WO2020007700A1

WO2020007700A1 - Antisense oligonucleotides targeting spi1

Info

Publication number: WO2020007700A1
Application number: PCT/EP2019/067141
Authority: WO
Inventors: Eva Marie Lindholm; Steffen Schmidt
Original assignee: Roche Innovation Center Copenhagen A/S
Priority date: 2018-07-02
Filing date: 2019-06-27
Publication date: 2020-01-09

Abstract

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to Spi1 pre-mRNA sequences, which are capable of inhibiting the expression of Spi1 protein. Inhibition of Spi1 expression is beneficial for a range of medical disorders including infections, like HIV and herpes viruses; respiratory diseases, like asthma; cancers, like leukemia and colon cancer; and in situations requiring hematopoietic cell renewal or transplantation.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING SPI1

FIELD OF INVENTION

The present invention relates to antisense oligonucleotides (oligomers) complementary to Spi1 pre-mRNA intron and exon sequences, which are capable of inhibiting the expression of Spi1. Inhibition of Spi1 expression is beneficial for a range of medical disorders including: infections, like HIV and herpes viruses; respiratory diseases, like asthma; cancers, like leukemia and colon cancer; and in situations requiring hematopoietic cell renewal or transplantation.

BACKGROUND

The Spi1 proto-oncogene (herein referred to as Spi1 ) encodes the transcriptional factor PU.1 which is normally expressed in all haematopoietic cell lineages except in T cell lines. It is an ETS-domain transcription factor involved in hematopoietic stem cell and progenitor cell self- renewal and is essential for the development of myeloid and B-lymphoid cells.

Spi1 plays a role in the development and progression of various types of human cancers. Indeed, similar to other differentiation-associated transcription factors, inappropriate Spi1 expression is oncogenic. Constitutive overexpression of Spi1 leads to pre-leukemic cells that have a shortened S phase duration with an increased replication fork speed and increased mutability in the absence of DNA breaks (Rimmilee et al. Oncotarget. 8(23):37104-371 14, 2017).

Acute myeloid leukemia (AML) is a malignant disease that affects the myeloid lineage and progresses from normal to pre-leukemic and leukemic stages due to the accumulation of mutations over time. Rimmilee et al. (Oncotarget. 8(23):37104-37114, 2017) showed that the S phase checkpoint protein CHK1 is maintained in a low phosphorylation state in Spi1- overexpressing cells and proposed a model in which Spi1 overexpression promotes cell transformation and contributes to leukemic progression by accelerating DNA replication, thus increasing the mutation load in pre-leukemic cells.

Zhao et al. (Oncol Rep. 2013 Oct;30(4): 1782-92. doi: 10.3892/or.2013.2627) demonstrated that inhibition of Spi1 suppresses colon cancer stem cell growth and induces apoptosis in vitro and in nude mouse xenografts. They also demonstrated that the percentage of

CD44+/CD166+ cells was significantly downregulated both in vivo and in vitro following Spi1 inhibition. Seki et al. (Nat Genet 49(8):1274-1281 , 2017) found that a proportion of relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) patients possessed fusions involving Spi1 , which retained transcriptional activity, and when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in maturation block.

Qian et al. (J Mol Cell Biol 7(6):557-567, 2015) found that Spi 1 serves as a critical regulator of alternatively activated macrophage (AAM) polarization and promotes the pathological progress of asthmatic airway inflammation. Spi1 -deficient mice displayed attenuated AAM, including decreased alveolar eosinophil infiltration and reduced production of IgE.

Lodie et al. (J Immunol. 161 (1 ): 268-276, 1998) demonstrated a role for Spi 1 (referred to there in as PU.1 ) in activation of human immunodeficiency virus (HIV) -1 long terminal repeat (LTR) by lipopolysaccharide (LPS) and that phosphorylation of PU.1 at serine 148 by CK2 was required for this activation.

W02010/120262 discloses nucleic acid-based therapeutics (NABTs) against >200 target genes, including Spi1. Therein, they propose that NABTs against Spi 1 can be used to treat diseases such as: viral infections, such as herpes virus infections, aberrant programming diseases, such as cancer, Parkinson’s, heart failure, Alzheimer’s disease and autoimmune diseases. They also speculate that the NABTs, including those against Spi-1 , can be used in re-programming normal cells (self-renewal), such as directing induced pluripotent cells (iPCs), embryonic stem cell or haematopoietic stem cells (HSCs) to differentiate into desired cell types; or generating populations of cell, such as lymphocytes, granulocytes or megakaryocytes, such as might be required to treat anemia or osteoporosis or to fight infection or to replace damaged normal cell types and numbers, such as in vitro or in vivo for subsequent harvesting and transplantation to a patient in need thereof.

Spi1 is therefore implicated in a number of diseases and is therefore a target for therapeutic treatment of a number of diseases, such as: infections, like HIV and herpes viruses;

respiratory diseases, like asthma; and, cancers, like leukemia and colon cancer. Antisense oligonucleotides against Spi-1 can also be useful for self-renewal, e.g. in the treatment of diseases or conditions requiring self-renewal of haematopoietic cells, such as osteoporosis, anemia and in immunocompromised situations such as after chemotherapy or radiotherapy or viral infection. In such situations, haematopoietic stem or pluripotent cells can be induced to differentiate into cells needed to fight infections, or for use in transplantation, and the like. The inventors designed and screened 39 LNA gapmer antisense oligonucleotides targeting human and/or mouse Spi1 and identified sequences and compounds which are particularly potent and effective at specifically targeting Spi1.

OBJECTIVE OF THE INVENTION

The inventors have identified regions of the Spi1 pre-mRNA and mRNA for antisense inhibition in vitro or in vivo. The invention therefore provides for antisense oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the Spi1 pre-mRNA or mature mRNA. The present invention provides oligonucleotides which inhibit mammalian, such as human, Spi1 which are useful in the treatment of a range of medical disorders including viral infection, respiratory diseases and cancers, and in situations requiring hematopoietic cell renewal or transplantation.

STATEMENT OF THE INVENTION

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 to any of SEQ ID Nos: 1 1 - 21 wherein the antisense oligonucleotide is capable of inhibiting the expression of Spi1 in a cell which is expressing Spi1.

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%

The invention provides for an 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%

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 to any of SEQ ID Nos: 1 1 - 21 wherein the antisense oligonucleotide is capable of inhibiting the expression of Spi1 in a cell which is expressing Spi1. The oligonucleotides targeting Spi1 are antisense oligonucleotides, i.e. are complementary to their Spi1 nucleic acid target.

In an advantageous embodiment, the antisense oligonucleotide of the invention is capable of inhibiting human Spi1 in a cell which is expressing human Spi1. By virtue of the sequence identity of particular target regions in different species, in particular mammalian species, certain of the oligonucleotides of the invention can function to inhibit Spi1 from different species. In some embodiments, the antisense oligonucleotide of the invention is capable of inhibiting the expression of human Spi1 in a human cell and at least one other mammalian Spi1 in a cell from that mammal. In some embodiments the other mammalian Spi1 target that the oligonucleotides of the invention can inhibit is selected from: monkey (such as cynomolgus monkey), rat or mouse. In a suitable embodiment, the oligonucleotide of the invention is capable of inhibiting the expression of human and mouse Spi1 , when in the cell from the particular mammal.

In a suitable embodiment, the oligonucleotide of the invention is capable of inhibiting the expression of human, mouse and cynomolgus monkey Spi1 , when in the cell from the particular mammal.

In some embodiments, the antisense oligonucleotide of the invention is capable of inhibiting the expression of Spi1 in a cell which is expressing said Spi1.

In one 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 to SEQ ID NO 16, 11 , 12 or 13. In suitable embodiments, the antisense oligonucleotide is capable of inhibiting the expression of human Spi1 transcript in a cell which is expressing human Spi1 transcript.

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

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

In one aspect, 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. In one aspect, the invention provides for an in vivo or in vitro method for modulating Spi1 expression in a target cell which is expressing Spi1 , said method comprising administering an oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount to said cell.

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

In some embodiments, the disease is one that is mediated by or associated with increased Spi1 expression.

In some embodiments, the disease is selected from the group consisting of: cancer, viral infection, respiratory disease and diseases or conditions requiring self-renewal and/or differentiation of haematopoietic cells.

In some embodiments, the disease or condition is one that will benefit from reducing Spi 1 expression.

In blood malignancies such as leukaemias, malignant cells disrupt the bone marrow (BM) microenvironment and compromise haematopoietic stem cell (HSC) functions (Miraki-Moud et al., Proc Natl Acad Sci U S A. 110(33): 13576-13581 , 2013). Furthermore, it has been shown that HSC depletion correlates with the aggressive blood malignancy progression (Wang et al., Haematologica. 102(9): 1567-1577, 2017). The collapse of haematopoiesis is frequently manifested by bleeding, anaemia and severe infections, which significantly contribute to morbidity and mortality of leukaemia patients (Lowenberg et al., N Engl J Med. 341 (14): 1051-1062, 1999). It is therefore of a major clinical importance to enhance HSC functions and boost normal haematopoiesis during the course of leukemia, such as AML..

The oligonucleotides, conjugates or pharmaceutical compositions of the invention can be used to increases in vivo expansion of the patient’s HSCs.

In one aspect of the invention there is provided a method to boost normal haematopoiesis during the course of leukaemogenesis in leukemia patients comprising administering to a patient in need thereof an effective amount of the oligonucleotides, conjugates or

pharmaceutical compositions of the invention.

Patients with reduced amounts of haematopoietic stem cells or mature haematopoietic cells (e.g. B-cell and other blood cells), such as those with anemia or having undergone ablative therapy (chemo or radiotherapy) can be treated with the molecules of the invention or via stem cell/blood transplantation of cell populations treated with the molecules of the invention. Thus, the oligonucleotides, conjugates or pharmaceutical compositions of the invention can be used to enhance self-renewal and differentiation ability of haematopoietic stem cells (and pluripotent cells) directly in a patient, or ex vivo for subsequent transplantation into a patient in need thereof.

In one aspect, the invention provides for a method for inducing self-renewal and/or differentiation of haematopoietic stem or pluripotent cells comprising administering an effective amount of an oligonucleotide or a conjugate or a pharmaceutical composition of the invention to a subject with diminished haematopoietic cells.

One problem with stem cell transplantation is that it does not restore mature haematopoietic cells immediately after transplantation (Nikiforow and Ritz, Cell Stem Cell. 18(1 ): 10-12, 2016). Due to the time required to generate mature cells from reinfused stem cells, there is a lag during which the patient remains immunocompromised. One proposed solution has been to expand the purified stem cells ex vivo to generate a cell population having both stem cells and slightly more differentiated cells (i.e. primitive progenitor cells), which would be able to provide both short- and long-term haematopoietic recovery.

In one aspect, the invention provides for a method for inducing self-renewal and/or differentiation of haematopoietic stem or pluripotent cells, comprising (i) administering an effective amount of an oligonucleotide or a conjugate or a pharmaceutical composition to a haematopoietic stem cell population ex vivo ; (ii) inducing stem cells self-renew and/or differentiation into mature haematopoietic cells; and, (iii) optionally, transplanting some or all of the cells from step (ii) to a patient in need thereof.

In other aspects, the invention provides for the oligonucleotide, the conjugate or the pharmaceutical composition of the invention for use in medicine.

In other aspects, the invention provides for the oligonucleotide, the conjugate or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease selected from the group consisting of: cancer, viral infection, respiratory disease and diseases or conditions requiring self-renewal of haematopoietic cells.

In other aspects, the invention provides the oligonucleotide, the conjugate or the

pharmaceutical composition of the invention for use in inducing self-renewal and/or differentiation of haematopoietic stem or pluripotent cells in a patient in need thereof.

pharmaceutical composition of the invention for use in a method of cell transplantation, the method comprising (i) administering an effective amount of an oligonucleotide or a conjugate or a pharmaceutical composition to a haematopoietic stem cell population ex vivo ; (ii) inducing stem cells self-renew and/or differentiation into mature haematopoietic cells; and, (iii) transplanting some or all of the cells from step (ii) to a patient in need thereof. In other aspects, the invention provides for the use of the oligonucleotide, the 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: cancer, viral infection, respiratory disease and diseases or conditions requiring self-renewal and/or differentiation of haematopoietic cells.

In other aspects, the invention provides for the use of the oligonucleotide, the conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for for inducing self-renewal and/or differentiation of haematopoietic stem or pluripotent cells.

In other aspects, the invention provides for the use of the oligonucleotide, the conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for use in a method for inducing self-renewal and/or differentiation of haematopoietic stem or pluripotent cells, comprising (i) administering an effective amount of an oligonucleotide or a conjugate or a pharmaceutical composition to a haematopoietic stem cell population ex vivo; (ii) inducing stem cells self-renew and/or differentiation into mature haematopoietic cells; and, (iii) transplanting some or all of the cells from step (ii) to a patient in need thereof.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : Testing in vitro efficacy of various antisense oligonucleotides targeting human and mouse Spi 1 mRNA in THP-1 and RAW264.7 cell lines at single concentration.

Figure 2: Comparison of in vitro efficacy for antisense oligonucleotides targeting human and mouse Spi 1 mRNA in THP-1 and RAW264.7 cell lines at single concentration shows good correlation. Two motifs (A and B) with very efficient targeting are highlighted.

Figure 3: Testing selected oligonucleotides targeting human (and mouse) SPI1 mRNA in vitro for concentration dependent potency and efficacy in THP-1 cell line.

Figure 4: Testing selected oligonucleotides targeting (human and) mouse Spi1 mRNA in vitro for concentration dependent potency and efficacy in RAW264.7 cell line.

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 an oligonucleotide capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. Suitably, 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 or hybridizes to the target nucleic acid. Although this region of the oligonucleotide is complementary to the target sequence, in some embodiments, not every nucleobase within the contiguous sequence need be complementary. Provided the contiguous nucleotide sequence can hybridize to the target sequence and inhibit its expression then a mismatch, or in some embodiments more than 1 mismatch may exist. Suitably, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid. The term“contiguous nucleotide sequence” 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.

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. Further, all the internucleoside linkages in the oligonucleotide sequence may be 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 (i.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 Lacroi. Oligonucleotides 13:515-537, 2003). 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 al.,

2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements. AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:1 1211-11216 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 Spi 1 and may for example be a gene, a Spi1 RNA, a mRNA, a pre-mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an Spi 1 target nucleic acid.

Suitably, the target nucleic acid encodes an Spi1 protein, in particular mammalian Spi 1 , such as the human Spi 1 encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 16, 11 , 12 or 13.

In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO: 11 to 21 , or naturally occurring variants thereof (e.g. Spi1 sequences encoding a mammalian Spi1 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 Spi 1 target nucleic acid in a cell which is expressing the Spi1 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the Spi1 target nucleic acid, as measured across the length of the oligonucleotide, optionally with no more than one mismatch, excluding the optional 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 Spi 1 protein, such as human Spi1 , e.g. the human Spi1 pre-mRNA sequence, such as that disclosed as SEQ ID NO 11 , or Spi1 mature mRNA, such as that disclosed as SEQ ID NO 12 or SEQ ID NO 13 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).

Table 1.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 1 1.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 12.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 13.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 12 and at least one of, such as two or three of SEQ ID NO 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21. In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 12 and 13.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 14 and 16.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 15 and 16.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 17, 20 and 21.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 18, 20 and 21.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 11 , 19, 20 and 21.

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. 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 Spi1 pre-mRNA (SEQ ID NO 11 ) 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, 11 , 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 oligonucleotides that are particularly potent against human and murine Spi1 at particular locations (motifs). With respect to the position in SEQ ID NO 11 , motif A (SEQ ID NO: 14) is from position 230-246, Motif B (SEQ ID NO: 15) is from position 220 - 236, motif C (SEQ ID NO: 16) covers both motifs A and B and runs from position 220- 246. Motif D (SEQ ID NO: 17) is from position 23191-23205, motif E (SEQ ID NO: 18) is from position 23235-23249, motif F (SEQ ID NO: 19) is from 23130-23145. The

oligonucleotides targeting motifs D, E and F are located very close together and so motif G (SEQ ID NO: 20) represents the region from position 23191-23249 and motif H (SEQ ID NO: 21 ) the region from 23130 and 23249 of SEQ ID NO 1 1. All positions mentioned are inclusive.

The inventors have also identified the following target sequence regions, listed in tables 2 & 3. Each region listed in the table is listed with its position on SEQ ID NO 1 1.

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 to any of target sequence regions R_1 - R_630 (listed in table 2). Table 2 Further exemplary regions of SEQ ID NO 11 which may be targeted by the oligonucleotides of the invention.

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 to any of target sequence regions S_1 - S_57 (listed in table 3).

Table 3: Further exemplary regions of SEQ ID NO 1 1 which may be targeted by the oligonucleotides of the invention.

By way of example, region S_4 (positions 219 - 253 of SEQ ID NO: 11 ) is a particularly useful target region, and indeed is one that many of the exemplified antisense

oligonucleotides disclosed in the Examples herein target.

The S_54 region (positions 23129 -23250 of SEQ ID NO: 11 ) is another particularly useful target region, and indeed is one that certain of the exemplified antisense oligonucleotides disclosed in the Examples herein target.

In a particular embodiment, the target nucleic acid comprises the sequence from positions 219 - 253, inclusive, of SEQ ID NO:11.

In a particular embodiment, the target nucleic acid comprises the sequence from positions 23129 -23250, inclusive, of SEQ ID NO: 11.

In some embodiments the target sequence is selected from the group consisting of R_1 - R_ 630 (Table 2).

In some embodiments, the target sequence is selected from the group consisting of S_1 - S_57 (Table 3) 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 Spi1 mRNA, such as the Spi1 pre- mRNA, e.g. SEQ ID NO 1 1 , or Spi1 mature mRNA (e.g. SEQ ID NO 12 or 13). The poly A tail of Spi 1 mRNA is typically disregarded for antisense oligonucleotide targeting.

Naturally occurring variant

The term“naturally occurring variant” refers to variants of Spi1 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. According to the Ensembl database entry ENST00000378538.7, the Spi 1 transcript has 5 exons, is annotated with 17 domains and features, and is associated with 250 variations. 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. Currently, there are 4 known transcripts (splice variants) of Spi1 .

The homo sapiens Spi1 gene is located at chromosome 1 1 : 47,354,860-47,378,576, reverse strand.

In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian Spi1 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 1 1 , 12 or 13. In some embodiments the naturally occurring variants have at least 99% homology to the human Spi1 target nucleic acid of SEQ ID NO: 1 1 .

The key database entries for Spi1 , including the exon start and end locations of one splice variant, are disclosed in Table 4 below:

Table 4:

Spi1 Exon and Introns:

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 11 , selected from the group consisting if Ex_1 - Ex_5.

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 11 , selected from the group consisting of 186 - 268, 2845 - 2944, 18537 - 18724, 19571 - 19733, 23031 - 23664, 1 - 268, 2848 - 2944, 23031 - 23717, 54 - 268, 23031 - 23476, 90 - 268 and 19571 - 20396.

These positions represent the exon regions in the known splice variant forms of Spi1.

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 11 , selected from the group consisting of lnt_1 - lnt_4.

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 11 , selected from the group consisting 268 - 2845, 2944 - 18537, 18724 - 19571 ,

19733 - 23031 , 268 - 2848 and 268 - 23031.

These positions represent the intron regions in the known splice variant forms of Spi 1. In some embodiments, the oligonucleotide of the invention targets a region within exon 1 , defined by positions 186-268, 1-268, 54-268 or 90-268, according to the position in SEQ ID NO 1 1.

In some embodiments, the oligonucleotide of the invention targets a region within exon 5 defined by positions 23031 - 23664, 23031 - 23717, 23031 - 23476 or 19571 - 20396, according to the position in SEQ ID NO 1 1.

Inhibition of expression

The term“Inhibition of expression” as used herein is to be understood as an overall term for an oligonucleotide’s ability to reduce the amount of Spi1 protein or Spi1 mRNA when compared to the amount of Spi 1 or Spi1 mRNA prior to administration of the oligonucleotide. Alternatively, the inhibition 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). Types of oligonucleotide induced inhibition, include an oligonucleotide’s ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of Spi1 , e.g. by degradation of Spi1 mRNA.

Suitably, the oligonucleotides of the invention are capable of inhibiting the expression of Spi1 mRNA in a cell which is expressing Spi 1 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.

2'-OMe ?'F P NL 2'F-ANA

2'-O-M0E 2'-0-AI!yl 2'-0-Elhylai-ine

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. Various gapmer designs are described herein. 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 be 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 10 to 30 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to 18, such as 15 to17 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

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 al., 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 nucleoside 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

Fl-8-[D3-4-El - D 3-₄]-F ^* ₁ -8

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, 11 , 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-s-[region G] -[LNA]i-s, 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_-8-[Region G]-[MOE] i_-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 WO2015/1 13922, 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:

F-G-F’; in particular Fi-8-G5-i6-F’2-8

D’-F-G-F’, in particular D’i_-3-Fi-8-G5-i6-F’2-8

F-G-F’-D”, in particular Fi-8-G5-i6-F’2-8-D”i_-3

D’-F-G-F’-D”, in particular D’_I-3- Fi-8-G5-i6-F’2-8-D”i_-3

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 modifies or enhances the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular, the 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. 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. 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, capable of inhibiting the expression of Spi1.

The oligonucleotides of the invention targeting Spi 1 are capable of hybridizing to and inhibiting the expression of a Spi1 target nucleic acid in a cell which is expressing the Spi 1 target nucleic acid.

The Spi1 target nucleic acid may be a mammalian Spi1 mRNA or pre-mRNA, such as a human Spi1 mRNA or pre-mRNA, for example a pre-mRNA or mRNA originating from the Homo sapiens Sp1-1 proto-oncogene(SpM ), see the following database entries:

Ensembl:ENSG00000066336; MIM:165170; Vega:OTTHUMG00000150150. The gene is located on Chromosome 11 : 47,354,860-47,378,576 reverse strand and is exemplified by Ensembl Reference Sequence: ENSG00000066336| (SEQ ID NO 11 ).

A mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO: 12 or 13. SEQ ID NO: 12 corresponds to reference sequence NM_001080547.1 ;

SEQ ID NO: 13 corresponds to reference sequence NM_003120.

The oligonucleotides of the invention are capable of inhibiting the expression of Spi 1 target nucleic acid, such as the Spi1 mRNA, in a cell which is expressing the target nucleic acid, such as the Spi1 mRNA.

In some embodiments, oligonucleotides of the invention are capable of inhibiting the expression of Spi1 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of Spi1 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 Spi1 target nucleic acid (e.g. the mRNA) in the cell. Suitably the cell is selected from the group consisting of THP-1 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 oligonucelotide, 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 16, 11 , 12 or 13.

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 any of SEQ ID Nos: 16, 14, 15, 17, 18 or 19.

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, such as one selected from SEQ ID Nos 11-21 The inventors have identified particularly effective sequences of the Spi1 target nucleic acid which may be targeted by the oligonucleotide of the invention.

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

In some embodiments the target sequence is SEQ ID NO 15.

In some embodiments the target sequence is SEQ ID NO 16.

In some embodiments the target sequence is SEQ ID NO 17.

In some embodiments the target sequence is SEQ ID NO 18.

In some embodiments the target sequence is SEQ ID NO 19.

In some embodiments the target sequence is SEQ ID NO 20.

In some embodiments the target sequence is SEQ ID NO 21.

SEQ ID NO: 14, 15 and 16 are present within exon 1 of Spi1. SEQ ID NO: 17, 18, 19, 20 and 21 , are located in exon 5 of Spi1.

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 16.

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 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 15. 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 17.

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 18.

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 19.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully

complementary to SEQ ID NO 1 1.

complementary to SEQ ID NO 12.

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, such as at least 16 contiguous nucleotides, such as at least 17 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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 15.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 16.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 17.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 18.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 19.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 20.

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, such as at least 16 contiguous nucleotides, such as at least 17 contiguous nucleotides, which is fully complementary to SEQ ID NO 21.

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 14 - 17 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 14.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 15.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 16.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 17.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 18.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 19.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 20. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 21.

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.

Table 5 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 a superscript m before a lower case c represent a 5-methyl cytosine DNA nucleoside, and all internucleoside linkages are phosphorothioate internucleoside linkages.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 1 , 3 or 8.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 2, 5, 6 or 9.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 1 , 2, 3, 5, 6, 8 or 9.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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 4.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 7.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 10.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 nucleosides 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, such as at least 16, such as at least 17, such as at least 18 contiguous nucleotides present in SEQ ID NO 4, 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 LNA gapmers as depicted in Table 5.

The invention provides antisense oligonucleotides selected from the group consisting of: CAttttgcacgcCTG, CGcctgtaacatcCAG, CCattttgcacgcCT, ATCttcttgcggtTG,

ACgcctgtaacatcCA, CGCctgtaacatcCA, TCTtgccgtagttGC, TCcattttgcacgcCT,

ACgcctgtaacatccAG and GAcgagaactggAAGG; 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.

ACgcctgtaacatcCA, CGCctgtaacatcCA, TCTtgccgtagttGC, TCcattttgcacgcCT,

ACgcctgtaacatccAG and GAcgagaactggAAGG; 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.

ACgcctgtaacatcCA, CGCctgtaacatcCA, TCTtgccgtagttGC, TCcattttgcacgcCT,

ACgcctgtaacatccAG and GAcgagaactggAAGG; wherein a capital letter is a beta-D-oxy-LNA nucleoside, wherein all LNA cytosines 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 salts

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.

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. In some embodiments, the oligonucleotide of the invention is administered at a dose of 10 - 1000pg.

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 11 , 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 SpH 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 Spi1 expression in a target cell which is expressing Spi1 , 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 such as a human, cynomolgus monkey or murine 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 Spi1 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.

For therapeutics, the oligonucleotides of the invention can be used to modulate the expression of Spi1 in an animal (e.g. a human) suspected of having a disease or disorder mediated by or associated with aberrant Spi 1 expression.

In a particular embodiment, an oligonucleotide of the invention is used to inhibit the expression of Spi1 in an animal suspected of having a disease or disorder mediated by or associated with aberrant Spi1 expression. In an embodiment, the disease or disorder is one mediated by or associated with elevated expression of Spi1 in the affected cells. For example, the affected cells could be tumour/cancer cells that express higher than normal amounts of Spi 1. A patient (e.g. animal) with diseased cells that express higher than normal levels of Spi1 is considered to be have a disease mediated by or associated with aberrant (e.g. elevated) Spi1 expression.

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, or an oligonucleotide conjugate or a pharmaceutical composition 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 or pharmaceutical composition as defined or described herein for the manufacture of a medicament for the treatment of a disease or disorder as referred to herein, or for a method of the treatment of a disease or disorder as referred to herein.

The disease or disorder, as referred to herein, is associated with expression of Spi 1. In some embodiments the disease or disorder may be associated with a mutation in the Spi 1 gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the Spi1 sequence.

The methods of the invention may be employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of Spi1.

The methods of the invention may be employed for treatment or prophylaxis against diseases caused by elevated levels and/or activity of Spi1. By elevated we mean greater than the level typically found in normal tissues. The degree of elevated expression indicative of a diseased cell/tissue can be determined by a clinician. However, the amount of increase in expression relative to normal cells/tissues could by an increase of 5%, 10%, 15%, .20%, 25%, 30%, 50%, 75%, 90%, 100%, 150%, 175%, 200%, 250% or more above normal levels. Such typical“normal” levels can be determined by measurement of levels in normal (non- diseased) cells or from a reference data set. A reference normal level, be it from direct measurements or from a reference data set, is usually one that is an average from multiple (e.g. >5) measurements.

In a particular embodiment, the patient is identified as having a disease or condition characterised by elevated SpH expression prior to administration of the oligonucleotide, conjugate or pharmaceutical composition according to the invention. Such identification can be carried out according to a variety of methods as described herein. In certain

embodiments, the level of SpH expression is determined from a biological sample previously isolated from the patient/subject .Such sample, could be a biopsy (such as tumour tissue) or fluid (such as blood) sample.

There are various well-known methods for determining the amount of protein or mRNA in a cell. Immunohistochemistry, ELISA, or mass spectroscopy methods, such as liquid- chromatography mass spectroscopy (LC-MS) are particularly suitable methods.

For mRNA determination, methods involving hybridisation to the target mRNA using a complementary nucleic acid can be employed. Various adaptations of reverse transcription polymerase chain reaction (RT-PCR), such as quantitative PCR or competitive RT-PCR, are suitable quantitative methods for determining the relative amount of a mRNA species in a normal cell versus an aberrant cell. The person skilled in the art is able to employ a suitable method for detection of the amount or protein or mRNA in the cell or cells. 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 Spi1. As noted above, such abnormal levels may be elevated levels.

In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from: cancer, viral infection, respiratory disease and diseases or conditions requiring self-renewal of haematopoietic cells.

Suitable cancers include: colon cancer and haematopoietic cancers, such as acute lymphocytic leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL).

Suitable viral infections include herpes simplex viruses, (such as HSV-1 and Varicella Zoster Virus) and HIV.

Suitable conditions or diseases requiring enhancement of the immune system, such as via enhancement of haematopoietic cells in the body, include anemia, infection, cancer and patients with ablated immune systems (such as following chemotherapy or radiotherapy).

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- 6021 14" (Athero-B-Cell)

EXAMPLES

Example 1 : Testing in vitro efficacy of antisense oligonucleotides targeting human (and mouse) SPI1 mRNA in THP-1 (and RAW264.7) cell lines at single concentration.

All oligonucleotides were made by standard automated phosphoramidite oligonucleotide synthesis.

THP-1 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% CO2. For assays, 50.000 cells/well (THP-1 ) 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: SPI1 , Hs00231368_m1 (Mm00488140_m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 (Mm99999915_g1 ) [VIC- MGB] All primer sets were purchased from Thermo Fisher Scientific. The relative SPI1 mRNA expression level in the table is shown as percent of control (PBS-treated cells).

Table 6 - Selected oligonucleotides used:

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, DNA cytosines preceded with a superscript ^m represents a 5-methyl C-DNA nucleoside. All internucleoside linkages are phosphorothioate internucleoside linkages.

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 to any of SEQ ID Nos: 16, 11 , 12 or 13, wherein the antisense oligonucleotide is capable of inhibiting the expression of human Spi 1 in a cell which is expressing human Spi1 ; or a pharmaceutically acceptable salt thereof.

2. The antisense oligonucleotide according to claim 1 , wherein the contiguous nucleotide sequence is fully complementary to any of SEQ ID Nos:11 to 21.

3. The antisense oligonucleotide according to claim 1 , wherein the contiguous nucleotide sequence is fully complementary to a region in SEQ ID NO 1 1 recited in Tables 2 or 3.

4. The antisense oligonucleotide according to any one of claims 1 - 3, 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.

5. The antisense oligonucleotide according to claim 4, 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.

6. The antisense oligonucleotide according to claim 4 or 5, wherein region G comprises 5 - 16 contiguous DNA nucleosides.

7. The antisense oligonucleotide according to any one of claims 1 - 6, wherein the

antisense oligonucleotide is a LNA gapmer oligonucleotide.

8. The antisense oligonucleotide according to any one of claims 4 - 7, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.

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

internucleoside linkages between the contiguous nucleotide sequence are

phosphorothioate internucleoside linkages.

10. The antisense oligonucleotide according to any one of claims 1 - 9, 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: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:10.

11. The antisense oligonucleotide according to any one of claims 1 - 10, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence:

CAttttgcacgcCTG (SEQ ID NO 1 )

CGcctgtaacatcCAG (SEQ ID NO 2)

CCattttgcacgcCT (SEQ ID NO 3)

ACgcctgtaacatcCA (SEQ ID NO 5)

CGCctgtaacatcCA (SEQ ID NO 6)

TCcattttgcacgcCT (SEQ ID NO 8)

ACgcctgtaacatccAG (SEQ ID NO 9)

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

12. The antisense oligonucleotide according to any one of claims 1 - 11 , wherein the

oligonucleotide comprises or consists of a contiguous nucleotide sequence:

CAttttgca^mcgcCT G (SEQ ID NO 1 )

CGcctgtaacatcCAG (SEQ ID NO 2)

CCattttgca^mcgcCT (SEQ ID NO 3)

ACgcctgtaacatcCA (SEQ ID NO 5)

CGCctgtaacatcCA (SEQ ID NO 6)

TCcattttgca^mcgcCT (SEQ ID NO 8)

ACgcctgtaacatccAG (SEQ ID NO 9)

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 the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.

13. A conjugate comprising the oligonucleotide according to any one of claims 1 - 12, and at least one conjugate moiety covalently attached to said oligonucleotide.

14. A pharmaceutical composition comprising the oligonucleotide of claim 1-12 or the

conjugate of claim 13 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

15. An in vivo or in vitro method for modulating Spi1 expression in a target cell which is expressing Spi1 , said method comprising administering an oligonucleotide of any one of claims 1-12, the conjugate according to claim 13, or the pharmaceutical composition of claim 14 in an effective amount to said cell.

16. 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 - 12 or the conjugate according to claim 13 or the pharmaceutical composition of claim 14 to a subject suffering from or susceptible to the disease.

17. The method of claim 16, wherein the disease is selected from the group consisting of infections, like HIV and herpes viruses; respiratory diseases, like asthma; cancers, like leukemia and colon cancer; and in situations requiring hematopoietic cell renewal or transplantation.

18. The oligonucleotide of any one of claims 1 - 12 or the conjugate according to claim 13 or the pharmaceutical composition of claim 16 for use in medicine.

19. The oligonucleotide of any one of claims 1 - 12 or the conjugate according to claim 13 or the pharmaceutical composition of claim 14 for use in the treatment or prevention of a disease selected from the group consisting of infections, like HIV and herpes viruses; respiratory diseases, like asthma; cancers, like leukemia and colon cancer; and in situations requiring hematopoietic cell renewal or transplantation.

20. Use of the oligonucleotide of claim 1 - 12 or the conjugate according to claim 13 or the pharmaceutical composition of claim 14, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of infections, like HIV and herpes viruses; respiratory diseases, like asthma; cancers, like leukemia and colon cancer; and in situations requiring hematopoietic cell renewal or

transplantation.