Chitinase antisense oligonucleotides

Abstract

The present disclosure relates to the field of oligonucleotides. More particularly, the present disclosure relates to antisense oligonucleotides for the specific binding and inhibition of translation of Chitinase (CHIT1) encoding mRNA. Additionally, this disclosure relates to methods of using such oligonucleotides in preventing or treating diseases, disorders or conditions associated with CHIT1, such as neurodegenerative or neuroinflammatory diseases, disorders or conditions like ALS.

Description

[0001] Chitinase antisense oligonucleotides

[0002] Related Application Data

[0003] The present application claims priority from Australian Patent Application No. 2024902912 filed on 13 September 2024 entitled “Chitinase antisense oligonucleotides”. The entire contents of which is hereby incorporated by reference.

[0004] Sequence Listing

[0005] The present application is filed with a Sequence Listing, which has been submitted electronically in XML format form and is hereby incorporated by reference in its entirety. Said XML copy, created on 15 September 2025, is named “208590PCT” and is 53 kb in size.

[0006] Technical field

[0007] The present disclosure relates to the field of oligonucleotides. More particularly, the present disclosure relates to antisense oligonucleotides for the specific binding and inhibition of translation of Chitinase (CHIT1) encoding mRNA. Additionally, this disclosure relates to methods of using such oligonucleotides in preventing or treating diseases, disorders or conditions associated with CHIT1, such as neurodegenerative or neuroinflammatory diseases, disorders or conditions like ALS.

[0008] Background

[0009] One of the few treatments available for MND, Riluzole, has limited efficacy. While this drug can extend life by a few months, it mildly delays rather than treats the disease and does not target the underlying cause of ALS. The emerging availability of gene-based therapies on the market and in clinical trials, such as QALSODY for SOD1-ALS, Ulefnersen for FUS-ALS, and others, is a positive step forward, however, these therapies only target approximately 2% of the total MND population. Given the failures of many experimental MND therapies, such as the recent failure of Relyvrio to meet its phase 3 clinical trial endpoints, there is a pressing need for new therapies, and more particularly generalised therapies which effectively target disease mechanisms with measurable benefits for as many people living with MND as possible.

[0010] Summary

[0011] The present disclosure is based on the surprising finding that particular CHIT1 antisense oligonucleotides (ASOs) can specifically bind to CHIT1 mRNA, including precursor and mature versions thereof, and inhibit CHIT1 mRNA and protein expression in microglial cells and subsequent inflammatory cytokine release therefrom. Given the established role for CHIT1 in the pathogenesis of neurodegenerative diseases, disorders and conditions, such antisense oligonucleotides may be utilised for the treatment of such diseases, disorders and conditions.

[0012] In a first aspect, the present disclosure provides an antisense oligonucleotide for reducing CHIT1 expression, the antisense oligonucleotide comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region of a CHIT1 mRNA, wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof.

[0013] Suitably, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40 or a fragment, variant or derivative thereof and wherein one or more of the U nucleotides thereof may be a T and / or one or more of the T nucleotides thereof may be a U.

[0014] In some examples, the target region comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 29 or a variant thereof. In this regard, the targeting sequence suitably comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 37, or a fragment, variant or derivative thereof.

[0015] In other examples, the target region comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 31 or a variant thereof. For such examples, the targeting sequence suitably comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 38, or a fragment, variant or derivative thereof.

[0016] Suitably, one or a plurality of nucleotides of the antisense oligonucleotide of the present aspect are modified. For certain examples, the modification of the one or plurality of nucleotides is selected from the group consisting of a morpholino modification, a LNA modification, a HNA modification, a CeNA modification, a 2 ’-methoxy ethyl modification, a 2’-O-alkyl modification, a 2’-O-allyl modification, a 2’-C- allyl modification, a 2’ -fluoro modification, a 2’-deoxy modification, and any combination thereof. In particular examples, the modification of the one or plurality of nucleotides is a morpholino modification.

[0017] Suitably, one or a plurality of intemucleotide linkages of the antisense oligonucleotide of the present aspect are modified. In some examples, the modification of the one or plurality of intemucleotide linkages is selected from the group consisting of a phosphorothioate modification; a non-bridging oxygen atom substituting a sulfur atom modification; a phosphonate, such as a methylphosphonate; a phosphodiester modification; a phosphoromorpholidate modification; a phosphoropiperazidate modification; an amide modification; a methylene or methylamino modification; a fromacetal modification; a thioformacetal modification; a peptide nucleic acid modification; or a phosphoroamidate modification; such as a phosphorodiamidate morpholino (PMO), an N3’-P5’ phosphoramidite or a thiophosphoroamidite modification. Referring to certain examples, the modification of the one or plurality of intemucleotide linkages is a phosphoroamidate modification.

[0018] Suitably, the modification of the one or plurality of nucleotides and / or intemucleotide linkages is a phosphorodiamidate morpholino modification. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a fragment, variant or derivative thereof, and wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In other examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 38, or a fragment, variant or derivative thereof, and wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0019] In a second aspect, the present disclosure provides a vector encoding the antisense oligonucleotide of the first aspect.

[0020] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide of the first aspect or the vector of the second aspect and a pharmaceutically-acceptable carrier, diluent or excipient.

[0021] In a fourth aspect, the present disclosure provides a method of reducing or inhibiting CHIT1 expression in a cell, said method including the step of contacting the cell with an effective amount of the antisense oligonucleotide of the first aspect, the vector of the second aspect and / or the pharmaceutical composition of the third aspect to thereby reduce or inhibit CHIT1 expression therein.

[0022] In a fifth aspect, the present disclosure provides a method of treating, preventing or ameliorating a disease, disorder or condition associated with CEHT1 in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide of the first aspect, the vector of the second aspect and / or the pharmaceutical composition of the third aspect to thereby treat, prevent or ameliorate the disease, disorder or condition.

[0023] In a sixth aspect, the present disclosure relates to the use of the antisense oligonucleotide of the first aspect, the vector of the second aspect and / or the pharmaceutical composition of the third aspect in the manufacture of a medicament for treating, preventing or ameliorating a disease, disorder or condition associated with CHIT1 in a subject.

[0024] In some examples, the disease, disorder or condition of the two above aspects is or comprises a neurodegenerative disease, disorder or condition.

[0025] In a seventh aspect, the present disclosure provides a method of treating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide of the first aspect, the vector of the second aspect and / or the pharmaceutical composition of the third aspect to thereby treat, prevent or ameliorate the neurodegenerative disease, disorder or condition.

[0026] In an eighth aspect, the present disclosure relates to the use of the antisense oligonucleotide of the first aspect, the vector of the second aspect and / or the pharmaceutical composition of the third aspect in the manufacture of a medicament for treating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject.

[0027] Referring to the above aspects, the neurodegenerative disease, disorder or condition is suitably associated with CHIT1. For some examples, the neurodegenerative disease, disorder or condition is selected from the group consisting of: Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease, Huntington’s disease, Multiple sclerosis and Parkinson’s disease. In particular examples, the neurodegenerative disease, disorder or condition is ALS.

[0028] In a ninth aspect, the present disclosure provides a method for designing, selecting or identifying an antisense oligonucleotide for reducing or inhibiting CFHT1 expression and / or activity, said method including the steps of:

[0029] (a) providing one or more candidate antisense oligonucleotides comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region of a CHIT1 mRNA, wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof;

[0030] (b) contacting a cell expressing CHIT1 with the one or more candidate antisense oligonucleotides;

[0031] (c) testing the ability of the one or more candidate antisense oligonucleotides to reduce or inhibit CHIT1 expression and / or activity in the cell, and

[0032] (d) selecting an antisense oligonucleotide which reduces or inhibits CFHT1 expression and / or activity in the cell. In certain examples, the target region comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 29 or a variant thereof. In various examples, the target region comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31 or a variant thereof.

[0033] In a tenth aspect, the present disclosure relates to an antisense oligonucleotide designed, selected or identified by the method of the ninth aspect.

[0034] Brief description of the drawings

[0035] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0036] Figure 1: CHIT! regulation of TGF-b signalling (from S. Y. Lee et al., 2022).

[0037] Figure 2: Hypothesis of mechanisms of CHITl-induced neurotoxicity in ALS (from Varghese et al., 2020).

[0038] Figure 3: ALS-associated gene modules in the cervical, thoracic, and lumbar spinal cord. (A) Expression of gene expression module in the cervical (left panel), thoracic and lumbar spinal cord (not shown) is significantly associated with ALS diagnosis. (B) CHIT1 gene expression is negatively correlated with retrospective disease duration.

[0039] Figure 4: Treatment of THP-1 monocytes with PMA induces differentiation into macrophages and expression of CHIT1. (A) Morphological changes of PMA treated monocytes from day 1-3 with characteristic transformation into macrophages by day 3. (B) Amplification of CEHT1 mRNA transcripts using different primer sets for the expression of CHIT1 mRNA following monocyte differentiation into macrophages.

[0040] Figure 5: 2-O-Methyl ASO-induced knockdown of CHIT1. RT-PCR demonstrating targeted exon skipping and / or knockdown of CHIT1 mRNA in PMA-treated THP-1 cells following treatment with 2-0-Me AOs targeting exon 3 (A), exon 5 (B), exon 6 (C), exon 7 (D) or exon 10 (E) of CHIT 1.

[0041] Figure 6: CHIT1 is strongly knocked down by high doses of PMOs targeting exon 5 and exon 6. (A) RT-PCR of CHIT1 and SMN mRNAs in PMA-treated THP-1 cells following treatment with 50 or 100 pM H6A(-15+10) or H7A(+20+44). (B) Quantification of the density of CHIT1 mRNAin cells treated with 50 or 100 pMH6A(- 15+10) or H7A(+20+44) normalised to SMN mRNA. (C) RT-PCR of CHIT1 and SMN mRNAs in PMA-treated THP-1 cells following treatment with 50 or 100 pM H5A(+17+41) or H7A(+49+73). (D) Quantification of the density of CHIT1 mRNA in cells treated with 50 or 100 pMH5A(+17+41) orH7A(+49+73) normalised to SMN mRNA. For quantification in (B, D), normalised CHIT1 levels are compared with the control group, which consists of the UT, ZAP and GTC samples. *Note: this replicate did not have detectable CHIT1 or 5MVmRNA and was excluded from analysis.

[0042] Figure 7: CHIT1 is strongly knocked down by lower doses of PMO targeting exon

[0043] 5. (A) RT-PCR of CHIT1 and SMN mRNAs in PMA-treated THP-1 cells following treatment with 5, 10 or 25 pM H5A(+17+41). (B) Quantification of the density of CHIT1 mRNA in cells treated with 5, 10 or 25 pM H5A(+17+41) normalised to 5MVmRNA. (C) RT-PCR of CHIT1 and SMN mRNAs in PMA-treated THP-1 cells following treatment with 5, 10 or 25 pM H6A(- 15+10). (D) Quantification of the density of CHIT1 mRNA in cells treated with 5, 10 or 25 pM H6A(-15+10) normalised to SMN mRNA. (E) RT-PCR of CHIT1 and SMN mRNAs in PMA- treated THP-1 cells following treatment with 5, 10 or 25 pM H7A(+49+73). (F) Quantification of the density of CHIT1 mRNA in cells treated with 5, 10 or 25 pM H7A(+49+73) normalised to SMN mRNA. (G) Levels of TBP mRNA in cells treated with 5, 10 or 25 pM H5A(+17+41), H6A(-15+10) or H7A(+49+73) as measured by quantitative PCR (qPCR) normalised to GTC. For quantification in (B, D, F), normalised CHIT1 levels are compared with the control group, which consists of the UT, ZAP and GTC samples.

[0044] Figure 8: MDMi derived from ALS cases exhibit canonical pathological features. Compared with MDMi generated from healthy donor PBMCs, ALS MDMi exhibit mislocalised cytoplasmic aggregates of TDP-43 (A), reduced phagocytosis and uptake of pHrodo-tagged E. coli particles (B), and increased secretion of inflammatory cytokines (C). Figures derived from Quek et al., 2022

[0045] Figure 9: CHIT1 in MDMi is knocked down by PMOs targeting exon 6 (H6A(-15+10) or E6-PMO) and exon 5 (H5A(+17+41) or E5-PMO). CHIT1 mRNA was significantly knocked down in MDMi by 50 and 25 pM E6-PMO (A-B) and E5-PMO (C-D). MDMi differentiation was comparable between Zap transfection control cells (E) and PMO-treated cells (F, 50 pM E5-PMO treatment shown).

[0046] Figure 10: E5-PMO significantly knocked down CHIT1 mRNA in HC (A) and ALS (B) MDMi (expressed as fold change of (7 / / 77 / 18S over ZAP control). *, p < 0.05; **, p < 0.01. Figure 11: Inflammatory cytokine production is decreased by CHIT1 knockdown in HC and ALS MDMi. CHIT1 knockdown by 50-10 pM E5-PMO in HC (circle symbols) and ALS (triangle symbols) MDMi resulted in reduced levels of IL-8 (A), IL-6 (B), TNF (C) and IL-18 (D). Cytokine levels normalized to ZAP control. *,p < 0.05 compared to ZAP control.

[0047] Figure 12: RT-PCR gels showing robust CHIT1 mRNA knockdown after transfection with PMOs targeting exon 5 (A) and exon 10 (B).

[0048] Figure 13: The evaluation of PMO targeting exon 7 has shown nearly complete knockdown of the CHIT1 transcript as determined by RT-PCR.

[0049] Figure 14. Key cytokine responses following CHIT1 knockdown in healthy controls and ALS patients. (A) IL-8 and TNF-a levels measured in MDMi from healthy controls (n = 3) and ALS patients (n = 3). The top graphs represent healthy controls (HC) and the bottom graphs represent ALS patients. (B) IL-6 and IL-10 levels measured under the same conditions. H6A was delivered via Setting #2 (10 pM, 15 pM, 25 pM), and H5A via Setting #4 (10 pM, 25 pM, 50 pM), while unrelated control mSODl PMO (10 pM) were used across both electroporation settings. Results are normalised as fold change relative to their respective Zap-only controls.

[0050] Figure 15. Less robust cytokine responses following CHIT1 knockdown in healthy controls and ALS patients. (A) IL-ip, IL-18, and IFN-y levels measured in MDMi from healthy controls (n = 3) and ALS patients (n = 3). The top graphs represent healthy controls and the bottom graphs represent ALS patients. H6A was delivered via Setting #2 (10 pM, 15 pM, 25 pM), H5A via Setting #4 (10 pM, 25 pM, 50 pM), and the unrelated control mSODl PMO (10 pM) was transfected under both settings. Results are normalised as fold change relative to their respective Zap-only controls.

[0051] Figure 16. Cytokines unaffected by CHIT1 knockdown in healthy controls and ALS patients. (A) IL-23, IL-33, IL-17A and (B) IFN-a2, IL-12p70, and MCP-1 levels in MDMi from healthy controls (n=3) and ALS patients (n = 3). H6A (Setting #2), H5A (Setting #4), and mSODl PMO (10 pM) were tested. Results are shown as raw data to depict trends. No significant cytokine changes were observed.

[0052] Figure 17. Detection of CHIT1 protein secretion in healthy controls and ALS MDMi. Western blot showing three paired samples of healthy controls (n=3) and ALS patients (n=3). Samples were probed with anti-CHITl antibody, detecting a specific band at approximately 50 kDa. Membranes were also stained using a reversible protein dye to assess total protein loading for normalisation. Each gel included recombinant human CHIT1 (rhCHIT-1) and supernatant spiked with cell lysate as positive controls to validate detection and assess transfer efficiency. This experiment represents a repeat, as the first MND40 sample exhibited significant cell death and undetectable protein loading. In this repeat, MND40 showed improved cell viability and protein recovery due to increased cell numbers at the time of harvest.

[0053] Figure 18. Densitometric analysis of CHIT1 secretion following PMO treatment in healthy controls (blue) and ALS MDMi (Green) supernatant. Densitometric analysis of Western blot bands from Figure 17 was performed using Image! CHIT1 band intensities were first normalised to total protein loading (reversible dye signal) and then further normalised to rhCHIT-1 or supernatant spiked with cell lysate controls on each gel. Quantitative analysis revealed a dose-dependent decrease in CHIT1 secretion against Zap control, correlating with increased CHIT1 knockdown following PMO treatment. Notably, 25 pM and 50 pM CHIT1 PMO treatments resulted in inhibition of CHIT1 secretion in both healthy controls and ALS MDMi.

[0054] Figure 19. LDH assay to assess cytotoxicity following CHIT1 PMO transfection in healthy controls and ALS MDMi. LDH levels were measured in the supernatants of MDMi from healthy controls (blue, n=6) and ALS patients (green, n=8) following CHIT1 PMO transfection. Results were normalised against UT-T (transfection buffer only) and Zap #4 electroporation without PMO using Setting #4) controls. Across all conditions, no significant increase in LDH release was observed, indicating no detectable cytotoxicity following transfection.

[0055] Figure 20. Nitric oxide production following CHIT1 PMO transfection in healthy controls and ALS MDMi. Nitric oxide (NO) levels were measured in the supernatants of MDMi from healthy controls (blue) and ALS patients (brown) following CHIT1 PMO transfection. Results were normalised to UT-T (transfection buffer only) control. No significant changes in nitric oxide production were observed across all transfected conditions, indicating that CHIT1 PMO transfection did not induce oxidative stress in either healthy or ALS MDMi.

[0056] Figure 21. CHIT1 expression following H5A PMO transfection in healthy controls and ALS MDMi. CHIT1 expression was assessed by RT-PCR in MDMi from healthy controls (blue, n = 6) and ALS patients (purple, n = 8) following transfection with H5A at 25 pM. Expression levels were normalised to Zap controls (electroporation only, no PMO). H5A transfection resulted in a significant knockdown of CHIT1 expression in both healthy controls and ALS MDMi. No changes were observed in mSODl PMO-treated cells, confirming the specificity of H5A-mediated CHIT1 knockdown. Rapid ALS patients are highlighted in orange and 50% knockdown is annotated with a dotted line for visualisation. Figure 22. TNF-a cytokine expression following H5A PMO transfection in healthy controls and ALS MDMi. TNF-a levels were measured in the supernatants of MDMi from healthy controls and ALS patients following H5A (25 pM) transfection. A decrease in TNF-a secretion was observed in H5 A-transfected cells compared to Zap only control, consistent with reduced inflammatory signalling following CHIT1 knockdown (Figure 21). No significant reduction in TNF-a levels was observed in non-transfected or control PMO-treated cells. Rapid ALS patients are highlight in orange for visualisation.

[0057] Key to the Sequence Listing

[0058] SEQ ID NO: 1 Nucleotide sequence of CHIT1_H3A(-15+1O) of Table 1

[0059] SEQ ID NO: 2 Nucleotide sequence of CHIT1 H3 A(+15+39) of Table 1

[0060] SEQ ID NO: 3 Nucleotide sequence of CHHT H3 A(+74+98) of Table 1

[0061] SEQ ID NO: 4 Nucleotide sequence of CHIT1_H3A(+138+162) of Table 1

[0062] SEQ ID NO: 5 Nucleotide sequence of CHHT_H3D(+22-03) of Table 1

[0063] SEQ ID NO: 6 Nucleotide sequence of OUT l_H3D(-01-25) of Table 1

[0064] SEQ ID NO: 7 Nucleotide sequence of CHHT_H5A(-16+09) of Table 1

[0065] SEQ ID NO: 8 Nucleotide sequence of CHHT_H5A(+ 17+41) of Table 1

[0066] SEQ ID NO: 9 Nucleotide sequence of CHITl_H5A(+68+72) of Table 1

[0067] SEQ ID NO: 10 Nucleotide sequence of CHHT_H5A(+106+130) of Table 1

[0068] SEQ ID NO: 11 Nucleotide sequence of CHHT_H5A(+136+160) of Table 1

[0069] SEQ ID NO: 12 Nucleotide sequence of CHHT_H5A(+08-17) of Table 1

[0070] SEQ ID NO: 13 Nucleotide sequence of CHHT_H6A(-15+10) of Table 1

[0071] SEQ ID NO: 14 Nucleotide sequence of CHHT_H6A(+13+37) of Table 1

[0072] SEQ ID NO: 15 Nucleotide sequence of CHHT_H6A(+84+108) of Table 1

[0073] SEQ ID NO: 16 Nucleotide sequence of CHHT_H6D(+17-08) of Table 1

[0074] SEQ ID NO: 17 Nucleotide sequence of CHHT_H7A(-06+19) of Table 1

[0075] SEQ ID NO: 18 Nucleotide sequence of CHHT_H7A(+20+44) of Table 1

[0076] SEQ ID NO: 19 Nucleotide sequence of CHITl_H7A(+49+73) of Table 1

[0077] SEQ ID NO: 20 Nucleotide sequence of CHITl_H7A(+87+l 11) of Table 1

[0078] SEQ ID NO: 21 Nucleotide sequence of CHIT1_H7D(+O9-16) of Table 1

[0079] SEQ ID NO: 22 Nucleotide sequence of CHITl_H10A(-22+03) of Table 1

[0080] SEQ ID NO: 23 Nucleotide sequence of CHHT_H10A(+08+32) of Table 1

[0081] SEQ ID NO: 24 Nucleotide sequence of CHITl_H10A(+35+59) of Table 1

[0082] SEQ ID NO: 25 Nucleotide sequence of CHHT_H10A(+83+107) of Table 1 SEQ ID NO: 26 Nucleotide sequence of CE0TT_H10D(+15-10) of Table 1

[0083] SEQ ID NO: 27 Nucleotide sequence of target region of CHIT 1_H3A(+ 15+39)

[0084] SEQ ID NO: 28 Nucleotide sequence of target region of CHIT1 H3 A(+74+98)

[0085] SEQ ID NO: 29 Nucleotide sequence of target region of CHIT 1_H5A(+ 17+41)

[0086] SEQ ID NO: 30 Nucleotide sequence of target region of CHITl_H5A(+106+130)

[0087] SEQ ID NO: 31 Nucleotide sequence of target region of CHIT1_H6A(-15+1O)

[0088] SEQ ID NO: 32 Nucleotide sequence of target region of CHITl _H6A(+13+37)

[0089] SEQ ID NO: 33 Nucleotide sequence of target region of CHITl_H7A(+20+44)

[0090] SEQ ID NO: 34 Nucleotide sequence of target region of CHITl_H7A(+49+73)

[0091] SEQ ID NO: 35 Nucleotide sequence of target region of CHITl_H10A(+08+32)

[0092] SEQ ID NO: 36 Nucleotide sequence of target region of CHITl_H10A(+35+59)

[0093] SEQ ID NO: 37 Nucleotide sequence of CHIT 1_H5A(+ 17+41) of Table 2

[0094] SEQ ID NO: 38 Nucleotide sequence of CHIT1_H6A(-15+1O) of Table 2

[0095] SEQ ID NO: 39 Nucleotide sequence of CHITl_H7A(+20+44) of Table 2

[0096] SEQ ID NO: 40 Nucleotide sequence of CHITl_H7A(+49+73) of Table 2

[0097] SEQ ID NO: 41 Amino acid sequence of CEHT1 protein

[0098] SEQ ID NO: 42 Nucleotide sequence of CEHT1 cDNA

[0099] SEQ ID NO: 43 Nucleotide sequence of CEHT1 pre-mRNA

[0100] Detailed description

[0101] General Techniques and Definitions

[0102] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0103] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppi -22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu etal, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0104] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0105] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0106] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0107] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0108] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0109] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0110] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5. 5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0111] As used herein, the terms “about” or “approximately”, unless stated to the contrary, refers to + / - 10% (e.g., + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or any range therein), more preferably + / -5%, even more preferably + / -1%, of the designated value.

[0112] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0113] By “consisting essentially of’, in the context of: (a) an amino acid sequence, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- and / or C-terminus thereof; or (b) a nucleic acid sequence, is meant the recited nucleic acid sequence together with an additional one, two or three nucleic acids at the 5’ and / or 3’ thereof.

[0114] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0115] For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0116] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0117] Antisense oligonucleotides

[0118] The present inventors have surprisingly shown that antisense oligonucleotides that hybridize to or are complementary to particular target regions or sequences in CHIT1 precursor mRNA (pre-mRNA) and / or mature mRNA can be utilised to bind and functionally decrease expression of full length CHIT1 mRNA and protein either by direct knockdown or inducing exon skipping. Decreases in inflammatory cytokine expression and release are also advantageously associated with such knockdown. Accordingly, in one broad form, the present disclosure provides an antisense oligonucleotide that includes a targeting sequence that hybridizes to or is complementary to at least a portion of a target sequence or region within a CHIT1 mRNA molecule, inclusive of precursor or mature versions thereof. More particularly, the targeting sequence of the antisense oligonucleotide hybridizes to or is complementary to at least a portion of a target sequence or region within exon 3, exon 5, exon 6, exon 7 or exon 10, inclusive of adjacent introns, of a mRNA molecule encoding CHIT 1. One of skill in the art can determine the corresponding exon and / or intron number in any isoform of CHIT1 using the numbers provided in reference to the mRNA sequence at ENST00000367229.6 or NM_003465.3.

[0119] Therefore, in one form, the present disclosure provides an antisense oligonucleotide for reducing CHIT1 expression, the antisense oligonucleotide comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region or sequence of a CHIT1 mRNA (e.g., a CHIT1 pre-mRNA and / or a CHIT1 mRNA), wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof.

[0120] In a related form, the present disclosure provides an antisense oligonucleotide for reducing CHIT1 expression, the antisense oligonucleotide comprising a targeting sequence that that comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 26 and 37 to 40 or a fragment, variant or derivative thereof (or more particularly SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40 or a fragment, variant or derivative thereof) and wherein one or more of the U nucleotides thereof may be a T and / or one or more of the T nucleotides thereof may be a U.

[0121] In the context of this disclosure, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA), deoxyribonucleic acid (DNA) or combinations thereof, wherein the polymer or oligomer of nucleotide monomers contains any combination of nucleobases (referred to in the art and herein as simply as “base”), modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as “internucleotide linkage”). Oligonucleotides can be single-stranded or double-stranded or a combination thereof. A single-stranded oligonucleotide can have doublestranded regions and a double-stranded oligonucleotide can have single-stranded regions (such as a microRNA or shRNA).

[0122] The terms “antisense oligo”, “antisense oligonucleotide”, “antisense oligomer” or “ASO” are used interchangeably herein and refer to a nucleic acid molecule that is capable of modulating expression of a target gene by hybridizing to a target nucleic acid or polynucleotide (typically an RNA molecule), in particular to a contiguous sequence on a target nucleic acid, by Watson-Crick base pairing. Antisense oligonucleotides are typically not double stranded and are therefore not siRNAs or shRNAs. In certain examples, the antisense oligonucleotides disclosed herein are single stranded. It is understood that single stranded oligonucleotides disclosed herein can form antisenses 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 antisense oligonucleotide. The antisense oligonucleotides disclosed herein are suitably modified oligonucleotides, and more particularly chemically modified oligonucleotides, as described in more detail herein.

[0123] Such an antisense oligonucleotide can be designed to block or inhibit translation of mature mRNA or to inhibit natural pre-mRNA splice processing, or induce degradation of targeted mRNAs, and may be said to be specifically directed to or targeted against a target region or sequence with which the ASO in question hybridizes. The term “pre-mRNA” refers to any precursor mRNA species that contains at least one intron, whilst the term “mature mRNA” refers to mRNA that has undergone processing, such as splicing or modification, and is in a form suitable for protein synthesis. In certain examples, the target sequence is a region surrounding or including an AUG start codon of an mRNA, a 3’ or 5’ splice site of a pre- processed or precursor mRNA (i.e., an intron-exon boundary), or a branch point of an mRNA. The target region may be within an exon or within an intron or a combination thereof. The target region for a splice site may include an mRNA sequence having its 5’ end at 1 to about 25 base pairs downstream of a normal splice acceptor junction in a pre-mRNA. An exemplary target sequence for a splice site is any region of a pre-mRNA that includes a splice site or is contained entirely within an exon coding sequence or spans a splice acceptor or donor site (e.g., spans an intron-exon boundary).

[0124] The term “nucleic acid” as used herein designates single- or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and / or thiouridine, although without limitation thereto.

[0125] The terms “target sequence” or “target region” are used interchangeably herein and refer to a portion of the target mRNA against which the antisense oligonucleotide is directed, that is, the sequence to which the oligonucleotide will hybridize by Watson-Crick base pairing of a complementary sequence. In certain examples, the target sequence may be a contiguous region of a pre-mRNA that includes both intronic and exonic target sequence. For other examples, the target sequence will consist exclusively of either intronic or exonic sequences. Exemplary target sequences are set forth in SEQ ID NOs: 27 to 36. It is appreciated that variation may be observed in the target region, such as natural genomic variation that may be observed within a population of subjects, of the CHIT1 mRNA. Thus, in some examples, the target region provided herein have at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or any range therein), and more particularly at least about 95% sequence identity, with the exemplary target regions as disclosed herein, such as those set forth in SEQ ID NOs: 27 to 36 below.

[0126] Exemplary antisense sequences for targeting the human CHIT1 pre-mRNA are shown in Tables 1 and 2 below. Antisense oligonucleotides of the present disclosure can comprise all or a portion of these targeting sequences. The term “targeting sequence” refers to the sequence in an antisense oligonucleotide that is complementary or substantially complementary to a portion of the target sequence or region in the RNA genome. The entire sequence, or only a portion thereof, of the antisense oligonucleotide may be complementary to the target sequence. In this regard, it is contemplated that the antisense oligonucleotide described herein may include additional sequence at a 5’ and / or 3’ end of the targeting sequence therein. Suitably, the antisense oligonucleotide provided herein may comprise a targeting sequence comprising about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55 or 60 bases (or any range therein, such as 10- 15 bases, 10-20 bases, 15-25 bases, 15-30 bases, 20-30 bases, 20 to 40 bases), inclusive of contiguous and non-contiguous bases, that are complementary or at least partly complementary to the target region of interest (e.g., one of SEQ ID NOs: 27 to 36). For example, in an antisense oligonucleotide containing 20 to 30 bases, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases (or any range therein, such as 10-15 bases, 10-20 bases, 15-25 bases, 15-30 bases, 20-30 bases) may be of a targeting sequence that is complementary or partly complementary to the target region (e.g., one of SEQ ID NOs: 27 to 36). Typically, the targeting sequence is formed of contiguous bases, but may alternatively be formed of non-contiguous sequences that when placed together, such as from opposite ends of the oligonucleotide, constitute a targeting sequence that spans the target sequence of the CHIT1 mRNA. Exemplary targeting sequences are set forth in SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40.

[0127] Oligomers, such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid (e.g., a target region of a CEUTl mRNA) to hybridize. In certain examples, antisense oligonucleotides can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence (e.g., a CHIT1 mRNA) to which they are targeted. For example, an antisense oligonucleotide in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining non- complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using alignment programs, as are well known in the art and described herein.

[0128] Suitably, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in one of SEQ ID NOs: 27 to 36 or a variant thereof. In some examples, the antisense oligonucleotide described herein comprises a nucleotide sequence (i.e., a targeting sequence) that is fully complementary to the target region set forth in SEQ ID NO: 27 or a variant thereof. In other examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 28 or a variant thereof. In certain examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 29 or a variant thereof. In particular examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 30 or a variant thereof. In various examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 31 or a variant thereof. In some examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 32 or a variant thereof. In certain examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 33 or a variant thereof. In various examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 34 or a variant thereof. In other examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 35 or a variant thereof. In certain examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to the target region set forth in SEQ ID NO: 36 or a variant thereof. In alternative examples, the antisense oligonucleotide described herein comprises a nucleotide sequence that is fully complementary to a portion (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 bases or any range therein) of the target region set forth in one of SEQ ID NOs: 27 to 36 or a variant thereof.

[0129] Suitably, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 26 and 37 to 40 or a fragment, variant or derivative thereof. Referring to certain examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40 or a fragment, variant or derivative thereof. For such examples, one or more of the U nucleotides thereof may be a T and / or one or more of the T nucleotides thereof may be a U. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 2 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 3 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 8 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 10 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 13 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 14 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 18 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 19 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 23 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 24 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 37 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 38 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 39 or a fragment, variant or derivative thereof. In some examples, the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 40 or a fragment, variant or derivative thereof.

[0130] In view of the foregoing, it is contemplated that an antisense oligonucleotide of the present disclosure need not hybridize to all nucleobases in a target region of a CHIT1 mRNA and the nucleobases to which it does hybridize may be contiguous or noncontiguous. Antisense oligonucleotides may hybridize over one or more segments of a pre-mRNA or mature RNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or antisense structure may be formed). In certain examples, an antisense oligonucleotide hybridizes to noncontiguous nucleobases in a target polynucleotide. For example, an antisense oligonucleotide can hybridize to nucleobases in a CHIT1 mRNA that are separated by one or more nucleobases to which the antisense oligonucleotide does not hybridize.

[0131] As described in more detail herein, variants of the antisense oligonucleotides of the present disclosure are contemplated. To this end, a “targeting sequence” may have “near” or “substantial” complementarity to the target sequence and still function for the purpose of the present disclosure, that is, still be “complementary to” or “hydridize to” a target region or sequence of an mRNA molecule. Suitably, the antisense oligonucleotides described herein have at most one, two or three mismatches with the target sequence out of 10 nucleotides (e.g., 10 contiguous nucleotides), and more particularly at most one, two, three or four mismatches with the target sequence out of 20 nucleotides (e.g., 20 contiguous nucleotides). Such mismatches may include, for example, a nucleotide suitable for wobble pairing, such as G-U pairing, with the target region of interest. Accordingly, one or more (e.g., 1, 2, 3, 4, 5 etc) adenine nucleotides of the targeting sequence (e.g., SEQ ID NOs: 1 to 26 and 37 to 40) may be replaced by a guanine nucleotide and complementarity and hybridization with the target region is suitably maintained by way of the formation of G-U wobble pairing. Thus, in some examples, the targeting sequence of the antisense oligonucleotide and the target region of the CHIT1 mRNA hybridize to one another to form a duplex having 70% to 100% complementarity (e.g., 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100% complementarity) with one another. In other examples, the antisense oligonucleotides provided herein or the targeting sequences thereof have at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or any range therein), and more particularly at least about 95% sequence identity, with the exemplary targeting sequences as disclosed herein, such as those set forth in SEQ ID NOs: 1 to 26 and 37 to 40, or more particularly SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40.

[0132] An antisense oligonucleotide can be considered to specifically hybridize or bind to a target polynucleotide (e.g., a CHIT1 mRNA) if the antisense oligonucleotide hybridizes to the target polynucleotide, or target region thereof, under physiological conditions, with a melting temperature (Tm) of at least about 45°C (e.g., at least about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 °C or any range therein), more particularly at least about 50°C, more particularly at least about 55°C, more particularly at least about 60°C, and even more particularly between about 50°C to about 90°C, between about 50°C to about 80°C, between about 50°C to about 70°C or between about 60°C to about 80°C. Such hybridization suitably corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of a targeting sequence of an antisense oligonucleotide hybridizes to a complementary target region of a target polynucleotide (e.g., a CHIT1 mRNA). Again, such hybridization may occur with near or substantial complementarity of the antisense oligonucleotide (or the targeting sequence thereof) to the target sequence of the CHIT1 mRNA, as well as with exact complementarity.

[0133] Oligonucleotides generally refer to relatively short sequences of nucleotides, typically with twenty or fewer bases (or nucleotide units), but can also be significantly longer, such as up to about 160 to about 200 nucleotides. By way of example, the antisense oligonucleotide provided herein is at least about 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides, or any range therein, in length as a single stranded molecule. More particularly, the antisense oligonucleotide is suitably about 3 to about 75 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, or 75 nucleotides, or any range therein) in length as a single stranded molecule. In various examples, the antisense oligonucleotide is about 15 to about 40, more particularly about 20 to about 35, more particularly about 15 to about 35, more particularly about 20 to about 50, more particularly about 20 to about 40, more particularly about 20 to about 30, even more particularly about 22 to about 28, nucleotides in length as a single stranded molecule. In some examples, the antisense oligonucleotide is about 24 nucleotides in length as a single stranded molecule. For other examples, the antisense oligonucleotide is about 25 nucleotides in length as a single stranded molecule. According to certain examples, the antisense oligonucleotide is about 26 nucleotides in length as a single stranded molecule.

[0134] The antisense oligonucleotides herein may include single-stranded DNA and / or RNA (e.g., RNA, DNA or RNA-DNA hybrids). In some examples, the antisense oligonucleotides herein include single-stranded DNA (e.g., the antisense oligonucleotides of SEQ ID NOs: 37 to 40). In other examples, the antisense oligonucleotides herein include single-stranded RNA (e.g., the antisense oligonucleotides of SEQ ID NOs: 1 to 26). DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. In this regard, the antisense oligonucleotide may be a DNA-RNA hybrid. An antisense oligonucleotide of the present disclosure comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and / or thiouridine, although without limitation thereto. It is further contemplated that one or more T nucleobases of the nucleic acids, and more particularly the antisense oligonucleotides, described herein may be replaced with a U nucleobase without substantially altering the functionality thereof. Similarly, one or more U nucleobases of the nucleic acids, and more particularly the antisense oligonucleotides, described herein may be replaced with a T nucleobase without substantially altering the functionality thereof.

[0135] Contemplated herein are “variant” antisense oligonucleotides that may include, for example, a targeting sequence that is complementary to a naturally occurring (e.g., allelic) variants and orthologs (e.g., from a different species) of, for example, a target region of a CHIT1 mRNA. As used herein, a nucleic acid “variant” shares a definable nucleotide sequence relationship with a reference nucleic acid sequence (e.g., SEQ ID NOs: l to 40). The “variant” nucleic acid may have one or a plurality of nucleic acids of the reference nucleic acid sequence deleted or substituted by different nucleic acids. It is well understood in the art that some nucleic acids of a DNA / RNA-based binding or recognition site may be substituted or deleted without changing (or only having minimal change to) the affinity of the target polynucleotide therefor. Suitably, nucleic acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated nucleic acid of the present disclosure (e.g., SEQ ID NOs: 1 to 40). Percent sequence identity may be determined by any method known in the art, such as that described herein.

[0136] Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleic acids / proteins may each comprise (1) only one or more portions of a complete nucleic acid / protein sequence that are shared by the nucleic acids / proteins, and (2) one or more portions which are divergent between the nucleic acids / proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA; Clustal Omega (https: / / www.ebi. ac.uk / jdispatcher / msa / clustalo?stype=protein) incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0137] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0138] Also contemplated herein are nucleic acid fragments, such as oligonucleotide fragments. A “fragment” is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100% of the nucleotide sequence. A non-limiting example is an amplification product or a primer or probe. In particular examples, a nucleic acid fragment may comprise, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 (inclusive of any range therein) contiguous nucleotides of said nucleic acid (e.g., SEQ ID NOs: 1 to 40 and 42 and more particularly SEQ ID NOs: 1 to 26 and 37 to 40).

[0139] Further contemplated herein are nucleic acid derivatives, inclusive of oligonucleotide derivatives. Such oligonucleotide derivatives may include, for example, one or more modifications and / or conjugates as described herein. By way of example, the antisense oligonucleotides may be conjugated to one or more moieties or groups which enhance the activity, cellular distribution or cellular uptake thereof. These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups. Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes / labels (e.g., Cy5 dye to determine cellular uptake and / or localisation of the antisense oligonucleotide).

[0140] Referring to certain examples, the antisense oligonucleotide described herein is linked, coupled or otherwise associated with one or more moieties, conjugates, substances or molecules, such as those known in the art to promote cellular uptake and / or penetration or transport across the blood-brain barrier. To this end, antisense oligonucleotides can be conjugated, linked, coupled or otherwise associated with a viral vector (e.g., an AAV vector), a conjugated peptide (e.g., a cell penetrating peptide, such as those described in U.S. Application No. 2010 / 0016215, which is incorporated by reference in its entirety), an antibody or antibody fragment (e.g., an antibody to the transferrin receptor, an anti-CD44 antibody, an anti-EphA2 antibody), a binding ligand (e.g., aptamers and acetylgalactosamine (GalNAc)), a nanoparticle (e.g., a lipid nanoparticle (LNP), a glucose-coated polymeric nanoparticle), a lipid-based particle (e.g., a liposome, an LNP) or an extracellular vesicle (e.g., an exosome). In embodiments, the antisense oligonucleotide is linked with a viral vector, to increase cellular uptake and / or increase transport across the blood-brain barrier of the antisense oligonucleotide. Conjugates can be chemically linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, such as by using a linker. Linkers can include a bivalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3’ end of the antisense oligonucleotide. Exemplary methods of preparing oligonucleotide conjugates are described in U.S. Pat. No. 8,450,467, which is incorporated by reference herein.

[0141] Suitably, the antisense oligonucleotides of the present disclosure are capable of inhibiting or reducing an expression of a target mRNA and / or a target protein, and more particular a CHIT1 mRNA and / or a CHIT1 protein. As used herein, the phrases “reduce CHIT1 expression”, “inhibit CEUTl expression” or the like refers to an antisense oligonucleotide of the present disclosure reducing the ability of a CHIT1 mRNA to be translated into a corresponding CHIT1 protein. A decrease or reduction in CHIT1 expression may be a statistically significant as compared to the expression of CHIT1 produced in the absence of the antisense oligonucleotide in question or a control composition. This may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in CHIT1 expression, including all integers in between and ranges therein, in a cell (e.g., a microglial cell), tissue (e.g., CNS tissue) or a biological sample (e.g., a CSF sample) of the subject , such a cell or tissue contacted with the antisense oligonucleotide versus a control cell or tissue in the absence of the oligonucleotide or a biological sample from a subject prior to receiving the antisense oligonucleotide versus a further biological sample from the subject after receiving the antisense oligonucleotide. In particular examples, CHIT1 expression (inclusive of mRNA and / or protein expression thereof) is decreased by the antisense oligonucleotide described herein, such as in a microglial cell or CSF sample of a subject, by about 1.1 to about 10 fold (e.g., about 1.5 to about 10 fold, about 2 to about 10 fold, about 3 to about 10 fold, about 4 to about 10 fold, about 1.1 to about 5 fold, about 1.1 to about 6 fold, about 1.1 to about 7 fold, about 1.1 to about 8 fold, about 1.1 to about 9 fold, about 2 to about 5 fold, about 2 to about 6 fold, about 2 to about 7 fold, about 2 to about 8 fold, about 2 to about 9 fold, about 3 to about 6 fold, about 3 to about 7 fold, about 3 to about 8 fold, about 3 to about 9 fold, about 4 to about 7 fold, about 4 to about 8 fold, about 4 to about 9 fold, at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 5 fold, or at least about 10 fold) compared to a CHIT1 expression in the absence of the antisense oligonucleotide (e.g., a control microglial cell in a subject not receiving the antisense oligonucleotide or a CSF sample in the subject prior to receiving the antisense oligonucleotide).

[0142] A reduction in expression of CHIT1 may be measured or assessed by mRNA and / or protein levels thereof, such as by standard means known in the art. By way of example, protein expression of CHIT1 may be detected or measured by western blotting, flow cytometry, immunoassays (e.g., ELISA), immunoprecipitation, immunocytochemistry or immunohistochemistry, typically by way of an antibody or antibody fragment which binds CHIT1. In other examples, CHIT1 gene expression may be measured by nucleic acid sequence amplification (e.g., PCR inclusive of quantitative and semi-quantitative PCR) or nucleic acid hybridization techniques, such as Northern blotting. Additionally, expression levels for CHIT1 can be measured indirectly by assessing a level of activation of cell signalling pathways associated therewith. For example, a level of CHIT1 expression may be determined by assessing a level of inflammation or inflammatory signalling.

[0143] Typically, an antisense oligonucleotide of the present disclosure will be synthesized in vitro, such as by chemical synthesis (e.g., solid-phase synthesis). However, in some instances, particularly where modified bases and a modified backbone are not required, they can be expressed in vitro or in vivo in a suitable system, such as by a recombinant virus or cell.

[0144] Suitably, the antisense oligonucleotides described herein are isolated. For the purposes of this disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material, such as the antisense oligonucleotides, may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0145] The targeting sequence of the antisense oligonucleotide may be complementary to or comprise the entirety of a target region described herein (e.g., SEQ ID NOs: 27 to 36), or a part thereof. It is also envisaged that the targeting sequence of the antisense oligonucleotide may be complementary to a variant of a consensus or reference sequence of a target region of CHIT1. The antisense oligonucleotide may of course comprise unrelated sequences, such as at a 5’ and / or 3’ end of a reference nucleic acid sequence (e.g., SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40), which may function to stabilize the molecule.

[0146] Modified Bases

[0147] Antisense oligonucleotides of the present disclosure may have nucleobase (“base”) modifications or substitutions. Such modifications can advantageously increase the binding specificity of the antisense oligonucleotide for the target region of the CHIT1 mRNA molecule and / or reduce or minimise the immunogenicity or antigenicity of the antisense oligonucleotide. It is contemplated that the antisense oligonucleotides described herein can employ a variety of antisense chemistries. Examples of oligomer chemistries include, without limitation, phosphoramidate morpholino oligomers and phosphorodiamidate morpholino oligomers (PMO), phosphorothioate modified oligomers, 2’ O-methyl modified oligomers, peptide nucleic acid (PNA), locked nucleic acid (LNA), phosphorothioate oligomers, 2’ O-MOE modified oligomers, 2’-fluoro-modified oligomer, 2'0,4'C-ethylene-bridged nucleic acids (ENAs), tricyclo-DNAs, tricyclo-DNA phosphorothioate nucleotides, 2'-O-[2-(N- methylcarbamoyl)ethyl] modified oligomers, morpholino oligomers, peptide-conjugated phosphoramidate morpholino oligomers (PPMO), phosphorodiamidate morpholino oligomers having a phosphorous atom with (i) a covalent bonds to the nitrogen atom of a morpholino ring, and (ii) a second covalent bond to a (1,4- piperazin)- 1-yl substituent or to a substituted (l,4-piperazin)-l-yl (PMOplus), and phosphorodiamidate morpholino oligomers having a phosphorus atom with (i) a covalent bond to the nitrogen atom of a morpholino ring and (ii) a second covalent bond to the ring nitrogen of a 4-aminopiperdin-l-yl (i.e., APN) or a derivative of 4-aminopiperdin-l-yl (PMO-X) chemistries, including combinations of any of the foregoing.

[0148] In particular examples, one or more bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc bases inclusive of any range therein, such as 5 to 30 modified bases, 10 to 25 modified bases, 10 to 20 modified bases, 15 to 30 modified bases) of the antisense oligonucleotide described herein are modified. For some examples, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases of the antisense oligonucleotide described herein are modified. For other examples, no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases of the antisense oligonucleotide described herein are modified. In certain examples, one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end of the antisense oligonucleotide are modified. In other examples, one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 3’ end of the antisense oligonucleotide are modified. In various examples, one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end of the antisense oligonucleotide are modified and one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 3’ end of the antisense oligonucleotide are modified. In some examples, all bases of the antisense oligonucleotide described herein are modified. In alternative examples, no bases of the antisense oligonucleotide described herein are modified. In certain examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the bases of the antisense oligonucleotide are modified.

[0149] The modified base may be any known in the art, such as those reviewed in Ochoa and Milam (Molecules 2020, 25(20), 4659). Examples of modified bases include oligonucleotides comprising one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. In one example, the antisense oligonucleotide comprises one of the following at the 2' position: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10.

[0150] Further examples of modified oligonucleotides include one or more nucleotides comprising one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.

[0151] In particular examples, the modification is selected from the group consisting of a 2'- O-methyl, 2'-O-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino, fluoroarabinonucleotide, threose nucleic acid or 2'-O— (N-methlycarbamate). In some examples, the modified base comprises a 2'-O-methyl, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'- CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-amino, fluoroarabinonucleotide, threose nucleic acid, 2'-O— (N-methlycarbamate) and any combination thereof.

[0152] Suitably, the modification includes 2'-methoxy (2'-O-CH3or 2’0Me), that is, an alkoxyalkoxy group. In certain examples, the antisense oligonucleotide includes one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end and / or a 3’ thereof that are 2’0Me modified. In particular examples, the antisense oligonucleotide includes at least one nucleotide at a 5’ end and a 3’ thereof that are 2’0Me modified. In some examples, the antisense oligonucleotide includes at least one nucleotide at a 5’ end thereof that is 2’0Me modified. In other examples, the antisense oligonucleotide includes at least one nucleotide at a 3’ end thereof that is 2’0Me modified.

[0153] In other examples, the modification includes 2' -methoxy ethoxy (2'-O-CH2CH2OCH3 (also known as 2'-O-(2-methoxyethyl) or 2'-M0E) (Martin et al., 1995). In further examples, the modification includes 2'-dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2group (also known as 2'-DMA0E), or 2'-dimethylaminoethoxy ethoxy (also known in the art as 2'-O- dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), that is, 2'-O-CH2-O-CH2-N(CH3)2. Other modifications include 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'- O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In certain examples, a 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0154] Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US 4,981,957, US 5,118,800, US 5,319,080, US

[0155] 5,359,044, US 5,393,878, US 5,446,137, US 5,466,786, US 5,514,785, US 5,519,134, US

[0156] 5,567,811, US 5,576,427, US 5,591,722, US 5,597,909, US 5,610,300, US 5,627,053, US

[0157] 5,639,873, US 5,646,265, US 5,658,873, US 5,670,633, US 5,792,747, and US 5,700,920.

[0158] A further modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In one example, the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98 / 39352 and WO 99 / 14226.

[0159] Modified nucleobases include other synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-CC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-m ethyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3- deazaadenine, mxA(l -methyladenosine); m2A(2 -methyladenosine); Am (2’-O- methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A(N6- threonylcarbamoyladenosine); ms2t6A(2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2’-O-ribosyladenosine(phosphate)); I (inosine); mfl - methylinosine); m1Im(l,2’-O-dimethylinosine); m3C(3 -methylcytidine); Cm(2’-0- methylcytidine); s2C(2-thiocytidine); ac4C(N4-acetylcytidine); f^C (5-formylcytidine); m5Cm(5,2’-O-dimethylcytidine); ac4Cm(N4-acetyl-2’-O-methylcytidine); k2C(lysidine); m1G(l -methylguanosine); m2G(N2-methylguanosine); m7G(7-methylguanosine); Gm(2’-0- methylguanosine); m22G(N2,N2-dimethylguanosine); m2Gm(N2,2’-O-dimethylguanosine); m22Gm(N2,N2,2’-O-trimethylguanosine); Gr(p)(2’-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxy queuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G+(archaeosine); D (dihydrouridine); m5Um(5,2’-O-dimethyluridine); s4U(4-thiouridine); m5s2U(5-methyl-2- thiouridine); s2Um(2-thio-2’-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U(5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U(uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U(5- methoxy carbonylmethyluridine); mcm5Um(5-methoxycarbonylmethyl-2’-O-methyluridine); mcm5s2U(5-methoxycarbonylmethyl-2 -thiouridine); nm5s2U(5-aminomethyl-2 -thiouridine); mnm5U(5-methylaminomethyluridine); mnm5s2U(5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U(5-carbamoylmethyluridine); ncm5Um(5-carbamoylmethyl-2’-O-methyluridine); cmnm5U(5- carboxymethylaminomethyluridine); cmnm5Um(5-carboxymethylaminomethyl-2’-O- methyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6- dimethyladenosine); Im(2’-O-methylinosine); m4C(N4-methyl cytidine); m4Cm(N4,2’-O- dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U(3 -methyluridine); cm5U(5- carboxymethyluridine); m6Am(N6,2’-O-dimethyladenosine); m62Am (N6,N6,O-2’- trimethyladenosine); m2,7G(N2,7-dimethylguanosine); m2’2’7G(N2,N2,7-trimethylguanosine); m3Um(3,2’-O-dimethyluridine); m5D(5-methyldihydrouridine); f^Cm (5-formyl-2’-O- methylcytidine); m 'Gm (l,2’-O-dimethylguanosine); m1Am(l,2’-O-dimethyladenosine); Tm5U(5-taurinomethyluridine); Tm5s2U(5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2(isowyosine); or ac6A(N6-acetyladenosine).

[0160] Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), and phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as, for example, a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).

[0161] Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in US 3,687,808, those disclosed in J.I. Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), those disclosed by Englisch et al. (1991), and those disclosed by Y.S. Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, S.T. Crooke, B. Lebleu (editors), CRC Press, 1993.

[0162] Certain of these nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotide. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C. In certain examples, these nucleobase substitutions are combined with 2'-O-methoxy ethyl sugar modifications.

[0163] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, US 3,687,808, US 4,845,205, US 5,130,302, US 5,134,066, US 5,175,273, US 5,367,066, US 5,432,272, US 5,457,187, US 5,459,255, US 5,484,908, US 5,502,177, US 5,525,711, US 5,552,540, US 5,587,469, US 5,594,121, US 5,596,091, US 5,614,617, US 5,645,985, US 5,830,653, US 5,763,588, US 6,005,096, US 5,681,941 and US 5,750,692.

[0164] In particular examples, the modification of the one or plurality of nucleotides of the antisense oligonucleotide described herein is selected from the group consisting of a morpholino modification, a LNA modification, a HNA modification, a CeNA modification, a 2’ -methoxy ethyl modification, a 2’-O-alkyl modification, a 2’-O-allyl modification, a 2’-C- allyl modification, a 2’ -fluoro modification, a 2’ -deoxy modification, and any combination thereof.

[0165] Suitably, the modification of the one or plurality of nucleotides is a morpholino-based modification. Even more particularly, the modification of the one or plurality of nucleotides is suitably a PMO modification or a PMO-based modification (e.g., PPMO, PMO-X, PM0+), as are known in the art. In certain examples, the antisense oligonucleotide includes one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 etc nucleotides) at a 5’ end and / or a 3’ thereof that are PMO modified. In particular examples, the antisense oligonucleotide includes at least one nucleotide at a 5’ end and a 3’ thereof that are PMO modified. In some examples, the antisense oligonucleotide includes at least one nucleotide at a 5’ end thereof that is PMO modified. In other examples, the antisense oligonucleotide includes at least one nucleotide at a 3’ end thereof that is PMO modified. In further examples, all nucleotides of the antisense oligonucleotide are PMO modified. In certain examples, at least 70%, 75%, 80%, 85%, 90% or 95% of nucleotides of the antisense oligonucleotide are PMO modified.

[0166] Unless stated to the contrary, reference to an A, T, G, U or C base herein can either mean a naturally occurring base or a modified version thereof, such as those described herein.

[0167] Backbones

[0168] Oligonucleotides of the present disclosure include those having modified backbones or non-natural internucleotide linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Such modifications, inclusive of a phosphorothioate backbone, may advantageously protect the antisense oligonucleotide from digestion by nucleases and / or increase the affinity of the antisense oligonucleotide to the target mRNA. This increased affinity may be due, at least in part, to additional electrostatic and London forces.

[0169] Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0170] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, US 3,687,808, US 4,469,863, US 4,476,301, US 5,023,243, US 5,177,196, US 5,188,897, US 5,264,423, US 5,276,019, US 5,278,302, US 5,286,717, US 5,321,131, US 5,399,676, US 5,405,939, US 5,453,496, US 5,455,233, US 5,466,677, US 5,476,925, US 5,519,126, US 5,536,821, US 5,541,306, US 5,550,111, US 5,563,253, US 5,571,799, US 5,587,361, US 5,194,599, US 5,565,555, US 5,527,899, US 5,721,218, US 5,672,697 and US 5,625,050.

[0171] Modified oligonucleotide backbones that do not include a phosphorus atom therein include, for example, backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0172] Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, US 5,034,506, US 5,166,315, US 5,185,444, US 5,214,134, US 5,216,141, US 5,235,033, US 5,264,562, US 5,264,564, US 5,405,938, US

[0173] 5,434,257, US 5,466,677, US 5,470,967, US 5,489,677, US 5,541,307, US 5,561,225, US

[0174] 5,596,086, US 5,602,240, US 5,610,289, US 5,602,240, US 5,608,046, US 5,610,289, US

[0175] 5,618,704, US 5,623,070, US 5,663,312, US 5,633,360, US 5,677,437, US 5,792,608, US

[0176] 5,646,269 and US 5,677,439.

[0177] Suitably, the antisense oligonucleotide described herein at least partly comprises a modified backbone. Exemplary modified backbones useful for the invention can include those which comprise a phosphorothioate modification; a non-bridging oxygen atom substituting a sulfur atom modification; a phosphonate, such as a methylphosphonate; a phosphodiester modification; a phosphoromorpholidate modification; a phosphoropiperazidate modification; an amide modification; a methylene or methylamino modification; a fromacetal modification; a thioformacetal modification; a peptide nucleic acid modification; or a phosphoroamidate modification; such as a morpholino phosphorodiamidate (PMO), N3’-P5’ phosphoramidite or thiophosphoroamidite. In some examples, one or a plurality of internucleotide linkages of the antisense oligonucleotide are modified with a phosphoroamidate modification or more particularly a phosphorodiamidate modification. Suitably, one or a plurality of internucleotide linkages of the antisense oligonucleotide are modified with a morpholino phosphorodiamidate (PMO) modification.

[0178] Morpholino-based oligonucleotides refer to an oligonucleotide comprising morpholino subunits supporting a nucleobase and, instead of a ribose, contains a morpholine ring. Exemplary internucleoside linkages include, for example, phosphoramidate or phosphorodiamidate intemucleoside linkages joining the morpholine ring nitrogen of one morpholino subunit to the 4’ exocyclic carbon of an adjacent morpholino subunit. Each morpholino subunit comprises a purine or pyrimidine nucleobase effective to bind, by basespecific hydrogen bonding, to a base in an oligonucleotide.

[0179] Morpholino-based oligomers (including antisense oligomers) and their synthesis are detailed, for example, in U.S. Patent Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,185,444; 5,521,063; 5,506,337 and U.S. Patent Application Nos. 12 / 271,036; 12 / 271,040; and PCT Publication No. W02009 / 064471 and W02012 / 043730 and Summerton et al.1997, Antisense and Nucleic Acid Drug Development, 7: 187-195, which are hereby incorporated by reference in their entirety. Within the oligomer structure, the phosphate groups are commonly referred to as forming the “internucleotide linkages” of the oligomer. The naturally occurring intemucleotide linkage of RNA and DNA is a 3’ to 5’ phosphodiester linkage. A“phosphoramidate” group comprises phosphorus having three attached oxygen atoms and one attached nitrogen atom, while a “phosphorodiamidate” group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms. In the uncharged or the cationic intersubunit linkages of morpholino-based oligomers described herein, one nitrogen is always pendant to the backbone chain. The second nitrogen, in a phosphorodiamidate linkage, is typically the ring nitrogen in a morpholine ring structure.

[0180] In particular examples, the oligonucleotide comprises a 5’ region comprising one or more bases which are modified and / or which have a modified backbone and a 3’ region comprising one or more bases which are modified and / or which have a modified backbone. In some examples, all intemucleotide linkages of the antisense oligonucleotide described herein are modified or comprise a modification, such as a phosphorothioate modification or a morpholino-based modification. In alternative examples, no intemucleotide linkages of the antisense oligonucleotide described herein are modified or comprise a modification. In particular examples, the antisense oligonucleotide comprises one or a plurality of phosphorothioate intemucleotide linkages. More particularly, all intemucleotide linkages of the antisense oligonucleotide comprise a phosphorothioate modification. In particular examples, the antisense oligonucleotide comprises one or a plurality of morpholino-based intemucleotide linkages. More particularly, all intemucleotide linkages of the antisense oligonucleotide comprise a morpholino-based modification. In certain examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the intemucleotide linkages of the antisense oligonucleotide are modified or non-natural. In certain examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the internucleotide linkages of the antisense oligonucleotide are modified with a phosphorothioate modification. In other examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the internucleotide linkages of the antisense oligonucleotide are modified with a morpholino-based modification.

[0181] In particular examples, at least a portion of the oligonucleotide has / is a ribonucleic acid, deoxyribonucleic acid, a DNA phosphorothioate, an RNA phosphorothioate, 2'-O-methyl- oligonucleotide, 2'-O-methyl-oligodeoxyribonucleotide, 2'-O-hydrocarbyl ribonucleic acid, 2'- O-hydrocarbyl DNA, 2'-O-hydrocarbyl RNA phosphorothioate, 2'-O-hydrocarbyl DNA phosphorothioate, 2'-F-phosphorothioate, 2'-F-phosphodiester, 2'-methoxyethyl phosphorothioate, 2-methoxyethyl phosphodiester, deoxy methylene(methylimino) (deoxy MMI), 2'-O-hydrocarby MMI, deoxy-methylphos-phonate, 2'-O-hydrocarbyl methylphosphonate, morpholino, 4'-thio DNA, 4'-thio RNA, peptide nucleic acid, 3'-amidate, deoxy 3'-amidate, 2'-O-hydrocarbyl 3'-amidate, locked nucleic acid, cyclohexane nucleic acid, tricycle-DNA, 2'fluoro-arabino nucleic acid, N3'-P5' phosphoroamidate, carbamate linked, phosphotriester linked, a nylon backbone modification and any combination thereof.

[0182] Suitably, the antisense oligonucleotide, in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- AGGUCUGACGGUUGUUGGCCGUGGC-3' (SEQ ID NO: 8; Table 1) or a fragment, variant or derivative thereof. Again, it will be understood that one or more of the U nucleotides of SEQ ID NO: 8 may be a T. It is contemplated that one or more of the nucleotides and / or one or more of the internucleotide linkages of SEQ ID NO: 8 can be modified, such as described herein. More particularly, at least about 70%, 80% or 90% of the nucleotides and / or at least about 70%, 80% or 90% of the intemucleotide linkages of SEQ ID NO: 8 can be modified, such as with a 2’0Me modification and a phosphorothioate modification respectively. In particular examples, all of the internucleotide linkages of SEQ ID NO: 8 comprise a phosphorothioate modification. In certain examples, all of the nucleotides of SEQ ID NO: 8 comprises a 2’0Me modification. For particular examples, all of the internucleotide linkages of SEQ ID NO: 8 comprise a phosphorothioate modification and all of the nucleic acid bases of SEQ ID NO: 8 comprise a 2’0Me modification (e.g., 5'- mA*mG*mG*mU*mC*mU*mG*mA*mC*mG*mG*mU*mU*mG*mU*mU*mG*mG*mC *mC*mG*mU*mG*mG*mC-3'; phosphorothioate modified intemucleotide linkages are indicated by and 2’0Me modified nucleic acid bases are indicated by “m”).

[0183] Suitably, the antisense oligonucleotide, in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- UGGCCAAGUCCUGUGGGAGUGGAGC-3' (SEQ ID NO: 13; Table 1) or a fragment, variant or derivative thereof. Again, it will be understood that one or more of the U nucleotides of SEQ ID NO: 13 may be a T. It is contemplated that one or more of the nucleotides and / or one or more of the internucleotide linkages of SEQ ID NO: 13 can be modified, such as described herein. More particularly, at least about 70%, 80% or 90% of the nucleotides and / or at least about 70%, 80% or 90% of the internucleotide linkages of SEQ ID NO: 13 can be modified, such as with a 2’0Me modification and a phosphorothioate modification respectively. In particular examples, all of the internucleotide linkages of SEQ ID NO: 13 comprise a phosphorothioate modification. In certain examples, all of the nucleotides of SEQ ID NO: 13 comprises a 2’0Me modification. For particular examples, all of the intemucleotide linkages of SEQ ID NO: 13 comprise a phosphorothioate modification and all of the nucleic acid bases of SEQ ID NO: 13 comprise a 2’0Me modification (e.g., 5'- mA*mG*mG*mU*mC*mU*mG*mA*mC*mG*mG*mU*mU*mG*mU*mU*mG*mG*mC *mC*mG*mU*mG*mG*mC-3'; phosphorothioate modified intemucleotide linkages are indicated by and 2’0Me modified nucleic acid bases are indicated by “m”).

[0184] Suitably, the antisense oligonucleotide, in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- AGGTCTGACGGTTGTTGGCCGTGGC-3' (SEQ ID NO: 37; Table 2) or a fragment, variant or derivative thereof. Again, it will be understood that one or more of the T nucleotides of SEQ ID NO: 37 may be a U. It is contemplated that one or more of the nucleotides and / or one or more of the intemucleotide linkages of SEQ ID NO: 37 can be modified, such as described herein. More particularly, at least about 70%, 80% or 90% of the nucleotides and / or at least about 70%, 80% or 90% of the intemucleotide linkages of SEQ ID NO: 37 can be modified, such as with a PMO modification or a PMO-based modification and a phosphoroamidate modification respectively. In particular examples, one or more of the nucleotides of SEQ ID NO: 37 comprise a PMO modification or a PMO-based modification. In certain examples, all of the nucleotides of SEQ ID NO: 37 comprise a PMO modification or a PMO-based modification. For particular examples, all of the intemucleotide linkages of SEQ ID NO: 37 comprise a phosphoroamidate modification, or more particularly a phosphorodiamidate modification, and all of the nucleic acid bases of SEQ ID NO: 37 comprise a morpholino modification (e g., 5'- mA*mG*mG*mT*mC*mT*mG*mA*mC*mG*mG*mT*mT*mG*mT*mT*mG*mG*mC* mC*mG*mT*mG*mG*mC-3'; phosphorodiamidate modified internucleotide linkages are indicated by and morpholino modified nucleic acid bases are indicated by “m”). In other words, the targeting sequence of the antisense oligonucleotide suitably comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 37 or a fragment, variant or derivative thereof, and wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0185] Suitably, the antisense oligonucleotide, in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- TGGCCAAGTCCTGTGGGAGTGGAGC-3' (SEQ ID NO: 38; Table 2) or a fragment, variant or derivative thereof. Again, it will be understood that one or more of the T nucleotides of SEQ ID NO: 38 may be a U. It is contemplated that one or more of the nucleotides and / or one or more of the internucleotide linkages of SEQ ID NO: 38 can be modified, such as described herein. More particularly, at least about 70%, 80% or 90% of the nucleotides and / or at least about 70%, 80% or 90% of the intemucleotide linkages of SEQ ID NO: 38 can be modified, such as with a PMO modification or a PMO-based modification and a phosphoroamidate modification respectively. In particular examples, one or more of the nucleotides of SEQ ID NO: 38 comprise a PMO modification or a PMO-based modification. In certain examples, all of the nucleotides of SEQ ID NO: 38 comprise a PMO modification or a PMO-based modification. For particular examples, all of the internucleotide linkages of SEQ ID NO: 38 comprise a phosphoroamidate modification, or more particularly a phosphorodiamidate modification, and all of the nucleic acid bases of SEQ ID NO: 38 comprise a morpholino modification (e g., 5'- mT*mG*mG*mC*mC*mA*mA*mG*mT*mC*mC*mT*mG*mT*mG*mG*mG*mA*mG* mT*mG*mG*mA*mG*mC-3'; phosphorodiamidate modified internucleotide linkages are indicated by and morpholino modified nucleic acid bases are indicated by “m”). In other words, the targeting sequence of the antisense oligonucleotide suitably comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 38 or a fragment, variant or derivative thereof, and wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0186] Vectors Vectors that encode and express the antisense oligonucleotides of the present invention are contemplated for the present disclosure. As such, the methods provided herein can be adapted to contacting a vector that encodes the antisense oligonucleotide to a cell, so that the antisense oligonucleotide binds to a CHIT1 mRNA and modulates the processing and / or expression of the CHIT1 mRNA.

[0187] Suitably, the vector comprises a viral vector encoding the antisense oligonucleotide. In some embodiments, the viral vector comprises an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a Herpes Simplex Virus (HSV) viral vector, or a retroviral vector. In particular examples, the vector is an AAV vector.

[0188] Methods of treatment

[0189] The present inventors have also surprisingly shown that the antisense oligonucleotides described herein can be utilised to bind CHIT1 mRNA in cells and thereby inhibit expression of this proinflammatory molecule in macrophages. As such, the antisense oligonucleotides may be administered to a subject in need thereof to prevent a disease, disorder or condition associated with or dependent on increased activity and / or expression of CHIT1.

[0190] Therefore, in one form, the present disclosure provides for the use of the antisense oligonucleotide, the vector and / or the pharmaceutical composition described herein in therapy or medical treatment.

[0191] In another form, the present disclosure provides a method of reducing or inhibiting CHIT1 expression, inclusive of expression at an mRNA and / or protein level, in a cell, said method including the step of contacting the cell with the antisense oligonucleotide, the vector and / or the pharmaceutical composition described herein to thereby reduce or inhibit CHIT1 expression therein.

[0192] In a related form, the present disclosure provides for the use of the antisense oligonucleotide, the vector or the pharmaceutical composition provided herein in the manufacture of a medicament for reducing or inhibiting CHIT1 expression in a cell.

[0193] In another related form, the present disclosure provides an antisense oligonucleotide, a vector or a pharmaceutical composition described herein for use in reducing or inhibiting CHIT1 expression in a cell.

[0194] Referring to another broad form, the present disclosure provides a method of inhibiting or treating inflammation in a CNS or neuronal cell or tissue, said method including the step of contacting the CNS or neuronal cell or tissue with an effective amount of an antisense oligonucleotide, a vector and / or a pharmaceutical composition described herein to thereby inhibit or treat inflammation therein.

[0195] In a related form, the present disclosure provides for the use of the antisense oligonucleotide, the vector or the pharmaceutical composition provided herein in the manufacture of a medicament for inhibiting or treating inflammation in a CNS or neuronal cell or tissue.

[0196] In another related form, the present disclosure provides an antisense oligonucleotide, a vector or a pharmaceutical composition described herein for use in inhibiting or treating inflammation in a CNS or neuronal cell or tissue.

[0197] It is contemplated that such methods may be performed in relation to cells in vitro, in vivo and / or ex vivo. In some examples, the method is performed in vitro, such as with microglial isolated from a subject, an organoid or a stem cell-derived (e.g., induced pluripotent stem cell(iPSC)-derived) cell, as described herein. Accordingly, in some examples, the present method includes the earlier step of isolating the cells expressing CHIT1 from a subject. In other examples, the present method is performed in vivo in a subject.

[0198] Suitably, the cell described herein is a CNS cell or a CNS-derived cell. In some examples, the cell is an immune cell and more particularly an inflammatory cell. In particular examples, the cell is a glial cell, such as an astrocyte, an oligodendrocyte or a microglia. More particularly, the cell suitably is or comprises a microglial cell.

[0199] As used herein, the term “effective amount” and the like refers to an amount of the antisense oligonucleotide, the vector or the pharmaceutical composition that is sufficient to induce a desired physiological outcome (e.g., at least partly inhibiting a target protein’s activity and / or expression). An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc.

[0200] In another form, the present disclosure provides a method of preventing, ameliorating or treating a disease, disorder or condition associated with CHIT1 in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein to thereby prevent, ameliorate or treat the disease, disorder or condition.

[0201] In a related form, the present disclosure also provides for the use of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with CHIT1 in a subject.

[0202] In a further related form, the present disclosure also provides for an antisense oligonucleotide, a vector or a pharmaceutical composition described herein for use in preventing, ameliorating or treating a disease, disorder or condition associated with CHIT1 in a subject.

[0203] In other forms, the present disclosure provides a method of treating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein to thereby treat, prevent or ameliorate the neurodegenerative disease, disorder or condition.

[0204] In a related form, the present disclosure provides for the use of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein in the manufacture of a medicament for treating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject.

[0205] In another related form, the present disclosure provides an antisense oligonucleotide, a vector or a pharmaceutical composition described herein for use in preventing, ameliorating or treating a neurodegenerative disease, disorder or condition associated in a subject.

[0206] In other forms, the present disclosure provides a method of treating, preventing or ameliorating a neuroinflammatory disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein to thereby treat, prevent or ameliorate the neuroinflammatory disease, disorder or condition.

[0207] In a related form, the present disclosure provides for the use of the antisense oligonucleotide, the vector or the pharmaceutical composition described herein in the manufacture of a medicament for treating, preventing or ameliorating a neuroinflammatory disease, disorder or condition in a subject.

[0208] In another related form, the present disclosure provides an antisense oligonucleotide, a vector or a pharmaceutical composition described herein for use in preventing, ameliorating or treating a neuroinflammatory disease, disorder or condition associated in a subject.

[0209] With respect to the aspects described herein, the term “subject”, “patient” and “individual” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Suitably, the subject is a human. In some examples, the subject is a female human. In other examples, the subject is a male human.

[0210] As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0211] As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

[0212] The term “ameliorating” as used herein refers to any improvement in the disease state of a patient suffering from the disease, disorder or condition described herein by administering to a subject in need thereof an antisense oligonucleotide, a vector or a pharmaceutical composition according to the present disclosure. Such an improvement may also be seen as a slowing or stopping of the progression of the disease, disorder or condition in the subject.

[0213] Typically, the antisense oligonucleotide is used to modify a trait of an animal, more typically to treat or prevent a disease, disorder or condition. In some examples, the disease, disorder or condition will benefit from inhibition of CHIT1 following administration of the antisense oligonucleotide. To this end, the disease, disorder or condition may be at least partly caused by or associated with an increased or elevated expression and / or activity of CHIT1. Put another way, the disease, disorder or condition described herein may be at least partly caused by or associated with CHIT1.

[0214] Diseases, disorders and conditions, which can be treated or prevented using an antisense oligonucleotide of the present disclosure include, but are not limited, to neuromuscular and neurological diseases (for example, spinal muscular atrophy, Amyotrophic Lateral Sclerosis, Duchenne muscular dystrophy, Huntington’s disease, Batten disease, Parkinson’s disease, amyotrophic lateral sclerosis, Ataxia-telangiectasia, cerebral palsy) and autoimmune and inflammatory diseases (for example arthritis, lupus, pouchitis, psoriasis, asthma). In particular examples, the disease, disorder or condition associated with CHIT1 is a neurodegenerative disease, disorder or condition. In other examples, the disease, disorder or condition associated with CHIT1 is a neuroinflammatory disease, disorder or condition. More particularly, the disease, disorder or condition associated with CHIT1, the neurodegenerative disease, disorder or condition or the neuroinflammatory disease, disorder or condition can be one of Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease, Huntington’s disease, Multiple sclerosis and Parkinson’s disease. Even more particularly, the disease, disorder or condition associated with CHIT1, the neuroinflammatory disease, disorder or condition or the neurodegenerative disease, disorder or condition can be Amyotrophic Lateral Sclerosis (ALS).

[0215] In the context of the present disclosure, by “a neurodegenerative disease, disorder or condition” is meant any disease, disorder and / or condition that comprises a progressive decline and / or deterioration in the structure, function, signalling and / or population of the neurons or neural tissue in an animal. In particular, the neurodegenerative disease, disorder or condition can be or comprise a neuromuscular disease, disorder or condition. As used herein, “a neuromuscular disease, disorder or condition” refers to any disease, disorder and / or condition that comprises a progressive decline and / or deterioration in the structure, function, signalling and / or population of the neurons or neural tissue that innervate and / or communicate, whether directly or indirectly, with the muscles of an animal.

[0216] The aetiology of a neurodegenerative disease, disorder or condition may involve, but is not limited to, inflammation, genetic mutations, protein misfolding and / or aggregation, autoimmune disorders, mitochondrial dysfunction, defective axonal transport, aberrant apoptosis and / or autophagy and elevated oxidative stress and / or reactive oxygen species (ROS) production.

[0217] Without limitation, neurodegenerative diseases, disorders or conditions can include Parkinson’s disease and related disorders, Huntington’s disease, Alzheimer’s disease and other forms of dementia, Spinocerebellar ataxia, Friedreich ataxia, Tay-Sachs disease, Lewy body disease, Parkinson’s disease and related disorders, Prion diseases (e.g. Creutzfeldt-Jakob disease), Multiple sclerosis (MS), Pick disease, Shy-Drager syndrome, pontocerebellar hypoplasia, neuronal ceroid lipofuscinoses, Gaucher disease, neurodegeneration with brain iron accumulation, spastic ataxia / paraplegia, supranuclear palsy, mesolimbocortical dementia, thalamic degeneration, cortical-striatal-spinal degeneration, corti cal -basal ganglionic degeneration, cerebrocerebellar degeneration, Leigh syndrome, post-polio syndrome, hereditary muscular atrophy, encephalitis, neuritis, hydrocephalus and the motor neurone diseases, such as ALS.

[0218] For the present disclosure, the subject with a neurodegenerative disease, disorder or condition may be undergoing a treatment regimen (preventative and / or therapeutic). In this regard, the subject may have been determined to either: (i) have an existing neurodegenerative disease, disorder or condition; or (ii) be predisposed to such a disease, disorder or condition.

[0219] Suitably, the neurodegenerative disease, disorder or condition described herein is a motor neurone disease (MND). Broadly, MNDs are a form of neurodegenerative diseases that typically involve the motor neurons of an affected subject. As will be readily understood by a skilled artisan, motor neurons are nerve cells that control the voluntary muscles of the trunk, limbs and phalanges, as well as those muscles that influence speech, swallowing and respiration. Accordingly, the clinical symptoms of a MND may include muscle weakness and / or wasting, muscle cramps, dysphagia, slurred speech, muscle tremors / fasciculations, reduced cognition, dyspnoea, respiratory failure, fatigue and weight loss without limitation thereto. MNDs include, but are not limited to, amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig’s disease), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), pseudobulbar palsy and spinal muscular atrophy (SMA).

[0220] In light of the foregoing, the MND may be ALS, PLS, PMA, PBP, pseudobulbar palsy or SMA. Suitably, the MND is ALS. The ALS may be familial or sporadic ALS. In one example, the ALS is sporadic ALS. In another example, the ALS is familial ALS.

[0221] The term “neuroinflammatory disease, disorder or condition” refers to a disease, disorder or condition caused by the damage of neural tissues due to inflammatory responses, and more specifically, refers to neurological damage and nervous system degeneration, caused by inflammatory responses. Exemplary neuroinflammatory diseases, disorders and conditions include Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, diffuse Lewy body disease, leukencephalitis, temporal lobe epilepsy, and spinal cord injury.

[0222] The term “therapeutically effective amount” describes a quantity of a specified agent, such as an antisense oligonucleotide, a composition or vector described herein, sufficient to achieve a desired effect in a subject being treated with that agent or composition. For example, this can be the amount of an antisense oligonucleotide and optionally one or more further therapeutic agents, necessary to reduce, alleviate and / or prevent a disease, disorder or condition associated with the target protein in question. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of such a disease, disorder or condition. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression. In some examples, the methods described herein provide a reduction of at least about 25%, at least about 50%, at least about 75% or at least about 80%, in probability of a clinical worsening event during the treatment period.

[0223] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and / or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition (e.g., disease progression), and the manner of administration of the therapeutic composition.

[0224] Suitably, the methods described herein further include the step of administering a further therapeutic agent to the subject (i.e., in addition to the antisense oligonucleotide). As such, the antisense oligonucleotide may be administered alone (i.e., monotherapy) or alternatively be administered in combination with the further therapeutic agent (e.g., Riluzole, an antioxidant, such as Edaravone, sodium phenylbutyrate / taurursodiol, tofersen) which aims to treat or prevent a disease, disorder or condition described herein. In certain examples, the antisense oligonucleotide described herein may be co-administered with (simultaneously or sequentially) a further treatment of a neurodegenerative disease, disorder or condition. Nonlimiting examples of such treatments include drug therapy, antibody, nucleic acid and other biomolecular therapies, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto.

[0225] The methods of the present disclosure may further include the earlier or initial step of identifying the presence of the disease, disorder or condition in the subject. Accordingly, the methods of this disclosure may include the initial step of determining an activity level and / or an expression level of CHIT1 in one or more cells or tissues of the subject in question, such as from a sample (e.g., a biopsy sample or a biological sample, such as a CSF sample) obtained from the subject. More particularly, the present methods may include identifying those subjects demonstrating an increased or elevated expression and / or activity of CHIT1, such as in the CNS or CSF thereof. Such a determining step may be performed by any means or method of testing known in the art.

[0226] CHIT1

[0227] CHIT1 encodes a chitotriosidase protein that degrades chitin, chitotriose, and chitobiose. It is most commonly secreted by reactive macrophages upon infection with fungi given the abundance of chitinous structures produced by this type of pathogen (Kanneganti, Kamba, & Mizoguchi, 2012). In many cell types, including primary alveolar macrophages, PBMCs or human brain endothelial cells (HBECs), stimulation with CHIT1 causes inflammation and promotes secretion of inflammatory cytokines (Correale & Fiol, 2011; Letuve et al., 2010).

[0228] Although the precise mechanisms of action of CHIT1 are not well understood, there is evidence that CHIT1 augments the TGF-b signalling pathway (Figure 1). Together with TGF- b, CHIT1 augments TGF-b-induced activation of SMAD2 / 3, MAPKZERK and AKT pathways, increases expression of TGFbRl and TGF-bR2, and inhibits the production of inhibitory SMAD7 (C. G. Lee et al., 2012; C. M. Lee et al., 2019). CHIT1 also interacts with TGFBRAP1, which augments TGF-b signalling by acting as a chaperone for SMALM (C. M. Lee et al., 2019). In addition, CHIT1 reduces the levels of nuclear FOXO3 / pFOXO3, a transcription factor which mediates TGF-b-induced expression of SMAD7 (C. M. Lee et al., 2019).

[0229] In the CNS, expression of CHIT1 is limited to microglia and infiltrating peripheral macrophages. Previous findings suggest that CHIT1 is a central factor in the CSF of ALS cases which mediates a positive feedback loop of microgliosis, inducing the overproduction of neuroinflammatory stimuli which drive oxidative stress, apoptosis, exci totoxi city and inflammation, culminating in motor neuron degeneration (Mishra et al., 2017) (Figure 2). As such, CHIT1 can be a positive regulator of neuroinflammation and its actions contribute to the degeneration of motor neurons in vivo. CHIT1 may be present at elevated levels in the neurological tissue and cerebrospinal fluid of people with ALS and its elevated levels have been associated with rapid disease progression.

[0230] By “protein” is meant an amino acid polymer. The amino acids may be natural or nonnatural amino acids, D- or L-amino acids, as are well understood in the art. A “peptide” is generally referred to as a protein typically having no more than fifty (50) amino acids and a polypeptide is generally referred to as a protein typically having more than fifty (50) amino acids.

[0231] The protein sequence of CHIT1 or Chitotriosidase-1 is publicly available (see, e.g., UniProt Accession No. Q13231). An exemplary amino acid sequence is set forth in SEQ ID NO: 41. Thus, a CHIT1 amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 41 or a fragment or derivative thereof.

[0232] MVRSVAWAGFMVLLMIPWGSAAKLVCYFTNWAQYRQGEARFLPKDLDPSLCTHLIYAFAG

[0233] MTNHQLSTTEWNDETLYQEFNGLKKMNPKLKTLLAIGGWNFGTQKFTDMVATANNRQTFV NSAIRFLRKYS FDGLDLDWEYPGSQGSPAVDKERFTTLVQDLANAFQQEAQTSGKERLLL SAAVPAGQTYVDAGYEVDKIAQNLDFVNLMAYDFHGSWEKVTGHNSPLYKRQEESGAAAS LNVDAAVQQWLQKGTPASKLILGMPTYGRS FTLASSSDTRVGAPATGSGTPGPFTKEGGM LAYYEVCSWKGATKQRIQDQKVPYI FRDNQWVGFDDVES FKTKVSYLKQKGLGGAMVWAL DLDDFAGFSCNQGRYPLIQTLRQELSLPYLPSGTPELEVPKPGQPSEPEHGPSPGQDTFC QGKADGLYPNPRERSS FYSCAAGRLFQQSCPTGLVFSNSCKCCTWN

[0234] (UniProt Accession No: Q13231-1; SEQ ID NO: 41)

[0235] An exemplary cDNA sequence encoding a full-length protein sequence of human CHIT1 is provided in SEQ ID NO: 42 below. Thus, a CHIT1 nucleic acid sequence may be a polynucleotide which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 42 or a fragment or derivative thereof.

[0236] ATGGTGCGGTCTGTGGCCTGGGCAGGTTTCATGGTCCTGCTGATGATCCCATGGGGCTCT GCTGCAAAACTGGTCTGCTACTTCACCAACTGGGCCCAGTACAGACAGGGGGAGGCTCGC TTCCTGCCCAAGGACTTGGACCCCAGCCTTTGCACCCACCTCATCTACGCCTTCGCTGGC AT GAG C AAC GAG GAG C T GAG GAG GAG T GAG T G GAAT GAG GAGAC T C T C T AC GAG GAG T T C AATGGCCTGAAGAAGATGAATCCCAAGCTGAAGACCCTGTTAGCCATCGGAGGCTGGAAT TTCGGCACTCAGAAGTTCACAGATATGGTAGCCACGGCCAACAACCGTCAGACCTTTGTC AACTCGGCCATCAGGTTTCTGCGCAAATACAGCTTTGACGGCCTTGACCTTGACTGGGAG TACCCAGGAAGCCAGGGGAGCCCTGCCGTAGACAAGGAGCGCTTCACAACCCTGGTACAG GACTTGGCCAATGCCTTCCAGCAGGAAGCCCAGACCTCAGGGAAGGAACGCCTTCTTCTG AGTGCAGCGGTTCCAGCTGGGCAGACCTATGTGGATGCTGGATACGAGGTGGACAAAATC GCCCAGAACCTGGATTTTGTCAACCTTATGGCCTACGACTTCCATGGCTCTTGGGAGAAG GTCACGGGACATAACAGCCCCCTCTACAAGAGGCAAGAAGAGAGTGGTGCAGCAGCCAGC CTCAACGTGGATGCTGCTGTGCAACAGTGGCTGCAGAAGGGGACCCCTGCCAGCAAGCTG ATCCTTGGCATGCCTACCTACGGACGCTCCTTCACACTGGCCTCCTCATCAGACACCAGA GTGGGGGCCCCAGCCACAGGGTCTGGCACTCCAGGCCCCTTCACCAAGGAAGGAGGGATG CTGGCCTACTATGAAGTCTGCTCCTGGAAGGGGGCCACCAAACAGAGAATCCAGGATCAG AAGGTGCCCTACATCTTCCGGGACAACCAGTGGGTGGGCTTTGATGATGTGGAGAGCTTC AAAACCAAGGTCAGCTATCTGAAGCAGAAGGGACTGGGCGGGGCCATGGTCTGGGCACTG GACTTAGATGACTTTGCCGGCTTCTCCTGCAACCAGGGCCGATACCCCCTCATCCAGACG CTACGGCAGGAACTGAGTCTTCCATACTTGCCTTCAGGCACCCCAGAGCTTGAAGTTCCA AAACCAGGTCAGCCCTCTGAACCTGAGCATGGCCCCAGCCCTGGACAAGACACGTTCTGC CAGGGCAAAGCTGATGGGCTCTATCCCAATCCTCGGGAACGGTCCAGCTTCTACAGCTGT GCAGCGGGGCGGCTGTTCCAGCAAAGCTGCCCGACAGGCCTGGTGTTCAGCAACTCCTGC AAAT GC T GCACC T GGAAT T GA

[0237] (Ensembl Accession No: ENST00000367229.6; SEQ ID NO: 42)

[0238] An exemplary pre-mRNA sequence encoding a full-length protein sequence of human CHIT1 is provided in SEQ ID NO: 43 below. The 5’ and 3’ UTRs are underlined, the exonic sequence is in capitals and the intronic sequence is in lower case. As noted herein, examples of the antisense oligonucleotides of the present disclosure are designed to target an intron-exon junction of the pre-mRNA transcript of CHIT1. Thus, a CHIT1 nucleic acid sequence may be a polynucleotide which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 43 or a fragment or derivative thereof.

[0239] GGTTTGTATGGGCTGCAGCCTGCCGCTGAGCTGCATCATGGTGCGGTCTGTGGCCTGGGCAG gtgagccgtcggatagtgggctgggtctcgggagctgtggggaggtggggatgtggatgttc agggaccacttgctaccatttgcaaggaagcaggaggactcaggagaacttcagagggtcat tttatccctctccatgcctcctggcttgcagtcccttgattacctttcctgtgggctccccc ttaggctctttcagcccaagagacttcatgttgcaagggaaggcttttctcaacaggctgaa gatgacttgactttcctctctctgtcctgaaagcccagagtcaatgaggcccctgcactggg tctttgtgtctttcccatgttcttaggtccctcctgttggccttatccaagaagcgcccact gccaagaacctcttctccaacctcatattgtctgtcctgcaagagggcagcctggggccaag atcctgggcatccttgcaaattttcatggaggttctattaggttgacagagaagtccaaggt caccaggtgaaagagatctcttcgaaggagtcctgtcagaccctaggtctccaaggctgtgg ttgcctaccacagccaggtagcggaaaggcaacaggaagccataaacgatgggagcagaggc acaggccctccccaggagatgaactctctccatgcagcccttccagattgattctttctagc cccaaaacaggcttgtttcttgttccgaaagagaaactatctctctaaataactcacaagtc agcagtttttccacaatcttcctgaatccacactgccctttactgaggtatagagagggggc agcctcagcccatggaactttccatcagcttctaggaccaacccaggccaggaagagacccc cgtggtctggtctgcatgaagggatgggcacagtgccctcctgggtcccttctgaaaggaga aagcttgatttgtatgaaatcagccctccctgaccacctcctgtgcttccgtaagctgtggg gcctgggaaggtgagaatggcagcataaccctggccttctctcttgtgttgctccagGTTTC ATGGTCCTGCTGATGATCCCATGGGgtaagtagcctctttcttggctgcctcagccccttcc ctgggctctgcttaatgtcccaaaacaccttccctgccagacctccccaagagctccagatt tcagtggggactctcttccaaagctcagacccctgcaaatgtccccatctgctaccagccct cccaggaaggctcttgcctccttatccattagctgagctcctgcacatctgtggggccagct tgcgcatcagcctctttcagggctttcctttcctggggttgctggctccctcctctgcactc tgtgctcttgcagcacctgtgtgttttacagtacagtactgcctctcattattggcttaggt gtctctctgcaccagtctcagactccaagttgctgcaggtaaggaacctaagtctcatctgg ttcagcatccccagccccttagctctgcgaggacaacacacttgcttctggtctgttgaact gatcagatgtggagcttgagagaggcaggagggagctccttgttgtgtcaaatggaaaagag acctgggggaccttcagcaacttgaactcctggtccccgtcttgtcccaccccattccctgg gcatcagtggcctgggaatgctggcctctcagaacttgtcctgcgactgtgcctctcacgct gggctgtagcattggaggtacatttccctctcagcctcataggatggaacagccgggggcac agaaacagcaaacatggggaagatgagaggtggaagatttctcaaaaagctttagtccaggc tacttctgggctttctgcagtatagtaataatagtaggactttgtgtggactaagggttaat atatgtgaagcacctagaacagaacttcttccttgcagtgttcctccgagtgttttacatat attaactttagcccacactggggtcctgtgacacacattgtggaaggtccaatggtctcccc gtttcacacaggaggaagctgagtcgcacagcgagtaagttgcaaagttgggatttcaaccc agacactgacatgccagagtccctgctgttaaccactgcactttattttctccatagtacag aaattcataacaatatttgatatgcgacagcatgacatgatagaaaaagagcttcctggctc cttcctacatggcctgggggatgagttatctggtttcatcccctccttcctttcttttcctt tggggagttcaccaattctcctgtatcattgcaggctacaccccagccccactagcaagctg cactcctgggtgacctctggttagagacagtgaccactccggggccacgtgtatgcccaagg tcagccctccat get ctcgggttctagttgaggcctgcct gagegg tctcttcccaaaactc tcctatagacactttgcacccctcctcttcctcgtcccatccctgtccatgtgggcattgga gggggtacagacagaccctgctggggcaaagggaagtcccaggttctagacaccagccacag gcacgctccagtccaaactcactgctccatcaagatcttggcataaaatgttggctcataca aaccacaccatcatagattgagagactggaagagaccccacagaccacacagtcattatcag atggggccgggattgaagatgagatagacccaaagtctatttttggcagatgctggaatgaa gactagcattcttgcttcctggtgtcctggtcaaccctgcacagggctgacctcagtggggg tcatcaccaccgagccctcactgggaatagaccatcccacccctgcaaagtcttttcacaca ctgtagggtactctagagacaagtaccctacagtgtgtggggtacttagagacaagatgcct tctcctagataaaattagctctattagaatctcagcagtgatattgataaacgtagattatc tacaagggataacaagcaaatgcaaatggctttaatgcaagactaaagaaacataaacccag gcctgtaactggggcctcctctctgctacttatctgtttgcattttaacgaagtattctgag gt attccaaaccgt tttt gaaagt tct gcct gccttccttct gcaga t gagcat gccatgtc taattccctctaggcct cat gttggggctgcctggggct acct tctgtcaggtttcagggcc tggaggggagaccctaactggggatagggagacctgcctgccagaccctgatcctgggagct cggactctgagactagcaactttcaaggatgtcaaggagtcatgagacttgggtcgcagcac ctctgctctgccctcttagcagccatgctgatggggacgggccccaagctcgtgagcctcaa cttgcttcccgggaaaagaaggcttagaagatgatggtcctgcccactcagtgttctgagga tcccatgtctggcctctgaaaatgctttcccaactgccagactccatccacacaaaagcaaa tgccatt tct gcct gtcgggtccctccactgactccaggtgtagaagagggaggtccaagga cattgcctaccccagatggagggtgaccagtccccagaaggtcctgctgacagctatccctt ttctttactccaacgggccagGCTCTGCTGCAAAACTGGTCTGCTACTTCACCAACTGGGCC CAGTACAGACAGGGGGAGGCTCGCTTCCTGCCCAAGGACTTGGACCCCAGCCTTTGCACCCA CCTCATCTACGCCTTCGCTGGCATGACCAACCACCAGCTGAGCACCACTGAGTGGAATGACG AGACTCTCTACCAGGAGTTCAATGGCCTGAAGAAGATgtgagccaaagcagaggagcagggt gggtgggatgtggtggggtgacagataacctcctcccagagccagacacctctccagcacta aggtcctgccacagacccactgtgtggctttgggcaagttgtatcacctctctgggctctcc tgtgcttaatgaaagaggtagggtttgcttaggggatcccaaaggtcccttccagtttaggt ctaagtgtgatcaggattgtgagactctttctgtctcccatgaagccccttcctcattatga aattgatccacccttcagctccagtgttctgggcgccttcacaaagcctcttgagaatctgc cccaatccttgcttcctgagcccacacagttagacaggtggttctgtgagtcaggaacgtga cttcccttttccaatctacagctgtccccagaatgcctaatacagtctctgcacataggcac tttcacacgtccccacccctgttgttcctccccactgtaacatccctgccagggtgccctgg gagctggcagagtaaatcctgctgtgtctcctgtctttcccagGAATCCCAAGCTGAAGACC CTGTTAGCCATCGGAGGCTGGAATTTCGGCACTCAGAAgttagttgactgtggtcacctgtg tggtaaagctcctggatgggaacctggtgcactggccagagggagccctggtcagggaatca ggccaggctgaggtgtggtcaggggaaaggggaggggagctgaactctgagggctgaatttc cttctgggttcttggcacctcagctggagggcagcacagtgagctttgctccaggctgtttc agatccagtcttgccagaagcataggtctaaacacagcagagggctcagacctctccaagcc tcagtctcgtcttccatgaaatgagaagagggagggtcccaactaatcctgacaacatgcag tgtctatgttttctgagctcagaaatggggtaccttgtcccagaaagagtgtttattccact tctgagtacaaaaggtaatctgtgagattgagcctggatgccagagtattggcatttggggg acattttttaaatgatttatggagaaaatggaatggagtatttgaaacttggtgttttctag aaaatccagaatatagggctcccaaagctctaaacagtctttctaaaacttttttggatcat gaacccttttgggaatctgatgaaagtgattactcatcttccaaaataatgcttatatacag actgtttattgaaatgcagcaggagtttttagcctggtgtcctcggactaaattaataaaaa taaaataggatcaatctgtaaagaacttgagaccccctcaagaagttcagggacttcccact aaggaaggaagatgtggagaggtggttcttggggcagcttggggattttgacgaactctgga ctagtaacagcaacaaaaacacaaagcgctctacgggtgtgagttaaaagaaagatgaccag gttaacctgccaggaaactaaaacagtgggctccccaggtctttactaaccttagtaaagga attagagaaattctaattagagaaaaatcttacttaaagaaaaaccttacaagaggaattag agaaactaattccttttcttttcaaacctccattctgagggtgtcaactagttctggtttgg ggtgggacaattcaagggcttggaggaagcctctggagggaattttgggaaaggctcctagg gtgtccatgtgcccctggtgatgtcgatgttcgggggtagaaagtgctctggatggtcccct ttcctcaacccggaccataactcaccagcaaatatccctgcagcctctgaagcagccctccc agtgcccctcctagcccagacacagagacgcactgtcctgagtgttttcttgcctgcttctg agtaagagtggcctcctgtctggtccaaggccggcccttactccccttcatcacctcacagG TTCACAGATATGGTAGCCACGGCCAACAACCGTCAGACCTTTGTCAACTCGGCCATCAGGTT TCTGCGCAAATACAGCTTTGACGGCCTTGACCTTGACTGGGAGTACCCAGGAAGCCAGGGGA GCCCTGCCGTAGACAAGGAGCGCTTCACAACCCTGGTACAGgtatggctgggcggggcgggg atcaccagtctgtgcagggacccagagctggagcccccacagcacatgtgggggctgctctg cccccagagccagctgcttcacgtatctgcatggcacagatcagccctgtggccagctctca gacttctacaagtgaacatctacctggggctcacaggggctgcctgcctggtgcgcgcccct gtgttgactctgagctccactcccacagGACTTGGCCAATGCCTTCCAGCAGGAAGCCCAGA

[0240] CCTCAGGGAAGGAACGCCTTCTTCTGAGTGCAGCGGTTCCAGCTGGGCAGACCTATGTGGAT

[0241] GCTGGATACGAGGTGGACAAAATCGCCCAgtgagtctcaggcagatggcttggagctgtgct ctctgagggaagagaggccaagggcagactgagctttcccatgggtagagcaggtgttcatc tacaaggcatttggaaagtcccatccttacaccagaacaaaagcagaaaattccctgctttt gtactcagggtaaaactaagacacatccagaaaggctttgctcagtgtccactgctgccctg ttccagatccttcattttggatctctgtcctcttcctcgggaactcattcttaaggtgaaaa tgcctctgagtgggtagtgaagccattgtcttcaagaaaatgggttttctggatccttgctc ctcaaagtacagcatgtggactcgcagccctcattaaaaccaggtgctcattagaattatgg actctcaggccccaccccaggccttctgaagaaccttcattttagtgagattcccaggtgat ctgtgtgcacactgaagtgggagaaaccctaagctagagttccctccagaatagcaatgtag caggagttttcttgagtggaatagggagggcctgggggtgctgaggggacagtggggtgagg gtgacaagtttgagtgagaatgagaatggccttcataagggggctggctgggactgctgtct gggtcaccttctgcagagccaggtgagcctgccacctttgcctctgaccccagtggttcctc cctaggccatgcactgagggggtaggagtgagaaagaagggctaccccacccccaatccctt gtttctcacctcctcttccatgcctcctgcagGAACCTGGATTTTGTCAACCTTATGGCCTA

[0242] CGACTTCCATGGCTCTTGGGAGAAGGTCACGGGACATAACAGCCCCCTCTACAAGAGGCAAG

[0243] AAGAGAGTGGTGCAGCAGCCAGCCTCAACGTGgtacgtgtggcaggagggctgaggcaggac tcccctgtccagcaggccctgtttggtggcttcttttgagccccgaggccctcatgggaaaa aagacacagggcactgagaaacaagaagcttttttgtttctttgcttgcttgttttgttcta ttttcctggtccgtgcccctgctgaggggaaacctcacttctgtggagcatcttggctgatg gtagaggcagcaaatcagtttgatgacaaacaagtgaccaatggggaagcccactttgagct tgagtctccagccagcaggcctgctgcttccctcacacacccccaccccatttccttcaaga cagtgggcagacttgtgctccatggtcacactggttgaccttctggaaggtcctttcaggga caggagctttcccaagttgataacatctctgcagatgtcactcagggtggaagtactcccag cacctgcaaagggcaggcttggtttttagactagggtaggttggggtttcctaacttttcct cattgagcaatcaagacacttttaattttttttaattttatatatttattcttaagagacag aatcttgttctgtcaccaaggagggtatgcagtggcatgatcatagctcactgcagcttcat cctcccaggcttaagcgatcctcctgccccagcctcctgagtaactgggatcactatgacca gctaattttttttaagtttgtagagatggagtcttgctatattgcccaggatggtctcaaac tcctgccctcaatcaatcctcctggctcagcctcccaaagtgttggaattataggcacaagc caacatgcccagcctagatcctttttataagccctctatatatcttaaaatatagagagggt ttataaaaccctgcagagagggcttataataactacattatttttctttcttaaaacaagca aaaccaaacaacaaagaaaaagccctaaattaaaatgctttttacttttggagagcccacat ctctgtatgtcagctggagtgggacatctttgattttctggctctttcatgaataaagagca cctctttgagccagtgagccaggcacccacatttcacactgagcctggcctctccatccctg tctggaagactgtgtgagttttggaggaagctgtatccctgtgctggtggggagacacagaa agaaatttagctgagaggtgagtggggtgggagtccctccagcaccctgctttccacattgt gtaccttgaatccaccgaggaatggaggccatcttattcctacaaagaggccttctactcac cacacagtgtgcccggggacaggttctaaccagatgggtgacttttcccaacttgcacaaaa gtgctataacagatcaggagaagccccgacttctgccttcagaaagcacccatctgatcaaa gataagttgtatagatggacaaaaagaaatatatttaaataaatgtagtgaaagctgttctt t acct aagt get aagtgggctcagagtaactagacctgacaaaactagggcagcttctgggg gaaagtgaaccatactcttggcttaaggaatgaggcttcattattcattaatttatcatact tgtaccgggtaccttctcccgtcacaggcctgggggtgttacaccaagtaatatataatcag gggaaggcct t at tcaaaggtgggtaaggaagcctttgttct at ct gagcat caggtgt cat tctagacactgaggatacagagataaataaagcagacccaattccctgcttttgtactcagg gtaaaactaagacaacaatcatgataacgatgatgaccacagcgaccctttattgcgttgtg ctgaccttttacatgcattataacctttaattctcacaatgccctaagagatggatattttc cttatttccatgttacagacagaacaccaaggttcagtgaggttaaataagctgctgggtaa ggcagggatctgtgtctgccttgttcactgatgtgtctgaagcaccatggacagcgcctggc acacagtgggccatcagtacacatttgttcagtgaatggttgtctgcgtccgaagcccacgc tctctggttatcatgccatgcagcttcttcactggggtcacgggtctaaggcaggctctaaa gaagatggggtagggctggaatcctaacaaaggcccgtagagctccagctgaggagcccctg ccctcctagctctgcctctggatcccccagacttagataatcagaaccaggctgagggctga gaaaattaaccctgccctcttccaccagGATGCTGCTGTGCAACAGTGGCTGCAGAAGGGGA CCCCTGCCAGCAAGCTGATCCTTGGCATGCCTACCTACGGACGCTCCTTCACACTGGCCTCC TCATCAGACACCAGAGTGGGGGCCCCAGCCACAGGGTCTGGCACTCCAGGCCCCTTCACCAA GGAAGGAGGGATGCTGGCCTACTATGAAgtaggaaaacccataaccaccctgggtattgtga gggtcagggggtgcctgagagaacagaggtttccttctcccaccgtttctgagtaaggatgc caattgcagagaattctggccatgtaaattccatctcatccatgccatcttgaatttatcct taagcccaggtgcctcaagctgagtccagaattctaggtcactgcaaagaacctcctgattc tccaggcttcctcagacaggaattttccaatctctagtctcctcctggtgaggaagggaagg tgcctgcaagagcctccctcctcctccccagtgctgccttctgctcccacagGTCTGCTCCT GGAAGGGGGCCACCAAACAGAGAATCCAGGATCAGAAGGTGCCCTACATCTTCCGGGACAAC CAGTGGGTGGGCTTTGATGATGTGGAGAGCTTCAAAACCAAGgtgaggcccagctctgctgg cagaggggtggtcctagtgaacaagtcctgtcaatgacatggaatgaggacagttcaggacc tcaagggtgagtctcctttaaatgtcccaacatttcataaatctccacgtgatagtattttg tacctttactacctattgactcagaaaagacaaaatgacaaaaaattgctctacatttagta gcactctggaaagtatttctattttattaaataccatacaaattctacagacatatgaagag gagaaatacacatggt gagacat at agctttcttcagcccattggcaat at ctggtgcatgt ccaaattacagaccttttgctttacttataggacccccttctcctgcccaatattggtgcat ttacctggtttcatgccattttcccctgtgaaactgttagctcctgaagacggatgcttcac tagtttatctgtacatcccacatcaactatcatgtgtggctgacagcggcagcttctagagg tggacaattgcaggggggcacgatcctggctggggagatgggagaaggggtcacggtggaag ggtatttcagataagtcctaaagcatagaaggtggccattcttaggggtgagggagcgggca actgcacaagcaaagacgcgggtgcagagaaagtaaaggctggactctgaaaacaaagtgta ggcccacatagctggacccccaagcagggcaggtagaggaagggagctagctgaaagggagg ctggggccagcaagggatcaagcaccatggccccctggcagggttagaggacagagaatccc actctgagggaacacacagtcataccctcaggaagcctcaggaatctggtggagggaagatg gctctcagggaagacaaagacatgaagacagagaagcaagccttgattgagatcttttggaa atctgagttgttccctatggtaactggacttaccttgaccttgaccttgaccagcatacaat acccttaaataaagcagctcagcccacacatgaccaagcggtgaagggttcccacttaggga ggtgcacacaactgacactattcctcccgactgcacccaccccaccccaggtcatgcttacg ttaagacaaggagactgcaaaatgactgatgtccaagcagcatgaggactcagcatccaggc tcccagcccaaggctgtctccagaatctacagccactcacaggcctgaggctgctggagcaa aaagggccctgtccagaagaggtagccaggctctggggtggccgggtcaggcttccccgggc tcctccactgcatcccctccctgcacagGTCAGCTATCTGAAGCAGAAGGGACTGGGCGGGG CCATGGTCTGGGCACTGGACTTAGATGACTTTGCCGGCTTCTCCTGCAACCAGGGCCGATAC CCCCTCATCCAGACGCTACGGCAGGAACTGAgtaagtaaggggcccagggccagtggtggaa tttggaggtggggtacaaacacagctccatgcatccaatgaactgtaggtcccgggtccttc tgaggaaatgactttgccccaatactgtaatttcccaagcttcatgtacccgcaggagctaa ggaaactgtcaaaacaattttcctttttgcggtctttctcttcctggttgtagcaatgcctg cccagggttgttgtaaccgtggcttccctccagagggcccccagactgggagcagcaccttg ttcctctcttacctgggagaaagcctccccttagcctgcagcgtggcacccccacctgccct cagcagtatatggttgtgcttgcagatgggtaaagccttaactgcctgtcacatgtgaggcc aggtgttgcctgtggcactgtgcttcagctgtaggtatggctgttgtctggctgttctctgc ctggggtatctggctgtactcagcctgtagggcatgtggtgctgggctgctgccagccaatg gttgctgcctgtgtgctctgcctgtgggtttgggatctcttcgggggagccttggttgaatc ttcactgtcttcccagGTCTTCCATACTTGCCTTCAGGCACCCCAGAGCTTGAAGTTCCAAA ACCAGGTCAGCCCTCTGAACCTGAGCATGGCCCCAGCCCTGGACAAGACACGTTCTGCCAGG GCAAAGCTGATGGGCTCTATCCCAATCCTCGGGAACGGTCCAGCTTCTACAGCTGTGCAGCG GGGCGGCTGTTCCAGCAAAGCTGCCCGACAGGCCTGGTGTTCAGCAACTCCTGCAAATGCTG GAG C T G GAAT T GAGTCGCTAAAGCCCCTCCAGTCCCAGCTTTGAGGCTGGGCCCAGGATCAC TCTACAGCCTGCCTCCTGGGTTTTCCCTGGGGGCCGCAATCTGGCTCCTGCAGGCCTTTCTG TGGTCTTCCTTTATCCAGGCTTTCTGCTCTCAGCCTTGCCTTCCTTTTTTCTGGGTCTCCTG GGCTGCCCCTTTCACTTGCAAAATAAATCTTTGGTTTGTGCCCCTCTTCCCAAAGATGTGGT GACTTAAGAGGCTCTCTAAGCACACTGTTGACTCCAAAACATCCGCAGGTCAGAGCCAGGTG GGAAGGTGGTCCGTGCAGGATGTGCCAGGCCCTGTGGCAGGTCTTGCCCCATGAGTCCATAT GCCAGTGGGCAGTTTCCAGTACACAGGTGATGGCAGCCAGAGCGCGGCCCCAAGTGCAACAC GTTCTTTGGTTATTGGCTCAGAAGCCTGGAAACAGGAGCTTCTGCGGACCTTCACGCACGTG TTATTCCCAAAATGCCACTAGATGGCAGCAGAGAAGAACTTTTCCCTGATTTAAGCGCTCTG CGAGCAGCCACTCAGCATTACACTAAGAATTCAGACCCCTGGTACTCAGGGGTCTTCGAGAT AAATCCTGTTTGGGCCCTTTGCAGGCCCTATCTCACCTACTGCCCCCGTTCACCAGATGAAG AAGGAACAAT GAT TAT T CT GAG T T TATAGGCAT T TAAAC T GAAGAC T GAT T C TAACCAAT T C TGAGTCCAGAACCAAGGCCTGAAATGGGCCAGAGAGCATGCACACGGTGTCCTGGTTAAATA AAG T C AT C C C AAC AT T C

[0244] In view of the above, the antisense oligonucleotides provided herein can inhibit CHIT1 activity or function, such as CHIT 1 -associated inflammation or neuroinflammation. As used herein, the phrase “inhibits CHIT1 activity” or variations thereof means that after administration of an antisense oligonucleotide described herein, such as that of SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40, to a subject, tissue or a cell, an activity of CHIT1 in the subject, tissue or cell, is abrogated or reduced. In various examples, the activity of CHIT1 is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the activity thereof in the absence of the antisense oligonucleotide or prior to administration of the antisense oligonucleotide.

[0245] Formulation

[0246] In a particular form, the pharmaceutical compositions described herein comprise an acceptable carrier, diluent or excipient.

[0247] By “acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluents and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.

[0248] A useful reference describing acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.

[0249] Antisense oligonucleotides of the disclosure may be admixed, encapsulated, conjugated (such as fused) or otherwise associated with other molecules, molecule structures or mixtures of compounds, resulting in, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and / or absorption. Representative United States patents that teach the preparation of such uptake, distribution and / or absorption-assisting formulations include, but are not limited to, US 5,108,921, US 5,354,844, US 5,416,016, US 5,459,127, US 5,521,291, US 5,543,158, US 5,547,932, US

[0250] 5,583,020, US 5,591,721, US 4,426,330, US 4,534,899, US 5,013,556, US 5,108,921, US

[0251] 5,213,804, US 5,227,170, US 5,264,221, US 5,356,633, US 5,395,619, US 5,416,016, US

[0252] 5,417,978, US 5,462,854, US 5,469,854, US 5,512,295, US 5,527,528, US 5,534,259, US

[0253] 5,543,152, US 5,556,948, US 5,580,575, and US 5,595,756.

[0254] The antisense oligonucleotides may be formulated as pharmaceutically acceptable salts, esters, or salts of the esters, or any other compounds which, upon administration are capable of providing (directly or indirectly) the biologically active metabolite. The term “pharmaceutically acceptable salts” as used herein refers to physiologically and pharmaceutically acceptable salts of the oligonucleotide that retain the desired biological activities of the parent compounds and do not impart undesired toxicological effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860.

[0255] It is further envisaged that the antisense oligonucleotides provided herein may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bio equivalents. The term “prodrugs” as used herein refers to therapeutic agents that are prepared in an inactive form that is converted to an active form (i.e., a drug) upon administration by the action of endogenous enzymes or other chemicals and / or conditions. In particular, prodrug forms of the oligonucleotide of the disclosure are prepared as SATE [(S acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93 / 24510, WO 94 / 26764 and US 5,770,713.

[0256] A prodrug may, for example, be converted within the body, such as by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series (1976); "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985; and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate".

[0257] In some examples, the antisense oligonucleotides of the present disclosure can be complexed with a complexing agent to increase cellular uptake thereof. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells.

[0258] The term “cationic lipid” includes lipids and synthetic lipids having both polar and nonpolar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general, cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g, from 1 to about 25 carbon atoms. Exemplary straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., C1-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

[0259] Examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECT AMINE™ (e.g, LIPOFECT AMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), andEufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3.beta.-[N— (N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g, steryl-poly (L-lysine). Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., US 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al., 1996; Hope et al., 1998). In addition to those listed above, other lipid compositions are also known in the art and include, for example, those taught in US 4,235,871; US 4,501,728; 4,837,028; 4,737,323.

[0260] In some examples, lipid compositions can further comprise agents (e.g., viral proteins) to enhance lipid-mediated transfections of oligonucleotides. In further examples, N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides into tissues and cells.

[0261] In various examples, a composition for delivering antisense oligonucleotides of the present disclosure comprises a peptide having from between about one to about four basic residues. These basic residues can be located, e.g., on the amino terminal, C-terminal, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (can also be considered non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Suitably, a preponderance of neutral amino acids with long neutral side chains are used.

[0262] Suitably, the antisense oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide”. In one example, the composition includes an oligonucleotide and a covalently attached transporting peptide.

[0263] In further examples, the antisense oligonucleotide is attached to a targeting moiety such as N-acetylgalactosamine (GalNAc), an antibody, an antibody-like molecule or aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2018)).

[0264] Administration

[0265] Any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intranasal, intraocular, intraperitoneal, intracerebroventricular, intrathecal, topical, mucosal and transdermal administration, although without limitation thereto. In particular examples, the antisense oligonucleotide, the vector or the composition described herein is administered intravenously. In other examples, the antisense oligonucleotide, the vector or the composition described herein is administered subcutaneously. In various examples, the antisense oligonucleotide, the vector or the composition described herein is administered intrathecally.

[0266] In one example, the oligonucleotide of the disclosure is administered systemically. As used herein “systemic administration” is a route of administration that is either enteral or parenteral.

[0267] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and poly glycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and / or microspheres.

[0268] Compositions may be presented as discrete units such as capsules, sachets, functional foods / feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0269] The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as effective. The dose administered to a subject, in the context of the present disclosure, should be sufficient to effect a beneficial response in a subject over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. Design and Testing of Candidate Oligonucleotides

[0270] In another form, the present disclosure provides a method for designing, selecting or identifying an antisense oligonucleotide for reducing or inhibiting CHIT1 expression and / or activity, said method including the steps of:

[0271] (a) providing or producing one or more candidate antisense oligonucleotides comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region of a CHIT1 mRNA, wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof;

[0272] (b) contacting a cell expressing CHIT1 with the one or more candidate antisense oligonucleotides;

[0273] (b) testing the ability of the one or more candidate antisense oligonucleotides to reduce or inhibit CHIT1 expression and / or activity in the cell, and

[0274] (c) selecting an antisense oligonucleotide which reduces or inhibits CHIT1 expression and / or activity in the cell.

[0275] Suitably, the antisense oligonucleotide, inclusive of modifications thereof, and the CHIT1 mRNA are that hereinbefore described. In particular examples, the target region comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 29 or a variant thereof. In other examples, the target region comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31 or a variant thereof.

[0276] Suitably, the cell described herein is a neural cell, such as a neuronal cell or neurone or a glial cell (e.g., of the central nervous system or the peripheral nervous system). More particularly, the cell can be a microglial cell (e.g., a CNS-resident microglial cell or a monocyte-derived microglia-like cell), such as a microglial cell derived from a subject having a neurodegenerative disease, disorder or condition described herein (e.g., ALS). In some examples, the cell is part of or in the form of an organoid, such as a neural or neuronal organoid.

[0277] In addition to design elements of the present disclosure, there are many known factors to be considered when producing an antisense oligonucleotide. The specifics depend on the purpose of the oligonucleotide but include features such as strength and stability of the oligonucleotide-target polynucleotide interaction, such as the secondary structure of the antisense oligonucleotide, thermodynamic stability and / or functional motifs.

[0278] In view of the above, the present method may include measuring a change in expression level (e.g., a protein and / or mRNA expression level) and / or activity level of CHIT1 in the cell (such as by those methods described herein) in response to the candidate oligonucleotide(s). In some examples, the expression level and / or activity level of CHIT is reduced to about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the expression level and / or activity level of CHIT1 prior to administration of the candidate oligonucleotide (e.g., untreated control cells).

[0279] Accordingly, the level of CHIT1 expression and / or activity may also be compared to a reference or threshold level thereof. Thus, any of the methods disclosed herein may comprise a step of establishing a reference or threshold level of expression and / or activity of CHIT1.

[0280] Once synthesized, candidate antisense oligonucleotides can be tested for their desired activity using standard procedures in the art. This may involve administering the candidate antisense oligonucleotides to cells in vitro expressing CHIT1 mRNA and measuring binding and / or inhibition thereof by the candidate antisense oligonucleotide (e.g., analysing the amount of transcription and / or translation of CHIT1, such as mature mRNA and / or protein levels) and / or utilising one or more functional assays (e.g., cell growth and proliferation, apoptosis, cellular differentiation, inflammation including neuroinflammation) to determine whether the activity thereof changes after exposure to a candidate antisense oligonucleotide. In another example, the candidate oligonucleotide is administered to an animal, and the animal screened for the amount of CHIT1 inhibition. In other examples, the candidate antisense oligonucleotide is tested for its ability to bind or hybridize to the target region of the CHIT1 mRNA.

[0281] In some examples, inhibitory activity of the candidate antisense oligonucleotides can be assessed by mRNA reverse transcription quantitative real-time PCR (RT-qPCR) to determine expression of the CHIT1 mRNA. For example, RNA can be extracted and purified from cells which have been incubated with a candidate oligonucleotide. cDNA is then synthesized from isolated RNA and RT-qPCR can be performed, using methods and reagents known the art. Protein products and / or downstream cell signalling (and / or one or more markers thereof) of CHIT1 may alternatively or additionally be determined, such as by any means known in the art, including, but not limited to, ELISA, western blot, mass spectrometry, proteomics, immunoprecipitation and immunostaining.

[0282] Suitably, the candidate antisense oligonucleotide possesses or displays little or no significant off-target and / or nonspecific effects.

[0283] Suitably, the candidate antisense oligonucleotide is at least partly capable of crossing the blood brain barrier. Accordingly, the present method may include the step of testing whether the candidate antisense oligonucleotide is capable of crossing the blood brain barrier. It is envisaged that any method known in the art to assess whether the candidate agent crosses the blood-brain barrier of the subject, such as those described herein, may be utilised for the present method. Such methods may be carried out using in silico, in vitro and / or ex vivo assays, such as in in vitro models of the blood-brain barrier, as are known in the art, such as a computational model (e.g., an in silico model), a 2D transwell model, a 3D organoid model, and a microfluidic model (see, e.g., Chaulagain et al., Int J Mol Sci. 2023 Feb; 24(3): 2710). Alternatively, the present method may be performed in vivo, for example, in animal subjects to assess the permeability of candidate agents in respect of the blood-brain barrier (e.g., permeability of labelled, such as fluorescently labelled or radiolabelled, conjugates of the candidate agent, pharmacokinetic studies).

[0284] Additionally, the present method may further include one or more of the steps of: selecting the candidate antisense oligonucleotide that binds (e.g., specifically binds with high affinity) and / or reduces the expression and / or the activity of CHIT1; isolating or purifying the candidate antisense oligonucleotide; formulating the candidate antisense oligonucleotide into a pharmaceutical formulation; and adding the candidate antisense oligonucleotide or the pharmaceutical formulation to packaging and / or a container.

[0285] Accordingly, in another form, the present disclosure provides an antisense oligonucleotide designed, selected or identified by the present methods.

[0286] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples.

[0287] Examples

[0288] Example 1.

[0289] This Example tested the ability of a range of CHIT 1 -targeted antisense oligonucleotide to bind and functionally inhibit expression and / or exon skipping of CHIT1 mRNA in macrophages expressing CHIT1.

[0290] Weighted gene co-expression network analysis

[0291] The present inventors performed weighted gene co-expression network analysis (WGCNA) using bulk RNA-seq data from post-mortem brain and spinal cord tissues supplied by the New York Genome Center (NYGC) ALS Consortium. These studies identified a module of 100 genes that were upregulated in the spinal cord of individuals with ALS, which were particularly prevalent in the cervical and thoracic regions (Figure 3A). The expression of this gene module was negatively correlated with retrospective disease duration. Importantly, the most statistically significant member of this gene expression module was CHIT1 (Figure 3B). A recent publication recapitulated the findings and identified CHIT1 as the strongest negatively correlated gene with disease duration in the cervical and lumbar spinal cord (Humphrey et al., 2023).

[0292] These findings suggest that CHIT1 is a driver of neuroinflammation that promotes the death of motor neurons in ALS and contributes to disease progression. CHIT1 may be directly neurotoxic.

[0293] Optimising conditions for inducing CHIT 1 expression in human monocytes

[0294] CHIT1 is not significantly expressed in human fibroblasts and therefore an alternative cell model was established to allow the evaluation of ASOs designed to induce exon skipping and transcript knockdown of CHIT1 mRNA. The in vitro model for CHIT1 expression was selected from a publication which demonstrated increased expression of CHIT1 following the differentiation of human THP-1 monocytes into macrophages after stimulation with phorbol 12-myri state- 13 -acetate (PMA) (Pham et al., 2007). Following treatment with PMA, the suspension cultures of THP-1 monocytes underwent morphological changes as they differentiated into adherent macrophages (Figure 4A). In addition, PMA-treated THP-1 cells expressed increased levels of CHIT1 mRNA compared with DMSO- or untreated cells (Figure 4B).

[0295] Evaluating knockdown of CHIT1 mRNA transcript using antisense oligonucleotide sequences with 2-O-Methyl chemistry

[0296] A total of 26 ASOs were synthesised using 2-O-Methyl chemistry (Table 1). These ASOs were designed to target exons which carry partial codons at their exon boundaries in order to induce exon skipping and transcript knockdown.

[0297] Table 1. 2-O-Methyl ASO sequences for CHIT1 exon 3, 5, 6, 7, and 10.

[0298]

[0299] THP-1 monocytes were treated with 200 ng / mL PMA for 72 hours to induce macrophage-like differentiation and CHIT1 expression. Differentiated cells were then transfected for 24 hours with 2-O-methyl ASOs targeting exons 3, 5, 6, 7 and 10 of CHIT1 and RNA was isolated for RT-PCR. A number of 2-0-Me ASOs targeting exon 3 (H3A(+15+39) and H3A(+74+98)), exon 5 (H5A(+17+41) and H5A(+106+130)), and exon 7 (H7A(+20+44) and H7A(+49+73)) induced exon skipping and knockdown of CHIT1 transcript, as indicated by the presence of two distinct bands with reduced density compared with scramble ASO- treated cells (Figure 5A, B, D). Certain 2-0-Me ASOs targeting exon 6 (H6A(-15+10) and H6A(+13+37)) and exon 10 (H10A(+08+32) and H10A(+35+59)) induced knockdown of CHIT1, as indicated by the reduced density of the CHIT1 band (Figure 5C, E). Assessing knockdown of CHIT1 mRNA transcript using PMOs which target exon 5, 6, and 7 of CHIT1

[0300] The ASOs CHHT_H5A(+17+41), CHIT1_H6A(-15+1O), CHITl_H7A(+20+44) CHITl_H7A(+49+73) were selected based on their successful knockdown of CHIT1. Their sequences were then synthesised using phosphorodiamidate (PDA) morpholino oligomers (PMO) chemistry (Table 2). PMOs have higher serum stability, higher resistance to nuclease degradation, a more favourable safety profile, and a wider therapeutic window than 2-0- Methyl ASOs (Rinaldi & Wood, 2018).

[0301] Table 2. Nucleotide sequences for CHIT1 exon 5, 6, and 7-targeting PMOs.

[0302] The efficacy of the PMOs was evaluated by treating differentiated THP-1 cells with 50 or 100 pM H6A(-15+10), H7A(+20+44), H5A(+17+41) or H7A(+49+73) for 72 hours. Compared with control cells, THP-1 macrophages treated with 50 pM H6A(-15+10) had significantly reduced levels of CHIT1 mRNA (Figure 6A-B). Treatment with 100 pM H6A(- 15+10) also reduced levels of CHIT1 mRNA, but did not reach statistical significance (p = 0.0607). By contrast, CHIT1 mRNA levels in H7A(+20+44)-treated cells were comparable to controls. In addition, treatment with 50 or 100 pM H6A(-15+10) significantly reduced the CHIT1 mRNA levels in THP-1 macrophages (Figure 6C-D). While 100 pM H7A(+49+73) led to an apparent decrease in CHIT1 mRNA levels (Figure 6D), statistical testing was not performed, as one replicate did not have detectable CHIT1 or SMN mRNA and was excluded, reducing this sample group to two biological replicates (Figure 6C).

[0303] In addition to observing significant CHIT1 knockdown with the exon 5 and 6-targeting PMOs, we also noted that the levels of SMN appeared lower in samples with strong CHIT1 knockdown (Figure 6 A, C).

[0304] Differentiated THP-1 macrophages were treated with 5, 10 or 25 pM H6A(-15+10), H7A(+20+44), H5A(+17+41) or H7A(+49+73) for 72 hours. Compared with controls and the lower concentrations of PMO, cells treated with 25 pM H5A(+17+41) demonstrated significantly reduced levels of CHIT1 mRNA (Figure 7A-B). By contrast, H6A(-15+10), H7A(+20+44), H5A(+17+41) and H7A(+49+73) did not regulate CHIT1 mRNA levels at concentrations of 5-25 pM (Figure 7C-F). In addition, SMN levels in cells with strong CHIT1 knockdown appeared comparable to other conditions (Figure 7A). Similarly, analysis of a different housekeeper gene, TBP, via qPCR demonstrated that TBP levels were comparable for control and PMO-treated samples (Figure 7G).

[0305] In summary, the above data demonstrates that the use of splice-switching ASOs to suppress the expression of CHIT1 protein expression is feasible and appears to modulate CHIT1 expression. In particular, H5A(+ 17+41) and H6A(-15+10) were shown to strongly knock down CHIT1 mRNA levels at 25 pM and 50 pM, respectively.

[0306] BLAST analysis of off-target effects of H5A(+ 17+41) and H6A(-15+ 10)

[0307] The sequences for H5A(+ 17+41) and H6A(-15+10) were submitted to nucleotide Basic Local Alignment Search Tool (BLAST) to assess hybridisation-dependent off-target binding. BLASTN was performed with the following databases: Nucleotide collection (nr / nt) and excluding models (XM / XP), and Reference RNA sequence database (refseq rna) and excluding models (XM / XP). Default settings were used for all other parameters. Matches of 15 contiguous nucleotides (nt) or above, with the reverse complementary sequence of the PMO, in the correct strand of DNA containing the gene were reported. In addition, matches of 15 nt or above with 1 or 2 mismatches, with the reverse complementary sequence of the PMO, in the correct strand of DNA containing the gene were reported.

[0308] For the H5A(+ 17+41) PMO, two off-target matches were identified, with 15 contiguous nt matching in exon 8 of TPCN2, and 22 nt with 1 mismatch in the 3’ UTR of TMEM108. For the H6A(-15+10) PMO, two off-target matches were identified, with 15 contiguous nt matching in exon 14 of the MANE transcript of GRIN2A (NM_001134407.3) and 15 contiguous nt spanning 1 nt of exon 14 and 13 nt of intron 13 in a different isoform of GRIN2A (NM_001134408.2). The PMO is therefore unlikely to bind or have activity in genomic sites with substantial mismatches and overhanging bases.

[0309] Target engagement of ASOs in MDMi cells

[0310] PMO-based versions of the H5A(+17+41) and H6A(-15+10) (also referred to as E5- PMO and E6-PMO respectively) therapeutic candidates have further been tested in monocyte- derived microglia (MDMi) derived from controls and ALS cases to demonstrate target engagement and therapeutic effect.

[0311] The molecular and cellular phenotype of MDMi cells are remarkably similar to human microglia in vivo. Theoretically, MDMi cells retain aging-related and epigenetic information, circumventing the limitations associated with iPSC-derived microglia, which exhibit a nascent, embryonic-like state. A robust and reproducible approach to transdifferentiate PBMCs into MDMi over 14 days has been previously developed (Quek, H., et al., STARProtoc, 2022. 3(4): p. 101747).

[0312] MDMi derived from ALS cases exhibit pathognomonic features of disease, including cytoplasmic inclusions of non-phosphorylated and phosphorylated TDP-43 (Fig. 8A), impaired phagocytosis (Fig. 8B), increased secretion of inflammatory cytokines IL-8, IL-18 and TNF (Fig. 8C), and morphologies consistent with a neuroinflammatory phenotype (Quek, H., et al., J Neuroinflammation, 2022. 19(1): p. 58). Importantly, these cells also express CHIT1 and are therefore an optimal ALS cell model for investigating the biological effects of CHIT1 knockdown.

[0313] Robust target engagement has been confirmed for both of the H5A(+ 17+41) and H6A(- 15+10) antisense oligonucleotides in healthy control MDMi. Immediately following CHIT1 PMO transfection of healthy control PBMCs using Neon electroporation, cells were transdifferentiated into MDMi over 14 days. On day 11, cells were observed via phase-contrast microscopy and on day 14 were harvested for RT-PCR analysis. Compared with control cells, MDMi treated with PMO-based H5A(+17+41) and H6A(-15+10) antisense oligonucleotides exhibited significant reductions in CHIT1 mRNA levels (Fig. 9A-D). Importantly, transfection with or without PMOs did not alter MDMi differentiation and gave rise to healthy, microglialike cells (Fig. 9E-F).

[0314] Target engagement was further confirmed for the E5-PMO in healthy control (HC) and ALS MDMi. Immediately following transfection with or without PMOs of HC and ALS PBMCs using Neon electroporation (ZAP), cells were transdifferentiated into MDMi over 14 days and then harvested for RT-PCR analysis. E5-PMO gave rise to healthy, microglia-like cells (Fig. 9), indicating that E5-PMO may be a promising candidate for therapeutic applications due to its ability to maintain cell viability while effectively reducing CHIT1 mRNA levels. The selected lead E5-PMO was validated in HC and ALS MDMi, demonstrating significant knock down of CHIT1 mRNA at 25pM (p < 0.05 in both HC and ALS; Fig 10). Transfection with a negative control PMO (mouse SOD1 PMO), predicted to have no binding in human cells, showed no significant difference to ZAP and UT controls (data not shown). Interestingly, ALS patient-derived MDMi also exhibited greater variability in baseline CHIT1 levels than healthy control-derived MDMi.

[0315] The E5-PM0 also demonstrates positive biological efficacy with CHIT1 knockdown on the secretion of inflammatory cytokines associated with ALS (Fig. 11). Secretion of IL-8 was significantly reduced in ALS MDMi treated with E5-PM0 (10-50 pM; Fig. 11 A). Levels of IL-6 were significantly lowered by 50pM E5-PMO (Fig. 1 IB), while TNF levels were decreased by 25-50 pM E5-PMO in both HC and ALS MDMi (Fig. 11C). Additionally, ALS MDMi treated with 10 pM E5-PMO secreted significantly lower levels of IL-18 (Fig. 1 ID).

[0316] Example 2

[0317] The present prophetic Example outlines additional proposed studies for the antisense oligonucleotides of the present disclosure.

[0318] / . Dosage dependency

[0319] ALS patient PBMCs will be transfected with the lead CEHT1 PMO via Neon electroporation (0.1-100 pM; n=4 lines / concentration). Immediately following transfection, PBMCs will be transdifferentiated into MDMi using published protocols (Quek, H., et al., STAR Protoc, 2022. 3(4): p. 101747). At day 14, cell culture supernatant will be collected for ELISA and / or Western blot analysis. Cells will be harvested and lysed for RNA extraction. The EC50 of the CHIT1 PMO will be determined by quantification of:

[0320] • CHIT1 mRNA levels via RT-PCR;

[0321] • CHIT1 protein levels via ELISA and / or Western blot.

[0322] 2. In vitro toxicology

[0323] Compared with ASOs using other chemistries, PMOs are biologically tolerable and demonstrate a better safety profile. Administration in humans will be via intrathecal injection to increase efficacy in the spinal cord and limit off-target effects, therefore, to establish feasibility and reasonable safety of the CHIT1 PMO, in vitro toxicology will be performed using cell lines reflective of the CNS, including MDMi, iPSC-derived motor neuron and iPSC- derived astrocyte monocultures.

[0324] ALS patient PBMCs will be transfected with the lead CHIT1 PMO (0.1-100 pM; n=4 MDMi lines / concentration) and differentiated into MDMi. At day 14, cell culture supernatant will be collected and cells harvested. The LC50 of the CHIT1 PMO will be determined by:

[0325] • LDH assay as a quantitative measure of MDMi health. The effective optimal dose will be tested in iPSC-derived motor neurons and astrocytes using established methods. ALS patient iPSC lines (n=6) will be differentiated into 90% HB9+ and ChAT+ spinal motor neurons, using established protocols (see, e.g., Bax, M., et al., 2019. 8(6): p. 581). CHIT1 PMOs will be transfected on day 18 using Neon electroporation, and differentiated for a further 10 days. iPSC-derived astrocytes will be generated from ALS patient iPSCs cell lines (n=6) using established protocols (see, e.g., Wasielewska, J.M., et al., Theranostics, 2022. 12(16): p. 6826-6847). CHIT1 PMOs will be transfected into iPSC-derived astrocytes after 60 days using Neon electroporation, followed by culture in astrocyte medium supplemented with BMP4 and CNTF for a further 7 days to maintain astrocyte maturation. At the experimental endpoints, cell culture supernatant will be collected, and cells harvested.

[0326] With cell culture supernatant and harvested cells from MDMi, iPSC-derived motor neurons and astrocytes, the following cell viability assays will be performed:

[0327] • LDH assay as a quantitative measure of cell health.

[0328] RNA-seq analysis of a separate set of CHIT1 PMO-treated iPSC-derived motor neurons and astrocytes (n=6 lines / cell type / treatment) will be undertaken. RNA will undergo quality control and sent to the Australian Genome Research Facility (AGRF) for sequencing (Illumina HiSeq 2500 or Pacbio Iso-seq). Bioinformatic analysis will be performed to assess off-target binding of CHIT1 PMO, and compared to that from BLAST.

[0329] 3. Large scale screen of sporadic and familial ALS MDMi lines

[0330] The CHIT1 PMO antisense oligonucleotides will be tested in ALS patient (n=50; sporadic and familial) and healthy control (n=10) PBMC lines using an optimal effective CHIT1 PMO dose. Study design will balance for gender, age, and bulbar / spinal ALS. Immediately following transfection, PBMCs will be transdifferentiated into MDMi. At day 14, CHIT1 PMO effects on phenotype assays will be compared with controls (untreated and PMO transfection controls) across some ALS-relevant parameters that may including, as most appropriate:

[0331] • CHIT1 RNA and protein levels measured via RT-PCR and ELISA and / or Western blot;

[0332] • Microglial cell viability by LDH assay;

[0333] • Detection of secreted chemokines, cytokines, and reactive oxygen species (ROS) using LEGENDplex™ Human Inflammation Panel 1 multi-plex kit and Griess assay;

[0334] • Phagocytosis assay;

[0335] • Immunofluorescent staining of TDP-43 and pTDP-43 to assess mislocalisation and aggregation. 4. Development of LC-MS / MS methods for quantification of CHIT1 PMO in rat fluids and tissues.

[0336] To facilitate in vivo ADME / PK / PD studies, liquid chromatography / tandem mass spectrometric (LC-MS / MS) methods will be developed for the quantification of PMO in rat fluids and tissues. An LC-MS / MS method will be developed for the quantification of the PMO in rat plasma, faeces, urine and tissue homogenate (brain / spinal cord, kidney, liver). Appropriate chromatographic, mass spectrometric, and sample extraction procedures will be developed to achieve the sensitivity and specifications needed and can be used to support nonregulated studies.

[0337] This development will enable the next phase of ADME / PK / PD studies in rats to inform the expected maximum tolerated dose of CHIT1 PMO and administration frequency, and IND- enabling studies of GLP in vivo nonclinical testing.

[0338] Example 3

[0339] The present Example provides some initial preliminary data for PMO-based antisense oligonucleotides of the present disclosure.

[0340] Evaluation of PMOs targeting CHIT1 exon 5 and exon 10

[0341] Initially identified candidate sequences to skip exon 5, exon 7 and exon 10 were synthesised as PMOs and assessed both for CHIT1 mRNA (Figures 12A and 12B; Figure 13).

[0342] The PMO-based antisense oligonucleotides, including H5A(+17+41), H5A(+106+130), H7A (+49+73), H10A(+08+32), and H10A(+35+59) all demonstrate CHIT1 knockdown. The data generated and observed in this Example suggests that the use of spliceswitching AOs to suppress the expression of CEHT1 protein expression is feasible and appears to modulate CHIT1 expression.

[0343] Example 4: Impact of cytokine release following CHIT1 knockdown in healthy and ALS MDMi (samples from Example 1)

[0344] Supernatants were collected from MDMi cultures following CHIT1 PMO transfection (from Example 1) to assess cytokine release using the LEGENDplex™ Human Inflammation Panel 1 (13-plex). The panel included detection of IL-ip, IFN-a2, IFN-y, TNF-a, MCP-1, IL- 6, CXCL8 (IL-8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33. Cytokine levels were normalised to the corresponding RNA concentrations measured by nanodrop to account for cell loss or variability between samples.

[0345] Key cytokines of interest following CHIT1 knockdown were identified as IL-8, TNF- a, IL-6, and IL-10 (Figure 14), with lesser changes observed in IL-ip, IL-18, and IFN-y (Figure 15). Expression levels of IL-23, IL-33, IL-17A, IFN-a2, IL-12p70, and MCP-1 were comparable between H6A- and H5A-transfected samples and controls, indicating that CHIT1 knockdown does not significantly alter baseline cytokine production (Figure 16).

[0346] Overall, greater CHIT1 knockdown correlated with reduced cytokine release, particularly in ALS MDMi compared to healthy controls.

[0347] Example 5. Detection of CHIT1 Protein Secretion in Healthy Control and ALS MDMi Supernatants

[0348] CHIT1 protein secretion was evaluated in the supernatants of healthy controls and ALS MDMi (samples collected from Example 1).

[0349] Using the workflow — loading 30 pg of protein, employing a reversible PVDF stain for total protein normalisation, and detecting with the Sigma anti-CHITl antibody — CEHT1 was successfully detected as a specific band at approximately 50 kDa. A recombinant human CHIT1 (rhCHITl) positive control (0.5 ng) was included on each gel to confirm CHIT1 protein specificity (Figure 17).

[0350] To ensure consistency across donors and experiments, each gel was loaded with a paired healthy control and ALS sample side-by-side. Furthermore, all gels were exposed under identical conditions for both the protein stain and the CHIT1 signal, enabling reliable comparison of CHIT1 secretion levels between samples.

[0351] Densitometric analysis of CHIT1 bands (Figure 18) revealed a dose-dependent decrease in CHIT1 secretion correlating with increased CHIT1 knockdown following PMO treatment. Notably, 25 pM and 50 pM CHIT1 PMO treatments resulted in significant inhibition of CHIT1 secretion in healthy controls and partial inhibition in ALS MDMi. These findings validate that CHIT1 secretion can be reliably detected and quantified in supernatants from MDMi cultures and that PMO-mediated CHIT1 knockdown effectively reduces secreted CHIT1 levels.

[0352] Example 6: Additional assays performed • LDH assay: Assessment of cytotoxicity and membrane integrity in MDMi cultures. (Figure 19)

[0353] • Nitric oxide assay: Measurement of nitric oxide production as a marker of oxidative stress and inflammation. (Figure 20)

[0354] • CHIT1 knockdown validation via RT-PCR (Figure 21)

[0355] • TNF-a cytokine secretion (Figure 22)

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Claims

CLAIMS:

1. An antisense oligonucleotide for reducing CHIT1 expression, the antisense oligonucleotide comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region of a CHIT1 mRNA, wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof.

2. The antisense oligonucleotide of Claim 1, wherein the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 8, 10, 13, 14, 18, 19, 23, 24 and 37 to 40 or a fragment, variant or derivative thereof and wherein one or more of the U nucleotides thereof may be a T and / or one or more of the T nucleotides thereof may be a U.

3. The antisense oligonucleotide of Claim 1 or Claim 2, wherein the target region comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 29 or a variant thereof.

4. The antisense oligonucleotide of Claim 3, wherein the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 37, or a fragment, variant or derivative thereof.

5. The antisense oligonucleotide of Claim 1 or Claim 2, wherein the target region comprises, consists of, or consists essentially of the nucleotide sequence set forth in SEQ ID NO: 31 or a variant thereof.

6. The antisense oligonucleotide of Claim 5, wherein the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 38, or a fragment, variant or derivative thereof.

7. The antisense oligonucleotide of any one of the preceding claims, wherein one or a plurality of nucleotides thereof are modified.

8. The antisense oligonucleotide of Claim 7, wherein the modification of the one or plurality of nucleotides is selected from the group consisting of a morpholino modification, a LNA modification, a HNA modification, a CeNA modification, a 2 ’-methoxy ethyl modification, a 2’-O-alkyl modification, a 2’-O-allyl modification, a 2’-C- allyl modification, a 2’-fluoro modification, a 2’-deoxy modification, and any combination thereof.

9. The antisense oligonucleotide of Claim 7 or Claim 8, wherein the modification of the one or plurality of nucleotides is a morpholino modification.

10. The antisense oligonucleotide of any one of the preceding claims, wherein one or a plurality of intemucleotide linkages thereof are modified.

11. The antisense oligonucleotide of Claim 10, wherein the modification of the one or plurality of internucleotide linkages is selected from the group consisting of a phosphorothioate modification; a non-bridging oxygen atom substituting a sulfur atom modification; a phosphonate, such as a methylphosphonate; a phosphodiester modification; a phosphoromorpholidate modification; a phosphoropiperazidate modification; an amide modification; a methylene or methylamino modification; a fromacetal modification; a thioformacetal modification; a peptide nucleic acid modification; or a phosphoroamidate modification; such as a phosphorodiamidate morpholino (PMO), an N3’-P5’ phosphoramidite or a thiophosphoroamidite modification.

12. The antisense oligonucleotide of Claim 10, wherein the modification of the one or plurality of internucleotide linkages is a phosphoroamidate modification.

13. The antisense oligonucleotide of any one of Claims 7 to 12, wherein the modification of the one or plurality of nucleotides and / or internucleotide linkages is a phosphorodiamidate morpholino modification.

14. The antisense oligonucleotide of Claim 13, wherein the targeting sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a fragment, variant or derivative thereof and wherein the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

15. A vector encoding the antisense oligonucleotide of any one of Claims 1 to 14.

16. A pharmaceutical composition comprising the antisense oligonucleotide of any one of Claims 1 to 14 or the vector of Claim 15 and a pharmaceutically-acceptable carrier, diluent or excipient.

17. A method of reducing or inhibiting CHIT1 expression in a cell, said method including the step of contacting the cell with an effective amount of the antisense oligonucleotide of any one of Claims 1 to 14, the vector of Claim 15 and / or the pharmaceutical composition of Claim 16 to thereby reduce or inhibit CHIT1 expression therein.

18. A method of treating, preventing or ameliorating a disease, disorder or condition associated with CHIT1 in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide of any one of Claims 1 to 14, the vector of Claim 15 and / or the pharmaceutical composition of Claim 16 to thereby treat, prevent or ameliorate the disease, disorder or condition.

19. Use of the antisense oligonucleotide of any one of Claims 1 to 14, the vector of Claim 15 and / or the pharmaceutical composition of Claim 16 in the manufacture of a medicament for treating, preventing or ameliorating a disease, disorder or condition associated with CHIT1 in a subject.

20. The method of Claim 18 or the use of Claim 19, wherein the disease, disorder or condition is or comprises a neurodegenerative disease, disorder or condition.

21. A method of treating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject, said method including the step of administering to the subject a therapeutically effective amount of the antisense oligonucleotide of any one of Claims 1 to 14, the vector of Claim 15 and / or the pharmaceutical composition of Claim 16 to thereby treat, prevent or ameliorate the neurodegenerative disease, disorder or condition.

22. Use of the antisense oligonucleotide of any one of Claims 1 to 14, the vector of Claim 15 and / or the pharmaceutical composition of Claim 16 in the manufacture of a medicament fortreating, preventing or ameliorating a neurodegenerative disease, disorder or condition in a subject.

23. The method or use of any one of Claims 20 to 22, wherein the neurodegenerative disease, disorder or condition is selected from the group consisting of: Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease, Huntington’s disease, Multiple sclerosis and Parkinson’s disease.

24. The method or use of Claim 23, wherein the neurodegenerative disease, disorder or condition is ALS.

25. A method for designing, selecting or identifying an antisense oligonucleotide for reducing or inhibiting CHIT1 expression and / or activity, said method including the steps of:(a) providing one or more candidate antisense oligonucleotides comprising a targeting sequence that hybridizes to or is complementary to at least a portion of a target region of a CHIT1 mRNA, wherein the target region comprises, consists of, or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27 to 36 or a variant thereof;(b) contacting a cell expressing CHIT1 with the one or more candidate antisense oligonucleotides;(c) testing the ability of the one or more candidate antisense oligonucleotides to reduce or inhibit CHIT1 expression and / or activity in the cell, and(d) selecting an antisense oligonucleotide which reduces or inhibits CEHT1 expression and / or activity in the cell.

26. An antisense oligonucleotide designed, selected or identified by the method of Claim 25.

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