Pabpn1 isoforms for use in therapy

Abstract

Short expansion mutations in PABPN1 cause adult-onset, autosomal dominant oculopharyngeal muscular dystrophy (OPMD). The molecular hallmark of OPMD is the presence of insoluble nuclear aggregates of the expanded (exp)PABPN1. The aggregation of PABPN1 results in reduced levels of functional PABPN1. In OPMD, PABPN1 levels are under a critical threshold which leads to muscle weakness. Interestingly, reduced levels of PABPN1 are also found in ageing muscles, where these reduced levels lead to muscle atrophy and has also been found to be associated with other adult-onset pathologies affecting tissues other than skeletal muscles, such as cancers, including bladder cancer, and heart diseases.

Description

[0001] PABPN1 ISOFORMS FOR USE IN THERAPY

[0002] Short expansion mutations in PABPN1 cause adult-onset, autosomal dominant oculopharyngeal muscular dystrophy (OPMD). The molecular hallmark of OPMD is the presence of insoluble nuclear aggregates of the expanded (exp)PABPNI . The aggregation of PABPN1 results in reduced levels of functional PABPN1. In OPMD, PABPN1 levels are under a critical threshold which leads to muscle weakness. Interestingly, reduced levels of PABPN1 are also found in ageing muscles, where these reduced levels lead to muscle atrophy and has also been found to be associated with other adult-onset pathologies affecting tissues other than skeletal muscles, such as cancers, including bladder cancer, and heart diseases.

[0003] PABPN1 is a master regulator of RNA processing and its limited availability in skeletal muscles causes atrophy and weakness. In OPMD, PABPN1 forms nuclear aggregates whereby levels of functional PABPN1 are below a critical level leading to genome-wide transcriptional shifts affecting protein translation efficiency and impaired homeostasis, impacting muscle function.

[0004] Solving the problem of age-associated muscle weakness, particularly in diseases like OPMD, is crucial for improving the quality of life for affected individuals. OPMD and similar neuromuscular disorders cause progressive muscle degeneration, leading to difficulties in mobility, swallowing, and other daily activities. With no current cure available, patients face significant challenges in managing symptoms and maintaining functionality.

[0005] Restoring PABPN1 expression levels by overexpression of functional PABPN1 or via aggregate disaggregation are considered therapeutic options for OPMD. A gene therapy approach to correct the expression of expPABPNI is in clinical trials. Yet, overexpression of PABPN1 is not without risk as it can lead to an increase in aggregation of PABPN1 with consequent depletion of functional PABPN1 rather than restore PABPN1 function. With current international research focusing on gene therapy approaches, there is growing momentum towards developing novel therapeutic interventions for OPMD. However, concerns regarding the safety and efficacy of existing strategies highlight the need for alternative approaches.

[0006] To try and address the problem of overexpression and aggregation, Malerba et al., 20177have developed a dual action therapy method, where the endogenous PABPN1 is depleted using RNAi and a replacement codon-optimized PABPN1 that is resistant to siRNA degradation is expressed. The present invention is particularly advantageous over this approach as it does not require the initial clearance of endogenous PABPN1. This may reduce safety risks associated with depletion of the endogenous protein and an over-expression of the codon- optimized variant. This therapy is based on the expression on natural PABPN1 isoform therefore, reduce the risk of toxicity and immunogenicity, therefore is expected to be safer and simpler.

[0007] To address the unmet needs discussed above, the current invention aims to elevate expression levels of PABPN1 , without driving protein aggregation. This could open therapeutic options for adult-onset pathologies driven by a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 , including, but not limited to, OPMD. Although PABPN1 is primarily linked to OPMD and muscle ageing5, recent studies have demonstrated its causative involvement in bladder cancer6. In both settings, reduced PABPN1 levels drive the pathology. In OPMD, this reduction occurs alongside protein aggregation, while in bladder cancer the loss of functional PABPN1 appears to be the key driver, therefore the present invention appears to have utility in diseases with loss of functional PABPN1 such as bladder cancer.

[0008] Brief summary of the disclosure

[0009] The present invention is based on the inventors’ finding that a truncated isoform of PABPN1 (tr-PABPN1) lacking exon 1 encoding for the aggregation domain is naturally expressed in skeletal muscle, and that its expression levels correlate with PABPN1 molecular function in OPMD mice, cellular models and in OPMD muscle biopsies. The inventors then made the surprising finding that tr-PABPN1 is soluble and stable and that overexpression of tr-PABPN1 in muscle cells does not form nuclear aggregates, thus making tr-PABPN1 a suitable candidate for treating diseases or conditions associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 .

[0010] The inventors have further shown that loss of functional PABPN1 appears to be a key driver of pathology in cancer. Interestingly, the inventors have shown that Tr-PAB PABPN1 (Tr-PAB) expression in cancer cells does not repress endogenous PABPN1 levels and expression of Tr-PAB rescue PABPN1 function, including a shift from APA towards distal PAS usage and restores cell growth and migration of bladder cancer. Therefore, demonstrating that the present invention is not only therapeutically relevant to muscular pathology, but also diseases associated with a reduction in the level of PABPN1 , such as bladder cancer. Advantageously, isoforms of PABPN1 lacking the aggregation domain can restore PABPN1 function without contributing to aggregation, thus minimising risk of enhanced aggregation as seen with therapeutic delivery of full-length PABPN1 . Such isoforms also do not interfere with cellular metabolism and may enhance muscle cell differentiation. Even further, as tr-PABPN1 is a naturally occurring protein, tr-PABPN1 has the additional advantage of a reduced risk of toxicity and / or immunogenicity.

[0011] The data presented herein shows that the naturally occurring tr-PABPN1 isoform 207 (SEQ ID NO: 1) does not form nuclear aggregates when overexpressed in human muscle cells and can restore PABPN1 molecular function. Even further, the data shows that this effect is not limited to muscle cells, and that tr-PABPN1 can be expressed in bladder cancer cells. Cancer cells are known to have elevated levels of mitochondrial activity, and the data presented herein shows thattr-PABPNI results in a reduction mitochondrial activity compared to vehicle control. Additionally, as demonstrated herein, expression of tr-PABPN1 in cancer cells also reduced cellular migration and slow down growth rate. Thus, the use of a truncated PABPN1 has utility beyond OPMD.

[0012] The invention is not limited to tr-PABPN1 , as the inventors have identified further nonaggregating variants of PABPN1 . The invention therefore provides functional non-aggregating variants of PABPN1 for use as a medicament. These variants are described in more detail below. These variants are described as “PABPN1 polypeptides disclosed herein”, but may also be interchangeably referred to as “truncated PABPN1 polypeptides” (e.g. truncated compared to SEQ ID NO:2), PABPN1 derivatives (e.g. derivatives of SEQ ID NO:2), or PABPN1 variants (particularly variants or truncated variants of SEQ ID NO:2). They may also be referred to by function, as “non-aggregating PABPN1 polypeptides”.

[0013] Accordingly, in one aspect the invention provides a poly(A) binding protein nuclear 1 (PABPN1 ) polypeptide ora nucleic acid encoding the PABPN1 polypeptide for use in a method for treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; does not comprise an intrinsically disordered region according to SEQ ID NO:9; does not comprise a coiled coil domain according to SEQ ID NO: 4.

[0014] In some embodiments, the PABPN1 polypeptide is a truncated isoform or variant of SEQ ID NO:2, wherein the PABPN1 polypeptide does not comprise at least one of: the N-terminal alanine tract according to SEQ ID NO:3; the intrinsically disordered region according to SEQ ID NO:9; the coiled coil domain according to SEQ ID NO: 4. In some embodiments, a truncated isoform or variant of PABPN1 comprises no more than 300 amino acids of SEQ ID NO: 2, no more than 290 amino acids of SEQ ID NO: 2, no more than 280 amino acids of SEQ ID NO: 2, no more than 270 amino acids of SEQ ID NO: 2, no more than 260 amino acids of SEQ ID NO: 2, no more than 250 amino acids of SEQ ID NO: 2, no more than 240 amino acids of SEQ ID NO: 2, no more than 230 amino acids of SEQ ID NO: 2, no more than 220 amino acids of SEQ ID NO: 2, no more than 210 amino acids of SEQ ID NO: 2, no more than 200 amino acids of SEQ ID NO: 2, or no more than 150 amino acids of SEQ ID NO: 2.

[0015] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0016] In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise at least one of: (i) the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2,

[0017] (ii) the intrinsically disordered region corresponding to amino acids 1 to 115 of SEQ ID NO:2,

[0018] (iii) the coiled coil domain corresponding to amino acids 115 to 151 of SEQ ID NO:2.

[0019] In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise: (i) the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2, (ii) the intrinsically disordered region corresponding to amino acids 1 to 115 of SEQ ID NO:2, and / or (iii) the coiled coil domain corresponding to amino acids 115 to 151 of SEQ ID NO:2.

[0020] In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 3 and does not comprise SEQ ID NO:9. In one preferred embodiment, the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 4 and does not comprise SEQ ID NO: 3. In yet another preferred embodiment, the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 4, does not comprise SEQ ID NO: 3 and does not comprise SEQ ID NO:9.

[0021] The full-length amino acid sequence of PABPN1 polypeptide is shown in SEQ ID NO: 2. This is also referred to as the “wild-type” amino acid sequence of PABPN1 herein. This may be used interchangeably with wt-PABPN1 . In some embodiments, the disease or condition is further associated with an increase in alternative polyadenylation (APA) and / or alternative last exon (ALE) usage. Suitably, in some embodiments the disease or condition is further associated with a shift to proximal pA site usage. Suitably, in some embodiments the disease or condition is further associated with a shift to proximal APA. Suitably, in some embodiments the shift is from a distal polyadenylation (pA) to a proximal pA in the same 3’UTR.

[0022] In some embodiments of the present invention, the disease or condition is cardiac disease, cancer, autoimmune disease, ageing and / or muscle pathology. Suitably, in some embodiments the cancer is non-bladder cancer, bladder cancer, or small cell lung cancer; or the muscle pathology is oculopharyngeal muscular dystrophy (OPMD), facioscapulohumeral muscular dystrophy (FSHD), or sarcopenia.

[0023] In some embodiments, the disease or condition is associated with a reduction in the level of PABPN1 by at least 30% compared to levels of PABPN1 polypeptide in a healthy subject.

[0024] In some embodiments, the disease or condition is associated with a reduction in the level of PABPN1 by 30-90% compared to levels of PABPN1 polypeptide in a healthy subject.

[0025] In some embodiments, the PABPN1 polypeptide restores cellular defects, and optionally wherein the PABPN1 polypeptide restores 3’-UTR length or ALE usage. Suitably, in some embodiments the cellular defects are caused by APA and / or alternative last exon (ALE) usage. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein restores PABPN1 level and / or activity, optionally wherein the PABPN1 polypeptide disclosed herein restores mitochondrial activity caused by PABPN1 dysfunction and / or increases differentiation in muscle tissue.

[0026] Suitably in some embodiments of the present invention, the method comprises administering or expressing the PABPN1 polypeptide disclosed herein or the nucleic acid encoding the PABPN1 polypeptide disclosed herein in the subject in need thereof. Suitably, in some embodiments of the present invention, the PABPN1 polypeptide disclosed herein is administered to the subject in need thereof. Suitably, in some embodiments of the present invention, the nucleic acid sequence encoding the PABPN1 polypeptide disclosed herein is administered to the subject in need thereof.

[0027] In some embodiments, the PABPN1 polypeptide (or nucleic acid) disclosed herein restores the level of PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject. Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 2-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0028] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein is expressed in the subject by providing the nucleic acid encoding the PABPN1 polypeptide disclosed herein to the subject.

[0029] Suitably, in some embodiments the method further comprises administering or expressing an inhibitory RNA to suppress the endogenous aggregating mutant form of PABPN1 . Suitably, in some embodiments the inhibitory RNA suppresses or inhibits both the endogenous aggregating mutant form of PABPN1 and the wild-type form of PABPN1. In some embodiments the inhibitory RNA does not suppress or inhibit the wild-type form of PABPN1. In some embodiments, the inhibitory RNA is an siRNA or an antisense oligonucleotide.

[0030] In some embodiments of the present invention, the inhibitory RNA is expressed in the subject by providing a nucleic acid encoding the inhibitory RNA to the subject.

[0031] In some embodiments of the present invention, the method further comprises administering or expressing a gene editing system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1. Suitably, in some embodiments the gene editing system is a CRISPR-Cas system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1 . Suitably, in some embodiments the gene editing system is a CRISPR-Cas9 system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1. Suitably, in some embodiments the CRISPR-Cas9 system comprises a gRNA targeting exon 1 of PABPN1 . Suitably, in some embodiments the gRNA is encoded by SEQ ID NO: 22.

[0032] In some embodiments, the nucleic acid encoding the PABPN1 polypeptide as disclosed herein and / or the nucleic acid encoding the inhibitory RNA are comprised in a vector, optionally a viral vector. Suitably, the nucleic acid encoding the PABPN1 polypeptide disclosed herein and the nucleic acid encoding the inhibitory RNA are comprised in the same or different vectors.

[0033] In some embodiments, the vector is a viral vector, preferably an adeno-associated viral vector (AAV) or an adenoviral vector particle (AdVP). Suitably, in some embodiments the AAV has muscle tropism. Suitably, in some embodiments the AAV has cardiac and skeletal muscle tropism. Suitably, in some embodiments the AAV has cardiac muscle tropism. Suitably, in some embodiments the AAV has skeletal muscle tropism. Suitably, in some embodiments the AAV serotype is selected from any one of serotypes 1 , 6, 8, 9, and 10. Suitably, in one embodiment the AAV is AAV9. In some embodiments the AAV is a myotropic AAV. In some embodiments the myotropic AAV is AAVMYO, AAVMYO2 or AAVMYO3.

[0034] In some embodiments, the nucleic acid encoding the PABPN1 polypeptide disclosed herein is inserted into the genome of the subject in need thereof using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system. Suitably, in some embodiments the CRISPR-Cas system may comprise an endonuclease and a template sequence. Suitably, in some embodiments the CRISPR-Cas system comprises an endonuclease, a template sequence and a guide RNA (gRNA). Suitably, in some embodiments the endonuclease is Cas9 nuclease. Suitably, in some embodiments the template is homology dependent repair (HDR) template sequence. Suitably, in some embodiments the nucleic acid encoding the PABPN1 polypeptide comprises a HDR template sequence. In some embodiments, the gRNA is a single guide RNA (sgRNA). It is known in the art that a sgRNA is a synthetic RNA molecule created by fusing the crRNA (which contains the target DNA sequence) and tracrRNA (which binds to Cas9).

[0035] In one aspect of the invention there is provided a method of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 , wherein the method comprises administering a poly(A) binding protein nuclear 1 (PABPN1) polypeptide or a nucleic acid encoding the PABPN1 polypeptide to a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4.

[0036] In one aspect of the present invention there is provided use of a poly(A) binding protein nuclear 1 (PABPN1 ) polypeptide or a nucleic acid encoding the PABPN1 polypeptide for the manufacture of a medicament for treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N- terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4.

[0037] In one aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective level of a PABPN1 polypeptide or a nucleic acid encoding the PABPN1 polypeptide or a vector comprising the nucleic acid sequence encoding a PABPN1 polypeptide and a pharmaceutically acceptable diluent or carrier, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; does not comprise a coiled coil domain according to SEQ ID NO:4.

[0038] Suitably, in some embodiments the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1 .

[0039] In a further aspect of the present invention, there is provided a method of identifying a subject that will benefit from treatment with a poly(A) binding protein nuclear 1 (PABPN1) polypeptide or a nucleic acid encoding the PABPN1 polypeptide, wherein the method comprises:

[0040] - testing the subject for PABPN1 level and / or activity to establish a test value;

[0041] - comparing the test value to a reference value, wherein the reference value is PABPN1 level and / or activity of a healthy subject;

[0042] - where the test value is less than the reference value, recommending treatment with a PABPN1 polypeptide, a nucleic acid, a vector and / or a pharmaceutical composition according to any aspect or embodiment as described herein, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4.

[0043] Suitably, in some embodiments the method further comprises a step of administering or expressing the PABPN1 polypeptide disclosed herein or the nucleic acid encoding the PABPN1 polypeptide disclosed herein in the subject in need thereof.

[0044] Suitably in some embodiments the method further comprises a step of administering or expressing an inhibitory RNA to suppress the endogenous aggregating mutant form of PABPN1. Suitably, in some embodiments the inhibitory RNA is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO).

[0045] In some embodiments, the method further comprises administering or expressing a CRISPR- Cas system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1. In some embodiments, the method further comprises administering or expressing a CRISPR-Cas9 system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1 . Suitably, in some embodiments the CRISPR-Cas9 system comprises a gRNA targeting exon 1 of PABPN1.

[0046] Suitably, in some embodiments the gRNA is encoded by SEQ ID NO: 22.

[0047] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0048] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0049] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0050] Various aspects of the invention are described in further detail below.

[0051] Brief description of the Figures

[0052] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0053] Figure 1 : PABPN1 function and correlation with tr-PABPN1 isoform. A. Volcano plot of APA-shifts in the A17.1 mouse model (a mouse model for OPMD which expresses mutant PABPN1 ). A shift to proximal polyadenylation site usage is dominating over distal shifts. B. A schematic presentation of Pabpnl most abundance isoforms in mouse, the first exon encoding for the poly(alanine) track, exon encoding for the coil-coil domain (CCD), aggregationessential, and the exons encoding for the RNA-binding domain (RBP). The last exon is followed by the 3' UTR. The tr-PABPN1 isoform in mouse is iso-208, the full length isoforms are 201 and 202. The bar chart shows expression levels (CPM) of Pabpnl transcript isoforms in FVB (WT) and A17.1 (OPMD mouse model). Statistical difference was assessed with ANOVA, adjusted for multiple corrections. C. Schematic representation of the most abundant human PABPN1 isoforms. The human and mouse PABPN1 are similarly structured. Isoforms 201 and 202 are full-length, and iso-207 is the truncated-isoform, lacking exon 1 and translation starts from methionine in exon 2 (incomplete CCD). The exons encoding for the RNA binding motif are unchanged in these three isoforms. Primer sets specific to each isoform are denoted with arrows (left panel). RT-qPCR results with isoform specific primers are from control and OPMD muscles (right panel). D. Heatmaps of Spearman correlation (r) between APA-shift ratio (log 2) in the APA-shift significant transcripts and expression levels (Iog2) of the three most abundant PABPN1 isoforms (Iso) in mouse A17.1. Correlation analysis was carried out for transcripts with APA shift to proximal pA sites. The percentage of the significantly associated transcripts from total transcripts with APA-shift is denoted (left). APA- shift to proximal is mostly correlated with Iso-208.

[0054] Figure 2: A schematic representation of PABPN1 protein domains and secondary structures. A. A schematic representation of PABPN1 deletions (AALA, Al DR, ACCD), and the natural form tr-PAB. The protein features, protein stability and nuclear aggregation, are summarized on the right. CCD facilitates aggregation and RRB RNA binding motif, the PAP2A binding site is crucial for PABPN1 function. B. AlphaFold3 prediction of N-terminus IDR+CCD monomer (upper row) or tetramer (lower row). Amino-acids number at each end are denoted. C. AlphaFold3 prediction of N-terminus IDR+CCD tetramer. The alanine-stretch is after the first methionine (1). The pIDDT scale indicate confidence in prediction. The IDR in a tetramer has a higher confidence than in a monomer, and a higher confidence in Ala16 higher than in Ala10. In Ala16 the Ala stretch is entangled with the CCD.

[0055] Figure 3: mRNA analyses of PABPN1 endogenous and truncated isoforms expression levels in human muscle cells. A. RT-qPCR using primers to exon 3-4 sequence (RNA recognition motif region), Flag sequence at the C-terminus, and the 3-UTR. Average and statistical significance are from N=3 replicates. B.RNA-sequencing analysis of endogenous PABPN1-201 (full length) or the 207 isoform. Average and statistical significance are from N=4 replicates. *: p<0.05 , ***: p<0.005 , ****: p<0.001.

[0056] Figure 4: Protein expression of PABPN1 deletions in human bladder cancer cells. A. A Western blot of all PABPN1 deletions using anti-Flag antibody. B. Immunofluorescence with anti-Flag and PAB2 antibodies. The percentage of Flag positive cells is denoted under each representative image. C. Western blots of PABPN1 deletions and tr-PAB, excluding DCCD using anti-Flag or PAB2 antibodies. A16, overexpression of Ala16-PABPN1 , is used as a control. GAPDH is a loading control. PAB2 does not detect tr-PAB. D. Immunofluorescence with anti-Flag and PAB2 antibodies. The black and white insert in the upper right shows Flag staining. The dot-plot graph shows Flag fluorescence accumulation over time (left) or Flag signal in spots (puncta), measuring PABPN1 aggregation. Ala16 is used as a control for PABPN1 aggregation. Figure 5. Expression of tr-PABPN1 (tr-PAB) in human muscle cells. A. Western blot showing the tr-PAB-YFP and A16-YFP protein detected with anti-GFP antibodies. B. By induction A16 (mutant PABPN1) in cells the YFP signal can be detected after 16 hrs, and nuclear aggregates are formed in 3 days (upper panel). Nuclear aggregates are not formed by overexpression of tr-PAB (lower panel). C. SC35 marks nuclear speckles. Cell imaging shows co-localization of SC35 and overexpressed A16-YFP or tr-PAB-YFP in a nucleus. D. Image quantification of YFP puncta mean fluorescence intensity (MFI) in undifferentiated and fused muscle cells shows that co-expression of tr-PAB-YFP in A16 cells reduces YFP puncta intensity. (*** indicates p<0.005). Puncta are marginally formed in tr-PAB-YFP cells. E. Mitochondrial activity assessment with TMRM in cells expressing tr-PAB. Expression of tr- PAB(±YFP) restores mitochondrial activity in A16(±YFP) expressing cells. Experiments were carried out in stable A16(±YFP) +Dox that were transduced with tr-PAB(±YFP) lentivirus. Analysis was carried out 3 days after transduction. Averages and standard deviation are from N=3. statistical significance was assessed with the student’s t-test, p<0.05 is depicted with *; and p<0.005 is depicted with **.

[0057] Figure 6: tr-PABPN1 (tr-PAB) effect on the expanded PABPN1 (A16) in co-cultures differentiated human muscle cells. A. A western blot of protein lysate from parental, A16- FLAG and tr-PAB-FLAG cells. A blot with anti-FLAG antibody is in the upper panel, a blot with PAB2 antibody is in the middle panel. Tubulin and total protein stain show loading control. B. Schematic presentation of the flag fusion at the C-terminus of the protein and the location of the PAB2 epitope at the CCD. The PAB2 differentiates between endogenous PABPN1 / A16 and tr-PAB protein. C. A summary of the experimental design: 50% tr-PAB and 50% Ala16 or parental cells we mixed before seedings, followed by Doxycycline treatment and differentiation. After 5 days in differentiation cells were fixed stained with PAB2 and FLAG antibodies and imaged. D. FLAG staining specifically detects tr-PAB protein, and not in controls: without Dox treatment or without co-culturing. In co-cultures, the % of FLAG objects is reduced, as expected. E. Tr-PAB-FLAG co-culturing reduces A16-YFP aggregation compared with Ala16-YFP single culture. Aggregation is measured by the area of YFP puncta and YFP mean fluorescence intensity (MFI). F and G. Tr-PAB-FLAG co-culturing elevates muscle cell differentiation. Fusion index (F) is measured by the proportion of myonuclei in cells expressing the myosin heavy chain protein (a differentiation marker). Differentiated cells are multinucleated forming large multinucleated objects. The area of multinucleated objects is elevated in co-cultures with tr-PAB cells (G). H. Representative images of differentiated cells. The A16 is detected with the PAB2 antibodies. A16 aggregates are seen as fluorescence puncta, which are reduced in fused cells co-cultured with tr-PAB-FLAG. Expression tr-PAB in Ala16 expressing cells results in reduced aggregation, and enhanced muscle cell fusion. Figure 7: Tr-PAB co-expression with Ala16-PABPN1 in differentiated muscle cells restores PABPN1 molecular function. A. Schematic summary of the workflow for studying PABPN1 function in fused cells. In brief, A16 and tr-PAB stable cells were mixed 1 :1 during seeding. Cells were induced for differentiation while the transgene overexpression was induced by Doxcycline (Dox). After 4 or 5 days in differentiation conditions, cells were detached using trypsin and large cells were separated from small cells using a 45mm strainer. Subsequently RNA was isolated, cDNA was synthesized and APA-shift or expression levels were determined using qRT-PCR and gene specific primers. B. Bar chart shows APA-shift for 4 transcripts ASB5, CYBRD1, TMEM123 and PSTPIP2 and a control mRNA (LRP1). Coexpression of tr-PAB restores A16 mediated APA-shift. C. Bar chart shows normalized expression levels (ddCT normalized to HPRT and parental) for PABPN1 (exon3-4) RNA experiments were carried out in N=3 biological replicates. Statistical assessment was made with one-way Anova.

[0058] Figure 8: Tr-PAB co-expression with Ala16-PABPN1 in differentiated human muscle cells restores mRNA subcellular localization. A. Representative images of oligo-dT staining and YFP signal in multinucleated cells. Multinucleated regions are encircled with a dashed line. B. and C. show analysis of oligo-dT mean fluorescence intensity (MFI) in the nucleus. In all cells (B) or only multinucleated objects (C). D. plot shows oligo-dT MFI ratio between nuclear to cytosolic. E. Plot shows tr-PAB effect on A16-YFP puncta area. Averages and SD are from N=3 biological replicates. Statistical assessment was made with one-way Anova.

[0059] Figure 9: tr-PAB expression in bladder human cancer cells. A. Immunofluorescence with anti-Flag and anti-PABPN1 antibody to the CCD shows nuclear co-localisation of tr-PAB-Flag with endogenous PABPN1. After 5 days almost 100% of the cells express tr-PAB (middle graph). PABPN1 levels are stabilised 45 hours after Dox induction. Importantly, tr-PAB does not lead to downregulation of PABPN1. B. RT-qPCR shows the mRNA expression levels of endogenous PABPN1 and Flag overexpression. Notably, tr-PABPN1 restores PABPN1 mRNA levels. C. RT-qPCR shows the distal to proximal pA site (PAS) ratio at the 3'UTR of four genes, including the master cell cycle regulator CDKN1A. tr-PAB reverses the distal to proximal pA site (PAS) ratio.

[0060] Figure 10: tr-PAB effect of human bladder cancer cell function. A. Growth rate as measured by cell confluence. The graph shows the ratio between dox-treated and vehicle cell cultures in 2 cell seeding, 8K and 4K. Imaging was started 24 hours after induction. The right graph shows the slope for the cell culture at 8K and 4K. The experiment shows that tr-PAB slows down cell density. B. Cell migration was measured by wound size over 18 hours. Cancer cells are denoted grey and cancer cells with Tr.PAB are black. The experiment was performed in cells treated with dox for 48 days. A complete wound area equals zero. The experiment shows that tr-PAB inhibits cell migration. C. Images of TMRM staining (in red) in normal and cancer cells. TMRM labels mitochondrial activity. Images of cancer+tr-PAB were taken after immunofluorescence with anti-Flag. A cell without Flag staining shows high TMRM staining. Images are from cells treated with dox for 24 hours. D. TMRM mean fluorescence intensity (MFI) in vehicle-treated cells. Cancer cells have higher mitochondrial activity. Right graph: TMRM MFI in Dox-treated cells normalised to vehicle-treated cells. Expression of Tr-PAB reduces TMRM MFI. E. Cell area is represented by the percentage of large cells in the cell culture. Cancer cells are significantly smaller than normal cells. Tr-PAB restores cell size. All experiments were performed in biological triplicates. Statistical significance is calculated by ANOVA.

[0061] Figure 11 : PABPN1 gene effect in cancer cell lines (Chronos). A. Scatter plot shows PABPN1 Chronos score (x-axis) and PABPN1 expression levels (y-axis). B. Bar plot shows the Chronos score per tissue type. The dotted line marks the average PABPN1 Chronos score in bladder cancer. The dashed line marks PABPN1 pathogenicity (average Chronos). Bladder cancer is highlighted in bold. Cancers with predicted Chronos like, or lower than, bladder cancer are depicted. Cancers with predicted PABPN1 Chronos score lower (1 standard deviation) than bladder cancer are boxed.

[0062] Figure 12: Experimental design and TA mass recovery after AAV9-tr-PAB injection. A. Schematic of the experimental timeline. AAV9-tr-PAB (5.53 x 10A13 vg mlA-1) was injected into the TA of 10-12-week-old male A17.1 / + mice. In vivo physiological measurements were performed 12 weeks later, followed by muscle harvest for ex-vivo analysis. Harvested muscles were lost during shipment and ex-vivo assays could not be performed for this cohort (see Results). B. TAweight normalized to body weight (mg / g) in tr-PAB-treated and control A17.1 / + mice (n = 8 per group). Data are mean ± SEM. *p < 0.05 by [one-way Anova],

[0063] Figure 13: tr-PAB expression in bladder cancer cells. Experiments were performed in bladder cancer cells (cancer) that stably express tr-PAB-Flag under inducible promoter (+Dox). Normal cells refer to normal epithelial bladder cells. A. Immunofluorescence with antiFlag and anti-PABPN1 shows the nuclear localisation of tr-PAB-Flag (left panel). Images were taken 120 hours after Dox induction, showing no nuclear aggregation of tr-PAB (anti-Flag) or endogenous PABPN1 (PAB2). Tr-PAB PABPN1 levels are stabilised 24 hours after Dox induction (left panel). And PABPN1 levels in tr-PAB cells are also stabilized after 24 hours (right panel). This indicates that tr-PAB does not represses endogenous PABPN1 levels. B. RT-qPCR shows the mRNA expression levels of endogenous PABPN1 and Flag overexpression, exon = exon 3-4 to the RNA recognition motif amplifies both endogenous and transgene; 3’UTR only the endogenous, and FLAG only tr-PAB. PABPN1 levels are lower in the cancer cells compared with normal cells (PABPN1 exon 3-4 and the 3’-UTR). Notably, tr- PABPN1 restores PABPN1 mRNA levels. C. RT-qPCR shows the distal to proximal ratio at the 3'UTR of four cell cycle genes, including the master cell cycle regulator CDKN1 A. tr-PAB reverses the distal to proximal ratio. *: p<0.05; **p<0.01.

[0064] Figure 14: Tr-PAB function in cancer cells. A. shows cell growth after doxorubicin (Dox) induction (t = 0). B. Cell toxicity was measured using the CellTox™ real-time cell death assay (Promega) over the course of 40 hours of cell culture. C. Cell Toxicity over the course of 24 hours of cell culture.

[0065] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0066] Various aspects of the invention are described in further detail below.

[0067] Detailed Description

[0068] Non-aggregating isoforms of PABPN1 polypeptides are provided herein for use in the present invention. Suitably, the PABPN1 polypeptide or a nucleic acid encoding the PABPN1 polypeptide for use in the present invention does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3, an intrinsically disordered region according to SEQ ID NO:9; does not comprise a coiled coil domain according to SEQ ID NO: 4. These polypeptides are referred to as “PABPN1 polypeptides disclosed herein” throughout.

[0069] Suitably, in some embodiments the PABPN1 polypeptide does not comprise an N-terminal alanine tract according to SEQ ID NO:3.

[0070] Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 to 2 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to the sequence AAAAAAAAAA (SEQ ID NO: 3). Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 3.

[0071] The alanine tract of the OPM D PABPN1 comprises an extended N-terminal alanine tract of 11 to 18 alanine amino acids. Suitably, a PABPN1 polypeptide disclosed herein for use in the present invention, does not comprise an extended N-terminal alanine tract, suitably a PABPN1 polypeptide disclosed herein for use in the present invention do not comprise an N-terminal alanine tract of 11 to 18 amino acids.

[0072] In some embodiments the PABPN1 polypeptide disclosed herein does not comprise an intrinsically disordered region according to SEQ ID NO:9. Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise the intrinsically disordered region corresponding to amino acids 1 to 115 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the intrinsically disordered region corresponding to amino acids 2 to 115, 3 to 115, 4 to 115, 5 to 115, 6 to 115, 7 to 115, 8 to 115, 9 to 115, 10 to 115, 11 to 115, 12 to 115, 13 to 115, 14 to 115, 15 to 115, 20 to 115, 30 to 115, 40 to 115, 50 to 115, 60 to 115, 70 to 115, 80 to 115, 90 to 115, 100 to 115, 110 to 115 of SEQ ID NO:2.

[0073] Suitably, in some embodiments an alanine tract is comprised within the IDR. Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise an N-terminal alanine tract according to SEQ ID NO:3 and does not comprise the IDR according to SEQ ID NO:9. In some embodiments, the PABPN1 polypeptide disclosed herein comprises the IDR according to SEQ ID NO:9 but does not comprise an N-terminal alanine tract according to SEQ ID NO:3.

[0074] Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO:9. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 3 and does not comprise SEQ ID NO:9.

[0075] The coiled coil domain (CCD) of PABPN1 is required for aggregation of the PABPN1 protein. It may be advantageous in some embodiments for the PABPN1 polypeptides disclosed herein for use in the present invention to retain all or part of the CCD. Alternatively, it may also be advantageous in some embodiments for the PABPN1 polypeptide disclosed herein to have a complete deletion of the CCD. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the coiled coil domain according to SEQ ID NO:4. In some embodiments, the PABPN1 polypeptide disclosed herein does comprise a coiled coil domain (e.g. it comprises the CCD according to SEQ ID NO:4) Suitably, in some embodiments the PABPN1 polypeptide disclosed herein comprises a part of a coiled coil domain (e.g. it comprises part (but not all) of the CCD shown in SEQ ID NO:4).

[0076] A ’part’ as referred to herein may refer to a portion or fragment of the CCD shown in SEQ ID NO:4. A part may comprise any amino acid residues of amino acids 115 to 151 of SEQ ID NO:2. A part may comprise amino acids 129 to 151 of SEQ ID NO:2. It is advantageous to the present invention that any part of the CCD that is retained in the PABPN1 polypeptide disclosed herein for use in the methods of treatment as described herein, does not result in a nuclear aggregate of PABPN1 .

[0077] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein for use in the present invention comprises the amino acid sequence MEEEAEKLKELQNEVEKQMNMSP (SEQ ID NO: 8).

[0078] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4.

[0079] In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an N-terminal alanine tract (e.g. SEQ ID NO:3). In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO: 4 and does not comprise an IDR (e.g. SEQ ID NO:9). Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO: 4, does not comprise an N- terminal alanine tract (e.g. SEQ ID NO:3) and does not comprise an IDR (e.g. SEQ ID NO:9).

[0080] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO: 4 and does not comprise an N-terminal alanine tract according to SEQ ID NO: 3.

[0081] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO: 4 and does not comprise an IDR according to SEQ ID NO:9.

[0082] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according SEQ ID NO: 4, does not comprise an N-terminal alanine tract according to SEQ ID NO: 3 and does not comprise an IDR according to SEQ ID NO:9. In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0083] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0084] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0085] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0086] As used herein, ‘derivative thereof’ refers to any (poly)peptide sequence that is derived from the reference sequence (in this example, SEQ ID NO: 1 ). Such derivatives may include fragments of the reference sequence. In the context of SEQ ID NO: 1 , which is 177 amino acids long, a derivative of SEQ ID NO:1 may therefore be a fragment of less than 177 consecutive amino acids of SEQ ID NO:1. The fragment may have at least 100 consecutive amino acids from SEQ ID NO:1 ; it may have at least 115, 120, 130, 140, 140, 145, 150, 155, 160, 165, 170 or 175 (and up to 176) consecutive amino acids of SEQ ID NO:1. It may have between about 100 to 176 consecutive amino acids of SEQ ID NO:1 , or between about 150 to 176 consecutive amino acids of SEQ ID NO:1.

[0087] Derivatives may also include a functional variant of the polypeptide sequence. The term ‘functional variant’ is commonly known in the art to mean a variant of a reference sequence (e.g., such as SEQ ID NO: 1) that retains the ability to function in the same way as the reference sequence. Alternative terms for such functional variants include “biological equivalents” or “equivalents”. Functional variants may include conservative amino acid variants.

[0088] A person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of a reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc). Homologues of a reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc) can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0089] As one example, the variant may comprise an amino acid sequence with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions compared to the reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc.), wherein the variant retains PABPN1 activity. It may have no more than 5, no more than 4, no more than 3, for example no more than 2, or no more than 1 amino acid substitutions, insertions or deletions compared to the reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc.), wherein the variant retains PABPN1 activity. In some examples, the variant comprises 0 to 10 (or 0 to 5) conservative amino acid substitutions compared to the reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc.), wherein the variant retains PABPN1 activity. In some examples, the variant has no more than 5, no more than 4, no more than 3, for example no more than 2, or no more than 1 conservative amino acid substitutions compared to the reference sequence (such as SEQ ID NO:1 , SEQ ID NO:7, or SEQ ID NO:8 etc.), wherein the variant retains PABPN1 activity.

[0090] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, variants can be introduced randomly along all or part of coding sequences, such as by saturation mutagenesis, and the resultant variants can be screened for PABPN1 activity to identify variants that retain activity. Following mutagenesis of the reference sequence (such as SEQ ID NO:1 , SEQ ID NOT, or SEQ ID NO:8 etc.), the encoded proteins can be expressed recombinantly and the biological activity of the protein can be determined.

[0091] The variants described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequence of the reference sequence (such as SEQ ID NO:1 , SEQ ID NOT, or SEQ ID NO:8 etc.), wherein the variant retains PABPN1 activity. The terms “sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

[0092] The person skilled in the art would further appreciate that the PABPN1 polypeptide of the present invention may comprise additional amino acids at the C-terminal or N-terminal of the polypeptide chain. Such amino acids may correspond to, but are not limited to, peptide or protein tags such as a FLAG-tag or a polyhistidine tag. The PABPN1 polypeptide may also comprise protein modifications such as post-translational modifications (e.g. any of phosphorylation, glycosylation, methylation and acetylation).

[0093] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0094] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0095] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0096] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0097] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0098] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0099] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0100] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0101] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0102] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0103] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0104] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0105] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof. In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0106] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0107] In some embodiments of the present invention, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0108] The full length amino acid sequence of PABPN1 is provided in Figure 2A. This isoform may also be referred to as the ‘wild-type’ isoform of PABPN1. The wild-type or full length isoform of PABPN1 is a 306 amino acid protein. The amino acid numbering as used herein corresponds to the numbering as used with respect to the wild type amino acid sequence, i.e., amino acids 1 to 306 of SEQ ID NO:2.

[0109] In some embodiments, the PABPN1 polypeptide as disclosed herein restores the level of PABPN1 in the target subject to the level of functional PABPN1 polypeptide as observed in a healthy subject. The term “restores” as used herein refers to a complete or partial restoration of the level or function of PABPN1 as observed in a healthy subject. A complete restoration may refer to having an equivalent level of PABPN1 as typically observed in a healthy subject. Complete restoration may also refer to having an equivalent function of PABPN1 as typically observed in a healthy subject. A partial restoration may refer to having an level or function of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% PABPN1 as typically observed in a healthy subject.

[0110] In some embodiments, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0111] In one embodiment, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 2-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject. The polypeptides disclosed herein may be a “natural polypeptide” i.e. a polypeptide composed of natural amino acids. Such polypeptides are composed of conventional amino acids defined by the genetic code, linked to each other by a normal peptide bond. Natural polypeptides may, for example, be produced by a cell (via protein expression, e.g. using a nucleic acid or vector described herein), or they may be made synthetically (i.e. outside of a cell, using chemical synthesis).

[0112] Alternatively, the polypeptide may be a “synthetic polypeptide”. A synthetic polypeptide may comprise a mix of natural amino acids and amino acids other than conventional amino acids defined by the genetic code (“synthetic amino acids”). Alternatively, it may be composed of synthetic amino acids only. Examples of synthetic amino acids are well known in the literature.

[0113] Natural polypeptides and synthetic polypeptides may be modified. In other words, the polypeptide may comprise amino acids modified by natural processes, such as post- translational maturation processes or by chemical processes, which are well known to a person skilled in the art. Such modifications are fully detailed in the literature. These modifications can appear anywhere in the polypeptide: in the polypeptide skeleton, in the amino acid chain or at the carboxy- or amino-terminal ends. Non-limiting examples of polypeptide modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination. Such modifications are fully detailed in the literature. Accordingly, the terms “peptide”, “polypeptide”, “protein” may include for example lipopeptides, lipoproteins, glycopeptides, glycoproteins and the like. As a further non-limiting example, the polypeptide can be branched following ubiquitination or be cyclic with or without branching. This type of modification can be the result of natural or synthetic post-translational processes that are well known to a person skilled in the art.

[0114] Nucleic acids Encoding Non-aggregating Isoforms of PABPN1

[0115] Nucleic acid sequences encoding non-aggregating isoforms of PABPN1 and their uses are encompassed by the present invention. The skilled person will appreciate that the nucleic acid sequence may encode any of the non-aggregating PABPN1 polypeptide isoforms as described above (also referred to as “PABPN1 polypeptides disclosed herein”). It will be appreciated that the nucleic acid encoding the PABPN1 polypeptide disclosed herein may be codon optimised. Suitably, in some embodiments there is provided a nucleic acid encoding a PABPN1 polypeptide disclosed herein, wherein the encoded PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4.

[0116] Suitably, in some embodiments there is provided a nucleic acid encoding a PABPN1 polypeptide disclosed herein, wherein the encoded PABPN1 polypeptide does not comprise an N-terminal alanine tract according to SEQ ID NO:3. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or2 to 2 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to the sequence AAAAAAAAAA (SEQ ID NO: 3). Suitably, in some embodiments the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein that does not comprise SEQ ID NO: 3.

[0117] In one embodiment, the nucleic acid comprises or consists of SEQ ID NO: 16.

[0118] Suitably, in some embodiments there is provided a nucleic acid encoding a PABPN1 polypeptide disclosed herein, wherein the encoded PABPN1 polypeptide does not comprise an intrinsically disordered region according to SEQ ID NO:9. Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise the intrinsically disordered region corresponding to amino acids 1 to 115 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the intrinsically disordered region corresponding to amino acids 2 to 115, 3 to 115, 4 to 115, 5 to 115, 6 to 115, 7 to 115, 8 to 115, 9 to 115, 10 to 115, 11 to 115, 12 to 115, 13 to 115, 14 to 115, 15 to 115, 20 to 115, 30 to 115, 40 to 115, 50 to 115, 60 to 115, 70 to 115, 80 to 115, 90 to 115, 100 to 115, 110 to 115 of SEQ ID NO:2. Suitably, in some embodiments an N-terminal alanine tract is comprised within the IDR. Suitably, in some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise an N-terminal alanine tract according to SEQ ID NO:3 and does not comprise an IDR according to SEQ ID NO:9. In some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an IDR according to SEQ ID NO:9 but does not comprise an N-terminal alanine tract according to SEQ ID NO:3. Suitably, in some embodiments the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise SEQ ID NO:9. Suitably, in some embodiments the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein that does not comprise SEQ ID NO:3 and does not comprise SEQ ID NO:9.

[0119] In one embodiment, the nucleic acid comprises or consists of SEQ ID NO:18.

[0120] Suitably, in some embodiments there is provided a nucleic acid encoding a PABPN1 polypeptide disclosed herein, wherein the encoded PABPN1 polypeptide does not comprise an intrinsically disordered region according to SEQ ID NO:9 and / or does not comprise a coiled coil domain according to SEQ ID NO:4.

[0121] Suitably, in some embodiments the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise the coiled coil domain according to SEQ ID NO:4. In some embodiments, the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein that comprises the coiled coil domain according to SEQ ID NO:4. Suitably, in some embodiments the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein that comprises a part (but not all) of the coiled coil domain shown in SEQ ID NO:4.

[0122] Suitably, in some embodiments, nucleic acid encodes a PABPN1 polypeptide for use in the present invention that comprises MEEEAEKLKELQNEVEKQMNMSP (SEQ ID NO: 8). In some embodiments of the present invention, the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4.

[0123] In some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an N-terminal alanine tract (e.g. according to SEQ ID NO:3). In some embodiments, nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an IDR (e.g. according to SEQ ID NO:9). Suitably, in some embodiments nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4, does not comprise an N-terminal alanine tract (e.g. according to SEQ ID NO:3) and does not comprise an IDR (e.g. according to SEQ ID NO:9). Suitably, in some embodiments, nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an N-terminal alanine tract according to SEQ ID NO:3.

[0124] Suitably, in some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an IDR according to SEQ ID NO:9.

[0125] Suitably, in some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4, does not comprise an N-terminal alanine tract according to SEQ ID NO:3 and does not comprise an IDR according to SEQ ID NO:9.

[0126] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein which comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0127] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein which comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0128] In some embodiments of the present invention, the nucleic acid sequence encodes a PABPN1 polypeptide disclosed herein which comprises an amino acid sequence according to SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0129] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein which consists of an amino acid sequence according to SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0130] In some embodiments of the present invention, the nucleic acid comprises or consists of a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NQ:10.

[0131] In some embodiments, the nucleic acid may also further comprise additional flanking sequences, e.g. additional flanking sequences that do not induce nuclear aggregation of the PABPN1 polypeptide.

[0132] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 10.

[0133] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0134] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0135] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence according to SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0136] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that consists of an amino acid sequence according to SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0137] In some embodiments of the present invention, the nucleic acid comprises or consists of a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:11. T1

[0138] In some embodiments, the nucleic acid may also further comprise additional flanking sequences, e.g. additional flanking sequences that do not induce nuclear aggregation of the PABPN1 polypeptide.

[0139] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 11 .

[0140] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0141] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0142] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence according to SEQ ID NO:19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0143] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that consists of an amino acid sequence according to SEQ ID NO:19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0144] In some embodiments of the present invention, the nucleic acid comprises or consists of a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:13.

[0145] In some embodiments, the nucleic acid may also further comprise additional flanking sequences, e.g. additional flanking sequences that do not induce nuclear aggregation of the PABPN1 polypeptide.

[0146] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 13. In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0147] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0148] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence according to SEQ ID NQ:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0149] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that consists of an amino acid sequence according to SEQ ID NQ:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0150] In some embodiments of the present invention, the nucleic acid comprises or consists of a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:14.

[0151] In some embodiments, the nucleic acid may also further comprise additional flanking sequences, e.g. additional flanking sequences that do not induce nuclear aggregation of the PABPN1 polypeptide.

[0152] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 14.

[0153] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0154] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0155] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that comprises an amino acid sequence according to SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0156] In some embodiments of the present invention, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that consists of an amino acid sequence according to SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0157] In some embodiments of the present invention, the nucleic acid comprises or consists of a sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:15.

[0158] In some embodiments, the nucleic acid may also further comprise additional flanking sequences, e.g. additional flanking sequences that do not induce nuclear aggregation of the PABPN1 polypeptide.

[0159] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 15.

[0160] In some embodiments, the nucleic acid encoding PABPN1 polypeptide as disclosed herein restores the level of PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject. The term “restores” as used herein refers to a complete or partial restoration of the level or function of PABPN1 as observed in a healthy subject. A complete restoration may refer to having an equivalent level or function of PABPN1 as typically observed in a healthy subject. A partial restoration may refer to having an level or function of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of PABPN1 as typically observed in a healthy subject. In some embodiments, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that is expressed at a level that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0161] In one embodiment, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that is expressed at a level that is at least 2-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0162] In one embodiment, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that is expressed at a level that is at least 3-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0163] In one embodiment, the nucleic acid encodes a PABPN1 polypeptide disclosed herein that is expressed at a level that is at least 4-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0164] Nucleic acids which encode a PABPN1 polypeptide disclosed herein for use in the invention may be naturally occurring or wholly or partially synthetic and may include, but are not limited to, DNA, cDNA and RNA. Nucleic acid sequences encoding the PABPN1 polypeptide disclosed herein for use in the invention can be readily prepared by the skilled person using techniques which are well known to those skilled in the art, such as those described in Sambrook et al. "Molecular Cloning", A laboratory manual, Cold Spring Harbor Laboratory Press, Volumes 1-3, 2001 (ISBN-0879695773), and Ausubel et al. Short Protocols in Molecular Biology. John Wiley and Sons, 4th Edition, 1999 (ISBN - 0471250929). Said techniques include (I) the use of the polymerase chain reaction (PCR) to amplify samples of nucleic acid, (ii) chemical synthesis, or (iii) preparation of cDNA sequences. DNA encoding PABPN1 polypeptide disclosed herein for use in the invention may be generated and used in any suitable way known to those skilled in the art, including taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The excised portion may then be operably linked to a suitable promoter and expressed in a suitable expression system, such as a commercially available expression system. Alternatively, the relevant portions of DNA can be amplified by using suitable PCR primers. Modifications to the DNA sequences can be made by using site directed mutagenesis. As used herein “nucleic acid sequence”, “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence. The term nucleic acid sequence may therefore be replaced by the term nucleic acid herein. The nucleotide sequence may be of genomic, synthetic or recombinant origin, and may be double-stranded or single-stranded (representing the sense or antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g. mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. In one example, the nucleotide sequence lacks introns. In other words, it is an intronless nucleic acid sequence. For example, the nucleotide sequence may be a DNA sequence that does not comprise intron sequences.

[0165] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).

[0166] The nucleic acid of the invention may be a non-naturally occurring nucleic acid sequence (e.g. it may be that the entire sequence does not occur in its entirety in nature). For example, the nucleic acid of the invention may be operably linked to a promoter, wherein the promoter is not naturally associated with equivalent human nucleic acid sequences in nature; i.e. it is not the entire promoter that is naturally associated with the nucleic acid in its natural environment. In this context, such promoters may be considered exogenous promoters. Examples of appropriate promoters are described elsewhere.

[0167] Nucleic acid encoding a PABPN1 polypeptide disclosed herein for use in the invention may be provided as expression constructs in the form of a plasmid, vector, transcription or expression cassette which comprises at least one nucleic acid as described above operably liked to one or more expression control sequences, e.g. a promoter, an enhancer, a poly-A sequence, an intron or suchlike. Suitably the expression control sequences are sufficient to provide expression of the PABPN1 polypeptide disclosed herein in a target cell. The expression may be constitutive or regulatable.

[0168] Accordingly, in a further aspect there is provided an expression construct comprising a nucleic acid as set out above. Suitably the expression construct is a vector, e.g. an expression vector adapted for expression in a eukaryotic or prokaryotic cell.

[0169] RNA interference

[0170] It may be desirable in some instances to not only provide a PABPN1 polypeptide disclosed herein to a subject in need thereof, but also to decrease the abundance of the mutated aggregating form of PABPN1 , such as, without limitation, the abundance of OPMD PABPN1. Similarly, it may be desirable in some instances to control or regulate the abundance of wt- PABPN1 (e.g. full length PABPN1 protein according to SEQ ID NO: 2). Such instances may include, without limitation, cancer. In cancerous cells, an excess of wt-PABPN1 may lead to PABPN1 aggregation. In such cases, it is desirable to decrease the abundance of wt-PABPN1 . However, as shown by the inventors herein, in some cancer types such as bladder cancer, loss of functional wt-PABPN1 is observed. In such cases, it may be less desirable to decrease the abundance of wt-PABPN1 . Therefore, in some instances it is important to restore PABPN1 function using the non-aggregating isoform.

[0171] The skilled person will appreciate that the term ‘abundance’ refers to the level or concentration of PABPN1 found in a cell. The level or concentration may refer to the mutated aggregating form of PABPN1 or may refer to the level or concentration of wt-PABPN1 (e.g. full length PABPN1 protein according to SEQ ID NO: 2). Reducing the abundance of the mutated aggregating form of PABPN1 or wt-PABPN1 may be achieved by any suitable means in the art. One such approach is RNA interference (RNAi) to silence the gene encoding the mutated aggregating form of PABPN1 or the gene encoding wt-PABPN1 (e.g. full length PABPN1 protein according to SEQ ID NO: 2).

[0172] The term “RNA interference” and the term “RNAi” are synonymous and refer to the process by which a polynucleotide (a miRNA or siRNA) comprising at least one polyribonucleotide unit exerts an effect on a biological process. The process includes, but is not limited to, gene silencing by degrading mRNA, attenuating translation, interactions with tRNA, rRNA, hnRNA, cDNA and genomic DNA, as well as methylation of DNA with ancillary proteins. An “inhibitory RNA” is an RNA molecule capable of RNA interference, either as is or after cellular processing. Thus, the term includes, inter alia, RNA precursors that are processed to form a miRNA, siRNA, or other RNA that can degrade target mRNA.

[0173] The term “gene silencing” refers to a process by which the expression of a specific gene product is lessened or attenuated by RNA interference. The level of gene silencing (also sometimes referred to as the degree of “knockdown” or reduction of expression) can be measured by a variety of means, including, but not limited to, measurement of transcript levels by Northern Blot Analysis, B-DNA techniques, transcription-sensitive reporter constructs, expression profiling (e.g. DNA chips), qRT-PCR and related technologies. Alternatively, the level of silencing can be measured by assessing the level of the protein encoded by a specific gene. This can be accomplished by performing a number of studies including antibody-based detection such as Western Analysis and flow cytometry, measuring the levels of expression of a reporter protein that has e.g. fluorescent properties (e.g., GFP) or enzymatic activity (e.g. alkaline phosphatases), or several other procedures. Typically, the level of silencing or reduction is compared to the level of expression of a given protein or mRNA in a control cell, as appropriate.

[0174] The terms “microRNA”, “miRNA”, or “miR” are synonymous and all refer to non-coding RNAs of, for example, about 19-24 nucleotides in length (and also, as the context will indicate, to DNA sequences that encode such RNAs) that are capable of entering the RNAi pathway and regulating gene expression. “Primary miRNA” or “pri-miRNA” represents the non-coding transcript prior to Drosha processing and includes the stem-loop structure(s) as well as flanking 5' and 3' sequences. “Precursor miRNAs” or “pre-miRNA” represents the non-coding transcript after Drosha processing of the pri-miRNA. The term “mature miRNA” can refer to the double stranded product resulting from Dicer processing of pre-miRNA or the single stranded product that is introduced into RISC following Dicer processing. In some cases, only a single strand n miRNA enters the RNAi pathway. In other cases, both strands of a miRNA are capable of entering the RNAi pathway. miRNAs are found in a wide range of organisms (e.g. insects, mammals, plants, nematodes) and have been shown to play a role in development, homeostasis, and disease aetiology.

[0175] The term “silencing RNA form” or “silencing RNAs” or “silencing RNA molecule”, “sRNA” or “trigger sequence / RNA” refers to the mature small RNA being capable of hybridizing with a target RNA (or fragment thereof) and engage the RNAi pathway.

[0176] The term “antisense oligonucleotides (ASOs)” is well known in the art. ASOs as used herein refer to short chain oligonucleotides, typically 15-25 nucleotides that target messenger RNA (mRNA). ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. ASOs may be DNA or RNA. In some embodiments, the ASO may be combined with a cell -penetrating moiety, preferably a muscle-cell penetrating moiety. Suitably, in some embodiments, the ASO may be combined with a homing moiety. Suitable moieties are well known in the art.

[0177] The term “target RNA” refers to a specific RNA that is targeted by the RNAi pathway, resulting in a decrease in the functional activity of the RNA. In some cases, the RNA target is a mRNA (typically, but not limited to, OPMD PABPN1 mRNA) whose functional activity is its ability to be translated. In such cases, the RNAi pathway will decrease the functional activity of the mRNA by translational attenuation or by cleavage. In the instant disclosure, target RNAs are targeted by non-naturally occurring miRNAs. The term “target” can also refer to DNA.

[0178] Suitably, in some embodiments of the present invention, the method further comprises administering or expressing an inhibitory RNA to suppress or inhibit the endogenous aggregating mutant form of PABPN1 in the subject. In some embodiments, the method further comprises administering or expressing a nucleic acid encoding an inhibitory RNA to suppress or inhibit the endogenous aggregating mutant form of PABPN1 .

[0179] Suitably, in some embodiments the inhibitory RNA is a small interfering RNA (siRNA). In some embodiments the inhibitory RNA is a microRNA (miRNA). In some embodiments the inhibitory RNA is an antisense oligonucleotide (ASO). Suitably, in some embodiments the inhibitory RNA is an siRNA or an antisense oligonucleotide. Suitable inhibitory RNAs are known in the art.

[0180] In some embodiments, the inhibitory RNA targets a nucleic acid sequence encoding wt- PABPN1 . In some embodiments, the inhibitory RNA targets a nucleic acid sequence encoding an endogenous aggregating mutant form of PABPN1. Suitably, in some embodiments, the inhibitory RNA targets a nucleic acid sequence encoding a OPMD PABPN1 polypeptide. Suitably, in some embodiments the OPMD polypeptide comprises an extended alanine tract of 11 to 18 alanine amino acids.

[0181] Suitably, in some embodiments, the method further comprises administering or expressing an inhibitory RNA to suppress or inhibit both the endogenous aggregating mutant form of PABPN1 and the wild-type form of PABPN1 .

[0182] It will be appreciated that the inhibitory RNA may be provided to the subject in need thereof, in addition to the PABPN1 polypeptide disclosed herein and / or in addition to the nucleic acid encoding the PABPN1 disclosed herein.

[0183] Suitably, in some embodiments the method comprises administering or expressing the PABPN1 polypeptide disclosed herein or the nucleic acid encoding the PABPN1 polypeptide disclosed herein in the subject in need thereof. Suitably, in some embodiments the method further comprises administering or expressing an inhibitory RNA to suppress or inhibit the endogenous aggregating mutant form of PABPN1 In a preferred embodiment, the inhibitory RNA is a small interfering RNA (siRNA). It may be desirable in some instances to provide the inhibitory RNA or the sequence encoding the inhibitory RNA in a vector. Suitable vectors are described in detail below.

[0184] The skilled person will appreciate that reducing the abundance of the wt-PABPN1 or mutated aggregating form of PABPN1 may be achieved by any suitable means in the art. Such alternative methods may include excision of exon 1 of PABPN1 from the mutated aggregating form of PABPN1 or wt-PABPN1. Excision may be achieved using zinc-finger nucleases, TALENs or CRISPR-Cas based systems. It will be appreciated that such methods may include targeting the wild type or disease-causing allele (i.e. the gene encoding wt-PABPN1 or the mutated aggregating form of PABPN1 ). Any such methods of excision known in the art may be used.

[0185] Suitably, in some embodiments, the methods of the invention further comprise excision of exon 1 of mutated aggregating PABPN1. Suitably, in some embodiments, the methods of the invention further comprise excision of exon 1 of wt-PABPN1. Suitably, in some embodiments, the method comprises excision of exon 1 of wt-PABPN1 using a CRISPR-Cas system. Suitably, in some embodiments, the method comprises excision of exon 1 ofwt-PABPN1 using a CRISPR-Cas9 system. Suitably, in some embodiments, the method comprises excision of exon 1 of mutated aggregating PABPN1 using a CRISPR-Cas9 system. Suitably, in some embodiments the mutated aggregating PABPN1 may be OPMD PABPN1.

[0186] In some embodiments the CRISPR-Cas system comprises at least one guide RNA (gRNA). The skilled person would appreciate that gRNAs targeting exon 1 of wt-PABPN1 (and / or the mutated aggregating isoform of PABPN1 target sequences) are designed to not target nonaggregating PABPN1 polypeptides as provided herein. Suitable examples of gRNAs would be those that target outside the transgene sequence of SEQ ID NO: 1. Suitably, the gRNA may target intronic sequences of PABPN1 (e.g. as provided in SEQ ID NO: 12) to avoid targeting non-aggregating PABPN1 polypeptides as provided herein. Suitably, in some embodiments, the gRNA targets upstream of position 1 (i.e. Met) of SEQ ID NO: 1. Suitably, in some embodiments, the gRNA targets exon 1 of the sequence encoding full length PABPN1 (e.g. SEQ ID NO:2). Suitably, in some embodiments, the gRNA targets exon 1 and the intron connecting exon 1 and exon 2 of the sequence encoding full length PABPN1 (e.g. SEQ ID NO:2 or 17, encoded by SEQ ID NO: 12). Suitably, it will be understood that the gRNA does not target the methionine at position 129 of the full length PABPN1.

[0187] The methods of the invention may further comprise administering or expressing a CRISPR- Cas system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1. Suitably, in some embodiments the CRISPR-Cas system is a CRISPR-Cas9 system. Suitably, in some embodiments the CRISPR-Cas system comprises a gRNA targeting exon 1 of PABPN1 . Suitably, in some embodiments the gRNA is encoded by SEQ ID NO: 22 or SEQ ID NO: 23. In a preferred embodiment, the gRNA is encoded by SEQ ID NO: 22.

[0188] Vectors

[0189] In some embodiments, the nucleic acid encoding the PABPN1 polypeptide disclosed herein for use in the present invention may be comprised in a vector. In some embodiments, the nucleic acid encoding the inhibitory RNA for use in the present invention may be comprised in a vector. Suitably, in some embodiments, the vector is a gene therapy vector, suitably an AAV vector, an adenoviral vector, an adenoviral vector particle (AdVP), a retroviral vector, a herpes simplex vector or a lentiviral vector.

[0190] Lentiviral vectors have been extensively used as a gene transfer tool. They are beneficial as they have relatively large cloning capacity and do not express viral genes. A particularly preferred lentiviral vector system is based on HIV-1 .

[0191] AAV vectors have been extensively discussed in the art. AAV vectors are of particular interest as AAV vectors do not typically integrate into the genome and do not elicit an immune response. Without wishing to be bound by theory, AAV8 is believed to be the most efficient serotype for transducing skeletal and cardiac muscles when delivered systemically. AAV8 can cross the blood vessel barrier. AAV9 is thought to be the most efficient serotype for transducing muscle stem cells. AAV9 preferentially transduces ischemic muscle. AAV6 is also believed to have a higher transduction efficiency than AAV9 and AAV6 can transduce mature myofibers in vivo, but doesn't effectively transduce muscle satellite cells. AAV rh.10 has been shown to display strong muscle tropism following intraperitoneal delivery. Other AAV serotypes may also be of interest. For example, AAV1 , AAV2 / 8 and AAV9, PHP.S and AAV- retro have all been shown to successfully transduce cells in the bladder or kidney. Other AAV serotypes of interest may include: AAV3B and AAV5. Myotropic AAVs may include any AAVs known in the art, such as those disclosed in Weinmann J, et al., 2020, Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat Commun.;11(1):5432. doi: 10.1038 / s41467-020-19230-w. PMID: 33116134; PMCID: PMC7595228, El Andari J, et al., 2022, Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. Sci Adv. 2022 Sep 23;8(38):eabn4704. doi: 10.1126 / sciadv.abn4704. Epub. PMID: 36129972; PMCID: PMC9491714 and Liu J, et al., 2023, Progress in Bioengineering of Myotropic Adeno- Associated Viral Gene Therapy Vectors. Hum Gene Then May; 34(9-10): 350-364. doi: 10.1089 / hum.2023.057. PMID: 37082964. Exemplary myotropic AAVs include but are not limited to AAVMYO (an AAV9 mutant), AAVMYO2 and AAVMYO3. Suitably, in some embodiments the AAV is AAV6. Suitably, in some embodiments the AAV is AAV9. Suitably, in some embodiments the AAV is AAV1 . Suitably, in some embodiments the AAV is AAV2 / 8. Suitably, in some embodiments the AAV is AAV2. Suitably, in some embodiments the AAV is AAV8.

[0192] Suitably, in some embodiments, the vector for use in the present invention may be an AAV vector. Suitably, the AAV is a recombinant AAV viral vector which is replication defective, lacking sequences encoding functional Rep and Cap proteins within the viral genome. These defective AAV vectors may lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, a tissue, an organ or an organism. Suitably AAV vectors for use herein comprises a virus that has been reduced to the minimum components necessary for transduction of the nucleic acid encoding the PABPN1 polypeptide disclosed herein and / or the inhibitory RNA as described herein. In this manner, AAV vectors are engineered as vehicles for specific delivery while lacking the deleterious replication and / or integration features found in wild-type viruses. In one embodiment, the AAV particle of the present invention is an scAAV. In another embodiment, the AAV particle of the present invention is an ssAAV. Methods for producing and / or modifying AAV particles are disclosed extensively in the art (see e.g. W02000 / 28004; W02001 / 23001 ; W02004 / 112727; WO 2005 / 005610 and WO 2005 / 072364, which are incorporated herein by reference). In some embodiments the AAV is a myotropic AAV. In some embodiments the myotropic AAV is AAVMYO, AAVMYO2 or AAVMYO3.

[0193] Methods of making AAV vectors are well known in the art and are described in e.g., U.S. Patent Nos. US6204059, US5756283, US6258595, US6261551 , US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International Publication Nos. WO1996039530, WO1998010088, WO 1999014354, WO1999 / 015685, W01999 / 047691 , W02000 / 055342, W02000 / 075353 and W02001 / 023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al, Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J Fir.63:3822-8 (1989); Kajigaya et al, Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al, Vir., 219:37-44 (1996); Zhao et al, Vir.272: 382-93 (2000); the contents of each of which are herein incorporated by reference. Viral replication cells commonly used for production of recombinant AAV viral particles include but are not limited to HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.

[0194] In some embodiments the vector is a non-viral vector, for example using cationic polymers or cationic lipids, as is known in the art. Various non-viral vectors are discussed in Selene Ingusci et al. (Gene Therapy Tools for Brain Diseases. Front. Pharmacol. 10:724. doi: 10.3389)

[0195] In some embodiments, there is provided a virion (viral particle) comprising a vector, suitably a viral vector according to the present invention. In some embodiments the virion is an AAV virion.

[0196] The invention thus further provides recombinant virions (viral particles) comprising a vector as described above.

[0197] Suitably, in some embodiments, the vector is an adenoviral vector particle (AdVP).

[0198] The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid as described herein in accordance with the invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC), or human (HAC)) may be used to accommodate larger inserts. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno-associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replicationdeficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

[0199] Suitably, in some embodiments the vector comprises a sequence encoding an inhibitory RNA that suppresses or inhibits the sequence encoding the endogenous aggregating mutant form of PABPN1 . Suitably, in some embodiments the inhibitory RNA is a miRNA. Suitably, in some embodiments the inhibitory RNA is an siRNA. The skilled person will appreciate that when the inhibitory RNA is an antisense oligonucleotide, the ASO is not comprised in a vector. ASOs may delivered to a subject by any suitable means such as by direct delivery or using nanocarriers such as lipid-based nanoparticles (e.g. LNPs). Direct delivery will be understood to also include delivery of the ASO when conjugated to a cell penetrating moiety, such as a muscle cell penetrating moieties, or homing moiety. Such moieties may include transferrin receptor (TfR) ligands and antibody carriers.

[0200] Suitably, in some embodiments the vector comprises a sequence according to SEQ ID NO: 10, 11 , 13, 14 or 15.

[0201] Suitably, in some embodiments, the vector comprises a sequence encoding the siRNA.

[0202] It will be appreciated that the sequence encoding the inhibitory RNA and the sequence encoding the PABPN1 polypeptide disclosed herein may be comprised in the same vector or in a different vector.

[0203] Suitably, in some embodiments, the method comprises providing or administering a subject in need thereof a vector comprising a nucleic acid encoding a PABPN1 polypeptide as disclosed herein. Suitably, in some embodiments, the method comprises providing or administering a subject in need thereof a vector comprising a nucleic acid encoding a PABPN1 polypeptide as disclosed herein and a sequence encoding an inhibitory RNA as described herein. Suitably, in some embodiments, the nucleic acid encoding the PABPN1 polypeptide disclosed herein and the sequence encoding an inhibitory RNA are provided or administered to the subject in a single vector. Suitably, in some embodiments, the nucleic acid encoding the PABPN1 polypeptide disclosed herein and the nucleic acid encoding an inhibitory RNA are provided or administered to the subject in different vectors. In one embodiment, there is provided a vector that comprises a nucleic acid encoding the PABPN1 polypeptide disclosed herein for use in the present invention. Suitably, in some embodiments the nucleic acid comprises any one of: SEQ ID NO: 10, 11 , 13, 14 and 15.

[0204] Suitably, in some embodiments the vector comprises a nucleic acid having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 10. Suitably, in one embodiment the vector comprises a nucleic acid according to SEQ ID NO: 10.

[0205] Suitably, in some embodiments the vector comprises a nucleic acid having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 11 . Suitably, in one embodiment the vector comprises a nucleic acid according to SEQ ID NO: 11 .

[0206] Suitably, in some embodiments the vector comprises a nucleic acid having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 13. Suitably, in one embodiment the vector comprises a nucleic acid according to SEQ ID NO: 13.

[0207] Suitably, in some embodiments the vector comprises a nucleic acid having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 14. Suitably, in one embodiment the vector comprises a nucleic acid according to SEQ ID NO: 14.

[0208] Suitably, in some embodiments the vector comprises a nucleic acid having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 15. Suitably, in one embodiment the vector comprises a nucleic acid according to SEQ ID NO: 15.

[0209] In one embodiment, there is provided a vector that comprises a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1. In some embodiments, the inhibitory RNA is an siRNA. In one embodiment, there is provided a vector that comprises a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the wt-PABPN1 (e.g. full length PABPN1 according to SEQ ID NO: 2). In some embodiments, the inhibitory RNA is an siRNA.

[0210] In a particularly preferred embodiment, the vector comprises a sequence encoding the PABPN1 polypeptide disclosed herein for use in the present invention and a sequence encoding an siRNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1.

[0211] Suitably, in some embodiments the vector comprises a nucleic acid encoding the PABPN1 polypeptide according to SEQ ID NO: 1 and a nucleic acid encoding the siRNA.

[0212] It is understood that it some embodiments, the host cell is contacted with the vector (e.g. viral vector) in vitro, ex vivo, and in some embodiments, the host cell is contacted with the vector (e.g. viral vector) in vivo. The term "host cell" includes any cell into which the nucleic acid sequences or vectors described herein may be introduced (e.g. transduced). Once a nucleic acid molecule or vector has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule or vector is introduced into the host cell, the resultant modified cell should be capable of expressing the encoded polypeptide.

[0213] The nucleic acid composition or vector system may be introduced into the cell using any conventional method known in the art. For example, the nucleic acid composition or vector system may be introduced using CRISPR technology. Other conventional methods such as transfection, transduction or transformation of the cell may also be used.

[0214] The term “modified cell” refers to a genetically altered (e.g. transformed, transduced or transfected) cell. The modified cell includes at least one exogenous nucleic acid sequence (i.e. a nucleic acid sequence that is not naturally found in the host cell). The term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0215] Pharmaceutical composition

[0216] In another aspect the present invention there is provided a pharmaceutical composition comprising a therapeutically effective amount of a PABPN1 polypeptide disclosed herein, a nucleic acid encoding said PABPN1 polypeptide, a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1 and / or wt- PABPN1 or a vector comprising a nucleic acid sequence encoding said PABPN1 polypeptide and / or said inhibitory RNA. Such a composition typically comprises at least one pharmaceutically acceptable diluent or carrier.

[0217] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0218] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the pharmaceutical composition is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure. Various other conventional pharmaceutical ingredients may be provided in the pharmaceutical composition, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0219] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable cells.

[0220] Suitably in one aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a PABPN1 polypeptide disclosed herein or a nucleic acid encoding the PABPN1 polypeptide ora vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide as disclosed herein and / or a nucleic acid sequence encoding an inhibitory RNA as described herein and a pharmaceutically acceptable diluent or carrier.

[0221] Suitably, in some embodiments the pharmaceutical composition comprises a therapeutically effective amount of a PABPN1 polypeptide disclosed herein (that does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3, an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4) and a pharmaceutically acceptable diluent or carrier.

[0222] Suitably, in some embodiments the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid encoding the PABPN1 polypeptide disclosed herein, (wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4) and a pharmaceutically acceptable diluent or carrier.

[0223] Suitably, in some embodiments the pharmaceutical composition comprises a therapeutically effective amount of a vector comprising a nucleic acid encoding a PABPN1 polypeptide disclosed herein, (wherein the PABPN1 polypeptide does not comprise at least one of: an N- terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO: 4) and a pharmaceutically acceptable diluent or carrier.

[0224] Suitably, in one embodiment the pharmaceutical composition comprises a therapeutically effective amount of a vector comprising a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1 and a pharmaceutically acceptable diluent or carrier. In some embodiments, the inhibitory RNA is an siRNA.

[0225] Suitably, in one embodiment the pharmaceutical composition comprises a therapeutically effective amount of a vector comprising a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the wt-PABPN1 and a pharmaceutically acceptable diluent or carrier. In some embodiments, the inhibitory RNA is an siRNA.

[0226] Suitably, in one embodiment the pharmaceutical composition comprises a therapeutically effective amount of a vector comprising a nucleic acid encoding an inhibitory RNA that suppresses or inhibits the wt-PABPN1 , an inhibitory RNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1 and a pharmaceutically acceptable diluent or carrier. In some embodiments, the inhibitory RNA is an siRNA.

[0227] Suitably, in one embodiment the pharmaceutical composition comprises a therapeutically effective amount of a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein, (wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; does not comprise an intrinsically disordered region according to SEQ ID NO:9; does not comprise a coiled coil domain according to SEQ ID NO: 4) and a vector comprising a nucleic acid sequence encoding an inhibitory RNA that suppresses or inhibits the endogenous aggregating mutant form of PABPN1 and a pharmaceutically acceptable diluent or carrier. In some embodiments, the inhibitory RNA is an siRNA. Suitably, in some embodiments, the nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein and the nucleic acid sequence encoding the inhibitory RNA are comprised in the same or different vectors.

[0228] In some embodiments the pharmaceutical composition comprises a therapeutically effective amount of a PABPN1 polypeptide disclosed herein or a nucleic acid encoding the PABPN1 polypeptide or a vector comprising the nucleic acid encoding the PABPN1 , wherein the PABPN1 polypeptide does not comprise an N-terminal alanine tract according to SEQ ID NO:3. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or2 to 2 of SEQ ID NO: 2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise the N-terminal alanine tract corresponding to the sequence AAAAAAAAAA (SEQ ID NO: 3). Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO: 3.

[0229] The N-terminal alanine tract of the OPMD PABPN1 comprises an extended alanine tract of 11 to 18 alanine amino acids. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise an extended N-terminal alanine tract. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise an N-terminal alanine tract of 11 to 18 amino acids.

[0230] In some embodiments the PABPN1 polypeptide disclosed herein does not comprise an intrinsically disordered region according to SEQ ID NO:9. Suitably, the intrinsically disordered region (IDR) corresponds to amino acids 1 to 115 of SEQ ID NO:2. Suitably, in some embodiments the IDR corresponds to amino acids 2 to 115, 3 to 115, 4 to 115, 5 to 115, 6 to 115, 7 to 115, 8 to 115, 9 to 115, 10 to 115, 11 to 115, 12 to 115, 13 to 115, 14 to 115, 15 to 115, 20 to 115, 30 to 115, 40 to 115, 50 to 115, 60 to 115, 70 to 115, 80 to 115, 90 to 115, 100 to 115, 110 to 115 of SEQ ID NO:2.

[0231] Suitably, in some embodiments an N-terminal alanine tract is comprised in the IDR. Suitably, in some embodiments, PABPN1 polypeptide disclosed herein does not comprise an N- terminal alanine tract according to SEQ ID NO:3 and does not comprise an IDR according to SEQ ID NO:9 . In some embodiments, the PABPN1 polypeptide comprises the IDR according to SEQ ID NO:9 but does not comprise an N-terminal alanine tract (e.g. that according to SEQ ID NO:3).

[0232] Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise SEQ ID NO:9. Suitably, in some embodiments the PABPN1 polypeptide does not comprise SEQ ID NO:3 and does not comprise SEQ ID NO:9.

[0233] Suitably, in some embodiments the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein, or a nucleic acid sequence encoding the PABPN1 polypeptide, or a vector comprising the nucleic acid encoding the PABPN1 , that does not comprise the coiled coil domain according to SEQ ID NO:4. In some embodiments, the PABPN1 polypeptide disclosed herein comprises a coiled coil domain e.g. the CCD according to SEQ ID NO:4. Suitably, in some embodiments the PABPN1 polypeptide disclosed herein comprises a part of a coiled coil domain (e.g. part (but not all) of the CCD according to SEQ ID NO:4).

[0234] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising MEEEAEKLKELQNEVEKQMNMSP (SEQ ID NO:8), a nucleic acid encoding a PABPN1 polypeptide disclosed herein comprising MEEEAEKLKELQNEVEKQMNMSP (SEQ ID NO:8) or a vector comprising a nucleic acid encoding a PABPN1 polypeptide disclosed herein comprising MEEEAEKLKELQNEVEKQMNMSP (SEQ ID NO:8).

[0235] In some embodiments of the present invention, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4, a nucleic acid encoding a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4 or a vector comprising a nucleic acid encoding a PABPN1 polypeptide disclosed herein that does not comprise a coiled coil domain according to SEQ ID NO:4.

[0236] In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an N-terminal alanine tract (e.g. that of SEQ ID NO:3). In some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an IDR (e.g. that of SEQ ID NO:9). Suitably, in some embodiments the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4, does not comprise an N-terminal alanine tract (e.g. that of SEQ ID NO:3) and does not comprise an IDR (e.g. that of SEQ ID NO:9). Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an N-terminal alanine tract according to SEQ ID NO:3.

[0237] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4 and does not comprise an IDR according to SEQ ID NO:9.

[0238] Suitably, in some embodiments, the PABPN1 polypeptide disclosed herein does not comprise a coiled coil domain according to SEQ ID NO:4, does not comprise an N-terminal alanine tract according to SEQ ID NO:3 and does not comprise an IDR according to SEQ ID NO:9.

[0239] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0240] In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0241] In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0242] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0243] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof. In some embodiments, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0244] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0245] In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0246] In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0247] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0248] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0249] In some embodiments, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO:7 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0250] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0251] In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0252] In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0253] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0254] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0255] In some embodiments, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO: 19 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0256] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NQ:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0257] In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0258] In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0259] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NQ:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0260] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0261] In some embodiments, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NQ:20 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0262] In some embodiments, the pharmaceutical composition comprises a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0263] In some embodiments, the pharmaceutical composition comprises a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0264] In some embodiments, the pharmaceutical composition comprises a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide disclosed herein comprising an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0265] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0266] In some embodiments, the PABPN1 polypeptide disclosed herein comprises an amino acid sequence according to SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0267] In some embodiments, the PABPN1 polypeptide disclosed herein consists of an amino acid sequence according to SEQ ID NO:21 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

[0268] Suitably, in some embodiments, the pharmaceutical composition is suitable to restore cellular defects in a cell. Suitably, in some embodiments the cellular defects are caused by APA and / or ALE usage. Suitably, in some embodiments the pharmaceutical composition restores 3’-UTR length or ALE usage.

[0269] Restoring cellular defects may refer to restoring cellular function such as restoration of genome-wide mRNA expression levels, restoration of mRNA nuclear export and restoration of translation efficiency. Suitably, in some embodiments, the pharmaceutical composition as described herein may be for use in methods of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject, wherein the pharmaceutical composition restores mRNA levels in a cell to that of a healthy cell. Suitably, in some embodiments, the pharmaceutical composition as described herein may be for use in methods of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject, wherein the pharmaceutical composition restores mRNA nuclear export in a cell. Suitably, in some embodiments, the pharmaceutical composition as described herein may be for use in methods of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject, wherein the pharmaceutical composition restores translation efficiency in a cell.

[0270] In some embodiments, the pharmaceutical composition restores the level of PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject. The term “restores” as used herein refers to a complete or partial restoration of the level PABPN1 as observed in a healthy subject. A complete restoration may refer to having an equivalent level of PABPN1 as typically observed in a healthy subject. A partial restoration may refer to having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of PABPN1 as typically observed in a healthy subject.

[0271] A “healthy” cell as used herein may refer to any cell that does not comprise an endogenous aggregating mutant form of PABPN1. The healthy cell may refer to any cell that has normal PABPN1 function and / or normal level of PABPN1.

[0272] It will be understood that where the pharmaceutical composition comprises a vector comprising a nucleic acid encoding a PABPN1 polypeptide disclosed herein and a pharmaceutically acceptable diluent or carrier, the nucleic acid sequence may encode any PABPN1 polypeptide as described herein. The vector may also comprise a nucleic acid sequence encoding an inhibitory RNA. The nucleic acid may encode any inhibitory RNA as described herein. The skilled person will appreciate that the nucleic acid encoding the PABPN1 polypeptide and the nucleic acid encoding the inhibitory RNA can be provided in the same or different vectors.

[0273] The pharmaceutical composition of the present invention may comprise any vector as detailed above. In particularly preferred embodiments, the vector is a viral vector that is suitable for use in gene therapy applications, e.g. a gene therapy vector.

[0274] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0275] Therapeutic and Other Methods and Uses

[0276] In one aspect of the present invention there is provided a method of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 , wherein the method comprises administering a poly(A) binding protein nuclear 1 (PABPN1 ) polypeptide, a nucleic acid sequence encoding the PABPN1 polypeptide, a vector comprising a nucleic acid sequence encoding the PABPN1 polypeptide or a pharmaceutical composition according to any aspect or embodiment as described herein to a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; does not comprise a coiled coil domain according to SEQ ID NO:4.

[0277] In another aspect, there is provided use of a poly(A) binding protein nuclear 1 (PABPN1) polypeptide, a nucleic acid sequence encoding the PABPN1 polypeptide, a vector comprising a nucleic acid sequence encoding the PABPN1 polypeptide or a pharmaceutical composition according to any aspect or embodiment as described herein for the manufacture of a medicament treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

[0278] In any of the aspects or embodiments as described herein, the PABPN1 polypeptide disclosed herein or nucleic acid disclosed herein may restore PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject when used in a method of treatment as described herein. Suitably, in some embodiments the vector comprising said nucleic acid may restore PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject when used in a method of treatment as described herein. Pharmaceutical compositions of the present invention may restore PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject when used in a method of treatment as described herein.

[0279] In some embodiments, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0280] In one embodiment, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 2-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0281] In one embodiment, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 3-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject. In one embodiment, the PABPN1 polypeptide disclosed herein is expressed at a level that is at least 4-fold compared to the level of wild-type PABPN1 polypeptide observed in a healthy subject.

[0282] It will be appreciated that the methods and uses as described herein may further comprise administration or expression of an inhibitory RNA to suppress the endogenous aggregating mutant form of PABPN1 and / or the wild type form of PABPN1. In most preferred embodiments, the inhibitory RNA is an siRNA.

[0283] Method of gene therapy

[0284] It will be evident to the skilled person that nucleic acid sequences encoding the PABPN1 polypeptides as disclosed herein and vectors comprising said nucleic acid sequences may be used for gene therapy. Accordingly, the use of such nucleic acid constructs in gene therapy form part of the present invention.

[0285] The nucleic acid sequences, expression cassettes, vectors or virions as described herein, may be for use in gene therapy in a subject, preferably gene therapy through muscle-specific expression of an expression product, preferably a PABPN1 polypeptide as disclosed herein. Suitably, in some embodiments muscle-specific expression may refer to skeletal muscle specific expression, smooth muscle specific expression or cardiac muscle specific expression. The skilled person will appreciate that muscle specific expression may be achieved by administering a vector or virion with enhanced muscle tropism or by selective use of a muscle specific promoter.

[0286] The nucleic acid sequences, expression cassettes, vectors or virions as described herein, may be for use in gene therapy in a subject, preferably gene therapy through cancer cellspecific expression of an expression product, preferably a PABPN1 polypeptide as disclosed herein. Suitably, in some embodiments cancer cell-specific expression may refer to expression in bladder cancer cells, non-bladder cancer cells, lung cancer cells, biliary tract cancer cells, bowel cancer cells, kidney cancer cells, liver cancer cells, lymphoid cancer cells, pleura cancer cells, prostate cancer cells or testicular cancer cells.

[0287] The nucleic acids, expression cassettes, vectors or virions as described herein may be for use in gene therapy through bladder-specific expression of an expression product, preferably a PABPN1 polypeptide as disclosed herein. It will be understood that it may be desirable in some instances that expression is cell-type specific. Suitably, in some embodiments, expression of an expression product in the bladder may be limited to bladder cancer cells and not expressed in non-cancerous bladder cells. Alternatively, it may be desirable in some instances that the expression product is expressed in non-cancerous bladder cells. Suitably, in some embodiments expression of an expression product in the bladder is limited to non- cancerous bladder cells.

[0288] Disclosed herein is also a method of gene therapy of a subject, preferably a human, in need thereof, the method comprising:

[0289] - administering to the subject a nucleic acid encoding a PABPN1 polypeptide disclosed herein, a vector comprising said nucleic acid sequence, a virion comprising said vector or pharmaceutical composition comprising said nucleic acid, vector or virion.

[0290] The method suitably comprises expressing a therapeutic amount of the PABPN1 polypeptide disclosed herein from the gene in the muscle of said subject. Various conditions and diseases that can be treated and suitable diseases are discussed below.

[0291] In some embodiments, the vector is a viral gene therapy vector, for example an AAV vector. Suitable vectors are described in more detail above.

[0292] In some embodiments, the method comprises administering the viral gene therapy vector systemically. Systemic administration may be enteral (e.g. oral, sublingual, and rectal) or parenteral (e.g. injection). Preferred routes of injection include intravenous, intramuscular, subcutaneous, intra-arterial, intra-articular, intrathecal, and intradermal injections.

[0293] In some embodiments, the viral gene therapy vector may be administered concurrently or sequentially with one or more additional therapeutic agents or with one or more saturating agents designed to prevent clearance of the vectors by the reticular endothelial system.

[0294] Where the vector is an AAV vector, the dosage of the vector may be from 1x1 O10gc / kg to 1x1015gc / kg or more, suitably from 1x1012gc / kg to 1x1014gc / kg, suitably from 5x1012gc / kg to 5x1013gc / kg (gc / kg stands for genome copies per kilogram).

[0295] In general, the subject in need thereof will be a mammal, and preferably primate, more preferably a human. Typically, the subject in need thereof will display symptoms characteristic of a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1. The method typically comprises ameliorating the symptoms displayed by the subject in need thereof, by expressing the therapeutic amount of the expression product, suitably a therapeutic expression product. Gene therapy protocols for therapeutic gene expression in target cells in vitro and in vivo, are well-known in the art and will not be discussed in detail here. Briefly, they include intramuscular injection, interstitial injection, instillation in airways, application to endothelium, intra-hepatic parenchyme, and intravenous or intra-arterial administration (e.g. intra-hepatic artery, intra- hepatic vein) of plasmid DNA vectors (naked or in liposomes) or viral vectors. Various devices have been developed for enhancing the availability of DNA to the target cell. While a simple approach is to contact the target cell physically with catheters or implantable materials containing the relevant vector, more complex approaches can use jet injection devices and suchlike.

[0296] Gene transfer into mammalian muscle cells has been performed using both ex vivo and in vivo procedures. The ex vivo approach typically requires harvesting of the muscle cells, in vitro transduction with suitable expression vectors, followed by reintroduction of the transduced myocytes into the muscle. In vivo gene transfer has been achieved by injecting DNA or viral vectors into the muscle. In some preferred embodiments, the preferred route of administration is intravenous. Intravenous delivery is particularly preferred for gene therapy targeting the muscle. In some preferred embodiments, the preferred route of administration is intracoronary. Intracoronary delivery is particularly preferred for gene therapy targeting the cardiac muscle. Similar gene transfer methods may be used for any desired cell or tissue type, for example bladder cells.

[0297] It may be desirable in some instances to insert the nucleic acid encoding the PABPN1 polypeptide disclosed herein into the genome of the subject in need thereof. Similarly, in some embodiments it may be desirable to insert the nucleic acid encoding the inhibitory RNA as described herein into the genome of the subject in need thereof. Any suitable method known in the art may be used. In a preferred embodiment, the nucleic acid is inserted into the genome of the subject in need thereof using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system, preferably a CRISPR-Cas9 system. Such systems are described in detail above.

[0298] Suitably, in some embodiments the nucleic acid encoding the PABPN1 polypeptide and / or the inhibitory RNA as disclosed herein is inserted into the genome of the subject in need thereof using a method comprising:

[0299] (a) introducing into a cell of the subject a sequence-specific nuclease that cleaves the genome at an insertion site;

[0300] (b) introducing into the cell a donor construct comprising the nucleic acid encoding the PABPN1 polypeptide and / or the nucleic acid encoding the inhibitory RNA; and

[0301] (c) introducing into the cell a guide RNA (gRNA) recognising the insertion site, wherein the donor construct is a linear nucleic acid or cleaved within the cell to produce a linear nucleic acid, wherein the linear nucleic acid comprises a 5' homology arm, the nucleic acid encoding the PABPN1 polypeptide and / or nucleic acid encoding the inhibitory RNA and a 3' homology arm, wherein the 5' homology arm is homologous to a sequence upstream of the nuclease cleavage site on the genome and wherein the 3' homology arm is homologous to a sequence downstream of the nuclease cleavage site on the genome; wherein the 5' homology arm and the 3' homology arm are proximal to the 5' and 3' ends of the linear nucleic acid, respectively, and wherein the nucleic acid encoding the PABPN1 polypeptide and / or inhibitory RNA is inserted into the genome at the insertion site through homologous recombination. In some embodiments, the method may further comprise a step of introducing into the cell an exonuclease.

[0302] Suitably, in some embodiments, the sequence-specific nuclease is Streptococcus pyogenes Cas9 (spCas9) or Staphylococcus aureus Cas9 (saCas9).

[0303] According to some preferred embodiments, the methods set out above may be used for the treatment of a subject with a disease as discussed below, e.g. OPMD or cancer.

[0304] Disease or condition

[0305] The disease or condition according to any aspect or embodiment as described herein may be any disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1. In preferred embodiments, the disease is a disease which can be alleviated by providing or expressing a non-aggregating isoform of PABPN1 as described herein.

[0306] The term “disease or condition associated with a reduction in the level of PABPN1” will be understood to mean a disease or condition which can be characterised by a reduced function or lower level of PABPN1 expression compared to the normal or healthy subject. The amount may vary in a particular subject, but it will be generally understood that the reduced function or lower level of PABPN1 expression is less than a normal or healthy subject and at a level that will result in a disease or condition. Suitably, in some embodiments the disease or condition associated with a reduction in the level of PABPN1 , wherein PABPN1 is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10% or at least 5% compared to the levels of wildtype PABPN1 polypeptide in a healthy subject. It will be appreciated that in some diseases or conditions such as OPMD, PABPN1 levels are reduced due to aggregation of PABPN1 .

[0307] In some embodiments, the disease or condition is associated with a reduction in the level of PABPN1 by at least 30% compared to levels of PABPN1 polypeptide in a healthy subject.

[0308] In some embodiments, the reduction in the level of PABPN1 is 30-90% compared to levels of PABPN1 polypeptide in a healthy subject. In some embodiments, the reduction in the level of PABPN1 is 30-80% compared to levels of PABPN1 polypeptide in a healthy subject. In some embodiments, the reduction in the level of PABPN1 is 30-70% compared to levels of PABPN1 polypeptide in a healthy subject. In some embodiments, the reduction in the level of PABPN1 is 30-60% compared to levels of PABPN1 polypeptide in a healthy subject. In some embodiments, the reduction in the level of PABPN1 is 30-50% compared to levels of PABPN1 polypeptide in a healthy subject. In some embodiments, the reduction in the level of PABPN1 is 30-40% compared to levels of PABPN1 polypeptide in a healthy subject.

[0309] The term “impairment in the function of PABPN1” will be understood to mean that PABPN1 does not function or have the same activity as the wildtype PABPN1 protein or has lower function or activity in a subject when compared to a normal or healthy subject. In some embodiments, impairment in the function of PABPN1 may result in impaired mitochondrial activity. In some embodiments, impairment in the function of PABPN1 may result in impaired muscle tissue differentiation.

[0310] In some embodiments, the disease or condition is further associated with an increase in alternative polyadenylation (APA) and / or ALE (alternative last exon) usage. A disease or condition associated with APA will be understood to mean any disease or condition that is caused by dysregulated APA or where APA is considered a contributing factor to the disease or condition pathology or aetiology. APA can alter messenger RNA (mRNA) and affect the stability, localisation, and the way proteins are made, therefore influencing pathways important for normal health. Therefore, diseases and conditions where there is an increase in APA, may be considered a disease or condition associated with increased APA where the increase in APA contributes to the disease pathology or aetiology. Similarly, a disease or condition associated with ALE will be understood to mean any disease or condition where increased levels of ALE compared to a normal or healthy subject or cell is observed and may be contributing factor to the disease or condition pathology of aetiology. Alternative last exon usage allows for the production of multiple protein isoforms from a single gene, which can differ in functional or regulatory regions. Increased ALE usage may result in an alternative 3' untranslated region (UTR), which contain regulatory elements that control mRNA stability, localisation, and translation efficiency and nonsense-mediated decay (NMD). In some cases, the selection of an alternative exon can introduce a premature stop codon, triggering the degradation of the mRNA transcript through NMD, effectively preventing the production of a functional protein. Increased ALE usage is a known cause or contributing factor to diseases such as cancer and muscle pathologies.

[0311] The skilled person will appreciate that the maturation of nascent RNAs is a key step in transcription. For mRNA, the maturation of messenger RNA precursors (pre-mRNAs), involves the processing of 3’termini, where the 3’end of nascent mRNA is cleaved, followed by addition of a poly(A) tail (i.e., polyadenylation). Cleavage or polyadenylation can generate transcript isoforms which differ in their coding regions or 3’UTRs. This phenomenon, which gives rise to various transcript isoforms, is termed as alternative polyadenylation (APA). Alternative polyadenylation (APA) results in numerous transcripts with differing 3’ends, thus greatly expanding the diversity of mRNAs and of proteins derived from a single gene. As a key molecular mechanism, APA is involved in various gene regulation steps including mRNA maturation, mRNA stability, cellular RNA decay, and protein diversification. APA is frequently dysregulated in cancers leading to changes in oncogenes and tumour suppressor gene expressions.

[0312] In some embodiments, the disease or condition is further associated with a shift to proximal pA site usage. The term “shift to proximal pA site usage” will be understood to mean there is preferential selection by the cell for a polyadenylation site closer to the coding regions over more distal sites in the same gene. In some instances, the proximal pA site is located within an intron or a coding exon, which can lead to a change in the protein's C-terminal domain or produce a truncated protein isoform. In some embodiments, the disease or condition is further associated with a shift to proximal APA. Shifts to proximal APA may affect mRNA stability or processing of the involved transcript and hence biological function relevant for the disease process. Shorter mRNA are more stable but less well translated and as such, a shift to proximal APA may also impact the cell proteome.

[0313] Alternative last exon usage (ALE) is a mechanism that produces multiple mRNA and protein isoforms by using different last exons in a gene. Aberrant ALE can be associated with a variety of diseases, including neurological disorders and cancers.

[0314] Suitably, in some embodiments the disease or condition is associated with ALE (alternative last exon) usage. It will be appreciated that an increase in APA and / or an increase in ALE may refer to an abnormal or elevated level compared to a healthy cell. Typically, an increase in APA and / or ALE may refer to any level that results in or is associated with disease.

[0315] The skilled person will appreciate that the level of APA and / or ALE may be assessed by any suitable means known in the art. Such methods include those described in the Example section below and as published in Shademan M, Mei H, van Engelen B, Ariyurek Y, Kloet S, Raz V. PABPN1 loss-of-function causes APA-shift in oculopharyngeal muscular dystrophy. HGG Adv. 2024 Apr 11;5(2): 100269. doi: 10.1016 / j.xhgg.2024.100269. Epub 2024 Jan 11. PMID: 38213032; PMCID: PMC10840355. Without limitation, the level of APA and / or ALE may be determined by RNA sequencing and calculating the ratio between reads at distal part and proximal part of the 3'UTR (APA) or the ratio between reads at two alternative last exons (ALE). A change may be determined by the calculation the ratio in normal condition versus the ratio in disease or OPMD condition.

[0316] In some embodiments, the disease or condition is cardiac disease, cancer, autoimmune disease, ageing and / or muscle pathology.

[0317] In some embodiments, the disease or condition is cardiac disease, cancer, autoimmune disease, and / or muscle pathology.

[0318] Suitably, in some embodiments the disease or condition is ageing. The term ageing will be understood to include diseases or conditions typically associated with or have increase prevalence with increased age.

[0319] Suitably, in some embodiments the disease or condition is autoimmune disease. Autoimmune diseases may include any one of the following: rheumatoid arthritis, multiple sclerosis, scleroderma, lupus and Sjogren’s syndrome.

[0320] Suitably, in some embodiments the disease or condition is cancer. Suitably, in some embodiments cancer is any one selected from the following: bladder cancer, non-bladder cancer, lung cancer, biliary tract cancer, bowel cancer, kidney cancer, liver cancer, lymphoid cancers, pleura cancer, prostate cancer or testicular cancer.

[0321] Suitably, in some embodiments, the cancer is non-bladder cancer, bladder cancer or small cell lung cancer. In some preferred embodiments, the disease or condition is bladder cancer.

[0322] In some embodiments, the disease or condition is muscle pathology. Suitably, the muscle pathology is oculopharyngeal muscular dystrophy (OPMD), facioscapulohumeral muscular dystrophy (FSHD), or sarcopenia. Suitably in some embodiments the disease or condition is muscular dystrophy. In some embodiments, the muscular dystrophy is oculopharyngeal muscular dystrophy (OPMD).

[0323] In some embodiments, the disease or condition is a cardiac disease. In some preferred embodiments, the disease or condition is heart failure. In some preferred embodiments, the disease or condition is congestive heart failure.

[0324] The PABPN1 polypeptides disclosed herein, nucleic acids encoding said PABPN1 polypeptides, vectors, virions and pharmaceutical compositions may restore cellular defects such as those described above.

[0325] Suitably, in some embodiments the PABPN1 polypeptide disclosed herein or nucleic acid encoding the PABPN1 polypeptide restores cellular defects. Suitably, in some embodiments a vector comprising a nucleic acid encoding the PABPN1 polypeptide disclosed herein and / or a nucleic acid sequence encoding the inhibitory RNA restores cellular defects. Suitably, in some embodiments the pharmaceutical composition of the present invention restores cellular defects. Suitably, cellular defects may be any cellular defect as described above, most preferably cellular defects associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1. In another preferred embodiment, the cellular defects are caused by APA, and / or ALE usage. Suitably, in some embodiments the 3’ UTR length is restored. Suitably, in some embodiments the last exon usage is restored. Suitably, in some the PABPN1 polypeptide disclosed herein restores mitochondrial activity caused by PABPN1 dysfunction and / or increases differentiation in muscle tissue.

[0326] Methods of identifying a subject in need of treatment

[0327] In a further aspect, there is provided herein a method of identifying a subject that will benefit from treatment with a poly(A) binding protein nuclear 1 (PABPN1) polypeptide disclosed herein or a nucleic acid encoding the PABPN1 polypeptide; wherein the method comprises:

[0328] - testing the subject for PABPN1 level and / or activity to establish a test value;

[0329] - comparing the test value to a reference value, wherein the reference value is PABPN1 level and / or activity of a healthy subject; - where the test value is less than the reference value, recommending treatment with a PABPN1 polypeptide, a nucleic acid, a vector and / or a pharmaceutical composition according to any aspect or embodiment as described herein, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

[0330] In another aspect, there is provided herein a method of identifying a subject that will benefit from treatment with a poly(A) binding protein nuclear 1 (PABPN1) polypeptide disclosed herein or a nucleic acid encoding the PABPN1 polypeptide; wherein the method comprises:

[0331] - testing the subject for oculopharyngeal muscular dystrophy;

[0332] - where the subject is positive for oculopharyngeal muscular dystrophy, recommending treatment with a PABPN1 polypeptide, a nucleic acid, a vector and / or a pharmaceutical composition according to any aspect or embodiment as described herein, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

[0333] A subject that will benefit from treatment will be understood to mean a subject that may have an undiagnosed or diagnosed disease or condition that is caused by or associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1. The skilled person will understand that a subject that will benefit from treatment with a poly(A) binding protein nuclear 1 (PABPN1) polypeptide disclosed herein or a nucleic acid encoding the PABPN1 polypeptide is a subject that has a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 and when provided said treatment their condition or disease will improve or be expected to improve upon treatment.

[0334] Suitably in some embodiments, testing the subject for oculopharyngeal muscular dystrophy is genetic testing for mutated versions of the PABPN1 gene. The wild type PABPN1 gene typically contains 10 repeats of the DNA sequence GCN, but the mutated versions of the PABPN1 gene that cause OPMD contain an expanded number (11-18) of GCN repeats. Suitably, in some embodiments, testing the subject for oculopharyngeal muscular dystrophy comprises genetic screening for extended GCN repeats. A positive result for oculopharyngeal muscular dystrophy may be indicated by the presence of extended GCN repeats. Suitably, in some embodiments, a positive result of OPMD is 11 or more GCN repeats in the PABPN1 gene.

[0335] Suitably, in some embodiments of the methods described herein further comprise obtaining a blood sample from the subject.

[0336] As referred to herein, ‘reference value’ refers to the level, concentration, amount, abundance or activity of PABPN1 in a healthy subject or healthy cell, e.g., a subject or cell that is substantially free of disease. Such examples of substantially free of disease may include subjects or cells that do not comprise PABPN1 aggregates or endogenous mutated forms of PABPN1. This includes, but is not limited to OPMD PABPN1.

[0337] The test value may refer to the level (e.g., concentration or abundance or amount) or activity of PABPN1 as determined in a sample. In some preferred embodiments the test value is the concentration of PABPN1 in a sample. In some preferred embodiments the test value is the activity of PABPN1 in a sample. Suitably, the sample may be cells or blood obtained from the subject.

[0338] The term ‘less than’ as used herein refers to a negative deviation from the reference value. Less than may refer to a decrease in the level, concentration, abundance or activity of PABPN1 compared to the reference value. It is particularly preferred that the level of or activity of PABPN1 when compared to the reference value is of clinical significance, e.g. at such levels that cause disease in a subject.

[0339] Suitable methods to determine the concentration and / or activity of PABPN1 in a sample e.g., a cell, are well known in the art and exemplified in the Example section below. The skilled person will appreciate that any such suitable methods may be used.

[0340] Where the test value is less than the reference value, treatment with a PABPN1 polypeptide, a nucleic acid sequence, a vector and / or a pharmaceutical composition according to any aspect or embodiment as described herein is recommended. Suitably, in such embodiments the method further comprises a step of administering or expressing in the subject in need thereof the PABPN1 polypeptide disclosed herein or the nucleic acid encoding the PABPN1 polypeptide, the vector comprising a sequence encoding the PABPN1 polypeptide or a pharmaceutical composition of the present invention. Suitably, in some embodiments the method further comprises a step of administering or expressing an inhibitory RNA or a nucleic acid encoding an inhibitory RNA to suppress or inhibit the endogenous aggregating mutant form of PABPN1. In some embodiments, the method further comprises a step of administering or expressing an inhibitory RNA or nucleic acid encoding an inhibitory RNA to suppress or inhibit wt-PABPN1. In some embodiments, the inhibitory RNA is an siRNA or an antisense oligonucleotide (ASO).

[0341] The method may further comprise administering or expressing a CRISPR-Cas9 system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1 . Such systems are described in detail above.

[0342] Suitably, in some embodiments the CRISPR-Cas9 system comprises a gRNA targeting exon 1 of PABPN1. Suitably, in some embodiments wherein the gRNA is encoded by SEQ ID NO: 22 or SEQ ID NO: 23. In one preferred embodiment, the gRNA is encoded by SEQ ID NO: 22.

[0343] General Definitions

[0344] The terms "identity" and "identical" and the like refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules or between two amino acid sequences such as between two peptide chains. Sequence alignments and determination of sequence identity can be done, e.g., using the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250).

[0345] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881- 90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31 ; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0346] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the "help" section for BLAST™ . For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method. Typically, the percentage sequence identity is calculated over the entire length of the sequence.

[0347] For example, a global optimal alignment is suitably found by the Needleman-Wunsch algorithm with the following scoring parameters: Match score: +2, Mismatch score: -3; Gap penalties: gap open 5, gap extension 2. The percentage identity of the resulting optimal global alignment is suitably calculated by the ratio of the number of aligned bases to the total length of the alignment, where the alignment length includes both matches and mismatches, multiplied by 100.

[0348] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0349] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2,

[0350] 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of

[0351] 4, and a frameshift gap penalty of 5.

[0352] Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0353] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>.

[0354] The terms "peptide", "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0355] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991 ) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole.

[0356] As used here in the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.

[0357] Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0358] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

[0359] EXAMPLES

[0360] Background The processing of mRNA into mature transcripts involves a cleavage step followed by addition of a poly(A) tail that stabilizes the transcript, ensuring nuclear export. The cleavage is carried out by a protein complex that recognizes the polyadenylation site (PAS or pA sites) in the DNA, which is most often at the 3’-UTR of transcripts. Alternative transcripts are made when alternative PASs within a 3’UTR are utilized, resulting in transcripts that impact translation efficiency and subsequently cellular processes. Alternative last exon usage (ALE) is a mechanism that produces multiple mRNA and protein isoforms by using different last exons in a gene. Aberrant ALE can be associated with a variety of diseases, including neurological disorders and cancers. Alternative PolyAdenylation (APA) is recognized by specific RNA sequencing procedures and was showed to be involved in a wide range of pathologies, including heart failure, cancer, dementia, immune-diseases, and muscle pathologies.

[0361] APA is regulated by a protein complex in which poly(A) binding protein nuclear 1 (PABPN1 ) plays a central role, as reduced PABPN1 levels causes APA. In several pathologies, like nonsmall cell lung cancer and oculopharyngeal muscular dystrophy (OPMD), APA is associated with low PABPN1 levels. Low PABPN1 expression levels are also associated with aging, especially in skeletal muscles and neuronal cells. PABPN1 is a vital protein that is prone to aggregation. Due to its aggregation, functional (soluble) PABPN1 reaches below-critical levels which causes genome-wide alterations in mRNA expression levels, and defects in mRNA nuclear export and translation efficiency. In OPMD, a rare autosomal dominant myopathy, PABPN1 levels are below critical levels due to mutations in PABPN1 that make the protein even more aggregation-prone. And in OPMD reduced PABPN1 levels strongly correlate with APA (Fig. 1). Thus, restoring PABPN1 levels by simple overexpression could open a therapeutical window for OPMD. This is challenging, however, due to the aggregation- proneness of PABPN1 which depletes the levels of its soluble and functional form.

[0362] Overexpression of a naturally occurring isoform of PABPN1 to restore pathologies driven by Alternative Polyadenylation

[0363] The inventors have identified a naturally occurring shorter PABPN1 isoform which expression strongly correlates with PABPN1 function, and its expression level is associated with PABPN1- mediated muscle pathology in human and mouse (Fig. 1 ). This naturally expressed PABPN1 isoform is conserved in human and mouse, and it lacks the aggregation domain in PABPN1 but contains the entire RNA binding domain and C-terminus that is crucial for PABPN1 function (Figs 1 & 2). The PABPN1 protein comprises a poly-alanine stretch within the N-terminal intrinsically disordered region (IDR), a coiled coil domain (CCD), and a C-terminal RNA recognition motif (RRM) (Figure 2A). Structural (Logicoil) predictions show that the CCD to form a tetrameric coiled coil. The CCD is critical for aggregation but the N-terminal alanine stretch alone does not, suggesting that folding of the contiguous IDR+CCD region may form a stable structure leading to aggregation. AlphaFold3 prediction of the monomeric IDR+CCD predicts, with low confidence, that the alanine tract in Ala16 folds on the CCD (Figure S3B), which may be more stable than the Ala10 structure. Taking the Logicoil prediction of a stable CCD tetramer, AlphaFold3 predicts that the wild type (Ala10) I DR N-terminus is parallel to the tetrameric coiled-coil domain. However, the Ala16 expansion is consistently predicted to intercalate with the tetrameric CCD (Figure 2C). These predictions hint at a structural role for the Ala16 expansion that might lead to pathogenesis, although the mechanism is still unclear. Here it is shown that ectopic expression of this PABPN1 isoform (named tr-PABPN1 or tr- PAB) does not result in nuclear aggregates (as expected) (Figs 3 & 4) and that it restores cellular defects caused by aggregated PABPN1 (Fig. 5-8). Thus tr-PABPN1 can hold therapeutic promise in conditions with reduced or otherwise dysfunctional PABPN1 levels.

[0364] The advantage of the invention: tr-PABPN1 overexpression does not aggregate and does not accumulate in PABPN1 aggregates while showing functional restoration of A16 pathology. The tr-PABPN1 is a natural isoform, therefore there is little risk of toxicity due to its overexpression / ectopic expression.

[0365] Overexpression of the full-length PABPN1 is in clinical trials (silence and replace strategy in which endogenous aggregating mutant PABPN1 is silenced by RNAi while functional PABPN1 is added by expression of an RNAi-resistant copy of PABPN1 ). But PABPN1 overexpression readily forms aggregates, thus that approach is higher risk.

[0366] MATERIALS AND METHODS

[0367] PABPN1 constructs and lentivirus production

[0368] The expanded PABPN1 (Ala16) and tr-PAB were cloned into the pCW57-MCS1-2A-MCS2 doxycycline (Dox) inducible lentiviral vector (Addgene plasmid #71782). The GFP fusion was published in [1], The FLAG fusion was made by in vitro mutagenesis. Cloning was confirmed by Sanger sequencing. Lentivirus production was performed as detailed in (Carlotti, Bazuine et al. 2004). The tr-PAB will be cloned into the AAV cassette using restriction enzymes.

[0369] Cell culture

[0370] Immortalized muscle cells were cultured in growth medium (F10 (Gibco) medium supplemented with 15% FCS, 1 ng / ml bFGF, 10 ng / ml EGF and 0.4 pg / ml Dexamethasone). Cells were propagated in confluence 50-80%. Cell cultures did not reach 100% confluence to avoid spontaneous differentiation. Cell differentiation was induced at high confluency (85- 95%) in DMEM+2% horse serum for 3-5 days. Cells were transduced with lentiviruses encoding Ala16 and stable cell cultures were created using puromycin selection. The transgene was induced with 4 mg / ml doxycycline hyclate (D5207, Sigma Aldrich). For high content screening (HCS), cells were seeded in a Nunc 96 well plate.

[0371] Protein extraction and Western blot analysis

[0372] Bulk protein extraction of soluble and insoluble fractions a cell pellet was collected from a 12 well o using a lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCI, 5 mM EDTA, 0.1% NP40, and 1 mM DTT and 1x protease inhibitor cocktail), after sonication and spin down, the supernatant was. Insoluble proteins were extracted from the pellet with lysis buffer +2% SDS. For equal loading on SDS-PAGE, protein amount was determined in the soluble fraction, and the proportional aliquots from the paired insoluble fraction. Protein aliquots were separated on 10% SDS-PAGE. Western blotting was carried out with a PVDF membrane. Bulk proteins were visualized with the No-Stain Protein Labeling Reagent (#A44717, ThermoFisher) and imaged using the iBright Imaging System (ThermoFisher). The membrane was blocked with 5% dried milk powder (T145.2, Carl Roth), first antibody incubation was carried out at 4 degrees overnight, and secondary antibody incubation at room temperature for one hour. Antibodies are listed in Table S1 . An Odyssey CLx Infrared imaging system (LiCOR, NE. USA) was used to detect the fluorescent signal. Quantification of protein abundance was done using Imaged. Values were corrected for background and normalized to loading controls. Western blot quantification was carried out with Imaged. Normalization was made for both the No-Stain and house-keeping signal.

[0373] RNA isolation and qRT-PCR

[0374] RNA isolation was made with the RNAeasy kit (Qiagen) according to the manufacturer protocol. cDNA synthesis was made with RevertAid First Strand cDNA Synthesis Kit (ThermoFisher) according to the manufacturer protocol. qRT-PCR was conducted with SYBR Green qPCR Master Mix (ThermoFisher) according to the manufacturer protocol. Primers for qPCR for APA-shift were designed from the proximal and distal part of the 3’-UTR close to the most abundant distal or proximal PAS (pA sites), which was determined based on the RNAsequencing analysis. Primers for qPCR to determine gene expression were diseased from the last two exons. Primer design was performed in Primer3.

[0375] Immunofluorescence and staining

[0376] PAB2, FLAG and MF20 staining: cells were fixed with 4% Paraformaldehyde (PFA) (T.J. Baker) in PBS for 5 minutes. Permeabilization was carried out with 1% Triton-X100 for 10 minutes, followed by PBS washing and first antibody incubation. The first antibody mix (Rabbit- anti-PAB2 and mouse-FLAG or mouse-MF20) was incubated for 1 hour and the secondary (anti-Rabbit-cy5 and anti-mouse-alexa594) for 30 minutes. Antibodies were washed with PBS- T (PBS + 0.05% tween). All incubations were carried out at room temperature. DAPI nuclear counterstaining was added to the secondary antibody mix. Cells were left in PBS during imaging.

[0377] Cellular Assays

[0378] TMRM staining was carried out in living cells according to the manufacturer instruction (33342 ThermoFisher) was added to the growth medium and incubated for 30 minutes at 37°C. Cells were washed once with PBS and were kept in growth medium during imaging. JC-1 was imaged with 488, and 560nm filters.

[0379] The Protein Synthesis Assay was performed with the protein synthesis assay kit (Cayman Chemicals #601100) was employed according to the manufacturer protocol, with the following modifications: azido-O-propargyl-puromycin (OPP)-488 (named here OPP). For the negative control, 30 minutes pre incubation with 20pM cycloheximide was used. Hoechst was added after fixation.

[0380] RNA hybridization with oligo(dT): cell cultures were fixed using 3.7% FA for 15 minutes at RT. After two PBS washes the cells were incubated in Protease III diluted 1 :30 in PBS (#322337 Advanced Cell Diagnostics) for 15 minutes at RT. After twice PBS washes cells were incubated in hybridization buffer (#10369 Cepham Life Sciences) for 15 minutes at RT. Incubation with 5’-Cy5-Oligo(dT)12-18 probe (#26-4400-02 Gene Link), diluted 1 :1000 in hybridization buffer, was carried out overnight at 40 degrees in a humidified chamber. The following day, washes were carried out at 40 degrees for 5 minutes with 4x, 2x, 1x SSC buffer, and with PBS. Finally, the cells were incubated with Hoechst and kept in PBS during imaging.

[0381] Imaging and Image quantification

[0382] The Cellinsight CX7 LZR high content screening (HCS) platform was used for high- content imaging. A cell-based analysis was performed with the accompanied HCS Platform spot detector and co-localization toolbox (ThermoFisher Scientific).

[0383] Imaging for calculation of differentiation index was made with a 10x objective covering over 12,000 nuclei per well, and imaging for nuclear PABPN1 quantification with a 20x objective, covering at least 5000 nuclei per well. The co-localization toolbox was used for the quantification of differentiation index by the percentage of myonuclei without MyHC objects or FLAG objects, and the spot detection toolbox for the PABPN1 puncta.

[0384] Mouse experiments:

[0385] Example 1: The effect of tr-PAB in A17.1 / + TA muscles.

[0386] Materials and Methods

[0387] To determine the effect of tr-PAB in the OPMD mouse model, A17.1 , the tr-PAB was subcloned into an AAV9 vector, and virus particles were produced at a concentration of 5.53E+13 vp / ml. The AAV particles were injected into the TA muscles of 8-10-week-old male mice, and physiological measurements were taken 12 weeks after injection, followed by muscle harvesting, per condition, 8 A17.1 mice and 8 FvB 8 week-old male mice were used. Vectors were injected in both TA muscles of 8 mice, while the same volume of saline was injected in both TAs of 8 control or A17.1 mice. The muscles were then harvested for ex vivo analysis (Figure 12A). However, the TA muscles were not ready for ex vivo analysis, and the histological investigation is delayed.

[0388] Experimental design:

[0389] Time point 1 : 3 mice per group (n=6 / group) at 8 weeks; time point 2:n=10 / group at 20 weeks.

[0390] • 8 A17 control (saline both legs)

[0391] • 8 A17 treated tr-PAB (both legs)

[0392] • 8 FvB control (saline both legs)

[0393] RESULTS

[0394] Based on the inventors RNA sequencing studies in the mouse OPMD model A17-1 and OPMD patients, APA in OPMD is caused by reduced levels of PABPN1 , including the full-length isoforms 201 and 202 and the truncated natural isoform 207 or 208 in human or mouse, respectively (Figure 1A-C, REF: DOI: 10.1016 / j.xhgg.2O24.100269). It is important to note that in a correlation assessment between PABPN1 isoform levels and APA shift, both isoform 201 and the truncated isoform 208 showed similar correlation values, suggesting that the truncated isoform is functional (Figure 1 D, REF: DOI: 10.1016 / j.xhgg.2024.100269).

[0395] PABPN1 cDNA shares >95% similarity between human and mouse (ENSG00000100836 and ENSMUSG00000022194, respectively), and five major isoforms are predicted to form polypeptides in human and eight polypeptides in mouse. The isoforms encode the found protein isoforms: #1. the full-length isoforms 201 and 202 in human and 201 , 203 and 202 and 207 in mouse); #2. an isoform starting at exon 2 (207 in human and 208 in mouse); #3. an isoform lacking the last exon (208 in human and 205 and 203 in mouse); and #4. a short isoform starting at the middle of exon 4 and lacking the last exon (205 in human and 209 in mouse). In humans, polypeptide #2 is also produced by BCL2L2-PABPN1 ENSG00000258643.

[0396] Based on the inventors RNA sequencing in skeletal muscle, the isoforms coding for polypeptides #3 and #4 are expressed at low levels that are below a confidence threshold, these polypeptides lacking the last exon does not contain domains for PABPN1 function. Together, they are not considered (Figure 1 ). In contrast, polypeptides #1 and #2 contain the essential regions for PABPN1 function (RNA recognition motif and PAP2A binding; Figure 2A). polypeptide #2 lacks the first exon and part of the coiled-coil domain, the latter being critical for aggregation. AlphaFold prediction suggests that the expansion mutation in OPMD folds with CCD and increases aggregation (Figure 2B). Thus, deletion of the N-terminus, alanine stretch - I DR and the CCD, may hold a therapeutic option in conditions caused by limited PABPN1 expression levels. We propose that the natural isoform 207 in humans, here named tr-PAB, is functional and when overexpressed does not form aggregates and overexpression is not toxic.

[0397] To first determine the stability and expression of endogenous PABPN1 and truncated isoforms in human muscle cells mRNA analysis was performed to determine PABPN1 endogenous and truncated isoforms expression levels. mRNA confirms expression of all truncated isoforms and shows that dALA mRNA is the most stable compared to dIDR and dCCD, while dCCD mRNA expression levels are similar to dIDR (Figure 3A). The endogenous 201 isoform is expressed at similar levels in cells expressing the truncated forms. The 207 isoform is expressed at higher levels in dIDR cells and could suggest an additional regulation layer (Figure 3B). Protein expression was also assessed showing the ACCD protein is unstable and therefore was excluded (Figure 4A & B), AIDR and tr-PAB are more stable compared with AALA (Figure 4A- C). PABPN1 puncta are detected in AALA, AIDR, not in tr-PAB (Figure 4D). Collectively, these data show that the natural isoform outperforms all PABPN1 deletion constructs in terms of stability and non-aggregating properties.

[0398] To determine whether tr-PAB is prone to aggregation, tr-PAB fused to YFP was overexpressed in muscle cells. A Western blot revealed a stable polypeptide (Figure 5A) that was nuclear localized and did not form protein aggregates (Figure 5B) in contrast to the expanded PABPN1 (Ala16). Furthermore, tr-PAB colocalized with SC35, a marker of RNA export in nuclear speckles, whereas the A16 puncta did not localize to SC35 (Figure 5C). Importantly, coexpression of tr-PAB with Ala16-YFP significantly reduces YFP puncta (Figure 5D), indicating reduced Alai 6 aggregation by tr-PAB. In addition, tr-PAB reverses the negative effect of Ala16 on mitochondrial activity (Figure 5E). Furthermore, tr-PAB did not induce cell toxicity based on mitochondrial activity (Figure 5E). Taken together, this suggests that tr-PAB expression in differentiating muscle cells is not toxic and may be beneficial to counteract the Ala16 effect.

[0399] Next, the inventors examined the effect of tr-PAB without YFP fusion. A Western blot showed that the 34 kD of tr-PAB FLAG was not recognized by the PAB2 antibody (Figure 6A). The PAB2 antibody is directed against the CCD and can discriminate between full-length PABPN1 and tr-PAB (Figure 6B). To assess the effect of tr-PAB in differentiated cell cultures, we induced transgene expression under the tetracycline promoter, which is activated by doxycycline (Dox) treatment in tr-PAB and Ala16 co-cultures (Figure 6C). To evaluate the effect of tr-PAB on muscle cell differentiation, Dox treatment was performed at the same time as the differentiation conditions. Image quantification of cell cultures stained with FLAG antibodies and image quantification in fused cells showed that transgene expression was completely dependent on Dox treatment (Figure 6D). In the single cell line or co-expression with the parental line tr-PAB-FLAG staining was found in -85% of the cells, in co-culture with Alai 6 or Alai 6-YFP cells 40-60% of the cells were FLAG positive. This indicates that the fused cells contained both tr-PAB and Ala16 nuclei. We then evaluated the fluorescence intensity and area of Alai 6-YFP puncta in fused cells and found that both YFP puncta were more than 2-fold reduced by tr-PAB (Figure 6E). The Ala16-driven reduced fusion index was restored by tr-PAB expression (Figure 6F). The fusion index of tr-PAB was higher than in parental cells, and tr-PAB expression resulted in larger myofibers (Figure 6G). Representative images of tr- PAB-FLAG and Alai 6-YFP co-cultures are shown in Figure 6H). Together, tr-PAB, in contrast to Ala16, is beneficial for muscle cell differentiation.

[0400] After co-culture and differentiation, cells were sorted by size and RNA was extracted from the large cells (Figure 7A). PABPN1 function was assessed by APA shift using two sets of primers to the 3' UTR of genes whose APA is activated in Ala16 cells. Reversed APA shift by tr-PAB co-culture was found in ASB5, CYBRD1 , TMEM123 and PSTPIP2 transcripts (Figure 7B). LRP1 is a control gene whose APA is not affected by PABPN1 (Figure 7B). PCR for PABPN1 exon3-4 confirmed Ala16 and tr-PAB mRNA overexpression (Figure 7C).

[0401] In addition to APA activation, PABPN1 also modulates nuclear export of mRNA, and mRNA nuclear export is mediated by Ala16-PABPN1 nuclear aggregation (Figure 8A). Ala16 coculture with tr-PAB resulted in reduced mRNA nuclear export, and the effect of tr-PAB is enhanced in multinucleated objects (Figure 8B and 8C). Furthermore, the mRNA nuclear to perinuclear ratio is reduced by tr-PAB (Figure 8D) and a stronger effect was found in multinucleated objects (Figure 8E). This suggests that co-expression of tr-PAB with Ala16 restores nuclear export of mRNA and, relevant to skeletal muscle, the effect of tr-PAB is greater in fused cells. Taken together, the present results in muscle cells demonstrate that tr- PAB is beneficial for muscle cell biology and restores cellular and molecular defects caused by the expression of expanded (pathogenic) PABPN1 .

[0402] Taken together, these results show that tr-PAB expression can restore PABPN1 function and cell function in both differentiated human muscle cells.

[0403] Mouse Experiment

[0404] The effect of tr-PAB was then investigated in the A17-1 mouse model by intramuscular injection of AAV into leg muscles (tibialis anterior) andPABPNI nuclear aggregates, APA shift and muscle leg physiology were analysed. Analysis of muscle weight normalised to body weight showed significant muscle weight recovery in the tr-PAB-injected group (Figure 12B).

[0405] As discussed above, the shipment of the TA muscles were lost during shipment, therefore ex vivo analysis could not be carried out. In the A17-1 mouse model, the expanded PABPN1 is expressed under a strong muscle promoter resulting in high overexpression. Therefore, the A17 mRNA will be downregulated to PABPN1 by siRNA. tr-PAB will reduce PABPN1 aggregates and restore muscle physiology in A17-1 mice.

[0406] Example 2: Tr-PAB function in human cancer cells

[0407] Background

[0408] Although PABPN1 is historically linked to OPMD and muscle ageing5, recent studies have demonstrated its causative involvement in bladder cancer6. In both settings, reduced PABPN1 levels drive the pathology. In OPMD, this reduction occurs alongside protein aggregation, while in bladder cancer the loss of functional PABPN1 appears to be the key driver. PABPN1 disease risk is high (negative Chronos values), and less expression levels are predicted to have a higher disease risk (Figure 11 A). PABPN1 is predicted as more pathogenic in several cancers including bladder cancer (Figure 11 B).

[0409] To investigate the role of loss of functional PABPN1 in bladder cancer, the present inventors investigated further the expression of PABPN1 in bladder cancer cells.

[0410] Figure 13A shows the nuclear localisation of tr-PAB-Flag in human bladder cancer cells (left panel). Images were taken 120 hours after Dox induction, showing no nuclear aggregation of tr-PAB (anti-Flag) or endogenous PABPN1 (PAB2). Tr-PAB PABPN1 levels are stabilised 24 hours after Dox induction (left panel). PABPN1 levels in tr-PAB cells are also stabilized after 24 hours (right panel). This indicates that tr-PAB does not represses endogenous PABPN1 levels. Figure 13B shows the mRNA expression levels of endogenous PABPN1 and Flag overexpression. PABPN1 levels are lower in the cancer cells compared with normal cells (PABPN1 exon 3-4 and the 3’-UTR). Notably, tr-PABPN1 restores PABPN1 mRNA levels. Figure 13C shows the distal to proximal ratio at the 3'UTR of four cell cycle genes, including the master cell cycle regulator CDKN1A. tr-PAB reverses the distal to proximal ratio. This experiment shows APA in cancer cells with lower PABPN1 levels. In cells expressing tr- PAB, endogenous PABPN1 levels are elevated APA is shifted back towards distal PAS usage.

[0411] The effect of tr-PAB expression on cancer growth was then investigated. It was then investigated whether tr-PAB could reverse cell function caused by reduced expression levels. Reduced PABPN1 levels play a critical role in bladder cancer (DOI: 10.1186 / s13578-023- 00997-6). Overexpression of tr-PAB in bladder cancer cells did not form nuclear aggregates and showed similar localisation to endogenous PABPN1 (Figure 9A). tr-PAB expression was stable over time (Figure 9B) and did not lead to reduced PABPN1 levels (Figure 9C), which could indicate cell toxicity. Using RT-qPCR, reduced PABPN1 mRNA levels and APA utilisation were confirmed in the bladder cancer cells (Figure 9D and 9E). Importantly, expression of tr- PAB increased the levels of endogenous PABPN1 as measured by primer set to the 3 -UTR (Figure 9D). Furthermore, APA utilisation was significantly restored by tr-PAB expression (Figure 9E). Finally, it was investigated whether tr-PAB affects the phenotypes of bladder cancer cells. Cell growth and migration and mitochondrial activity are hallmarks of cancer cells. Cell growth was measured by cell density over time and a significantly slower growth rate was found in cancer cells expressing tr-PAB (Figure 10A). Expression of tr-PABPN1 also slowed down cell migration (Figure 10B). Mitochondrial activity was also restored by tr-PAB expression without toxicity (Figure 10C and 10D). Finally, the morphology of cancer cells is small and round, while normal epithelial cells are larger (Figure 10C). The size of cancer cells expressing tr-PAB was significantly larger compared to cancer cells (Figure 10E).

[0412] Finally, the inventors then demonstrated that trPAB reduces cancer cell growth without cell toxicity and outperforms the PABPN1 deletion variants (Figure 14). The growth rate in the dIDR cell culture is different from that of the parental bladder cancer cells (Figure 14A). dAla accelerates the growth rate, while the growth rate slows down in the trPAB cell culture. There was no difference in cell toxicity between the parental bladder cancer cells and the PABPN1 overexpression line (Figure 14B).

[0413] SEQUENCES

[0414] Truncated PABPN1 - isoform 207 (SEQ ID NQ:1) MEEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSIEEKMEADARSIYVGNVDYGATAEEL

[0415] EAHFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGRQIKVIPKRT

[0416] NRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARATSWYSPY

[0417] RNA binding domain - shown in bold

[0418] POLA2A interaction domain - shown in underline

[0419] Truncated PABPN1 - isoform 207 (SEQ ID NO:1Q) (DNA sequence) atggaggaagaagctgagaagctaaaggagctacagaacgaggtagagaagcagatgaatatgagtccacctccaggcaatgct ggcccggtgatcatgtccattgaggagaagatggaggctgatgcccgttccatctatgttggcaatgtggactatggtgcaacagcaga agagctggaagctcactttcatggctgtggttcagtcaaccgtgttaccatactgtgtgacaaatttagtggccatcccaaagggtttgcgt atatagagttctcagacaaagagtcagtgaggacttccttggccttagatgagtccctatttagaggaaggcaaatcaaggtgatcccaa aacgaaccaacagaccaggcatcagcacaacagaccggggttttccacgagcccgctaccgcgcccggaccaccaactacaaca gctcccgctctcgattctacagtggttttaacagcaggccccggggtcgcgtctacaggggccgggctagagcgacatcatggtattccc cttactaa

[0420] Full length PABPN1 protein (SEQ ID NO: 2)

[0421] MAAAAAAAAAAGAAGGRGSGPGRRRHLVPGAGGEAGEGAPGGAGDYGNGLESEELEPE

[0422] ELLLEPEPEPEPEEEPPRPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGDGAIEDPE

[0423] LEAIKARVREMEEEAEKLKELQNEVEKQMNMSPPPGN AGPVIMSIEEKMEADARS I YVG NV

[0424] DYGATAEELEAHFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRG

[0425] RQIKVIPKRTNRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARAT

[0426] SWYSPY

[0427] RNA binding domain - shown in bold

[0428] POLA2A interaction domain - shown in underline

[0429] Alanine tract -.shown in. underline

[0430] Coiled coil domain (CCD) - shown in underline

[0431] PABPN1 Isoform 201 Q86U42 (SEQ ID NO: 17)

[0432] MAAAAAAAAAAGAAGGRGSGPGRRRHLVPGAGGEAGEGAPGGAGDYGNGLESEELEPE

[0433] ELLLEPEPEPEPEEEPPRPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGDGAIEDPE

[0434] LEAIKARVREMEEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSIEEKMEADARSIYVGNV

[0435] DYGATAEELEAHFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGR

[0436] QIKVIPKRTNRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARATS

[0437] WYSPY

[0438] PABPN1 Isoform 201 (DNA sequence) (SEQ ID NO: 12)

[0439] Atggcggcggcggcggcggcggcagcagcagcgggggctgcgggcggtcggggctccgggccggggcggcggcgccat cttgtgcccggggccggtggggaggccggggagggggccccggggggcgcaggggactacgggaacggcctggagtctg aggaactggagcctgaggagctgctgctggagcccgagccggagcccgagcccgaagaggagccgccccggccccgcgc ccccccgggagctccgggccctgggcctggttcgggagcccccggcagccaagaggaggaggaggagccgggactggtc gagggtgacccgggggacggcgccattgaggacccggagctggaagctatcaaagctcgagtcagggagatggaggaag aagctgagaagctaaaggagctacagaacgaggtagagaagcagatgaatatgagtccacctccaggcaatgctggcccg gtgatcatgtccattgaggagaagatggaggctgatgcccgttccatctatgttggcaatgtggactatggtgcaacagcagaag agctggaagctcactttcatggctgtggttcagtcaaccgtgttaccatactgtgtgacaaatttagtggccatcccaaagggtttgc gtatatagagttctcagacaaagagtcagtgaggacttccttggccttagatgagtccctatttagaggaaggcaaatcaaggtga tcccaaaacgaaccaacagaccaggcatcagcacaacagaccggggttttccacgagcccgctaccgcgcccggaccacc aactacaacagctcccgctctcgattctacagtggttttaacagcaggccccggggtcgcgtctacaggggccgggctagagcg acatcatggtattccccttactaa

[0440] Alanine tract (SEQ ID NO: 3)

[0441] AAAAAAAAAA

[0442] DNA sequence encoding alanine tract (SEQ ID NO: 16) gcggcggcggcggcggcggcagcagcagcggcg

[0443] Coil coil domain (CCD) (SEQ ID NO: 4)

[0444] EDPELEAIKARVREMEEEAEKLKELQNEVEKQMNMSP

[0445] Q86U42-2 - isoform 202 (SEQ ID NO: 5)

[0446] MAAAAAAAAAAGAAGGRGSGPGRRRHLVPGAGGEAGEGAPGGAGDYGNGLESEELEPE

[0447] ELLLEPEPEPEPEEEPPRPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGDGAIEDPE LEAIKARVREMEEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSIEEKMEADARSIYVGNV DYGATAEELEAHFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGR QIKVIPKRTNRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRSG

[0448] H0YJH9 - isoform 205 (SEQ ID NO: 6)

[0449] XTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGRQIKVIPKRTNRPGISTTDRGFPRARYRAR TTNYNSSRSRFYSGFNSRPRGRVYRSG

[0450] G3UWS5 - isoform 208 (SEQ ID NO: 7) (murine)

[0451] MEEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSLEEKMEADARSIYVGNVDYGATAEELEAHFHG CGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGRQIKVIPKRTNRPGISTTDRGFPR SRYRARTTNYNSSRSRFYSGFNSRPRGRIYRGRARATSWYSPY

[0452] B4DEH8 - isoform 208 (SEQ ID NO: 11) (DNA sequence) (murine) atggaggaagaggctgagaagctaaaggagctacaaaacgaggtagagaagcagatgaatatgagtccacccccaggcaatgctggcccagtg atcatgtctcttgaggagaagatggaggctgatgcccgctctatctacgttggcaatgtggactatggtgcaacagcagaagagctggaagcccattttc atggctgtggttcagtcaaccgtgttactatactctgtgacaaatttagtggccatcccaaagggtttgcatatatagagttctcggacaaagagtcagtga ggacgtccctggccttagatgagtccctgttcagaggaagacaaatcaaggtgattcccaaacgaaccaacagaccaggcatcagcacaacagac cggggtttcccgcgctcccgataccgtgcccggactaccaactacaacagctcccgatctcgattctacagtggttttaacagcaggccccggggtcg aatctacaggtcaggatag

[0453] Retained CCD (SEQ ID NO: 8)

[0454] MEEEAEKLKELQNEVEKQMNMSP

[0455] IDR (SEQ ID NO: 9)

[0456] MAAAAAAAAAAGAAGGRGSGPGRRRHLVPGAGGEAGEGAPGGAGDYGNGLESEELEPEELLL

[0457] EPEPEPEPEEEPPRPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGDGAIE

[0458] IDR (DNA sequence) (SEQ ID NO: 18) atgggggctgcgggcggtcggggctccgggccggggcggcggcgccatcttgtgcccggggccggtggggaggccgggga gggggccccggggggcgcaggggactacgggaacggcctggagtctgaggaactggagcctgaggagctgctgctggagc ccgagccggagcccgagcccgaagaggagccgccccggccccgcgcccccccgggagctccgggccctgggcctggttcg ggagcccccggcagccaagaggaggaggaggagccgggactgg

[0459] Lacking Ala-tract (SEQ ID NO: 13)

[0460] Atgggggctgcgggcggtcggggctccgggccggggcggcggcgccatcttgtgcccggggccggtggggaggccggggaggg ggccccggggggcgcaggggactacgggaacggcctggagtctgaggaactggagcctgaggagctgctgctggagcccgagcc ggagcccgagcccgaagaggagccgccccggccccgcgcccccccgggagctccgggccctgggcctggttcgggagcccccg gcagccaagaggaggaggaggagccgggactggtcgagggtgacccgggggacggcgccattgaggacccggagctggaagc tatcaaagctcgagtcagggagatggaggaagaagctgagaagctaaaggagctacagaacgaggtagagaagcagatgaatat gagtccacctccaggcaatgctggcccggtgatcatgtccattgaggagaagatggaggctgatgcccgttccatctatgttggcaatgt ggactatggtgcaacagcagaagagctggaagctcactttcatggctgtggttcagtcaaccgtgttaccatactgtgtgacaaatttagt ggccatcccaaagggtttgcgtatatagagttctcagacaaagagtcagtgaggacttccttggccttagatgagtccctatttagaggaa ggcaaatcaaggtgatcccaaaacgaaccaacagaccaggcatcagcacaacagaccggggttttccacgagcccgctaccgcg cccggaccaccaactacaacagctcggctagagcgacatcatggtattccccttactaa

[0461] Lacking Ala-tract (SEQ ID NO: 19)

[0462] MGAAGGRGSGPGRRRHLVPGAGGEAGEGAPGGAGDYGNGLESEELEPEELLLEPEPEPE

[0463] PEEEPPRPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGDGAIEDPELEAIKARVREM EEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSIEEKMEADARSIYVGNVDYGATAEELEA HFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKESVRTSLALDESLFRGRQIKVIPKRTNRP GISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSRPRGRVYRGRARATSWYSPY

[0464] Lacking Ala-tract+IDR (SEQ ID NO: 14)

[0465] Atggagggtgacccgggggacggcgccattgaggacccggagctggaagctatcaaagctcgagtcagggagatggaggaaga agctgagaagctaaaggagctacagaacgaggtagagaagcagatgaatatgagtccacctccaggcaatgctggcccggtgatc atgtccattgaggagaagatggaggctgatgcccgttccatctatgttggcaatgtggactatggtgcaacagcagaagagctggaag ctcactttcatggctgtggttcagtcaaccgtgttaccatactgtgtgacaaatttagtggccatcccaaagggtttgcgtatatagagttctc agacaaagagtcagtgaggacttccttggccttagatgagtccctatttagaggaaggcaaatcaaggtgatcccaaaacgaaccaa cagaccaggcatcagcacaacagaccggggttttccacgagcccgctaccgcgcccggaccaccaactacaacagctcggctaga gcgacatcatggtattccccttactaa

[0466] Lacking Ala-tract+IDR (SEQ ID NO: 20)

[0467] MVEGDPGDGAIEDPELEAIKARVREMEEEAEKLKELQNEVEKQMNMSPPPGNAGPVIMSIE EKMEADARSIYVGNVDYGATAEELEAHFHGCGSVNRVTILCDKFSGHPKGFAYIEFSDKES VRTSLALDESLFRGRQIKVIPKRTNRPGISTTDRGFPRARYRARTTNYNSSRSRFYSGFNSR P RG RVYRG R ARATS WYS P Y

[0468] Lacking Ala-tract+IDR+CCD (SEQ ID NO: 15) atgccaggcaatgctggcccggtgatcatgtccattgaggagaagatggaggctgatgcccgttccatctatgttggcaatgtgga ctatggtgcaacagcagaagagctggaagctcactttcatggctgtggttcagtcaaccgtgttaccatactgtgtgacaaatttagt ggccatcccaaagggtttgcgtatatagagttctcagacaaagagtcagtgaggacttccttggccttagatgagtccctatttaga ggaaggcaaatcaaggtgatcccaaaacgaaccaacagaccaggcatcagcacaacagaccggggttttccacgagcccg ctaccgcgcccggaccaccaactacaacagctcccgctctcgattctacagtggttttaacagcaggccccggggtcgcgtctac aggggccgggctagagcgacatcatggtattccccttactaa

[0469] Lacking Ala-tract+IDR+CCD (SEQ ID NO: 21)

[0470] MPPGNAGPVIMSIEEKMEADARSIYVGNVDYGATAEELEAHFHGCGSVNRVTILCDKFSGH

[0471] PKGFAYIEFSDKESVRTSLALDESLFRGRQIKVIPKRTNRPGISTTDRGFPRARYRARTTNYN SS RS RF YS GFNSRPRG RVYRG R ARATS WYS P Y qRNA to PABPN1 qRNA (G)3:

[0472] TATGTTGGCAATGTACGTACTGG (SEQ ID NO: 22) qRNA(G)2:

[0473] TGATCACCGGGCCAGCTATCAGG (SEQ ID NO: 23)

[0474] References

[0475] 1. Raz, V., et al. Nucleus, 2011. 2(3): p. 208-218.

[0476] 2. Harish, P., et al., Inhibition of myostatin improves muscle atrophy in oculopharyngeal muscular dystrophy (OPMD). J Cachexia Sarcopenia Muscle, 2019. 10(5): p. 1016-1026.

[0477] 3. Malerba, A., et al., PABPN1 gene therapy for oculopharyngeal muscular dystrophy. Nat

[0478] Commun, 2017. 8: p. 14848.

[0479] 4. Zhang, Y., Liu, L., Qiu, Q. etal. Alternative polyadenylation: methods, mechanism, function, and role in cancer. J Exp Clin Cancer Res 40, 51 (2021 ). https: / / doi.org / 10.1186 / s13046-021- 01852-7.

[0480] 5.Riaz M, Raz Y, van Putten M, Paniagua-Soriano G, Krom YD, Florea Bl, et al. (2016)

[0481] PABPN1 -Dependent mRNA Processing Induces Muscle Wasting. PLoS Genet 12(5): e1006031 . https: / / doi.org / 10.1371 / journal.pgen.1006031 .

[0482] 6. Chen, L., Dong, W., Zhou, M. et al. PABPN1 regulates mRNA alternative polyadenylation to inhibit bladder cancer progression. Cell Biosci 13, 45 (2023). https: / / doi.org / 10.1186 / s13578-023-00997-6.

[0483] 7. Malerba, A., Klein, P., Bachtarzi, H. et al. PABPN1 gene therapy for oculopharyngeal muscular dystrophy. Nat Commun 8, 14848 (2017). https: / / doi.org / 10.1038 / ncomms14848.

Claims

1. Claims1. A poly(A) binding protein nuclear 1 (PABPN1 ) polypeptide or a nucleic acid encoding the PABPN1 polypeptide for use in a method for treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

2. The PABPN1 polypeptide or nucleic acid for use according to claim 1 , wherein the PABPN1 polypeptide comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

3. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide does not comprise at least one of: (i) the N-terminal alanine tract corresponding to amino acids 2 to 11 of SEQ ID NO:2, (ii) the intrinsically disordered region corresponding to amino acids 1 to 115 of SEQ ID NO:2, (iii) the coiled coil domain corresponding to amino acids 115 to 151 of SEQ ID NO:2.

4. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide does not comprise SEQ ID NO:3 and does not comprise SEQ ID NO:9.

5. The PABPN1 polypeptide or nucleic acid for use according to claim 4, wherein the PABPN1 polypeptide does not comprise SEQ ID NO:4, does not comprise SEQ ID NO:3 and does not comprise SEQ ID NO:9.

6. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the disease or condition is further associated with an increase in alternative polyadenylation (APA) and / or ALE (alternative last exon) usage.

7. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the disease or condition is further associated with a shift to proximal pA site usage.

8. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the disease or condition is cancer, muscle pathology cardiac disease, autoimmune disease, and / or ageing .

9. The PABPN1 polypeptide or nucleic acid for use according to claim 8, wherein the cancer is bladder cancer, non-bladder cancer, , or small cell lung cancer; or the muscle pathology is oculopharyngeal muscular dystrophy (OPMD), facioscapulohumeral muscular dystrophy (FSHD), or sarcopenia.

10. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the reduction in the level of PABPN1 is at least 30% compared to the level of PABPN1 polypeptide in a healthy subject.11 . The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:1 or a derivative or a functional variant (e.g. conservative amino acid variant) thereof.

12. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide restores cellular defects, optionally wherein the PABPN1 polypeptide restores 3’-UTR length or ALE usage.

13. The PABPN1 polypeptide or nucleic acid for use according to claim 12, wherein the cellular defects are caused by APA, and / or ALE usage.

14. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide restores PABPN1 level and / or activity, optionally wherein the PABPN1 polypeptide restores mitochondrial activity caused by PABPN1 dysfunction and / or increases differentiation in muscle tissue.

15. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the method comprises administering or expressing the PABPN1 polypeptide or the nucleic acid encoding the PABPN1 polypeptide in the subject in need thereof.

16. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide or nucleic acid restores the level of PABPN1 to the level of functional PABPN1 polypeptide as observed in a healthy subject.

17. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the method further comprises administering or expressing an inhibitory RNA to suppress or inhibit the endogenous aggregating mutant form of PABPN1 .

18. The PABPN1 polypeptide or nucleic acid for use according claim 17, wherein the inhibitory RNA suppresses or inhibits both the endogenous aggregating mutant form of PABPN1 and the wild-type form of PABPN1 .

19. The PABPN1 polypeptide or nucleic acid for use according to claims 17 or 18, wherein the inhibitory RNA is a small interfering RNA (siRNA) or an antisense oligonucleotide.

20. The PABPN1 polypeptide or nucleic acid for use according to any one of claims 17 to 19, wherein the inhibitory RNA is expressed in the subject by providing a nucleic acid encoding the inhibitory RNA to the subject.

21. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the method further comprises administering or expressing a CRISPR-Cas system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1 .

22. The PABPN1 polypeptide or nucleic acid for use according to claim 21 , wherein the CRISPR-Cas9 system comprises a gRNA targeting exon 1 of PABPN1 .

23. The PABPN1 polypeptide or nucleic acid for use according to claim 22, wherein the gRNA is encoded by SEQ ID NO: 22.

24. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the PABPN1 polypeptide is expressed in the subject by providing a nucleic acid encoding the PABPN1 polypeptide to the subject.

25. The PABPN1 polypeptide or nucleic acid for use according to any one of the previous claims, wherein the nucleic acid encoding the PABPN1 polypeptide and / or the nucleic acid encoding the inhibitory RNA are comprised in a vector, optionally a viral vector.

26. The PABPN1 polypeptide or nucleic acid for use according to claim 22, wherein the nucleic acid encoding the PABPN1 polypeptide and the nucleic acid encoding the inhibitory RNA are comprised in the same or different vectors.

27. The PABPN1 polypeptide or nucleic acid for use according to claims 22 or 23, wherein the viral vector is an adeno-associated viral vector (AAV) or an adenoviral vector particle (Ad VP).

28. The PABPN1 polypeptide or nucleic acid for use according to any previous claim, wherein the nucleic acid encoding the PABPN1 polypeptide is inserted into the genome of the subject in need thereof using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system.

29. A method of treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 , wherein the method comprises administering a poly(A) binding protein nuclear 1 (PABPN1 ) polypeptide or a nucleic acid encoding the PABPN1 polypeptide to a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3, an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

30. Use of a poly(A) binding protein nuclear 1 (PABPN1) polypeptide or a nucleic acid encoding the PABPN1 polypeptide for the manufacture of a medicament treating or preventing a disease or condition associated with a reduction in the level of PABPN1 and / or impairment in the function of PABPN1 in a subject in need thereof, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3, an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

31. A pharmaceutical composition comprising a PABPN1 polypeptide or a nucleic acid encoding the PABPN1 polypeptide or a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide and a pharmaceutically acceptable diluent or carrier, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3, an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

32. The pharmaceutical composition according to claim 31 , wherein the PABPN1 polypeptide comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1.

33. A method of identifying a subject that will benefit from treatment with a poly(A) binding protein nuclear 1 (PABPN1) polypeptide or a nucleic acid encoding the PABPN1 polypeptide; wherein the method comprises:- testing the subject for PABPN1 level and / or activity to establish a test value;- comparing the test value to a reference value, wherein the reference value is PABPN1 level and / or activity of a healthy subject;- where the test value is less than the reference value, recommending treatment with a PABPN1 polypeptide, a nucleic acid sequence encoding a PABPN1 polypeptide, a vector comprising a nucleic acid sequence encoding a PABPN1 polypeptide and / or a pharmaceutical composition of claims 28 or 29, wherein the PABPN1 polypeptide does not comprise at least one of: an N-terminal alanine tract according to SEQ ID NO:3; an intrinsically disordered region according to SEQ ID NO:9; a coiled coil domain according to SEQ ID NO:4.

34. The method according to claim 33, wherein the method further comprises a step of administering or expressing the PABPN1 polypeptide or the nucleic acid encoding the PABPN1 polypeptide in the subject in need thereof.

35. The method according to claim 34, wherein the method further comprises a step of administering or expressing an inhibitory RNA to suppress the endogenous aggregating mutant form of PABPN1 .

36. The method according to claim 35, wherein inhibitory RNA is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO).

37. The method according to any one of claims 33-36, wherein the method further comprises administering or expressing a CRISPR-Cas system to suppress or inhibit the endogenous aggregating mutant form of PABPN1 and / or the wild-type form of PABPN1 .

38. The PABPN1 polypeptide or nucleic acid for use according to claim 37, wherein the CRISPR-Cas system comprises a gRNA targeting exon 1 of PABPN1 .

39. The PABPN1 polypeptide or nucleic acid for use according to claim 38, wherein the gRNA is encoded by SEQ ID NO: 22.

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