PNA aptamers and uses thereof

Abstract

The invention provides modified oligonucleotides with improved binding properties for pathological TAR DNA-binding protein 43 (TDP-43) aggregates associated with various neurological diseases. The aptamers find utility in diagnosis of diseases associated with pathological TDP-43 aggregation.

Description

[0001] 008884231

[0002] 1

[0003] PNA aptamers and uses thereof

[0004] Field of the Invention

[0005] The present invention relates to modified aptamers which specifically bind to TDP-43 and their uses, and particularly, although not exclusively, to anti-TDP-43 peptide nucleic acid (PNA) aptamers. The invention further relates to the use of the modified anti-TDP-43 aptamers for example in diagnostic tools.

[0006] Background

[0007] TAR DNA-binding protein 43 (TDP-43) is an RNA-binding protein which functions to repress cryptic exon inclusion during splicing events. TDP-43 consists of four domains: an N-terminal domain with a well-defined fold necessary for dimerization / oligomerisation; two RNA binding motifs; and an unstructured C-terminal domain comprising a glycine-rich region (GRR), or Prion-like Domain (PLD).

[0008] Under physiological conditions, TDP-43 is soluble and localises predominately in the nucleus, shuttling between nucleus and cytoplasm in a transcription dependent manner. However, TDP-43 is prone to aggregation in both the nucleus and cytoplasm. These aggregation events can occur over a prolonged period of time and form pathological aggregates which increase in size. Pathological cytoplasmic and nuclear TDP-43 aggregates are a hallmark of a number of diseases, including Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, Pick’s disease and Limbic-predominant age-related TDP-43 encephalopathy (LATE). C9orf72 is the most common genetic cause of ALS and carriers display the characteristic TDP-43 pathology at post-mortem. Pathological TDP-43 aggregates can also be seen, at post-mortem, in asymptomatic individuals in an age-dependent manner.

[0009] Antibodies have been developed to detect TDP-43 and broadly encompass two distinct types: (i) antibodies that bind to the C-terminus and (ii) those that target, disease-related, post-translational modifications (e.g. phospho-TDP-43). While C-terminal binding antibodies are generally very sensitive, they lack specificity for pathological TDP-43, so it is difficult to discriminate between physiological and pathological TDP-43, especially nuclear aggregation events that are obscured by normal TDP-43 localisation in the nucleus. In contrast, antibodies which bind specifically to disease-related posttranslation modifications are very specific but lack sensitivity and do not detect all aggregation events. WO 2023 / 217742 (which is hereby incorporated by in its entirety) discloses an alternative approach for the detection of TDP-43 aggregates using RNA aptamers specific for TDP-43. The aptamers disclosed therein are capable of distinguishing between TDP-43 structures, including a wide size range of TDP-43 aggregates. This was confirmed in Spence et al. (2023) which further demonstrated that the aptamer008884231

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[0011] TDP-43aP‘ has improved sensitivity and specificity for pathogenic TDP-43 aggregates compared to TDP-43 antibodies, including that TDP-43aP‘ can detect early aggregation events that are not discriminated by classical antibodies. While TDP-43aP‘ shows clear advantages over classical antibody approaches, these RNA-based molecules have a number of drawbacks to their use. Firstly, RNA aptamers are not stable at room temperature, making them unsuitable for use in room temperature detection systems, including point of care devices. Moreover, as shown in Spence et al. (2023), to achieve antigen detection by TDP-43aP‘ an overnight paraformaldehyde fixation step is required and therefore further limiting their suitability for use in point of care devices. The requirement for an overnight fixation step also prevents the RNA aptamers use in many other assays, including single cell sorting assays (e.g. FACS), single nuclei sorting techniques (e.g. FANS), live imaging, and spatial sequencing.

[0012] It is also recognised that iPSC lines do not recapitulate TDP-43 pathology unless stress is caused to the cells, which is not physiological. However, RNA-sequencing of unstressed iPSC-derived cells reveals evidence of TDP-43 loss of function, including cryptic exon inclusion, suggesting that occult TDP-43 pathology is present under basal conditions. Although the RNA aptamer can detect such inclusions, again, the requirement for overnight paraformaldehyde fixation limits assay choice.

[0013] The present invention has been devised in light of the above considerations.

[0014] Summary of the Invention

[0015] Currently, no reliable point of care diagnostic tests exists which can distinguish between physiological and pathological TDP-43 to aid in the diagnoses of TDP-43 proteinopathies.

[0016] At its broadest, the present invention provides modified RNA or DNA oligonucleotides comprising or consisting of a TDP-43 binding oligonucleotide sequence. The modified oligonucleotides of the disclosure may be used in point of care diagnostic methods, kits and reagents. The modified oligonucleotides of the invention comprise RNA or DNA analogues.

[0017] Thus, in a first aspect, the disclosure provides a modified TDP-43-binding oligonucleotide. The oligonucleotide comprises at least four G nucleotides, wherein at least two of the four G nucleotides are consecutive. In some embodiments, the modified oligonucleotide is a modified DNA oligonucleotide. In some embodiments, the modified DNA oligonucleotide comprises at least two TG dinucleotides, or at least two GT dinucleotides, or at least one TG dinucleotide and at least one GT dinucleotide.

[0018] In some embodiments, the modified oligonucleotide is a modified RNA oligonucleotide. In some embodiments the modified RNA oligonucleotide comprises at least two UG dinucleotides, or at least two GU dinucleotides, or at least one UG dinucleotide and at least one GU dinucleotide.

[0019] In some embodiments, the modified oligonucleotides comprise RNA or DNA analogues comprising: i) a modified backbone; and / or

[0020] ii) a modified ribose ring or a modified deoxyribose ring, respectively.008884231

[0021] 3

[0022] In some embodiments, the RNA or DNA analogue comprises a modified backbone. In some embodiments, the modified backbone comprises a peptide nucleic acid (PNA) backbone, for example an aegPNA backbone (Figure 1). In some embodiments, the modified backbone has a neutral charge or a positive charge. In some embodiments, the neutral backbone comprises / V-(2-aminoethyl)glycine linkages, diaminopyrrolidine linkages, or phosphorodiamidate linkages. The / V-(2-aminoethyl)glycine linkages, diaminopyrrolidine linkages, or phosphorodiamidate linkages are in place of a sugar-phosphate backbone. In some embodiments, the RNA or DNA analogue is a peptide nucleic acid (PNA) comprising / V-(2-aminoethyl)glycine linkages. In some embodiments, the PNA oligonucleotide comprises dapPNA monomers. In some embodiments, the PNA oligonucleotide is a dapPNA aptamer (i.e. the dapPNA monomers comprise covalent formed between the exo-amine of one dapPNA monomer and the carboxylic acid of a second dapPNA monomer). In some embodiments, the PNA oligonucleotide is a secdapPNA aptamer (i.e. the dapPNA monomers comprise covalent formed between the endo-amine of one dapPNA monomer and the carboxylic acid of a second dapPNA monomer).

[0023] In some embodiments, the modified oligonucleotide comprises a modified ribose ring comprising a methylene bridge linking the 2’ ribose oxygen and the 4’ ribose carbon.

[0024] In some embodiments, the modified RNA oligonucleotide comprises a modified ribose ring. In some embodiments the modified DNA oligonucleotide comprises a modified deoxyribose ring. In some embodiments, the modified oligonucleotide comprises a methylene bridge linking the 2’ ribose oxygen and the 4’ ribose carbon. In some embodiments, the modified oligonucleotide is a locked nucleic acid (LNA) comprising a methylene bridge linking the 2’ ribose oxygen and the 4’ ribose carbon. In some embodiments, the modified ribose ring comprises a morpholine ring. In some embodiments, the modified oligonucleotide comprises a modified ribose ring comprising a morpholine ring and a modified backbone comprising phosphorodiamidate.

[0025] In some embodiments, the modified oligonucleotide is a PNA aptamer. For example, the modified oligonucleotide may comprise / V-(2-aminoethyl)glycine in the backbone in place of a sugar phosphate. In some embodiments, the modified oligonucleotide is an LNA aptamer. For example, the modified oligonucleotide may comprise nucleotides wherein the 2’ oxygen of the ribose rings are covalently linked to the 4’ carbon of the ribose rings. In some embodiments, the modified oligonucleotide is a morpholino aptamer. For example, the modified oligonucleotide may comprise phosphorodiamidate in the backbone in place of sugar phosphate and may further comprise a morpholine ring in place of a ribose sugar. In some embodiments, the modified oligonucleotide comprises an RNA sequence having at least 80%, at least 90% or 100% sequence identity to CGGUGUUGCU (SEQ ID NO: 1), GUGGUCCCCG (SEQ ID NO: 2), CGCUGUGGUC (SEQ ID NO: 3), AGCUGUGGCC (SEQ ID NO: 4), CGCUGGUGCU (SEQ ID NO: 5), CGCUGUGGCU (SEQ ID NO: 6), CGGCGUUGUU (SEQ ID NO: 7), CGGUGUAGGU(SEQ ID NO: 8), CUCUGUGGUG (SEQ ID NO: 9), or GUGGUCGCUG (SEQ ID NO: 10). In some embodiments, the modified oligonucleotide comprises an RNA sequence having at least 80% sequence identity to SEQ ID008884231

[0026] 4

[0027] In some embodiments, the modified oligonucleotide comprises a DNA sequence having at least 80%, at least 90% or 100% sequence identity to CGGTGTTGCT (SEQ ID NO: 11), GTGGTCCCCG (SEQ ID NO: 12), CGCTGTGGTC (SEQ ID NO: 13), AGCTGTGGCC (SEQ ID NO: 14), CGCTGGTGCT (SEQ ID NO: 15), CGCTGTGGCT (SEQ ID NO: 16), CGGCGTTGTT (SEQ ID NO: 17), CGGTGTAGGT (SEQ ID NO: 18), CTCTGTGGTG (SEQ ID NO: 19), GTGGTCGCTG (SEQ ID NO: 20). In some embodiments, the modified oligonucleotide comprises a DNA sequence having at least 80%, at least 90% or 100% sequence identity to a DNA sequence having at least 80% sequence identity to SEQ ID NO: 11.

[0028] In some embodiments, the modified oligonucleotide may comprise up to 20 nucleotides, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides. In some embodiments, the modified oligonucleotide consists of 10 nucleotides. In some embodiments, the modified oligonucleotide consists of 10 nucleotides as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 20.

[0029] In some embodiments, the modified oligonucleotide comprises SEQ ID NO: 1 and the modified oligonucleotide comprises PNA nucleotides comprising / V-2-aminoethylglycine. In some embodiments, the modified oligonucleotide comprises SEQ ID NO: 1 and the modified oligonucleotide consists of PNA nucleotides comprising / V-(2-aminoethyl)glycine. In some embodiments, the modified oligonucleotide consists of SEQ ID NO: 1 and the modified oligonucleotide comprises PNA nucleotides comprising N-2-aminoethylglycine. In some embodiments, the modified oligonucleotide consists of SEQ ID NO: 1 and the modified oligonucleotide consists of PNA nucleotides comprising / V-2-aminoethylglycine. In further embodiments, the modified oligonucleotide may be conjugated to a detection agent. In some embodiments, the detection agent is selected from the group comprising a fluorophore, a quantum dot, a radioisotope, a nucleic acid polymer, an amino acid polymer, a hybrid nucleic acid / amino acid polymer, a gold colloid, biotin, streptavidin or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin, a peroxidase, a horseradish peroxidase, or any combination thereof. In some embodiments, the detection agent is the specific fluorophore Atto488.

[0030] In a second aspect, the invention provides an in vitro method of measuring TDP-43 aggregates in a sample. In some embodiments, the method comprises:

[0031] i) providing a modified oligonucleotide according to the first aspect immobilized on a substrate; ii) bringing the sample into contact with the substrate; and

[0032] iii) measuring the levels or amount of TDP-43 aggregate bound to the substrate.

[0033] In some embodiments, the method comprises:

[0034] i) providing a substrate with an immobilized TDP-43 binder;

[0035] ii) bringing a sample into contact with the substrate to immobilise TDP-43 aggregates in the sample on the substate;

[0036] iii) bringing the substrate of ii) into contact with a modified oligonucleotide according the first aspect; and

[0037] iv) measuring the level or amount of TDP-43 aggregate bound to the substrate.008884231

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[0039] In some embodiments, the method comprises:

[0040] i) immobilising a sample of a substrate;

[0041] ii) bringing the immobilized sample into contact with a modified oligonucleotide according to the first aspect; and

[0042] iii) measuring the amount of modified oligonucleotide bound to the sample.

[0043] In some embodiments, the modified oligonucleotide is conjugated to a first member of a specific binding pair and the substrate comprises a second member of the specific binding pair. In such embodiments, the modified oligonucleotide immobilized on the substrate is prepared by bringing the first member and the second member of the specific binding pair into contact with each other. In some embodiments, the TDP-43 binder is conjugated to a first member of a specific binding pair and the substrate comprises a second member of the specific binding pair. In such embodiments, the TDP-43 binder immobilized on the substrate is prepared by bringing the first member and the second member of the specific binding pair into contact with each other. In some embodiments, the specific binding pair is comprised of (i) biotin and (ii) streptavidin. In some embodiments, the binding pair is comprised of (i) biotin and (ii) a protein that is functionally equivalent to streptavidin which is also capable of specifically binding to biotin. In some embodiments, the first member of the binding pair is biotin. In some embodiments, the second member of the binding pair is streptavidin, or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin. In some embodiments, the first member of the binding pair is streptavidin, or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin. In some embodiments, the second member of the binding pair is biotin.

[0044] Any suitable method known in the art may be used to measure the amount of TDP-43 bound to the substrate. For example, in some embodiments of the second aspect, the TDP-43 aggregates bound to substrate are measured using a method selected from any one of optical microscopy, electron microscopy, colorimetric analysis, for example using a lateral flow device, electro-optical analysis, electrochemical detection, or biochemical detection, or any combination thereof.

[0045] In some embodiments of the second aspect, the substrate may comprise a capture membrane (104) of a lateral flow device (100), an ELISA plate, or a magnetic bead.

[0046] In some embodiments of the second aspect, the sample is a sample previously obtained from a patient. For example, the sample may be a blood sample, serum sample, stool sample, urine sample, saliva sample, a cell sample or tissue sample previously obtained from a patient.

[0047] In a third aspect, the invention provides a lateral flow device (100). The lateral flow device comprises a sample pad (102) and a capture membrane (104). The capture membrane (104) may be positioned downstream of the sample pad (102). In some embodiments, the capture membrane (104) comprises an immobilised capture agent comprising a TDP-43 binder. In some embodiments, the TDP-43 binder comprises a modified oligonucleotide according to the first aspect of the invention. In some embodiments, the device further comprises a conjugate pad (110) interposed between the sample pad (102) and the capture membrane (104). In some embodiments, a detection agent (112) is deposited on008884231

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[0049] the conjugate pad (110). In some embodiments, the detection agent comprises a modified oligonucleotide according to the first aspect of the invention. In some embodiments, the detection agent comprises an anti-TDP-43 antibody.

[0050] In a related fourth aspect, the invention provides a lateral flow device (100) comprising a sample pad (102), a capture membrane (104) positioned downstream of the sample pad (102) and a conjugate pad (110) interposed between the sample pad (102) and the capture membrane (104). The capture membrane (104) comprises an immobilised capture agent comprising a TDP-43 binder. The conjugate pad (110) comprises a detection agent deposited thereon. The detection agent comprises a modified oligonucleotide according to the first aspect of the invention. In some embodiments, the TDP-43 binder may comprise an oligonucleotide according to the first aspect of the invention. In other embodiments, the TDP-43 binder may comprise an anti-TDP-43 antibody.

[0051] In a related fifth aspect, the invention provides a kit comprising a lateral flow device (100) and a modified oligonucleotide according to the first aspect of the invention. The lateral flow device (100) comprises a capture agent comprising a TDP-43 binder immobilized on a capture membrane (104), and the modified oligonucleotide is suitable for detecting TDP-43 aggregates bound to the capture agent. In some embodiments, the TDP-43 binder may comprise an oligonucleotide according to the first aspect of the invention. In other embodiments, the TDP-43 binder may comprise an anti-TDP-43 antibody.

[0052] In some embodiments of the third, fourth and fifth aspects of the invention, the capture agent (106) comprises a specific binding pair. In some embodiments, the first member (106) of the specific binding pair is conjugated to a TDP-43 binder and the second member (120) of the specific binding pair is immobilised on the capture membrane (104). In some embodiments, the specific binding pair is comprised of (i) biotin and (ii) streptavidin. In some embodiments, the binding pair is comprised of (i) biotin and (ii) a protein that is functionally equivalent to streptavidin which is also capable of specifically binding to biotin. In some embodiments, the first member of the binding pair is biotin. In some embodiments, the second member of the binding pair is streptavidin, or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin. In some embodiments, the first member of the binding pair is streptavidin, or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin. In some embodiments, the second member of the binding pair is biotin.

[0053] In some embodiments of the third, fourth and fifth aspects of the invention, the detection agent comprises a colorimetric detection agent. In some embodiments, the detection agent comprises gold colloid, a fluorophore, a peroxidase, or a horseradish peroxidase. In some embodiments, the lateral flow device (100) further comprises a control antibody (116) immobilised on the capture membrane (104) for binding a control antigen. Any suitable control antibody may be used and may depend on the sample to be analysed. That is, a control antibody may be selected based on the presence of a target control antigen normally present in the sample type. For example, in some embodiments wherein the sample to be analysed is a serum or blood sample, the control antibody may specifically bind to albumen. In some008884231

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[0055] embodiments wherein the sample to be analysed is a stool sample, the control antibody may specifically bind to CK7.

[0056] In a related sixth aspect, the invention provides a use of a lateral flow device according to the third or fourth aspect, or the use of a kit according to the fifth aspect, in a method of detecting TDP-43 aggregates in a sample. The method comprises providing a lateral flow device or kit according to any one of aspects three to five, applying a sample to the sample pad (102) and allowing the sample to migrate through the capture membrane (104) to contact the capture agent (106). The presence of TDP-43 aggregates in the sample is indicated by a detectable signal generated by the detection agent (112).

[0057] In a related seventh aspect, the invention provides the oligonucleotide according to the first aspect, the lateral flow device of the third or fourth aspect, or the kit of the fifth aspect, for use in a method of diagnosing a TDP-43 proteinopathy. In some embodiments, the TDP-43 proteinopathy is selected from the group consisting of Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, Pick’s disease and Limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0058] In an eighth aspect, the invention provides an imaging composition comprising the modified oligonucleotide according to the invention for use in an in vivo method of medical imaging or experimental imaging. In a related ninth aspect, the invention provides a method of detecting TDP-43 aggregates in vivo. In a related tenth aspect, the invention provides a method of diagnosing a TDP-43 proteinopathy. The compositions for use and methods according to the eighth, ninth and tenth aspects of the invention comprise administering to a subject an imaging composition comprising the modified oligonucleotide of the invention. In some embodiments, the subject is subjected a bioimaging technique selected from positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT) scanning, or any combination thereof. In some embodiments, the modified oligonucleotide comprises a radioisotope. In some embodiments, the radioisotope is selected from fluorine-18, gallium-68, copper-64 and zirconium-89. In some embodiments, the radioisotope is provided as a chelated metal complex. In some embodiments, the chelating moiety is selected from the group consisting of DOT A, NOTA, DTPA and desferrioxamine (DFO).

[0059] In some embodiments of the above aspects, the modified oligonucleotide may be functionalised by any suitable method known in the art. In some embodiments of the above aspects, the modified oligonucleotide may be a PNA oligonucleotide and may be functionalised. A PNA oligonucleotide may be functionalised at any position along the peptide backbone, at the C terminus, and / or at the N-terminus using any suitable conjugation chemistry well known in the art, for example click-chemistry utilising Cu-catalyzed azide alkyne cycloaddition (CuAAC) . In some embodiments, the PNA oligonucleotide may be functionalised using any suitable conjugate comprising a natural or non-natural amino acid, for example as described in deGruyte et al (2017) which is hereby incorporated by reference. In some embodiments, the PNA oligonucleotide may be functionalised using a conjugate comprising lysine, cysteine, aspartate,008884231

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[0061] glutamate, tyrosine, histidine, and / or arginine. In some embodiments, the modified oligonucleotide may be functionalised via the epsilon nitrogen of an amino acid. In some embodiments, the C-terminus may be functionalised using a conjugate comprising lysine, cysteine, aspartate, glutamate, tyrosine, histidine, arginine. In some embodiment, the modified oligonucleotide is a PNA oligonucleotide and may be functionalised at the / V-terminus. In some embodiment, the PNA oligonucleotide may be functionalised directly or using a linker, for example a peptide linker, for example an AEEA peptide linker.

[0062] In some embodiments, the modified oligonucleotide comprises a contrast dye. In some embodiments, the modified oligonucleotide is conjugated to a contrast dye. In some embodiments, the modified oligonucleotide comprises a paramagnetic metal complex, e.g. a paramagnetic metal suitable for use in MRI, for example56Fe or Gd( 111 )-DOTA. In some embodiments, the modified oligonucleotide is a PNA comprising Gd(lll)-DOTA.

[0063] In an eleventh aspect, the invention provides the modified oligonucleotide according to the first aspect for use in a method of treatment. In a related twelfth aspect, the invention provides the modified oligonucleotide according to the first aspect for use in a method of treating a TDP-43 proteinopathy. In some embodiments of the eighth, ninth, tenth, eleventh, and twelfth aspects, the TDP-43 proteinopathy is selected from the group comprising or consisting of Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, Pick’s disease and Limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0064] In a thirteenth aspect, the invention provides a method of imaging TDP-43 aggregates in vitro or ex vivo. In some embodiments, the imaging is achieved through the detection of the detectable label of the modified oligonucleotide. In some embodiments, the imaging technique is selected from the group consisting of: fluorescence imaging, fluorescent immunohistochemistry, optical microscopy, confocal microscopy or electron microscopy. In some embodiments, the method of imaging is performed on a population of cells which have not underwent a fixation step. For example, the sample of cells may not be fixed using paraformaldehyde prior to incubation with the modified oligonucleotide of the first aspect. In some embodiments, the cells are incubated with the modified oligonucleotide for at least 20, 30, 40, 50 or 60 minutes. In some embodiments, the cells are incubated with the modified oligonucleotide for at least 1.5 hours, 2 hours or 3 hours.

[0065] In a fourteenth aspect, the invention provides a method of isolating a population of cells or isolated nuclei comprising TDP-43 aggregates by a sorting method. The method comprises incubating a first population of cells or nuclei with the modified oligonucleotide according to the first aspect and isolating from the first population a subpopulation that comprises cells or isolated nuclei containing TDP-43 aggregates. In some embodiments, the sorting method is selected from the group consisting of: fluorescence-activated cell sorting (FACS), fluorescence-activated nuclei sorting (FANS), laser-capture single-cell microdissection, and microfluidic single-cell sorting. In some embodiments, the method is performed on a population of cells which have not underwent a fixation step. For example, the sample of cells may not be fixed using008884231

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[0067] paraformaldehyde prior to incubation with the modified oligonucleotide of the first aspect. In some embodiments, the cells are incubated with the modified oligonucleotide for at least 20, 30, 40, 50 or 60 minutes. In some embodiments, the cells are incubated with the modified oligonucleotide for at least 1.5 hours, 2 hours or 3 hours.

[0068] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0069] Summary of the Figures

[0070] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0071] Figure 1: Structural comparison of an aeg peptide nucleic acid (aegPNA) and RNA backbones.

[0072] Figure 2. Schematic diagram showing of a lateral flow device for detecting aggregated TDP-43.

[0073] Figure 3. Schematic diagrams showing three configurations of lateral flow devices for detecting aggregated TDP-43. Configuration 1 shows a lateral flow device using an anti-TPD-43 antibody as a capture agent and an oligonucleotide of the invention as a TDP-43 binder conjugated to gold colloid. Configuration 2 shows a lateral flow device using a capture agent comprising an oligonucleotide of the invention conjugated to biotin which interacts with streptavidin immobilised on the capture membrane, and an anti-TDP-43 antibody as a TDP-43 binder conjugated to gold colloid. Configuration 3 shows a lateral flow device using an oligonucleotide of the invention as both a TDP-43 binder and as part of the capture agent as in configuration 2.

[0074] Figure 4. Micrographs comparing staining of tissue samples using RNA-based aptamers in tissue sections processed without (A) and with (B) an overnight PFA fixation, and PNA aptamers in tissue sections processed without the need for fixation (C). No signal is detectable in tissue samples without fixation using RNA aptamers, whereas abundant pathological TDP-43 is detected in tissue sections derived from the same patient tissue after overnight fixation. In contrast, PNA aptamers show abundant staining of pathological TDP-43 aggregates without the need for an overnight fixation step. White and closed black arrows show neuronal cytoplasmic and nuclear pathology and black open arrows show glial cytoplasmic pathology.

[0075] Figure 5. Micrographs showing that PNA aptamer, TDP-43pnapt, can detect pathological TDP-43 in fluorophore-based immunohistochemistry (IHC) assays. The top row (No PNA control) shows minimal background TDP-43pnaptstaining. Sections treated with TDP-43pnaptconjugated to both008884231

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[0077] 3'-Atto-488 and 5'-Atto-488 (middle and bottom rows, respectively) demonstrate clear cytoplasmic localisation of the aptamer. White dashed lines mark the cell boundaries, “N” indicates the nucleus, and grey arrowheads show the punctate TDP-43pnaptsignal.

[0078] Figure 6. Micrographs showing Immunofluorescence staining of TDP-43 in human IPSC-derived motor neurons using Atto488 conjugated TDP-43pnapt. Cytoplasmic TDP-43 aggregates are observed in C9orf72 patient lines under non-stressed conditions (three independent lines: BS, M2 and DN); whereas no cytoplasmic aggregates are observed in their paired isogenic controls or in two unrelated healthy control lines. White arrowheads indicate cytoplasmic TDP-43 pathology. Scale bars, 20 pm.

[0079] Figure 7. Micrographs showing immunofluorescent staining of TDP-43 in human IPSC-derived motor neurons grown as organoids using Atto488 conjugated TDP-43pnapt. Cytoplasmic TDP-43 aggregates (green) are consistently observed in C9orf72 patient lines under non-stressed conditions (five independent organoids from one iPSC line); while unrelated healthy controls show no cytoplasmic aggregates (top panel).

[0080] Figure 8: ELISA-based quantification of TDP-43 in human biofluid samples using antibody-based detection (total TDP-43; orange (left) bars) or PNA-aptamer-based detection (pathological TDP-43; purple (right) bars). PNA aptamer-based signal is selectively observed in ALS samples (2-7), whereas no signal is observed for the control, non-ALS TDP-43 sample (1).

[0081] Detailed Description of the Invention

[0082] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0083] Modified oligonucleotides of the invention

[0084] The present disclosure provides modified oligonucleotides which are, surprisingly, capable of binding to TDP-43 aggregates without the need for a fixation step of the protein complex prior to introduction of the modified oligonucleotide. Due to this, and the improved accuracy, precision, stability, and affinity of the modified oligonucleotides for unfixed pathological TDP-43 aggregates, the modified oligonucleotides of the invention may be used in a variety of research and diagnostic applications (including point of care diagnostic methods, immunohistochemical analysis, immunofluorescence imaging, cell sorting, live cell imaging, spatial sequencing and as PET agent for in vivo imaging), and in pharmaceutical compositions.008884231

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[0086] The modified oligonucleotides may comprise an RNA or a DNA sequence. The modified oligonucleotides can detect TDP-43 with improved accuracy, precision, stability, and affinity compared to currently available alternative reagents, such as antibodies, RNA aptamers and DNA aptamers.

[0087] The modified oligonucleotides of the invention may be referred to as aptamers. Nucleic acid aptamers are reviewed e.g. in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202. They may be identified and / or produced by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEX), or by developing SOMAmers (slow off-rate modified aptamers) (Gold L et al. (2010) PLoSONE 5(12): e15004). Aptamers and SELEX are described in Tuerk and Gold, Science (1990) 249(4968): 505-10, and in WO 91 / 19813. Aptamers may comprise short oligonucleotides or deoxyribonucleotides, and may be single-stranded or double-stranded. Aptamers bind with high affinity and specificity to their target protein, peptide or small molecule. Aptamers may have secondary or tertiary structure and thus fold into diverse and intricate molecular structures. In some embodiments, the modified oligonucleotides of the invention are single stranded or double stranded. In preferred embodiments, the modified oligonucleotides are single stranded.

[0088] It is generally understood that RNA aptamers and DNA aptamers which share corresponding nucleotide sequences have the same or similar binding properties and, as such, the choice of RNA or DNA based aptamers can be selected in view of the specific intended use (Amero P. et al., 2021; Kuo, PH et al., 2009).

[0089] Aptamers may comprise chemically modified nucleic acids (nucleic acid analogues), for example in which the sugar (i.e. the ribose or deoxyribose ring) and / or the phosphate (referred to herein as the ‘backbone’) and / or base is chemically modified. Such modifications may improve the stability of the aptamer and / or make the aptamer more resistant to degradation (Wang F. et al., 2022 which is hereby incorporated by reference in its entirety).

[0090] The oligonucleotides of the disclosure may be modified such that the oligonucleotide comprises one or more non-ionic backbone linkages in place of an anionic phosphodiester linkage present in a nonmodified RNA or DNA oligonucleotide. In some examples, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 non-ionic backbone linkages.

[0091] For example, the oligonucleotides of the present disclosure may comprise peptide nucleic acids “PNAs”. PNAs are synthetic mimics of RNA / DNA in which the ribose or deoxyribose phosphate backbone is replaced by a pseudo-peptide polymer to which the nucleobases are linked (reviewed in Pellestor, F., Paulasova, P. The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics. Eur J Hum Genet 12, 694-700 (2004)). For example, PNA monomers may comprise a N-(2-aminoethyl)glycine backbone, or a diaminopyrrolidine backbone. PNAs comprising diaminopyrrolidine comprise a positively charged backbone.

[0092] Diaminopyrrolidine based peptide nucleic acid (dapPNA) monomers may be present in the PNAs described herein. dapPNA monomer has the following structure, where T represents a nucleotide base:008884231

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[0094]

[0095] Accordingly, dapPNA monomer has two amine groups via which peptide bonds in the backbone of a PNA can be formed. For example, a peptide bond in the backbone of a dapPNA could be formed between a carboxylic acid and the amine group which is outside of the pyrrolidine ring (the exo-amine). Such a PNA is termed a dapPNA aptamer. Alternatively, a peptide bond in the backbone of a PNA could be formed between a carboxylic acid and the amine group which is a part of the pyrrolidine ring (the endo-amine). Such a PNA is termed a secdapPNA aptamer.

[0096] In some embodiments, the oligonucleotide of the present invention consists of PNA oligonucleotides. In some embodiments, the PNA oligonucleotide comprises an / V-(2-aminoethyl)glycine backbone. In some embodiments, the PNA oligonucleotide comprises dapPNA monomers. In some embodiments, the PNA oligonucleotide is a dapPNA aptamer (i.e. the dapPNA monomers comprise covalent formed between the exo-amine of one dapPNA monomer and the carboxylic acid of a second dapPNA monomer). In some embodiments, the PNA oligonucleotide is a secdapPNA aptamer (i.e. the dapPNA monomers comprise covalent formed between the endo-amine of one dapPNA monomer and the carboxylic acid of a second dapPNA monomer). In addition to, or instead of, modifications to the backbone phosphate, the oligonucleotides disclosed herein may comprise a modified sugar. For example, the oligonucleotide of the present disclosure may comprise ‘locked nucleic acids’ (LNAs), also known as ‘bridged nucleic acid’ (BNA). LNA are RNA nucleotide analogues wherein the ribose moiety is modified such that the 2’ ribose oxygen is covalently linked to the 4’ ribose carbon via a methylene bridge. The oligonucleotides of the present disclosure may comprise one or more LNA nucleotides. In some embodiments, the oligonucleotide of the present invention consists of LNA oligonucleotides.

[0097] In other examples, the oligonucleotide of the present disclosure may comprise morpholino nucleic acids. Morpholino nucleic acids comprise methylenemorpholine rings linked through phosphorodiamidate groups. In some embodiments, the oligonucleotide of the present invention consists of morpholino oligonucleotides.

[0098] It will be understood that the term “oligonucleotides” as used herein encompasses oligonucleotides comprising nucleic acid analogues, including analogues comprising modified sugars and / or backbones. The modified oligonucleotides of the disclosure are characterized by their ability to bind to the RNA recognition motif (RRM) of the TDP-43 protein. They are further characterised by shared structural characteristics, namely that the sequences include i) at least four “G” nucleotides, at least two of which are consecutive, and ii) at least two TG / GT dinucleotides or at least two UG / GU dinucleotides, as disclosed in WO2023217742A1 which is hereby incorporated by reference in its entirety.

[0099] It is to be understood that “at least two TG / GT dinucleotides” means that the modified oligonucleotide comprises at least two TG dinucleotides, at least two GT dinucleotides, or at least one TG dinucleotide008884231

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[0101] and at least one GT dinucleotide. Likewise, it is to be understood that “at least two UG / GU dinucleotides” means that the modified oligonucleotide comprises at least two UG dinucleotides, at least two UT dinucleotides, or at least one UG dinucleotide and at least one UT dinucleotide. The at least two TG / GT or UT / GU dinucleotides can be consecutive or non-consecutive. In some embodiments, the oligonucleotides of the invention comprise at least one C nucleotide.

[0102] In some embodiments, the protein-binding nucleotide sequence is 6 to 15 nucleotides in length, preferably 10 to 15 nucleotides in length.

[0103] In some embodiments, the modified oligonucleotides may comprise further modifications to increase the stability of the modified oligonucleotide. For example, in some embodiments, the modified oligonucleotide further comprises a modification to reduce degradation of the modified oligonucleotide by nucleases and / or to increase thermal stability. For example, in some embodiments, the modified oligonucleotide further comprises a fluorine atom linked at the 2’ position of the ribose or deoxyribose pentose ring.

[0104] In some embodiments, the oligonucleotides may comprise an RNA sequence having at least 70%, 80%, 90% or 100% sequence identity to any one of CGGUGUUUGCU (SEQ ID NO: 1 ), GUGGUCCCCG (SEQ ID NO:2), CGCUGUGGUC (SEQ ID NO:3), AGCUGUGGCC (SEQ ID NO:4), CGCUGGUGCU (SEQ ID NO: 5), CGCUGUGGCU (SEQ ID NO: 6), CGGCGUUGUU (SEQ ID NO: 7), CGGUGUAGGU(SEQ ID NO: 8), CUCUGUGGUG (SEQ ID NO: 9), or GUGGUCGCUG (SEQ ID NO: 10). In another example, the oligonucleotides may comprise a DNA sequence having at least 70%, 80%, 90% or 100% sequence identity to any one of CGGTGTTGCT (SEQ ID NO: 11), GTGGTCCCCG (SEQ ID NO: 12), CGCTGTGGTC (SEQ ID NO: 13), AGCTGTGGCC (SEQ ID NO: 14), CGCTGGTGCT (SEQ ID NO: 15), CGCTGTGGCT (SEQ ID NO: 16), CGGCGTTGTT (SEQ ID NO: 17), CGGTGTAGGT (SEQ ID NO: 18), CTCTGTGGTG (SEQ ID NO: 19), or GTGGTCGCTG (SEQ ID NO: 20).

[0105] The modified oligonucleotides of the disclosure may be conjugated to a “detection moiety” suitable for an intended use.

[0106] As used herein, the terms “conjugate” and “conjugated” refers to the association between molecules. The association can be by, for example, a covalent bond or a non-covalent bond. For example, the association between the oligonucleotide of the disclosure and the detection moiety may be covalent or non-covalent.

[0107] Detection agents include, but are not limited to, fluorophores, quantum dots, radioisotopes, nucleic acid polymers, amino acid polymers, hybrid nucleic acid / amino acid polymers, gold colloids, biotin, avidin, horseradish peroxidase, or any combination thereof. In some embodiments, the detection agent is the specific fluorophore Atto488. Those skilled in the art would understand that any suitable detection agent may be used in view of the intended use.

[0108] For example, for fluorescent immunohistochemistry (IHC) the detection agent may be selected from a fluorophore or quantum dot, while for chromogen-based IHC the detection agent may be selected from biotin, avidin, horseradish peroxidase, or a combination thereof.008884231

[0109] 14

[0110] It will be understood that a “detection agent” conjugated to the modified nucleic acid may not itself produce a detectable signal but may be a component of a detection system. For example, biotin may be considered a detection agent as when it is used in conjunction with an anti-Biotin-HRP conjugate, the association between biotin and the anti-Biotin-HRP conjugate allows a detectable signal to be produced using a chromogen (i.e. HRP converts the chromogen into a detectable signal).

[0111] Detection and diagnostic uses of the modified oligonucleotides of the invention

[0112] The disclosure also provides detection, diagnostic, and therapeutic uses of the modified oligonucleotides disclosed herein.

[0113] As discussed above, due to the improved accuracy, precision, stability, and affinity of the modified oligonucleotides of the disclosure for pathological TDP-43 aggregates, the modified oligonucleotides of the disclosure find use in many applications, including, but not limited to, point-of-care diagnostic tests, immunohistochemical analysis, immunofluorescence imaging, cell sorting, live cell imaging, spatial sequencing and as biomedical imaging agents, for example PET agents, MRI agent, and / or CT scan agents for in vivo imaging.

[0114] Thus, the disclosure further provides modified oligonucleotides for use in diagnostic methods. In particular, the disclosure provides methods of diagnosing TDP-43-related diseases, also termed TDP-43 proteinopathies.

[0115] TDP-43 proteinopathies are diseases characterised by a pathological increase in the presence of cytoplasmic TDP-43 aggregates. TDP-43 proteinopathies include, for example, Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease and Limbic-predominant age-related TDP-43 encephalopathy (LATE). The modified oligonucleotide of the disclosure may be used to detect TDP-43 aggregates in vitro using optical fluorescent microscopy. In such cases, the modified oligonucleotide of the disclosure may be conjugated to any suitable fluorophore known to those skilled in the art.

[0116] The modified oligonucleotide of the disclosure may also be used to detect TDP-43 aggregates in anti-and / or post-mortem biopsy samples. For example, the modified oligonucleotides find use in immunohistochemical analysis. In such cases, the modified oligonucleotides may be conjugated to any suitable detection moiety for producing a detectable signal, for example, biotin. Surprisingly, the modified oligonucleotides of the invention are able to detect pathological TDP-43 aggregates in biopsy tissues without the tissue needing to undergo a paraformaldehyde fixation step, significantly reducing the time and complexity required to make a diagnosis.

[0117] Thus, the disclosure provides methods of detecting TDP-43 aggregates in ex vivo tissue biopsy samples, optionally without a paraformaldehyde fixation step. The method may comprise chromogen-based008884231

[0118] 15

[0119] immunohistochemical analysis or fluorophore-based immunohistochemical analysis, directly conjugated or otherwise. The biopsy samples may have previously been obtained from living or deceased subjects. Also provided by the disclosure are methods of cell sorting using the modified oligonucleotides of the disclosure. In such cases, the modified oligonucleotide of the disclosure may be conjugated to any suitable detection agent suitable for allowing separation of cells comprising TDP-43 aggregates from cells that do not comprise TDP-43 aggregates. For example, the detection agent may comprise a fluorophore or a magnetic bead.

[0120] The modified oligonucleotides of the disclosure also find use in point-of-care diagnostic tests due to the demonstrated superior affinity and stability compared to, for example, unmodified RNA aptamers and their ability to bind unfixed TDP-43 aggregates. These properties allow the modified oligonucleotides to be used and stored at room temperature, allow their use in minimally processed samples (e.g. no fixation required for detecting sample in cells or tissues) and can be used to detect TDP-43 samples in liquid samples. Thus, the modified oligonucleotides may be used in methods of detecting TDP-43 aggregates.

[0121] Biomedical Applications

[0122] In a further aspect, the present invention provides methods of biological imaging comprising administering the modified oligonucleotide described herein to a subject to be imaged.

[0123] As used herein, “biological imaging” or “bioimaging” refers to a variety of techniques and imaging modalities used to visualise biological processes, tissues, cells and molecules. “Imaging” may refer to techniques for visualising internal structures and processes ( / .e. providing a visual representation of such structures and processes). Alternatively, “imaging” may refer to measurement and recording techniques that do not primarily produce images of structures or processes, but nonetheless provide information / data on biological processes, tissues etc. from a subject. Such information may be, for example, quantification of targeted compositions, or location data. This information may be suitably represented graphically rather than in an image perse.

[0124] In some embodiments, the present invention provides an imaging composition for use in a method of biological imaging. The imaging composition comprises the modified oligonucleotide of the invention. The method comprises administering the imaging composition to a subject to be imaged.

[0125] In some embodiments, the biological imaging comprises medical or experimental imaging.

[0126] As used herein, “medical imaging” refers to the imaging of biological processes, tissues, cells and molecules for clinical analysis (e.g. for aiding in the treatment / diagnosis of a patient, or for monitoring treatment of a patient, e.g. tracking the in vivo distribution of a cell therapy). Medical imaging includes techniques for quantifying the abundance of targeted compositions within structures / regions of a subject. That is, “medical imaging” does not exclusively refer to techniques resulting in the production of an image perse and may include other measurement and recording techniques for gathering information / data from a subject. “Medical imaging” encompasses imaging of humans or animals (e.g. veterinary imaging).008884231

[0127] 16

[0128] As used herein, “experimental imaging” refers to the imaging of biological processes, tissues, cells and molecules for research ( / .e. experimental) purposes. In some embodiments, “experimental imaging” encompasses imaging of humans and animals (e.g. laboratory animals). In some embodiments, “experimental imaging” encompasses imaging of laboratory animals (for example, mice, rats, rabbits, guinea pigs, pigs and non-human primates). The imaging compositions of the present invention may be useful as a research tool to screen therapies based on their ability to reduce TDP-43 aggregates within a subject.

[0129] The method of biological imaging (e.g. the method of medical or experimental imaging) may be in vivo or in vitro.

[0130] In some embodiments, the step of imaging the subject comprises PET scanning or magnetic resonance imaging. In some embodiments, the step of imaging comprises or additionally comprises single-photon emission computerized tomography (SPECT) imaging, photo acoustic imaging (e.g. with indocyanine green (ICG)) or near-infrared (NIR) imaging.

[0131] In some embodiments, the imaging composition is an imaging composition comprising a labelled modified oligonucleotide of the invention, as described herein. In some embodiments, wherein the labelled modified oligonucleotide comprises a paramagnetic metal complex such as56Fe or Gd( I ll)-DOTA, the imaging modality is MRI. In some embodiments, wherein the labelled complex comprises89Zr,18F,68Ga,11C or64Cu, the imaging modality is PET scanning.

[0132] In some embodiments, the step of imaging the subject comprises fluorescent imaging, including near infrared and infrared imaging. In such applications, the modified oligonucleotide may be conjugated to any suitable fluorophore known in the art. For example, the fluorophore may be a fluorophore that, upon excitation, emits light at a near infrared or infrared wavelength. Any suitable fluorophore known in the art may be used in such applications.

[0133] In a further aspect, the present invention provides a method of monitoring the treatment of a subject who has received treatment for a TDP-43 associated disease or disorder, wherein the method comprises administering to the subject an imaging composition comprising the modified oligonucleotide as described herein.

[0134] In some embodiments, the method of monitoring the treatment of a subject comprises a step of imaging the subject. In some embodiments, the step of imaging the subject comprises the use of PET scanning, MRI, or CT scanning.

[0135] In some embodiments, the imaging composition as described herein is introduced into the subject such that the imaging composition comes into contact with TDP-43 aggregates, whereby the modified oligonucleotide specifically binds to the TDP-43 aggregates. In such a way, the imaging compositions as described herein (which constitute modified oligonucleotides that specifically binds to TDP-43 aggregates) will accumulate in regions of the subject where TDP-43 aggregates are present, enabling detection of TDP-43 aggregates in a subject following the use of an appropriate imaging modality (e.g. PET scanning, MRI or CT scanning).008884231

[0136] 17

[0137] Imaging modalities

[0138] Aspects of the present invention relate to imaging compositions comprising the modified oligonucleotide as described herein.

[0139] As used herein, ‘imaging compositions’ refers to compositions useful for enhancing visualisation in any suitable imaging modality. Such imaging compositions comprise the compounds or labelled compositions as described herein. Such imaging compositions are used in conjunction with imaging modalities to improve detection or monitoring of target materials / cells / tissues / processes. Any suitable imaging modality may be used with the modified oligonucleotides of the invention for detecting TDP-43 and TDP-43 aggregates in vivo.

[0140] Such imaging modalities may include nuclear imaging modalities (e.g. positron emission tomography (PET) and single-photon emission computed tomography (SPECT)), optical imaging modalities (e.g. fluorescence imaging, photoacoustic imaging), and magnetic resonance imaging (MRI).

[0141] In some embodiments, the imaging modality is a nuclear imaging modality, e.g. PET or SPECT, or a nonnuclear imaging modality, e.g. MRI or optical imaging. In some embodiments, the imagine modality is PET. In some embodiments, the imaging modality is magnetic resonance imaging (MRI). In some embodiments, the imaging modality is computed tomography (CT) scanning.

[0142] As used herein, PET refers to positron emission tomography. PET is a nuclear imaging technique for visualising organs, tissues, and metabolic processes. PET uses radioactive tracers comprising a positronemitting isotope, which are introduced into the body to be imaged. As the radioactive tracer undergoes emission decay, it emits a positron. The positron interacts with an electron within the body, causing annihilation of both the positron and the electron and producing a pair of gamma photons moving in opposite directions. These two gamma photons are detected simultaneously, allowing for localisation of the positron annihilation event. This information is processed to create an image to represent the distribution of the radiotracer in the body. PET scanning is reviewed, for example, in Lameka et al. (2016). Handb Clin Neurol. 135:209-227.

[0143] In some embodiments, the imaging composition according to the present invention is a PET tracer composition. A PET tracer is a radioactive compound that emits positrons. In some embodiments, wherein the imaging composition is a PET tracer composition, the imaging composition comprises a labelled modified oligonucleotide. For example, in some embodiments, the labelled complex may comprise a compound coordinated with the nuclide89Zr,68Ga, or64Cu. In some embodiments, the labelled complex may comprise a compound linked to18F or11C. In some embodiments, the labelled complex may comprise a compound linked to18F or11C via any suitable prosthetic group known in the art, for example 2-[18F]fluoroethy I tosylate.

[0144] In some embodiments, the imaging modality is MRI.008884231

[0145] 18

[0146] As used herein, MRI refers to magnetic resonance imaging. MRI is a biological imaging technique for visualising anatomy and physiological processes. MRI uses magnetic fields that causes hydrogen protons found in water and fat within the body to align. Oscillating magnetic fields at radio frequencies are then used to temporarily disrupt the alignment. When the oscillating magnetic field stops, the protons ‘relax’ to their original state. MRI is reviewed, for example, in Nitz and Reimer et al. (1999). European Radiology.

[0147] 9:1032-1046. MRI contrast agents may be introduced into a subject to enhance visibility of certain tissues / structures. MRI contrast agents alter the magnetic properties of nearby tissues, altering their appearance on MRI images.

[0148] In some embodiments, the imaging composition according to the present invention is an MRI contrast agent composition. In some embodiments, wherein the imaging composition is an MRI contrast agent composition, the imaging composition comprises a labelled modified oligonucleotide. Any suitable MRI contrast agent may be conjugated to the modified nucleotide. For example, the labelled complex may comprise a modified oligonucleotide coordinated with the nuclide56Fe. In some embodiments, the modified oligonucleotide is conjugated to Gd(lll). In some embodiments, the modified oligonucleotide is conjugated to Gd(lll) comprising a chelator. In some embodiments, the modified oligonucleotide is conjugated to Gd(lll)-DOTA, Gd(lll)-DTPA, or Gd(lll)-DTPA-BMA (for example as described in Wang et al (2017) which is hereby incorporated by reference in its entirety).

[0149] In some embodiments, a method according to the present invention comprises a step of imaging a subject with a second imaging modality. The second imaging modality may be any suitable imaging modality. Such imaging modalities may include nuclear imaging modalities (e.g. positron emission tomography (PET) and single-photon emission computed tomography (SPECT)), optical imaging modalities (e.g. fluorescence imaging, photoacoustic imaging), and magnetic resonance imaging (MRI). In some embodiments, the first imaging modality is selected from PET / MRI, and the second imaging modality is selected from the group consisting of: PET, SPECT, fluorescence imaging, photoacoustic imaging and MRI.

[0150] Administration

[0151] Administration of the imaging compositions of the present invention may be by any appropriate route. For example, administration may be parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, pulmonary (e.g. via inhalation) or transdermal.

[0152] Administration may be by injection or infusion.

[0153] Administration of the imaging compositions of the present invention is preferably in an appropriate amount to enable imaging of the target tissues within a subject, via the appropriate imaging modality (e.g. PET or MRI). The actual amount administered, and rate and time-course of administration, will depend on the individual subject, the imaging modality selected, and the particular composition administered.

[0154] Prescription of agents, e.g., decisions on dosage etc., is within the responsibility of general practitioners008884231

[0155] 19

[0156] and other medical doctors, and typically takes account of the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0157] Multiple doses of the imaging compositions described herein may be provided.

[0158] Lateral flow devices

[0159] Lateral flow diagnostic devices are a commonly used diagnostic tools (reviewed in Omidfar K, Riahi F, Kashanian S. Lateral Flow Assay: A Summary of Recent Progress for Improving Assay Performance. Biosensors (Basel). 2023 Aug 23;13(9):837. doi: 10.3390 / bios13090837. PMID: 37754072; PMCID: PMC10526804). The disclosure further provides lateral flow devices and kits comprising the modified oligonucleotide disclosed herein for the detection of TDP-43 aggregates in a sample. Suitable lateral flow device configurations using the modified oligonucleotides capable of binding to TDP-43 aggregates disclosed herein include, for example, configurations as show in Figures 2 and 3.

[0160] A lateral flow device (100) of the disclosure may comprise a modified oligonucleotide according to the disclosure. In some embodiments, the lateral flow device comprises a capture membrane (104) comprising an immobilised capture agent (106), wherein the capture agent (106) comprises a modified oligonucleotide according to the disclosure. In some embodiments, the lateral flow device (100) comprises a conjugate pad (110), wherein the conjugate pad comprises a modified oligonucleotide according to the disclosure, optionally wherein the modified oligonucleotide is conjugated to a detection agent (112). In still further embodiments, the lateral flow device (100) comprises i) a capture membrane (104) comprising an immobilised capture agent (106), wherein the capture agent (106) comprises a modified oligonucleotide according to the disclosure and ii) a conjugate pad (110), wherein the conjugate pad comprises a modified oligonucleotide according to the disclosure, optionally wherein the modified oligonucleotide is conjugated to a detection agent (112).

[0161] The disclosure further provides a kit comprising lateral flow device (100) and a modified oligonucleotide according to the disclosure. The modified oligonucleotide may be comprised in a buffer, or it may be lyophilised. Instead of, or in addition to, a buffered or lyophilised oligonucleotide of the disclosure, the kit may comprise a lateral flow device (100) comprising a modified oligonucleotide immobilised on a capture membrane (104).

[0162] The disclosure further provides methods of detecting TDP-43 in liquid samples. In some embodiments, the methods comprise applying a liquid sample to a lateral flow device (100). A lateral flow device (100) comprises a sample pad (102) and a capture membrane (104) position downstream of the sample pad (102). In the context of a lateral flow device (100), the term “downstream” is to be understood as being in the direction of sample flow - i.e. away from the sample pad (102).

[0163] The capture membrane (104) further comprises an immobilised capture agent (106) capable of binding to TDP-43 aggregates (108). In some embodiments, the capture agent (106) may be conjugated to the008884231

[0164] 20

[0165] capture membrane (104) directly, or the capture agent (106) may be conjugated to an intermediary agent (120) which is in turn conjugated to the capture membrane (120).

[0166] After applying a sample to the sample pad (102) in the presence of a detection agent (112), the sample migrates through the capture membrane (104). If TDP-43 aggregates (108) are present in the sample, the capture agent (106) binds the TDP-43 aggregates (106) thereby immobilising the TDP-43 aggregates (106). Immobilised TDP-43 aggregates (108) are detected by a signal generated by the detection agent (112). At least one of the TDP-43 binder (114) or the capture agent (106) comprises a modified oligonucleotide of the disclosure.

[0167] The capture agent (106) may comprise biotin which can bind to avidin / streptavidin which acts as an intermediary agent (120) which is itself bound to the capture membrane (104). In such a configuration, the capture agent (106) may be present in the lateral flow device (100) before applying the sample to be analysed to the sample pad (102), or the capture agent (106) may be applied to the lateral flow device (100) at the same time as the sample to be analysed. The capture agent may be, for example, a modified oligonucleotide of the disclosure or an anti-TDP-43 antibody. In other embodiments, the capture agent (106) may be an anti-TDP-43 antibody directly conjugated to the capture membrane (104).

[0168] The TDP-43 binder (114) is conjugated to a detection agent (112) capable of producing a detectable signal when bound to TDP-43 aggregates (108). Any suitable detection agent (112) may be used to produce a detectable signal for determining the presence or absence of TDP-43 aggregates (112) in a liquid sample applied to the lateral flow device (100). For example, the detection agent (112) may be colloidal gold. In some embodiments, the TDP-43 binder (114) may be a modified oligonucleotide of the disclosure or an anti-TDP-43 antibody.

[0169] In some embodiments, the detection agent (112) / TDP-43 binder (114) conjugates may be present in the lateral flow device (100) before applying the sample to be analysed to the sample pad (102), or the detection agent (112) / TDP-43 binder (114) conjugates may be applied to the lateral flow device (100) with the sample to be analysed.

[0170] In certain embodiments, the lateral flow device (100) comprises a conjugate pad (110) interposed between the sample pad (102) and the capture membrane (104) such that a liquid sample applied to the sample pad (102) will flowthrough the conjugate pad (110) before flowing through the capture membrane (104). The capture pad (110) comprises the detection agent (112) / TDP-43 binder (114) conjugate. TDP-43 aggregates (108) present in the liquid sample will bind to the TDP-43 binder (114) as the liquid sample passes through the conjugate pad (110) and thereby transport the detection agent (112) / TDP-43 binder (114) conjugates to the capture membrane (104) where the detection agent ( 112) / TDP-43 binder (114) / TDP-43 aggregate (108) complexes are immobilised as discussed above.

[0171] A lateral flow device may further comprise an absorbent pad (118) downstream of the capture membrane (104).008884231

[0172] 21

[0173] By binding selectively to aggregated TDP-43, the modified oligonucleotides of the invention can be expected to, for example, destabilise pre-existing aggregates, inhibit further TDP-43 aggregation, or promote degradation of pre-existing aggregates. Thus, the modified oligonucleotides of the invention may find use in the treatment of TDP-43 proteinopathies.

[0174] The modified oligonucleotides of the invention may be formulated as pharmaceutical compositions for clinical use and may comprise a pharmaceutically acceptable carrier, diluent or adjuvant. The composition may be formulated for topical, parenteral, intravenous, intramuscular, intrathecal, intraocular, subcutaneous, oral, inhalational or transdermal routes of administration which may include injection. Injectable formulations may comprise the selected compound in a sterile or isotonic medium.

[0175] Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In some embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the US federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognised pharmacopeia for use in animals, and more particularly in humans.

[0176] The term “carrier” refers to diluents, binders, lubricants and dis integrants. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers. The pharmaceutical compositions provided herein may include one or more excipients, e.g., solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, antioxidants or antimicrobial preservatives. When used, the excipients of the compositions will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredients, i.e. the modified oligonucleotide used in the composition. Thus, the skilled person will appreciate that compositions are provided wherein there is no incompatibility between any of the components of the dosage form. Excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0177] The invention provides the modified oligonucleotide for use in a method of treatment. Relatedly, the invention provides the modified oligonucleotide for use in a method of treating a TDP-43 proteinopathy. The invention further provides a method of treating a TDP-43 proteinopathy, the method comprising administering an effective amount of the modified oligonucleotide to a subject in need thereof. The invention further provides the use of a modified oligonucleotide according to the invention for the manufacture of a medicament for the treatment of a TDP-43 proteinopathy.

[0178] The TDP-43 proteinopathy may be selected from the group comprising or consisting of Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders008884231

[0179] 22

[0180] (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, Pick’s disease and age-related TDP-43 encephalopathy (LATE).

[0181] Methods of isolating cells & nuclei

[0182] The invention provides a method of isolating a population of cells or nuclei comprising (i) incubating a first population of cells or nuclei with the modified oligonucleotide, and (ii) isolating from the first population a subpopulation that comprises cells or isolated nuclei containing TDP-43 aggregates using a sorting method. In some embodiments, the subpopulation may be isolated based on the presence of the detectable label of the modified oligonucleotide.

[0183] “Sorting methods” or “Sorting-based analytical methods” are widely used in biological research for isolating defined populations of cells or nuclei. Suitable sorting methods include fluorescence-activated cell sorting (FACS), fluorescence-activated nuclei sorting (FANS), laser-capture single-cell microdissection, and microfluidic single-cell sorting. FACS is a long-established flow-cytometric method that enables high-throughput isolation of labelled cells based on fluorescence and light-scatter parameters (reviewed in Kuhn TM, Paulsen M, Cuylen-Haering S. Accessible high-speed image-activated cell sorting. Trends Cell Biol. 2024;34(8):657-670. doi:10.1016 / j.tcb.2024.04.007). FANS is an analogous technique adapted for isolating nuclei. These techniques permit enrichment of cells or nuclei containing TDP-43 aggregates fordownstream molecular, imaging, or biochemical analyses. The skilled person will appreciate that such methods are routinely compatible with labelled oligonucleotides and may be performed under conditions that preserve the stability and functionality of the modified oligonucleotide. In some embodiments, the sorting method may be performed without a fixation step, for example without a paraformaldehyde fixation.

[0184] ***

[0185] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0186] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

[0187] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.008884231

[0188] 23

[0189] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0190] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0191] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0192] Examples

[0193] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of howto practise the invention, and are not intended to limit the scope of the invention.

[0194] EXAMPLE 1 - Exemplary sequences of the modified oligonucleotides of the invention

[0195] The nucleic acid sequences of exemplary modified oligonucleotides are set forth here:

[0196] CGGUGUUGCU (SEQ ID N0:1)

[0197] GUGGUCCCCG (SEQ ID N0:2)

[0198] CGCUGUGGUC (SEQ ID N0:3)

[0199] AGCUGUGGCC (SEQ ID N0:4)

[0200] CGCUGGUGCU (SEQ ID NO: 5)

[0201] CGCUGUGGCU (SEQ ID NO: 6)

[0202] CGGCGUUGUU (SEQ ID NO: 7)

[0203] CGGUGUAGGU (SEQ ID NO: 8)

[0204] CUCUGUGGUG (SEQ ID NO: 9)

[0205] GUGGUCGCUG (SEQ ID NO: 10)

[0206] CGGTGTTGCT (SEQ ID NO: 11)

[0207] GTGGTCCCCG (SEQ ID NO: 12)

[0208] CGCTGTGGTC (SEQ ID NO: 13)

[0209] AGCTGTGGCC (SEQ ID NO: 14)

[0210] CGCTGGTGCT (SEQ ID NO: 15)

[0211] CGCTGTGGCT (SEQ ID NO: 16)008884231

[0212] 24

[0213] CGGCGTTGTT (SEQ ID NO: 17)

[0214] CGGTGTAGGT (SEQ ID NO: 18)

[0215] CTCTGTGGTG (SEQ ID NO: 19)

[0216] GTGGTCGCTG (SEQ ID NO: 20)

[0217] The above nucleic acid sequences are characterized by their ability to bind to the RNA recognition motif (RRM) of the TDP-43 protein. They are further characterised by shared structural characteristics, namely that the sequences include i) at least four “G” nucleotides, at least two of which are consecutive, and ii) at least two TG / GT dinucleotides or at least two UG / GU dinucleotides.

[0218] EXAMPLE 2 - Improved detection of TDP-43 aggregates in chromophore- and immunofluorescencebased IHC

[0219] Methods

[0220] PNA aptamer staining with exemplary PNA aptamer TDP-43PnaP‘ was compared to RNA aptamer TDP-43aP‘ staining in human post-mortem tissue sections using DAB immunohistochemistry (IHC) with and without an overnight paraformaldehyde fix step as follows.

[0221] Paraffin embedded tissue sections were deparaffinised using two 3-minute xylene washes, two 2-minute alcohol wases and then rehydrated in water for 5 minutes. Antigen / Epitope retrieval was performed in a 10 mM citric acid (pH 6) solution using a pressure cooker for 15 minutes.

[0222] A 30-minute peroxidase block was performed using a 3% H2O2 solution (Leica Novolink polymer detection kit), then washed with TBS.

[0223] Avidin / biotin blocking steps were performed using the Abeam Avidin / Biotin Blocking Kit (Abeam, ab64212). Tissue sections were incubated in avidin block for 15-minutes, washed once with TBS (5 minutes) before incubation with biotin block (15 minutes).

[0224] Tissue sections were washed in dH2O for 5 minutes before being incubated with either TDP-43PnaP‘or TDP-43aptfor 1 hour.

[0225] Tissue sections were then washed in dH2O (5 min), anti-biotin / HRP (1:100) in dH2O (30 mins), washed with dH2O (5 min), and then incubated with DAB solution (50 L chromogen + 1 mL DAB substrate buffer; 5 min). Tissue sections were then extensively washed with running water.

[0226] Tissue sections were counterstained using haematoxylin (2 mins) and lithium carbonate (30 seconds) before being dehydrated, cleared and mounted using standard techniques.

[0227] Control tissue sections were also prepared wherein the sections underwent an overnight PFA before staining with RNA aptamer TDP-43aP‘.

[0228] Tissue samples were also prepared for immunofluorescence IHC using TDP-43PnaP‘. Paraffin embedded sections were deparaffinised using two 3-minute xylene washes, two 2-minute alcohol wases and then rehydrated in water for 5 minutes. Antigen / Epitope retrieval was performed in a 10 mM citric acid (pH 6) solution using a pressure cooker for 15 minutes.008884231

[0229] 25

[0230] Tissue sections were washed in dF for 5 minutes before being incubated at room temperature for 1 hour in the dark with TDP-43PnaP‘ conjugated with Atto488. Tissue sections were extensively wash with running water before mounting with Vectashield and DAPI.

[0231] Results

[0232] No detectable signal was present in unfixed tissue sections from a patient with confirmed TDP-43 proteinopathy using RNA aptamer TDP-43aP‘ (Figure 4A). In contrast, in tissue sections derived from the same patient which were fixed overnight in PFA, abundant signal was detected using TDP-43aP‘ (Fig 4B). These results confirm that in order for TDP-43aP‘ to cross-react with TDP-43 aggregates, a fixation step is required.

[0233] In contrast, the PNA aptamer TDP-43PnaP‘ is shown to bind strongly to TDP-43 under non-fixation conditions (Figure 4C). In particular, TDP-43PnaP‘ reveals pathogenic TDP-43 aggregates present in neuronal cytoplasm and nuclei (Figure 4C; which and black closed arrows, respectively) as well as pathological TDP-43 aggregates present in glial cytoplasm (Figure 4C; open black arrows).

[0234] In addition, TDP-43PnaP‘ is able to detect pathological TDP-43 in fluorophore-based IHC assays (Figure 5). Figure 5 demonstrates (i) neuronal cytoplasmic (dark arrows) TDP-43 aggregation and a control cell with no cytoplasmic staining (white arrow).

[0235] Together, these results demonstrate that TDP-43PnaP‘ is surprisingly able to detect TDP-43 pathology without a fixation step, and this is likely due to improved target affinity compared to TDP-43aP‘.

[0236] These results further indicate that PNA aptamers specific for the TDP-43 RRM domain (i.e. the modified oligonucleotides of the disclosure) can be used to detect TDP-43 pathology without the need for a fixation step in IHC methods.

[0237] These results further support the use of the modified oligonucleotides of the disclosure in diagnostic and research contexts where a fixation step is not required, or not possible, such as live cell imaging, cell sorting, IHC and point of care diagnostic tests.

[0238] EXAMPLE 3 - TDP-43PnaP‘ detects cytoplasmic TDP-43 aggregates in C9orf72 iPSC-derived motor neurons under non-stressed conditions

[0239] Methods

[0240] Human Induced Pluripotent Stem Cells (hiPSCs) were thawed, added to 9 ml_ of complete Essential 8 (E8) medium (85850, STEMCELL Technologies), and centrifuged at 1000 * g for 1 min. Cells were resuspended in 1 mL of complete E8 supplemented with 100 pM ROCK inhibitor (R&D Systems) and added dropwise to 1 mL of E8 containing 100 pM ROCK inhibitor in a 6-well plate coated with Geltrex™ (A1413302, Thermo Fisher Scientific). After 24 h, the medium containing ROCK inhibitor was removed and replaced with E8 medium alone.008884231

[0241] 26

[0242] hiPSCs were passaged upon reaching 70-85% confluency. Cells were then plated onto Geltrex™-coated plates and incubated overnight at 37°C in a humidified incubator with 5% CO2.

[0243] Motor neuron differentiation was performed using a protocol developed by the Patani Lab (Hall et al, 2017; the protocol is hereby incorporated by reference). hiPSCs were propagated as adherent monolayers in 2 mL of Essential 8 medium (85850, STEMCELL Technologies) on Geltrex™-coated 6-well plates (A1413302, Life Technologies). Cells were passaged at approximately 80% confluency using 0.5 M EDTA. All cultures were maintained at 37°C with 5% CO2. Prior to induction, all lines were STR-profiled and routinely tested for mycoplasma throughout the differentiation process.

[0244] For induction, hiPSCs were plated to 100% confluency in neural maintenance medium, a chemically defined medium consisting of DMEM / F12 GlutaMAX (10565018, Life Technologies) and Neurobasal medium without phenol red (12348017, Life Technologies), supplemented with N2 (17502048), B27 (17504044), L-glutamine (35050038), p-mercaptoethanol (21985023), insulin (I9278, Sigma), and non-essential amino acids (11140050, Life Technologies).

[0245] Neural induction was achieved through dual SMAD inhibition by culturing cells in neural induction medium supplemented from day O to day 7 with 1 M dorsomorphin (3093 / 10, Tocris Bioscience), 2 pM SB431542 (1614 / 10, Tocris Bioscience), and 3.3 pM CHIR99021 (4423 / 10, Tocris Bioscience).

[0246] CHIR99021 was included to prevent early forebrain specification. The neuroepithelial layer was enzymatically dissociated using dispase (1 mg / mL; GIBCO) between days 4 and 5.

[0247] From day 8, cells were patterned toward a ventral spinal cord fate using neural patterning medium I supplemented with 0.5 pM retinoic acid and 1 pM purmorphamine for 7 days. At day 14, spinal cord neural precursors were further treated with neural patterning medium II containing 0.1 pM purmorphamine for an additional 4 days. The neuroepithelium was again dissociated using dispase (1 mg / mL; GIBCO) between days 10 and 12.

[0248] Between days 25 and 30, cells were dissociated using Accutase and plated onto final assay plates.

[0249] Plates were coated with poly-L-ornithine overnight at 37°C, washed once with DPBS the following day, then coated with Geltrex™ and incubated at 37°C for 1 h. Cells were gently washed with DPBS and plated at the required densities in motor neuron medium, consisting of neural maintenance medium supplemented with Compound E. Medium was changed every three days.

[0250] TDP^SP^P* staining in cultured adherent cells

[0251] TDP-43PnaP‘ was fluorescently labelled with Atto488. Atto488 was either conjugated via the s-amino group of a lysine residue at a terminus of the PNA (AC-10mer-Lys(Atto488)-NH2), or Atto488 was conjugated via two amino-hexanoic acid (AEEA) linker moieties (Atto488-2*[AEEA]-10mer-NH2).

[0252] Protease III (1:15 dilution; BaseScope™ kit, Cat. 322381; Protease III Cat. 322337, Advanced Cell Diagnostics) was pre-warmed for 10 min at 40°C. Blocking buffer was prepared using 5% BSA and 0.3% Triton X-100 in 1 * DPBS. Coverslips or 96-well plates were treated with Protease III for 10 min at room temperature using the pre-warmed solution, followed by two washes in 1 * DPBS (2 min each).008884231

[0253] 27

[0254] Samples were then blocked in blocking buffer for 1 h at room temperature before incubation with Atto488-conjugated TDP-43PnaP‘ at a 1:100 dilution in DPBS (78 M) overnight. Following incubation, samples were washed twice with 1* DPBS, with the final wash containing DAPI at a 1:10,000 dilution for nuclear staining. Samples were washed twice more in 1 * DPBS and either mounted and sealed (coverslips) or stored in 1* DPBS (96-well plates).

[0255] TDP^SP^P* staining in cells grown as organoids in culture

[0256] Slides containing fixed organoid sections stored at -20 °C were removed and incubated on a 40 °C heat block for 10 min to promote tissue adhesion. Protease III (BaseScope™ kit; Advanced Cell Diagnostics) was diluted 1:15, pre-warmed at 40 °C for 10 min, and applied to the slides for 10 min at room temperature. Slides were then washed twice in 1 * DPBS for 2 min each. Samples were blocked for 10 min at room temperature in blocking buffer consisting of 5% BSA and 0.3% Triton X-100 in 1* DPBS, followed by incubation with TDP-43 PNA aptamer (1:100 dilution; 78 M) in blocking buffer for 1 h at room temperature. After incubation, slides were washed twice in 1* DPBS, with DAPI (1:5,000; Life

[0257] T echnologies) included in the final wash for nuclear staining. Samples were washed twice more in 1 * DPBS and mounted in Mowiol and sealed (coverslips).

[0258] Microscopy

[0259] Coverslips were imaged using either a Zeiss 880 Airyscan confocal microscope with a 63*, 1.4 NA oilimmersion objective or an EVOS M5000 microscope using 20* and 40* objectives. A minimum of five images per well were acquired. Z-stacks were captured using a pinhole diameter of one Airy unit, with a minimum of five optical slices per stack, and displayed as maximum-intensity projections. Image analysis was performed using Fiji (Imaged).

[0260] Ninety-six-well plates were imaged using the Opera Phenix™ High-Content Screening System (PerkinElmer) with 20* or 40* water-immersion objectives. A minimum of five fields per well were acquired. Z-stacks consisted of at least five optical sections and were displayed as maximum-intensity projections. Imaging and analysis parameters were standardised across experiments.

[0261] Results

[0262] Immunofluorescence staining using the Atto488-conjugated TDP-43PnaP‘ revealed robust cytoplasmic TDP-43 aggregates in human iPSC-derived motor neurons carrying C9orf72 expansions (three independent lines) under non-stressed basal culture conditions (Figure 6).

[0263] In contrast, no cytoplasmic TDP-43 aggregation was observed in the corresponding isogenic control lines (three lines) or in unrelated healthy control iPSC-derived motor neurons (two lines) (Figure 6). The signal was restricted to disease lines and displayed a cytoplasmic localisation consistent with pathological TDP-43 mislocalisation.008884231

[0264] 28

[0265] In addition, human iPSC-derived motor neuron organoids carrying C9orf72 expansions showed cytoplasmic TDP-43 inclusions under non-stressed conditions, whereas unrelated healthy control organoids lacked cytoplasmic aggregates (Figure 7).

[0266] The Atto488-conjugated TDP-43PnaP‘ had such high affinity that it could resolve these cytoplasmic inclusions with only a single 1-hour incubation at room temperature.

[0267] Together, these findings demonstrate that TDP-43PnaP‘ enables detection of disease-associated TDP-43 pathology in human iPSC and human iPSC-derived organoid models without the need for exogenous stress paradigms, thereby overcoming a limitation of conventional antibody-based approaches.

[0268] Furthermore, the rapid and selective target engagement in human iPSC-derived motor neuron organoids under non-stressed conditions supports the use of the modified oligonucleotide in therapeutic applications. By binding selectively to aggregated TDP-43, the modified oligonucleotides can be expected to, for example, destabilise pre-existing aggregates, inhibit further TDP-43 aggregation, or promote degradation of pre-existing aggregates.

[0269] EXAMPLE 4 - ELISA-based detection of disease-associated TDP-43 using a PNA aptamer Method

[0270] Normal / total TDP-43 protein levels were measured using a commercially available TDP-43 ELISA kit (Creative Diagnostics), in accordance with the manufacturer’s instructions (available at https: / / www.creative-diagnostics.com) except for the modifications below. Human biofluid samples comprised one control (sample 1) and six amyotrophic lateral sclerosis (ALS) cases (samples 2-7). Modifications:

[0271] In the sample addition step, 50 pL of undiluted sample was added to each well instead of 100 pL of diluted sample.

[0272] In the detection step, for assays of endogenous (total) TDP-43, the kit’s biotin-labelled antibody was used as supplied. For PNA-derived measurements, the antibody step was replaced with a biotin-labelled PNA aptamer (3'-biotin tag; 78 pM), 100 pL per well, with incubation at 37 °C for 60 minutes.

[0273] Results

[0274] Using the kit antibody, measurable concentrations of total TDP-43 were obtained in both control and ALS samples. Using the PNA aptamer, no signal was obtained for the kit standards comprising monomeric TDP-43, and no signal was obtained for the control non-ALS sample, whereas positive signals were recorded for ALS samples 2-7 (Figure 8).

[0275] These results demonstrate that TDP-43 can be detected in human biofluids from people affected by ALS at concentrations that are within the limit of detection of lateral flow tests. Additionally, under these assay conditions, the PNA aptamer selectively detects the disease-associated TDP-43 proteoforms present in ALS samples and shows no detectable binding to monomeric TDP-43.008884231

[0276] 29

[0277] References

[0278] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

[0279] Hall CE, Yao Z, Choi M, Tyzack GE, Serio A, Luisier R, Harley J, Preza E, Arber C, Crisp SJ, Watson PMD, Kullmann DM, Abramov AY, Wray S, Burley R, Loh SHY, Martins LM, Stevens MM, Luscombe NM, Sibley CR, Lakatos A, Ule J, Gandhi S, Patani R. Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS. Cell Rep. 2017 May 30; 19(9): 1739-1749. doi: 10.1016 / j.celrep.2017.05.024. PMID: 28564594; PMCID: PMC5464993.

[0280] Justine N. deGruyter, Lara R. Malins, and Phil S. Baran Biochemistry 201756 (30), 3863-3873 DOI: 10.1021 / acs. biochem.7b00536

[0281] Spence, H., Waldron, F.M., Saleeb, R.S. et al. RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS. Acta Neuropathol 147, 50 (2024). https: / / doi.org / 10.1007 / s00401-024-02705-1

[0282] Wang, F., Li, P., Chu, HC., Lo, PK. Nulciec Acids and Their Analogues for Biomedical Applications. Biosensors 12, 93 (2022). https: / / doi.org / 10.3390 / bios12020093

[0283] Xiaoxiao Wang, Mark Milne, Francisco Martinez, Timothy J. Scholl and Robert H. E. Hudson. Synthesis of a poly(Gd(lll)-DOTA)-PNA conjugate as a potential MRI contrast agent via post-synthetic click chemistry functionalization. RSC Adv., 2017, 7, 45222-45226.

[0284] For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Claims

00865116830Claims:

1. A modified oligonucleotide comprising or consisting of a TDP-43 binding oligonucleotide sequence, wherein the modified oligonucleotide comprises:i) at least four G nucleotides, at least two of which are consecutive; andii) at least two dinucleotides selected from TG or GT, or a combination thereof, or at least two dinucleotides selected from UG or GU, or a combination thereof;characterised in that the modified oligonucleotide comprises an RNA or DNA analogue.

2. The modified oligonucleotide of claim 1 , wherein the RNA or DNA analogue comprises a modified backbone and / or a modified ribose ring or a deoxyribose ring; optionallywherein the modified oligonucleotide comprises a peptide nucleic acid (PNA) backbone; and / or wherein the modified oligonucleotide comprises a neutral backbone or a positive backbone.

3. The modified oligonucleotide of claim 2, wherein the backbone comprises an N-(2-aminoethyl)glycine linkage, a diaminopyrrolidine linkage or a phosphorodiamidate linkage.

4. The modified oligonucleotide of any one of the preceding claims, wherein the modified oligonucleotide comprises a modified ribose ring comprising a methylene bridge linking the 2’ ribose oxygen and the 4’ ribose carbon; orwherein the modified oligonucleotide comprises a backbone comprising a phosphorodiamidate linkage and the modified ribose ring comprises a morpholine ring.

5. The modified oligonucleotide of any one of the preceding claims, wherein the modified oligonucleotide is:a PNA aptamer comprising / V-(2-aminoethyl)glycine linkages in the backbone;a PNA aptamer comprising diaminopyrrolidine linkages in the backbone, optionally a dapPNA or a secdapPNA aptamer;an LNA aptamer comprising methylene bridges linking the 2’ ribose oxygen and the 4’ ribose carbon of the ribose rings; ora morpholino aptamer comprising phosphorodiamidate linkages in the backbone and morpholine rings.

6. The modified oligonucleotide of any one of the preceding claims, wherein the modified oligonucleotide comprises:i) an RNA sequence having at least 80%, at least 90% or 100% sequence identity to CGGUGUUGCU (SEQ ID NO: 1), GUGGUCCCCG (SEQ ID NO: 2), CGCUGUGGUC (SEQ ID NO: 3), AGCUGUGGCC (SEQ ID NO: 4), CGCUGGUGCU (SEQ ID NO: 5), CGCUGUGGCU (SEQ ID NO: 6), CGGCGUUGUU (SEQ ID NO: 7), CGGUGUAGGU(SEQ ID NO: 8), CUCUGUGGUG (SEQ ID NO: 9), or GUGGUCGCUG (SEQ ID NO: 10); or00865116831ii) a DNA sequence having at least 80%, at least 90% or 100% sequence identity to CGGTGTTGCT (SEQ ID NO: 11), GTGGTCCCCG (SEQ ID NO: 12), CGCTGTGGTC (SEQ ID NO: 13), AGCTGTGGCC (SEQ ID NO: 14), CGCTGGTGCT (SEQ ID NO: 15), CGCTGTGGCT (SEQ ID NO: 16), CGGCGTTGTT (SEQ ID NO: 17), CGGTGTAGGT (SEQ ID NO: 18), CTCTGTGGTG (SEQ ID NO: 19), GTGGTCGCTG (SEQ ID NO: 20).

7. The modified oligonucleotide according to claim 6, wherein the RNA sequence comprises SEQ ID NO: 1 and wherein the modified oligonucleotide consists of PNA monomers comprising N-(2-aminoethyljglycine.

8. The modified oligonucleotide of any one of the preceding claims, wherein the modified oligonucleotide is conjugated to a detection agent; optionallywherein the detection agent is selected from the group comprising a fluorophore, a quantum dot, a radioisotope, for example11C,18F,68Ga,89Zr or64Cu, , optionally wherein89Zr,68Ga, and64Cu are provided as chelated metal complexes, a nucleic acid polymer, an amino acid polymer, a hybrid nucleic acid / amino acid polymer, a gold colloid, biotin, streptavidin or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin, a peroxidase, a horseradish peroxidase, a paramagnetic metal complex for MRI, for example56Fe or Gd( 11 l)-DOTA, or any combination thereof.

9. An in vitro method for measuring the level of TDP-43 aggregates in a sample, the method comprising:(a) providing a modified oligonucleotide according to any one of claims 1-8 immobilized on a substrate, bringing the sample and the substrate into contact with each other, and measuring the amount of TDP-43 aggregate bound to the substrate; or(b) providing a substrate with an immobilized TDP-43 binder, bringing the sample and the substrate into contact with each other such that TDP-43 aggregates in the sample are immobilized on the substrate, and bringing the sample into contact with a modified oligonucleotide according to any one of claims 1-8, and measuring the amount of TDP-43 aggregate bound to the substrate; or(c) immobilising a sample on a substrate, bringing the immobilised sample into contact with a modified oligonucleotide according to any one of claims 1-8 and measuring the amount of modified oligonucleotide bound to the sample.

10. The method according to claim 9, wherein the sample is a sample previously obtained from a patient, optionally a blood sample, serum sample, stool sample, urine sample, saliva sample, a cell sample or tissue sample.

11. The method according to claims 9 or 10, wherein the modified oligonucleotide or the TDP-43 binder is conjugated to a first member of a specific binding pair and wherein the substrate comprises a second member of the specific binding pair, such that the modified oligonucleotide or the anti-TDP-4300865116832binder immobilized on the substrate has been prepared by bringing the first member and the second member of the specific binding pair into contact with each other.12 The method according to claim 11, wherein the specific binding pair is comprised of (i) biotin and (ii) streptavidin or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin.

13. The method according to any one of claims 9 to 12, wherein the TDP-43 aggregates are measured using a method selected from any one of optical microscopy, electron microscopy, colorimetric analysis for example using a lateral flow device, electro-optical analysis, electrochemical detection, or biochemical detection, or any combination thereof.

14. The method according to any one of claims 9 to 13, wherein the substrate is a capture membrane (104) of a lateral flow device (100), an ELISA plate, or a magnetic bead.

15. A lateral flow device (100) comprising:(i) a sample pad (102);(ii) a capture membrane (104) positioned downstream of the sample pad (102), wherein the capture membrane (104) comprises an immobilised capture agent comprising a TDP-43 bindercharacterised in that the TDP-43 binder comprises a modified oligonucleotide according to any one of claims 1-8.

16. The lateral flow device (100) of claim 15, wherein the device further comprises a conjugate pad (110) interposed between the sample pad (102) and the capture membrane (104), and a detection agent (112) deposited on the conjugate pad (110).

17. The lateral flow device (100) according to claim 16, wherein the detection agent (112) comprises a modified oligonucleotide according to any one of claims 1-8 or an anti-TDP-43 antibody.

18. A lateral flow device (100) comprising:(i) a sample pad (102);(ii) a capture membrane (104) positioned downstream of the sample pad (102), wherein the capture membrane (104) comprises an immobilised capture agent comprising a TDP-43 binder; and(iii) a conjugate pad (110) interposed between the sample pad (102) and the capture membrane (104), and a detection agent (112) deposited on the conjugate pad (110) characterised in that the detection agent comprises a modified oligonucleotide according to any one of claims 1-8.0086511683319. A kit comprising a lateral flow device (100) and a modified oligonucleotide according to any one of claims 1-8, wherein the lateral flow device (100) comprises a capture agent comprising a TDP-43 binder immobilized on a capture membrane (104), and the modified oligonucleotide is suitable for detecting TDP-43 aggregates bound to the capture agent.

20. The lateral flow device (100) according to claim 18 or the kit according to claim 19, wherein the TDP-43 binder comprises an oligonucleotide according to any one of claims 1-8, or an anti-TDP-43 antibody.

21. The lateral flow device (100) according to any one of claims 15-18 or 20, or the kit according to claim 19 or 20, wherein the capture agent comprises a specific binding pair, wherein the first member (106) of the specific binding pair is conjugated to a TDP-43 binder and the second member (120) of the specific binding pair is immobilised on the capture membrane (104); optionally wherein the specific binding pair is comprised of (i) biotin and (ii) streptavidin or a protein that is functionally equivalent to streptavidin because it is also capable of binding to biotin, optionally wherein the first member of the specific binding pair is biotin; and / orwherein the detection agent comprises a colorimetric detection agent; and / orwherein the detection agent (112) comprises gold colloid, a fluorophore, a peroxidase, or a horseradish peroxidase.

22. Use of a lateral flow device according to any one of claims 15 to 18 or 20 to 22, or the kit according to claim 19 to 21, in a method of detecting TDP-43 aggregates in a sample, the method comprising the steps of:i) providing a lateral flow device or kit according to any one of claims 16 to 21 ; andii) applying the sample to the sample pad (102) and allowing the sample to migrate through the capture membrane (104) to contact the capture agent (106);wherein the presence of TDP-43 aggregates in the sample is indicated by a detectable signal generated by the detection agent (112).

23. The modified oligonucleotide according to any one of claims 1-8, the method according to any one of claims 9-14, or the lateral flow device or kit according to any one of 15-22, for use in the diagnosis of a TDP-43 proteinopathy; optionallywherein the TDP-43 proteinopathy is selected from the group comprising or consisting of Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease and age-related TDP-43 encephalopathy (LATE).

24. An imaging composition comprising the modified oligonucleotide according to any one of claims 1-13 for use in an in vivo method of medical imaging or experimental imaging.0086511683425. The imaging composition for use of claim 24, wherein the method of medical imagining or experimental imaging comprises positron emission tomography (PET) scanning, magnetic resonance imaging (MRI), or computer tomography (CT) scanning; optionallywherein the oligonucleotide comprises a radioisotope suitable for use in a method of medical imagining or experimental imaging, optionally wherein the radioisotope is selected from89Zr,18F,68Ga,64Cu,56Fe or Gd(lll)-DOTA.

26. A method of imaging TDP-43 aggregates in cells in vitro or ex vivo, the method comprising: i) incubating the cells with the modified oligonucleotide according to any one of claims 1-8; and ii) imaging the cells following incubation with the modified oligonucleotide.

27. The method of claim 26, wherein the method is performed without a fixation step, for example without a paraformaldehyde fixation.

28. The method of any one of claims 26 to 27, wherein the cells are incubated with the modified oligonucleotide for at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, or at least 3 hours.

29. The method of any one of claims 26 to 28, wherein the imaging is achieved through the detection of the detectable label of the modified oligonucleotide.

30. The method of any one of claims 26 to 29, wherein the imaging technique is selected from the group consisting of: fluorescence imaging, fluorescent immunohistochemistry, optical microscopy, confocal microscopy or electron microscopy.

31. A method of isolating a population of cells or nuclei comprising TDP-43 aggregates, the method comprising:i) incubating a first population of cells or nuclei with the modified oligonucleotide according to claim 8; andii) isolating from the first population a subpopulation that comprises cells or nuclei containing TDP- 43 aggregates.

32. The method of claim 31, wherein the method is performed without a fixation step, for example without a paraformaldehyde fixation step.

33. The method of any one of claims 31 to 32, wherein the sorting method is selected from the group consisting of: fluorescence-activated cell sorting (FACS), fluorescence-activated nuclei sorting (FANS), laser-capture single-cell microdissection, and microfluidic single-cell sorting.

34. A modified oligonucleotide according to any one of claims 1 to 8 for use in a method of treatment.0086577683535. A modified oligonucleotide according to any one of claims 1 to 8 for use in the treatment of a TDP-43 proteinopathy.

36. The modified oligonucleotide for use according to claim 35, wherein the TDP-43 proteinopathy is selected from the group comprising or consisting of: Amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis frontotemporal dementia spectrum disorders (ALSFTSD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease, Lewy body dementia, Huntington’s disease, argyrophilic grain dementia, Perry syndrome, progressive supranuclear palsy, corticobasal degeneration, Pick’s disease and Limbic-predominant age-related TDP-43 encephalopathy (LATE).

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