MODEL OF INFLAMMATORY BOWEL DISEASE

FIELD

[0001] The present disclosure relates generally to Mincle-binding DNA aptamers and uses thereof.

BACKGROUND

[0002] An expanding field of biological-therapy aims to target individual pattern recognition receptors (PRRs) due to their proposed roles in the initiation and continuation of chronic inflammatory diseases ^{1 4} . Indeed, many PRRs have been found to be dysregulated within the diseased host and described to contribute directly to the mounting inflammation through the spontaneous or uncontrolled production of inflammatory mediators ⁴⁵ . A subset of these receptors, which includes inducible-PRRs (iPRRs), are thought to play a distinct role in the pathogenesis of many diseases including; inflammatory bowel disease (IBD), psoriasis, multiple sclerosis, and some parasitic infections such as Leishmaniasis ^{5 8} . Macrophage inducible C-type lectin (CLEC4e/Mincle) was first identified in the late 1990s and was observed to be transcriptionally upregulated in macrophages following stimulation with a variety of inflammatory stimuli. During homeostasis, Mincle expression is low, but expression can be induced through the activation of several PRRs including TLR2 and TLR4 or inflammatory cytokines such as IFNy, TNFa, and IL-6 ⁹ . Importantly, the increase in Mincle expression is seemingly dependent on MyD88 and NFKB (P65/RELA). Mincle expression has been shown to be limited to a sub-set of cell types, predominantly myeloid cells such as neutrophils, monocytes, monocyte-derived macrophages, and dendritic cells 10-13

[0003] Mincle; through Syk-mediated signalling, mediates a variety of proinflammatory responses within the host, can promote or even prolong inflammation ¹³ . Interestingly, while Mincle has been described to serve as a receptor to a variety of: mycobacterial ¹⁴¹⁵ ; bacterial ¹⁶ ; and fungal pathogens ¹⁷ , it can be directly activated by several host-derived damaged-associated molecular patterns (DAMPs) suggesting an intimate involvement in a sterile inflammatory insult ¹⁸ . Specifically, previous studies have outlined that Mincle senses aberrant levels of cholesterol sulphate ⁷ and SAP130 ¹⁹ , DAMPs associated with cellular damage and necrosis, which, when bound to Mincle, trigger a pro-inflammatory cytokine response in the host. Furthermore, through the use of knockout mice, neutralizing Mincle antibodies, targeted-siRNA, or pan-Syk-inhibitors, the distinct role of Mincle in these disease models has been significantly investigated ⁵ - ⁸ ’ ¹⁶ ’ ¹⁸ . [0004] The expanding field of nucleic acid therapeutics have highlighted the potential use of aptamers as a direct substitute for commercially-available, but rather expensive, monoclonal antibodies while avoiding the non-specific nature of kinase- directed pharmacological inhibitors ²⁰ . The inherent nature of aptamers with their high- specificity and non-immunogenic qualities make them desirable for the targeted blockade of inflammation as an alternative to small immune-pathway chemical inhibitors and their possible off-target effects ²¹ . Promised for almost 20 years, Aptamer-based therapeutics, such as the anti-VEGF aptamer Pegaptanib (Macugen™) ²² , have begun to spill into the market. However, with the familiarity of monoclonal antibodies, the need for Aptamerbiosimilars is overlooked.

[0005] Inflammatory Bowel Disease (IBD), subdivided into Ulcerative Colitis (UC) and Crohn’s Disease (CD), is a rapidly expanding cohort of chronic inflammatory intestinal disease, predominant within the developed world ²³ . UC, is a chronic idiopathic disorder of the colon manifesting in contiguous mucosal inflammation that can extend from the distal ileum to anus. Bimodal in its age distribution, UC is the most common form of IBD and is a significant form of morbidity worldwide ²⁴ . Patients with UC, frequently experience acute-cyclical episodes of disease manifestation with symptoms including bloody diarrhoea, pain, fever, and weight loss. With several pro-inflammatory cytokines elevated during the disease flares, current biological therapies revolve almost entirely around modulating systemic TNFa, through the prohibitively expensive use of monoclonal antibody therapies such as Infliximab, Adalimumab, Certolizumab, or Golimumab ²⁵²⁶ . Furthermore, the underlying cause of the over-production of TNFa within these patients is still debated. It is with this incomplete understanding of disease pathogenesis that the discovery and investigation of new targets for therapeutic intervention must be made. PRRs, such as Mincle, present a novel and promising target upstream of the canonical neutralization of circulating inflammatory cytokines.

[0006] While Mincle has been implicated in the progression of several experimentally-induced inflammatory disease models; there has been a lack of commercially available, Mincle-directed-therapeutic-agents.

SUMMARY

[0007] In one aspect there is provided an aptamer that binds to Mincle polypeptide. [0008] In one aspect there is provided an aptamer that binds to Mincle polypeptide, comprising:

[0009] a) a nucleic acid comprising or consisting of any one of AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2), SEQ ID NO: 3, MApT7 (SEQ ID NO: 4), MApT1 SEQ ID NO: 5), MApT4 (SEQ ID NO: 6), MApT5 (SEQ ID NO: 7), MApT6 SEQ ID NO: 8), MApT2 SEQ ID NO: 9), MApT3 (SEQ ID NO: 10), or MApT8 (SEQ ID NO: 1);

[0010] b) a nucleic acid having at least 70% sequence identity to the nucleic acid sequence of a);

[0011] c) a nucleic acid that hybridizes with the complementary strand of the nucleic acid of a);

[0012] d) a nucleic acid that differs from a) by one or more nucleotides that are substituted, deleted, and/or inserted; or

[0013] e) a derivative of a), b), c), or d).

[0014] In one example, wherein in step (b) said nucleic acid has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identify.

[0015] In one example, wherein in step (c) said nucleic acid hybridizes with the complementary strand of the nucleic acid of a) under conditions of high stringency.

[0016] In one aspect there is provided a pharmaceutical compositions comprising an aptamer according to any one of claims 1 to 4, and pharmaceutically acceptable carrier.

[0017] In one aspect there is provided a method of treating a subject having, or suspected of having, an inflammatory disease, comprising: administering at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5.

[0018] In one example, wherein the inflammatory disease is Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, or Multiple Sclerosis

[0019] In one aspect there is provided a method of treating a subject having, or suspected of having, a Mincle-associated disease, comprising: administering at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5.

[0020] In one example, wherein the Mincle-associated disease is Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, or Multiple Sclerosis. [0021] In one example, wherein the route of administration, comprises: oral routes; gavage, topical routes, intranasal routes, vaginal routes, rectal routes, parenteral routes, intravenous routes, subcutaneous routes, intradermal routes, intramuscular routes, intraarticular routes, intrathecal routes, intraperitoneal routes, or topical routes. [0022] In one example, wherein said subject is a human.

[0023] In one aspect there is provided a use of at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5 for treating a subject having, or suspected of having, an inflammatory disease, comprising: administering at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5.

[0024] In one aspect there is provided a use of at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5, in the manufacture of medicament for treating a subject having, or suspected of having, an inflammatory disease, comprising: administering at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5.

[0025] In one example, wherein the inflammatory disease is Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, or Multiple Sclerosis

[0026] In one aspect there is provided a use of at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5 for treating a subject having, or suspected of having, a Mincle-associated disease.

[0027] In one aspect there is provided a use of at least one aptamer of any one of claims 1 to 4, or the pharmaceutical composition of claim 5 in the manufacture of a medicament for treating a subject having, or suspected of having, a Mincle-associated disease.

[0028] In one example, wherein the Mincle-associated disease is Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, or Multiple Sclerosis.

[0029] In one example, wherein the route of administration, comprises: oral routes; gavage, topical routes, intranasal routes, vaginal routes, rectal routes, parenteral routes, intravenous routes, subcutaneous routes, intradermal routes, intramuscular routes, intraarticular routes, intrathecal routes, intraperitoneal routes, or topical routes. [0030] In one example, wherein said subject is a human.

[0031] In one aspect there is provided a method of detecting Mincle polypeptide in a sample, comprising (a) contacting the sample with an aptamer of any one of claims 1 to 4 to allow Mincle polypeptide in the sample to bind to the aptamer, and (b) detecting the Mincle polypeptide bound to the aptamer.

[0032] In one aspect there is provided a method of purifying Mincle polypeptide from a sample, comprising (a) contacting the sample comprising Mincle with an aptamer of any one of claims 1 to 4 to allow Mincle polypeptide in the sample to bind to the aptamer, and (b) separating the Mincle polypeptide bound to the aptamer from the sample.

[0033] In one aspect there is provided a complex comprising an aptamer according to any one of claims 1 to 4 and a functional substance.

[0034] In one example, wherein the functional substance is a substance for labeling, an enzyme, a drug delivery vehicle or a drug.

BRIEF DESCRIPTION OF THE FIGURES

[0035] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0036] Fig. 1 : Characterization of the in vitro function of Aptamers with

Mincle affinity. A) Binding characteristics of AptMincle, and its modified counterparts, with rhMincle determined by ELONA. B) Graphical depiction of the modifications made to the aptamer sequences (Black - Original, White - Modified) C) The predicted secondary and tertiary structures and docking simulation of AptMincle ^CORE (Purple) with Mincle (White) (Box) highlighted predicted region of interaction surrounding a Calcium molecule. D) The predicted secondary and tertiary structures and docking simulation of AptMincle ^RND (Purple) with Mincle (White) (Box) highlighted predicted region of interaction surrounding a Calcium molecule. Dissociation constants were calculated on GraphPad Prism 8 using a non-linear regression binding analysis with assumed 1 -site target parameters.

[0037] Fig. 2: in vitro binding characteristics of aptamer-AptMincle, in comparison to antibody. A) Immunofluorescent imaging of the relative expression of pSyk ^Y525 and Mincle in unstimulated, TDB-stimulated (50μM), LPS-primed (10ng/ml - 24hrs), or LPS->TDB (10ng/ml LPS + 50μM) stimulated J774.1 macrophages (Left-right). B) Staining of a heterogenous population of control (Mincle ^low ) and LPS-primed (Mincle ^High ) J774.1 macrophages co-stained with anti-CLEC4E antibody (Invivogen, USA) and 5’-Cy3-conjugated AptMincle. C) Dose-dependent inhibition of Syk ^Y525 phosphorylation in J774.1 macrophages by anti-Mincle antibody (Invivogen) compared to AptMincle. D) Whole cell ELISA analysis of i) pSyk ^Y525 /Syk and ii) pP65/P65 relative expression in LPS-primed, TDB treated J774.1 macrophages when challenged with AptMincle (1 M). E) Whole blood assessment of AptMincle ^DRBL presence in mice via fully quantitative RT-PCR. IC50 was calculated on GraphPad Prism 8 using a non-linear fit Y=100/(1 +X/IC50). One-way Anova with Tukeys Posthoc tests were performed for multiple non-parametric data comparisons. ***P<0.001, ****P<0.0001.

[0038] Fig. 3: AptMincle ^DRBL suppresses DSS-induced colitis through a dose- dependent inhibition of colonic inflammation. A) Daily changes in disease activity score (DAI) over the onset of DSS-induced colitis B) MPO activity within colon C) Body weight percentage change at the end of the experiment D) Colon length and E) terminal DAI measured at Day 7 with relation to treatment groups with AptMincle ^DRBL (0.35, 1 or 3.5mg/kg) or AptMincle ^RND (3.5mg/kg). Immunoblot analysis of F) Syk or G) P65 phosphorylation of total colonic lysates. H) Representative photographs of colons from mice (DSS + AptMincle ^DRBL is 3.5mg/kg group only). These data are presented as mean ± SEM and were analysed by a repeated measures ANOVA with Tukeys post hoc test. Black asterisks indicate significance against sham group while red asterisks indicate significance compared to DSS group ***P<0.001 , ****P<0.0001. Mice are a composite of 10-week-old mice n=4-12, with experiments performed on separate days.

[0039] Fig. 4: The protective effect of AptMincle ^DRBL in DSS-induced colitis is sequence specific. A) Disease activity score (DAI) during the onset of DSS-induced colitis compared with treatment groups AptMincle ^{DRBL/RND/CORE/PORT} (3.5mg/kg) given at day 3, B) Presence of detectable AptMincle sequences within the serum of treated mice, C) overall body weight percentage change during the experiments, D) Colon length at termination, E) DAI measured at termination of day 7, F) MPO activity of colon of mice at day 7. G) 14-point histological assessment of terminal colon of groups and H) measured villi length of colon samples. These data are presented as mean ± SEM and were analysed by a repeated measures Anova with Tukeys post hoc test. Black stars indicated significance compared to sham control while red stars are significance against the DSS- group. **P<0.01 , ***P<0.001 , ****P<0.0001 . Mice are a composite of 10-week-old mice n=3, with experiments performed on separate days.

[0040] Fig. 5 - Clustal Omega sequence alignment and family analysis of most abundant anti-Mincle aptamer sequences. The 8 identified, highly recurrent aptamer sequences within the final round of SELEX are presented. A) Their sequence similarity and alignment determined by Clustal Omega. B) Their predicted family order delineating sequence similarity across species. [0041] Fig. 6 - Representative histological H&E section staining on colonic luminal cross-sections of DSS and treated mice. H&E staining and close ups of 10μM sections of colonic luminal cross sections in A) sham treatment (H ₂ 0), B) DSS (2.5% for 7 days), or C) DSS + AptMincle ^DRBL D) DSS + AptMincle ^CORE E) DSS + AptMincle ^PORT F) DSS + AptMincle ^RND (2.5% DSS + 3.5mg/kg AptMincle given I.P. on day 3). Scale bar = 50pm.

[0042] Fig. 7: TDB exacerbates DSS-colitis phenotype and repeated doses of AptMincle ^DRBL does not improve therapeutic terminal outcome in DSS mice compared with a single-dose. A) Disease activity index scoring (DAI) is not significantly improved by repeated dosage of AptMincle ^DRBL (DSS + DRBL 1dose/4dose) but is worsened significantly by addition of TDB gavage (1 mg/kg). B) MPO activity of AptMincle DSS mice does not improve with repeated doses. C-D) Body weight change of DSS mice with TDB is not significantly altered by TDB co-administration nor repeated doses of AptMincle ^DRBL . E) Terminal disease activity score is worsened by TDB co-administration with DSS treatment. Anova with Sidaks posthoc test **P<0.01 , ***P<0.001 , ****P<0.0001 . Black stars represent significance from Sham controls while red stars indicate significance against DSS groups. n=4-12.

DETAILED DESCRIPTION

[0043] In one aspect, there is described herein a Systematic Evolution of Ligands by Exponential enrichment (SELEX) method of producing one or more aptamers that binds to pattern recognition receptor Mincle (CLEC4E) polypeptide.

[0044] In some examples, the aptamers described herein may be useful for the treatment of a subject having, or suspect of having, an inflammatory disease.

[0045] As used herein, “inflammatory disease” refers to a disease or condition involving an inflammatory response. The inflammatory response may be acute and/or chronic. Non-limiting examples of inflammatory disease include, Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, and Multiple Sclerosis.

[0046] In some examples, the aptamers described herein may be useful for the treatment of a subject having, or suspect of having, a Mincle-associated disease. Nonlimiting examples of Mincle-associated diseases include, Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, and Multiple Sclerosis. [0047] Aptamers provide an attractive alternative to costly monoclonal antibody development. Highly specific, affordable, and modifiable, DNA aptamers can circumvent many issues associated with monoclonal antibody therapy. In addition to being non- immunogenic through simplistic modifications of size and structural integrity, aptamer delivery can be tailored to acute delivery and clearance from the biological system (occurring within hours), or protected from exonuclease action and escaping renal filtration through size modification (extending half-life to several days in vivo) ²⁷ . In this study, we developed, characterized and investigated an antagonistic Mincle-targeting ssDNA aptamer, and the protective effects within the chemically-induced mouse Dextran- Sodium Sulfate (DSS) model of UC.

[0048] The term "aptamer" as used herein refers to single-stranded nucleic acid molecules with secondary structures that facilitate high-affinity binding to a target molecule.

[0049] As used herein, "nucleic acid molecules" or "nucleic acid sequence" are interchangeable with the term "polynucleotide(s)" and it generally refers to any polyribonucleotide or poly- deoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or any combination thereof. As used herein, the term "nucleic acid(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "nucleic acids". The term "nucleic acids" as it is used herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including for example, simple and complex cells.

[0050] In a specific example, the aptamers herein comprise ssDNA.

[0051] In some examples, one or more nucleotides are modified at the base, sugar or backbone to make the nucleic acid more stable or less likely to be cleared e.g. phosphorothioate backbone, pegylated backbone.

[0052] A “target” molecule may be any compound upon which a nucleic acid can interact in a desired manner. For example, a target molecule may be a polypeptide, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, etc. A target also includes variations of a particular compound or molecule, such as, in the case of a polypeptide, for example, variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule.

[0053] In a specific example, the target is a polypeptide.

[0054] The term "polypeptide" refers to amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides may be isolated natural products, or may be produced partially or wholly using recombinant or synthetic techniques. Thus, the term “polypeptide” may also refer to “protein”.

[0055] The term "isolated" refers to sequences that are removed from their natural cellular or other naturally occurring biological environment or from the environment of the experiment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequences may be prepared by at least one purification step.

[0056] The term "purified" as used herein does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample.

[0057] In one example, the polypeptide is a Macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide. This may also be referred to as Mincle.

[0058] In one example, the polypeptide is a human Macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide.

[0059] In one example, the polypeptide is a mouse Macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide.

[0060] Selective Evolution of Ligands by Exponential enrichment (SELEX) refers to a process that combines the selection of nucleic acids that interact with a target in a desirable manner (e.g., binding to a protein) with the amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids that interact most strongly with the target from a pool that contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In some embodiments of the SELEX process, aptamers that bind non-covalently to their targets are generated. In other embodiments of the SELEX process, aptamers that bind covalently to their targets are generated.

[0061] The SELEX target is as described above and herein with respect to “target”. [0062] In one example, the SELEX target comprises or consists of a Macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide.

[0063] Methods of SELEX are known. A generalized SELEX method may be described as follow.

[0064] 1) A complex formation step in which a mixture of nucleic acids of differing sequence is prepared is contacted with a selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these conditions, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target complexes between the target and those nucleic acids having the strongest affinity for the target.

[0065] 2) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only a small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5 to 50%) are retained during partitioning.

[0066] 3) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.

[0067] 4) The partitioning and amplifying steps are repeated, and the newly formed candidate mixture contains fewer and fewer weakly binding sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.

[0068] Accordingly, a SELEX process may yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids sequences from the original candidate mixture which fold into a specific secondary and tertiary structure enabling the highest affinity interaction with the target molecule.

[0069] As described herein, there is provided a SELEX method of producing a nucleic acid aptamer that binds to a Macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide.

[0070] In one example, the nucleic acid aptamer may be a natural nucleic acid such as a DNA, an RNA, or a combination thereof. Also, the nucleic acid may partially or wholly comprise a non-natural nucleotide or a non-natural nucleic acid.

[0071] In a specific example, the nucleic acid is a DNA. Thus, in one example the aptamer is a ssDNA aptamer. [0072] The term “binds specifically” refers to high avidity and/or high affinity binding of an aptamer to a target, for example a macrophage inducible C-type lectin (CLEC4e/Mincle) polypeptide.

[0073] Aptamers which bind specifically to a target of interest may be capable of binding other polypeptides at weak, yet detectable, level. Such weak binding, or background binding, is readily discernable from the specific aptamer binding to the compound or polypeptide of interest, e.g., by use of appropriate controls, as would be known to the worker skilled in the art.

[0074] In one example, the following aptamers were identified using the methods described herein.

[0075] In one example, the “AptMincle” is MApT8, with the following sequence:

[0076] In one example, an aptamer further comprises a 5’ biotin-streptavidin, and 3’ iDT.

[0077] In one example, there is provided an aptamer referred to as AptMincle ^DRBL , with the following sequence:

[0078] 5’Biotin-

[0079] In another example, there is provided an aptamer with the following sequence:

[0080]

[0081] In one example there is provided an aptamer that binds to Mincle, comprising:

[0082] a) a nucleic acid comprising or consisting of any one of [0083] b) a nucleic acid having at least 70% sequence identity to the nucleic acid sequence of a);

[0084] c) a nucleic acid that hybridizes with the complementary strand of the nucleic acid of a);

[0085] d) a nucleic acid that differs from a) by one or more nucleotides that are substituted, deleted, and/or inserted; or

[0086] e) a derivative of a), b), c), or d).

[0087] In one example, wherein in step (b) said nucleic acid has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identify.

[0088] In another example, the aptamer is a “fragment” of one or more of the aptamers AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2); SEQ ID NO: 3; MApT7 (SEQ ID NO: 4), MApT1 (SEQ ID NO: 5), MApT4 (SEQ ID NO: 6), MApT5 (SEQ ID NO: 7), MApT6 (SEQ ID NO: 8), MApT2 (SEQ ID NO: 9), MApT3 (SEQ ID NO: 10), or MApT8 (SEQ ID NO: 1);

[0089] A “fragment” of the aptamer sequence may comprise a subsequence that binds to the same target as the full length sequence.

[0090] In one example, the aptamer is a derivative of one or more of the aptamers AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2); SEQ ID NO: 3; MApT7 (SEQ ID NO: 4), MApT1 (SEQ ID NO: 5), MApT4 (SEQ ID NO: 6), MApT5 (SEQ ID NO: 7), MApT6 (SEQ ID NO: 8), MApT2 (SEQ ID NO: 9), MApT3 (SEQ ID NO: 10), or MApT8 (SEQ ID NO: 1).

[0091] The term “derivative” as used herein refers to an aptamer which has a chemical structure which does not occur in natural DNA or RNA.

[0092] The nucleotide sequences of aptamers identified using the methods described herein, can be modified. In some examples, the aptamer contains one or more chemical modifications. The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.

[0093] In some examples, there is provided an aptamer which is derived from an aptamer comprising or consisting of one or more of AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2); SQE ID NO: 3, MApT7 (SEQ ID NO: 4), MApT1 (SEQ ID NO: 5), MApT4 (SEQ ID NO:6), MApT5 (SEQ ID NO: 7), MApT6 (SEQ ID NO: 8), MApT2 (SEQ ID NO: 9), MApT3 (SEQ ID NO: 10), or MApT8 (SEQ ID NO: 1), such that in one of the sequences one or nucleotides are substituted, removed (deleted), added within the sequence (inserted) and/or added at the 5' end and/or 3' end.

[0094] The term "sequence identity" as used herein, refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be 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, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, IIe, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) 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.

[0095] In some example, the aptamer has a nucleotide sequence that is at least 70% identical to the nucleotide sequence of one or more of AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2), SEQ ID NO: 3, MApT7, MApT1 , MApT4, MApT5, MApT6, MApT2, MApT3, or MApT8, wherein such an aptamer binds to Macrophage inducible C-type lectin (CLEC4e/Mincle). In one example, the sequence identity is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0096] In some examples, there is provided an aptamer that hybridizes with a nucleotide sequence of one or more of Macrophage inducible C-type lectin (CLEC4e/Mincle), or to their complementary strand, under stringency conditions described below.

[0097] As used herein, the term “hybridizes under low stringency, medium stringency, or high stringency” describes conditions for hybridization and washing. Aqueous and non-aqueous methods can be used.

[0098] The aptamers described herein may be used to assess presence or level of Mincle polypeptide in a biological sample, and/or to isolate Mincle polypeptide from a biological sample.

[0099] The term “sample” or "biological sample," as used herein, refers to any cell, body tissue, or body fluid sample obtained from a subject. Non-limiting examples of body fluids include, for example, lymph, saliva, blood, urine, and the like. Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods including, but not limited to, tissue biopsy, including punch biopsy and cell scraping, needle biopsy; or collection of blood or other bodily fluids by aspiration or other suitable methods.

[00100] In one example, said determination is in vivo determination.

[00101] In one example, said determination is in vitro determination.

[00102] As used herein “in vivo" refers to events that occur within a subject, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[00103] As used herein “in vitro" refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in cell culture, etc., rather than within a subject.

[00104] In one example, there is described a method for detecting Mincle polypeptide in a biological sample, comprising: contacting said sample with an aptamer of any one of AptMincle(SEQ ID NO: 1), AptMincleDRBL (SEQ ID NO: 2), SEQ ID NO: 3, MApT7 (SEQ ID NO: 4), MApT1 (SEQ ID NO: 5), MApT4 (SEQ ID NO: 6), MApT5 (SEQ ID NO: 7), MApT6 (SEQ ID NO: 8), MApT2 (SEQ ID NO: 9), MApT3 (SEQ ID NO: 10), or MApT8 (SEQ ID NO: 1), said aptamer comprising a detectable label, and determining whether an increase in the detectable label, wherein an increase in the detectable label is indicative of the presence of Mincle polypeptide in the sample.

[00105] The term “detectable label” as used herein refers to a chemical moiety that is directly or indirectly detectable (for example, due to its spectral properties, conformation or activity) when attached to a target or compound and used in the present methods, including reporter molecules, solid supports and carrier molecules. The detectable label can be directly detectable (fluorophore) or indirectly detectable (hapten or enzyme).

[00106] Detectable labels include, but are not limited to, radiolabels that can be measured with radiation-counting devices; pigments, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example. The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate. Further, MRI contrast agent labelled aptamer, or CT agent labelled aptamer, may be used.

[00107] The term detectable label can also refer to a "tag" or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, one can use biotin as a tag and then use an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent to detect the presence of HRP. Labels are known by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their colorimetric, fluorogenic and chemiluminescent substrates and other labels.

[00108] In some examples, Mincle may be detected in a biological sample using fluorescence, immunofluorescence, or histological staining.

[00109] In some examples, there is provided a method of detecting Mincle, comprising (a) contacting a sample with an aptamer as described herein, to allow Mincle polypeptide in the sample to bind to the aptamer, and (b) detecting the Mincle polypeptide bound to the aptamer.

[00110] In some examples, there is provided a method of purifying Mincle polypeptide, comprising (a) contacting a sample comprising Mincle polypeptide with an aptamer as described herein to allow Mincle in the sample to bind to the aptamer, and (b) separating the Mincle polypeptide bound to the aptamer from the sample.

[00111] In some examples, there is provided a complex comprising an aptamer as described herein, and a functional substance.

[00112] In some examples, the functional substance is a substance for labeling, an enzyme, a drug delivery vehicle or a drug.

[00113] The aptamers described herein may be useful for the treatment of a subject having, or suspect of having, an inflammatory disease.

[00114] As used herein, “inflammatory disease” refers to a disease or condition involving an inflammatory response. The inflammatory response may be acute and/or chronic. Non-limiting examples of inflammatory disease include, Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, and Multiple Sclerosis.

[00115] The aptamers described herein may be useful for the treatment of a subject having, or suspect of having, a Mincle-associated disease. Non-limiting examples of Mincle-associated disease include, Inflammatory Bowel Disease (IBD), Crohn’s disease, Colitis, Psoriasis, Leishmaniasis, Melanoma, and Multiple Sclerosis.

[00116] The term “subject” or “patient” as used herein, refers to any mammal or non-mammal that would benefit from determining the benefit from treatment, diagnosis, therapeutic monitoring and/or prognosis. In certain examples a subject or patient includes, but is not limited to, humans, farm animals, companion animals (such as cats, dogs and horses), primates and rodent (such as mice and rats). In a specific embodiment, the subject is a human. In an additional specific embodiment, the subject is female. In another example the subject is male.

[00117] The term “treat” or “treatment” as used herein, refers to clinical intervention in an attempt to alter the course of the subject or cell being treated. In non-limiting examples, treatment includes preventing or delaying recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[00118] It will be appreciated that in some examples, an aptamer is administered to a subject. In one example, a pharmaceutical composition comprising an aptamer is administered to a subject. A pharmaceutical composition may comprise an aptamer and a pharmaceutically acceptable excipient. In some examples a pharmaceutical composition comprising an aptamer as described herein may be used.

[00119] Depending on the intended mode of administration, the compositions used may be in the form of solid, semi-solid or liquid dosage forms, such, as for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, or the like, preferably in unit dosage forms suitable for single administration of precise dosages.

[00120] The phrase "pharmaceutically acceptable" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use. Each carrier must be "acceptable" in the sense of being compatible with other ingredients of the formulation and not unacceptably injurious to the patient.

[00121] In some examples, the pharmaceutically acceptable carrier is inert to the nucleic acid, which has no detrimental side effects or toxicity under the conditions of use.

[00122] In some examples, injectable formulations for parenteral administration may be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, but are not limited to, water, saline, dextrose, glycerol, ethanol or the like.

[00123] The aptamers may be administered by numerous routes of administration. Such routes of administration include, but are not limited to, oral routes; gavage, topical routes, such as intranasally, vaginally or rectally; and parenteral routes, such as intravenous, subcutaneous, intradermal, intramuscular, intraarticular and intrathecal administration, intraperitoneal, and topical administration. Suitable routes of administration may also be used in combination, such as intravenous administration followed by subcutaneous administration.

[00124] Oral dosage forms may be administered as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups or emulsions.

[00125] Topical dosage forms include creams, ointments, lotions, aerosol sprays and gels for intranasal vehicles, inhalants or transdermal patches.

[00126] Parenteral dosage forms include pre-filled syringes, and solutions and lyophilized powders that are reconstituted prior to administration.

[00127] Method of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[00128] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[00129] EXAMPLES

[00130] Abstract

[00131] Pattern recognition receptors such as Mincle (Clec4e), play a significant role in the regulation of inflammation. Enhanced signaling of Mincle through the release of damage associated molecular patterns during sterile inflammation has been shown to be important in the progression and manifestation of several diseases. A limitation to Mincle- targeted therapeutics is the feasibility of human-scale antibody therapy and the lack of alternate small-molecule inhibitors. Herein, we describe a highly specific neutralizing DNA-aptamer targeting Mincle and demonstrate its therapeutic potential. Our data demonstrates that AptMincle selective high-affinity binding to both human and mouse Mincle and is able to directly target and reduce Mincle activation. AptMincle can specifically reduce TDB-induced Syk and P65 phosphorylation in vitro in manner comparable to the commercially available neutralizing antibody in vitro. Moreover, a biostable modified, AptMincle ^DRBL was successful in reducing disease activity in the DSS- induced model of ulcerative colitis in a dose- and sequence-dependent manner. The results present an alternative, highly specific DNA aptamer with antagonistic function for use in the investigation of Mincle-associated diseases. The data also shows the translational potential for Mincle-targeting aptamers as a new category of biologic therapy in the treatment of IBD.

[00132] Results

[00133] Generation of Mincle specific DNA Aptamers

[00134] The full length amino-acid sequence of human and mouse Mincle are 65% identical (Uniprot codes CLC4E_Mouse Q9R0Q8, CLC4E_Human Q9ULY5). The recombinant fragment of extracellular Mincle used within this project, contains 85% overall amino-acid sequence identity. Given that functional studies are typically carried out in murine models, we examined whether a Human Mincle-targeting ssDNA aptamer, could function in multiple species while retaining immediate translational potential into human systems in the future.

[00135] Human Mincle-targeting aptamers were identified using nitrocellulose filter SELEX (Detailed in methods). The cloned and sequenced aptamers and recurrent sequences were analysed using Clustal Omega alignment algorithms and categorized into predicted familial groups based on their homology (Fig. 5). In total, 8 highly abundant sequences (72% of the sequenced community) were taken forward for in silico structural predictions and protein interaction modelling. Identification and validation of one of these sequences, herein referred to as AptMincle, was validated for specific binding to Mincle. AptMincle was synthesized with a 5’-biotin and 3’-inverted dT, this modified sequence is referred to as AptMincle ^DRBL , and using a modified ELONA protocol its binding efficiency was quantified (K _D =1.722nM). Through comparative-analysis and replacement of key portions of the sequence, it was determined that the initially random, n=40, region at the core of the sequence (AptMincle ^CORE - K _d =1.512nM) was responsible for its binding affinity towards Mincle. It was only though randomizing that core region (AptMincle ^RND - K _d = N/A), that was functionality lost. Replacement of the flanking primer sequences (AptMincle ^PORT K _D =1.376nM) had no discernible negative impact on binding affinity toward Mincle (Fig. 1A&B).

[00136] Secondary structures, predicted in silico by Vienna Webfold revealed that AptMincle ^CORE forms a long-stem stable hairpin structure. In silico 3D-structure predictions of AptMincle ^CORE in RNAcomposer, were combined to create docking simulations to the crystal structure of human Mincle (PDB identifier-3WH3). To note, as there is an absence of modelling software for the prediction of ssDNA tertiary structures, we have assumed the sequence can be modelled as ssRNA. The generated interactions suggested that the short hairpin structure of the core sequence of AptMincle ^CORE (40 bases in length) specifically nucleotides 18-30, potentially interact with Mincle in regions near to calcium binding domains suggesting a possible site of interference through by allosteric hindrance (amino acids Ser90-Val152) a highly conserved region between Human and Mouse Mincle (93.5% homology) (Fig. 1C). To further support the prediction, the n=40 scrambled oligonucleotide sequence of AptMincle ^RND was modelled in the same manner and was poorly predicted to bind to a non-descript region of the extracellular domain fragment (Fig. 1 D).

[00137] Functionality assessment of AptMincle in vitro

[00138] As an inducible PRR, Mincle is expressed at low levels in unstimulated macrophages (Fig. 2A - control). However, this expression can be greatly increased via overnight stimulation with the TLR4 agonist LPS (Fig. 2A - LPS). Within LPS-primed J774.1 macrophages (Mincle ^High ), stimulation with a ligand of Mincle, Trehalose-6,6- dibehenate (TDB), induces a robust phosphorylation of the Mincle adapter, Syk ^Y525 (Fig. 2A - pSyk). This phosphorylation is not found in control cells stimulated with TDB only (Fig. 2A - TDB alone). This LPS-induced increase in Mincle protein expression can be detected in the primed cells using commercially available monoclonal antibody immunostaining (Fig. 1A - Mincle). Combining two suspensions of J774.1 macrophages, one primed with LPS (Mincle ^High ) the other untreated (Mincle ^Low ), we formed a heterogenous population of Mincle-expressing cells. Immunofluorescence staining revealed that both the antibody, and a 5’-Cy3-conjugated AptMincle, could detected and discriminate between these cell populations, staining the primed murine lymphocytes in a highly conserved pattern (Fig. 2B). Determining that AptMincle specifically bound Mincle in vitro, we aimed to determine whether it could impede phosphorylation of Mincle adapter Syk ^Y525 in an antagonistic/blocking manner as structurally predicted in Fig. 1D. Phosphorylation of Syk ^Y525 in LPS-primed and TDB stimulated J774 macrophages could be effectively neutralized in a dose-dependent manner using both a commercially available Mincle-neutralizing antibody and the AptMincle sequence, from which the IC50 of each was calculated (Fig. 2C). The IC50 of AptMincle (IC50 - 0.05853pg/ml) was shown to be almost ten-times less than that of the commercial antibody (IC50 - 0.5274pg/ml) within this system. AptMincle displays continued downstream antagonistic activity toward murine Mincle, confirmed through analysis of both the phosphorylation of the Mincle adapter Syk and NFKB subunit P65 phosphorylation measured in TDB stimulated cells by whole-cell lysate direct ELISA (Fig. 2Di-ii). Highly abundant in exonucleases, the blood of mice, or humans, would rapidly degrade naked DNA sequence to prevent aberrant immune responses through other PRRs such as TLR9. This effect can be compounded in inflammatory settings, where serum and intracellular exonucleases can be altered in expression and function. Furthermore, the body’s natural filtration system, the nephron based renal exclusion system, has been previously shown to exclude unmodified aptamers due their relatively low molecular weight (<25kDa) ²⁸ . In an attempt to combat these eventualities, two modifications were added to protect the sequences. An inverted deoxythmidine base (iDT) was added at the 3’ terminus of the sequence, preventing degradation by 3’ exonucleases while simultaneously prevent extension by DNA polymerases ²⁹ . This significantly improved stability in isolated serum however, the viability within the host was only improved to between 24-48hrs, as the excess was likely filtered and excreted in the urine. The latter modification, an additive 5’ biotin, allowed for the direct conjugation of a variety of streptavidin-linked moieties (Fig. 2E). In this project we also wanted to increase the molecular weight of the sequence to improve its bioavailability by reducing renal filtration. This was achieved through addition of a 5’-streptavidin linker which increased AptMincle’s molecular weight to ~75kDa. In combination with the 3’ iDT, the 5’ streptavidin improved the bioavailability of AptMincle in vivo past 4 days (confirmed by quantitative analysis of AptMincle in whole blood). This data suggests that AptMincle significantly depletes endogenous TDB-induced Mincle Syk and P65 phosphorylation within macrophages (Fig. 2C &D) and that synthesis with 3’ iDT and biotin-streptavidin 5’ modification protect it from in vitro degradation. The aptamer will herein be referred to as AptMincle ^DRBL and used as the primary aptamer sequence for in vivo investigation.

[00139] AptMincle ^DRBL demonstrates dose-dependent protective effect in DSS-induced colitis

[00140] Having validated the AptMincle sequence and improved its stability in vivo, we aimed to test the therapeutic potential of the sequence in a characterized disease model previously shown to be regulated in part by Mincle ⁵ . We used the chemically induced Dextran-sodium sulfate (DSS) model of ulcerative colitis, resulting in an acute inflammation of the gastrointestinal tract, manifesting primarily within the colon and reproducing many of the pathological signs of human UC, such as; weight loss, diarrhoea, intestinal bleeding, colon shortening and submucosal inflammation ⁴³⁰³¹ . Previously, we have highlighted that the earliest DSS-induced colitis macroscopically displays within the 7-days DSS treatment, is day 3, whereby fecal consistency and weight loss is prominent. Therefore, at day 3 a single dose of AptMincle ^DRBL was injected intraperitoneally to mice (0.35, 1 , 3.5mg/kg) in a therapeutic-intervention rather than prophylactically. This treatment method is in line with our previously published data of the protective effect of TLR4 inhibition in the same model ⁴ . The data shows a protective effect of AptMincle ^DRBL , which was achieved in a dose dependent manner. Disease activity index (DAI) was significantly improved following the aptamer administration (Day 4, Fig. 3A - Black arrow). At the termination of the experiment (day 7), colonic inflammation, indicated by myeloperoxidase activity (MPO) was significantly reduced with AptMincle ^DRBL treatment (MPO reduced by: 0.35mg/mg = 37.2%, 1 mg/kg = 52.3%, 3.5mg/kg = 63.5%) (Fig. 3B). Loss of body weight, found accompanying DSS-treatment, was also significantly reduced at the highest dose of AptMincle ^DRBL (Fig. 3C, 3.5mg/kg, ANOVA P=0.0002) and was tending to significance at the lower doses. Interestingly, at all therapeutic concentrations of AptMincle ^DRBL , colonic shortening (an indicator of intestinal inflammation and fibrosis) was completely resolved (Fig. 3D & H, P<0.0001). All AptMincle ^DRBL treatment groups, regardless of dose, showed a significant resolution of day 7 DSS-associated DAI (Fig. 3E, P<0.0001) while the random-sequence oligonucleotide control, AptMincle ^RND had no protective effect in any assessed metric (Fig. 3E). Analysis of colon sample lysates using western-blot and band densitometry analysis, showed a trend toward a reduction of DSS-induced total Syk (P=0.068) and P65 phosphorylation within the tissue in mice treated with the highest dose of AptMincle ^DRBL (Fig. 3F & G). Finally, DSS-induced colon changes, as well as alterations of the luminal content of the colon, can be seen in representative examples of harvested tissue (Fig. 3H). Overall, these data highlight the in vivo protective effect of AptMincle ^DRBL at multiple therapeutic doses and that through the concurrent assessment and use of the AptMincle ^RND control sequence, we show the nucleotide sequence specificity of the aptamer core region and the gross anti-inflammatory action created by the active sequence.

[00141] AptMincle’s bio-protective effect in DSS-induced colitis is sequence specific

[00142] As shown throughout these data, it appeared the core oligonucleotide sequence of the AptMincle is solely responsible for the aptamer’s binding function in vitro. In an attempt to confirm whether the protective effect of AptMincle ^DRBL in vivo was too sequence specific, we evaluated the potential of the alternate sequences: AptMincle ^PORT (Alternative flanking primers but with conserved core n=40 sequence ³² ), AptMincle ^CORE (removal of flanking primers, overall having a reduced molecular weight) and AptMincle ^RND (scrambled n=40 core region, but with conserved flanking primers). As presented in Fig. 4A, by day 7, the single ‘high-dose’ (3.5mg/kg) of AptMincle ^DRBL or AptMincle ^PORT had a significant effect in attenuating the disease activity Index compared to DSS controls (ANOVA, P<0.0001). while AptMincle ^RND had no discernible effect (ANOVA, P=0.705). Interestingly, AptMincle ^CORE initially showed a potent preventative effect upon DAI in mice (Day 4, ANOVA, P=0.003), but was rapidly lost at day 6 (ANOVA, P=0.292) 3 days after injection. This correlated to the sudden loss of detectable AptMincle ^CORE within the serum of the treated mice determined by fully quantitative RT- PCR (Fig. 4B). We theorize that while the 3’ iDT present on the AptMincle ^CORE sequence shielded it from degradation within the host, the drastic reduction in molecular weight meant it fell short of the renal filtration threshold and was likely excreted from the system in the urine. Histological assessment of the colon of mice demonstrated that during DSS treatment there was significant: leukocyte infiltrate, goblet cell loss, crypt density & hyperplasia, muscle thickening and submucosal infiltrate compared to healthy controls with a loss of villi length, as detailed in ³³ . The histology scoring and villus length was shown to significantly improve in the DSS+AptMincle ^DRBL and DSS+AptMincle ^PORT groups compared to DSS counterparts (Fig. 4G&H and Fig. 6). The results compound the evidence that the size (molecular weight) and nucleotide sequence of AptMincle is important for its longevity and action respectively in vivo.

[00143] Discussion

[00144] In the current study, we have developed, characterized, and validated a DNA aptamer targeting the pattern recognition receptor Mincle (CLEC4E). Termed AptMincle, this oligonucleotide aptamer demonstrates specific binding and antagonistic activity toward both human and murine Mincle and subsequently, has a protective effect against experimental colitis in mice, a setting where Mincle has been previously demonstrated to play a crucial role in the maintenance of disease ⁵ . We first identified 8- highly enriched aptamer sequences that bound recombinant human Mincle, utilizing a modified nitrocellulose-based SELEX protocol (fig. 5). This protocol selected a community of putative Mincle-specific: agonistic; neutral; or antagonistic aptamers targeting a recombinant fragment of the extracellular domain of human Mincle (Accession number Q9ULY5, amino acids 41-219). In silico structural prediction using Vienna Webfold server suggested AptMincle formed a tight hair-pin structure specifically at its core region (n=40). Due to the lack of tertiary structure modelling software for ssDNA we have assumed the aptamer can be modelled as RNA. RNAcomposer successfully modelled AptMincle ^CORE (Fig. 1 C) ³⁴ . For subsequent protein-RNA association modelling, we combined the PDB crystal structure of Mincle (PDB: 3wh3) with the predicted tertiary structure of RNA- AptMincle ^CORE using PatchDock ³⁵³⁶ . It was noted that the proposed 3D model suggested a likelihood that AptMincle ^CORE bound to Mincle nearby calcium binding regions and proposed potential antagonistic properties of the aptamer sequence. As aptamers bind to proteins based on van der Waals, hydrogen bonding, and electrostatic interactions, it was promising to note that the confirmational structure of human and mouse Mincle was highly conserved. Using the alphafold structural prediction of murine Mincle (as no crystal structure exists as of the time writing this paper) we aligned the sequences using Pymol2.0. The predicted Root Mean Square Deviation (RSMD) was <1A (0.612). The authors highlight and acknowledge that this data is speculative and is provided for reference purposes only. Structural binding of AptMincle to Mincle must be performed in the future to support these claims.

[00145] When evaluated, the binding of AptMincle to recombinant Mincle was determined to be specific to the core 40-nucleotide (initially variable) region. This specificity was confirmed through post-SELEX truncation of the sequence removing the flanking primers (AptMincle ^CORE ), substituting the primer sequences for alternatives (AptMincle ^PORT ), or a scrambled control (AptMincle ^RND ). It was noted that only the AptMincle ^RND lost its binding capacity to rhMincle, while the AptMincle ^CORE and AptMincle ^PORT sequences-maintained function (Fig. 1). As previously mentioned, one of the main project goals was to develop an antibody alternative for targeting Mincle. Comparative analysis and use of commercially available anti-Mincle antibodies, allowed us to demonstrate similar, if not better functionality in neutralization assays and immunofluorescent imaging (Fig. 2). Based on our data the IC50 of AptMincle is significantly less than that of the MAb suggesting a much more powerful mechanism of inhibition. However, we accredit this to the aptamer’s vastly smaller molecular weight (~25kDa) and therefore a ~6x abundance of molecules within mol-mol equivalents (1 g/ml AptMincle = 40nM = 240x10 ¹⁴ molecules, while 1 g/ml MAb = 6.67nM = 40x10 ¹⁴ molecules). As AptMincle was able to be successfully used in an enzyme linked oligonucleotide assay (ELONA) and immunofluorescent (IF) imaging, it is feasible that the sequence can be used for all techniques where the confirmational structure of Mincle is maintained (Non-denaturing PAGE, Immunoprecipitation, Flow cytometry etc). These applications will require validation in the future. Serum exonucleases, combined with renal filtration and excretion, commonly results in rapid degradation and removal of oligonucleotides from the system ³⁷³⁸ . Thankfully, due to their highly-modifiable nature, the half-life of nucleic acid aptamers within a system can be readily and easily altered. We wanted AptMincle to be stable within the host to inhibit Mincle-induced pro- inflammatory responses during inflammation. However, the unmodified oligonucleotide was rapidly degraded in isolated serum and undetectable after 30 minutes exposure at room temperature (data not shown). In an attempt to circumvent these issues, modifications to the 5’ and 3’ ends of the AptMincle sequences were made to a) inhibit exonuclease digestion and b) limit renal secretion. Both of these modifications were made during the sequence synthesis process and allowed for flexible modification of the sequence for specific application (Fig. 2-4). The inclusion of an inverted dT on the 3’ end of the sequence alone, improved stability of AptMincle within the serum, but was still noted to be depleted in vivo, likely due to size exclusion during nephron filtration within the kidneys (Fig. 2 and 4). The addition of the 5’ biotin moiety and subsequent streptavidin conjugation, increased the molecular weight of AptMincle to ~50-75kDa, far exceeding the filtration threshold ²⁷ . This modification in combination with its exonuclease resistance allowed the aptamer to persist within the system for in excess of 4 days (Fig. 4). It is also important to discuss that without these modifications, any Aptamer’s biological function would likely be reduced to short (<1 hour) bursts following administration, a strategy that could aid in preventing long term or off-target cellular toxicities, although no such eventualities have currently been identified. Previous work has implicated Mincle in the pathogenesis of several diseases including models of ulcerative colitis ⁵ . We therefore wanted to validate the protective effect of AptMincle within the same model, murine DSS-induced colitis. The addition of the 5’ biotinstreptavidin and 3’ iDT allowed for treatment to consist of a single dose of AptMincle ^DRBL (0.5, 1 , 3.5mg/kg) due to minimal clearance from the host and no additional benefit with multiple doses (Fig. 7). Initial experimentation showed a clear dose-dependent therapeutic response of the aptamer with significant reduction in several disease markers (Fig. 5) with the data demonstrating a significant reduction in disease activity index, weight loss, colonic shortening, and colonic MPO. Interestingly, disease activity scoring which is a combination of 3 distinct parameters (weight loss, stool consistency & occult rectal bleeding) indicated that a majority of the reduction was due to improvements in weight and stool consistency but did not affect the presence of blood in the feces suggesting a Mincle-independent role of vascular leakage and rectal bleeding.

Furthermore, the significant reduction in DSS-induced colonic MPO, improved histological scoring and colon length, suggesting the primary mechanism of action is through the prevention of a Mincle-driven inflammatory response. In an attempt to further support the validity of the AptMincle sequence in vivo, multiple versions of the sequence were created. Previous data had shown that the CORE nucleotide sequence was solely responsible for binding to recombinant Mincle in vitro. We therefore assessed the biological impact of these alterations by comparing the protective capacity of each sequence to the DSS-colitis model. Data shows that AptMincle ^DRBL and AptMincle ^PORT (alternative flanking sequences) were equally effective in reducing severity of disease activity in treated mice. Interestingly, AptMincle ^CORE had a potent effect during the early stages of pathology, negating all external markers of disease activity until day 5 at which time all mice rapidly returned to similar DSS-control values. We predict that this phenomenon is driven by two intrinsic mechanisms. Firstly, as we treated mice with 3.5mg/kg of each modified Aptamer, it is possible the reduced size of the AptMincle ^CORE (MW 12451.10 Da/28.1 μM) vs AptMincle ^DRBL7PORT (23656.36 Da/14.8μM & 24354.87 Da/14.4μM, respectively) increased the overall concentration of the therapy by up to 2- fold. Combined with the previously determined half-life of the unmodified AptMincle sequence in vivo, we can hypothesise these are the key reasons the protective effect of AptMincle ^CORE is transient. Importantly, the efficacy of AptMincle in a model of ulcerative colitis presents and supports the use of this newly developed aptamer in patients unresponsive to conventional anti-inflammatory therapeutics. The absence of toxicity and tractable modifications made to the sequence make it an encouraging candidate for further clinical assessment in IBD and other Mincle-related diseases. While sequence homology between human and mouse Mincle is calculated at 65% (Uniprot alignment codes Q9ULY5/Q9R0Q8) the evidence presented here suggests a conserved activity of AptMincle on murine Mincle despite being raised against a recombinant human protein. Furthermore, AptMincle did not bind significantly to BSA, mouse serum components or recombinant CLEC4D (MCL) despite the latter sharing 35% sequence homology with CLEC4E (Mincle) and a common ligand TDB, further validating the specific targeting of Mincle by AptMincle. Other aptamers against pattern recognition receptors have been developed showing protective effects in disease relevant animal models ³⁹⁴⁰ . Rather than targeting secreted, serum or tissue born cytokines, as is the case with conventional antibody-based biologic therapies, it is proposed that the targeting of aberrant PRR activity is a more effective way to treat the inflammation at the causative source. The aptameric-targeting of inducible PRRs like Mincle however, has never been attempted. AptMincle’s development creates a novel avenue for intervention-medicine whereby the uncontrolled and sustained inflammatory response can be dampened, increasing the change of tissue regeneration and disease resolution. AptMincle, presented here, provides a tool for the investigation and treatment of Mincle-driven diseases including ulcerative colitis. Before the aptamer can be used in human settings, additional tests and validation methodologies will need to be performed in animal models characterizing biodistribution and long-term immunogenicity responses. Once fully validated, we envision that AptMincle be assessed for translational potential into human for the treatment of ulcerative colitis. Furthermore, investigation into the therapeutic benefits in mouse models of Crohn’s disease, psoriasis and multiple sclerosis are underway, diseases all shown to be exacerbated, or driven in part by Mincle. With limited effective treatments in all of the aforementioned disease groups, the addition of a new therapeutic is greatly needed. Furthermore, the stability, cost effectiveness, and low immunogenicity associated with nucleic acid-based aptamers, compared to current antibody-biologic therapies, provide an appealing alternative to both clinicians and patients.

[00146] Materials & Methods

[00147] Ethics Committee Approval and mouse use

[00148] All mice were housed at constant temperature (22°C) on a 12: 12-h lightdark cycle, with food and water ad libitum. Both male and female mice were used at 8-10 weeks of age. The animal handling and experiments were approved by the University of Calgary Animal Care and Ethics Committee and conformed to the guidelines established by the Canadian Council on Animal Care (Protocol Number AC17-0186).

[00149] Chemicals and oligonucleotides:

[00150] All oligonucleotides were synthesized by Integrated DNA technologies, Inc (Coralville, Iowa, USA). The sequences of all ssDNA anti-Mincle aptamers created by this project can be found in Fig. 1. All chemicals were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada) unless otherwise stated.

[00151] Aptamer selection protocol (SELEX)

[00152] Mincle-specific ssDNA aptamers were isolated and identified using nitrocellulose filter SELEX. The synthetic library of ssDNA was composed of 76- nucleotide-long, single stranded DNAs with a random region of 40 bases (RND40) flanked by conserved primer sequences.

[00153]

[00154] In the first round of the SELEX procedure, 1 nmol (6.022x10 ¹⁴ individual sequences) of RND40 population (1 Opl of stock 100μM) was added to 90pl of SELEX buffer (20 mM TrisHCI, pH 7.4; 150 mM NaCI; 1 mM MgCI2), was denatured at 90°C for 10 min, and then cooled on ice for 10 min. These aptamers are then termed “folded”. The folded RND pool was then mixed with 1 pg (14 pmol) of rhMincle (ab152015, Abeam, Toronto, ON, Canada) (reconstituted in phenol red free DMEM + 10% FCS) in 90 pl of SELEX buffer (200pl total) and incubated at 37°C for 1 hr (rounds 1-3) or 30 min (rounds 4-6).

[00155] The bound aptamer-rhMincle complexes were purified using nitrocellulose adherence as previously described ⁴¹ and, after washing three times with SELEX buffer, the ssDNA-protein complexes were boiled off of the membrane in 200 l of distilled H ₂ O and amplified by PCR. Simply, 20pl of the eluted aptamers from the previous round were amplified for 15-25 cycles using F’ and R’ primers (Tablet) in complete 2x PCR buffer under the conditions of 1 μM/primer, 250 μM dNTPs, in a final volume of 100 pl as above. Counterselection steps were performed before the initial round and after the 3rd and 6th selection rounds using the reconstitution media (DMEM + 10% FCS) and the nitrocellulose membrane as the non-specific targets. All aptamers that bound to the membrane at these points were discarded and the unbound fraction (Mincle-targeted) were retained. The PCR amplifications performed at the end of each round of selection showed increased amounts of DNA product after the 15 cycles but they did not reach saturation consistent with the molar-ratio of Mincle protein target used consistently throughout the process.

[00156] Primers used in serum analysis of aptamer

[00157] Detection of AptMincle within the blood was analysed using fully quantitative qPCR with an 8-part AptMincle standard curve. Briefly, 1 ul of collected whole blood from mice was used as a template for RT-PCR and quantified relative to the standard curve. Primers used to detect:

[00158]

[00159] Amplification protocol consisted of an initial denaturation step at 98°C, followed by 30 cycles of; 95°C for 30s, 55-59°C for 15s, 72°C for 30s, final extension 72°C for 5 mins. Amplicon specificity was determined by agarose gel electrophoresis.

[00160] Educated aptamer pool cloning and sequencing

[00161] The final pool of putative aptamer sequences was cloned into pJET1.2 blunt cloning vectors using the CloneJet PCR cloning kit (ThermoFisher Scientific, Burlington, ON, Canada). Successfully transformed DH10B competent E.coliwere grown on ampicillin (100ug/ml) LB selection plates after which colonies were picked and plasmids sequenced using pJET1.2 Forward and Reverse sequencing primers using the Sanger Sequencing Platform at the University of Calgary’s Centre for Health Genomics and Informatics Facility. Aptamer flanking primer regions were identified by hand and sequences compiled.

[00162] Aptamer modification

[00163] AptMincle (with biotin modification at 5’ end) was incubated in a 1 :4 molar ratio with streptavidin (Biolegend, San Diego, CA, USA) in SELEX buffer for 1 hr at 37°C. Confirmation of successful conjugation was confirmed by aptamer band retardation assessment within a 2.5% agarose gel.

[00164] Binding affinity determination (ELONA)

[00165] Recombinant human Mincle (0, 1 , 3.33, 10, 33.3, 100nM) was bound to an ELISA capture plate before being probed with 5’biotin-AptMincle (100nM) at 37°C for 1 hr. In addition, rhMincle (100nM) or BSA (100nM) were incubated and probed with AptMincle to confirm specificity of the aptamer. After binding of 5’biotin-AptMincle, samples were washed before being incubated with streptavidin-HRP for 30 min at room temperature. Samples were washed again before colorimetric assessment of binding via TMB substrate conversion and acid neutralization. The dissociated constant (K _d ) was calculated by Graphpad Prism 8, using the equation Y=Amax xX/ (K _d + X). The value of AptMincle binding to rhMincle were represented as % bound using PBS alone as baseline reference.

[00166] Mincle-Aptamer agonism/antagonism assessment in J774.1 macrophages

[00167] J774.1 murine macrophages were cultured and maintained in DMEM supplemented with 10% FCS, 1% Pen/Strep. In order to induce expression of Mincle within the resting macrophages, 1 ng/ml E.coli LPS was added to the stimulation media for 24hrs (termed J774-Mn ⁺ ). Induction of Mincle expression was confirmed through immunofluorescence imaging. The pre-stimulated J774-Mn ⁺ cells were then challenged with TDB (Invivogen, San Diego, CA, USA; 50pg/ml) or aptamer species (1 μM) for 30 min and phosphorylation of Syk and P65 assessed by western blot analysis as proxies of Mincle receptor activation. Agonistic effects of aptamers were assessed by incubation of J774-Mn ⁺ cells with 1 μM individual folded aptamer species while antagonistic effects were determined by preincubation with 1 μM of aptamer species followed by stimulation with 50pg/ml TDB (Invivogen, Cat No tlrl-tdb). [00168] Protein Collection and Quantification

[00169] Cell culture and tissue samples were prepared in lysis buffer as previously described ⁴² . Briefly, cells or cleaned and mechanically homogenised tissue were lysed in NP-40 lysis buffer (100mM NaCI, 200mM Tris-HCL (pH 8.0), 0.5% SDS and 1 mM EDTA supplemented with: phosphatase inhibitor cocktail (Cat N°: C755C25), 25mM Sodium Fluoride (1 M), 1 mM Sodium Orthovanadate, and 1% Triton X-100). Samples were sonicated to ensure complete cellular lysis and dissociation of proteins before being clarified at 12,000g for 10 min at 4°C. Protein concentration was then confirmed and normalised using the Pierce™ BCA protein assay kit (ThermoFisher Scientific, Burlington, ON, Canada).

[00170] Direct-Enzyme linked immunosorbent assay (ELISA)

[00171] Protein (10pg diluted in 1% Bovine Serum Albumin (BSA) in TBST (0.1% Tween-20)) was loaded in triplicate per sample into wells of a 96 well plate and incubated overnight at 4°C to allow for protein binding. All wells were subsequently blocked for 1 hr at RT in 2% BSA (TBST) before being washed three times with TBST. Sample wells were then incubated with primary antibodies for 2hrs at RT on an orbital shaker. After three more washes with TBST, relevant HRP-conjugated secondary antibodies were incubated in corresponding wells for 1 hr on an orbital shaker. Samples were washed for a final three times before being developed, acid-stopped (1 M HCI) and analysed using a ictorX4 spectramax plate-reader. Phosphorylated protein values were normalized to total protein for analysis and comparison.

[00172] Western blot

[00173] Previously collected protein lysates were combined with 5x loading buffer (20% SDS, 1 M Tris pH 6.8, bromophenol blue, p-mercaptoethanol, glycerol and water) and boiled for 10 minutes. Samples (30pg) were loaded and ran on 10% Tris-Glycine SDS-page gels made by hand according to standard protocols. Following resolution, proteins were transferred to a nitrocellulose membrane for 1 ,5hrs on ice. Ponceau staining confirmed efficient and complete transfer. Blots were washed and then blocked for 1 hr in 5% BSA or milk in TBST (0.1% Tween-20) at room temperature on an orbital shaker. Blots were subsequently incubated with primary antibodies as pre-determined manufacturer dilutions overnight under agitation at 4°C. Blots were washed, incubated with the appropriate HRP-conjugated secondary antibody (Jackson Immuno-Research Laboratories Inc, Westgrove, PA, USA) for 1 hr at room temperature. Finally, blots were incubated with extended duration ECL chemiluminescent substrate (ThermoFisher Scientific, cat no PIA34075) and visualized using Bio-Rad ChemiDoc MP. [00174] Immunofluorescent staining and imaging

[00175] The protocol used for the immunofluorescent staining of sectioned tissue or cells in culture are the same. Briefly, samples were fixed in 4% PFA for 20-60mins at room temperature before being transferred into PBS (containing 0.1% sodium azide). Colonic tissue samples were further processed by overnight fixation in 30% sucrose solution before imbedding in OCT and sectioning in 10pm sections on a cryotome. Slides were blocked with 2% BSA in PBST (0.1% Triton X-100) for 1 hr at RT before being incubated overnight with primary antibodies at 4°C. Samples were then washed three times for 5 min in PBST before being probed with secondary antibodies or complementary conjugated primary antibodies for 2hrs at RT under agitation. Samples were washed a further three times for 10 min before being sealed with Vectashield fluorescent mounting medium and a cover slip. All images were taken on an inverted Leica SP8 confocal microscope and analysed using Leica LASX software (LEICA microsystems Canada Inc, Richmond Hill, ON, Canada).

[00176] Induction of colitis and administration of treatments

[00177] Six-week-old C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Colitis was induced in these mice by administration of 2.5% (weight/vol) dextran sodium sulphate (DSS; Affymetrix, Cleveland, Ohio, USA) in drinking water for 7 days. Sham mice were given normal drinking water. I.P. injections of aptamer sequence variations of AptMincle (0.5-3.5 mg/kg) were administered at day 3 in 200 pl total volume saline, control mice received saline only. Mice were euthanized at day 7 by exposure to isoflurane (Pharmaceutical partners of Canada, Richmond Hill, ON, Canada) and cervical dislocation.

[00178] Disease Evaluation

[00179] In order to assess the severity of DSS-induced inflammation a multiparameter approach was used. Disease activity index (DAI) is a combinatory metrics consisting of weight loss, fecal consistency and fecal blood content. Calculation parameters of DAI are detailed in Table 1. Colon shortening, a common sign of inflammation-driven fibrosis, as well as myeloperoxidase assays were used as measures of colonic inflammation.

[00180] Myeloperoxidase assay

[00181] MPO activity was measured in colonic tissue samples by quantification of the absorbance of a coloured compound produced by MPO interacting with O-dianisidine and hydrogen peroxide. Absorbance readings were normalized to tissue weights.

[00182] Aptamer structure prediction and docking simulation [00183] The 2D-structure of AptMincle was predicted using the Vienna web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ). Due to a lack of a ssDNA 3D-structure prediction models, we have assumed that AptMincle could be modelled as an ssRNA aptamer. We used RNAcomposer (http://rnacomposer.cs.put.poznan.pl/) for 3D-structure prediction rendering. Docking simulations for AptMincle and Mincle (PDB 3WH3) itself, were performed using the PatchDock Server (http://bioinfo3d.cs.tau.ac.il/PatchDock/) and images were taken using PDB online viewer (https ://www . rcsb . org/3d- view) .

[00184] Statistics

[00185] Data are expressed as the mean ± one standard error of the mean (SEM). Experiments were performed as a minimum experimental triplicate with technical triplicates performed for each experiment. Statistical significance was assessed through the use of two-tailed unpaired Student’s t-test for parametric data, while Mann Whitney test performed for non-parametric data. Graphpad column analysis function was used to determine what previously mentioned tests were used on what data set due to normal distribution characteristics. Multiple analyses were performed using a one-way Anova with post-hoc Tukeys test where indicated. A P-value of 0.05 was considered significant. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 .

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Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[00230] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[00231] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.