Modulation of gene expression

Abstract

The present disclosure relates generally to methods for modulating the expression of a heterologous transgene administered to a cell, and to nucleic acid constructs useful in such methods. The present disclosure also relates to the use of the nucleic acid constructs for enhancing motor neuron survival, inhibiting motor neuron degeneration and treating neurodegenerative conditions.

Description

TITLE “MODULATION OF GENE EXPRESSION” RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Patent Application No. 2023902151 entitled "Modulation of Gene Expression" filed 5 July 2023, the contents of which are incorporated herein by reference in their entirety. FIELD

[0002] The present disclosure relates generally to methods for modulating the expression of a heterologous transgene administered to a cell, and to nucleic acid constructs useful in such methods. The present disclosure also relates to the use of the nucleic acid constructs for enhancing motor neuron survival, inhibiting motor neuron degeneration and treating neurodegenerative conditions. BACKGROUND

[0003] Gene therapy offers promising avenues for the treatment of myriad human diseases. Gene therapy has most commonly been investigated and achieved using viral vectors, with notable recent advances being based on adeno-associated virus (AAV)-based vectors. AAV vectors (also referred to as recombinant AAV, rAAV) that contain a genome that lacks some, most or all of the native AAV genome and instead contains one or more heterologous sequences flanked by the ITRs have been successfully used in gene therapy settings. These AAV vectors are widely used to deliver heterologous nucleic acid to cells of a subject for therapeutic purposes, serving for example to either overexpress a therapeutic transgene or inhibit expression of a pathogenic gene or allele. Recombinant AAV vectors are increasingly showing huge therapeutic promise in targeting organs and cells for in vivo gene delivery.

[0004] There is increasing interest in potential for gene therapy to treat diseases of the central nervous system, including spinal muscular atrophy (SMA) and motor neuron diseases (MND). Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease and refers to the selective degeneration of upper and lower motor neurons of the brain and spinal cord, respectively. ALS and the related disease frontotemporal dementia (FTD), the second most common form of early-onset dementia, sit within a spectrum of disease.15% of ALS patients demonstrate co-morbidity with FTD and more than 50% of ALS patients display cognitive changes.

[0005] The etiology of ALS and FTD remains poorly understood, however most ALS patients and more than half of all FTD patients share common histopathological features. Post- mortem analysis of ALS-patient brain and spinal cord tissue frequently reveals the presence of tau- negative, ubiquitin-positive aggregates which appear as round or skein-like inclusions, most commonly in the cytoplasm of affected neurons and glia. These inclusions are speckled with ubiquitin, sqstm1, ubiquilin 1 and ubiquilin 2 – proteins that are all involved in ubiquitin-mediated protein turnover, suggesting that defective proteasomal clearance is a contributing factor in ALS / FTD pathogenesis.

[0006] The major component of these ubiquitin-positive aggregates is Transactive Response DNA binding protein of 43 kDa (TDP-43). This predominantly nuclear protein is found to translocate from the nucleus to the cytoplasm in the brain and / or spinal cord of ALS / FTD patients. Characterization of sarkosyl-insoluble fractions from patient brain lysates reveals a striking shift in the biochemical profile of TDP-43. In patient lysates, TDP-43 is poly-ubiquitylated, hyper- phosphorylated and cleaved at the C-terminus. TDP-43 proteinopathy has now been identified in over 98% of ALS cases and in over 50% of FTD cases regardless of familial or sporadic origin, making TDP-43-positive cytoplasmic aggregates or inclusions a pathological hallmark of ALS / FTD.

[0007] Contrary to its insoluble, aggregated pathological form, soluble TDP-43 (sTDP- 43) is required for normal cellular function. In normal, healthy neurons TDP-43 remains soluble, predominantly resides in the nucleus, and participates in several aspects of RNA metabolism, such as pre-mRNA splicing, RNA stability, RNA transport and miRNA processing, and is necessary for neuronal viability. The presence of a nuclear localization sequence (NLS) and a nuclear export sequence in the N-terminus of the protein allow sTDP-43 to shuttle between the nucleus and cytoplasm. sTDP-43 is also known to regulate mRNAs involved in the development of neurons and embryos and is expressed throughout central nervous system (CNS) development into adulthood. As such, it is understood that sTDP-43 is an essential RNA binding protein. Alterations in its ability to carry out its cellular roles are toxic to neuronal cells. While a gain-of-function mechanism due to the accumulation of insoluble TDP-43 aggregates in the cytoplasm is thought to contribute to neurodegeneration, it has also been postulated that a loss of TDP-43 splicing repression, resulting from the depletion of nuclear-located TDP-43 leads to neuron loss in ALS and FTD.

[0008] While the prospect of being able to treat diseases such as ALS and FTD using gene therapy approaches gains increasing attention, there remains a need for the development of gene vectors allowing improved control over transgene expression. Currently, transgene expression is typically constitutive. While inducible systems are available, typically in which gene expression can be activated in the presence or absence of a chemical inducing agent, these approaches are not feasible for translation to the clinical setting as exogenous chemical inducing agents are either toxic or have undesirable off-target effects making their in vivo human use unfeasible. Alternative means of modulating expression of therapeutic transgenes are needed. There is also a need for the development of gene therapies that are disease-responsive, i.e. are regulated by the underlying disease mechanism associated with ALS and FTD, without requiring an external inducing agent to modulate gene expression. SUMMARY

[0009] In a first aspect, the present disclosure provides a method for modulating the expression of a heterologous transgene administered to a cell, wherein the heterologous transgene is located in a nucleic acid construct comprising a TDP-43 responsive binding element operably linked at the 5' end of the heterologous transgene, wherein the TDP-43 binding element and the transgene are operably linked to a promoter capable of driving expression of the transgene in the cell,wherein in the presence of nuclear TDP-43, the TDP-43 binds to the TDP-43 responsive binding element repressing expression of the transgene and wherein in the absence of nuclear TDP-43 the transgene is expressed, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein modulation of expression of the transgene is achieved by regulation of RNA splicing of the first and second nucleic acid sequences, such that in the absence of nuclear TDP-43 the first and second nucleic acid sequences are removed from the nucleic acid construct by RNA splicing resulting in expression of the transgene.

[0010] A second aspect of the present disclosure provides a nucleic acid construct comprising a heterologous transgene operably linked to a TDP-43 responsive binding element 5' to the transgene, wherein the transgene and the TDP-43 responsive binding element are operably linked to a promoter capable of driving expression of the transgene in a cell, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G.

[0011] In accordance with the above aspects the first and second nucleic acid sequences comprise intronic splice sites at the 5' and 3' ends. Typically, the first and second nucleic acid sequences comprise a GA dinucleotide at the 5' end. Typically, the first and second nucleic acid sequences comprise an AG dinucleotide or CAG trinucleotide at the 3' end.

[0012] In an embodiment of the above aspects, nuclear TDP-43 binds to one of the first and second nucleic acid sequences. In another embodiment, nuclear TDP-43 binds to both the first and second nucleic acid sequences.

[0013] In accordance with the above aspects, at least about 80%, 85%, 90% or 95% of the nucleotides of the region comprising at least about 30 consecutive nucleotides in the first nucleic acid sequence and / or second nucleotide sequence may be U or G. Said region may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. Said region may comprise one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. Said region may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. Said region may comprise two, three, four, five, six, seven or more of said consecutive motifs.

[0014] In accordance with the above aspects, at least about 80%, 85%, 90% or 95% of the first nucleic acid sequence may be U or G. The first nucleotide sequence may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. The first nucleotide sequence may comprise one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. The first nucleotide sequence may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. Thefirst nucleotide sequence may comprise two, three, four, five, six, seven or more of said consecutive motifs.

[0015] Alternatively, or in addition, at least about 80%, 85%, 90% or 95% of the second nucleic acid sequence may be U or G. The second nucleotide sequence may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. The second nucleotide sequence may comprise one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. The second nucleotide sequence may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. The second nucleotide sequence may comprise two, three, four, five, six, seven or more of said consecutive motifs.

[0016] In exemplary embodiments of the above aspects, the region comprising at least about 30 consecutive nucleotides in the first nucleic acid sequence and / or second nucleotide sequence may comprise or be located within the sequence set forth in SEQ ID NO:10, 15 or 19 or a sequence having at least about 80% sequence identity thereto.

[0017] In accordance with the above aspects, optionally the third nucleic acid sequence comprises a cryptic exon, wherein the cryptic exon does not contain any stop codons. The cryptic exon may encode a cryptic peptide. In exemplary embodiments, the cryptic exon may comprise the sequence set forth in SEQ ID NO:8, 13 or 17, or a sequence having at least about 80% sequence identity thereto.

[0018] In an embodiment in accordance with the above aspects in which the cryptic exon encodes a cryptic peptide, a nucleotide sequence encoding a degradation domain may be incorporated at the 3' end of the cryptic peptide-encoding sequence, and a linker is introduced at the 5' end of the heterologous transgene, such that expression of the cryptic peptide produces a fusion peptide comprising the cryptic peptide and the degradation domain, wherein the presence of the degradation domain facilitates auto-degradation of the fusion peptide.

[0019] In accordance with the method of the first aspect, the absence of nuclear TDP- 43 may be caused by the cytoplasmic accumulation of insoluble TDP-43.

[0020] In accordance with the method of the first aspect, the level of expression of the heterologous transgene may be responsive to levels of nuclear TDP-43, such that a reduction in the level of nuclear TDP-43 results in an increase in the level of expression of the transgene, and an increase in the level of nuclear TDP-43 result in a decrease in the level of expression of the transgene.

[0021] In exemplary embodiments of the method of the first aspect, the cell may be a cell of the central nervous system or peripheral nervous system. For example, the cell may be a neuron or a glial cell. Optionally, the promoter may be capable of driving expression in a cell of the central nervous system or peripheral nervous system, optionally a neuron, or a glial cell. Optionally the promoter is a constitutive promoter.

[0022] In accordance with the above aspects, the heterologous transgene may be a therapeutic transgene that encodes a gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43. The neurodegenerative condition may be selected from amyotrophic lateralsclerosis (ALS) and frontotemporal dementia (FTD). The heterologous transgene may encode a therapeutic gene product useful for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or for restoring nuclear TDP-43 function. In exemplary embodiments, the transgene may encode, for example, a cyclin F polypeptide.

[0023] In accordance with the above aspects, the heterologous transgene may encode a detectable and / or measurable marker useful for detecting nuclear TDP-43 depletion, and is therefore useful for diagnosing a neurodegenerative condition, impaired neuron function or neuronal degeneration. In exemplary embodiments, the transgene may encode, for example, a neuronally-expressed protein that is detectable in the blood and / or in the cerebrospinal fluid.

[0024] In accordance with the above aspects, the nucleic acid construct may comprise more than one heterologous transgene, operably linked to the same or different promoters. For example, the nucleic acid construct may comprise two heterologous transgenes, operably linked to the same or different promoters. By way of example only, a first heterologous transgene may encode a therapeutic gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or for restoring nuclear TDP-43 function, and a second heterologous transgene may encode a detectable and / or measurable marker useful for detecting nuclear TDP-43 depletion, and is therefore useful for diagnosing a neurodegenerative condition, impaired neuron function or neuronal degeneration.

[0025] Another aspect of the invention provides a vector comprising a nucleic acid construct of the present invention. In an exemplary embodiment, the vector is a plasmid or a viral vector.

[0026] Another aspect of the invention provides a host cell comprising a nucleic acid construct or vector of the invention.

[0027] Another aspect of the invention provides a method for treating a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising administering to a subject in need thereof a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, and wherein nuclear depletion of soluble TDP-43 results in expression of the transgene, thereby treating the neurodegenerative condition.

[0028] Other aspects of the invention provide methods for enhancing survival of a neuron, for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, and for restoring nuclear TDP-43 function, the methods comprising contacting or exposing the neuron to a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a gene product useful for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, or inhibiting aggregated or insoluble TDP-43accumulation in a neuron and restoring nuclear TDP-43 function, and wherein nuclear depletion of soluble TDP-43 results in expression of the transgene.

[0029] A further aspect of the invention provides a method for diagnosing in a subject a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, for diagnosing or detecting impaired neuron function or for diagnosing or detecting neuronal degeneration, comprising: (a) administering to the subject a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a detectable and / or measurable marker; and (b) obtaining from the subject a biological sample and analysing the sample to detect the presence or absence of the marker, wherein the presence of the marker in the sample is indicative of nuclear depletion of soluble TDP-43 and of the subject having the neurodegenerative condition, or having impaired neuron function or neuronal degeneration.

[0030] In exemplary embodiments, the biological sample comprises a blood sample or cerebrospinal fluid. In exemplary embodiments, the marker comprises a neuronally-expressed protein or polypeptide.

[0031] Another aspect of the invention provides a method for monitoring the response of a subject undergoing therapeutic treatment for a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising the steps of: (a) administering to the subject a nucleic acid construct or vector of the present disclosure before or after commencement of the therapeutic treatment, wherein the heterologous transgene encodes a detectable and / or measurable marker; (b) obtaining from the subject a first biological sample at a first time point and analysing said sample for the presence or amount of said marker; (c) obtaining from the subject at least a second biological sample at a subsequent time point and analysing said sample for the presence or amount of said marker; and (d) comparing the levels of the marker from the first and second samples, wherein a reduction in the level of the marker in the second sample relative to the first sample is indicative of a positive response of the subject to the therapeutic treatment.

[0032] The method may further comprise obtaining from the subject one or more further biological samples at one or more subsequent time points and comparing levels of the marker in said samples to the levels in the first and second samples.

[0033] In exemplary embodiments, the first and second biological samples, and optionally the one or more further samples, comprise blood samples or cerebrospinal fluid. In exemplary embodiments, the marker comprises a neuronally-expressed protein or polypeptide.

[0034] Another aspect of the invention provides a method for treating a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising:(a) administering to the subject a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a gene product useful in the treatment of the neurodegenerative condition; and (b) administering to the subject a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a detectable and / or measureable marker, wherein the presence or level of the marker of (b) in at least one biological sample, optionally a blood sample, from the subject is indicative of the efficacy of the product of the transgene of (a) in the treatment of the neurodegenerative condition.

[0035] In an embodiment, the transgene encoding the gene product useful in the treatment of the neurodegenerative condition and the transgene encoding the marker are present in the same nucleic acid construct, such that steps (a) and (b) are simultaneous and comprise the administration of a single nucleic acid construct or vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Embodiments of the present invention are described herein, by way of non- limiting example only, with reference to the following figures.

[0037] Figure 1 - Schematic representation of TDP-43 regulated expression of cryptic exons. In normal (healthy) cells nuclear TDP-43 represses inclusion of a cryptic exon (CE) in the final spliced isoform of an mRNA molecule by virtue of binding to intronic regions flanking the CE. In this scenario, the full intact mRNA encoding the gene is expressed, leading to translation of the complete protein. In the case of ALS / FTD (MND state), TDP-43 is depleted from the nucleus (forming insoluble cytoplasmic aggregates (inclusions), thereby exposing the cryptic exon for splicing inclusion. The cryptic exon typically contains multiple stop codons (in-frame UAA, UAG and UGA nucleotides) preventing translation of the complete protein. The mRNA containing the cryptic exon sequence may then be recognised by the cellular translation machinery as nonsense sequence and subsequently the mRNA is degraded via nonsense-mediated decay (Option 1), or alternatively protein may be produced but is truncated due to the presence of the premature stop codons in the CE (Option 2). Recent studies have reported a newly identified type of cryptic exon where there are no internal stop codons, which results in the expression of the cryptic sequence (referred to as a cryptic peptide) within the protein (Option 3).

[0038] Figure 2 – (A) Schematic representation of the operation of a gene expression modulation switch of the present disclosure regulating cyclin F-mediated clearance of cytoplasmic TDP-43. (B) Schematic describing depletion of nuclear TDP-43 in a disease state (top panel) and restoration of nuclear TDP-43 upon activation of a gene expression modulation switch of the present disclosure (bottom panel). TO-cyclin F, therapeutically-optimised cyclin F.

[0039] Figure 3 – Schematic overview of an exemplary gene expression modulation switch based on inclusion of a Cryptic Exon (CE) within a reporter cassette. (A) The CE and surrounding flanking intronic regions from a gene (e.g. CE and flanking introns between exon 20 and 21 of UNC13A) are inserted between an upstream reporter (e.g. an mCherry reporter) and a downstream therapeutic transgene and an eGFP reporter. T2A, a self-cleaving peptide is positioned at the 5' end of the therapeutic transgene to enable translational separation from the mCherryreporter and a cryptic peptide encoded by the CE. The entire transgene cassette is regulated by a CMV promoter for constitutive expression. (B) In the healthy state, binding of TDP-43 prevents inclusion of the CE during splicing, and transcription is terminated at a premature stop codon incorporated out of frame within the T2A sequence. The only product is the mCherry reporter protein. (C) In the disease (MND) state, nuclear TDP-43 is depleted resulting in a lack of binding and inclusion of the CE in the final mRNA product. This shifts the premature stop codon out of frame resulting in transcription of all coding domains, resulting in one product comprising mCherry- cryptic peptide-T2A and another product comprising the transgene product and eGFP. The skilled person will appreciate that in the clinical setting the upstream and downstream reporters are optional.

[0040] Figure 4 - Schematic representations of three alternate gene expression modulation switch cassettes (Series 1 - Series 3) of the present disclosure as described herein. CE, cryptic exon; CP, cryptic peptide; DD-FKBP and DD, degradation domain; T2A, self-cleaving peptide.

[0041] Figure 5 - Schematic representation of three exemplary alternate gene expression modulation switch cassettes (Series 1 - Series 3) demonstrating the expression of a biomarker transgene, with and without a therapeutic transgene, of the present disclosure as described herein. CE, cryptic exon; CP, cryptic peptide; DD-FKBP and DD, degradation domain; T2A, self-cleaving peptide.

[0042] Figure 6 - Exemplary neuron-specific cryptic exons and flanking UG rich introns (from Jeong et al., 2017, Mol Neurodegener 12:13).

[0043] Figure 7 - STMN2 cryptic exon inclusion in ALS post-mortem brain tissue. RNA sequencing (RNA-seq) was performed upon post-mortem tissue from six CNS regions from 25 sporadic ALS cases and 11 healthy controls (n = 184 samples). Expression of the STMN2 cryptic exon was detected by the presence of truncated STMN2 mRNA within the RNA-seq dataset. Cases are considered positive if the truncated STMN2 mRNA is present above a pre-set threshold of 2 or more uniquely mapped reads. ALS cases were subcategorised by their TDP-43 pathology stage. Six CNS regions varying in their presentation of TDP-43 pathology were examined: motor cortex and spinal cord (pathology in stages 1-4), prefrontal cortex (pathology in stages 2-4), hippocampus (pathology in stage 4), occipital cortex and cerebellum (rare to nil TDP-43 pathology). The number of examined cases per condition is shown to the right of the dotted line.

[0044] Figure 8 – Images of cells transfected with a construct containing the UNC13A- CE with eGFP reporter (left panel) versus untransfected cells (right panel). Note that the UNC13A- CE sequence has been modified to remove 5 internal stop codons and an unwanted alternative splice site. Under basal conditions, low-level eGFP expression was observed (confirmed in three independent experiments), demonstrating that the UNC13A-CE construct works in achieving splicing and readthrough of the CE.

[0045] Figure 9 – Cells expressing UNC13A-CE-eGFP (via prior transfection) were treated with siRNA to knockdown endogenous TDP-43 (right panel) results in a 53% increase in mean eGFP expression (measured by flow cytometry analysis) versus scrambled siRNA (control; left panel).

[0046] Figure 10 - Endogenous TDP-43 is depleted by siRNA (TARDBP) over 72 hours. HEK293T cells with endogenous mCherry tagged TDP-43 were treated with 100nM of siRNA (TARDBP) or siRNA (scrambled) and continuously monitored over 72 hours using Incucyte® live- cell imaging. The red channel measured endogenous TDP-43-mCherry. Results are shown as TDP- 43-mCherry fluorescence intensity of cells treated with siRNA (TARDBP) (green) or siRNA (scrambled) (red). Two biological replicates per condition were included in analysis.

[0047] Figure 11 - UNC13A-CE switch expresses eGFP in more cells following siRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressing mCherry-UNC13A-CE switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or siRNA (TARDBP) and monitored continuously over 92 hours via Incucyte® live-cell imaging. The UNC13A-CE switch was activated from 20 hours onwards. Results are shown as the number of eGFP-positive cells following siRNA (TARDBP) treatment (green) or siRNA (scrambled) treatment (red).

[0048] Figure 12 - UNC13A-CE switch expresses higher eGFP levels in cells following siRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressing mCherry-UNC13A-CE switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or siRNA (TARDBP) and monitored continuously over 92 hours via Incucyte® live-cell imaging. The UNC13A-CE switch was activated from 20 hours onwards. The increase in the mean eGFP-fluorescence intensity in eGFP-positive cells following siRNA (TARDBP) treatment versus siRNA (scrambled).

[0049] Figure 13 - SYT7-CE switch expresses eGFP in more cells following siRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressing mCherry-SYT7-CE switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or siRNA (TARDBP) and monitored continuously over 92 hours via Incucyte® live-cell imaging. The SYT7-CE switch was activated from 20 hours onwards. Results are shown as the number of eGFP- positive cells following siRNA (TARDBP) treatment (green) or siRNA (scrambled) treatment (red).

[0050] Figure 14 - SYT7-CE switch expresses higher eGFP levels in cells following siRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressing mCherry-SYT7-CE switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or siRNA (TARDBP) and monitored continuously over 92 hours via Incucyte® live-cell imaging. The SYT7-CE switch was activated from 20 hours onwards. Results are shown as the mean eGFP-fluorescence intensity in eGFP-positive cells following siRNA (TARDBP) treatment (green) or siRNA (scrambled) treatment (red).

[0051] Figure 15 – Endogenous TDP-43 is depleted by shRNA (TARDBP) over 8 days. Endogenously labelled TDP-43-mCherry HEK293T cells were transduced with lentivirus-shRNA (TARDBP), and treated with 0.1µg / ml tetracycline to induce shRNA expression. mCherry-TDP-43 fluorescence was continuously monitored over 8 days with Incucyte® live-cell imaging. Control cells transduced with the lentivirus-shRNA (TARDBP) but not treated with tetracycline were also included. Results are shown as the number of mCherry-positive cells following treatment with tetracycline (green) or no treatment (red) (normalized to initial level).

[0052] Figure 16 – UNC13A-CE switch expresses eGFP in more cells following shRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressingmCherry-UNC13A CE switch-eGFP and stably with lentivirus-shRNA (TARDBP), were treated with 0.1µg / ml tetracycline and monitored continuously over 8 days via Incucyte® live-cell imaging. Control cells transiently transfected with a plasmid expressing mCherry-UNC13A CE switch-eGFP and stably with the lentivirus-shRNA (TARDBP), but not treated with tetracycline were also included. Results are shown as the number of eGFP-positive cells following treatment with tetracycline (green) or no treatment (red).

[0053] Figure 17 – UNC13A-CE switch expresses higher eGFP levels in cells following shRNA knockdown of TDP-43 levels. HEK293T cells transiently transfected with a plasmid expressing mCherry-UNC13A CE switch-GFP and stably with lentivirus-shRNA (TARDBP), were treated with 0.1µg / ml tetracycline and monitored continuously over 8 days via Incucyte® live-cell imaging. Control cells transiently transfected with a plasmid expressing mCherry-UNC13A CE switch-eGFP and stably with the lentivirus-shRNA (TARDBP) but not treated with tetracycline were also included. The results are shown as the mean eGFP-fluorescence intensity in treated cells (green) versus untreated cells (red).

[0054] Figure 18 – shRNA knockdown of TDP-43 levels over an 8-day timecourse. HEK293T were prior transduced with lentivirus expressing NIR-shRNA (TARDBP). These cells were treated with 0.1µg / ml tetracycline on day 0, and refreshed at 2 days and 18 hrs. The cells were transfected with the plasmid expressing mCherry-UNC13A CE switch-eGFP at 2 days and 18 hrs. Expression of the NIR-tagged shRNA (TARDBP) was detected and shown as mean fluorescence intensity within a field of view.

[0055] Figure 19 - UNC13A-CE switch expresses eGFP in more cells following shRNA knockdown of TDP-43 levels over an 8-day timecourse. HEK293T were prior transduced with lentivirus expressing NIR-shRNA (TARDBP). These cells were treated with 0.1µg / ml tetracycline on day 0, and refreshed at 2 days and 18 hrs. The cells were transfected with the plasmid expressing mCherry-UNC13A CE switch-eGFP at 2 days and 18 hrs. Expression of eGFP reporter (expressed when the UNC13A-CE switch is activated) was detected, with results shown as the number of eGFP-positive cells within a field of view.

[0056] Some figures and text contain color representations or entities. Color illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office. TABLE 1 BRIEF DESCRIPTION OF EXEMPLARY SEQUENCES S I 1 C A G C A GG T CAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCMV promotor, DNA CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC sequence CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA A G A C G G C A C C T C C T C C T C C T C C G A G A T T A T A T GT C AAGTATTGATAATTGGGTTCATAATTTCTGAATATTTAGATGGATGGTTGTGAGTGGCTGGTGGACAGACGAAAAATGGATGGTTGGATAAATTGATGGG TGGATGGATGGTTGGTTGTATGAAAGAATGAATGATTGGGTAGGTGGATT T T A C G U U A U A U G U C G G U U U A C G C C A C A G C G C T C C T A G T T C C G A G T C G A G CG A GCTGTTGGGATTCTTCAGCCTGCTCTGGGTGGCACAAGGCAAAGGCATGGAGGGACTTCACTGTAGTCGCTGCTGTCTCTTCCAAGACCCTGTCAGCTGC GAGATCTGAGGACATGCCCTCTCCCAGCTTGGCAAAGGAGGCCCTATCAT G G G C T T G T G U C G A G C G A G C U G G G C U U G U G T T C G T T C C G G C G G A G T A C A G T A C C C U G Gunderlined), RNA G sequence CCCACUGUGUGCCCGGCCCCGGGGUCCUGGUCCCUUCUGCCCCCGCAGGACUUGCAGUUCUUGUUUCUUCUUGUAUCUCCCUGUUUUCUCCCCUCUCUCU CUGCCCCUCCAG A A G A T C C T G T G G G G C C T C T A C T A C T C C C G C T A G G A G G G C A A A G T T G G E F W E Y S Y A A D PL Q Q C QESSDEEACPEDKGPQDPQALALDTQIPATPGPKPLVRTSREPGKDVTTSGYSSVSTASPTSSVDGGLGALPQPTSVLSLDSDSHTQPCHHQARKSCLQC RPPSPPESSVPQQQVKRINLCIHSEEEDMNLGLVRL A A G A T C C T G T G G G G C C T C T A C T A C T C C C G C T A G G A G G G C A A A G T T G G a E F W E Y S Y A A DP >MRL>AAA region Q Q L LGSFLDWSLDCCSGYEGDQESEGEKEGDVTAPSGILDVTVVYLNPEQHCCAAYIA (SEQ ID QESSDEEACPEDKGPQDPQALALDTQIPATPGPKPLVRTSREPGKDVTTS NO:38) GYSSVSTASPTSSVDGGLGALPQPTSVLSLDSDSHTQPCHHQARKSCLQC T A G C A C C T C A A C A C G G(For use with +2bp CEs like UNC13A)27 T2A, DNA sequenceGGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGA GAATCCTGGCCCA 2 C 2 A A A G G T 3 A G C G T C A G C T C C A G 3 3 3 3 3 3 3 3 3 4 4(nucleic acid) DETAILED DESCRIPTION Definitions

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0058] The articles “a”, “an” and “the” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless context clearly indicates otherwise.

[0059] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0060] Further, the terms “about” and “approximate”, as used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variations of ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like.

[0061] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0062] As used herein, the term “administered” refers to the placement of an agent described herein, into a cell or subject by a method or route which results in at least partial localization of the compound at a desired site. An agent described herein can be administered by any appropriate route which results in effective treatment, i.e., administration results in delivery to a desired location in the cell or subject. Exemplary modes of administration to a cell include, but are not limited to transfection, transformation, transduction and conjugation. Exemplary modes of administration to a subject include, but are not limited to, injection, infusion, instillation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0063] The term “agent” includes a molecule, compound or construct that induces a desired pharmacological and / or physiological effect. The term “agent” is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents. Thus, the term “agent” extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells as described herein, as well as to transgenes contained in said nucleic acid constructs and vectors and to the products of said transgenes. Exemplary agents include nucleic acid constructs and vectors of the presentdisclosure, as well as to the products of transgenes contained therein, such as cyclin F polypeptides and nucleic acids encoding cyclin F polypeptides.

[0064] The term “construct” in the context of a nucleic acid construct refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present disclosure will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the methods of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rdedition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0065] In the context of the present disclosure, the term "cryptic exon" refers to a sequence located within an intron that can become erroneously incorporated into a mRNA transcript as a result of a TDP-43-dependent splicing error. Cryptic exons often include premature stop codons or introduce frameshift mutations, which disrupt translation and can lead to nonsense- mediated decay. In the context of the present disclosure, and specifically the use of cryptic exon sequences in nucleic acid constructs described herein, any such stop codons are removed from the cryptic exon prior to introducing the cryptic exon into the nucleic acid construct to ensure translation (under the appropriate conditions) and to prevent nonsense-mediated decay. In some cases, cryptic exons do not naturally include stop codons or introduce frameshift errors, andinstead encode cryptic peptides which can result in an elongated polypeptide gene product including the cryptic peptide sequence.

[0066] In the context of the present disclosure the term "flanking introns" refers to introns located on either side of a cryptic exon sequence. In the context of the present disclosure, the first nucleic acid sequence and the second nucleic acid sequence of nucleic acid constructs of the invention may represent flanking introns.

[0067] By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene or for the final mRNA product of a gene (e.g. the mRNA product of a gene following splicing). By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene or for the final mRNA product of a gene.

[0068] As used herein, the term “condition” includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.

[0069] The term “contacting” or “contact” as used herein in connection with contacting a neuron or neuron surrogate cell includes subjecting the neuron or surrogate cell to an appropriate culture media which comprises the indicated compound and / or agent. Where the neuron or surrogate cell is in vivo, “contacting” or “contact” includes administering the compound and / or agent in a pharmaceutical composition to a subject via an appropriate administration route such that the compound and / or agent contacts the neuron or surrogate cell in vivo. In specific embodiments, the contacted neuron or surrogate cell are assayed for cell survival. Measurement of cell survival can be based on the number of viable cells after period of time has elapsed after contacting of cells with a compound or agent. For example, number of viable cells can be counted after about at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days or more and compared to number of viable cells in a non-treated control.

[0070] The terms “decrease”, “reduce” or “inhibit” and their grammatical equivalents are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, the terms “decrease”, “reduce” or “inhibit” and their grammatical equivalents mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, where the decrease is less than 100%. In one embodiment, the decrease includes a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0071] As used herein, the term “effective amount” means an amount of the compound and / or agent which is effective to promote the survival of motor neuron cells or to inhibit or slow the death of such cells. Determination of an effective amount is well within the capability of those skilled in the art. Generally, an effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other agents that inhibit pathological processes in neurodegenerative conditions.

[0072] As used herein, the terms “encode”, “encoding” and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to “encode” a polypeptide if it can be transcribed and / or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms “encode”, “encoding” and the like include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0073] As used herein, the phrase “enhancing motor neuron survival” refers to an increase in survival of motor neuron cells as compared to a control. In some embodiments, contacting of a motor neuron with an agent described herein results in at least about 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more increase in motor neuron survival relative to non-treated control. Motor neuron survival can be assessed by for example (i) increased survival time of motor neurons in culture; (ii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase, acetylcholinesterase and cyclin F; (iii) reduced abnormal accumulation of proteins including TDP-43 in culture or in vivo; or (iv) decreased symptoms of motor neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In one non-limiting example, increased survival of motor neurons may be measured by the method described by Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; reduced abnormal accumulation of proteins may be assayed through detection of aggregated proteins in aggresomes and inclusion bodies as described for example by Shen et al. (2011, Cell Biochem Biophys 60:173–185), and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder. In one embodiment, the increase in motor neuron survival can be assessed by measuring the increase in cyclin F levels. Cell survival can also be measured by uptake of calcein AM, an analog of the viable dye, fluorescein diacetate. Calcein is taken up by viable cells and cleaved intracellularly to fluorescent salts which are retained by intact membranes of viable cells. Microscopic counts of viable neurons correlate directly with relative fluorescence values obtained with the fluorometric viability assay. This method thus provides a reliable and quantitative measurement of cell survival in the total cell population of a given culture (Bozyczko-Coyne et al., J. Neur. Meth.50:205-216, 1993). Other methods of assessing cell survival are described in U.S. Pat. Nos.: 5,972,639; 6,077,684 and 6417,160, contents of which are incorporated herein by reference. In vivo motor neuron survival can be assessed by an increase in motor neuron, neuromotor or neuromuscular function in a subject. In one non-limiting example, motor neuron survival in a subject can be assessed by reversion, alleviation, amelioration, inhibition, slowing down or stopping of the progression, aggravation or severity of a condition associated with motor neuron dysfunction or death in a subject, e.g., ALS or FTD.

[0074] As used herein, the term “exogenous” refers to a molecule (e.g., a nucleic acid, carbohydrate, lipid or polypeptide) that is introduced into a host cell. In specific embodiments, an exogenous polypeptide refers to a polypeptide that is expressed from a polynucleotide which is foreign to the cell into which it has been introduced, or a polynucleotide that is homologous to a sequence in the cell into which it is introduced but in a position within the host cell nucleic acid in which the polynucleotide is not normally found.

[0075] The term “expression” with respect to a gene sequence refers to transcription of the gene to produce a RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.) and, as appropriate, translation of a resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a coding sequence results from transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0076] As used herein, the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like, and in some embodiments, polypeptide. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements including promoters, enhancers, termination sequences and 5’ and 3’ untranslated regions). In certain embodiments, the term “gene” includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5’ and 3’ non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control sequences such as promoters, enhancers, termination and / or polyadenylation signals that are naturally associated with a given gene, or heterologous control sequences. The gene sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for introduction into a host.

[0077] The term "heterologous", for the purposes of the present disclosure, refers to a molecule (e.g., a nucleic acid or polypeptide) that is not naturally present and / or naturally expressed within a host organism or cell thereof, or a domain or region that is not naturally present within a gene, nucleic acid or protein.

[0078] The terms “increase”, “enhance”, or “activate” and their grammatical equivalents are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increase”, “enhance”, or “activate” and their grammatical equivalents mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10- fold or greater as compared to a reference level.

[0079] As used herein, the phrase “inhibiting motor neuron degeneration” refers to reducing loss of motor neuron viability, reducing loss of motor neuron function and / or reducing loss of the number of motor neurons. In some embodiments, contacting of a motor neuron with an agent described herein results in at least about 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%,90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more decrease in motor neuron degeneration relative to non-treated control. Motor neuron degeneration can be assessed by for example by assaying oxidative stress or endoplasmic reticulum stress or apoptosis or neuronal death in general.

[0080] As used herein, the term “modulate” means to cause or facilitate a qualitative or quantitative change, alteration, or modification in a molecule, a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, a change in binding characteristics, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon.

[0081] As used herein, the phrase “motor neuron degeneration” or “degeneration of motor neuron” means a condition of deterioration of motor neurons, wherein the neurons die or change to a lower or less functionally-active form.

[0082] The term “neurodegenerative condition” is an inclusive term encompassing acute and chronic conditions, disorders or diseases of the central or peripheral nervous system and is generally caused by or associated with the deterioration of cells or tissues of the nervous system. A neurodegenerative condition may be age-related, or it may result from injury or trauma, or it may be related to a specific disease or disorder. Acute neurodegenerative conditions include, but are not limited to, conditions associated with neuronal cell death or compromise including cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain damage, spinal cord injury or peripheral nerve trauma, e.g., resulting from physical or chemical burns, deep cuts or limb severance. Examples of acute neurodegenerative disorders are: cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well as intracranial hemorrhage of any type (such as epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (such as contusion, penetration, shear, compression and laceration), as well as whiplash and shaken infant syndrome. Chronic neurodegenerative conditions include, but are not limited to, Alzheimer's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), degenerative ataxias, cortical basal degeneration, ALS- Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease / spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay- Sach's disease, Sandhoff disease, familial spastic disease, Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome), and prion diseases (including, but not limited to Creutzfeldt-Jakob, Gerstmann- Straussler-Scheinker disease, Kuru and fatal familial insomnia), demyelination diseases and disorders including multiple sclerosis and hereditary diseases such as Leukodystrophies. In specific embodiments, the neurodegenerative condition is selected from ALS and FTD.

[0083] As used herein, the term “neuron” includes a neuron and a portion or portions thereof (e.g., the neuron cell body, an axon, or a dendrite). The term “neuron” as used herein denotes nervous system cells that include a central cell body or soma, and two types of extensions or projections: dendrites, by which, in general, the majority of neuronal signals are conveyed to the cell body, and axons, by which, in general, the majority of neuronal signals are conveyed from the cell body to effector cells, such as target neurons or muscle. Neurons can convey information from tissues and organs into the central nervous system (afferent or sensory neurons) and transmit signals from the central nervous systems to effector cells (efferent or motor neurons). Other neurons, designated interneurons, connect neurons within the central nervous system (the brain and spinal column). The neurons may be any neurons, including without limitation sensory, sympathetic, parasympathetic, or enteric, e.g., dorsal root ganglia neurons, motor neurons, and central neurons, e.g., neurons from the spinal cord. Certain specific examples of neuron types that may be subject to treatment or methods according to the present disclosure include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a motor neuron.

[0084] The terms “neuron degeneration” and “neuronal degeneration” are used interchangeably herein to refer to any pathological changes in neuronal cells, including, without limitation, death or loss of neuronal cells, any changes that precede cell death, and any reduction or loss of an activity or a function of the neuronal cells. The pathological changes may be spontaneous or may be induced by any event and include, for example, pathological changes associated with apoptosis. The neurons may be any neurons, including without limitation sensory, sympathetic, parasympathetic, or enteric, e.g., dorsal root ganglia neurons, motor neurons, and central neurons, e.g., neurons from the spinal cord. Neuronal degeneration or cell loss is a characteristic of a variety of neurological diseases or disorders, e.g., neurodegenerative diseases or disorders. In some embodiments, the neuron is a sensory neuron. In some embodiments, the neuron is a motor neuron.

[0085] The term “neurotropic viral vector” refers to a viral vector that selectively infects neuronal cells, including motor neurons.

[0086] The term “operably connected” or “operably linked” as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) “operably linked” to a nucleotide sequence of interest (e.g., a coding and / or non-coding sequence) refers to positioning and / or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non-coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0087] The terms “patient”, “subject”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the present disclosure include, but are not restricted to, any member of thesubphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. A preferred subject is a human in need of increasing the level or activity of cyclin F and / or treatment of a neurodegenerative condition. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0088] As used here, the term “pharmaceutically acceptable refers to those compounds, agents, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0089] As used herein, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0090] The term “polynucleotide” is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides. Typically, a polynucleotide of present disclosure is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single-stranded molecule) are provided. “Polynucleotide sequence” as used herein can refer to the polynucleotide material itself and / or to the sequence information (e.g., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

[0091] The term “polypeptide” as used herein refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. Oneor more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a non-polypeptide moiety covalently or non-covalently associated therewith is still considered a “polypeptide”. Exemplary modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term “polypeptide sequence” or “amino acid sequence” as used herein can refer to the polypeptide material itself and / or to the sequence information (e.g., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0092] The term “promoter” refers to a nucleotide sequence, usually upstream (5’) to a transcribable sequence, which controls the expression of the transcribable sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short nucleic acid sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which control elements (e.g., cis-acting elements) are added for control of expression. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus control elements (e.g., cis-acting elements) that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific nucleic acid-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic nucleic acid segments. A promoter may also contain nucleic acid sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as “minimal or core promoters.” In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and / or an initiator. The term “regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and / or spatially-regulated manner, and include both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in host cells are constantly being discovered. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity. Illustrative regulated promoters include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible systems, promoters derived from pathogen-inducible systems, promoters derived from carbohydrate inducible systems, promoters derived from hormone inducible systems, promoters derived from antibiotic inducible systems, promoters derived from metal inducible systems, promoters derived from heat shock inducible systems, and promoters derived from ecdysone-inducible systems.

[0093] “Regulatory sequences”, “regulatory elements” and the like refer to nucleotide sequences located upstream (5’ non-coding sequences), within, or downstream (3’ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence, either directly or indirectly. Regulatory elements include enhancers, promoters, translation leader sequences, introns, Rep recognition element, intergenic regions and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

[0094] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The present disclosure contemplates the use in the methods disclosed herein of full-length cyclin F polypeptides as well as their biologically active fragments. Typically, biologically active fragments of a full-length cyclin F polypeptide may participate in an interaction, for example, an intra-molecular or an inter- molecular interaction.

[0095] “Similarity” refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Tables 1 and 2 supra. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al.1984, Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0096] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window”, “sequence identity,” “percentage of sequence identity” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides or polypeptides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide or polypeptide sequence) that is similar between the two polynucleotides or polypeptides, and (2) a sequence that is divergent between the two polynucleotides or polypeptides, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the twopolynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Chapter 15. In one example, where the sequence of a modified cyclin F polypeptide is aligned with a wild-type cyclin F polypeptide, the comparison window comprises the full length of the modified cyclin F polypeptide. As would be appreciated, where the modified cyclin F polypeptide is truncated is compared to a wild-type cyclin F polypeptide, the comparison window will be less than the full length of the wild- type cyclin F polypeptide (e.g.40%, 50%, 60%, 70%, 80%, or 90% of the length of the wild-type cyclin F polypeptide).

[0097] The term “TDP-43 proteinopathy" is used herein to describe neurodegenerative conditions linked to the deposition of TDP-43, including but not limited to amyotrophic lateral sclerosis (ALS), argyrophilic grain disease, frontotemporal dementias (such as FTD-TDP-43 and FTD-tau), ALS-Parkinsonism dementia complex of Guam, corticobasal degeneration, Dementia with Lewy bodies, Huntington's disease (HD), Lewy body disease, motor neuron disease, frontotemporal lobar degeneration (FTLD), frontotemporal dementia, frontotemporal lobar degeneration with ubiquitin-positive inclusions, hippocampal sclerosis, inclusion body myopathy, inclusion body myositis, Parkinson's disease (PD), Parkinson's disease dementia, Parkinson-dementia complex in Kii peninsula, Pick's disease, Machado-Joseph disease and the like. Further details of TDP-43 proteinopathies are described in Gendron et al., 2010, Neuropathol. Appl. Neurobiol.36:97-112 and Lagier-Tourenne et al., 2010, Hum. Mol. Gen.19(1):R46-R64; the disclosures of which are incorporated herein by reference. In specific embodiments, the TDP-43 proteinopathy is associated with TDP-43 deposition in neurons, which referred to herein as a “neuronal TDP-43 proteinopathy”.

[0098] As used herein the terms "treating", "treatment", and grammatical equivalents refer to any and all uses which remedy the stated neurodegenerative condition, prevent, retard or delay the establishment of the condition, or otherwise prevent, hinder, retard, or reverse the progression of the condition. Thus the terms "treating" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. Where the condition displays or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms. Thus, “treatment of a neurodegenerative condition” includes within its scope delaying or preventing the onset of such a condition (e.g. death of motor neurons), at reversing, alleviating, ameliorating,inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of such a condition. In one embodiment, the symptom of a neurodegenerative condition is alleviated by at least 20%, at least 30%, at least 40%, or at least 50%. In one embodiment, the symptom of a neurodegenerative condition is alleviated by more than 50%. In one embodiment, the symptom of a neurodegenerative condition is alleviated by 80%, 90%, or greater. Treatment also includes improvements in neuromuscular function. In some embodiments, neuromuscular function improves by at least about 10%, 20%, 30%, 40%, 50% or more.

[0099] By “vector” is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra- chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably a viral or viral-derived vector, which is operably functional in animal and preferably mammalian cells. Non-limiting viral vectors that are useful for the practice of the present disclosure include adeno-associated viral vectors (AAV), lentiviral vectors, adenoviral vectors and herpes simplex viral vectors. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are known to those of skill in the art and include the nptII gene that confers resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene which confers resistance to the antibiotic hygromycin B.

[0100] The terms “wild-type”, “native” and “naturally occurring” are used interchangeably herein to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type, native or naturally occurring gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene or gene product.

[0101] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise. Modulation of transgene expression

[0102] It has previously been identified that TDP-43 binds to specific intronic regions of RNA, allowing repression of the inclusion of a nearby exon (termed a cryptic exon) in the final spliced isoform of the resulting mRNA. An overview of the mechanism by which TDP-43 regulatesexpression of cryptic exons is presented in Figure 1.In the absence of soluble TDP-43, which may be the consequence of nuclear depletion of TDP-43, and / or the RNA-binding loss-of-function of cytoplasmic TDP-43, the cryptic exon is not repressed, and instead is transcribed and incorporated into the final mRNA transcript. However cryptic exons frequently contain multiple stop codons, disrupting the translation of the cryptic mRNA and leading to either nonsense-mediated decay or production of a truncated protein. Alternatively, where the cryptic exon does not include premature stop codons, translational readthrough produces a protein product including a cryptic peptide (encoded by the cryptic exon).

[0103] TDP-43-responsive cryptic exons have been identified in a number of genes identified as risk factors for amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD), including stathmin 2 (STMN2; e.g. see Melamed et al., 2019, Nat Neurosci 22:180-190), unc-13 homolog A (UNC13A; e.g. see Ma et al., 2022, Nature 603:1240-130) and hepatoma- derived growth factor-related protein 2 (HDGFL2; e.g. see Irwin et al., 2023, bioRxiv 2023 2023.2001.2023.525202). The skilled addressee will appreciate that the scope of the present disclosure is not limited by reference to any specific individual gene containing a TDP-43- responsive cryptic exon.

[0104] As described herein the inventors have identified that TDP-43-responsive cryptic exon in STMN2 displays brain-specific expression and are specifically expressed in ALS patients. Expression is absent from healthy controls. Furthermore, the incidence of STMN2 cryptic exon expression is more widespread across the brain in ALS patients with greater regional spread of TDP-43 pathology.

[0105] Predicated at least in part on these findings, the inventors have developed a novel mechanism for modulating gene expression, responsive to the presence, and levels, of TDP- 43, taking advantage of the ability of TDP-43 to bind to intronic regions flanking cryptic exons. Thus, provided herein is a gene expression modulation switch responsive to a disease state, in which expression of a transgene is repressed (switched off) in normal, healthy cells in the presence of nuclear-located soluble TDP-43 and is activated (switched on) in diseased cells in the absence of nuclear-located soluble TDP-43.

[0106] The gene expression modulation switch represents a nucleic acid construct or cassette, which can be inserted into a suitable vector for introduction into a cell. The nucleic acid construct or cassette may be employed in the form of a DNA or RNA molecule, delivered to cells using a variety of suitable delivery vehicles as described hereinbelow.

[0107] As further described hereinbelow, in particular embodiments of the invention, the transgene is a therapeutic transgene, preferably a therapeutic transgene encoding a product that degrades cytoplasmic TDP-43, such as, for example, a cyclin F polypeptide. In other embodiments as further described hereinbelow, the transgene may be a biomarker transgene encoding a detectable and / or measurable marker in a biological sample, wherein said marker can act as a surrogate marker for nuclear depletion of soluble TDP-43 and hence for a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43. In further embodiments as described hereinbelow, the gene expression modulation switch may comprise two or more transgenes, for example a first, therapeutic transgene and a second, biomarker transgene.

[0108] A schematic representation illustrating the operation of a gene expression modulation switch regulating cyclin F-mediated TDP-43 clearance according to an embodiment of the present invention is shown in Figure 2. In accordance with this embodiment, the switch is designed such that it turns on therapeutic transgene expression when pathogenic (cytoplasmic) TDP-43 accumulates and nuclear TDP-43 is depleted (such as occurs in ALS patients) and the switch is turned off when pathogenic TDP-43 levels are reduced and nuclear TDP-43 levels are restored. It will be understood that in this embodiment the switch is not directly responsive to pathogenic TDP-43 levels, rather it is directly responsive to the levels of nuclear TDP-43, as a surrogate marker of early stages of neural dysfunction in ALS. It will also be understood that the switch is not completely on or off in terms of transgene expression, but rather transgene expression is graded in response to levels of nuclear TDP-43. When the switch is paired with a therapeutic transgene that targets clearance of pathogenic TDP-43 and degrades TDP-43 (such as cyclin F), this pairing creates a novel gene therapy, producing bi-phasic transgene expression. That is, transgene expression is turned on in response to accumulation of cytoplasmic TDP-43 / nuclear depletion of TDP-43, which causes expression of the therapeutic TDP-43 degrader that clears the pathogenic TDP-43, subsequently restoring nuclear TDP-43 to “healthy” levels and resulting in the promoter being turned off. The switch henceforth operates as a feedback-loop (see Figure 2) with further transgene expression again turned on in response to nuclear depletion of TDP-43.

[0109] Embodiments of the present disclosure provide a nucleic acid construct comprising a heterologous transgene operably linked to a TDP-43 responsive binding element 5' to the transgene, wherein the transgene and the TDP-43 responsive binding element are operably linked to a promoter capable of driving expression of the transgene in a cell, wherein the TDP-43 responsive binding element comprises at least a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein one or both of the first and second nucleic acid sequences comprise a region to which TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G.

[0110] Embodiments of the present disclosure also provide a method for modulating the expression of a heterologous transgene administered to a cell, wherein the heterologous transgene is located in a nucleic acid construct comprising a TDP-43 responsive binding element operably linked at the 5' end of the heterologous transgene, wherein the TDP-43 binding element and the transgene are operably linked to a promoter capable of driving expression of the transgene in the cell, wherein in the presence of soluble TDP-43 the TDP-43 binds to the TDP-43 responsive binding element repressing expression of the transgene and wherein the in absence of soluble TDP-43 the transgene is expressed, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein one or both of the first and second nucleic acid sequences comprise a region to which TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, andwherein modulation of expression of the transgene is achieved by regulation of RNA splicing of the first and second nucleic acid sequences, such that in the absence of nuclear TDP-43 the first and second nucleic acid sequences are removed from the nucleic acid construct by RNA splicing resulting in expression of the transgene.

[0111] In the context of the present disclosure reference herein to the binding of TDP- 43 to the TDP-43 responsive binding element should be understood to mean binding of soluble, non-pathogenic nuclear TDP-43, as is expressed in normal, healthy neurons. In neurons in which soluble, non-pathogenic TDP-43 is depleted, and insoluble TDP-43 accumulates in the cytoplasm, typically forming agglomerates (i.e. in 'disease state' neurons), the ability of soluble, non- pathogenic TDP-43 to bind to the TDP-43 responsive binding element is reduced or eliminated as levels of the soluble, non-pathogenic TDP-43 diminish.

[0112] In gene expression modulation switches of the present disclosure, soluble TDP- 43 is capable of binding to at least one of the first nucleic acid sequence and the second nucleic acid sequence within the TDP-43 responsive binding element. In some embodiments, soluble TDP- 43 is capable of binding to both the first and second nucleic acid sequences.

[0113] In exemplary embodiments at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides of the region at least about 30 consecutive nucleotides in the first nucleic acid sequence and / or second nucleotide sequence are U or G. In exemplary embodiments, the region may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. The region may comprise, for example one, two, three, four or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. Said stretches of consecutive nucleotides may comprise, for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. The region may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. For example, the region may comprise two, three, four, five, six, seven or more of said consecutive motifs. Exemplary U-rich, G-rich sequences that may be used in or may comprise the above-described regions are shown in Figure 6, representing sequences found in introns flanking cryptic exons in various genes.

[0114] The first and / or second nucleic acid sequence of the TDP-43 responsive binding element may comprise, consist or consist essentially of said region comprising (or consisting or consisting essentially of) at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G.

[0115] Thus in some embodiments, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the first nucleic acid sequence may be U or G. The first nucleotide sequence may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. The first nucleotide sequence may comprise one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. The first nucleotide sequence may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. The first nucleotide sequence may comprise two, three, four, five, six, seven or more of said consecutive motifs.

[0116] Alternatively, or in addition, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the second nucleic acid sequence may be U or G. The second nucleotide sequence may comprise one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues. The second nucleotide sequence may comprise one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues. The second nucleotide sequence may comprise one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG. The second nucleotide sequence may comprise two, three, four, five, six, seven or more of said consecutive motifs.

[0117] The first and second nucleic acid sequences of the TDP-43 responsive binding element function as intronic sequences allowing removal of specific sequences from a primary RNA transcript by RNA splicing. According, the first and second nucleic acid sequences of the TDP-43 responsive binding element typically comprise intronic splice sites at the 5' and 3' ends. Typically therefore, the first and second nucleic acid sequences each comprise a GA dinucleotide at the 5' end and an AG dinucleotide or CAG trinucleotide at the 3' end. Adjacent to or near the 3' end of the first and second sequences there will typically be a splicing branch point where the 5' splice site reconnects with the 3' splice site.

[0118] Where only one of the first and second nucleic acid sequences comprises the region comprising (or consisting or consisting essentially of) at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G. The other of said sequences may comprise a sequence of at least about 20 nucleotides.

[0119] The first and second nucleic acid sequences flank a third nucleic acid sequence, optionally wherein the third nucleic acid sequence comprises a cryptic exon. The cryptic exon may be modified so as to remove stop codons. The cryptic exon may encode a cryptic peptide.

[0120] In exemplary embodiments, the TDP-43 responsive binding element may comprise or consist of one or more intronic sequences from a gene, optionally wherein expression of the gene is associated with, or is a risk factor for, a neurodegenerative condition associated with the cytoplasmic accumulation of insoluble TDP-43, such as ALS and / or FTD. The TDP-43 responsive binding element may comprise or consist of one or more intronic sequences from a gene recognised by TDP-43, which intronic sequences, in the context of the gene in which they are found, flank a cryptic exon or cryptic peptide. By way of non-limiting example only, exemplary genes possessing cryptic exons recognised by, and subject to alternative splicing mediated by, TDP-43 include UNC13A, STMN2, SYT7, EPB41L4A, CEP72, INSR, FAM114A2, PFKP, ST5, RNFT2, ALX1, AGRN, ATG4B, SETD5, KDELC2, MUC16, PKN1, IRF9, UPF2, GPSM2, XPO4, RASA4, RASA4B, PARP6, KRT7, TRAPPC12, RANBP1, HERC6, BLZF1, ZFP91, HDGFRP2, MAP3K8, SSFA2, CENPK, ITPR3, KYNU, IRF9, COL4A6 and KYNU (see Ling et al., 2015, Science 349: 650-655). By way of non-limiting example only, exemplary genes possessing cryptic peptides resulting from TDP-43- mediated alternative splicing include MYO1C, KCNQ2, HDGFL2, DNM1, MYO18A, CAMK2B, ACTL6B, RSF1, KALRN, SYT7, AGRN and ADGRB1 (see Seddighi et al., 2023, bioRxiv 2023.01.23.525149; Irwin et al., 2023, bioRxiv 2023.01.23.525202). The skilled addressee will appreciate that the above lists of genes containing cryptic exons and cryptic peptides is not an exhaustive list.

[0121] By way of example, the TDP-43 responsive binding element may comprise or consist of one or more intronic sequences from the UNC13A gene, or one or more UG rich regions derived from a UNC13A intronic sequence, recognised by TDP-43, which intronic sequences, in the context of the UNC13A gene, flank a cryptic exon. The introns reside between exons 20 and 21 of the UNC13A gene. In an exemplary embodiment, the TDP-43 responsive binding element may comprise or consist of the sequence set forth in SEQ ID NO:10, or a U and G rich region of at least about 30 consecutive nucleotides within SEQ ID NO:10, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The TDP-43 responsive binding element may further comprise the cryptic exon from the UNC13A gene. The cryptic exon recognised by TDP-43 normally residing in the UNC13A gene contains multiple stop codons. The cryptic exon may comprise the sequence set forth in SEQ ID NO:8, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, in which stop codons from the wild type cryptic exon (see SEQ ID NO:7) have been removed. Stop codons may be removed by any method as will be well known to those skilled in the art. The UNC13A cryptic exon in the nucleic acid construct may be flanked by a 5' flanking intron having or comprising a sequence set forth in SEQ ID NO:5 or 6, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The UNC13A cryptic exon in the nucleic acid construct may be flanked by a 3' flanking intron having or comprising a sequence set forth in SEQ ID NO:9, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. TDP-43 may or may not be capable of directly binding to the sequence of the cryptic exon. Thus, in the case of a normal, healthy neuron soluble TDP-43 need not bind directly to each component of the TDP-43 responsive binding element, and may for example bind to the first nucleic acid sequence, the second nucleic acid sequence and / or the cryptic exon sequence.

[0122] In another example, the TDP-43 responsive binding element may comprise or consist of one or more intronic sequences from the SYT7 gene, or one or more U and G rich regions derived from a SYT7 intronic sequence, recognised by TDP-43, which intronic sequences, in the context of the SYT7 gene, flank a cryptic exon. In an exemplary embodiment, the TDP-43 responsive binding element may comprise or consist of the sequence set forth in SEQ ID NO:19, or a U and G rich region of at least about 30 consecutive nucleotides within SEQ ID NO:19, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The TDP- 43 responsive binding element may further comprise the cryptic exon from the SYT7 gene having the sequence set forth in SEQ ID NO:17, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The SYT7 cryptic exon in the nucleic acid construct may be flanked by a 5' flanking intron having or comprising a sequence set forth in SEQ ID NO:16, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The SYT7 cryptic exon in the nucleic acid construct may be flanked by a 3' flanking intron having or comprising a sequence set forth in SEQ ID NO:18, or a sequence having at least or about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. TDP-43 may or may not be capable of directly binding to the sequence of the cryptic exon. Thus, in the case of a normal, healthy neuron soluble TDP-43 need not bind directly to each component of the TDP-43 responsive binding element, and may for example bind to the first nucleic acid sequence, the second nucleic acid sequence and / or the cryptic exon sequence.

[0123] In an alternative embodiment, the TDP-43 responsive binding element may comprise or consist of one or more intronic sequences from the MYO18A gene, or one or more UG rich regions derived from a MYO18A intronic sequence, recognised by TDP-43, which intronic sequences, in the context of the MYO18A gene, flank a cryptic exon. In an exemplary embodiment, the TDP-43 responsive binding element may comprise or consist of the sequence set forth in SEQ ID NO:15, or a U and G rich region of at least about 30 consecutive nucleotides within SEQ ID NO:15, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The TDP-43 responsive binding element may further comprise the cryptic exon from the MYO18A gene. The cryptic exon may comprise the sequence set forth in SEQ ID NO:13, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, wherein the sequence of SEQ ID NO:13 includes a 1bp or 2bp insertion relative to the wild type MYO18A cryptic exon (SEQ ID NO:12) to induce a reading frame shift. The MYO18A cryptic exon in the nucleic acid construct may be flanked by a 5' flanking intron having or comprising a sequence set forth in SEQ ID NO:11, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The MYO18A cryptic exon in the nucleic acid construct may be flanked by a 3' flanking intron having or comprising a sequence set forth in SEQ ID NO:14, or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. TDP-43 may or may not be capable of directly binding to the sequence of the cryptic exon. Thus, in the case of a normal, healthy neuron soluble TDP-43 need not bind directly to each component of the TDP-43 responsive binding element, and may for example bind to the first nucleic acid sequence, the second nucleic acid sequence and / or the cryptic exon sequence.

[0124] Figure 3 provides a schematic illustration of an exemplary nucleic acid construct (gene expression modulation switch) of the present disclosure and the use of this construct for modulating expression of a transgene located in the nucleic acid construct. In the exemplary schematic illustration of Figure 3, a segment of a gene (e.g. a segment of the UNC13A gene as described above) including the cryptic exon ('CE', engineered to remove stop codons) and flanking introns (residing between exons 20 and 21 in UNC13A) (representing a TDP-43 responsive binding element of the present disclosure) are positioned in the nucleic acid construct downstream of a reporter (mCherry) and upstream of a therapeutic transgene and a green fluorescent protein (eGFP) transgene. T2A, a self-cleaving peptide is positioned at the 5' end of the therapeutic transgene to enable post-translational separation from the mCherry reporter. The components of the construct are operably linked to (i.e. under the control of) the constitutive CMV promoter. In a normal healthy neuron (healthy state), soluble TDP-43 is available in the nucleus to bind to the CE and / or flanking intron(s), repressing inclusion of the CE in a translated product. Thus, only themCherry reporter peptide is translated (an in-frame premature stop codon ensures eGFP is not translated). However in a disease state, TDP-43 loses its CE repressive function as it leaves the nucleus and forms cytoplasmic aggregates. This failure to bind the CE allows for an in-frame readthrough into the therapeutic transgene and eGFP.

[0125] In the exemplary construct shown in Figure 3, exemplified as noted above by inclusion of a cryptic exon and flanking introns residing between exons 20 and 21 of the UNC13A gene, a suitable nucleic acid (DNA) construct may comprise 5'-3': a CMV promoter (e.g. SEQ ID NO:2); a non-coding spacer (e.g. SEQ ID NO:3 or 4), an mCherry coding sequence (SEQ ID NO:24); UNC13A 5' flanking intron (e.g. SEQ ID NO:5 or 6); UNC13A cryptic exon with stop codons removed (e.g. SEQ ID NO:8); UNC13A 3' flanking intron (e.g. SEQ ID NO:9); a T2A spacer (e.g. SEQ ID NO:26); a therapeutic transgene, for example encoding a cyclin F (e.g. SEQ ID NO:20 or 22, encoding a cyclin F polypeptide having sequence SEQ ID NO:21 or 23, respectively); and eGFP (e.g. SEQ ID NO:30). Those skilled in the art will appreciate that it is important that frameshifting occurs immediately after the intron-cryptic exon-intron component of the construct to ensure that there is a stop codon if the readthrough is from mcherry to T2A (i.e. in the event that TDP-43 is bound and suppresses expression of the cryptic exon). This needs to work in such a way that when the full readthrough is allowed (i.e. when TDP-43 is absent) there is a clean in-frame read through from mcherry through the cryptic exon and into the transgene. For this reason, the linker sequence (e.g. T2A as shown in Figure 3) may require a +1bp frameshift (e.g. see SEQ ID NO:25) or a +2bp frameshift (e.g. SEQ ID NO:26). The choice of the +1bp or +2bp frameshift may depend on the cryptic exon employed (for example a +1bp frameshift in the linker may be required where the cryptic exon is from MYO18A or SYT7, while a +1bp frameshift in the linker may be required where the cryptic exon is from UNC13A). The skilled person will be able to determine the suitable frameshift to employ in any specific circumstance without need for further experimentation and without undue burden.

[0126] In the schematic shown in Figure 3, the construct is illustrated as including a gene encoding a green fluorescent protein (eGFP) as a reporter protein, and the construct further encodes a mCherry reporter polypeptide to facilitate detection. It will be immediately apparent to the skilled person that in a clinical setting the upstream reporter and the eGFP transgene are unnecessary. Thus, in embodiments of the present disclosure in which nucleic acid constructs are employed for the treatment of neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, or for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron or restoring or maintaining functional levels of soluble TDP-43 in the nucleus, those skilled in the art will appreciate that the mCherry reporter becomes unnecessary and the construct may only include a therapeutic transgene as described hereinbelow. In embodiments of the present disclosure in which nucleic acid constructs are employed for the diagnosis of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, for detecting impaired neuronal function or neuronal degeneration, or for monitoring the response of a subject to a therapeutic treatment, the mCherry reporter becomes unnecessary and the construct may only include a biomarker transgene as described hereinbelow.

[0127] Exemplary alternate embodiments of gene expression modulation switch constructs according to the present invention are illustrated in Figure 4 (represented as 'Series 1' – 'Series 3'). In each exemplary Series construct shown in Figure 4 the promoter could be any suitable promoter as discussed herein below, including, for example, a constitutive promoter (e.g. CMV promoter) or a cell-specific (e.g. neuron-specific) promoter, such as synapsin. In each exemplary Series construct shown in Figure 4, 'Reporter #1' refers to a gene product used to confirm that a cell has been transduced with the gene expression modulation switch construct. As exemplified herein, the Reporter #1 may be mCherry. The skilled addressee will appreciate that in a therapeutic context, Reporter #1 need not be present, and may instead be replaced by a nucleotide sequence including an in-frame start codon. In each exemplary Series construct shown in Figure 4, 'Reporter #2' refers to a gene product used to confirm the expression of the transgene. As exemplified herein, the Reporter #2 may be eGFP. Those skilled in the art will appreciate that in a therapeutic context, Reporter #2 need not be present. In each exemplary Series construct shown in Figure 4, 'Flanking intron' refers to intronic sequences that flank a cryptic exon (CE) as described herein. Flanking introns located on, or derived from, each side of a CE may be included in the construct, and each may be of variable length, provided that at least one of the flanking intron sequences represents or includes a TDP-43 responsive binding element as defined herein, comprising a region of at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G. Thus, while the exemplary Series shown in Figure 4 illustrate two flanking introns, the skilled person will appreciate that only one need be present, provided it represents or includes a TDP-43 responsive binding element as defined herein. In each exemplary Series construct shown in Figure 4, 'Therapeutic transgene' refers to any suitable therapeutic gene, such as a gene encoding a product capable of degrading TDP-43 (e.g. a cyclin F polypeptide).

[0128] With reference to the exemplary Series 1 construct in Figure 4, a cryptic exon (CE) is used, in which any stop codons that may normally be present in the CE are removed to allow translational read through, and including a number of bases so as to create a reading frame shift. The CE may encode a cryptic peptide. The therapeutic gene should start in-frame with the end of the CE. The transgene should also include a synonymous mutation in this reading frame to introduce a stop codon in the alternative reading frame in the event of translational read through directly from Reporter #1 to the transgene, skipping the CE. The CE may be any suitable CE derived from a CE-containing gene as described herein.

[0129] With reference to the exemplary Series 2 construct in Figure 4, construct features are the same as described above for Series 1, with the exception that a degradation domain sequence and a linker (T2A) are incorporated at the 5' end of the transgene. To overcome any potential off-target effects of the expressed CE, the Series 2 construct drives the auto- degradation of a cryptic peptide (CP) encoded by the CE. As illustrated in Figure 4, the degradation domain DD-FKBP is used to drive auto-degradation of a CP-DD-FKBP fusion peptide, while the T2A linker ensures the production of two separate products, one being the CP-DD-FKBP fusion peptide to be degraded, and one comprising the therapeutic gene product and reporter #2.

[0130] DD-FKBP (Banaszynski et al, 2006, Cell 126: 995-1004) is merely one suitable exemplary degradation domain that may be employed. Variants of DD-FKBP and other suitable degradation domains are described in the literature and would be known to the person skilled in the art (e.g. see Banaszynski et al, 2006, Cell 126: 995-1004; Chu et al., 2008, Bioorg Med ChemLett 18: 5941-5944). A nucleotide sequence encoding the exemplary DD-FKBP degradation domain is set forth in SEQ ID NO:29.

[0131] The nucleotide sequences of exemplary linkers T2A and P2A are set forth in SEQ ID NO:27 and SEQ ID NO:28, respectively. Optionally, the T2A and P2A sequences may be modified by the addition of one or two nucleotides, optionally at the 5' end of the linker sequence, to induce a frameshift.

[0132] With reference to the exemplary Series 3 construct in Figure 4, construct features are the same as described above for Series 1, with the exception that the CE is replaced with a 5' portion of the therapeutic transgene.

[0133] While a TDP-43 responsive binding element of the present disclosure may be paired with any suitable therapeutic transgene for treatment of conditions associated with cytoplasmic accumulation of insoluble TDP-43 as described hereinbelow, in particular embodiments as described herein, the transgene encodes a therapeutic product that degrades pathogenic TDP- 43, such as a modified or unmodified cyclin F polypeptide. This pairing creates a novel disease- specific, bi-phasic gene therapy approach. That is, transgene expression is switched on in response to accumulation of pathogenic TDP-43 / nuclear depletion of soluble TDP-43, which causes expression of the therapeutic product that degrades or clears the pathogenic TDP-43, subsequently restoring nuclear soluble TDP-43 levels, and resulting transgene expression being switched off. Hence a feedback loop is generated, with further transgene expression only again switched on in response to nuclear depletion of TDP-43. Promoters

[0134] In typical embodiments of the present disclosure the TDP-43 responsive binding element and the transgene are operably linked to a suitable promoter. The promoter may be derived from a vector in which the nucleic acid construct is located (as described hereinbelow) or may be derived from any suitable source. In specific embodiment, the promoter is a constitutive promoter. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter.

[0135] Tissue-specific promoters can also be used. Non-limiting examples of tissue- specific promoters include, central nervous system (CNS) specific promoters such as, neuron- specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA, 86:5473-77 (1989)) and glial specific promoters (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991)). In specific embodiments, the promoter is tissue specific and is essentially not active outside the central nervous system, or the activity of the promoter is higher in the central nervous system that in other systems. For example, a promoter specific for the spinal cord, brainstem, (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (corpus striatum, cerebral cortex, or within the cortex, the occipital, temporal, parietal or frontal lobes), or a combination thereof may be selected. The promoter may be specific for particular cell types, such as neurons or glial cells in the CNS. If it is active in glial cells, it may be specific for astrocytes, oligodendrocytes, ependymal cells, Schwanncells, or microglia. If it is active in neurons, it may be specific for particular types of neurons, e.g., motor neurons, sensory neurons, or interneurons. Additionally, it may be specific for neurons with a specific phenotype, e.g., dopamine-producing neurons, serotonin-producing neurons, etc. In certain embodiments, the promoter is specific for cells in particular regions of the brain, for example, the cortex, striatum, nigra, and hippocampus.

[0136] Suitable neuronal specific promoters include, but are not limited to, synapsin promoter, neuron specific enolase (NSE) (Olivia et al., Genomics, 10:157-65 (1991). GenBank Accession No: X51956), and human neurofilament light chain promoter (NEFL) (Rogaev et al., Human Molecular Genetics, 1:781(1992), GenBank Accession No: L04147). Glial specific promoters include, but are not limited to, glial fibrillary acidic protein (GFAP) promoter (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991), GenBank Accession No: M65210), S100 promoter (Morii et al., Biochemical & Biophysical Research Communications, 175:185-91 (1991), GenBank Accession No: M65210) and glutamine synthase promoter (Van den et al., Biochimica Biophysica Acta, 2:249-51(1991), GenBank Accession No: X59834). In a preferred embodiment, the gene is flanked upstream (i.e., 5') by the neuron specific enolase (NSE) promoter. In another preferred embodiment, the gene of interest is flanked upstream (i.e., 5') by the elongation factor 1 alpha (EF) promoter. Suitable phenotype-specific promoters include, but are not limited to, tyrosine hydroxylase promoter, dopamine beta-hydroxylase, acetylcholinesterase promoter, choline acetyltransferase promoter, dopamine receptor I and II promoters, dopamine transporter promoter, vesicular monoamine transporter promoter, neuopsin promoter, and vesicular acetylcholine transporter promoter.

[0137] As described herein, methods and constructs of the present invention make it possible to modulate the expression of a transgene in a disease-specific manner, in response to the physiological state of TDP-43. One advantage of the disclosure therefore is that it is unnecessary to employ an inducible promoter. Notwithstanding, inducible promoters can be employed in nucleic acid constructs of the disclosure, allowing a further level of regulation of gene expression in response to, for example, exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state such as a particular differentiation state of the cell, or in replicating cells only. Non-limiting examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin- inducible system. Therapeutic transgenes

[0138] In accordance with the present disclosure, the heterologous transgene can be any gene that encodes a gene product, the expression of which is desirable in a neuron in which soluble, nuclear TDP-43 is depleted and typically where insoluble, pathogenic TDP-43 predominates (i.e. with cytoplasmic accumulation of insoluble TDP-43), in particular in situations where the neuron resides in a subject suffering from, or at risk of developing a neurodegenerative condition associated with the nuclear depletion of soluble TDP-43, and optionally the cytoplasmic accumulation of insoluble TDP-43, such as ALS and / or FTD. Thus, the transgene may be any gene that encodes a therapeutic expression product (e.g. protein or nucleic acid) that can correct adefect in a neuron with nuclear depletion of TDP-43 and cytoplasmic accumulation of insoluble TDP-43. Transgenes can include genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, fusion proteins, and mutants that maintain some or all of the therapeutic function of the full-length polypeptide encoded by the transgene.

[0139] In particular exemplary embodiments, the transgene encodes a cyclin F polypeptide. The cyclin F polypeptide may be unmodified (i.e. wild-type) or modified, such as a cyclin F modified or optimized for therapeutic use. As example of unmodified cyclin F polypeptide is the wild-type cyclin F set forth in SEQ ID NO:21 or a cyclin F polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The transgene encoding the unmodified cyclin F may comprise the nucleotide sequence set forth in SEQ ID NO:20 or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0140] The transgene may encode a modified cyclin F polypeptide, for example a modified polypeptide displaying increased cytoplasmic targeting relative to the wild-type cyclin F. Thus, the modified cyclin F may be optimized for therapeutic use, for example demonstrating improved cytoplasmic targeting and an improved ability to ability to effectively bind and clear insoluble cytoplasmic TDP-43, while leaving the nuclear form of TDP-43 (which is required for normal cell function) essentially untouched. Examples of suitable modified cyclin F polypeptides are described, for example, in International Patent Application No. PCT / AU2022 / 050366, the disclosure of which is incorporated herein in its entirety.

[0141] For example, the transgene may encode a modified cyclin F polypeptide with the sequence set forth in SEQ ID NO:23 or a cyclin F polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The transgene encoding the modified cyclin F may comprise the nucleotide sequence set forth in SEQ ID NO:22 or a sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Biomarker transgenes

[0142] As described herein, in certain embodiments, the heterologous transgene may encode a molecule that acts as a surrogate marker for nuclear depletion of soluble TDP-43 and hence as a marker for a neurodegenerative condition associated with the nuclear depletion of TDP- 43 and / or the cytoplasmic accumulation of insoluble TDP-43, for impaired neuron function or for neuronal degeneration. The term "biomarker" as used in the context of a "biomarker transgene" means that the transgene encodes a gene product representing a biomarker useful in the diagnosis of disease as well as in the monitoring of disease progression, and assessing, determining or monitoring the efficacy of a treatment.

[0143] Typically the marker is one that is detectable or measurable (i.e. optionally quantifiable) in a biological sample from a subject, such as a blood sample or cerebrospinal fluid. Typically the marker will comprise a polypeptide or protein that is specifically, selectively or preferentially expressed in neurons or glial cells. Also typically the marker will comprise apolypeptide or protein that that is specifically, selectively or preferentially expressed in neurons or glial cells and is detectable in blood or cerebrospinal fluid, e.g. by immunoassay or other suitable detection method.

[0144] Exemplary polypeptides and proteins expressed in neurons and detectable in the blood include, but are not limited to, arginine vasopressin (AVP), oxytocin / neurophysin I prepropeptide (OXT), pro-melanin concentrating hormone (PMCH), VGF nerve growth factor (VGF) and agouti related neuropeptide (AGRP).

[0145] Exemplary polypeptides and proteins expressed in neurons and detectable in cerebrospinal fluid include, but are not limited to, agouti related neuropeptide (AGRP), carbonic anhydrase 10 (CA10), cerebellin 3 precursor (CBLN3), cerebellin 4 precursor (CBLN4), cell adhesion molecule L1-like (CHL1), cellular repressor of E1A stimulated genes 2 (CREG2), hyaluronan and proteoglycan link protein 4 (HAPLN4), IgLON family member 5 (IGLON5), KIAA0319, leucine zipper protein 2 (LUZP2), neurocan (NCAN), neuronal pentraxin 1 (NPTX1), prodynorphin (PDYN), protein tyrosine phosphatase receptor type Z1 (PTPRZ1), secretogranin 3 (SCG3), serpin family I member 1 (SERPIN1), SLIT and NTRK like family member 1 (SLITRK1), tenascin R (TNR), and tetratricopeptide repeat domain 9B (TTC9B).

[0146] Other suitable polypeptides and proteins will be known to those skilled in the art, and can be determined, for example, by reference to suitable materials including The Human Protein Atlas (www.proteinatlas.org).

[0147] For use in diagnostic embodiments, the transgene encoding the polypeptide or protein to be used as a marker may be modified, using methods well known to those skilled in the art, to modify, reduce or remove any unwanted biological activity or function associated with the polypeptide or protein. Delivery vehicles

[0148] The present disclosure also contemplates delivery vehicles for delivering the nucleic acid constructs of the disclosure to a neuron (e.g., a motor neuron). Suitable delivery vehicles for nucleic acid molecules are well known in the art and may, for example, be viral or non- viral vectors.

[0149] Suitable viral vectors for delivery of nucleic acid constructs include, but are not limited to adeno-associated viral vectors (AAV), lentiviral vectors, adenovirus vectors and herpes simplex viral vectors, and in specific embodiments are neurotropic viral vectors.

[0150] The use of AAV vectors to deliver genes into the central nervous system is well known in the art (See. e.g., U.S. Pat. Nos.8,198,257 and 7,534,613, U.S. patent application Ser. No.13 / 881,956, each of which is incorporated by reference). AAV vectors for delivering transgenes to motor neurons are also known in the art (See, for example, U.S. Pat. No.7,335,636, incorporated by reference). AAV vectors can be constructed using known techniques to provide at least the operably connected components of control elements including a transcriptional initiation region (e.g., a promoter), a transcriptional termination region and optionally at least one post- transcriptional regulatory sequence. The control elements are selected to be functional in the targeted cell. The resulting construct which contains the operably connected components istypically flanked at the 5' and 3' region with functional AAV inverted terminal repeat sequences (ITRs).

[0151] The nucleotide sequences of AAV ITR regions are known. The ITR sequences for AAV2 are described, for example, by Kotin et al. Human Gene Therapy, 5:793-01 (1994); Fields & Knipe, Fundamental Virology, “Parvoviridae and their Replication” (2d ed.1986). The skilled artisan will appreciate that AAV ITR's can be modified using standard molecular biology techniques (e.g., Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). Accordingly, AAV ITRs used in the vectors of the present disclosure need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9, and the like. Furthermore, the 5' and 3' ITRs, which flank a selected nucleotide sequence in an AAV expression vector, need not necessarily be identical or derived from the same AAV serotype or isolate, so long as the ITR's function as intended, i.e., to allow for excision and replication of the bounded nucleotide sequence of interest when AAV rep gene products are present in the cell.

[0152] An AAV vector harboring a nucleic acid construct of the disclosure, and any suitable post-transcriptional regulatory sequences (PRE) flanked by AAV ITRs, can be constructed by directly inserting the nucleotide sequence(s) of interest and the PRE into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, as long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. These constructs can be designed using techniques well known in the art. (See, e.g., Lebkowski et al., Molecular & Cellular Biology, 8:3988-96 (1988); Vincent et al., Vaccines 90 (Cold Spring Harbor Laboratory Press, 1990); Carter, Current Opinion Biotechnology, 3:533-39 (1992); Muzyczka, Current Topics Microbiology & Immunology, 158:97- 29 (1992); Kotin, Human Gene Therapy, 5:793-01(1994); Shelling et al., Gene Therapy, 1:165-69 (1994); and Zhou et al., J Experimental Medicine, 179:1867-75 (1994)). Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Green & Sambrook (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)). Several AAV vectors are available from the American Type Culture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

[0153] In order to produce recombinant AAV particles, an AAV vector can be introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art (See, e.g., Graham et al., Virology, 52:456 (1973); Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); Davis et al., Basic Methods Molecular Biology, (Elsevier, 1986); and Chu et al., Gene, 13:197 (1981)). Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al., Virology, 52:456-67 (1973)), direct microinjection into cultured cells (Capecchi, Cell, 22:479-88 (1980)), electroporation (Shigekawa et al., BioTechniques, 6:742-51 (1988)), liposome mediated gene transfer (Mannino et al., BioTechniques, 6:682-90 (1988)), lipid-mediated transduction (Feigner et al., Proceedings Nat'l Acad. Sci. USA, 84:7413-17(1987)), and nucleic acid delivery using high- velocity microprojectiles (Klein et al., Nature 327:70-73 (1987)).

[0154] Suitable host cells for producing recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an exogenous nucleic acid molecule. Thus, a “host cell” as used herein generally refers to a cell which has been transfected with an exogenous nucleic acid molecule. The host cell includes any eukaryotic cell or cell line so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system chosen or the fermentation system employed. Non-limiting examples include CHO DHFR-minus cells (Urlaub and Chasin Proceedings Nat'l Acad. Sci. USA, 77:4216-420 (1980)), 293 cells (Graham et al., J. General Virology 36:59-72 (1977)), or myeloma cells like SP2 or NSO (Galfre & Milstein, Methods Enzymology, 73:3-46 (1981)).

[0155] In some embodiments, the host cells are cells from the stable human cell line, 293 (readily available through, e.g., the ATCC under Accession No. ATCC CRL 1573), which is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al., J. General Virology, 36:59-72 (1977)), and expresses the adenoviral E1a and E1 b genes (Aiello et al., Virology, 94:460-69 (1979)). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce AAV virions.

[0156] Host cells containing the above-described AAV vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the expression cassette flanked by the AAV ITRs to produce recombinant AAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV vectors. Thus. AAV helper functions include one, or both of the major AAV open reading frames (ORFs), namely the rep and cap coding regions, or functional homologues thereof.

[0157] The AAV rep coding region of the AAV genome encodes the replication proteins Rep 78, Rep 68, Rep 52, and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other exogenous) promoters. The Rep expression products are collectively required for replicating the AAV genome. The AAV cap coding region of the AAV genome encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV vector comprising the expression cassette, AAV helper constructs are thus used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and plM29+45 which encode both Rep and Cap expression products (See, e.g., Samulski et al., J. Virology, 63:3822-28 (1989); McCarty et al., J. Virology, 65:2936-45 (1991)). A number of other vectors have been described which encode Rep and / or Cap expression products (See. e.g., U.S. Pat. No.5,139,941, incorporated by reference).

[0158] As a consequence of the infection of the host cell with a helper virus, the AAV Rep and / or Cap proteins are produced. The Rep proteins also serve to duplicate the AAV genome.The expressed Cap proteins assemble into capsids, and the AAV genome is packaged into the capsids. This results the AAV being packaged into recombinant AAV particles comprising the expression cassette. Following recombinant AAV replication, recombinant AAV particles can be purified from the host cell using a variety of conventional purification methods, such as CsCl gradients. The resulting recombinant AAV particles are then ready for use for gene delivery to various cell types.

[0159] In some embodiments, the number of viral vector and / or virion particles administered to a subject may be on the order ranging from 103to 1015particles / mL, or any values therebetween, such as for example, about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015particles / mL. In some embodiments, vector and / or virion particles of higher than 1013particles / mL are administered. Volumes between 1 µL and 10 mL may be administered such that the subject receives between 102and 1016total vector and / or virion particles. Thus, in some embodiments, about 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or 1016vector and / or virion particles are administered.

[0160] In the practice of the methods of the present disclosure, an AAV of any serotype can be used. The serotype of the viral vector used in certain embodiments of the invention is selected from the group consisting from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 (see, e.g., Gao et al., 2002, PNAS 99:11854-11859; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Other serotypes besides those listed herein can be used. Furthermore, pseudotyped AAV vectors may also be utilized in the methods described herein. Pseudotyped AAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example, an AAV vector that contains the AAV2 capsid and the AAV1 genome or an AAV vector that contains the AAV5 capsid and the AAV 2 genome (Auricchio et al., 2001. Hum. Mol. Genet.10(26):3075-81). AAV vectors are derived from single- stranded (ss) DNA parvoviruses that are nonpathogenic for mammals (reviewed in Muzyscka, 1992. Curr. Top. Microb. Immunol.158:97-129). Briefly, recombinant AAV-based vectors have the rep and cap viral genes that account for 96% of the viral genome removed, leaving the two flanking 145-basepair (bp) inverted terminal repeats (ITRs), which are used to initiate viral DNA replication, packaging and integration. In the absence of helper virus, wild-type AAV integrates into the human host-cell genome with preferential site-specificity at chromosome 19q 13.3 or it may be maintained episomally. A single AAV particle can accommodate up to 5 kb of ssDNA, therefore leaving about 4.5 kb for a transgene and regulatory elements, which is typically sufficient. However, trans-splicing systems as described, for example, in U.S. Pat. No.6,544,785, may nearly double this limit.

[0161] In certain instances, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and CSp3.

[0162] AAV-based gene therapy vectors targeted to cells of the CNS have been described for example in U.S. Pat. Nos.6,180,613 and 6,503,888. Additional exemplary AAV vectors are recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 7, AAV2 / 8 and AAV2 / 9 serotype vectors encoding human protein. In specific embodiments, the AAV is a neurotropic AAV selected from rAAV2 / 1, rAAV2 / 8 and rAAV2 / 9, as described for example in Ayers et al. (2015, Mol Ther.23(1): 53–62).

[0163] Alternatively, a vector of the present disclosure can be a virus other than the adeno-associated virus, or portion thereof. For example, replication defective retroviruses, adenoviruses, herpes simplex viruses, and lentivirus can be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel et al., Current Protocols in Molecular Biology §§9.10-9.14 (Greene Publishing Associates, 1989) and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art. Examples of suitable packaging virus lines include Crip, Cre, 2 and Am. The genome of adenovirus can be manipulated such that it encodes and expresses the protein of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (See e.g., Berkner et al., BioTechniques, 6:616-29 (1988); Rosenfeld et al., Science, 252:431-34 (1991); Rosenfeld et al., Cell 68:143-55 (1992)). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.

[0164] The production of suitable lentiviral vectors is well known in the art (See, e.g., U.S. patent application Ser. No.13 / 893,920, incorporated by reference). A lentiviral vector according to the present disclosure may be derived from or may be derivable from any suitable lentivirus. A recombinant lentiviral particle is capable of transducing a target cell with a nucleotide of interest. Once within the cell the RNA genome from, the vector particle is reverse transcribed into DNA and integrated into the genome of the target cell.

[0165] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin et al., Retroviruses 758-763 (Cold Spring Harbor Laboratory Press, 1997). In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SrV). The non-primate lentiviral group includes the prototype “slow virus” visnaimaedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV).

[0166] Lentiviruses differ from other members of the retrovirus family in that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al., EMBO J., 11:3053-58 (1992)); Lewis & Emerman, J Virology, 68:510-16 (1994)). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

[0167] A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. That component part may be involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated. The basic structure of retrovirus and lentivirus genomes share many common features such as a 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components, which are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as the rev and rev response element (RRE) sequences, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible forproviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. The LTRs themselves are identical sequences that can be divided into three elements, which are called “U3,” “R” and “U5.” U3 is derived from the sequence unique to the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different viruses.

[0168] In a defective lentiviral vector genome gag, pol and env may be absent or non- functional. The R regions at both ends of the RNA are repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.

[0169] In a typical lentiviral vector of the present disclosure, at least part of one or more protein coding regions essential for replication may be removed from the virus, which makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a nucleic acid in order to generate a vector comprising the nucleic acid which is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome. In one embodiment, the lentiviral vectors are non-integrating vectors as described in U.S. patent application Ser. No.12 / 138,993 (herein incorporated by reference).

[0170] In a further embodiment, the vectors have the ability to deliver a sequence which is devoid of or lacking viral RNA. A heterologous binding domain (heterologous to gag) may be located on the RNA to be delivered and a cognate binding domain on gag or pol can be used to ensure packaging of the RNA to be delivered. Both of these vectors are described in U.S. patent application Ser. No.12 / 139,035 (herein incorporated by reference). The lentiviral vector may be a “non-primate” vector, i.e., derived from a virus which does not primarily infect primates, especially humans.

[0171] The examples of non-primate lentivirus may be any member of the family of Lentiviridae which does not naturally infect a primate and may include a feline immunodeficiency virus (FIV), a bovine immunodeficiency virus (BIV), a caprine arthritis encephalitis virus (CAEV), a Maedi-visna virus (MW) or an equine infectious anemia virus (EIAV).

[0172] In some embodiments, the viral vector is derived from EIAV. EIAV has the simplest genomic structure of the lentiviruses. In addition to the gag, pol and env genes, EIAV encodes three other genes: tat, rev, and S2. Tat acts as a transcriptional activator of the viral LTR (Derse & Newbold, Virology, 194:530-36(1993); Maury et al., Virology, 200:632-42(1994)). Rev regulates and coordinates the expression of viral genes through rev-response elements (RRE) (Martarano et al., J. Virology, 68:3102-11 (1994)). The mechanisms of action of these two proteins are thought to be broadly similar to the analogous mechanisms in the primate viruses (Martarano et al., J. Virology, 68:3102-11 (1994)). The function of S2 is unknown. In addition, an EIAV protein, Ttm, has been identified that is encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.

[0173] The viral vector may be manipulated to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell (See, e.g., U.S. Pat. No. 6,669,936, incorporated by reference). In some embodiments, the genome is limited to sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packagingcomponents, into a viral particle capable of infecting a target cell. Infection of the target cell may include reverse transcription and integration into the target cell genome. The lentiviral vector carries non-viral coding sequences which are to be delivered by the vector to the target cell. In some embodiments, the vector is incapable of independent replication to produce infectious lentiviral particles within the final target cell. Usually the recombinant lentiviral vector lacks a functional gag-pol and / or env gene and / or other genes essential for replication. A vector of the present disclosure may be configured as a split-intron vector (See, e.g., U.S. Pat. No.7,303,910, incorporated by reference).

[0174] The vector may be a self-inactivating vector. Self-inactivating retroviral vectors may be constructed by deleting the transcriptional enhancers or the enhancers and promoter in the U3 region of the 3' LTR. After a round of vector reverse transcription and integration, these changes are copied into both the 5' and the 3' LTRs producing a transcriptionally inactive provirus (Yu et al., Proceedings Nat'l Acad. Sci. USA, 83:3194-98 (1986); Dougherty and Temin et al., Proceedings Nat'l Acad. Sci. USA, 84:1197-01 (1987): Hawley, Proceedings Nat'l Acad. Sci. USA, 84:2406-10 (1987); Yee et al., Proceedings Nat'l Acad. Sci. USA, 91:9564-68 (1994)). However, any promoter(s) internal to the LTRs in such vectors will still be transcriptionally active. This strategy has been employed to eliminate effects of the enhancers and promoters in the viral LTRs on transcription from internally placed genes. Such effects include increased transcription (Jolly et al., Nucleic Acids Research, 11:1855-72 (1983)) or suppression of transcription (Emerman & Temin, Cell, 39:449-67 (1984)). This strategy can also be used to eliminate downstream transcription from the 3' LTR into genomic DNA (Herman & Coffin, Science, 236:845-48 (1987)). This is of particular concern in human gene therapy where it is of critical importance to prevent the adventitious activation of an endogenous oncogene.

[0175] A plasmid vector used to produce the viral genome within a host cell / packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell / packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed lentiviral sequence, i.e. the 5' U3 region, or they may be a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral genomes require additional sequences for efficient virus production. For example, in the case of HIV, the rev and RRE sequences are preferably included; however the requirement for rev and RRE may be reduced or eliminated by codon optimization (See U.S. patent application Ser. No.12 / 587,236, incorporated by reference). Alternative sequences which perform the same function, as the rev / RRE system are also known. For example, a functional analogue of the revIRRE system is found in the Mason Pfizer monkey virus. This is known as the constitutive transport element (CTE) and comprises an RRE-type sequence in the genome which is believed to interact with a factor in the infected cell. The cellular factor can be thought of as a rev analogue. Thus, CTE may be used as an alternative to the reviRRE system. Any other functional equivalents which are known or become available may be relevant to the methods of the present disclosure. For example, the Rex protein of HTLV-1 can functionally replace the Rev protein of HIV-1. It is also known that Rev and Rex have similar effects to IRE-BP.

[0176] In certain embodiments, the lentiviral vector is a self-inactivating minimal lentiviral vector, derived from Equine Infectious Anemia Virus (EIAV), from which a CCNFpolynucleotide is expressible. The vector may be produced by the transient transfection of cells (e.g. HEK293T cells) with three plasmids, encoding for: (1) the recombinant EIAV PROSAVIN (Oxford BioMedica pic, Oxford UK) vector genome (Farley et al., J. Gen. Med., 9:345-56 (2007); U.S. Pat. No.7,259,015, incorporated by reference); (2) the synthetic EIAV gag / pol expression vector (pESGPK, U.S. patent application Ser. Nos.13 / 893,920 and 12 / 587,236, incorporated by reference) and (3) the VSV-G envelope expression vector (pHGK).

[0177] Herpes simplex virus (HSV) vectors may also be utilized to deliver and express a CCNF polynucleotide in cells of the nervous system, including neurons (e.g., motor neurons). The genome of the type-1 (HSV-1) is about 150 kb of linear, double-stranded DNA, featuring about 70 genes. Many viral genes may be deleted without the virus losing its ability to propagate. The “immediately early” (IE) genes are transcribed first. They encode trans-acting factors which regulate expression of other viral genes. The “early” (E) gene products participate in replication of viral DNA. The late genes encode the structural components of the virion as well as proteins that turn on transcription of the IE and E genes or disrupt host cell protein translation.

[0178] The HSV vector may be a plasmid-based system, whereby a plasmid vector (termed an amplicon) is generated that contains a nucleotide sequence encoding the gene and two cis-acting HSV recognition signals. The recognition signals are the origin of DNA replication and the cleavage packaging signal, which encode no HSV gene products. Thus, helper virus is required to replicate the amplicon and package it into an HSV coat. The vector therefore expresses no viral gene products within the recipient cell, and recombination with or reactivation of latent viruses by the vector is limited due to the minimal amount of HSV DNA sequence present within the defective HSV vector genome.

[0179] Examples of HSV-mediated gene therapy are well known in the art (Breakefield & DeLuca. New Biologist, 3:203-18 (1991); Ho & Mocarski, Virology, 167:279-93 (1988); Palella, et al., Molecular & Cellular Biology, 8:457-60 (1988); Palella et al., Gene, 80:137-44 (1988); Andersen et al., Human Gene Therapy, 3:487-99 (1992); Kaplitt et al., Current Topics Neuroendocrinology, 11:169-91 (1993); Spade & Frenkel, Cell, 30:295-04 (1982); Kaplitt et al., Molecular & Cellular Neuroscience, 2:320-30 (1991); Federoff et al., Proceedings Nat. Acad. Sci. USA, 89:1636-40 (1992)).

[0180] Adenovirus vectors may be utilized to deliver and express CCNF polynucleotides in cells of the nervous system, including neurons (e.g., motor neurons). The adenovirus genome consists of about 36 kb of double-stranded DNA. Adenoviruses target airway epithelial cells, but are also capable of infecting neurons. Recombinant adenovirus vectors have been used as gene transfer vehicles for non-dividing cells. These vectors are similar to recombinant HSV vectors, since the adenovirus E1a immediate-early gene is removed but most viral genes are retained. Since the Ela gene is small (roughly 1.5 kb) and the adenovirus genome is approximately one-third of the size of the HSV genome, other non-essential adenovirus genes are removed in order to insert a foreign gene within the adenovirus genome.

[0181] Examples of adenovirus-mediated gene therapy are well known in the art (Akli et al., Nature Genetics, 3:224-28 (1993); La Salle et al., Science, 259:988-90 (1993), La Salle, Nature Genetics, 3:1-2 (1993); Neve, Trends Biochemical Sci., 16:251-53 (1993)).

[0182] Nucleic acid constructs of the disclosure can also be delivered using virus-like particles (VLPs) and virus-like nanoparticles (VLNPs). VLPs and VLNPs comprise one or more virus- derived structural proteins, but lacking viral genetic material, thereby forming self-assembling structures with the ability to be recognised and taken up by host cells (e.g. see Chung et al., Advanced Drug Delivery Rev, 156:214-235 (2020); Nooraei et al., J Nanobiotech, 19:59 (2021)). VLPs and VLNPs can be exploited as efficient vehicles for delivery of a range of compounds and molecules, including small molecules, peptides, proteinaceous molecules and nucleic acids, including both DNA and RNA, cells and subjects in vivo. As such, VLPs and VLNPs can be utilized for gene therapy applications, vaccine delivery, diagnostics and theranostics. In the context of the present disclosure, VLPs and VLNPs can be used for the delivery of DNA and RNA nucleic acid constructs of the disclosure.

[0183] VLPs and VLNPs can be generated using structural proteins (e.g. capsid, envelope and / or core proteins) derived from any one or more suitable viruses, such as those hereinbefore described, and / or recombinant viral proteins, using methods known to those skilled in the art. It will also be understood by those skilled in the art that a range of symmetrical particles, with an organized underlying geometry, formed from non-viral and artificial proteins can also be considered VLPS and VLNPs (e.g. see Heddle et al., Curr Opin Struct Biol, 43:148-155 (2017)). The scope of the present disclosure is not considered to be limited by reference to any one form, type or size of VLP or VLNP.

[0184] Nucleic acid constructs of the disclosure can also be delivered using a non-viral delivery system. Any delivery method or system for delivery of nucleic acid molecules known in the art can be used, including for example delivery to the desired tissues in colloidal dispersion systems that include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Other delivery systems include exosomes, virosomes, nanoparticles (including gold or silica nanoparticles), polymers (e.g. dendrimers, polymeric nanogels, etc.) Suitable delivery reagents for nucleic acid include, but are not limited to, e.g., the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Research, 32:e109 (2004); Hanai et al. Annals N.Y. Acad. Sci., 1082:9-17 (2006); Kawata et al. Molecular Cancer Therapeutics, 7:2904-12 (2008).

[0185] In one example, a liposome is used as the delivery vehicle. Liposomes suitable for delivery of the nucleic acid constructs described herein can be formed from standard vesicle- forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. Examples of suitable lipids liposomes production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Additional examples of lipids include, but are not limited to, polylysine, protamine., sulfate and 3.beta.-[N--(N',N'-dimethylaminoethane) carbamoyl] cholesterol. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al., Annual Rev. Biophysics & Bioengineering, 9:467-08 (1980); and U.S. Pat. Nos.4,235,871; 4,501,728; 4,837,028; and 5,019,369, which are herein incorporated by reference.

[0186] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms, containing an aqueous solution in the core.

[0187] In another example, non-viral delivery of DNA and RNA nucleic acid constructs of the disclosure may comprise encapsulation or entrapment of a construct within lipid nanoparticles (LNPs). LNPs are typically less than 100 nm in diameter, comprising a mixture of four lipid components, ionizable (e.g. cationic) lipids, 'helper' lipids (e.g. phospholipids), PEGylated lipids and cholesterol in specific ratios. A nucleic acid payload may be entrapped or encapsulated by mixing the nucleic acid molecule(s) with the lipids at an acid pH. Particularly suitable for the delivery of RNA, LNPs can protect the RNA from in vivo degradation by nucleases while facilitating intracellular delivery. LNPs can be generated using a variety of techniques well known to those skilled in the art. Methods of treatment and diagnosis

[0188] Neurons (e.g., motor neurons) can be contacted with a nucleic acid construct of the disclosure in a cell culture e.g., in vitro or ex vivo, or via administration to a subject, e.g., in vivo. In some embodiments, the nucleic acid construct can be administered to a subject to treat or inhibit the development of neurodegenerative conditions, in particular those associated with cytoplasmic accumulation of insoluble, pathogenic TDP-43 (neuronal TDP-43 proteinopathy) and neuronal cell death, such as ALS, FTD and Alzheimer's disease.

[0189] For in vitro methods, neurons can be obtained from different sources. For example, neurons can be obtained from a subject. In some embodiments, the neuron is a whole cell. In some embodiments, the subject is suffering from a neurodegenerative condition, suspected of having a neurodegenerative condition or at risk of developing a neurodegenerative condition. In some embodiments, motor neuron cells are derived from a subject's embryonic stem cells (ESCs). In some embodiments, the subject is human. For in vivo methods, an effective amount of a nucleic acid construct can be administered to a subject. Methods of administering agents to a subject are known in the art and easily available to one of skill in the art.

[0190] Certain aspects of the present disclosure relate to methods for treating neurodegenerative conditions, particularly ones that are associated with cytoplasmic accumulation of insoluble, pathogenic TDP-43 (neuronal TDP-43 proteinopathy) and conditions characterized by neuronal degeneration. Accordingly, an aspect of the present disclosure relates to a method of treating or inhibiting the development of a neurodegenerative condition associated with the cytoplasmic accumulation of insoluble TDP-43, comprising administering to a subject in need thereof a nucleic acid construct or vector of the present disclosure, wherein the transgene encodes a gene product useful in the treatment of a neurodegenerative condition associated with the cytoplasmic accumulation of insoluble TDP-43, and wherein cytoplasmic accumulation of insoluble TDP-43 results in expression of the transgene, thereby treating the neurodegenerative condition. Also provided is a method for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or restoring nuclear TDP-43 function, the method comprising contacting or exposing the neuron to a nucleic acid construct or vector of the present disclosure, wherein thetransgene encodes a gene product useful for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, or inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, and wherein cytoplasmic accumulation of insoluble TDP-43 results in expression of the transgene.

[0191] Where, for example, the transgene encodes a cyclin F polypeptide (the "agent"), suitably the transgene enhances or increases the level or activity of cyclin F and enhances neuronal survival (e.g., a motor neuron survival) and / or inhibits neuronal degeneration (e.g., a motor neuron degeneration) in the subject. In some embodiments, the agent ameliorates at least one symptom associated with the neurodegenerative condition in the subject. In some embodiments, the agent treats the subject's neurodegenerative condition. In some embodiments, the agent prevents the subject from developing a neurodegenerative condition. In some embodiments, the agent prevents the subject's neurodegenerative condition from progressing.

[0192] In some embodiments, the agent decreases the amount of cytoplasmic TDP-43 and enhances neuronal survival (e.g., a motor neuron survival) and / or inhibits neuronal degeneration (e.g., a motor neuron degeneration) in the subject. In some embodiments, the agent decreases the amount of cytoplasmic TDP-43 and ameliorates at least one symptom associated with the neurodegenerative condition in the subject. In some embodiments, the agent decreases the amount of cytoplasmic TDP-43 and treats the subject's neurodegenerative condition. In some embodiments, the agent decreases the amount of cytoplasmic TDP-43 and prevents the subject from developing a neurodegenerative condition. In some embodiments, the agent decreases the amount of cytoplasmic TDP-43 and prevents the subject's neurodegenerative condition from progressing.

[0193] In particular embodiments, the subject is a human.

[0194] In some embodiments, the subject selected for treatment has a neurodegenerative condition, or a condition characterized by motor neuron degeneration. In some embodiments, the subject is at risk of developing a neurodegenerative condition, particularly one that is associated with a neuronal TDP-43 proteinopathy, or a condition characterized by motor neuron degeneration. In some embodiments, the subject is suspected of having a neurodegenerative condition, particularly one that is associated with a neuronal TDP-43 proteinopathy, or a condition characterized by motor neuron degeneration. In some embodiments, the subject is suffering from a neurodegenerative condition, particularly one that is associated with a neuronal TDP-43 proteinopathy. The neurodegenerative condition can be any neurodegenerative condition described herein. In some embodiments, the neurodegenerative condition is marked by motor neuron degeneration. In some embodiments, the neurodegenerative condition is a motor neuron disease. In some embodiments, the neurodegenerative condition is ALS. In some embodiments, the neurodegenerative condition is FTD. In some embodiments, the neurodegenerative condition includes neuronal degeneration other than motor neuron degeneration. some embodiments, the neurodegenerative condition is Alzheimer's disease.

[0195] Cytoplasmic accumulation of insoluble, pathogenic TDP-43 (neuronal TDP-43 proteinopathy) may also be associated with other disease such as Perry syndrome, chronic traumatic encephalopathy, Parkinsonism-dementia complex of Guam, hippocampal sclerosis and multisystem proteinopathy, frontotemporal lobar degeneration, corticobasal degeneration,progressive supranuclear palsy, Gerstmann Straussler Scheinker, neurodegeneration with brain iron accumulation, globular glial tauopathies, primary age-related tauopathy, age-related tau astrogliopathy, post-encephalitic parkinsonism, subacute sclerosis panencephalitis, pantothenate kinase-associated neurodegeneration, chronic traumatic encephalopathy, Down syndrome, myotonic dystrophy, lipofuscinosis, Niemann-Pick disease, type C, Alexander disease, Cockayne syndrome, ganglioglioma / gangliocytoma, pilocytic astrocytoma, lead encephalopathy, traumatic brain injury (acute) and Inclusion body myositis, as presented for example in Chornenkyy et al. (Laboratory Investigation 99:993–1007 (2019)).

[0196] In some embodiments, an additional therapeutic agent is also administered to the subject. Such additional therapeutic agent is typically administered concurrently with the nucleic acid construct of the present disclosure for the treatment of a neurodegenerative condition. For example, the additional therapeutic agent may be selected from hydrogenated pyrido [4,3-b] indoles or pharmaceutically acceptable salts thereof, COX-2 inhibitors, poly(ADP- ribose)polymerase-1 (PARP-I) inhibitors, 3OS ribosomal protein inhibitors, NMDA antagonists, NMDA receptor antagonists, sodium channel blockers, glutamate release inhibitors, K(V)4.3 channel blockers, anti-inflammatory agents, 5-HT1A receptor agonists, neurotrophic factor enhancers, agents that promote motoneuron phenotypic survival and / or neuritogenesis, agents that protect the blood brain barrier from disruption, inhibitors of the production or activity of one or more proinflammatory cytokines, immunomodulators, neuroprotectants, modulators of the function of astrocytes, antioxidants (such as small molecule catalytic antioxidants), free radical scavengers, agents that decrease the amount of one or more reactive oxygen species, agents that inhibit the decrease of non-protein thiol content, stimulators of a normal cellular protein repair pathway (such as agents that activate molecular chaperones), neurotrophic agents, inhibitors of nerve cell death, stimulators of neurite growth, agents that prevent the death of nerve cells and / or promote regeneration of damaged brain tissue, cytokine modulators, agents that reduce the level of activation of microglial cells, cannabinoid CB1 receptor ligands, nonsteroidal anti-inflammatory drugs, cannabinoid CB2 receptor ligands, creatine, creatine derivatives, stereoisomers of a dopamine receptor agonist such as pramipexole hydrochloride, ciliary neurotrophic factors, agents that encode a ciliary neurotrophic factor, glial derived neurotrophic factors, agents that encode a glial derived neurotrophic factor, neurotrophin 3, agents that encode neurotrophin 3, or any combination thereof.

[0197] In some embodiments, the ancillary therapeutic agent may include, for example, one or more of antibiotics (e.g., Aminoglycosides, Cephalosporins, Chloramphenicol, Clindamycin, Erythromycins, Fluoroquinolones, Macrolides, Azolides, Metronidazole, Penicillins, Tetracyclines, Trimethoprim-sulfamethoxazole, Vancomycin), steroids (e.g., Andranes (e.g., Testosterone)), Cholestanes (e.g., Cholesterol), Cholic acids (e.g., Cholic acid), Corticosteroids (e.g., Dexamethasone), Estraenes (e.g., Estradiol), Pregnanes (e.g., Progesterone), narcotic and non- narcotic analgesics (e.g., Morphine, Codeine, Heroin, Hydromorphone, Levorphanol, Meperidine, Methadone, Oxydone, Propoxyphene, Fentanyl, Methadone, Naloxone, Buprenorphine, Butorphanol, Nalbuphine, Pentazocine), anti-inflammatory agents (e.g., Alclofenac, Alclometasone Dipropionate, Algestone Acetonide, alpha Amylase, Amcinafal, Amcinafide, Amfenac Sodium, Amiprilose Hydrochloride, Anakinra, Anirolac, Anitrazafen, Apazone, Balsalazide Disodium, Bendazac, Benoxaprofen, Benzydamine Hydrochloride, Bromelains, Broperamole, Budesonide, Carprofen, Cicloprofen, Cintazone, Cliprofen, Clobetasol Propionate, Clobetasone Butyrate,Clopirac, Cloticasone Propionate, Cormethasone Acetate, Cortodoxone, Decanoate, Deflazacort, Delatestryl, Depo-Testosterone, Desonide, Desoximetasone, Dexamethasone Dipropionate, Diclofenac Potassium, Diclofenac Sodium, Diflorasone Diacetate; Diflumidone Sodium, Diflunisal, Difluprednate, Diftalone, Dimethyl Sulfoxide, Drocinonide, Endrysone, Enlimomab, Enolicam Sodium, Epirizole, Etodolac, Etofenamate, Felbinac, Fenamole, Fenbufen, Fenclofenac, Fenclorac, Fendosal, Fenpipalone, Fentiazac, Flazalone, Fluazacort, Flufenamic Acid, Flumizole, Flunisolide Acetate, Flunixin, Flunixin Meglumine, Fluocortin Butyl, Fluorometholone Acetate, Fluquazone, Flurbiprofen, Fluretofen, Fluticasone Propionate, Furaprofen, Furobufen, Halcinonide, Halobetasol Propionate, Halopredone Acetate, Ibufenac, Ibuprofen, Ibuprofen Aluminum, Ibuprofen Piconol, Ilonidap, Indomethacin, Indomethacin Sodium, Indoprofen, Indoxole, Intrazole, Isoflupredone Acetate, Isoxepac, Isoxicam, Ketoprofen, Lofemizole Hydrochloride, Lomoxicam, Loteprednol Etabonate, Meclofenamate Sodium, Meclofenamic Acid, Meclorisone Dibutyrate, Mefenamic Acid, Mesalamine, Meseclazone, Mesterolone, Methandrostenolone, Methenolone, Methenolone Acetate, Methylprednisolone Suleptanate, Morniflumate, Nabumetone, Nandrolone, Naproxen, Naproxen Sodium, Naproxol, Nimazone, Olsalazine Sodium, Orgotein, Orpanoxin, Oxandrolane, Oxaprozin, Oxyphenbutazone, Oxymetholone, Paranyline Hydrochloride, Pentosan Polysulfate Sodium, Phenbutazone Sodium Glycerate, Pirfenidone, Piroxicam, Piroxicam Cinnamate, Piroxicam Olamine, Pirprofen, Prednazate, Prifelone, Prodolie Acid, Proquazone, Proxazole, Proxazole Citrate, Rimexolone, Romazarit, Salcolex, Salnacedin, Salsalate, Sanguinarium Chloride, Seclazone, Sermetacin, Stanozolol, Sudoxicam, Sulindac, Suprofen, Talmetacin, Talniflumate, Talosalate, Tebufelone, Tenidap, Tenidap Sodium, Tenoxicam, Tesicam, Tesimide, Testosterone, Testosterone Blends, Tetrydamine, Tiopinac, Tixocortol Pivalate, Tolmetin, Tolmetin Sodium, Triclonide, Triflumidate, Zidometacin, Zomepirac Sodium), or anti-histaminic agents (e.g., Ethanolamines (like diphenhydrmine carbinoxamine), Ethylenediamine (like tripelennamine pyrilamine), Alkylamine (like chlorpheniramine, dexchlorpheniramine, brompheniramine, triprolidine), other anti-histamines like astemizole, loratadine, fexofenadine, Bropheniramine, Clemastine, Acetaminophen, Pseudoephedrine, Triprolidine).

[0198] Also provided herein are methods for diagnosing a neurodegenerative condition associated with nuclear depletion of TDP-43 and / or cytoplasmic accumulation of insoluble TDP-43, or for diagnosing impaired neuron function or neuronal degeneration, wherein the methods employ a gene expression modulation switch of the present disclosure in which the, or at least one, heterologous transgene encodes a molecule that acts as a surrogate marker for nuclear depletion of soluble TDP-43 and hence as a marker for a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, for impaired neuron function or for neuronal degeneration.

[0199] An embodiment of the disclosure provides a method for diagnosing in a subject a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising: (a) administering to the subject a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a detectable and / or measurable marker; and (b) obtaining from the subject a biological sample and analysing the sample to detect the presence or absence of the marker, wherein the presence of the marker in the sample is indicative of nuclear depletion of soluble TDP-43 and of the subject having the neurodegenerative condition, or having impaired neuron function or neuronal degeneration.

[0200] Also provided herein is a method for monitoring the response of a subject undergoing therapeutic treatment for a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising the steps of: (a) administering to the subject a nucleic acid construct or vector of the present disclosure before or after commencement of the therapeutic treatment, wherein the heterologous transgene encodes a detectable and / or measurable marker; (b) obtaining from the subject a first biological sample at a first time point and analysing said sample for the presence or amount of said marker; (c) obtaining from the subject at least a second biological sample at a subsequent time point and analysing said sample for the presence or amount of said marker; and (d) comparing the levels of the marker from the first and second samples, wherein a reduction in the level of the marker in the second sample relative to the first sample is indicative of a positive response of the subject to the therapeutic treatment.

[0201] The method may further comprise obtaining from the subject one or more further biological samples at one or more subsequent time points and comparing levels of the marker in said samples to the levels in the first and second samples.

[0202] Suitable exemplary transgenes for use in diagnostic embodiments are described herein above. The transgene may be barcoded or otherwise tagged, using methods well known to those skilled in the art, to facilitate detection or measurement and to distinguish the polypeptide or protein representing a marker, from the endogenous polypeptide or protein expressed by cells in the subject.

[0203] Preferably, the marker is detectable or measurable in one or more biological samples obtained from the subject. Any suitable biological sample can be used depending on the expression and secretion profile of the marker. In exemplary embodiments, the sample may comprise a blood sample, optionally blood serum and / or plasma. In other exemplary embodiments, the sample may comprise cerebrospinal fluid. The skilled person will appreciate that the scope of the present disclosure is not limited by reference to any specific type of biological sample or method of obtaining a biological sample.

[0204] The marker may be detected, assayed or otherwise measured or quantified in biological samples by any suitable methodology known to those skilled in the art, including, for example, immunoassays. The immunoassay may detect or measure both the endogenous polypeptide or protein upon which the marker is based and the diagnostic marker, or may be designed (e.g. using suitable designed antibodies) to detect or measure only the diagnostic marker.

[0205] Utilising both therapeutic and diagnostic aspects of the present disclosure, also contemplated herein are methods enabling both (i) the diagnosis or monitoring of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, of impaired neuron function or of neuronal degeneration, and for (ii) treating said condition, enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or restoring nuclear TDP-43 function.

[0206] For example, in one embodiment there is provided a method comprising: (a) administering to the subject a nucleic acid construct or vector of the present disclosure, whereinthe heterologous transgene encodes a gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, or useful for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or restoring nuclear TDP-43 function; and (b) administering to the subject a nucleic acid construct or vector of the present disclosure, wherein the heterologous transgene encodes a detectable and / or measurable marker, wherein the presence or level of the marker of (b) in at least one biological sample, optionally a blood sample, from the subject is indicative of the efficacy of the product of the transgene of (a) in the treatment of the neurodegenerative condition or the efficacy of the product of the transgene of (a) for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal protein accumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or restoring nuclear TDP-43 function.

[0207] In particular embodiments the transgene of (a) and the transgene of (b) are located in the same nucleic acid construct, operably linked to the same or different promoters. Thus, in an embodiment, step (a) and step (b) are simultaneous and comprise the administration of a single nucleic acid construct.

[0208] Schematic illustrations of exemplary gene expression modulation switch constructs according to the present invention, comprising biomarker transgenes are illustrated in Figure 5 (represented as 'Series 1' – 'Series 3'). Series 1 is the same as Series 1 of Figure 4, with the exception that the transgene is a biomarker rather than therapeutic transgene. In Series 2, the construct comprises both a therapeutic transgene and a biomarker transgene separated by a suitable linker, such as self-cleaving peptide (e.g. T2A). In Series 3, the therapeutic transgene and the biomarker transgene are separated by a linker (T2A) as in Series 2, and a degradation domain sequence and a linker (T2A) are incorporated at the 5' end of the first transgene. The degradation domain DD-FKBP is used to drive auto-degradation as described above for the Series 2 construct in Figure 4. DD-FKBP (Banaszynski et al, 2006, Cell 126: 995-1004) is merely one suitable exemplary degradation domain that may be employed. Variants of DD-FKBP and other suitable degradation domains are described in the literature and would be known to the person skilled in the art (e.g. see Banaszynski et al, 2006, Cell 126: 995-1004; Chu et al., 2008, Bioorg Med Chem Lett 18: 5941-5944).

[0209] In the Series 2 and 3 constructs of Figure 5 the therapeutic transgene is shown located 5' to the biomarker transgene. Those skilled in the art will recognise that this is exemplary only, and the order of the transgenes in the construct may be altered. Compositions

[0210] The present disclosure also provides for compositions, including pharmaceutical compositions, comprising a nucleic acid construct or vector of the disclosure. In some embodiments, pharmaceutical compositions comprise an effective amount of the nucleic acid construct or vector and a pharmaceutically acceptable carrier. For instance, in certain embodiments, the pharmaceutical composition comprises an effective amount of a vector and a pharmaceutically acceptable carrier. An effective amount can be readily determined by thoseskilled in the art based on factors such as body size, body weight, age, health, sex of the subject, ethnicity, and viral titers.

[0211] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. For example, a vector may be formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. Methods for the formulation of compounds with pharmaceutical carriers are known in the art and are described in, for example, in Remington's Pharmaceutical Science, (17th ed. Mack Publishing Company, Easton, Pa.1985); and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th Edition, McGraw-Hill Professional, 2005); the disclosures of each of which are hereby incorporated herein by reference in their entirety.

[0212] Pharmaceutically acceptable carriers suitable for inclusion within any pharmaceutical composition include water, buffered water, saline solutions such as, for example, normal saline or balanced saline solutions such as Hank's or Earle's balanced solutions), glycine, hyaluronic acid etc. The pharmaceutical composition may be formulated for parenteral administration, such as intravenous, intramuscular or subcutaneous administration. Pharmaceutical compositions for parenteral administration may comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, solvents, diluents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and mixtures thereof, vegetable oils (such as olive oil), injectable organic esters (e.g. ethyl oleate).

[0213] The pharmaceutical compositions may comprise penetration enhancers to enhance their delivery. Penetration enhancers may include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, reclineate, monoolein, dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, mono and di-glycerides and physiologically acceptable salts thereof. The compositions may further include chelating agents such as, for example, ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g. sodium salicylate, 5-methoxysalicylate, homovanilate).

[0214] The pharmaceutical compositions may comprise a nucleic acid construct or vector disclosed herein in an encapsulated form. For example, the vectors may be encapsulated within a nanocapsule, such as a nanocapsule comprising one or more biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). In some embodiments, the vectors are encapsulated within polymeric nanocapsules. In other embodiments, the vectors are encapsulated within biodegradable and / or erodible polymeric nanocapsules. In some embodiments, the polymeric nanocapsules are comprised of two different positively charged monomers, at least one neutral monomer, and a crosslinker.

[0215] For administration to a subject, nucleic acid constructs and vectors of the present disclosure can be administered orally, parenterally, for example, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. One method for targeting the nervous system, such as spinal cord glia, is by intrathecal delivery. including CNS delivery, see Ho et al., Curr. Opin. Mol. Ther. (1999), 1:336-3443; Groothuis et al., J. Neuro Virol. (1997), 3:387-400; and Jan, Drug Delivery Systmes: Technologies and Commercial Opportunities, Decision Resources, 1998, content of all which is incorporate herein by reference.

[0216] Examples of injectable forms include solutions, suspensions and emulsions. Injectable forms also include sterile powders for extemporaneous preparation of injectable solutions, suspensions or emulsions. The agents of the present invention can be injected in association with a pharmaceutical carrier such as normal saline, physiological saline, bacteriostatic water, Cremophor.TM. EL (BASF, Parsippany, N.J.), phosphate buffered saline (PBS), Ringer's solution, dextrose solution, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, and other aqueous carriers known in the art. Appropriate non-aqueous carriers may also be used and examples include fixed oils and ethyl oleate. In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. A suitable carrier is 5% dextrose in saline. Frequently, it is desirable to include additives in the carrier such as buffers and preservatives or other substances to enhance isotonicity and chemical stability.

[0217] The agents can also be administered parenterally. Solutions or suspensions of these agents can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Kits

[0218] The nucleic acid constructs and vectors of the present disclosure (i.e. “the agent”) can be provided in a kit, typically with informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and / or the use of the agent for the methods described herein. For example, the informational material describes methods for administering the agent to enhance motor neuron survival, treat or inhibit the development of a neurodegenerative condition, particularly one that isassociated with cytoplasmic accumulation of insoluble TDP-43 or neuronal TDP-43 proteinopathy (e.g., ALS, FTD, AD, etc.), or at least one symptom of the neurodegenerative condition.

[0219] In one embodiment, the informational material can include instructions to administer the agent in a suitable manner, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions for identifying a suitable subject, e.g., a human, e.g., an adult human. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and / or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the modulator and / or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

[0220] In addition to the agent, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and / or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the agent. In such embodiments, the kit can include instructions for admixing the agent and the other ingredients, or for using the agent together with the other ingredients.

[0221] The agent can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the agent be substantially pure and / or sterile. When the agent is provided in a liquid solution, the liquid solution preferably is an aqueous solution.

[0222] The kit can include one or more containers for the composition containing the compound and / or agent. In some embodiments, the kit contains separate containers, dividers or compartments for the agent (e.g., in a composition) and informational material. For example, the agent (e.g., in a composition) can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the agent (e.g., in a composition) is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agent (e.g., in a composition). For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the agent. The containers of the kits can be air tight and / or waterproof.

[0223] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0224] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

[0225] Throughout the specification the aim has been to describe the preferred embodiments of the disclosure without limiting the disclosure to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

[0226] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES Example 1 – ALS-specific expression of cryptic exons

[0227] The genes STMN2 and UNC13A, both considered risk factors for ALS and FTD, have previously been identified as containing cryptic exons regulated by TDP-43 (see Melamed et al., 2019, Nat Neurosci 22:180-190; Ma et al., 2022, Nature 603:1240-130).

[0228] The cryptic exon from STMN2 displays CNS-specific expression in ALS patients. RNA sequencing (RNA-seq) was performed upon post-mortem tissue from five brain regions from 25 sporadic ALS cases and 11 healthy controls (see Figure 7), and expression of the STMN2 cryptic exon was detected by the presence of truncated STMN2 mRNA within the RNA-seq dataset. Cases were considered positive if the truncated STMN2 mRNA is present above a pre-set threshold of 2 or more uniquely mapped reads. ALS cases were subcategorised by their TDP-43 pathology stage. Six CNS regions varying in their presentation of TDP-43 pathology were examined: motor cortex and spinal cord (pathology in stages 1-4), prefrontal cortex (pathology in stages 2-4), hippocampus (pathology in stage 4), occipital cortex and cerebellum (rare to nil TDP-43 pathology). Example 2 – TDP-43 responsive gene expression switch

[0229] Cells were transfected with a nucleic acid construct 'UNC13A-CE-eGFP'; GenScript. The construct contains the UNC13A cryptic exon (CE) (SEQ ID NO:8, with stop codons from the wild-type cryptic exon sequence removed) and flanking introns (SEQ ID NOs:5 and 9) located between a FLAG-tag and an eGFP gene (SEQ ID NO:30) preceded by a single G nucleotide to induce a frameshift, under the control of the CMV promoter (SEQ ID NO:2) separated from FLAG by a non-coding spacer (SEQ ID NO:3 or 4).

[0230] UNC13A was chosen because disease-causing genetic variants in UNC13A have been identified in ALS and FTD patients, and there are recent reports describing that the UNC13A cryptic exon is regulated by TDP-43 binding to the adjoining intron. The UNC13A cryptic exon sequence was modified to remove five internal stop codons and an unwanted alternative splice site. Cells were analysed for eGFP 24 hours post transfection. eGFP expression can only occur in the absence of TDP-43 expression (or where TDP-43 levels are insufficient to repress all of the CE present in the transfected construct). In preliminary experiments shown in Figure 8, under basal conditions, a low-level eGFP expression was observed, demonstrating that the UNC13A-CE-eGFP construct works in achieving splicing and readthrough of the CE. Without wishing to be bound by theory, the inventors suggest that the low level of eGFP expression was seen in basal conditionsbecause there is insufficient endogenous TDP-43 to fully suppress the overexpressed UNC13A-CE- eGFP.

[0231] The inventors have also transfected HEK293T cells with the UNC13A-CE-eGFP construct together with siRNA against the TDP-43 gene TARDBP or a scrambled siRNA control. After 24 hours, flow cytometry was used to analyse eGFP levels in individual cells. As shown in Table 2 and Figure 9, an approximately 53% increase in mean eGFP expression was observed in cells co- transfected with TARDBP siRNA versus scrambled siRNA (control). This demonstrates that reduction in endogenous nuclear TDP-43 levels causes activation of the UNC13A-CE-GFP switch. Table 2 Treatment Mean GFP si nal U U s1 Relative fluorescence units (RFU)

[0232] The inventors have also transfected HEK293T cells with the SYT7-CE-eGFP construct together with siRNA against the TDP-43 gene TARDBP or a scrambled siRNA control. After 24 hours, flow cytometry was used to analyse eGFP levels in individual cells. As shown in Table 3, an approximately 30% increase in mean eGFP expression (average of 3 replicates) was observed in cells co-transfected with TARDBP siRNA versus scrambled siRNA (control). Table 3 T S S1 Relative fluorescence units (RFU) Example 3 – Real time monitoring of TDP-43 responsive gene expression switch

[0233] To observe the responsiveness of the switch to TDP-43 levels over a longitudinal timecourse, continuous real-time monitoring of cells and fluorescence was performed using the Incucyte® platform. To confirm that both the UNC13A-CE and SYT7-CE switch described above (i.e. mCherry-UNC13A-CE-eGFP and mCherry-SYT7-CE-eGFP) are TDP-43 responsive, two different approaches to knockdown TDP-43 levels were utilized:siRNA (a pool of 4x siRNA against differentTDP-43 target sequences, i.e. different TARDBP-specific siRNAs); and a lentivirus expressing shRNA against the TDP-433’ UTR, i.e. endogenous specific TARDBP shRNA. A. Methods

[0234] siRNAs: TARDBP-specific siRNAs having the following sequences were generated: GCUCAAGCAUGGAUUCUAA (SEQ ID NO:31); CAAUCAAGGUAGUAAUAUG (SEQ ID NO:32); GGGCUUCGCUACAGGAAUC (SEQ ID NO:33); and CAGGGUGGAUUUGGUAAUA (SEQ ID NO:34) (Dharmacon L-012394-00-0005).

[0235] shRNA: The lentivirus expressing TARDBP-specific shRNA (lentivirus-shRNA (TARDBP)) is a pLV lentiviral vector encoding a Near-InfraRed (NIR) fluorophore (miRFP670nano), followed by the TDP-43 shRNA target from Fiesel et al. (The EMBO Journal, 2009, 29: 209 – 221) CACTACAATTGATATCAAAAGG (SEQ ID NO:35), which is excised from the NIR transcript by flanking with miR30 sequence. The expression of this transcript is under the control of a TRE3G tetracycline-inducible promoter.

[0236] HEK293T cells: A HEK293T cell line with endogenously labelled TDP-43-mCherry was used for experiments for transient knock down of TARDBP. Cells were maintained in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum in an incubator at 37⁰C and 5% CO2.

[0237] siRNA knockdown: Transient knock down of TARDBP was performed using 100nM of siRNA(targeting TARDBP) (or control siRNA(scrambled)) using lipofectamine2000 according to manufacturer’s protocol (Thermo Fisher Scientific) over at least three days. Subsequently, the cells were treated with siRNA and also transiently transfected with plasmids for mCherry-UNC13A-switch-eGFP or mCherry-SYT7-switch-eGFP, using lipofectamine2000.

[0238] shRNA knockdown: HEK293T cells and a HEK293T cell line with endogenously labelled TDP-43-mCherry were each transduced with a lentivirus expressing tetracycline-inducible shRNA targeting TARDBP containing a Near InfraRed (NIR) reporter, and used for experiments for inducible knock down of TARDBP. Cells were maintained in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum in an incubator at 37⁰C and 5% CO2. Inducible knock down of TARDBP was achieved with 0.1µg / ml tetracycline treatment (inducing the shRNA(TARDBP) and NIR reporter). Untreated (no tetracycline and hence not inducing the shRNA(TARDBP)) cells served as the control group. Subsequently, both tetracycline-treated and untreated cells were also transiently transfected with a plasmid for mCherry-UNC13A-switch-eGFP, using lipofectamine2000, on day 0 unless otherwise specified. Live cell imaging and analysis was conducted using Incucyte® SX5 (Sartorius). For experiments analyzing the NIR reporter, 25 µM of biliverdin was added to the media. B. Responsiveness of the switches to TDP-43 knockdown by siRNA

[0239] The knockdown of endogenous TDP-43 expression using TARDBP-specific siRNAs was first validated in HEK293T cells over 72 hours. Endogenously labelled TDP-43-mCherry HEK293T cells (i.e. HEK293T cells engineered to introduce a mCherry fusion-tag onto endogenous TDP-43) treated with 100nM of TARDBP-specific siRNA or control siRNA (scrambled) for 72 hours were continuously monitored with Incucyte® live-cell imaging. Red channel measured endogenous TDP-43-mCherry. As shown in Figure 10, there was an approximate 35% knockdown in TDP-43levels by TARDBP-specific siRNA between 20-70 hours post-transfection. siRNA (scrambled) had a minor transient effect on TDP-43 levels (~15%).

[0240] Responsiveness of the UNC13A-CE switch to siRNA knockdown of TDP-43 levels was then assessed. HEK293T cells transiently transfected with a plasmid expressing mCherry- UNC13A-CE switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or TARDBP-specific siRNA and monitored continuously over 92 hours via Incucyte® live-cell imaging. The UNC13A-CE switch was activated from 20 hours onwards, consistent with the knockdown in TDP-43 levels (as shown in Figure 10). The activation of the UNC13A-CE switch was confirmed by an increase in both the number of eGFP-positive cells (Figure 11) and the mean eGFP- fluorescence intensity in cells (Figure 12). Fluorescence (eGFP) images captured at 92 hours post- transfection confirmed the activation of the UNC13A-CE switch in a number of cells resulting in eGFP reporter expression, with some background autofluorescence observed in cells expressing the UNC13A-CE switch treated with the scrambled siRNA (data not shown).

[0241] Responsiveness of the SYT7-CE switch to siRNA knockdown of TDP-43 levels was then assessed. HEK293T cells transiently transfected with a plasmid expressing mCherry-SYT7 CE- switch-eGFP were treated with 100nM of control siRNA (scrambled sequence) or siRNA (TARDBP) and monitored continuously over 92 hours via Incucyte®live-cell imaging. The SYT7-CE switch was activated from 20 hours onwards, consistent with the knockdown in TDP-43 levels (as shown in Figure 10). The activation of the SYT7-CE switch was confirmed by an increase in both the number of eGFP-positive cells (Figure 13) and the mean eGFP-fluorescence intensity in cells (Figure 14). Fluorescence (eGFP) images captured at 92 hours post-transfection confirmed the activation of the SYT7-CE switch in a number of cells resulting in eGFP reporter expression, with some background autofluorescence observed in cells expressing the SYT7-CE switch treated with the scrambled siRNA (data not shown). C. Responsiveness of switch to TDP-43 knockdown by shRNA

[0242] The knockdown of endogenous TDP-43 expression in HEK293T cells using a lentivirus expressing a tet-inducible shRNA targeted against TARDBP was first validated. The shRNA was tagged with a Near InfraRed (NIR) reporter. Endogenously labelled TDP-43-mCherry HEK293T cells were transduced with the lentivirus-shRNA (TARDBP), and treated with 0.1µg / ml tetracycline to induce shRNA expression. TDP-43-mCherry fluorescence was continuously monitored over 8 days with Incucyte®live-cell imaging. Figure 15 shows ~60% knockdown in TDP-43 levels by shRNA (TARDBP) between 2-8 days post-tetracycline treatment, in comparison to untreated cells (where shRNA is not activated).

[0243] Responsiveness of the UNC13A-CE switch to shRNA knockdown of TDP-43 levels was then assessed. HEK293T cells transiently transfected with a plasmid expressing mCherry- UNC13A-CE switch-eGFP and lentivirus-shRNA (TARDBP) were treated with 0.1µg / ml tetracycline and monitored continuously over 8 days via Incucyte live-cell imaging. The UNC13A-CE switch was activated from 2 days onwards, consistent with the knockdown in TDP-43 levels (as shown in Figure 15). The activation of the UNC13A-CE switch was confirmed by an increase in both the number of eGFP-positive cells (Figure 16) and the mean eGFP-fluorescence intensity in cells (Figure 17).

[0244] To further confirm these observations and the responsiveness of the UNC13A CE switch to TDP-43, HEK293T were prior transduced with lentivirus expressing shRNA (TARDBP) with a Near-InfraRed (NIR) readout. These cells were treated with 0.1µg / ml tetracycline on day 0, and refreshed at 2 days and 18 hours. The cells were transfected with the plasmid expressing mCherry- UNC13A-CE switch-eGFP at 2 days and 18 hours. Expression of the NIR (shRNA (TARDBP)) was detected 5 days after tetracycline treatment by NIR fluorescence monitoring (Figure 18), peaking at 6 days and then expression levels declined up to 8 days. Importantly, this coincided with the activation of the UNC13A-CE switch which expresses eGFP between 5-8 days (Figure 19), which follows a similar time-course of expression to the NIR (shRNA (TARDBP)).

Claims

CLAIMS:

1. A method for modulating the expression of a heterologous transgene administered to a cell, wherein the heterologous transgene encodes a cyclin F polypeptide and is located in a nucleic acid construct comprising a TDP-43 responsive binding element operably linked at the 5' end of the heterologous transgene, wherein the TDP-43 responsive binding element and the transgene are operably linked to a promoter capable of driving expression of the transgene in the cell, wherein in the presence of nuclear TDP-43, the TDP-43 binds to the TDP-43 responsive binding element repressing expression of the transgene and wherein in the absence of nuclear TDP-43 the transgene is expressed, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein modulation of expression of the transgene is achieved by regulation of RNA splicing of the first and second nucleic acid sequences, such that in the absence of nuclear TDP-43 the first and second nucleic acid sequences are removed from the nucleic acid construct by RNA splicing resulting in expression of the transgene.

2. A method for modulating the expression of a heterologous transgene administered to a cell, wherein the heterologous transgene is located in a nucleic acid construct comprising a TDP-43 responsive binding element operably linked at the 5' end of the heterologous transgene, wherein the TDP-43 responsive binding element and the transgene are operably linked to a promoter capable of driving expression of the transgene in the cell, wherein in the presence of nuclear TDP-43, the TDP-43 binds to the TDP-43 responsive binding element repressing expression of the transgene and wherein in the absence of nuclear TDP-43 the transgene is expressed, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein modulation of expression of the transgene is achieved by regulation of RNA splicing of the first and second nucleic acid sequences, such that in the absence of nuclear TDP-43 the first and second nucleic acid sequences are removed from the nucleic acid construct by RNA splicing resulting in expression of the transgene.

3. The method of claim 1 or 2, wherein at least about 80%, 85%, 90% or 95% of the nucleotides of the region comprising at least about 30 consecutive nucleotides in the first nucleic acid sequence and / or second nucleotide sequence are U or G.

4. The method of any one of claims 1 to 3, wherein said region comprises one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues.

5. The method of any one of claims 1 to 4, wherein said region comprises one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues.

6. The method of any one of claims 1 to 5, wherein said region comprises one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG, optionally wherein said region comprises two, three, four, five, six, seven or more of said consecutive motifs.

7. The method of any one of claims 1 to 6, wherein the third nucleic acid sequence comprises a cryptic exon, wherein the cryptic exon does not contain any stop codons.

8. The method of claim 7, wherein the cryptic exon encodes a cryptic peptide.

9. The method of claim 7 or 8, wherein a nucleotide sequence encoding a degradation domain is incorporated at the 3' end of the cryptic exon or cryptic peptide-encoding sequence, and a linker is introduced at the 5' end of the heterologous transgene, such that expression of the cryptic exon or cryptic peptide produces a fusion peptide comprising a product of the cryptic exon and the degradation domain or comprising the cryptic peptide and the degradation domain, wherein the presence of the degradation domain facilitates auto-degradation of the fusion peptide.

10. The method of any one of claims 1 to 9, wherein the absence of nuclear TDP-43 is caused by the cytoplasmic accumulation of insoluble TDP-43.

11. The method of any one of claims 1 to 10, wherein the level of expression of the heterologous transgene is responsive to levels of nuclear TDP-43, such that a reduction in the level of nuclear TDP-43 results in an increase in the level of expression of the transgene, and an increase in the level of nuclear TDP-43 results in a decrease in the level of expression of the transgene.

12. The method of any one of claims 1 to 11, wherein the cell is a neuron, optionally a motor neuron, or a glial cell.

13. The method of any one claims 1 to 12, wherein the promoter is capable of driving expression in a neuron, optionally a motor neuron, or a glial cell, and optionally wherein the promoter is a constitutive promoter.

14. The method of any one of claims 2 to 13, wherein the heterologous transgene is a therapeutic transgene that encodes a gene product useful in the treatment of a neurodegenerativecondition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, and / or a gene product useful for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or for restoring nuclear TDP-43 function.

15. The method of claim 14, wherein the neurodegenerative condition is selected from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

16. The method of claim 14 or 15, wherein the transgene encodes a cyclin F polypeptide.

17. The method of any one of claims 2 to 15, wherein the heterologous transgene encodes a detectable and / or measurable marker useful for detecting nuclear TDP-43 depletion and for diagnosing a neurodegenerative condition, impaired neuron function or neuronal degeneration.

18. The method of claim 17, wherein the transgene encodes a neuronally-expressed protein.

19. The method of claim 17 or 18, wherein the marker is detectable in the blood and / or in the cerebrospinal fluid.

20. The method of any one of claims 2 to 19, wherein the nucleic acid construct comprises two heterologous transgenes, operably linked to the same or different promoters, optionally wherein the first heterologous transgene encodes a therapeutic gene product as defined in any one of claims 15 to 17 and the second heterologous transgene encodes a detectable and / or measurable marker as defined in any one of claims 18 to 20.

21. A nucleic acid construct comprising a heterologous transgene, wherein the heterologous transgene encodes a cyclin F polypeptide and is operably linked to a TDP-43 responsive binding element that is 5' to the transgene, wherein the transgene and the TDP-43 responsive binding element are operably linked to a promoter capable of driving expression of the transgene in a cell, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, said region comprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G.

22. A nucleic acid construct comprising a heterologous transgene operably linked to a TDP-43 responsive binding element that is 5' to the transgene, wherein the transgene and the TDP-43 responsive binding element are operably linked to a promoter capable of driving expression of the transgene in a cell, wherein the TDP-43 responsive binding element comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are separated by a third nucleotide sequence, and wherein one or both of said first and second nucleic acid sequences comprises a region to which nuclear TDP-43 binds, said regioncomprising at least about 30 consecutive nucleotides in which at least about 75% of the nucleotides are U or G.

23. The nucleic acid construct of claim 21 or claim 22, wherein at least about 80%, 85%, 90% or 95% of the nucleotides of the region comprising at least about 30 consecutive nucleotides in the first nucleic acid sequence and / or second nucleotide sequence may be U or G.

24. The nucleic acid construct of any one of claims 21 to 23, wherein said region comprises one or more stretches of at least 5 consecutive nucleotides consisting of U and G residues.

25. The nucleic acid construct of any one of claims 21 to 24, wherein said region comprises one or more stretches of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides consisting of U and G residues.

26. The nucleic acid construct of any one of claims 21 to 25, wherein said region comprises one or more consecutive motifs selected from UG, GU, UU, GG, UGU and GUG., optionally wherein said region comprises two, three, four, five, six, seven or more of said consecutive motifs.

27. The nucleic acid construct of any one of claims 21 to 26, wherein the third nucleic acid sequence comprises a cryptic exon, wherein the cryptic exon does not contain any stop codons.

28. The nucleic acid construct of claim 27, wherein the cryptic exon encodes a cryptic peptide.

29. The nucleic acid construct of claim 27 or 28, wherein a nucleotide sequence encoding a degradation domain is incorporated at the 3' end of the cryptic exon or cryptic peptide-encoding sequence, and a linker is introduced at the 5' end of the heterologous transgene, such that expression of the cryptic exon or cryptic peptide produces a fusion peptide comprising a product of the cryptic exon and the degradation domain or comprising the cryptic peptide and the degradation domain, wherein the presence of the degradation domain facilitates auto-degradation of the fusion peptide.

30. The nucleic acid construct of any one of claims 22 to 29, wherein the heterologous transgene is a therapeutic transgene that encodes a gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, and / or a gene product useful for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or for restoring nuclear TDP-43 function.

31. The nucleic acid construct of claim 30, wherein the neurodegenerative condition is selected from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

32. The nucleic acid construct of claim 30 or 31, wherein the transgene encodes a cyclin F polypeptide.

33. The nucleic acid construct of any one of claims 22 to 29, wherein the heterologous transgene encodes a detectable and / or measurable marker useful for detecting nuclear TDP-43 depletion and for diagnosing a neurodegenerative condition, impaired neuron function or neuronal degeneration.

34. The nucleic acid construct of claim 33, wherein the transgene encodes a neuronally- expressed protein.

35. The nucleic acid construct of claim 33 or 34, wherein the marker is detectable in the blood and / or in the cerebrospinal fluid.

36. The nucleic acid construct of any one of claims 21 to 35, wherein the nucleic acid construct comprises two heterologous transgenes, operably linked to the same or different promoters, optionally wherein the first heterologous transgene encodes a therapeutic gene product as defined in any one of claims 30 to 32 and the second heterologous transgene encodes a detectable and / or measurable marker as defined in any one of claims 33 to 35.

37. The nucleic acid construct of any one claims 21 to 36, wherein the promoter is capable of driving expression in a neuron, optionally a motor neuron, or a glial cell, and optionally wherein the promoter is a constitutive promoter.

38. A vector comprising the nucleic acid construct of any one of claims 21 to 37.

39. The vector of claim 38, wherein the vector is a plasmid or a viral vector.

40. A host cell comprising the nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39.

41. A method for treating a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising administering to a subject in need thereof a nucleic acid construct of any one of claims 21 to 37 or a vector of claim 38 or 39, wherein the heterologous transgene encodes a gene product useful in the treatment of a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, and wherein nuclear depletion of soluble TDP-43 results in expression of the transgene, thereby treating the neurodegenerative condition.

42. The method of claim 41, wherein the neurodegenerative condition is selected from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

43. A method for enhancing survival of a neuron, for inhibiting degeneration of a neuron, for inhibiting abnormal protein accumulation in a neuron, for inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or for restoring nuclear TDP-43 function, the method comprising contacting or exposing the neuron to a nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39, wherein the heterologous transgene encodes a gene product useful for enhancing survival of a neuron, inhibiting degeneration of a neuron, inhibiting abnormal proteinaccumulation in a neuron, inhibiting aggregated or insoluble TDP-43 accumulation in a neuron, or restoring nuclear TDP-43 function, and wherein nuclear depletion of soluble TDP-43 results in expression of the transgene.

44. A method for diagnosing in a subject a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, for diagnosing or detecting impaired neuron function or for diagnosing or detecting neuronal degeneration, comprising: (a) administering to the subject a nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39, wherein the heterologous transgene encodes a detectable and / or measurable marker; and (b) obtaining from the subject a biological sample and analysing the sample to detect the presence or absence of the marker, wherein the presence of the marker in the sample is indicative of nuclear depletion of soluble TDP- 43 and of the subject having the neurodegenerative condition, or having impaired neuron function or neuronal degeneration.

45. The method of claim 44, wherein the biological sample comprises a blood sample or cerebrospinal fluid.

46. A method for monitoring the response of a subject undergoing therapeutic treatment for a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising the steps of: (a) administering to the subject a nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39 before or after commencement of the therapeutic treatment, wherein the heterologous transgene encodes a detectable and / or measurable marker; (b) obtaining from the subject a first biological sample at a first time point and analysing said sample for the presence or amount of said marker; (c) obtaining from the subject at least a second biological sample at a subsequent time point and analysing said sample for the presence or amount of said marker; and (d) comparing the levels of the marker from the first and second samples, wherein a reduction in the level of the marker in the second sample relative to the first sample is indicative of a positive response of the subject to the therapeutic treatment.

47. The method of claim 46, further comprising obtaining from the subject one or more further biological samples at one or more subsequent time points and comparing levels of the marker in said samples to the levels in the first and second samples.

48. The method of claim 46 or 47, wherein the first and second biological samples, and optionally the one or more further samples, comprise blood samples or cerebrospinal fluid.

49. The method of any one of claims 44 to 48, wherein the marker comprises a neuronally-expressed protein or polypeptide.

50. A method for treating a neurodegenerative condition associated with the nuclear depletion of TDP-43 and / or the cytoplasmic accumulation of insoluble TDP-43, comprising: (a) administering to the subject a nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39, wherein the heterologous transgene encodes a gene product useful in the treatment of the neurodegenerative condition; and (b) administering to the subject a nucleic acid construct of any one of claims 21 to 37 or the vector of claim 38 or 39, wherein the heterologous transgene encodes a detectable and / or measurable marker, wherein the presence or level of the marker of (b) in at least one biological sample, optionally a blood sample, from the subject is indicative of the efficacy of the product of the transgene of (a) in the treatment of the neurodegenerative condition.

51. The method of claim 50, wherein the transgene encoding the gene product useful in the treatment of the neurodegenerative condition and the transgene encoding the marker are present in the same nucleic acid construct, such that steps (a) and (b) are simultaneous and comprise the administration of a single nucleic acid construct or vector.

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