and D” can be attached to the 5’ end of region F or the 3’ end of region F’, respectively to generate designs of the following formulas D’-F-G-F’, F-G-F’-D” or

D’-F-G-F’-D”. In this instance the F-G-F’ is the gapmer portion of the oligonucleotide and region D’ or D” constitute a separate part of the oligonucleotide.

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

In one embodiment the oligonucleotide of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes the gapmer.

In some embodiments, the oligonucleotide of the present invention can be represented by the following formulae:

In some embodiments the internucleoside linkage positioned between region D’ and region F is a phosphodiester linkage. In some embodiments the internucleoside linkage positioned between region F’ and region D” is a phosphodiester linkage. Conjugate

The term conjugate as used herein refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of the oligonucleotide of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particularthe conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. A the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.

In an embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, 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 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 or gapmer region F-G-F’ (region A).

In some embodiments of the invention the conjugate or oligonucleotide 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. DNA phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference) - see also region D’ or D” herein.

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 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 a preferred embodiment the linker (region Y) is a C6 amino alkyl group.

Gal N Ac Conjugates

In some embodiments, the conjugate moiety comprises or is an asialoglycoprotein receptor targeting moiety, which may include, for example galactose, galactosamine, N-formyl- galactosamine, Nacetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl- galactosamine, and N-isobutanoylgalactos-amine. In some embodiments the conjugate moiety comprises a galactose cluster, such as N-acetylgalactosamine trimer. In some embodiments, the conjugate moiety comprises a GalNAc (N-acetylgalactosamine), such as a mono-valent, di-valent, tri-valent of tetra-valent GalNAc. Trivalent GalNAc conjugates may be used to target the compound to the liver (see e.g. US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov-Dec;6(6):695-701 , W02009/126933, WO2012/089352,

WO2012/083046, WO2014/1 18267, WO2014/179620, & WO2014/179445), see also the exemplified example in figure 7. These GalNAc references and the specific conjugates used therein are hereby incorporated by reference.

In some embodiments the conjugate of the invention comprises the trilavent GalNAc conjugate disclosed in figure 7.

Exemplary conjugates of the invention include:

wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and ^mc is 5- methyl cytosine DNA, and wherein subscript s represents a phosphorothioate

internucleoside linkage, and a subscript o represents a phosphodiester internucleoside linkage, and GN2-C6 is a 5’ conjugate of formula:

wherein the wavy line represents the covalent bond to the phosphodiester linkage at the 5’ end of the oligonucleotide.

Conjugate 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 directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety to an oligonucleotide (e.g. the termini of region A or C).

In some embodiments of the invention the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region which is positioned between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and oligonucleotide is biocleavable.

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 a preferred embodiment the nuclease susceptible linker comprises between 1 and 10 nucleosides, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably between 2 and 6 nucleosides and most preferably between 2 and 4 linked nucleosides comprising at least two consecutive phosphodiester linkages, such as at least 3 or 4 or 5 consecutive phosphodiester linkages. Preferably the nucleosides are DNA or RNA. Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference).

Conjugates may also be linked to the oligonucleotide via non biocleavable linkers, or in some embodiments the conjugate may compise a non-cleavable linker which is covalently attached to the biocleavable linker. Linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety to an oligonucleotide or

biocleavable linker. Such linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. 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. Conjugate linker groups may be routinely attached to an oligonucleotide via use of an amino modified oligonucleotide, and an activated ester group on the conjugate group. 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.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to oligonucleotides, such as antisense oligonucleotides, targeting CERS5 expression.

The oligonucleotides of the invention targeting CERS5 are capable of hybridizing to and inhibiting the expression of a CERS5 target nucleic acid in a cell which is expressing the CERS5 target nucleic acid.

The CERS5 target nucleic acid may be a mammalian CERS5 mRNA or premRNA, such as a human CERS5 mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens ceramide synthase 5, RefSeqGene on chromosome 12, exemplified by NCBI Reference Sequence NC_000012.12: 50129306..50167533, GRCh38.p12 Primary

Assembly or Ensembl ENSG00000139624 (SEQ ID NO 15).

The human CERS5 pre-mRNA is encoded on Homo sapiens Chromosome 12,

NC_000012.12 (50129306..50167533, complement). GENE ID = 91012 ( CERS5 ).

A mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 16.

The oligonucleotides of the invention are capable of inhibiting the expression of CERS5 target nucleic acid, such as the CERS5 mRNA, in a cell which is expressing the target nucleic acid, such as the CERS5 mRNA.

In some embodiments, the oligonucleotides of the invention are capable of inhibiting the expression of CERS5 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of CERS5 target nucleic acid (e.g. the mRNA) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the expression level of the CERS5 target nucleic acid (e.g. the mRNA) in the cell. Suitably the cell is selected from the group consisting of A549, HeLa and RAW264.7 cells. Example 1 provides a suitable assay for evaluating the ability of the oligonucleotides of the invention to inhibit the expression of the target nucleic acid. Suitably the evaluation of a compounds ability to inhibit the expression of the target nucleic acid is performed in vitro, such a gymnotic in vitro assay, for example as according to Example 1.

An aspect of the present invention relates to an antisense oligonucleotide, such as an LNA antisense oligonucleotide gapmer which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity, such as is fully

complementary to SEQ ID NO 15, 16, 17, 18, 19, 20, 21 or 22.

In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 - 30 nucleotides, which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 11.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 19, such as 13, 14, 15, 16, 17, or 18 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 13.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 16, such as 13 14, or 15 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 14.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 23.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 15, such as 13 or 14 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 22, such as 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 25.

In some embodiments, the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer, such as an LNA gapmer, a mixed wing gapmer, or an alternating flank gapmer.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully

complementary to SEQ ID NO 11.

complementary to SEQ ID NO 12. In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 13.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 14.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 23.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 24.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 25.

In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is less than 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 24 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 22 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 18 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 16 nucleotides in length.

Advantageously, in some embodiments all of the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 13.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 14.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 23.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 25.

In some embodiments, the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

In some embodiments, the sugar modified nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’- O-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro- ANA and LNA nucleosides.

In some embodiments, region G comprises 5 - 16 contiguous DNA nucleosides. In some embodiments, wherein the antisense oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide.

In some embodiments, the LNA nucleosides are beta-D-oxy LNA nucleosides.

In some embodiments, the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

Sequence Motifs and Compounds of the Invention

In the compound column, capital letters are beta-D-oxy LNA nucleosides, and LNA C are all 5-methyl C, lower case letters are DNA nucleosides, and all internucleoside linkages are phosphorothioate internucleoside linkages.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24, such as 12 - 18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 14, such as at least 15 contiguous nucleotides present in any one of SEQ ID NO 1 to 10.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 1 , 2, 3, 4 or 9.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 7 or 10.

The invention provides LNA gapmers according to the invention comprising or consisting of a contiguous nucleotide sequence selected from SEQ ID NO 1 - 10.

The invention provides antisense oligonucleotides selected from the group consisting of: CATGctttcacagaaTT; ACatgctttcacagAATT; ACAtgctttcacagAAT; ATGctttcacagaaTTT; AGCAttggatttttCG; TTatcaagtgtaGGAG; CGACcagagaaatCT; AAttcaagatgcagCCA;

ACAtgctttcacagaatTT; and CCGaccagagaaaTC; wherein a capital letter is a LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.

ACAtgctttcacagaatTT; and CCGaccagagaaaTC wherein a capital letter is a beta-D-oxy-LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.

ACAtgctttcacagaatTT; and CCGaccagagaaaTC; wherein a capital letter is a beta-D-oxy-LNA nucleoside, wherein all LNA cytosinese are 5-methyl cytosine, and a lower case letter is a DNA nucleoside, wherein all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages, and optionally DNA cytosine may be 5-methyl cytosine.

Method of manufacture

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

Pharmaceutical Composition

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

In some embodiments the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50 - 300mM solution.

The compounds according to the present invention may exist in the form of their

pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non- toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In:

Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.

Suitable formulations for use in the present invention are found in Remington's

Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533,

1990). WO 2007/031091 provides further suitable and preferred examples of

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

Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of

pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

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

The oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.

In research, such oligonucleotides may be used to specifically modulate the synthesis of CERS5 protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.

The present invention provides an in vivo or in vitro method for modulating CERS5 expression in a target cell which is expressing CERS5, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.

In some embodiments, the target cell, is a mammalian cell in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal.

In diagnostics the oligonucleotides may be used to detect and quantitate CERS5 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.

For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of CERS5

The invention provides methods for treating or preventing a disease, comprising

administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

The invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.

The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.

The disease or disorder, as referred to herein, is associated with expression of CERS5. In some embodiments disease or disorder may be associated with a mutation in the CERS5 gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the CERS5 sequence.

The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of CERS5.

The invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of CERS5.

In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis,

cardiomyopathy, opioid drug addiction and colorectal cancer.

Administration

The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intra- muscular, intracerebral, intracerebroventricular or intrathecal).

In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration. In one embodiment the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1 - 15 mg/kg, such as from 0.2 - 10 mg/kg, such as from 0.25 - 5 mg/kg. The administration can be once a week, every 2^nd week, every third week or even once a month.

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. The therapeutic agent can for example be the standard of care for the diseases or disorders described above.

The work leading to this invention has received funding from the European Union Seventh Framework Programme [FP7-2007-2013] under grant agreement“HEALTH-F2-2013- 602222" (Athero-Flux) EXAMPLES

Example 1 : Testing in vitro efficacy of antisense oligonucleotides targeting human and mouse CERS5 (Ceramide Synthase 5) mRNA in A549, HeLa (and RAW264.7) cell lines at single concentration.

A549, HeLa and RAW264.7 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37°C with 5% C02. For assays, 3000 cells/well (A549; HeLa) or 2500 cells/well (RAW264.7) were seeded in a 96 multi well plate in culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS. Final concentration of oligonucleotides: 25 mM. 3 days after addition of oligonucleotides, the cells were harvested. RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and eluated in 50mI water. The RNA was subsequently diluted 10 times with DNase/RNase free Water (Gibco) and heated to 90°C for one minute.

For gene expressions analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex set up. The following TaqMan primer assays were used for qPCR: CERS5, Hs00332291_m1 [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 (Mm99999915_g1 ) [VIC-MGB] All primer sets were purchased from Thermo Fisher Scientific. The relative NFKB1 mRNA expression level in the table is shown as percent of control (PBS-treated cells).The CERS5 mRNA levels from cells treated with a selection of the compounds are shown in figure 1 and figure 2, evaluated in human HeLa and A549 cell lines. From the initial library screen 2 motifs on the CERS5 human transcript were identified which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 1 1 ), and Motif B (SEQ ID NO 12).

For Compounds: Capital letters represent LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methyl cytosine, lower case letters represent DNA nucleosides. All internucleoside linkages are phosphorothioate internucleoside linkages.

Example 2: Testing in vitro potency and efficacy of selected oligonucleotides targeting human CERS5 mRNA in A549, HeLa and RAW264.7 cell lines in a dose response curve.

A549 cell line and HeLa cell line was described in Example 1. The assay was performed as described in Example 1 . Concentration of oligonucleotides: from 50 mM, 1 :1 dilution, 8 points. 3 days after addition of oligonucleotides, the cells were harvested. RNA extraction and duplex One Step RT-qPCR were performed as described in Example 1. Determination of IC50 values was performed in GraphPad Prism6. The relative CERS5 mRNA level at treatment with 50 mM oligonucleotide is shown in the table as percent of control (PBS).

The concentration response curves in human HeLa, A549 cell lines as well as mouse RAW264.7 cell line are provided as Figures 3, 4, and 5, respectively.

Example 3: Mouse in vivo efficacy and tolerance study, 16 days of treatment,

Intravenous IV (tail vein).

Animals

Experiment was performed on female C57BL/6JBom mice. Five animals were included in each group of the study, including a saline control group.

Compounds and dosing procedures

Animals were dosed intravenous (tail vein) with 15mg/kg compound at day 0, 3, 7, 10, 14 until the study was terminated at day 16.

Euthanasia

At the end of the study (day 16) all mice were euthanized with C02 before tissue samples of liver, kidney and adipose tissue were dissected and snap frozen.

Quantification of Cers5 RNA expression

Tissue samples were kept frozen until lysed in MagNA Pure LC RNA Isolation Tissue Lysis Buffer (Product No. 03604721001 , Roche) and RNA extraction continued using the MagNA Pure 96 Cellular RNA Large Volume Kit (Product No. 05467535001 , Roche) on a MagNA Pure 96 Instrument (Roche) according to the user’s manual and RNA diluted to 5ng/pl in water.

For gene expressions analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex set up. The following TaqMan primer assays were used for qPCR: Cers5, Mm00510998_m1 (FAM-MGB) and endogenous control Gapdh, Mm99999915_g1 (VIC-MGB). All primer sets were purchased from Thermo Fisher Scientific. The relative mRNA expression levels are shown as % of saline treated control group (Figure 6).

Claims

1. An antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 15, wherein the antisense

oligonucleotide is capable of inhibiting the expression of human CERS5 in a cell which is expressing human CERS5 ; or a pharmaceutically acceptable salt thereof.

2. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1 1 or SEQ ID NO 12,

3. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to SEQ ID NO 13 and/or SEQ ID NO:

23.

4. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to SEQ ID NO 14, SEQ ID NO 24 and/or SEQ ID NO: 25.

5. An antisense oligonucleotide according to any one of claims 1 - 4, wherein the

contiguous nucleotide sequence is fully complementary to a region of SEQ ID NO 15, selected from the group consisting of 45 - 77; 85 - 106; 1 13 - 139; 144 - 220; 221 - 308; 320 - 355; 363 - 430; 478 - 499; 501 - 524; 526 - 547; 557 - 582; 584 - 600; 606 - 643; 645 - 664; 666 - 691 ; 700 - 733; 735 - 762; 764 - 799; 814 - 840; 849 - 881 ;

903 - 921 ; 932 - 1015; 1017 - 1038; 1054 - 1 115; 1147 - 1 174; 1179 - 1220; 1221 - 1243; 1248 - 1266; 1293 - 1328; 1319 - 1337; 1341 - 1359; 1360 - 1390; 1383 -

1402; 1391 - 1412; 1414 - 1529; 1531 - 1547; 1550 - 1579; 1579 - 1593; 1594 -

1642; 1644 - 1670; 1672 - 1690; 1698 - 1724; 1737 - 1793; 1821 - 1866; 1906 -

1928; 1932 - 1954; 1960 - 2003; 2001 - 2058; 2079 - 2098; 2087 - 2102; 2094 -

2108; 2100 - 21 15; 2106 - 2128; 2120 - 2159; 2159 - 2178; 2168 - 2188; 2180 -

2197; 2197 - 2211 ; 2218 - 2232; 2234 - 2252; 2257 - 2291 ; 2313 - 2334; 2341 -

2355; 2344 - 2380; 2438 - 2465; 2467 - 2515; 2517 - 2534; 2524 - 2552; 2567 -

2585; 2587 - 2617; 2619 - 2647; 2649 - 2686; 2689 - 2719; 2735 - 2774; 2776 -

2852; 2854 - 2876; 2881 - 2903; 2923 - 2957; 2959 - 2978; 2993 - 3015; 3066 -

3080; 3095 - 31 12; 3122 - 3147; 3174 - 3192; 3189 - 3205; 3194 - 3214; 3221 - 3235; 3245 - 3260; 3251 - 3271 ; 3276 - 3330; 3329 - 3401 ; 3403 - 3419; 3413 -

3429; 3427 - 3478; 3480 - 3500; 3490 - 3513; 3518 - 3535; 3538 - 3582; 3585 - 3599 3591 - 3632 3673 - 3689 3691 3706 3710-3731 3729 - 3746 3758-

3781 3787 - 3801 3853 - 3871 3953 3968 3963 - 3977 4049 - 4098 4095-

4121 4132-4172 4189-4255 4246 42654257 - 4278 4283 - 4298 4306-

4328 4324 - 4341 4342 - 4378 4377 4411 4433 - 4447 4451 - 4520 4522-

4536 4535 - 4552 4544 - 4563 4571 4638 4642 - 4681 4688-4710 4702-

4717 4711 -4773 4783 - 4836 4829 4843 4839 - 4869 4864 - 4899 4909-

4938 4938 - 4973 4975 - 4991 4985 5000 5016-5054 5057 - 5073 5102-

5160 5162-5190 5214-5260 5262 5297 5299 - 5327 5328 - 5351 5347-

5367 5359 - 5373 5369-5411 5423 5438 5430 - 5444 5451 - 5473 5481 -

5497 5490 - 5505 5500-5518 5521 5565 5558 - 5572 5567 - 5582 5584-

5624 5618 -5632 5626 - 5666 5721 5775 5775 - 5798 5829 - 5845 5835-

5849 5847 - 5863 5912-5935 5930 5959 5969 - 5988 5984 - 5998 5987-

6032 6030 - 6050 6109-6128 6183 6198 6200 - 6240 6242 - 6273 6275-

6302 6312 -6350 6352 - 6395 6396 6433 6476 - 6508 6513-6531 6548-

6594 6619 -6636 6638 - 6674 6676 6733 6744 - 6765 6779 - 6826 6858-

6880 6882 - 6929 6972 - 7024 7044 7065 7080 - 7099 7094-7112 7139-

7171 7174 -7203 7193-7214 7205 7249 7244 - 7259 7261 -7313 7304-

7344 7361 - 7407 7401 - 7422 7424 7441 7430 - 7454 7456 - 7491 7488-

7506 7662 - 7695 7706 - 7723 7715 7735 7737 - 7757 7758 - 7786 7795-

7846 7850 - 7865 7869 - 7903 7910 7924 8140-8155 8157-8173 8180-

8197 8205 - 8226 8258 - 8280 8290 8343 8334 - 8350 8352 - 8377 8379-

8396 8386 -8416 8418-8434 8438 8452 8454 - 8474 8475 - 8494 8495-

8511 8512-8545 8547 - 8562 8574 8591 8593-8615 8632 - 8655 8657-

8692 8724 - 8751 8759 - 8774 8801 8815 8817-8832 8834 - 8850 8859-

8878 8880 - 8897 8915-8933 8935 8962 8974 - 9090 9116-9133 9162-

9191 9204-9219 9225 - 9266 9268 9287 9289-9313 9318-9349 9351 -

9392 9428 - 9475 9481 - 9496 9502 9522 9519-9546 9549 - 9606 9598-

9614 9604 -9618 9611 -9636 9627 9642 9657 - 9683 9678 - 9709 9733-

9747 9759 - 9778 9780 - 9804 9823 9848 9878 - 9903 9906 - 9965 10003-

10046; 10048 - 10094; 10096 - 10126; 10160 - 10178; 10193 - 10230; 10282 10307; 10317 - 10339; 10342 - 10358; 10360 - 10381; 10400 - 10457; 10458 10489; 10511 - 10541; 10556- 10571; 10586 - 10613; 10630 - 10644; 10653 10669; 10671 - 10695; 10697 - 10715; 10714 - 10729; 10726 - 10761; 10797 10811; 10821 - 10835; 10832- 10866; 10883 - 10933; 10967 - 10984; 11014 11046; 11063 - 11117; 11114-11130; 11127- 11142; 11139- 11178; 11182 1 1197 1 1195 - 11213 11212 - 11228 11220 11237 11242 11259 11269

1 1288 1 1281 11312 11325 11349 11338 11407 11412 - 11442 11446

1 1478 1 1478 11504 11506 11533 11522 11540 11539 11559 11561

1 1613 1 1603 11620 11622 11645 11650 11664 11659 11684 11676

1 1705 1 1707 11748 11769 11810 11799 11841 11830 11845 11839

1 1864 1 1879 11913 11915 - 11936 11929 11952 11947 11968 1 1983

12014 12024 12059 12076 12091 12081 12108 12113 - 12138 12149

12182 12207 12233 12236 12341 12359 12386 12398 12469 12458

12502 12514 12530 12532 12555 12557 12584 12591 12620 12615

12639 12646 12662 12654 12684 12686 12702 12728 12746 12764

12847 12871 12931 12933 13019 13028 13046 13046 13062 13068

13097 13099 13124 13126 - 13164 13162 - 13177 13184 - 13199 13208

13229 13232 13246 13251 13266 13258 13296 13294 13308 13297

13311 13313 13327 13332 13375 13365 13382 13398 13413 13406

13420 13409 13444 13450 13484 13490 13519 13521 13535 13537

13554 13556 13601 13597 13615 13624 13663 13665 13687 13686

13702 13722 13744 13746 13772 13765 13807 13805 13836 13838

13852 13843 13862 13852 13870 13872 13925 13927 13949 13976

14008 14016 14033 14043 14065 14055 14097 14099 14113 14110

14129 14130 14180 14170 - 14192 14202 14226 14233 14266 14268

14320 14322 14342 14344 14370 14372 14403 14405 14422 14447

14481 14478 14531 14541 14592 14594 14614 14609 14642 14648

14693 14683 14698 14700 14739 14747 14766 14768 14782 14802

14822 14824 14842 14867 14890 14892 14937 14939 14972 14965

14994 14988 15031 15035 15063 15065 15093 15095 - 15145 15147

15171 15173 15191 15193 15248 15279 15390 15406 15421 15463

15501 15504 15576 15578 15592 15608 15632 15621 15640 15656

15682 15703 15720 15747 15763 15764 15778 15790 15817 15821

15852 15855 15877 15879 15905 15932 15950 15960 16003 16005

16062 16092 16126 16145 - 16193 16195 - 16246 16248 16280 16282

16316 16318 16349 16356 16388 16392 16420 16422 16476 16479

16508 16511 16530 16532 16552 16564 16613 16647 16663 16664

16689 16693 16719 16736 16755 16757 16778 16791 16809 16822

16890 16896 16933 16950 16969 17025 17063 17084 17099 17114

17133 17149 17178 17182 17240 17235 17250 17239 17279 17294 17313 17315 17331 17328 17349 17347 17397 17399 17423 17425

17444 17461 17477 17479 17500 17512 17539 17539 17553 17550

17568 17557 17577 17570 17584 17573 17588 17592 17645 17647

17673 17664 17684 17675 17690 17692 17708 17707 17726 17749

17766 17764 17779 17769 17791 17799 17874 17901 17940 17948

18030 18039 18069 18071 18094 18096 18115 18117 18165 18167

18202 18213 18240 18242 18264 18269 18286 18288 18310 18341

18363 18382 18401 18403 18506 18509 18529 18542 18556 18587

18606 18596 18612 18605 18642 18631 18646 18656 18679 18679

18695 18706 18732 18768 18815 18813 18828 18817 18838 18832

18861 18917 18935 18939 18953 18957 18981 18983 19006 19015

19043 19032 19047 19049 19063 19065 19180 19182 19212 19222

19249 19251 19279 19287 19305 19315 19395 19397 19412 19414

19442 19435 19461 19469 19523 19521 19581 19585 19662 19669

19718 19720 19768 19770 19786 19788 19807 19808 19849 19851

19904 19906 19938 19941 19966 19968 20013 20028 20045 20047

20063 20069 20093 20106 20137 20129 20147 20145 20159 20178

20199 20205 20227 20229 20258 20281 20348 20338 20360 20349

20364 20357 20407 20423 20442 20448 20469 20474 20488 20488

20518 20520 20538 20539 20582 20609 20628 20647 20664 20666

20703 20703 20731 20731 20780 20790 20817 20808 20824 20818

20873 20872 20903 20893 20945 20972 21036 21038 21064 21085

21124 21 126 21 144 21 162 21 194 21 198 21237 21239 21259 21296

21310 21313 21358 21374 21398 21430 21448 21450 21509 21521

21536 21537 21556 21565 21588 21590 21620 21647 21670 21674

21705 21725 21745 21747 21766 21774 21819 21831 21861 21875

21907 21909 21931 21933 21983 21996 22017 22032 22055 22062

22165 22166 22181 22221 22270 22272 22318 22331 22347 22347

22369 22374 22409 22423 22447 22441 22470 22473 22526 22516

22541 22547 22588 22604 22684 22686 22708 22710 22765 22767

22814 22816 22833 22847 22876 22878 22892 22912 22937 22939

23022 23029 23054 23056 23077 23085 23099 23101 23126 23128

23151 23171 2321 1 23213 23241 23245 23261 23253 23270 23263

23278 23297 23335 23337 23356 23358 23387 23389 23422 23458

23476 23478 23501 23504 23566 23577 23609 23619 23640 23664 23688 23711 23777 23789 23815 23817 23833 23851 23879 23881

23896 23913 23933 23944 23990 23992 2401 1 24000 24022 24029

24106 24108 24123 24135 24204 24205 24236 24237 24253 24326

24410 24412 24439 24441 24467 24478 24497 24499 24524 24536

24558 24560 24576 24578 24594 24596 24642 24682 24833 24835

24856 24871 24907 24909 24926 24966 24997 24994 25009 25000

25020 25013 25030 25037 25059 25065 25110 25115 25144 25145

25177 25179 25202 25206 25220 25209 25241 25238 25253 25255

25295 25307 25333 25343 25361 25385 25404 25416 25516 25518

25540 25543 25569 25563 25606 25603 25621 25622 25636 25636

25662 25670 25687 25689 25721 25729 25777 25767 25807 25796

25822 25814 25828 25823 25844 25853 25868 25878 25892 25884

25918 25914 25932 25934 25986 26008 26022 26031 26105 26107

26128 26144 26162 26164 26186 26189 2621 1 26205 26243 26252

26293 26283 26301 26292 26306 26338 26359 26372 26386 26384

26402 26400 26415 26438 26482 26490 26505 26507 26524 26558

26585 26607 26629 26640 26682 26678 26696 26713 26740 26745

26782 26797 26853 26855 26886 26909 26937 26960 27008 27020

27046 27061 27089 27104 27118 2711 1 27147 27141 27156 27159

27181 27197 2721 1 27217 27251 27254 27280 27282 27309 27308

27329 27342 27376 27378 27405 27437 27481 27483 27528 27564

27585 27591 27609 27622 27651 27667 27682 27698 27731 27721

27739 27731 27784 27786 27830 27832 27857 27850 27867 27870

27921 27944 27974 27977 28017 28007 28021 28035 28050 28055

28082 28076 28091 28096 28136 28138 28159 28167 28185 28197

28218 28210 28225 28218 28232 28249 28285 28290 2831 1 28306

28334 28336 28380 28397 28413 28437 28479 28486 28530 28535

28575 28582 28596 28612 28635 28637 28662 28698 28720 28722

28739 28759 28780 28792 28810 28817 28849 28855 28888 28890

28993 29030 29058 29060 29075 29104 29125 29133 29170 29172

29192 29194 29221 29256 29289 29291 29317 29328 29345 29347

29364 29368 29390 29388 29406 29417 29465 29467 29494 29496

29514 29531 29589 29591 29607 29609 29659 29682 29709 2971 1

29734 29736 29820 29844 29879 29881 29902 29904 29942 29944

30013 30015 30083 30093 30125 30138 30159 30159 30183 30200 30217 30219 30252 30254 30272 30274 30299 30301 30345 30363

30379 30393 30414 30416 30459 30461 30505 30507 30599 30601

30659 30671 30699 30701 30721 30726 30771 30781 30815 30817

30838 30843 30860 30915 30948 30976 31020 31023 31074 31077

31105 31105 31 129 31 144 31 189 31205 31236 31242 31287 31289

31333 31340 31356 31363 31377 31382 31398 31400 31420 31422

31508 31510 31543 31546 31604 31610 31628 31638 31654 31657

31703 31705 31721 31723 31751 31753 31781 31783 31855 31853

31904 31910 31950 31952 31966 31970 31998 32000 32041 32063

32087 32089 32113 32126 32144 32146 32185 32472 32509 32531

32548 32550 32564 32587 32630 32637 32673 32675 32700 32702

32761 32771 32785 32813 32922 32924 33016 33024 33047 33049

33124 33138 33155 33157 33195 33186 33210 33244 33293 33288

33318 33323 33350 33360 33374 33367 33382 33403 33418 33412

33466 33464 33482 33477 33503 33505 33529 33532 33546 33537

33582 33571 33591 33602 33630 33632 33671 33672 33689 33704

33737 33744 33774 33793 33826 33828 33877 33879 33914 33910

33929 33933 33956 33962 33993 3401 1 34031 34035 34054 34060

34080 34083 34097 34099 34136 34138 34165 34167 34196 34198

34234 34243 34304 34324 34358 34361 34402 34404 34454 34455

34491 34493 34604 34606 34630 34632 34664 34666 34707 34709

34732 34756 34777 34803 34835 34837 34855 34877 34944 34946

34962 34964 35003 35012 35065 35097 35116 35115 35129 35122

35138 35127 35141 35145 35176 35182 35204 35221 35262 35263

35287 35282 35313 35327 35357 35361 35405 35428 35444 35449

35517 35519 35544 35578 35608 35618 35657 35668 35696 35703

35718 35734 35759 35772 35791 35793 35810 35832 35894 35896

35928 35930 35944 35941 35955 35945 35996 36003 36025 36018

36049 36051 36102 36114 36129 36119 36163 36166 36201 36200

36214 36203 36255 36258 36273 36275 36334 36355 36422 36424

36443 36445 36463 36490 36527 36535 36560 36563 36599 36601

36619 36628 36647 36649 36672 36675 36690 36692 36714 36715

36775 36777 36791 36788 36802 36805 36835 36846 36916 36918

36958 36960 36999 37014 37031 37033 37071 37093 37119 37121

37144 37150 37170 37172 37199 37201 37218 37227 37253 37255 37271 ; 37282 - 37298; 37308 - 37349; 37373 - 37392; 37410 - 37441 ; 37451 - 37470; 37482 - 37529; 37533 - 37562; 37563 - 37599; 37601 - 37628; 37630 - 37664; 37666 - 37697; 37919 - 37953; 37967 - 37989; 38010 - 38036.

6. The antisense oligonucleotide according to any one of claims 1 - 5, wherein the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

7. The antisense oligonucleotide according to claim 6, wherein the sugar modified

nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’-0-methoxyethyl-RNA, 2’- amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA and LNA nucleosides.

8. The antisense oligonucleotide according to claim 6 or 7, wherein region G comprises 5 - 16 contiguous DNA nucleosides.

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

10. The antisense oligonucleotide according to any one of claims 6 - 9, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.

1 1. The antisense oligonucleotide according to any one of claims 1 - 10, wherein the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

12. The antisense oligonucleotide according to any one of claims 1 - 11 , wherein the oligonucleotide comprises a contiguous nucleotide 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 7, SEQ ID NO 9 and SEQ ID NO 10; or selected from the group consisting of SEQ ID NO 5, 6 and 8.

13. The antisense oligonucleotide according to any one of claims 1 - 12, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of: CATGctttcacagaaTT (SEQ ID NO 1 );

ACatgctttcacagAATT (SEQ ID NO 2); ACAtgctttcacagAAT (SEQ ID NO 3);

ATGctttcacagaaTTT (SEQ ID NO 4); AGCAttggatttttCG (SEQ ID NO 5);

TTatcaagtgtaGGAG (SEQ ID NO 6); CGACcagagaaatCT (SEQ ID NO 7);

AAttcaagatgcagCCA (SEQ ID NO 8); ACAtgctttcacagaatTT (SEQ ID NO 9); and CCGaccagagaaaTC (SEQ ID NO 10); such as selected from the group consisting of

CATGctttcacagaaTT (SEQ ID NO 1 );

ACatgctttcacagAATT (SEQ ID NO 2);

ACAtgctttcacagAAT (SEQ ID NO 3);

ATGctttcacagaaTTT (SEQ ID NO 4);

CGACcagagaaatCT (SEQ ID NO 7);

ACAtgctttcacagaatTT (SEQ ID NO 9); and

CCGaccagagaaaTC (SEQ ID NO 10)

wherein a capital letter represents a LNA nucleoside, a lower case letter represents a DNA nucleoside.

14. The antisense oligonucleotide according to any one of claims 1 - 13, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence selected from the group consisting of:

ACatgctttcacagAATT (SEQ ID NO 2); ACAtgctttcacagAAT (SEQ ID NO 3);

ATGctttcacagaaTTT (SEQ ID NO 4); AGCAttggatttttCG (SEQ ID NO 5);

TTatcaagtgtaGGAG (SEQ ID NO 6); CGACcagagaaatCT (SEQ ID NO 7);

AAttcaagatgcagCCA (SEQ ID NO 8); ACAtgctttcacagaatTT (SEQ ID NO 9); and CCGaccagagaaaTC (SEQ ID NO 10); such as selected from the group consisting of CATGctttcacagaaTT (SEQ ID NO 1 );

ACatgctttcacagAATT (SEQ ID NO 2);

ACAtgctttcacagAAT (SEQ ID NO 3);

ATGctttcacagaaTTT (SEQ ID NO 4);

CGACcagagaaatCT (SEQ ID NO 7);

ACAtgctttcacagaatTT (SEQ ID NO 9); and

CCGaccagagaaaTC (SEQ ID NO 10)

wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and wherein the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.

15. A conjugate comprising the oligonucleotide according to any one of claims 1 - 14, and at least one conjugate moiety covalently attached to said oligonucleotide.

16. The conjugate according to claim 15, wherein the conjugate moiety is a trilavent GalNAc conjugate moiety, such as a the conjugate moiety of formula

wherein the wavy line represents the covalent bond to the 5’ end of the

oligonucleotide.

17. The conjugate according to claim 15 or 16, wherein the compound is selected from the group consisting of

wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and mc is 5-methyl cytosine DNA, and wherein subscript s represents a phosphorothioate internucleoside linkage, and a subscript o represents a phosphodiester

internucleoside linkage, and GN2-C6 is a 5’ conjugate of formula:

18. A pharmaceutical composition comprising the oligonucleotide of claim 1 -14 or the conjugate of any one of claim 15-17 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

19. An in vivo or in vitro method for modulating CERS5 expression in a target cell which is expressing CERS5, said method comprising administering an oligonucleotide of any one of claims 1 -14, the conjugate according to any one of claims 15-17, or the pharmaceutical composition of claim 18 in an effective amount to said cell.

20. A method for treating or preventing a disease comprising administering a

therapeutically or prophylactically effective amount of an oligonucleotide of any one of claims 1 - 14 or the conjugate according to any one of claims 15-17 or the pharmaceutical composition of claim 18 to a subject suffering from or susceptible to the disease.

21 . The method of claim 21 , wherein the disease is selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.

22. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to any one of claims 15-17 or the pharmaceutical composition of claim 18 for use in medicine.

23. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to any one of claims 15-17 or the pharmaceutical composition of claim 18 for use in the treatment or prevention of a disease selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis,

cardiomyopathy, opioid drug addiction and colorectal cancer.

24. Use of the oligonucleotide of claim 1 - 14 or the conjugate according to any one of claims 15 to 17 or the pharmaceutical composition of claim 18, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of obesity, insulin resistance, diabetes such as type 2 diabetes, atherosclerosis, cardiomyopathy, opioid drug addiction and colorectal cancer.