Inducible gene expression system

Abstract

The present disclosure relates to the fields of molecular biology and nucleic acid technology, and provides polynucleotides encoding molecules of interest. The present disclosure also relates to therapy and prophylaxis of disease.

Description

[0001] Inducible Gene Expression System

[0002] Technical Field

[0003] The present disclosure relates to the fields of molecular biology and nucleic acid technology. The present disclosure also relates to therapy and prophylaxis of disease.

[0004] Background

[0005] Gene therapy as a modality aims at restoring disease-related phenotypes by using both viral and non- viral delivery systems. Significant efforts have been focusing on optimizing this therapy to be more efficient, non-immunogenic and less toxic while enabling a long-term expression of the gene of interest. One of the key challenges to achieve this goal is to regulate the expression of the transgene to meet the required level and to be expressed upon demand. For certain targets, where constitutive and unlimited expression leads to adverse events, regulated gene expression is inevitable to render gene therapy a viable option as a treatment modality.

[0006] Inducible gene expression systems allow for reversibility and flexibility and enable therapeutics’ production upon demand, thereby circumventing overdose-associated side effects. This regulation can be achieved using small molecules, with which transgene expression can be induced (generating ON- switches) or even repressed (OFF-switch). Among the existing inducible transcriptional gene regulatory systems, the tetracycline (Tet)-regulable system is the most widely exploited tool and can be used as an ON- or an OFF-switch. Tet-inducible systems, however, are derived from bacteria and require the expression of the regulator protein, e.g. the ON-switch components include the tetracycline-controlled transactivator (tTA), which consists of the bacterial tetR with a C-terminal domain of VP16 (virion protein 16), derived from the herpes simplex virus. The required co-expression of these components further increase the limitations in the size of the transgene for AAV packaging. Despite significant improvements over the years, this system is derived from bacteria, which can cause both silencing and potential immunogenicity effects when introduced into human tissue, which has hampered further translation into the clinics [1]. Riboswitches, RNA elements that are able to control gene expression in response to ligand binding, have a small genomic footprint and do not depend on other proteins for activity, which renders these types of regulated gene expression an attractive alternative to protein-based expression control systems [2]. However, RNA-based switches suffer from a poor dynamic range as well the high basal activity [3].

[0007] Previously, the SMN2 sequence has been engineered as a reporter gene-based system suitable for high throughput screening to identify small molecules that regulate SMN2 exon 7 splicing [6]. However, these systems are not suitable for gene therapy applications, as they have a very long switch sequence (>5 kbp) and are not designed to express a therapeutic protein but a reporter for pre-clinical assessments.

[0008] Summary

[0009] In a first aspect, the present disclosure provides a polynucleotide, comprising in 5’ to 3’ order:

[0010] (i) a nucleotide sequence comprising a 5’ splice site;

[0011] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13; and (iii) a nucleotide sequence comprising a 3’ splice site; wherein the polynucleotide comprises a nucleotide sequence encoding a molecule of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a molecule of interest; wherein the nucleotide sequence comprising a 5’ splice site is not derived from the 5’ splice site at human SMN2 exon 6-intron 6; and wherein nucleotide sequence comprising a 3’ splice site is not derived from the 3’ splice site at human SMN2 intron 7-exon 8.

[0012] In some embodiments, the polynucleotide does not comprise a nucleotide sequence having >80% sequence identity to SEQ ID NO:15.

[0013] In some embodiments, the human SMN2 exon 7-derived nucleotide sequence has at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 .

[0014] In some embodiments, the nucleotide sequence comprising a 5’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence formed by: the final 15 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 15 nucleotides of the intron immediately 3’ of the exon.

[0015] In some embodiments, the nucleotide sequence comprising a 3’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence formed by: the final 15 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 15 nucleotides of the 3’ exon.

[0016] In some embodiments, the gene region known to undergo alternative splicing via exon skipping is a gene region for which skipping of the relevant exon is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam.

[0017] In some embodiments, the gene region known to undergo alternative splicing via exon skipping is selected from: the region spanning exons 7 to 9 of human STRN3; the region spanning exons 5 to 6 of human RPS10; the region spanning exons 2 to 4 of human SLC25A17; the region spanning exons 8 to 10 of human FOXM1 ; the region spanning exons 6 to 8 of human APLP2; and the region spanning exons 20 to 22 of human MADD. In some embodiments, the gene region known to undergo alternative splicing via exon skipping is selected from: the region spanning exons 7 to 9 of human STRN3; the region spanning exons 5 to 6 of human RPS10; and the region spanning exons 2 to 4 of human SLC25A17.

[0018] In some embodiments, the nucleotide sequence comprising a 5’ splice site is derived from the 5’ splice site at human STRN3 exon 7-intron 7. In some embodiments, the nucleotide sequence comprising a 5’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:47, 46, 45, 44, 43, 42, 41 or 40. In some embodiments, the nucleotide sequence comprising a 3’ splice site is derived from the 3’ splice site at human STRN3 intron 8-exon 9. In some embodiments, the nucleotide sequence comprising a 3’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 94, 93, 92, 91 , 235, 234, 233, 238, 237, 236, 241 , 240, 239, 244, 243 or 242.

[0019] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:30, 29, 31 , 32, 33, 34, 35, 36, 37, 38 or 39 immediately 5’ to the human SMN2 exon 7-derived nucleotide sequence.

[0020] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:52, 51 , 53, 54, 55, 56, 57, 58, 59, 60 or 232 immediately 3’ to the human SMN2 exon 7-derived nucleotide sequence.

[0021] In some embodiments, the nucleotide sequence comprising a 5’ splice site is derived from the 5’ splice site at human RPS10 exon 5-intron 5. In some embodiments, the nucleotide sequence comprising a 5’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:158, 157, 156 or 155.

[0022] In some embodiments, the nucleotide sequence comprising a 3’ splice site is derived from the 3’ splice site at human RPS10 intron 5-exon 6. In some embodiments, the nucleotide sequence comprising a 3’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:90, 165, 164 or 163.

[0023] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:153 or 154 immediately 5’ to the human SMN2 exon 7-derived nucleotide sequence.

[0024] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:161 or 162 immediately 3’ to the human SMN2 exon 7-derived nucleotide sequence.

[0025] In some embodiments, the nucleotide sequence comprising a 5’ splice site is derived from the 5’ splice site at human SLC25A17 exon 2-intron 2. In some embodiments, the nucleotide sequence comprising a 5’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:199, 198, 197, 196 or 200.

[0026] In some embodiments, the nucleotide sequence comprising a 3’ splice site is derived from the 3’ splice site at human SLC25A17 intron 3-exon 4. In some embodiments, the nucleotide sequence comprising a 3’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:208, 207, 206 or 205. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:194 or 195 immediately 5’ to the human SMN2 exon 7-derived nucleotide sequence.

[0027] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:203 or 204 immediately 3’ to the human SMN2 exon 7-derived nucleotide sequence.

[0028] In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the human SMN2 exon 7-derived nucleotide sequence.

[0029] The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0030] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:28, 27 or 26;

[0031] (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 97, 96 or 95 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 at its 3’ end;

[0032] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NO:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 ;

[0033] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 112, 111 or 110 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:116, 115, 114, 113, 120, 119, 118, 117, 247, 246 or 245 at its 3’ end;

[0034] (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’ or ‘GG’, or (b) having at least 80% sequence identity to SEQ ID NO:63, 62 or 61 , or (c) encoding a molecule of interest, and comprising ‘TG’ or ‘GG’ at positions 1 and 2; and

[0035] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a molecule of interest.

[0036] In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NQ:30, 29, 31 , 32, 33, 34, 35, 36, 37, 38 or 39.

[0037] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:52, 51 , 53, 54, 55, 56, 57, 58, 59, 60 or 232.

[0038] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:147, 121 , 124, 128, 130, 132, 133, 122, 123, 125, 126, 127, 129, 131 , 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 148 or 149. In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:147, 121 , 124, 128, 130, 132 or 133. The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0039] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:152 or 151 ;

[0040] (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:169, 168, 167 or 166 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 at its 3’ end;

[0041] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0042] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:180, 179, 178, 177, 98, 183, 182 or 181 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:116, 186, 185 or 184 at its 3’ end;

[0043] (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’, or (b) encoding a molecule of interest, and comprising ‘TG’ at positions 1 and 2; and

[0044] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a molecule of interest.

[0045] In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NO:153 or 154.

[0046] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:161 or 162.

[0047] In some embodiments, the polynucleotide comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:187, 188, 189 or 190.

[0048] The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0049] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:193 or 192;

[0050] (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:212, 211 , 210 or 209 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218, 217 at its 3’ end;

[0051] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0052] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 221 , 220, 219, 183, 223, or 222 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:102, 226, 225 or 224 at its 3’ end;

[0053] (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’, or (b) encoding a molecule of interest, and comprising ‘TG’ at positions 1 and 2; and

[0054] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a molecule of interest. In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NO:194 or 195.

[0055] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:203 or 204.

[0056] In some embodiments, the polynucleotide comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:227, 228, 229 or 230.

[0057] The present disclosure also provides a polynucleotide, comprising in 5’ to 3’ order:

[0058] (i) a nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human STRN3 exon 7-intron 7;

[0059] (ii) a human STRN3 exon 8-derived nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NQ:50 or 49; and

[0060] (iii) a nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human STRN3 intron 8-exon 9; wherein the polynucleotide comprises a nucleotide sequence encoding a molecule of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a molecule of interest.

[0061] In some embodiments, the nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human STRN3 exon 7-intron 7 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:47, 46, 45, 44, 43, 42, 41 or 40.

[0062] In some embodiments, the nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human STRN3 intron 8-exon 9 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:67, 66, 65, 64, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69 or 68.

[0063] In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the human STRN3 exon 8-derived nucleotide sequence.

[0064] The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0065] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:27, 28 or 26;

[0066] (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 97, 96 or 95 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 at its 3’ end;

[0067] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:50 or 49;

[0068] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 112, 111 or 110 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 116, 115, 114, 113, 120, 119, 118, 117, 247, 246 or 245 at its 3’ end;

[0069] (v) a fifth nucleotide sequence: (a) having at least 80% sequence identity to SEQ ID NO:62, 63 or 61 , or

[0070] (b) consisting of the dinucleotide ‘TG’ or ‘GG’, or (c) encoding a molecule of interest, and comprising ‘TG’ or ‘GG’ at positions 1 and 2; and

[0071] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a molecule of interest.

[0072] In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NO:29, 30, 31 , 32, 33, 34, 35, 36, 37, 38 or 39.

[0073] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or 232.

[0074] In some embodiments, the polynucleotide comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:150.

[0075] The present disclosure also provides a polynucleotide, comprising in 5’ to 3’ order:

[0076] (i) a nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human RPS10 exon 5-intron 5;

[0077] (ii) a human RPS10 exon 5.5-derived nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NQ:160 or 159; and

[0078] (iii) a nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human RPS10 intron 5-exon 6; wherein the polynucleotide comprises a nucleotide sequence encoding a molecule of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a molecule of interest.

[0079] In some embodiments, the nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human RPS10 exon 5-intron 5 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:158, 157, 156 or 155.

[0080] In some embodiments, the nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human RPS10 intron 5-exon 6 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:90, 165, 164 or 163.

[0081] In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the human RPS10 exon 5.5-derived nucleotide sequence.

[0082] The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0083] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:152 or 151 ; (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:169, 168, 167 or 166 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 at its 3’ end;

[0084] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:160 or 159;

[0085] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:180, 179, 178, 177, 98, 183, 182 or 181 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:116, 186, 185 or 184 at its 3’ end;

[0086] (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’, or (c) encoding a molecule of interest, and comprising ‘TG’ at positions 1 and 2; and

[0087] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a molecule of interest.

[0088] In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NO:153 or 154.

[0089] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:161 or 162.

[0090] In some embodiments, the polynucleotide comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:191 .

[0091] The present disclosure also provides a polynucleotide, comprising in 5’ to 3’ order:

[0092] (i) a nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human SLC25A17 exon 2-intron 2;

[0093] (ii) a human SLC25A17 exon 3-derived nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NQ:202 or 201 ; and

[0094] (iii) a nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human SLC25A 17 intron 3-exon 4; wherein the polynucleotide comprises a nucleotide sequence encoding a molecule of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a molecule of interest.

[0095] In some embodiments, the nucleotide sequence comprising a 5’ splice site derived from the 5’ splice site at human SLC25A17 exon 2-intron 2 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:199, 198 197 or 196.

[0096] In some embodiments, the nucleotide sequence comprising a 3’ splice site derived from the 3’ splice site at human SLC25A 17 intron 3-exon 4 comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:208, 107, 106 or 205. In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the human SLC25A17 exon 3-derived nucleotide sequence.

[0097] The present disclosure also provides a polynucleotide comprising in 5’ to 3’ order:

[0098] (i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:193 or 192;

[0099] (ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:212, 211 , 210 or 209 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218 or 217 at its 3’ end;

[0100] (iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:202 or 201 ;

[0101] (iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 221 , 220, 219, 183, 223 or 222 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:102, 226, 225 or 224 at its 3’ end;

[0102] (v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’, or (c) encoding a molecule of interest, and comprising ‘TG’ at positions 1 and 2; and

[0103] (vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a), a sixth nucleotide sequence encoding a molecule of interest.

[0104] In some embodiments, the second nucleotide sequence has at least 80% sequence identity to SEQ ID NO:194 or 195.

[0105] In some embodiments, the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NQ:203 or 204.

[0106] In some embodiments, the polynucleotide comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:231 .

[0107] In some embodiments, when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.

[0108] In some embodiments, the polynucleotide further comprises a promoter sequence 5’ to a start codon. In some embodiments, the promoter sequence is a CMV promoter, CAG promoter, hEF1 a promoter, hUbiC promoter, RSV promoter, TK promoter, PGK promoter or CAG minimal promoter. In some embodiments, the promoter sequence is a CMV promoter, CAG promoter, hEF1a promoter, hUbiC promoter, RSV promoter or CAG minimal promoter. In some embodiments, the promoter sequence is a hEF1a promoter.

[0109] In some embodiments, the polynucleotide further comprises a polyadenylation sequence 3’ to the nucleotide sequence encoding a molecule of interest.

[0110] In some embodiments, the polynucleotide comprises an inverted terminal repeat (ITR) sequence at its 5’ end, and an ITR sequence at its 3’ end. The present disclosure also provides a vector comprising a polynucleotide according to the present disclosure.

[0111] In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0112] The present disclosure also provides a pharmaceutical composition comprising a polynucleotide or vector according to the present disclosure, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.

[0113] The present disclosure also provides a cell comprising a polynucleotide or vector according to the present disclosure.

[0114] In some embodiments, the cell further comprises a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

[0115] The present disclosure also provides a method for modifying a cell to express a molecule of interest, comprising:

[0116] (i) introducing a polynucleotide or vector according to the present disclosure into a cell; and

[0117] (ii) subsequently contacting the cell with a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

[0118] The present disclosure also provides a method for expressing a molecule of interest in a cell, comprising contacting a cell according to the present disclosure with a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

[0119] The present disclosure also provides a polynucleotide, a vector, or a pharmaceutical composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.

[0120] The present disclosure also provides a polynucleotide, a vector, or a pharmaceutical composition according to the present disclosure, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest.

[0121] The present disclosure also provides the use of a polynucleotide, a vector, or a pharmaceutical composition according to the present disclosure in the manufacture of a medicament for treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest. The present disclosure also provides a method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest, comprising administering to a subject a polynucleotide, a vector, or a pharmaceutical composition according to the present disclosure.

[0122] In some embodiments, treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

[0123] In some embodiments, the disease or condition is a disease or condition characterised by deficiency of the molecule of interest.

[0124] The present disclosure also provides a kit, comprising:

[0125] (i) a polynucleotide, a vector, or a pharmaceutical composition according to the present disclosure; and

[0126] (ii) a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

[0127] Description

[0128] The present disclosure relates to inducible transgene expression systems using small molecule splicing modifiers (SMSM), based on the described mechanism of action of the Survival of Motor Neuron 2 (human SMN2) splicing modifier risdiplam (and variants thereof), extending its applicability to other genes with alternative splice variants. The present disclosure provides engineered hybrid switches that integrate SMN2 sequences with those from other genes.

[0129] The SMSM binding sites for human SMN2 have previously been described to be two distinct sites within human SMN2 exon 7 pre-mRNA: the ESE2 region and the 5’ splice donor site (5’ss) [4]. Interaction of the small molecules with the mRNA-protein complex is thought to be critical for the high selectivity of the compound. This study suggested that both ESE2 and 5’ss regions are required for full activity of the SMSM compounds in a dose-dependent manner [4], Structural analysis using a compound from the same chemical class as risdiplam (SMN-C5) has demonstrated that the drug selectively promotes the recognition of the weak 5’ splice site of human SMN2 exon 7 by U1 snRNP by stabilizing an unpaired adenine at the exon-intron junction in the RNA helix [5].

[0130] The present disclosure provides novel constructs providing for the SMSM-inducible regulation of transgene expression, which are suitable to be employed for chemically-inducible regulation of the expression of gene therapies. In particular, the present disclosure provides hybrid switches comprising sequences derived from different genes.

[0131] In particular, the present disclosure provides constructs: (i) having a size permitting their application for the SMSM-inducible expression of molecules of interest (e.g. polypeptides) to be delivered as gene therapies, (ii) minimising undesirable expression of the molecules in the absence of the SMSM, and (iii) which are highly responsive to the SMSM, thus providing for strong induction of expression of the polypeptide in the presence of the SMSM, and (iv) that minimise the number of / completely remove extraneous amino acids at the N-terminus of the expressed polypeptide (in embodiments wherein the molecule of interest is a polypeptide of interest).

[0132] Polynucleotides

[0133] Aspects and embodiments of the present disclosure relate to polynucleotides. The polynucleotides of the present disclosure preferably provide for inducible regulation of expression of molecules of interest (e.g. polypeptides of interest) encoded by the polynucleotides, in the presence of a splicing modifier (e.g. as described herein).

[0134] A 'polynucleotide' refers to a polymer chain of a plurality of nucleotide monomers linked by bonds between the monomers, typically phosphodiester bonds (e.g. in the case of polynucleotides formed by naturally-occurring nucleotide monomers). Polynucleotides include oligonucleotides, which generally comprise <50 nucleotides. A polynucleotide may be single-stranded, or may be double-stranded ( / .e. may comprise a duplex formed by hydrogen-bonding between complementary nucleotides). Polynucleotides according to the present disclosure may comprise or consist of: single-stranded DNA, double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-stranded RNA, doublestranded RNA, RNA that is mixture of single- and double-stranded regions, single-stranded molecules comprising DNA and RNA, double-stranded molecules comprising DNA and RNA, and molecules comprising DNA and RNA having a mixture of single- and double-stranded regions.

[0135] In some embodiments, a polynucleotide comprises or consists of DNA. In some embodiments, a polynucleotide is a polydeoxyribonucleotide. In some embodiments, a polynucleotide comprises or consists of RNA. In some embodiments, a polynucleotide is a polyribonucleotide.

[0136] In aspects and embodiments wherein the polynucleotide of the present disclosure is defined by reference to a given nucleotide sequence, and wherein the given nucleotide sequence comprises or consists of RNA and / or is a polyribonucleotide, it will be appreciated that instances of T for thymidine in such sequences are replaced with ‘U ’, for uracil.

[0137] The present disclosure also contemplates polynucleotides comprising modified nucleotides, e.g. in which the phosphonate and / or ribose and / or base of a deoxyribonucleotide or ribonucleotide is / are chemically modified. Nucleotide modifications contemplated in accordance with the present disclosure include those described in Hu et al., Sig. Transduc. Tar. Ther. (2020) 5(101), which is hereby incorporated by reference in its entirety.

[0138] Phosphonate modifications may be selected from phosphorothioate (e.g. Rp isomer, Sp isomer), phosphorodithioate, methylphosphonate, methoxypropylphosphonate, 5'-(E)-vinylphosphonate, 5'- methylphosphonate, (S)-5'-C-methyl with phosphate, 5’-phosphorothioate, and peptide nucleic acid modifications. Ribose modifications may be selected from 2'-O-methyl, 2'-O-methoxyethyl, 2’-fluoro, 2’- deoxy-2’-fluoro, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-ally I, 2'-deoxy, 2'-hydroxyl, 2'-arabino-fluoro, 2’-0-benzyl, 2’-O-methyl-4-pyridine, locked nucleic acid, (S)-cEt-BNA, tricyclo-DNA, PMO, unlocked nucleic acid, hexitol nucleic acid and glycol nucleic acid modifications. Base modifications may be selected from pseudouridine, 2'-thiouridine, N6'-methyladenosine, 5’-methylcytidine, 5’-fluoro-2’- deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-phenylpyrrolo-cytosine, 2',4'-difluorotoluyl ribonucleoside and 5'-nitroindole modifications.

[0139] In some embodiments, a modified nucleotide may be selected from 2'-O-methyluridine-3'-phosphate, 2'- O-methyladenosine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphorothioate, 2'-O-methyladenosine-3'-phosphorothioate, 2'-O- methylguanosine-3'-phosphorothioate, 2'-O-methylcytidine-3'-phosphorothioate, 2'-fluorouridine-3'- phosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-fluorocytidine-3'- phosphate, 2'-fluorocytidine-3'-phosphorothioate, 2'-fluoroguanosine-3'-phosphorothioate, 2'- fluoroadenosine-3'-phosphorothioate, and 2'-fluorouridine-3'-phosphorothioate.

[0140] Nucleotide sequences of the polynucleotides of the present disclosure

[0141] Polynucleotides of the present disclosure are defined herein by reference to constituent nucleotide sequences of the polynucleotides.

[0142] It will be appreciated that the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided as subsequences of the complete sequence of the polynucleotide.

[0143] In some aspects and embodiments, the constituent nucleotide sequences of polynucleotides according to the present disclosure are provided in a particular order in the sequence of the polynucleotide, e.g. from 5’ to 3’. By way of illustration, in embodiments herein where a polynucleotide is described herein as comprising in 5’ to 3’ order e.g. a nucleotide sequence forming a 5’ splice site, a human SMN2 exon 7- derived nucleotide sequence and a nucleotide sequence forming a 3’ splice site, the nucleotide sequence forming a 5’ splice site is provided 5’ to ( / .e. upstream of) the human SMN2 exon 7-derived nucleotide sequence, in the context of the sequence of the polynucleotide. Similarly, the human SMN2 exon 7- derived nucleotide sequence is 5’ to the nucleotide sequence forming a 3’ splice site. By way of further illustration, in embodiments herein where a polynucleotide is described herein as comprising e.g. a first nucleotide sequence, a second nucleotide sequence and a third nucleotide sequence, the first nucleotide sequence is provided 5’ to ( / .e. upstream of) the second nucleotide sequence, in the context of the sequence of the polynucleotide. Similarly, the second nucleotide sequence is 5’ to the third nucleotide sequence, etc.

[0144] The constituent nucleotide sequences of the polynucleotides of the present disclosure are nonoverlapping. In some embodiments, constituent nucleotide sequences of the polynucleotides are provided in tandem in the context of the complete sequence of the polynucleotide. In some embodiments, constituent nucleotide sequences of the polynucleotide are immediately adjacent to one another ( / .e. the 3’ nucleotide of a given nucleotide sequence is followed immediately by the 5’ nucleotide of another given nucleotide sequence, in the context of the complete sequence of the polynucleotide). By way of illustration, in the polynucleotide of SEQ ID NO:147, positions 1 to 42 form a first nucleotide sequence according to the present disclosure, positions 43 to 367 form a second nucleotide sequence according to the present disclosure, positions 368 to 419 form a third nucleotide sequence according to the present disclosure, positions 420 to 744 form a fourth nucleotide sequence according to the present disclosure, and positions 745 and 746 form a fifth nucleotide sequence according to the present disclosure.

[0145] Nucleotide sequence comprising a 5’ splice site

[0146] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence comprising a 5’ splice site.

[0147] RNA splicing and alternative splicing are described e.g. in Marasco and Kornblihtt, Nat. Rev. Mol. Cell Biol. (2023) 24:242-254, Rogalska et al., Nat. Rev. Genet. (2023) 24:251-269, and Wilkinson et al., Ann. Rev. Biochem. (2020) 89:359-388, all of which are hereby incorporated by reference in their entirety.

[0148] A 5' splice site (5’ SS, sometimes also referred to as a donor site) refers to a nucleotide sequence formed by nucleotides at the junction of an exon and the intron immediately downstream of ( / .e. 3’ to) the exon. That is, a 5’ splice site comprises the final ( / .e. 3’) nucleotides of an exon, and the first ( / .e. 5’) nucleotides of the intron immediately downstream of the exon. By way of illustration, SEQ ID NO:19 is a nucleotide sequence comprising a 5’ splice site, in which positions 1 to 5 are derived from human SMN2 exon 6, and positions 6 to 10 are derived from human SMN2 intron 6.

[0149] It will be appreciated that in aspects and embodiments of the present disclosure wherein a polynucleotide of the disclosure is or comprises DNA (e.g. is a DNA molecule), ‘a nucleotide sequence comprising a 5’ splice site’ refers to a nucleotide sequence that when provided as the equivalent ribonucleotide sequence comprises a 5’ splice site. That is, in such DNA molecules, a nucleotide sequence comprising a 5’ splice site refers to a nucleotide sequence that when transcribed to RNA comprises a 5’ splice site. By way of illustration, a DNA polynucleotide comprising the nucleotide sequence of SEQ ID NO:19 comprises a 5’ splice site when provided as the corresponding polyribonucleotide ( / .e. comprising uracil nucleotides in place of thymine nucleotides).

[0150] A nucleotide sequence comprising a 5’ splice site according to the present disclosure refers to a nucleotide sequence that when provided as polyribonucleotide comprises a nucleotide sequence forming a site recognised by the U1 snRNA component of the spliceosome. In some embodiments, a 5’ splice site according to the present disclosure comprises the dinucleotide ‘GT’ - or when the polynucleotide comprising the 5’ splice site is provided as a polyribonucleotide ‘GU’ - e.g. at the first and second positions of the intronic sequence of the 5’ splice site. In some embodiments, a 5’ splice site according to the present disclosure comprises a nucleotide sequence conforming to the consensus shown in SEQ ID NO:248.

[0151] In aspects and embodiments described herein, a nucleotide sequence comprising a 5’ splice site according to the present disclosure may be derived from the nucleotide sequence of a reference 5’ splice site. A reference splice site may comprise or consist of the final ( / .e. 3’) nucleotides of an exon of a given gene, and the first ( / .e. 5’) nucleotides of the intron immediately downstream of the exon. In some embodiments, the final nucleotides of the exon may refer to the final 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides of the exon. In some embodiments, the first nucleotides of the intron immediately downstream of the exon may refer to the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides of the intron.

[0152] In some embodiments, the reference 5’ splice site is the 5’ splice site of a gene region known to undergo alternative splicing via exon skipping. Exon skipping is described e.g. in Marasco and Kornblihtt, Nat. Rev. Mol. Cell Biol. (2023) 24:242-254, Rogalska et al., Nat. Rev. Genet. (2023) 24:251-269, and Wilkinson et al., Ann. Rev. Biochem. (2020) 89:359-388, all of which are incorporated by reference hereinabove. Such gene regions may be referred to as a gene regions comprising a cassette exon. A cassette exon is an exon which is either included or excluded ( / .e. skipped) in the mature mRNA following splicing of pre-mRNA transcribed from the gene region.

[0153] In some embodiments, the reference 5’ splice site is the 5’ splice site of a gene region known to undergo alternative splicing via exon skipping, in which skipping of the relevant exon is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0154] Gene regions known to undergo alternative splicing via exon skipping, and in which skipping of the relevant exon is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam, are described e.g. in Singh et al., Neurosci Insights. (2020) 15:2633105520973985, which is hereby incorporated by reference in its entirety. Gene regions known to undergo alternative splicing via exon skipping, and in which skipping of the relevant exon is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam, include: the region spanning exons 6 to 8 of human SMN2 (e.g. positions 24713 to 32052 of NCBI Reference Sequence:NG_008728.1); the region spanning exons 7 to 9 of human STRN3 (e.g. positions 91076 to 97179 NCBI Reference Sequence:NC_000014.9); the region spanning exons 5 to 6 of human RPS10 (e.g. positions 12676 to 13646 of NCBI Reference Sequence:NG_023200.1); the region spanning exons 2 to 4 of human SLC25A17 (e.g. as determined with reference to NCBI Reference Sequence:NC_000022.11); the region spanning exons 8 to 10 of human F0XM1 (e.g. as determined with reference to NCBI Reference Sequence:NC_000012.12); the region spanning exons 6 to 8 of human APLP2 (e.g. positions 52412 to 56937 of NCBI Reference Sequence:NG_029770.1); and the region spanning exons 20 to 22 of human MADD (e.g. positions 26424 to 29797 of NCBI Reference Sequence:NG_029462.2).

[0155] In some embodiments, the reference 5’ splice site is the 5’ splice site at human STRN3 exon 7-intron 7. In some embodiments, the reference 5’ splice site is the 5’ splice site at human RPS10 exon 5-intron 5. In some embodiments, the reference 5’ splice site is the 5’ splice site at human SLC25A17 exon 2-intron 2. In some embodiments, the reference 5’ splice site is the 5’ splice site at human F0XM1 exon 8-intron 8. In some embodiments, the reference 5’ splice site is the 5’ splice site at human APLP2 exon 6-intron 6. In some embodiments, the reference 5’ splice site is the 5’ splice site at human MADD exon 20-intron 20. In some embodiments, the reference 5’ splice site is not the 5’ splice site at human SMN2 exon 6-intron 6.

[0156] In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 2 nucleotides of a given exon and the first 2 nucleotides of the intron immediately downstream of ( / .e. 3’ to) the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 5 nucleotides of a given exon and the first 5 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 10 nucleotides of a given exon and the first 10 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 15 nucleotides of a given exon and the first 15 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 20 nucleotides of a given exon and the first 20 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 25 nucleotides of a given exon and the first 25 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 30 nucleotides of a given exon and the first 30 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 40 nucleotides of a given exon and the first 40 nucleotides of the intron immediately downstream of the exon. In some embodiments, a 5’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 50 nucleotides of a given exon and the first 50 nucleotides of the intron immediately downstream of the exon.

[0157] As explained hereinbelow, a nucleotide sequence which is described as being ‘derived from’ a reference nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >80%, >85%, >90% or >95%) sequence identity to the reference nucleotide sequence. Accordingly, a nucleotide sequence comprising a 5’ splice site which is ‘derived from’ the nucleotide sequence of a reference 5’ splice site comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of the reference 5’ splice site.

[0158] In some embodiments, a 5’ splice site according to the present disclosure is not derived from the nucleotide sequence of a reference 5’ splice site. A nucleotide sequence comprising a 5’ splice site which is not ‘derived from’ the nucleotide sequence of a reference 5’ splice site does not comprise >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably does not comprise e.g. >80%, >85%, >90% or >95%) sequence identity to the reference nucleotide sequence. In some embodiments, a nucleotide sequence comprising a 5’ splice site which is not ‘derived from’ the nucleotide sequence of a reference 5’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to the nucleotide sequence of the reference 5’ splice site.

[0159] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 2 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 2 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 5 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 5 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 10 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 10 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 15 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 15 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 20 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 20 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 25 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 25 nucleotides of the intron immediately 3’ of the exon. In some embodiments, a nucleotide sequence comprising a 5’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 50 nucleotides of the 5’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 50 nucleotides of the intron immediately 3’ of the exon.

[0160] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human STRN3 exon 7-intron 7. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:47. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:46. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:45. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:44. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:43. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:42. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:41. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NQ:40.

[0161] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human RPS10 exon 5-intron 5. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:158. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:157. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:156. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:155.

[0162] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human SLC25A17 exon 2-intron 2. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:199. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:198. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:197. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:196. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NQ:200.

[0163] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is not derived from the 5’ splice site at human SMN2 exon 6-intron 6. In some embodiments, a nucleotide sequence comprising a 5’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:19. In some embodiments, a nucleotide sequence comprising a 5’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:18. In some embodiments, a nucleotide sequence comprising a 5’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:17. In some embodiments, a nucleotide sequence comprising a 5’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:16.

[0164] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91 %, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:19. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91 %, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:18. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91 %, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:17. In some embodiments, a nucleotide sequence comprising a 5’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91 %, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:16.

[0165] In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human F0XM1 exon 8-intron 8. In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human APLP2 exon 6-intron 6. In some embodiments, a nucleotide sequence comprising a 5’ splice site according to the present disclosure is derived from the 5’ splice site at human MADD exon 20-intron 20.

[0166] SMN2 exon 7-derived nucleotide sequence

[0167] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence derived from human SMN2 exon 7.

[0168] A human SMN2 exon 7-derived nucleotide sequence according to the present disclosure comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of human SMN2 exon 7.

[0169] In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence according to SEQ ID NO:13. It will be appreciated that ‘a nucleotide sequence according to SEQ ID NO:13’ refers to a nucleotide sequence conforming to the consensus nucleotide sequence of SEQ ID NO:13. Exemplary sequences conforming to the consensus nucleotide sequence of SEQ ID NO:13 include SEQ ID NOs:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

[0170] In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:12. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:2. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:3. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:4. In some embodiments, a human SMN2 exon 7- derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:5. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:6. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:8. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:9. In some embodiments, a human SMN2 exon 7-derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:10. In some embodiments, a human SMN2 exon 7- derived nucleotide sequence comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:11 .

[0171] In some embodiments, the human SMN2 exon 7-derived nucleotide sequence is a variant of human SMN2 exon 7. Accordingly, in some embodiments, the human SMN2 exon 7-derived nucleotide sequence does not consist of, or does not comprise, SEQ ID NO:1 . In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises, or consists of, a nucleotide sequence having <100% sequence identity to SEQ ID NO:1 .

[0172] In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘A’ at the position corresponding to position 2 of SEQ ID NO:13 (cf. e.g. position 2 of SEQ ID NOs:2, 3, 4, 5, 6, 7, 8, 9, 10 and 11). In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 2 of SEQ ID NO:13 (cf. e.g. position 2 of SEQ ID NO:1).

[0173] As explained herein, position(s) of a nucleotide sequence which ‘corresponds to’ specified position(s) of a reference nucleotide sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960). By way of illustration, positions 1 to 52 of SEQ ID NO:12 correspond to positions 368 to 419 of SEQ ID NO:147. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence (e.g. a human SMN2 exon 7-derived nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) comprises T at the position corresponding to position 3 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 3 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 4 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 4 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘A’ at the position corresponding to position 20 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 20 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 22 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘C’ at the position corresponding to position 22 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 25 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 25 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 26 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 26 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 34 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘C’ at the position corresponding to position 34 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘C’ at the position corresponding to position 35 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘A’ at the position corresponding to position 35 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘A’ at the position corresponding to position 36 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘C’ at the position corresponding to position 36 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘C’ at the position corresponding to position 37 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises T at the position corresponding to position 37 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence lacks a nucleotide at the position corresponding to position 40 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 40 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence lacks a nucleotide at the position corresponding to position 50 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘G’ at the position corresponding to position 50 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence comprises ‘A’ at the position corresponding to position 51 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence lacks a nucleotide at the position corresponding to position 51 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7- derived nucleotide sequence comprises T at the position corresponding to position 52 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence lacks a nucleotide at the position corresponding to position 52 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7- derived nucleotide sequence comprises ‘A’ at the position corresponding to position 53 of SEQ ID NO:13. In some embodiments, the human SMN2 exon 7-derived nucleotide sequence lacks a nucleotide at the position corresponding to position 53 of SEQ ID NO:13.

[0174] Nucleotide sequence comprising a 3’ splice site

[0175] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence comprising a 3’ splice site.

[0176] A 3' splice site (3’ SS, sometimes also referred to as an acceptor site) refers to a nucleotide sequence formed by nucleotides at the junction of an intron and the exon immediately downstream of ( / .e. 3’ to) the intron. That is, a 3’ splice site comprises the final ( / .e. 3’) nucleotides of an intron, and the first ( / .e. 5’) nucleotides of the exon immediately downstream of the intron. By way of illustration, SEQ ID NO:25 is a nucleotide sequence comprising a 3’ splice site, in which positions 1 to 5 are derived from human SMN2 intron 7, and positions 6 to 10 are derived from human SMN2 exon 8.

[0177] It will be appreciated that in aspects and embodiments of the present disclosure wherein a polynucleotide of the disclosure is or comprises DNA (e.g. is a DNA molecule), ‘a nucleotide sequence comprising a 3’ splice site’ refers to a nucleotide sequence that when provided as the equivalent ribonucleotide sequence comprises a 3’ splice site. That is, in such DNA molecules, a nucleotide sequence comprising a 3’ splice site refers to a nucleotide sequence that when transcribed to RNA comprises a 3’ splice site. By way of illustration, a DNA polynucleotide comprising the nucleotide sequence of SEQ ID NO:25 comprises a 3’ splice site when provided as the corresponding polyribonucleotide ( / .e. comprising uracil nucleotides in place of thymine nucleotides).

[0178] A nucleotide sequence comprising a 5’ splice site according to the present disclosure refers to a nucleotide sequence that when provided as polyribonucleotide comprises a nucleotide sequence forming a site recognised by the U2 auxiliary factor (U2AF) component of the spliceosome. In some embodiments, a 3’ splice site according to the present disclosure comprises the dinucleotide ‘AG’, e.g. at the penultimate and final positions of the intronic sequence of the 3’ splice site. In some embodiments, a 3’ splice site according to the present disclosure comprises a nucleotide sequence conforming to the consensus shown in SEQ ID NO:249. In some embodiments, a polypyrimidine tract is provided upstream of ( / .e. 5’ to) the 3’ splice site.

[0179] In aspects and embodiments described herein, a nucleotide sequence comprising a 3’ splice site according to the present disclosure may be derived from the nucleotide sequence of a reference 3’ splice site. A reference splice site may comprise or consist of the final ( / .e. 3’) nucleotides of an intron of a given gene, and the first ( / .e. 5’) nucleotides of the exon immediately downstream of the intron. In some embodiments, the final nucleotides of the intron may refer to the final 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides of the intron. In some embodiments, the first nucleotides of the exon immediately downstream of the intron may refer to the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides of the exon.

[0180] In some embodiments, the reference 3’ splice site is the 3’ splice site of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the reference 3’ splice site is the 3’ splice site of a gene region known to undergo alternative splicing via exon skipping, in which skipping of the relevant exon is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0181] In some embodiments, the reference 3’ splice site is the 3’ splice site at human STRN3 intron 8-exon 9. In some embodiments, the reference 3’ splice site is the 3’ splice site at human RPS10 intron 5-exon 6. In some embodiments, the reference 3’ splice site is the 3’ splice site at human SLC25A17 intron 3-exon 4. In some embodiments, the reference 3’ splice site is the 3’ splice site at human F0XM1 intron 9-exon 10. In some embodiments, the reference 3’ splice site is the 3’ splice site at human APLP2 intron 7-exon 8. In some embodiments, the reference 3’ splice site is the 3’ splice site at human MADD intron 21 -exon 22.

[0182] In some embodiments, the reference 3’ splice site is not the 3’ splice site at human SMN2 intron 7-exon 8.

[0183] In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 2 nucleotides of a given intron and the first 2 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 5 nucleotides of a given intron and the first 5 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 10 nucleotides of a given intron and the first 10 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 15 nucleotides of a given intron and the first 15 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 20 nucleotides of a given intron and the first 20 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 25 nucleotides of a given intron and the first 25 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 30 nucleotides of a given intron and the first 30 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 40 nucleotides of a given intron and the first 40 nucleotides of the exon immediately downstream of the intron. In some embodiments, a 3’ splice site refers to the nucleotide sequence comprising (from 5’ to 3’) the final 50 nucleotides of a given intron and the first 50 nucleotides of the exon immediately downstream of the intron. A nucleotide sequence comprising a 3’ splice site which is ‘derived from’ the nucleotide sequence of a reference 3’ splice site comprises at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of the reference 3’ splice site.

[0184] In some embodiments, a 3’ splice site according to the present disclosure is not derived from the nucleotide sequence of a reference 3’ splice site. A nucleotide sequence comprising a 3’ splice site which is not ‘derived from’ the nucleotide sequence of a reference 3’ splice site does not comprise >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably does not comprise e.g. >80%, >85%, >90% or >95%) sequence identity to the reference nucleotide sequence. In some embodiments, a nucleotide sequence comprising a 3’ splice site which is not ‘derived from’ the nucleotide sequence of a reference 3’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to the nucleotide sequence of the reference 3’ splice site.

[0185] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 2 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 2 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 5 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 5 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 10 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 10 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 15 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 15 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 20 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 20 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 25 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 25 nucleotides of the 3’ exon. In some embodiments, a nucleotide sequence comprising a 3’ splice site has at least 80% e.g. >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (preferably e.g. >90% or >95%) sequence identity to the nucleotide sequence formed by: the final 50 nucleotides of the intron immediately 5’ of the 3’ exon of a gene region known to undergo alternative splicing via exon skipping, and the first 50 nucleotides of the 3’ exon.

[0186] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human STRN3 intron 8-exon 9. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:90. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:89. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:88. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:87. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:86. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:85. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:84. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:83. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:82. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:81. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:80. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:79. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:78. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:77. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:76. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:75. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:74. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:73. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:72. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:71. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NQ:70. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:69. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:68. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:67. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:66. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:65. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:64. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:94. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:93. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:92. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:91. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:235. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:234. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:233. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:238. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:237. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:236. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:241. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NQ:240. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:239. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:244. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:243. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity (e.g. >80%, >85%, >90% or >95%) to SEQ ID NO:242.

[0187] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human RPS10 intron 5-exon 6. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NQ:90. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:165. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:164. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:163.

[0188] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human SLC25A 17 intron 3-exon 4. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:208. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NQ:207. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NQ:206. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises or consists of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NQ:205.

[0189] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is not derived from the 3’ splice site at human SMN2 intron 7-exon 8. In some embodiments, a nucleotide sequence comprising a 3’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:25. In some embodiments, a nucleotide sequence comprising a 3’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:24. In some embodiments, a nucleotide sequence comprising a 3’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:23. In some embodiments, a nucleotide sequence comprising a 3’ splice site does not comprise or consist of a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to a nucleotide sequence selected from: SEQ ID NO:22.

[0190] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91 %, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:25. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:24. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:23. In some embodiments, a nucleotide sequence comprising a 3’ splice site comprises less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to SEQ ID NO:22.

[0191] In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human F0XM1 intron 9-exon 10. In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human APLP2 intron 7-exon 8. In some embodiments, a nucleotide sequence comprising a 3’ splice site according to the present disclosure is derived from the 3’ splice site at human MADD intron 21 -exon 22.

[0192] First nucleotide sequence

[0193] Aspects and embodiments of the present disclosure relate to polynucleotides comprising specified first, second, third, fourth, fifth and optionally sixth nucleotide sequences.

[0194] In some embodiments, a first nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from an exon. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence derived from the 5’ exon ( / .e. the exon at the 5’ splice site) of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence derived from the 5’ exon ( / .e. the exon at the 5’ splice site) of a gene region known to undergo alternative splicing via exon skipping, in which exon skipping is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0195] In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human STRN3 exon 7. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:28. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:27. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:26.

[0196] In some embodiments, the first nucleotide sequence (e.g. a first nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:28, 27 or 26) comprises ‘C’ at the position corresponding to position 10 of SEQ ID NO:27. In some embodiments, the first nucleotide sequence comprises ‘C’ at the position corresponding to position 94 of SEQ ID NO:27. In some embodiments, the first nucleotide sequence comprises ‘C’ at the position corresponding to position 130 of SEQ ID NO:27. In some embodiments, the first nucleotide sequence comprises T at the position corresponding to position 138 of SEQ ID NO:27.

[0197] In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human RPS10 exon 5. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:152. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:151.

[0198] In some embodiments, the first nucleotide sequence (e.g. a first nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:152 or 151) does not comprise a nucleotide at the position corresponding to position 1 of SEQ ID NO:151. In some embodiments, the first nucleotide sequence does not comprise a nucleotide at the position corresponding to position 2 of SEQ ID NO:151.

[0199] In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human SLC25A 17 exon 2. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:193. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:192. In some embodiments, the first nucleotide sequence (e.g. a first nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:193 or 192) does not comprise a nucleotide at the position corresponding to position 17 of SEQ ID NO:192.

[0200] In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human F0XM1 exon 8. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human APLP2 exon 6. In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human MADD exon 20.

[0201] In some embodiments, the first nucleotide sequence comprises, or consists, a nucleotide sequence having less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to human SMN2 exon 6. In some embodiments, the first nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human SMN2 exon 6. In some embodiments, the first nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:14. In some embodiments, the first nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:15.

[0202] Second nucleotide sequence

[0203] In some embodiments, a second nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from an intron. In some embodiments, the second nucleotide sequence comprises, or consists, a nucleotide sequence derived from the intron immediately downstream of (i.e. 3’ to) the 5’ exon (i.e. the exon at the 5’ splice site) of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the second nucleotide sequence comprises, or consists, a nucleotide sequence derived from the intron immediately downstream of (i.e. 3’ to) the 5’ exon (i.e. the exon at the 5’ splice site) of a gene region known to undergo alternative splicing via exon skipping, in which exon skipping is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916). In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:97 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:96 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:95 at its 5’ end.

[0204] In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:101 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:100 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:99 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:109 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:108 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:107 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:106 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:105 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:104 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:103 at its 3’ end. In some embodiments, the second nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 97, 96 or 95 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 at its 3’ end.

[0205] As used herein, the ‘5’ end’ of a given nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 5’ to ( / .e. upstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence, or a subsequence of this region.

[0206] In a nucleotide sequence having an even number of nucleotides, the region 5’ to the ‘midpoint’ includes the nucleotide immediately preceding the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the region 5’ to the midpoint consists of positions 1 to 5. Similarly, in a nucleotide sequence having an even number of nucleotides, the region 3’ to the ‘midpoint’ includes the nucleotide immediately after the midpoint. For example, in a nucleotide sequence consisting of 10 nucleotides, the region 3’ to the midpoint consists of positions 6 to 10. In a nucleotide sequence having an odd number of nucleotides, the region 5’ to the ‘midpoint’ includes the nucleotides 5’ to ( / .e. upstream of) the nucleotide provided at the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the region 5’ to the midpoint consists of positions 1 to 4. Similarly, in a nucleotide sequence having an odd number of nucleotides, the region 3’ to the ‘midpoint’ includes the nucleotides 3’ to ( / .e. downstream of) the nucleotide provided at the midpoint. For example, in a nucleotide sequence consisting of 9 nucleotides, the region 3’ to the midpoint consists of positions 6 to 9.

[0207] By way of illustration, in SEQ ID NO:29, which consists of 400 nucleotides, a sequence provided at the 5’ end of SEQ ID NO:29 refers to a nucleotide sequence formed by positions 1 to 200 of SEQ ID NO:29, or a subsequence thereof. Conversely, the ‘3’ end’ of a given nucleotide sequence refers to the region of the nucleotide sequence formed by nucleotides 3’ to ( / .e. downstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence, or a subsequence of this region. By way of illustration, in SEQ ID NO:29, a sequence provided at the 3’ end of SEQ ID NO:29 refers to a nucleotide sequence formed by positions 201 to 400 of SEQ ID NO:29, or a subsequence thereof.

[0208] In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region of the nucleotide sequence formed by nucleotides 5’ to ( / .e. upstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:29 may comprise at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region formed by positions 1 to 200 of SEQ ID NO:29. In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region of the nucleotide sequence formed by nucleotides 3’ to ( / .e. downstream of, in the context of the complete sequence of the given nucleotide sequence) the midpoint of the given nucleotide sequence. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:29 may comprise at least 5%, e.g. one of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the region formed by positions 201 to 400 of SEQ ID NO:29.

[0209] In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 5’ nucleotide of the given nucleotide sequence. In some embodiments, a subsequence of a given nucleotide sequence provided at the 5’ end of the given nucleotide sequence includes the 5’ nucleotide of the given nucleotide sequence. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:29 may comprise one or more nucleotides provided within positions 1 to 25, e.g. within positions 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of SEQ ID NO:29. By way of illustration, a sequence provided at the 5’ end of SEQ ID NO:29 may comprise position 1 of SEQ ID NO:29.

[0210] In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes one or more nucleotides provided within 25 nucleotides, e.g. within one of 20, 15, 10 or 5 nucleotides, of the 3’ nucleotide of the given nucleotide sequence. In some embodiments, a subsequence of a given nucleotide sequence provided at the 3’ end of the given nucleotide sequence includes the 3’ nucleotide of the given nucleotide sequence. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:29 may comprise one or more nucleotides provided within positions 376 to 400, e.g. within positions 381 to 400, 386 to 400, 391 to 400, or 396 to 400 of SEQ ID NO:29. By way of illustration, a sequence provided at the 3’ end of SEQ ID NO:29 may comprise position 400 of SEQ ID NO:29.

[0211] In some embodiments the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 97, 96 or 95 in the region 5’ to its midpoint. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 in the region 3’ to its midpoint.

[0212] In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 97, 96 or 95 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 97, 96 or 95 comprises the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 comprises the 3’ nucleotide of the second nucleotide sequence.

[0213] In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:30. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:31 . In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:32. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:33. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:34. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:35. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:36. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:37. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:38. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:39.

[0214] In some embodiments, the second nucleotide sequence (e.g. a second nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NQ:30, 29, 31 , 32, 33, 34, 35, 36, 37, 38 or 39) comprises ‘C’ at the position corresponding to position 10 of SEQ ID NO:27. In some embodiments, the second nucleotide sequence comprises ‘C’ at the position corresponding to position 398 of SEQ ID NO:37. In some embodiments, the second nucleotide sequence comprises ‘C’ at the position corresponding to position 397 of SEQ ID NO:37. In some embodiments, the second nucleotide sequence comprises T at the position corresponding to position 395 of SEQ ID NO:37. In some embodiments, the second nucleotide sequence comprises T at the position corresponding to position 394 of SEQ ID NO:37. In some embodiments, the second nucleotide sequence comprises T at the position corresponding to position 393 of SEQ ID NO:37.

[0215] In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:169 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:168 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:167 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:166 at its 5’ end.

[0216] In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:173 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:172 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:171 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:170 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:106 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:176 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:175 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:174 at its 3’ end. In some embodiments, the second nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:169, 168, 167 or 166 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 at its 3’ end.

[0217] In some embodiments the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:169, 168, 167 or 166 in the region 5’ to its midpoint. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 in the region 3’ to its midpoint.

[0218] In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:169, 168, 167 or 166 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 169, 168, 167 or 166 comprises the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:173, 172, 171 , 170, 106, 176, 175 or 174 comprises the 3’ nucleotide of the second nucleotide sequence.

[0219] In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:153. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:154.

[0220] In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:212 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:21 1 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:210 at its 5’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:209 at its 5’ end.

[0221] In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:216 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:215 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:214 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:213 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:106 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:176 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:218 at its 3’ end. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:217 at its 3’ end.

[0222] In some embodiments, the second nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:212, 211 , 210 or 209 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218, 217 at its 3’ end.

[0223] In some embodiments the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:212, 211 , 210 or 209 in the region 5’ to its midpoint. In some embodiments, the second nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218, 217 in the region 3’ to its midpoint. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:212, 211 , 210 or 209 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:212, 211 , 210 or 209 comprises the 5’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218, 217comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the second nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:216, 215, 214, 213, 106, 176, 218, 217comprises the 3’ nucleotide of the second nucleotide sequence.

[0224] In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:194. In some embodiments, the second nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:195.

[0225] In some embodiments, the second nucleotide sequence consists of fewer than 500 nucleotides, e.g. <450 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides or <200 nucleotides.

[0226] Third nucleotide sequence

[0227] In some embodiments, a third nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from an exon. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence derived from the cassette exon ( / .e. the exon which is either skipped or not skipped) of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence derived from the cassette exon ( / .e. the exon which is either skipped or not skipped) of a gene region known to undergo alternative splicing via exon skipping, in which exon skipping is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0228] In some embodiments, a third nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from human SMN2 exon 7. In accordance with such embodiments, the nucleotide sequence derived from human SMN2 exon 7 may be a human SMN2 exon 7-derived nucleotide sequence as described hereinabove. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human STRN3 exon 8. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:50. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:49.

[0229] In some embodiments, the third nucleotide sequence (e.g. a third nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NQ:50 or 49) comprises ‘C’ at the position corresponding to position 9 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 16 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 19 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 20 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises T at the position corresponding to position 23 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises ‘G’ at the position corresponding to position 24 of SEQ ID NQ:50. In some embodiments, the third nucleotide sequence comprises ‘C’ at the position corresponding to position 40 of SEQ ID NQ:50.

[0230] In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human RPS10 exon 5.5. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NQ:160. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:159.

[0231] In some embodiments, the third nucleotide sequence (e.g. a third nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NQ:160 or 159) does not comprise a nucleotide at the position corresponding to position 32 of SEQ ID NO:159. In some embodiments, the third nucleotide sequence does not comprise a nucleotide at the position corresponding to position 54 of SEQ ID NO:159. In some embodiments, the third nucleotide sequence does not comprise a nucleotide at the position corresponding to position 88 of SEQ ID NO:159. In some embodiments, the third nucleotide sequence comprises ‘A’ at the position corresponding to position 61 of SEQ ID NO:159.

[0232] In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human SLC25A17 exon 3. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NQ:202. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NQ:201.

[0233] In some embodiments, the third nucleotide sequence (e.g. a third nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NQ:202 or 201) comprises T at the position corresponding to position 43 of SEQ ID NQ:202.

[0234] In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human F0XM1 exon 9. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human APLP2 exon 7. In some embodiments, the third nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human MADD exon 21 .

[0235] Fourth nucleotide sequence

[0236] In some embodiments, a fourth nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from an intron. In some embodiments, the fourth nucleotide sequence comprises, or consists, a nucleotide sequence derived from the intron immediately upstream of (i.e. 5’ to) the 3’ exon (i.e. the exon at the 3’ splice site) of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the fourth nucleotide sequence comprises, or consists, a nucleotide sequence derived from the intron immediately upstream of (i.e. 5’ to) the 3’ exon (i.e. the exon at the 3’ splice site) of a gene region known to undergo alternative splicing via exon skipping, in which exon skipping is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0237] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:112 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:111 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:110 at its 5’ end.

[0238] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:115 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:114 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:113 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:112 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:120 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:119 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:118 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:117 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:247 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:246 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:245 at its 3’ end.

[0239] In some embodiments, the fourth nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 1 12, 111 or 110 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 115, 114, 113, 120, 119, 118, 1 17, 247, 246 or 245 at its 3’ end.

[0240] In some embodiments the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 112, 1 11 or 110 in the region 5’ to its midpoint. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 1 15, 114, 113, 120, 119, 118, 117, 247, 246 or 245 in the region 3’ to its midpoint.

[0241] In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 112, 111 or 110 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 1 12, 111 or 1 10 comprises the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 115, 1 14, 113, 120, 119, 118, 117, 247, 246 or 245 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 115, 114, 113, 120, 119, 118, 117, 247, 246 or 245 comprises the 3’ nucleotide of the fourth nucleotide sequence.

[0242] In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:52. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:53. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:54. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:55. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:56. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:57. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:58. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:59. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID 1X10:60. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:232.

[0243] In some embodiments, the fourth nucleotide sequence (e.g. a fourth nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:52, 51 , 53, 54, 55, 56, 57, 58, 59, 60 or 232) comprises T at the position corresponding to position 197 of SEQ ID NQ:60. In some embodiments, the fourth nucleotide sequence comprises T at the position corresponding to position 392 of SEQ ID NO:232. In some embodiments, the fourth nucleotide sequence comprises T at the position corresponding to position 393 of SEQ ID NO:232.

[0244] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:180 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:179 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:178 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:177 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:183 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:182 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 181 at its 5’ end.

[0245] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:186 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:185 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO: 184 at its 3’ end.

[0246] In some embodiments, the fourth nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:180, 179, 178, 177, 98, 183, 182 or 181 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 186, 185 or 184 at its 3’ end.

[0247] In some embodiments the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:180, 179, 178, 177, 98, 183, 182 or 181 in the region 5’ to its midpoint. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 186, 185 or 184 in the region 3’ to its midpoint.

[0248] In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:180, 179, 178, 177, 98, 183, 182 or 181 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity SEQ ID NO: 180, 179, 178, 177, 98, 183, 182 or 181 comprises the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 186, 185 or 184 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:116, 186, 185 or 184 comprises the 3’ nucleotide of the fourth nucleotide sequence.

[0249] In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:161. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:162.

[0250] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:221 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:220 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:219 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:183 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:223 at its 5’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:222 at its 5’ end.

[0251] In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:226 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:225 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:224 at its 3’ end. In some embodiments, the fourth nucleotide sequence comprises: (a) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 221 , 220, 219, 183, 223 or 222 at its 5’ end, and (b) a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:102, 226, 225 or 224 at its 3’ end.

[0252] In some embodiments the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 221 , 220, 219, 183, 223 or 222 in the region 5’ to its midpoint. In some embodiments, the fourth nucleotide sequence comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102, 226, 225 or 224 in the region 3’ to its midpoint.

[0253] In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:98, 221 , 220, 219, 183, 223 or 222 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity SEQ ID NO:98, 221 , 220, 219, 183, 223 or 222 comprises the 5’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102, 226, 225 or 224 comprises one or more nucleotides provided within 25, e.g. within positions 20, 15, 10, or 5 nucleotides of the 3’ nucleotide of the fourth nucleotide sequence. In some embodiments the nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:102, 226, 225 or 224 comprises the 3’ nucleotide of the fourth nucleotide sequence.

[0254] In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:203. In some embodiments, the fourth nucleotide sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:202.

[0255] In some embodiments, the fourth nucleotide sequence consists of fewer than 500 nucleotides, e.g. <450 nucleotides, <400 nucleotides, <350 nucleotides, <300 nucleotides, <250 nucleotides or <200 nucleotides.

[0256] Fifth nucleotide sequence

[0257] In some embodiments, a fifth nucleotide sequence according to the present disclosure comprises, or consists, a nucleotide sequence derived from an exon. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence derived from the 3’ exon ( / .e. the exon at the 3’ splice site) of a gene region known to undergo alternative splicing via exon skipping. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence derived from the 3’ exon ( / .e. the exon at the 3’ splice site) of a gene region known to undergo alternative splicing via exon skipping, in which exon skipping is inhibited when splicing of a polyribonucleotide transcribed from the gene region is performed in the presence of risdiplam (RG-7916).

[0258] In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human STRN3 exon 9. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:63. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:62. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to the nucleotide sequence of SEQ ID NO:61 .

[0259] In some embodiments, the fifth nucleotide sequence (e.g. a fifth nucleotide sequence comprising or consisting of a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO:63, 62 or 61) comprises ‘A’ at the position corresponding to position 28 of SEQ ID NO:62. In some embodiments, the fifth nucleotide sequence comprises ‘C’ at the position corresponding to position 37 of SEQ ID NO:62.

[0260] In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human RPS10 exon 6. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human SLC25A17 exon 4. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human F0XM1 exon 10. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human APLP2 exon 8. In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having at least 60%, e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human MADD exon 22.

[0261] In some embodiments, the fifth nucleotide sequence comprises, or consists, a nucleotide sequence having less than 100%, e.g. one of <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%, <85%, <80%, <75%, <70%, <65% or <60% (preferably e.g. <95%, <90%, <85% or <80%) sequence identity to human SMN2 exon 8. In some embodiments, the fifth nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to human SMN2 exon 8. In some embodiments, the fifth nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:21 . In some embodiments, the fifth nucleotide sequence does not comprise, or consist of, a nucleotide sequence having >60%, e.g. >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%) sequence identity to SEQ ID NO:20.

[0262] In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TG’, ‘GG’ or ‘TT’ or ‘GA’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘TG’. In some embodiments, the fifth nucleotide sequence consists of the dinucleotide ‘GG’.

[0263] In some embodiments, the fifth nucleotide sequence encodes a molecule of interest (e.g. as described hereinbelow). In some embodiments, the fifth nucleotide sequence encodes a molecule of interest, and comprises ‘TG’, ‘GG’ or ‘TT’ or ‘GA’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a molecule of interest, and comprises ‘TG’ at positions 1 and 2. In some embodiments, the fifth nucleotide sequence encodes a molecule of interest, and comprises ‘GG’ at positions 1 and 2.

[0264] Nucleotide sequences encoding a molecule of interest

[0265] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence encoding a molecule of interest. In some embodiments, the fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a molecule of interest. Where present, the sixth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a molecule of interest.

[0266] In some embodiments, a molecule of interest is, or comprises, a polypeptide or a polynucleotide (e.g. a polyribonucleotide). In some embodiments, the nucleotide sequence encoding a molecule of interest comprises or consists of a sequence of deoxyribonucleotides that can be transcribed to a sequence of ribonucleotides, that can in turn be translated into an amino acid sequence forming a polypeptide of interest. In some embodiments, the nucleotide sequence encoding a molecule of interest comprises or consists of a sequence of ribonucleotides, that can be translated into an amino acid sequence forming a polypeptide of interest. In some embodiments, the nucleotide sequence encoding a molecule of interest comprises or consists of a sequence of deoxyribonucleotides that can be transcribed to a sequence of ribonucleotides forming a polynucleotide (that is, a polyribonucleotide) of interest.

[0267] In some embodiments, a molecule (e.g. a polypeptide or polynucleotide) of interest according to the present disclosure is suitable for use in therapy or prophylaxis of a disease / condition. In some embodiments, the molecule of interest is a detectable or has a detectable activity.

[0268] A molecule (e.g. polypeptide / polynucleotide) suitable for use in therapy or prophylaxis of a disease / condition may be any molecule whose administration is useful for the treatment or prevention of a disease / condition. In some embodiments, the molecule (e.g. polypeptide / polynucleotide) is a molecule for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition. In some embodiments, the molecule (e.g. polypeptide / polynucleotide) inhibits the expression and / or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition.

[0269] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence encoding a polypeptide of interest. In some embodiments, the fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest. Where present, the sixth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polypeptide of interest.

[0270] A polypeptide of interest may be any polypeptide.

[0271] In some embodiments, a polypeptide of interest according to the present disclosure may be an antigenbinding polypeptide, an aptamer, a constituent polypeptide of an antigen-binding polypeptide complex, an antibody / antigen-binding fragment or derivative thereof, a constituent polypeptide of an antibody / antigen- binding fragment or derivative thereof, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, a decoy receptor for a ligand, a decoy ligand for a receptor, an enzyme, an enzyme co-factor, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic, a transcription factor, an epigenetic modifier, a constituent protein of a site-specific nuclease nucleic acid editing system (e.g. a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, a CRISPR / C2c3 system, a ZFN system or a TALEN system), a constituent protein of a ribonucleoprotein, a viral protein (e.g. a capsid protein or a viral enzyme), or a protein useful for the production of a biomolecule.

[0272] In some embodiments, a polypeptide of interest is a polypeptide suitable for use in therapy or prophylaxis of a disease / condition. In some embodiments, a polypeptide of interest is a detectable polypeptide or a polypeptide having detectable activity. A polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be any polypeptide whose administration is useful for the treatment or prevention of a disease / condition. In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be a polypeptide for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition. By way of illustration, in some embodiments, the polypeptide of interest may be MeCP2. Deficiency of MeCP2 is associated with Rett syndrome.

[0273] In some embodiments, a polypeptide suitable for use in therapy or prophylaxis of a disease / condition may be a polypeptide which inhibits the expression and / or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition.

[0274] A detectable polypeptide may be or comprise a fluorescent polypeptide. Fluorescent polypeptides include green fluorescent protein and variants thereof (e.g. enhanced green fluorescent protein), yellow fluorescent protein (e.g. citrine), red fluorescent protein and variants thereof (e.g. mOrange, mCherry), blue fluorescent protein and variants thereof (e.g. TagBFP), cyan fluorescent protein and variants thereof (e.g. mTurquoise, cerulean), allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin.

[0275] A detectable polypeptide may be or comprise an epitope tag. Epitope tags include e.g. His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol).

[0276] A polypeptide having detectable activity may be or comprise an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

[0277] A polypeptide of interest expressed from a polynucleotide of the present disclosure may additionally comprises one or more extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. Such extraneous amino acids may be characterised as forming an N-terminal tag on the polypeptide of interest. It may be desirable to minimise the size of, or completely remove, such extraneous amino acids / N-terminal tags on the polypeptide of interest.

[0278] In some embodiments, the N-terminal tag consists of fewer than 50 amino acids, e.g. one of <40 amino acids, <30 amino acids, <25 amino acids, <20 amino acids, <15 amino acids, <10 amino acids, <9 amino acids, <8 amino acids, <7 amino acids, <6 amino acids, <5 amino acids, <4 amino acids, <3 amino acids, <2 amino acids or 1 amino acid. In some embodiments, the polypeptide of interest lacks an N-terminal tag.

[0279] In some embodiments, the polypeptide of the present disclosure comprises one or more cleavage sites. A cleavage site refers to a sequence of amino acids that acts as a substrate for an enzyme capable of cleaving peptide bonds. Many such cleavage sites are known to, and can be employed by, the person skilled in the art of molecular biology. In some embodiments, the cleavage sequence comprises an autocleavage site. Autocleavage sites include the 2A cleavage sequence from Picornavirus ‘NPGP’, which is cleaved at ’G / P’. Further autocleavage sites are described e.g. in Kim et al., PLoS ONE (2011) 6: e18556 (hereby incorporated by reference in its entirety), and include e.g. T2A, E2A, P2A and F2A cleavage sites. The amino acid sequences of T2A, E2A, P2A and F2A cleavage sites are shown in SEQ ID NOs:250, 251 , 252 and 253, respectively.

[0280] A cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of extraneous amino acids added at the N-terminus of the polypeptide, i.e. immediately upstream of the amino acid sequence of the polypeptide of interest. That is, a cleavage site may be included for the removal of an N-terminal tag as described hereinabove. Accordingly, in some embodiments, a polypeptide according to the present disclosure comprises a cleavage site adjacent to (J.e. in the amino acid sequence of the polypeptide, e.g. immediately downstream of) extraneous amino acid(s), e.g. extraneous amino acid(s) forming an N-terminal tag.

[0281] In some embodiments, a cleavage site according to the present disclosure is a 2A cleavage site, e.g. selected from a T2A, E2A, P2A and F2A cleavage site. In some embodiments, the cleavage site is a T2A cleavage site. In some embodiments, a polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:250, 251 , 252 or 253.

[0282] Aspects and embodiments of the present disclosure relate to polynucleotides comprising a nucleotide sequence encoding a polynucleotide of interest. In some embodiments, the fifth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polynucleotide of interest. Where present, the sixth nucleotide sequence according to the present disclosure comprises, or consists of, a nucleotide sequence encoding a polynucleotide of interest.

[0283] A polynucleotide of interest may be any polynucleotide. In some embodiments, a polynucleotide of interest is a polynucleotide suitable for use in therapy or prophylaxis of a disease / condition. In some embodiments, a polynucleotide of interest is a detectable polynucleotide or a polynucleotide having detectable activity.

[0284] A polynucleotide suitable for use in therapy or prophylaxis of a disease / condition may be any polynucleotide whose administration is useful for the treatment or prevention of a disease / condition. In some embodiments, a polynucleotide suitable for use in therapy or prophylaxis of a disease / condition may be a polynucleotide for which deficiency thereof is positively associated with, or implicated in the pathology of, a disease or condition. In some embodiments, a polynucleotide suitable for use in therapy or prophylaxis of a disease / condition may be a polynucleotide which inhibits the expression and / or activity of a factor whose expression or activity is positively associated with, or implicated in the pathology of, a disease or condition. In some embodiments, a polynucleotide of interest is a polyribonucleotide ( / .e. the polynucleotide of interest is an RNA of interest).

[0285] In some embodiments, the polynucleotide of interest is a polynucleotide capable of inducing / promoting silencing of a given target via RNA interference (RNAi). RNAi uses small double-stranded RNA molecules to cause degradation of target mRNA. In some embodiments, the molecule of interest is: an siRNA (or an siRNA derivative or pre-cursor), a short hairpin RNA (shRNA), a micro RNA (miRNA), a long primary miRNA transcript (pri-miRNA), a partially-processed 60-70 base pair hairpin miRNA transcript (pre- miRNA), a small activating RNA (saRNA) or a small nucleolar RNA (snoRNA). In some embodiments, the polynucleotide of interest is a nucleic acid aptamer.

[0286] Further nucleotide sequences of the polynucleotide

[0287] Polynucleotides according to the present disclosure may comprise additional nucleotide sequences and / or sequence features. For example, polynucleotides according to the present disclosure e.g. in addition to the first, second, third, fourth, fifth and / or sixth nucleotide sequences as described herein.

[0288] The polynucleotides of the present disclosure preferably comprise a start codon 5’ to ( / .e. upstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a molecule (e.g. polypeptide) of interest. The start codon is preferably the trinucleotide ‘ATG’.

[0289] In some embodiments, a start codon is provided in the polynucleotide such that following splicing (e.g. where the polynucleotide is a polyribonucleotide, or splicing of a polyribonucleotide transcribed from the polynucleotide where the polynucleotide is a polydeoxyribonucleotide), the start codon is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of a polypeptide of interest (e.g. encoded by the fifth or sixth nucleotide sequence; i.e. in embodiments wherein the molecule of interest is a polypeptide of interest).

[0290] In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein). By way of illustration, splicing of the polyribonucleotide having the nucleotide sequence of SEQ ID NO:121 in the presence of RG7800 / RG76196 results in the production of a mature mRNA molecule comprising an initiator codon for translation of a polypeptide of interest.

[0291] In some embodiments, the polynucleotide further comprises a Kozak sequence. In preferred embodiments, the Kozak sequence is provided immediately upstream of the start codon for initiating translation of a polypeptide of interest. In some embodiments, the Kozak sequence consists of a nucleotide sequence conforming to the consensus of SEQ ID NO:254. In some embodiments, the Kozak sequence consists of SEQ ID NO:255.

[0292] In some embodiments, the polynucleotide comprises a nucleotide sequence consisting of SEQ ID NO:256 5’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments, SEQ ID NO:256 is provided in the polynucleotide such that following splicing (e.g. where the polynucleotide is a polyribonucleotide, or splicing of a polyribonucleotide transcribed from the polynucleotide where the polynucleotide is a polydeoxyribonucleotide), the start codon of SEQ ID NO:256 is provided in the mature mRNA molecule in such a way as to serve as the initiator codon for translation of a polypeptide of interest (encoded by the fifth or sixth nucleotide sequence).

[0293] In some embodiments, the splicing referred to in the preceding paragraph is splicing performed in the presence of a splicing modifier (e.g. as described herein).

[0294] In some embodiments, the polynucleotide according to the present disclosure further comprises a promoter sequence. In embodiments wherein the polynucleotide comprises a nucleotide sequence comprising a 5’ splice site, the promoter sequence may be 5’ to the nucleotide sequence comprising a 5’ splice site. In embodiments wherein the polynucleotide comprises first nucleotide sequence as described herein, the promoter sequence may be 5’ to the first nucleotide sequence.

[0295] In some embodiments, the promoter is, or is derived from, a CMV promoter, CAG promoter, hEF1 a promoter, hUbiC promoter, RSV promoter, TK promoter, PGK promoter or CAG minimal promoter. In some embodiments, the promoter is, or is derived from, a CMV promoter, CAG promoter, hEF1a promoter, hUbiC promoter, RSV promoter or CAG minimal promoter. In some embodiments, the promoter is, or is derived from, a hEF1a promoter.

[0296] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 257. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 260 or 263. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 266. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 267. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 268. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 269. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 270.

[0297] In some embodiments, the polynucleotide further comprises one or more enhancer sequences. In embodiments wherein the polynucleotide comprises a nucleotide sequence comprising a 5’ splice site, the one or more enhancer sequences may be 5’ to the nucleotide sequence comprising a 5’ splice site. In embodiments wherein the polynucleotide comprises first nucleotide sequence as described herein, the one or more enhancer sequences may be 5’ to the first nucleotide sequence. In embodiments wherein the polynucleotide comprises a promoter sequence, the one or more enhancer sequences may be 5’ to the promoter sequence.

[0298] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 258. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 261 . In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 264.

[0299] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 259. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 262. In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 60% (e.g. one of >65%, >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% (e.g. >80%, >85%, >90% or >95%)) sequence identity to 265.

[0300] In some embodiments, the polynucleotide further comprises a stop codon. The stop codon is preferably provided immediately 3’ to ( / .e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the trinucleotide encoding the terminal amino acid of a polypeptide encoded by a nucleotide sequence encoding a polypeptide of interest.

[0301] In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence. In preferred embodiments, the polyadenylation signal sequence is provided 3’ to ( / .e. downstream of, in the context of the nucleotide sequence of the polynucleotide) the nucleotide sequence encoding a molecule (e.g. polypeptide) of interest. In some embodiments, the polyadenylation signal sequence is, or is derived from, bovine growth hormone (BGH). In some embodiments, the polyadenylation signal sequence comprises, or consists of, a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:271.

[0302] In some embodiments, the polynucleotide further comprises a terminator sequence. The terminator sequence is preferably 3’ to the nucleotide sequence encoding a molecule (e.g. polypeptide) of interest (and 3’ to the polyadenylation signal sequence, when present).

[0303] In preferred embodiments, the constituent nucleotide sequences of the polynucleotides of the present disclosure (e.g. the nucleotide sequence comprising a 5’ splice site, the human SMN2 exon 7 / human STRN3 exon 8 / human RPS10 exon 5.5 / human SLC25A17 exon 3-derived nucleotide sequence and the nucleotide sequence comprising a 3’ splice site as described herein; or the first, second, third, fourth, fifth and / or sixth nucleotide sequences as described herein) are provided immediately adjacent to one another. However, in some embodiments, the polynucleotide further comprises one or more linker nucleotide sequences between one or more of the constituent nucleotide sequences of the polynucleotide.

[0304] Linker nucleotide sequences may comprise, or consist of, 1-10, e.g. one of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- 3, or 1-2 nucleotides.

[0305] Where a polynucleotide according to the present disclosure comprises one more linker nucleotide sequences ( / .e. provided between the nucleotide sequence comprising a 5’ splice site, the human SMN2 exon 7 / human STRN3 exon 8 / human RPS10 exon 5.5 / human SLC25A17 exon 3-derived nucleotide sequence and the nucleotide sequence comprising a 3’ splice site as described herein; or provided between the first, second, third, fourth, fifth and / or sixth nucleotide sequences as described herein), the linker sequences are preferably selected such that they do not substantially affect post-transcriptional processing of the polynucleotide when the polynucleotide is a polyribonucleotide. In preferred embodiments, where a polynucleotide according to the present disclosure comprises one more linker sequences, splicing of the polynucleotide when the polynucleotide is a polyribonucleotide is substantially the same as splicing of an equivalent polyribonucleotide lacking the linker nucleotide sequence(s).

[0306] Similarly, where a polynucleotide according to the present disclosure comprises one more linker nucleotide sequences ( / .e. provided between the nucleotide sequence comprising a 5’ splice site, the human SMN2 exon 7 / human STRN3 exon 8 / human RPS10 exon 5.5 / human SLC25A17 exon 3-derived nucleotide sequence and the nucleotide sequence comprising a 3’ splice site as described herein; or provided between the first, second, third, fourth, fifth and / or sixth nucleotide sequences as described herein), the linker sequences are preferably selected such that they do not alter the amino acid sequence of a polypeptide encoded by the polynucleotide ( / .e., wherein the molecule of interest is a polypeptide of interest). In preferred embodiments, where a polynucleotide according to the present disclosure comprises one more linker sequences, the polynucleotide encodes the same polypeptide as the equivalent polyribonucleotide lacking the linker nucleotide sequence(s). In some embodiments, the polynucleotide further comprises inverted terminal repeat (ITR) sequences. In some embodiments, the polynucleotide comprises an ITR 5’ to the nucleotide sequence comprising a 5’ splice site (and 5’ to the promoter and / or enhancer sequences, when present). In some embodiments, the polynucleotide comprises an ITR 5’ to the first nucleotide sequence (and 5’ to the promoter and / or enhancer sequences, when present). In some embodiments, the polynucleotide comprises an ITR 3’ to the nucleotide sequence encoding a molecule (e.g. polypeptide) of interest (and 3’ to the stop codon, polyadenylation signal sequence and / or terminator sequence, when present).

[0307] In some embodiments, the polynucleotide comprises an ITR sequence at its 5’ end, and an ITR sequences at its 3’ end. In some embodiments, the first nucleotide of the ITR sequence provided at the 5’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the first nucleotide of the polynucleotide ( / .e. position 1 of the nucleotide sequence of the polynucleotide). In some embodiments, the final nucleotide of the ITR sequence provided at the 3’ end of the polynucleotide is provided within 1 to 25 nucleotides, e.g. within one of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides of the final nucleotide of the polynucleotide ( / .e. the terminal position of the nucleotide sequence of the polynucleotide).

[0308] In some embodiments, the polynucleotide of the present disclosure has a size permitting its delivery as a gene therapy, i.e. in a suitable vector. In some embodiments, the polynucleotide consists of a nucleotide sequence having a size within the packaging limit of a vector for delivering the polynucleotide.

[0309] In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector. In some embodiments, the polynucleotide has a size within the packaging limit of an AAV vector of one of the following serotypes: AAV9, AAV9.45, AAV-PHP.B, AAV1 , AAV2, AAV2i8, AAV5, AAV6, AAV8, AAV10 or AAVrh74.

[0310] In some embodiments, the polynucleotide of the present disclosure consists of fewer than 6,000 nucleotides, e.g. one of <5,000, <4,750, <4,500, <4,250, <4,000, <3,750, <3,500, <3,250, <3,000, <2,750, <2,500, <2,250, <2,000, <1 ,750, <1 ,500, <1 ,250 or <1 ,000 nucleotides.

[0311] The polynucleotide of the present disclosure may comprise 5’ cap, 5’ UTR, 3’ UTR and / or PolyA tail nucleotide sequences.

[0312] In some embodiments, the polynucleotide comprises a 5’ UTR 5’ to (i.e. upstream of, in the context of the nucleotide sequence of the polynucleotide) a start codon.

[0313] In some embodiments, the polynucleotide comprises a 3’ UTR 3’ to (i.e. downstream of, in the context of the nucleotide sequence of the polynucleotide) a stop codon. In some embodiments, the polynucleotide comprises a 3’ UTR 5’ to a polyadenylation signal sequence. In some embodiments, the polynucleotide comprises a 3’ UTR 3’ to a stop codon and 5’ to a polyadenylation signal sequence. In some embodiments, the polynucleotide of the present disclosure comprises one or more nucleotide sequences encoding a selectable marker, to facilitate identification and / or selection of cells comprising / expressing the polynucleotide. Selectable markers include proteins that confer resistance to antibiotics or other toxins, e.g., blasticidin, puromycin, ampicillin, neomycin, methotrexate, or tetracycline, and proteins that complement auxotrophic deficiencies.

[0314] In some embodiments, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an internal ribosome entry site (IRES). In some embodiments, the polynucleotide comprises a nucleotide sequence permitting two or more polypeptides to be translated separately from a single polyribonucleotide.

[0315] The polynucleotides of the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0316] Particular exemplary polynucleotides

[0317] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0318] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0319] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:12; and

[0320] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:90, 89, 88 or 87. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0321] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0322] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:28;

[0323] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:30;

[0324] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:12;

[0325] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:52;

[0326] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘TG’; and

[0327] (vi) a sixth nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:147.

[0328] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0329] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0330] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:4; and

[0331] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:67, 66, 65 or 64. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0332] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0333] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:27;

[0334] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29;

[0335] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:4;

[0336] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ;

[0337] (v) a fifth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:62; and

[0338] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0339] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:121 .

[0340] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0341] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0342] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:4; and (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:82, 81 , 80 or 79. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0343] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0344] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:27;

[0345] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29;

[0346] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:4;

[0347] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ;

[0348] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘GG’; and

[0349] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0350] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:124.

[0351] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0352] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0353] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2; and

[0354] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:89, 88, 87 or 86. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0355] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0356] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:27;

[0357] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29; (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2;

[0358] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ;

[0359] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘TG’; and

[0360] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0361] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:128.

[0362] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0363] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0364] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:3; and

[0365] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:82, 81 , 80 or 79. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0366] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0367] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:27;

[0368] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29;

[0369] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:3;

[0370] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ;

[0371] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘GG’; and

[0372] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0373] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:130.

[0374] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order): (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0375] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2; and

[0376] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:90, 89, 88 or 87. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0377] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0378] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:28;

[0379] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29;

[0380] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2;

[0381] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ;

[0382] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘TG’; and

[0383] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0384] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:132.

[0385] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0386] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0387] (ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2; and

[0388] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:90, 89, 88 or 87. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest. In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0389] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:28;

[0390] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:30;

[0391] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:2;

[0392] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:52;

[0393] (v) a fifth nucleotide sequence consisting of the dinucleotide ‘TG’; and

[0394] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0395] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:133.

[0396] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0397] (i) a nucleotide sequence comprising a 5’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:47, 46, 45 or 44;

[0398] (ii) a human STRN3 exon 8-derived nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:50; and

[0399] (iii) a nucleotide sequence comprising a 3’ splice site, comprising or consisting of a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:67, 66, 65 or 64. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a molecule of interest. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a polypeptide of interest.

[0400] In some embodiments, the polynucleotide comprises (in 5’ to 3’ order):

[0401] (i) a first nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:27;

[0402] (ii) a second nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:29;

[0403] (iii) a third nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NQ:50;

[0404] (iv) a fourth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:51 ; (v) a fifth nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:62; and

[0405] (vi) a sixth nucleotide sequence encoding a molecule of interest.

[0406] In some embodiments, the polynucleotide comprises nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:150.

[0407] In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 187, 188, 189, 190, 191 , 227, 228, 229, 230 or 231 . In some embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% (e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%) sequence identity to SEQ ID NO:147, 121 , 124, 128, 130, 132, 133 or 150.

[0408] Splicing modifiers

[0409] Aspects and embodiments of the present disclosure pertain to splicing modifiers. Splicing modifiers are molecules that influence splicing of polyribonucleotides. Such molecules are reviewed e.g. in Tang et al., Molecules (2021) 26(8):2263 and Schneider-Poetsch et al., The Journal of Antibiotics (2021) 74:603-616, both of which are hereby incorporated by reference in their entirety.

[0410] Splicing modifiers generally reduce / prevent association of factors required for normal post-transcriptional processing (e.g. components of the spliceosome) with RNA. Splicing modifiers typically bind to a nucleotide sequence of a polyribonucleotide and either inhibit or promote the association of RNA-binding proteins and / or non-coding RNAs which function as splicing activators or repressors. Through competitive inhibition of the recruitment of splicing activators and splicing repressors to the polyribonucleotide, splicing modifiers can alter the equilibrium of the splicing-regulatory RNA structures, and thus promote an increase or decrease in skipping or inclusion of an exon and / or retention or excision of an intron in the mature RNA molecule produced by splicing.

[0411] In some embodiments, a splicing modifier is a small molecule or a splice-switching nucleic acid (e.g. a splice-switching oligonucleotide).

[0412] A ‘small molecule’ refers to a low molecular weight (<1000 daltons, typically about 300 to about 700 daltons) organic compound.

[0413] Splice-switching nucleic acids are reviewed e.g. in Haves and Hastings, Nucleic Acids Res. (2016) 44(14): 6549-6563, which is hereby incorporated by reference in its entirety. Splice-switching nucleic acids include e.g. splice-switching oligonucleotides (SSOs). They disrupt the normal splicing of target RNA transcripts by blocking the RNA:RNA base-pairing and / or proteimRNA binding interactions that occur between components of the splicing machinery and pre-mRNA. Splice-switching nucleic acids may be designed to target a specific region of the target transcript, e.g. to promote skipping of exon(s) of interest and / or to promote inclusion of exon(s) of interest and / or to promote retention of introns of interest and / or to promote excision of introns of interest. SSOs often comprise alterations to oligonucleotide sugar-phosphate backbones in order to reduce / prevent RNAseh degradation, such as e.g. phosphorothioate linkages, phosphorodiamidate linkages such as phosphorodiamidate morpholino (PMOs), and may comprise e.g. peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g. 2'0-methyl (2'0Me) and 2'-O-methoxyethyl (MOE) ribose modifications and / or 5’-methylcytosine modifications.

[0414] Small molecule splicing modifiers contemplated in accordance with the present disclosure include RG- 7800 and RG-7916 (also known as risdiplam), and analogs thereof. RG-7800 and RG-7916 bind to the 5’ splice site of intron 7 and exonic splicing enhancer 2 of exon 7 of human SMN2, thereby stabilising the transient double-strand RNA structure formed by the human SMN2 pre-mRNA and U1 snRNP complex, and promoting inclusion of exon 7 in the mature RNA molecule obtained following splicing of pre-mRNA transcribed from human SMN2. RG-7800 and RG-7916 are useful to restore functional human SMN2 protein expression from human SMN2 alleles comprising the spinal muscular atrophy (SMA)-associated polymorphism c.840C>T (which potentiates skipping of exon 7). RG-7916 (DrugBank Acc. No. DB15305) is a close structural analog of RG-7800 having improved potency, pharmacokinetics and safety profile than RG-7800, and is approved by the FDA for the treatment of SMA. Further analogs of RG-7800 and RG-7916 having similar splicing modifier activity include SMN-C2, SMN-C3, SMN-C5 and TEC-1.

[0415] Branaplam (DrugBank Acc. No. DB14918; also known as LMI-070) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from human SMN2. Like RG- 7800 and RG-7916, branaplam binds to the 5’ splice site of intron 7 of human SMN2.

[0416] PK4C9 (also known as homocarbonyltopsentin) is another small molecule splicing modifier that promotes inclusion of exon 7 in the mature RNA expressed from human SMN2. It is thought to bind to the 5’ splice site of exon 7 and TSL2, and improve accessibility of the 5' splice site via stabilising a triloop structure of TSL2.

[0417] Small molecule splicing modifiers that promote inclusion of exon 7 in the mature RNA expressed from human SMN2 are described e.g. in WO 2015 / 173181 A1 and WO 2009 / 151546 A2, which are hereby incorporated by reference in their entirety. Further small molecule splicing modifiers that promote inclusion of exon 7 in the mature RNA expressed from human SMN2 are described e.g. in WO 2022 / 204471 A1 , which is hereby incorporated by reference in its entirety.

[0418] Nusinersen (DrugBank Acc. No. DB13161) is a splice-switching oligonucleotide that promotes exon 7 retention in mature RNA expressed from human SMN2. Nusinersen is an 18-mer 2’-MOe phosphorothioate antisense oligonucleotide that hybridises to intronic splicing silencer site 1 of intron 7, occupying the site and thereby inhibiting the association of the splicing suppressor ribonucleoproteins hnRNPs A1 / A2, thus promoting inclusion of exon 7 in the mature RNA molecule. The splicing modifier according to the present disclosure preferably promotes inclusion of human SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2. Such molecules increase the proportion of RNA molecules comprising human SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from human SMN2 (j.e. relative to the proportion obtained in the absence of the splicing modifier). For conciseness, ‘a splicing modifier that promotes inclusion of human SMN2 exon 7 in RNA obtained following splicing of pre-mRNA transcribed from human SMN2’ may be referred to herein simply as ‘a splicing modifier that promotes human SMN2 exon 7 inclusion’

[0419] In some embodiments, the splicing modifier promotes inclusion of exon 7 in RNA obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T. That is, in some embodiments, the splicing modifier increases the proportion of RNA molecules comprising human SMN2 exon 7 among RNA molecules obtained following splicing of pre-mRNA transcribed from an allele of human SMN2 comprising c.840C>T ( / .e. relative to the proportion obtained in the absence of the splicing modifier).

[0420] In some embodiments, the splicing modifier increases the level of human SMN2 protein comprising amino acids encoded by exon 7 of human SMN2 (i.e. relative to the level detected in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from human SMN2 j.e. relative to the proportion obtained in the absence of the splicing modifier). In some embodiments, the splicing modifier increases the proportion of polypeptides comprising amino acids encoded by exon 7 of human SMN2 among polypeptides expressed from an allele of human SMN2 comprising c.840C>T (j.e. relative to the proportion obtained in the absence of the splicing modifier).

[0421] In some embodiments, the splicing modifier promotes inclusion of the third nucleotide sequence in the product of splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide). In some embodiments, the splicing modifier increases the proportion of molecules comprising the third nucleotide sequence among molecules obtained following splicing of a polynucleotide according to the present disclosure (i.e. where the polynucleotide is a polyribonucleotide).

[0422] In some embodiments, the splicing modifier promotes inclusion of the nucleotide sequence consisting of SEQ ID NO:12 in molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:147. In some embodiments, the splicing modifier increases the proportion of molecules comprising the nucleotide sequence consisting of SEQ ID NO:12 among molecules obtained following splicing of a polyribonucleotide consisting of the sequence of SEQ ID NO:147.

[0423] Splicing modifiers having such functional properties can be identified by analysis e.g. in suitable in vitro assays. Such assays may comprise culturing cells in vitro in the presence or absence of a candidate splicing modifier, and analysing the RNA and / or protein produced after an appropriate period of time for an effect of the candidate splicing modifier to be observed. By way of illustration, cells can be transfected with a vector comprising DNA having the sequence of SEQ ID NO:147 and cultured in the presence or absence of a candidate splicing modifier for a suitable period of time (e.g. 24, 48, 72 hours). RNA can subsequently be isolated from the cells, and analysed (e.g. by qRT-PCR) to determine the level / proportion of mature RNA molecules comprising the nucleotide sequence consisting of SEQ ID NO:12.

[0424] In some embodiments, a splicing modifier that promotes human SMN2 exon 7 inclusion according to the present disclosure is a compound of formula (I) of WO 2015 / 173181 A1. In some embodiments, a splicing modifier that promotes human SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 39 of WO 2015 / 173181 A1. In some embodiments, a splicing modifier that promotes human SMN2 exon 7 inclusion according to the present disclosure is a compound selected from those listed in claim 40 of WO 2015 / 173181 A1 .

[0425] In some embodiments, the splicing modifier according to the present disclosure is selected from: RG- 7916, RG-7800, SMN-C2, SMN-C3, SMN-C5, TEC-1 , branaplam, PK4C9 and nusinersen. In some embodiments, the splicing modifier is selected from: RG-7916, RG-7800, SMN-C2, SMN-C3, SMN-C5 and TEC-1. In some embodiments, the splicing modifier is selected from RG-7916 and RG-7800. In preferred embodiments, the splicing modifier is risdiplam (RG-7916).

[0426] Functional properties of the polynucleotides

[0427] In aspects and embodiments of the present disclosure, the polynucleotides of the present disclosure may be characterised by reference to one or more functional properties.

[0428] In some embodiments, where the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence. In some embodiments, where the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides comprising the third nucleotide sequence.

[0429] In some embodiments, in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion, inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding the molecule of interest, and thus the level of the molecule of interest. In some embodiments, in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion, inclusion of the third nucleotide sequence is promoted in splicing of the polyribonucleotide, favouring the production of mature RNA molecules encoding the polypeptide of interest, and thus expression of the polypeptide of interest at the protein level. Such polynucleotides may be referred to herein as ‘ON-switch’ polynucleotides.

[0430] In some embodiments, cells comprising an ON-switch polynucleotide of the present disclosure substantially do not express the molecule of interest in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion. In some embodiments, cells comprising the ON-switch polynucleotide express the molecule of interest in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion. In some embodiments, cells comprising an ON-switch polynucleotide of the present disclosure substantially do not express the polypeptide of interest in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion. In some embodiments, cells comprising the ON-switch polynucleotide express the polypeptide of interest in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion.

[0431] Exemplary ON-switch polynucleotides according to the present disclosure include polynucleotides according to SEQ ID NOs:121 to 150, 187 to 191 and 227 to 231 .

[0432] Inclusion of the third nucleotide sequence as described herein in the products of splicing of a polyribonucleotide according to the present disclosure can be evaluated using methods that are well known to the person skilled in the art. Such methods include qRT-PCR based methods for the detection and / or quantification of RNA molecules comprising nucleotide sequences of interest.

[0433] For example, cells may be transduced in vitro with a vector comprising a polynucleotide according to the present disclosure and subsequently cultured in vitro in the presence or absence of a splicing modifier that promotes human SMN2 exon 7 inclusion for a period of time appropriate for an effect on splicing of a polyribonucleotide according to the present disclosure to be observed. After such period of time, total RNA may be isolated from the cells, cDNA may be prepared from the total RNA, and the number / proportion of mature RNA molecules comprising the third nucleotide sequence may be evaluated by qPCR using oligonucleotides providing for the specific amplification and / or detection of the products of splicing of the polyribonucleotide (e.g. oligonucleotides hybridising to nucleotide sequences spanning exomexon boundaries). The qPCR analysis may employ oligonucleotides enabling distinction between RNA molecules comprising the third nucleotide sequence, and RNA molecules lacking the third nucleotide sequence. Such qRT-PCR-based methods for analysing the products of splicing of a polyribonucleotide are described in the experimental examples of the present disclosure.

[0434] Expression of a molecule of interest can be evaluated using any suitable technique for the detection and / or quantification of the relevant molecule. Expression of a polypeptide of interest can be evaluated using any suitable technique for the detection and / or quantification of the relevant polypeptide. Such techniques include e.g. antibody-based methods, (for example flow cytometry, immunocytochemistry, western blot, ELISA), fluorescence microscopy and flow cytometry. In some embodiments, expression of a polypeptide of interest can be evaluated as described in the experimental examples of the present disclosure. In preferred embodiments, expression of a polypeptide of interest may be evaluated by flow cytometry.

[0435] Herein, within a plurality / population of polyribonucleotides obtained after splicing that ‘substantially lack’ the third nucleotide sequence, the third nucleotide sequence may be present in fewer than 15%, e.g. one of <10%, <5%, <4%, <3%, <2% or <1% of the polyribonucleotides. Conversely, within a plurality / population of polyribonucleotides obtained after splicing that ‘comprise’ the third nucleotide sequence, the third nucleotide sequence may be present in more than 80%, e.g. one of >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% of the polyribonucleotides. Herein, within a plurality / population of cells that ‘substantially do not express’ a molecule (e.g. polypeptide) of interest, the molecule may be expressed by fewer than 15%, e.g. one of <10%, <5%, <4%, <3%, <2% or <1 % of the cells. Conversely, within a plurality / population of cells that ‘express’ a molecule of interest, the molecule may be expressed by more than 80%, e.g. one of >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% of the cells.

[0436] In some embodiments, cells that ‘substantially do not express’ a molecule (e.g. polypeptide) of interest may display a level of expression of the molecule of interest which is less than 0.2 times, e.g. one of <0.1 times, <0.09 times, <0.08 times, <0.07 times, <0.06 times, <0.05 times, <0.04 times, <0.03 times, <0.02 times, or <0.01 times the level of expression by cells that express the molecule of interest. In some embodiments, cells that ‘express’ a molecule of interest may display a level of expression of the molecule of interest which is greater than 5 times, e.g. one of >10 times, >20 times, >50 times, >100 times, >1000 times, >5000 times or >10000 times level of expression by cells that ‘substantially do not express’ the molecule of interest.

[0437] In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13 in cells comprising a polynucleotide according to the present disclosure following culture in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion is greater than 1 times, e.g. one of >2 times, >3 times, >4 times, >5 times, >10 times, >20 times, >50 times, >100 times, >1000 times, >5000 times or >10000 times the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13 in equivalent cells cultured in the absence of the splicing modifier that promotes human SMN2 exon 7 inclusion.

[0438] In some embodiments, the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13 in cells comprising a polynucleotide according to the present disclosure following culture in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion is less than 100 times, e.g. one of <50 times, <20 times, <10 times, <5 times, <4 times, <3 times, <2 times or <1 times the level of RNA comprising a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13 in equivalent cells not comprising the polynucleotide.

[0439] In some embodiments, the level of a molecule (e.g. polypeptide) of interest in cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion is greater than 1 times, e.g. one of >2 times, >3 times, >4 times, >5 times, >10 times, >20 times, >50 times, >100 times, >1000 times, >5000 times or >10000 times the level in equivalent cells cultured in the absence of the splicing modifier that promotes human SMN2 exon 7 inclusion.

[0440] In some embodiments, the level of expression of a molecule (e.g. polypeptide) of interest by cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion is less than 100 times, e.g. one of <50 times, <20 times, <10 times, <5 times, <4 times, <3 times, <2 times or <1 times the level of the molecule of interest in equivalent cells not comprising the polynucleotide.

[0441] In some embodiments, the proportion of cells expressing a molecule (e.g. polypeptide) of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) cultured in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion is greater than 1 times, e.g. one of >2 times, >3 times, >4 times, >5 times, >10 times, >20 times, >50 times, >100 times, >1000 times, >5000 times or >10000 times the proportion of such cells expressing the molecule of interest cultured in the absence of the splicing modifier that promotes human SMN2 exon 7 inclusion.

[0442] In some embodiments, the proportion of cells expressing a molecule (e.g. polypeptide) of interest within a population of cells comprising a polynucleotide according to the present disclosure (e.g. an ON-switch polynucleotide) cultured in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion is less than 100 times, e.g. one of <50 times, <20 times, <10 times, <5 times, <4 times, <3 times, <2 times or <1 times the proportion of such cells expressing the molecule of interest cultured in the presence of the splicing modifier that promotes human SMN2 exon 7 inclusion.

[0443] The polynucleotides of the present disclosure possess novel and / or improved properties relative to reference transgene expression systems comprising a SMSM-mediated switch derived from human SMN2 exons 6 to 8. Such transgene expression systems comprising a SMSM-mediated switch derived from human SMN2 exons 6 to 8 are described e.g. in Zhang, et al., Gene Ther. (2001) 8: 1532-1538, WO 2022 / 204471 A1 , Monteys et al. Nature (2021) 596: 291-295 or WO 2021 / 163556 A1), and for conciseness may be referred to hereafter as reference human SMN2 exons 6 to 8-derived SMSM- mediated switches.

[0444] In some embodiments, the proportion of RNA molecules comprising the human SMN2 exon 7 / human STRN3 exon 8 / human RPS10 exon 5.5 / human SLC25A17 exon 3-derived nucleotide sequence or third nucleotide sequence according to the present disclosure produced by cells comprising a polynucleotide of the present disclosure cultured in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion is greater than (e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times) the proportion of RNA molecules comprising a human SMN2 exon 7-derived sequence produced by cells comprising a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch, and cultured under the same conditions. That is, in some embodiments the polynucleotides of the present disclosure are more effective at promoting inclusion of the cassette exon as compared to the reference human SMN2 exons 6 to 8- derived SMSM-mediated switch. That is, the polynucleotides of the present disclosure are more responsive to induction of inclusion of the cassette exon in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. It will be appreciated that the functional properties described herein are evaluated employing the same experimental conditions for the evaluation of cells comprising the different polynucleotides ( / .e. the polynucleotide of the present disclosure, and the given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch). For example, the same cell type, the same splicing modifier that promotes human SMN2 exon 7 inclusion, the same concentration of the splicing modifier that promotes human SMN2 exon 7 inclusion, etc. are used, the same culture period is provided, and the cells are analysed in order to determine the proportion of RNA molecules comprising the relevant nucleotide sequence and / or the level of the molecule (e.g. polypeptide) of interest in the same way.

[0445] In some embodiments, the proportion of RNA molecules comprising the human SMN2 exon 7 / human STRN3 exon 8 / human RPS10 exon 5.5 / human SLC25A17 exon 3-derived nucleotide sequence or third nucleotide sequence according to the present disclosure produced by cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion is less than (e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times) the proportion of RNA molecules comprising a human SMN2 exon 7-derived sequence produced by cells comprising a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch, and cultured under the same conditions. That is, the polynucleotides of the present disclosure are less ‘leaky’ with respect to cassette exon inclusion in the products of splicing in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. Leaky production of mature RNA molecules comprising the relevant cassette exon may also be referred to herein as ‘background’ production of such RNA molecules.

[0446] In some embodiments, cells comprising a polynucleotide of the present disclosure cultured in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion display an increased level of protein expression (e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1.1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times) of the molecule (e.g. polypeptide) of interest, compared to the level of protein expression of the molecule of interest by cells comprising a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch, and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at promoting protein expression of the molecule of interest in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. That is, the polynucleotides of the present disclosure are more responsive to induction of expression of the molecule of interest in the presence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. It will be appreciated that for the purposes of such comparison, the different polynucleotides ( / .e. the polynucleotide of the present disclosure, and the reference human SMN2 exons 6 to 8-derived SMSM- mediated switch) preferably encode the same molecule of interest.

[0447] In some embodiments, cells comprising a polynucleotide of the present disclosure cultured in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion display an reduced level of protein expression (e.g. one of <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times) of the molecule (e.g. polypeptide) of interest, compared to the level of protein expression of the molecule of interest by cells comprising a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch, and cultured under the same conditions. That is, in some embodiments the polynucleotide of the present disclosure is more effective at preventing protein expression of the molecule of interest in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. That is, the polynucleotides of the present disclosure are less ‘leaky’ with respect to expression of the molecule of interest in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion, compared to the reference human SMN2 exons 6 to 8-derived SMSM- mediated switch. Leaky expression of the molecule of interest ( / .e. in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion) may also be referred to herein as ‘background’ expression of the molecule.

[0448] In some embodiments, a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises fewer extraneous additional amino acids ( / .e. amino acids additional to the amino acid sequence of the polypeptide of interest) compared to a polypeptide of interest expressed from a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. In some embodiments, a polypeptide of interest expressed from a polynucleotide of the present disclosure comprises a smaller N- terminal tag ( / .e. formed of extraneous, additional amino acids in addition to the amino acid sequence of the polypeptide of interest) than a polypeptide of interest expressed from a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch. That is, in some embodiments, the polynucleotides of the present disclosure provide for the inducible expression of polypeptides of interest comprising fewer extraneous additional amino acids, relative to polypeptides of interest expressed from a given reference human SMN2 exons 6 to 8-derived SMSM-mediated switch.

[0449] Vectors

[0450] The present disclosure provides vectors comprising the polynucleotides according to the present disclosure. It will be appreciated that a vector is also a polynucleotide, and so in some embodiments a polynucleotide according to the present disclosure may be a vector.

[0451] A ‘vector’ as used herein refers to a polynucleotide used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell ( / .e. the vector may be an expression vector). Such vectors may include a promoter sequence operably linked to the nucleotide sequence to be expressed. Vectors may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used in a vector according to the present disclosure.

[0452] The term ‘operably linked’ may include the situation where nucleic acid encoding a molecule (e.g. polypeptide) of interest according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and / or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a molecule of interest under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. Where applicable, the resulting transcript(s) may then be translated into the desired polypeptide(s).

[0453] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno- associated virus vectors, vaccinia virus vectors, baculoviral vectors and herpesvirus vectors), transposonbased vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety.

[0454] In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. In some embodiments, the vector comprises a CMV (e.g. mCMV), SV40, RSV or PGK promoter. In some embodiments, the vector comprises a CMV promoter, CAG promoter, hEF1a promoter, hllbiC promoter, RSV promoter, TK promoter, PGK promoter or CAG minimal promoter. In some embodiments, the vector comprises a CMV promoter, CAG promoter, hEF1a promoter, hllbiC promoter, RSV promoter or CAG minimal promoter. In some embodiments, the vector comprises a hEF1a promoter.

[0455] In some embodiments, a vector is selected based on tropism for a cell type / tissue / organ to which it is desired to deliver the polynucleotide according to the present disclosure. In some embodiments, a vector is selected based on tropism for a cell type / tissue / organ in which it is desired to express the molecule (e.g. polypeptide) of interest. For example, it may be desired to deliver the polynucleotide to, and / or express the molecule of interest in, a cell type / tissue / organ affected by a disease / condition to be treated / prevented in accordance with the present disclosure (e.g. a cell type / tissue / organ in which the symptoms of the disease / condition manifest).

[0456] For example, it might be desirable to deliver a polynucleotide of the present disclosure encoding a molecule (e.g. polypeptide) of interest to neuronal cells / tissue, and vectors having a tropism for such cells / tissue may be employed in such instances (i.e. neurotropic vectors). In preferred embodiments, the vector is an adeno-associated virus (AAV) vector. Adeno-associated virus vectors and their use to vector gene therapy is reviewed e.g. in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378 and Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272, both of which are hereby incorporated by reference in their entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Wang et al., Nat. Rev. Drug Discov. (2019) 18: 358-378. In some embodiments, a vector may be an adeno-associated virus vector described in Li and Samulski, Nat. Rev. Genet. (2020) 12: 255-272.

[0457] In some embodiments, the vector is a self-complementary adeno-associated virus (scAAV) vector. Self- complementary adeno-associated virus vectors are described e.g. in McCarty, Mol Ther. (2008) 16(10):1648-56, which is hereby incorporated by reference in its entirety. Conventional AAV have a single-stranded DNA genome, and depend on the DNA replication machinery of a transduced cell to synthesise the complementary strand, delaying transgene expression. By contrast, scAAV contain complementary sequences that spontaneously anneal upon infection, eliminating the requirement for DNA synthesis in the transduced host cell. Compared to classical, single-stranded AAV vectors, scAAV vectors have been shown to provide for accelerated onset of transgene expression, and an increased level of transgene expression.

[0458] In some embodiments, a vector may be an adeno-associated viral vector of one of the following serotypes: AAV9 (including AAV9 variants AAV-PHP.B and AAV9.45), AAV1 , AAV2 (including AAV2 variant AAV2i8), AAV5, AAV6, AAV8, AAV10 or AAVrh74. In some embodiments, the vector is an AAV9 vector.

[0459] In some embodiments a vector comprises modification to increase binding to and / or transduction of a cell-type of interest ( / .e. as compared to the level of binding / transduction by the unmodified vector). In some embodiments modification is to a capsid protein.

[0460] In some embodiments a vector comprises a capsid protein comprising a cell-targeting peptide. In some embodiments the cell-targeting peptide is a cell-targeting peptide described in Buning and Srivastava, Molecular Therapy: Methods & Clinical Development (2019) 12: 248-265, which is hereby incorporated by reference in its entirety, e.g. a cell-targeting peptide shown in Table 1 , 2, 3 or 4 thereof.

[0461] In some embodiments a vector comprises a capsid protein comprising substitution to one or more tyrosine residues, e.g. one or more surface-exposed tyrosine residues. In some embodiments, one or more tyrosine residues of the capsid protein are substituted with phenylalanine. In some embodiments a vector comprises a capsid protein in which one or more tyrosine residues are substituted with another amino acid as described in lida et al., Biomed Res Int. (2013) 2013: 974819, which is hereby incorporated by reference in its entirety. In some embodiments, a vector may be an adeno-associated virus vector described in Buning and Srivastava, supra. In some embodiments, a vector may be an adeno-associated virus vector described in lida et al., supra.

[0462] In some embodiments the vector comprises a control element for inducible expression of the polynucleotide of the disclosure.

[0463] A sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide by cells of a particular type or tissue. For example, expression may be under the control of a cell type- or tissue-specific promoter.

[0464] Promoters for cell type- or tissue-specific expression of a polynucleotide in accordance with the present disclosure can be selected in accordance with a disease / condition to be treated / prevented. For example, the promoter may drive expression in a cell type / tissue / an organ affected by the disease / condition (e.g. a cell type / tissue / an organ in which the symptoms of the disease / condition manifest).

[0465] In some embodiments, a promoter may provide for expression of the polynucleotide in neuronal cells / tissue. In some embodiments, a promoter may be a neuron-specific promoter (e.g. a CaMKII, NSE or Synl-miniCMV promoter) In some embodiments, a promoter may provide for expression of the polynucleotide in muscle cells / tissue (e.g. cardiac and / or skeletal muscle cells / tissue). In some embodiments, a promoter may be a cardiac or cardiomyocte-specific promoter (e.g. a cTNT, a-MHC or MLC2v promoter). In some embodiments, a promoter may be a skeletal muscle / striated muscle cellspecific promoter (e.g. a MCK, MHCK7 or desmin promoter). In some embodiments, a promoter may be a vascular endothelial cell-specific promoter (e.g. a Tie2 promoter). In some embodiments, a promoter may be a vascular smooth muscle cell-specific promoter (e.g. a SM22a promoter). In some embodiments, a promoter may be a monocyte / macrophage-specific promoter (e.g. a LysM promoter).

[0466] A sequence for controlling expression of the polynucleotide may provide for expression of the polynucleotide in response to e.g. a given agent / signal. For example, expression may be under the control of inducible promoter. The agent may provide for inducible expression of the polynucleotide in vivo by administration of the agent to a subject having been administered with a modified cell according to the disclosure, or ex vivo / in vitro by administration of the agent to cells in culture ex vivo or in vitro.

[0467] In some embodiments a polynucleotide or vector according to the present disclosure may employ a conditional expression system for controlling expression of the polynucleotide by cells comprising the polynucleotide / vector. ‘Conditional expression’ may also be referred to herein as ‘inducible expression’, and refers to expression contingent on certain conditions, e.g. the presence of a particular agent. Conditional expression systems are well known in the art and are reviewed e.g. in Ryding et al. Journal of Endocrinology (2001) 171 , 1-14, which is hereby incorporated by reference in its entirety. Cells

[0468] The present disclosure also provides a cell comprising or expressing a polynucleotide according to the present disclosure. Also provided is a cell comprising or expressing vector according to the present disclosure.

[0469] A polynucleotide according to the present disclosure (e.g. a polyribonucleotide) may be produced within a cell by transcription from a polynucleotide (e.g. a polydeoxyribonucleotide) encoding the polynucleotide.

[0470] The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In preferred embodiments, the cell may be a human cell.

[0471] The present disclosure also provides a method for producing a cell comprising or expressing a polynucleotide / vector according to the present disclosure, the method comprising introducing a polynucleotide / vector of the present disclosure into a cell. In some embodiments, introducing a polynucleotide / vector according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. adeno-associated viral transduction). In some embodiments, the polynucleotide / vector is introduced to the cell in vivo, e.g. by administration of a vector according to the present disclosure (e.g. a viral vector, e.g. an adeno-associated viral vector) to a subject. In some embodiments, the polynucleotide / vector is introduced into cells in culture ex vivo or in vitro.

[0472] Any suitable method may be employed to produce a cell according to the present disclosure. Such methods may comprise nucleic acid transfer for permanent ( / .e. stable) or transient expression of the polynucleotide of the present disclosure. In some embodiments, following introduction into a cell, the polynucleotide may be integrated into or form part of the genomic DNA of the cell. In some embodiments, following introduction into a cell, the polynucleotide may be maintained extrachromosomally.

[0473] Any suitable genetic engineering platform may be used, and include gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol. (2014) 32:189-225, hereby incorporated by reference in its entirety. Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s) / vector(s) into cells include transduction, transfection and electroporation.

[0474] In some embodiments, the methods additionally comprise maintaining the cell under conditions suitable for expression of the polynucleotide / vector by the cell.

[0475] The present disclosure also provides cells obtained or obtainable by the methods according to the present disclosure. Compositions

[0476] The present disclosure also provides compositions comprising the polynucleotides, vectors and cells described herein. In particular, the present disclosure provides pharmaceutical compositions and medicaments comprising the polynucleotides, vectors and cells of the present disclosure.

[0477] Such compositions may comprise the relevant article ( / .e. the polynucleotide / vector / cell) in a formulation suitable for clinical use. The present disclosure is concerned in particular with pharmaceutical compositions / medicaments comprising polynucleotides and vectors according to the present disclosure.

[0478] The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0479] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed.(A)Adejare), 23rd Edition (2020), Academic Press.

[0480] The pharmaceutical compositions / medicaments according to the present disclosure may be formulated for administration to a subject, e.g. administration via a route of administration as appropriate for the nature of the therapeutic agent and the disease to be treated / prevented. In some embodiments, a pharmaceutical composition / medicament may be formulated for parenteral, systemic, topical, intracavitary, intravascular, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal,, oral or transdermal administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion. Medicaments and pharmaceutical compositions may be formulated for administration to a blood vessel, or to a tissue / organ of interest (e.g. a tissue / organ affected by a disease / condition, e.g. a tissue / organ in which symptoms of the disease / condition manifest).

[0481] The pharmaceutical compositions / medicaments may comprise the polynucleotide / vector / cell in a sterile or isotonic medium. The pharmaceutical compositions / medicaments may be provided in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via cannula) to a blood vessel, or a selected region of the human or animal body. The pharmaceutical compositions / medicaments may be provided in solid form, e.g. in lyophilised form.

[0482] The present disclosure also provides methods for producing pharmaceutical compositions / medicaments according to the present disclosure. Such methods may comprise mixing a polynucleotide / vector / cell described herein with a pharmaceutically-acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent. Such methods generally include the step of bringing into association the polynucleotide / vector / cell with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

[0483] Polynucleotides, vectors, cells and compositions according to the present disclosure may be modified and / or formulated to facilitate delivery to, and / or uptake by, a cell type / tissue / organ of interest (e.g. a cell type / tissue / organ in which symptoms of a disease / condition manifest).

[0484] Strategies for targeted delivery of polynucleotides are reviewed e.g. in Li et al., Int. J. Mol. Sci. (2015) 16: 19518-19536 and Fu et al., Bioconjug Chem. (2014) 25(9): 1602-1608, which are hereby incorporated by reference in their entirety.

[0485] In some embodiments, articles of the present disclosure may be encapsulated in a nanoparticle or a liposome. In some embodiments, articles of the present disclosure may be (covalently or non-covalently) associated with a cell-penetrating peptide (e.g. a protein transduction domain, trojan peptide, arginine-rich peptide, vectocell peptide), a cationic polymer, a cationic lipid or a viral carrier.

[0486] Nanoparticles may be organic, e.g. micelles, liposomes, proteins, solid-lipid particles, solid polymer particles, dendrimers, and polymer therapeutics. Nanoparticles may be inorganic, e.g. such as nanotubes or metal particles, optionally with organic molecules added. In some embodiments, a nanoparticle is a nanoparticle described in Chen et al., Mol Ther Methods Clin Dev. (2016) 3:16023, which is hereby incorporated by reference in its entirety. In some embodiments, a nanoparticle is a PLGA, polypeptide, poly(p-amino ester), DOPE, p-cyclodextrin-containing polycation, linear PEI, PAMAM dendrimer, branched PEI, chitosan or polyphosophoester nanoparticle. In some embodiments, polynucleotides and vectors according to the present disclosure comprise modification to incorporate one or more moieties facilitating delivery to, and / or uptake by, a cell type, organ or tissue of interest (e.g. a cell type / tissue / organ in which symptoms of a disease / condition manifest). In some embodiments, polynucleotides or vectors according to the present disclosure are linked (e.g. chemically conjugated to) one or more moieties facilitating delivery to, and / or uptake by, a cell type, tissue or organ of interest.

[0487] Moieties facilitating delivery to, and / or uptake by, cell types, tissues or organs of interest are described e.g. in Benizri et al., Bioconjug Chem. (2019) 30(2): 366-383, which is hereby incorporated by reference in its entirety. Such moieties include e.g. N-acetylgalactosamine (GalNAc), a-tocopherol, cell-penetrating peptides, nucleic acid aptamers, antibodies and antigen-binding fragments / derivatives thereof, cholesterol, squalene, polyethylene glycol (PEG), fatty acids (e.g. palmitic acid) and nucleolipid moieties.

[0488] Articles of the present disclosure may be formulated in a sustained release delivery system, in order to release the polynucleotide, vector, cell or composition at a predetermined rate. Sustained release delivery systems may maintain a constant drug / therapeutic / prophylactic concentration for a specified period of time. In some embodiments, articles of the present disclosure are formulated in a liposome, gel, implant, device, or drug-polymer conjugate e.g. hydrogel.

[0489] In some embodiments, a composition according to the present disclosure may further comprise a splicing modifier that promotes human SMN2 exon 7 inclusion (e.g. as described herein).

[0490] Therapeutic / prophylactic applications

[0491] The polynucleotides, vectors, cells and compositions of the present disclosure find use in therapy and prophylaxis.

[0492] Accordingly, the present disclosure provides a polynucleotide, vector, cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a polynucleotide, vector, cell or composition described herein for use in a method of treating or preventing a disease / condition described herein. Also provided is the use of a polynucleotide, vector, cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of a polynucleotide, vector, cell or composition described herein.

[0493] The intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition or reduce the pathology of a disease / condition. The intervention may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the intervention may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the intervention may prevent progression / development of the disease / condition a later stage (e.g. a chronic stage).

[0494] It will be appreciated that the polynucleotides, vectors, cells and compositions described herein may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from an increase in the level of the molecule (e.g. polypeptide) of interest ( / .e. the molecule of interest encoded by the polynucleotide).

[0495] For example, the disease / condition may be a disease / condition associated with and / or characterised by deficiency / insufficiency of the molecule (e.g. polypeptide) of interest. Deficiency / insufficiency of the molecule of interest may be positively associated with the onset, development or progression of the disease / condition, and / or positively associated with the severity of one or more symptoms of the disease / condition. Deficiency / insufficiency of the molecule of interest may be a risk factor for the onset, development or progression of the disease / condition. The disease / condition may be characterised by a decreased level of expression or activity of the molecule of interest, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition may be characterised by a decrease in the number / proportion / activity of cells expressing the molecule of interest, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue).

[0496] By way of illustration, in some embodiments, a polypeptide of interest may be MeCP2, and the disease / condition to be treated / prevented in accordance with the present disclosure may be a disease / condition caused by deficiency / insufficiency of MeCP2, e.g. Rett syndrome.

[0497] By way of further example, in embodiments wherein the molecule of interest is a molecule capable of inhibiting the expression and / or activity of a target antigen of interest, the disease / condition may be a disease / condition in which the target antigen, or cells comprising / expressing the target antigen are pathologically-implicated, e.g. a disease / condition in which an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing the target antigen is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing the target antigen may be a risk factor for the onset, development or progression of the disease / condition. The disease / condition may be characterised by an increase in the level of expression or activity of the target antigen, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition may be characterised by an increase in the number / proportion / activity of cells expressing the target antigen, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Therapeutic / prophylactic intervention in accordance with the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells comprising / expressing the target antigen.

[0498] By way of further example, the disease / condition may be a disease / condition to be treated by nucleic acid editing, and the molecule of interest may be a constituent protein of an appropriate site-specific nuclease nucleic acid editing system.

[0499] The present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering polynucleotides, vectors, cells and compositions according to the present disclosure to a subject (e.g. a subject in need of treatment). In some embodiments, the methods comprise administering to the subject a splicing modifier that promotes human SMN2 exon 7 inclusion (e.g. a splicing modifier that promotes human SMN2 exon 7 inclusion as described herein).

[0500] Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed.(A)Adejare), 23rd Edition (2020), Academic Press.

[0501] Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intraarterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and / or a tumor.

[0502] Multiple doses of an article of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.

[0503] In some embodiments, the present disclosure provides the articles of the present disclosure for use, uses of articles of the present disclosure, and methods comprising administering: (i) a polynucleotide, vector, cell or composition according to the present disclosure, and (ii) a splicing modifier that promotes human SMN2 exon 7 inclusion, to a subject (e.g. a subject in need of treatment). In embodiments in accordance with aspects of the preceding paragraph, provision of (i) and (ii) may be as a combination therapy. In some embodiments, (i) and (ii) may be provided simultaneously or sequentially.

[0504] Simultaneous administration refers to administration of the two or more agents (e.g. a polynucleotide, vector, cell or composition according to the present disclosure, and a splicing modifier that promotes human SMN2 exon 7 inclusion) together, for example as a pharmaceutical composition containing both agents ( / .e. as a combined preparation), or immediately after each other (e.g. within 1 , 4, 6, 8 or 12 hours), and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administra...

Claims

1. Claims:1 . A polynucleotide, comprising in 5’ to 3’ order:(i) a nucleotide sequence comprising a 5’ splice site;(ii) a human SMN2 exon 7-derived nucleotide sequence having at least 80% sequence identity to a nucleotide sequence according to SEQ ID NO:13; and(iii) a nucleotide sequence comprising a 3’ splice site; wherein the polynucleotide comprises a nucleotide sequence encoding a molecule of interest, and comprises a start codon 5’ to the nucleotide sequence encoding a molecule of interest; wherein the nucleotide sequence comprising a 5’ splice site is not derived from the 5’ splice site at human SMN2 exon 6-intron 6; and wherein nucleotide sequence comprising a 3’ splice site is not derived from the 3’ splice site at human SMN2 intron 7-exon 8.

2. The polynucleotide according to claim 1 , wherein the polynucleotide does not comprise a nucleotide sequence having >80% sequence identity to SEQ ID NO:15.

3. The polynucleotide according to claim 1 or claim 2, wherein the human SMN2 exon 7-derived nucleotide sequence has at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 .

4. The polynucleotide according to any one of claims 1 to 3, wherein the nucleotide sequence comprising a 5’ splice site is derived from the 5’ splice site at human STRN3 exon 7-intron 7, optionally wherein the nucleotide sequence comprising a 5’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:47, 46, 45, 44, 43, 42, 41 or 40.

5. The polynucleotide according to any one of claims 1 to 4, wherein the nucleotide sequence comprising a 3’ splice site is derived from the 3’ splice site at human STRN3 intron 8-exon 9, optionally wherein the nucleotide sequence comprising a 3’ splice site comprises, or consists of, a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:90, 89, 88, 87, 86, 85, 84, 83, 82, 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 94, 93, 92, 91 , 235, 234, 233, 238, 237, 236, 241 , 240, 239, 244, 243 or 242.

6. The polynucleotide according to any one of claims 1 to 5, wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NQ:30, 29, 31 , 32, 33, 34, 35, 36, 37, 38 or 39 immediately 5’ to the human SMN2 exon 7-derived nucleotide sequence.

7. The polynucleotide according to any one of claims 1 to 6, wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:52, 51 , 53, 54, 55, 56, 57, 58, 59, 60 or 232 immediately 3’ to the human SMN2 exon 7-derived nucleotide sequence.

8. The polynucleotide according to any one of claims 1 to 7, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the human SMN2 exon 7- derived nucleotide sequence.

9. A polynucleotide comprising in 5’ to 3’ order:(i) a first nucleotide sequence having at least 80% sequence identity to SEQ ID NO:28, 27 or 26;(ii) a second nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 97, 96 or 95 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NQ:102, 101 , 100, 99, 109, 108, 107, 106, 105, 104 or 103 at its 3’ end;(iii) a third nucleotide sequence having at least 80% sequence identity to SEQ ID NO:12, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 ;(iv) a fourth nucleotide sequence, comprising: (a) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:98, 112, 111 or 110 at its 5’ end, and (b) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:116, 115, 114, 113, 120, 119, 118, 117, 247, 246 or 245 at its 3’ end;(v) a fifth nucleotide sequence: (a) consisting of the dinucleotide ‘TG’ or ‘GG’, or (b) having at least 80% sequence identity to SEQ ID NO:63, 62 or 61 , or (c) encoding a molecule of interest, and comprising ‘TG’ or ‘GG’ at positions 1 and 2; and(vi) where the fifth nucleotide sequence is a nucleotide sequence according to (v)(a) or (v)(b), a sixth nucleotide sequence encoding a molecule of interest.

10. The polynucleotide according to claim 9, wherein the second nucleotide sequence has at least 80% sequence identity to SEQ ID NQ:30, 29, 31 , 32, 33, 34, 35, 36, 37, 38 or 39.11 . The polynucleotide according to claim 9 or claim 10, wherein the fourth nucleotide sequence has at least 80% sequence identity to SEQ ID NO:52, 51 , 53, 54, 55, 56, 57, 58, 59, 60 or 232.

12. The polynucleotide according to any one of claims 1 to 11 , wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:147, 121 , 124, 128, 130, 132, 133, 122, 123, 125, 126, 127, 129, 131 , 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 148 or 149; optionally wherein the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:147, 121 , 124, 128, 130, 132 or 133.

13. The polynucleotide according to any one of claims 9 to 12, wherein when the polynucleotide is a polyribonucleotide, splicing of the polyribonucleotide in the absence of a splicing modifier that promotes human SMN2 exon 7 inclusion yields polyribonucleotides substantially lacking the third nucleotide sequence.

14. The polynucleotide according to any one of claims 1 to 13, wherein the polynucleotide further comprises a promoter sequence 5’ to a start codon; optionally wherein the promoter sequence is a CMV promoter, CAG promoter, hEF1a promoter, hUbiC promoter, RSV promoter, TK promoter, PGK promoter or CAG minimal promoter.

15. A vector comprising a polynucleotide according to any one of claims 1 to 14, optionally wherein the vector is an adeno-associated virus (AAV) vector.

16. A vector comprising a polynucleotide according to any one of claims 1 to 15.

17. The vector according to claim 16, wherein the vector is an adeno-associated virus (AAV) vector.

18. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17, and a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant.

19. A cell comprising a polynucleotide according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17.

20. A method for modifying a cell to express a molecule of interest, comprising:(i) introducing into a cell a polynucleotide according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17; and(ii) subsequently contacting the cell with a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

21. A method for expressing a molecule of interest in a cell, comprising contacting a cell according to claim 19 with a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

22. A polynucleotide according to any one of claims 1 to 15, a vector according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18, for use in a method of medical treatment or prophylaxis.

23. A polynucleotide according to any one of claims 1 to 15, a vector according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18, for use in treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest.

24. Use of a polynucleotide according to any one of claims 1 to 15, a vector according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18, in the manufacture of a medicament for145treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest.

25. A method of treating or preventing a disease or condition that would derive therapeutic or prophylactic benefit from an increase in the level of expression of the molecule of interest, comprising administering to a subject a polynucleotide according to any one of claims 1 to 15, a vector according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18.

26. The polynucleotide, vector, or pharmaceutical composition for use according to claim 22 or 23, the use according to claim 24, or the method according to claim 25, wherein treating or preventing the disease or condition further comprises administering to the subject a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

27. The polynucleotide, vector, or pharmaceutical composition for use according to claim 22 or 23, the use according to claim 24, or the method according to claim 25, wherein the disease or condition is a disease or condition characterised by deficiency of the molecule of interest.

28. A kit, comprising:(i) a polynucleotide according to any one of claims 1 to 15, a vector according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18; and(ii) a splicing modifier that promotes human SMN2 exon 7 inclusion, optionally wherein the splicing modifier that promotes human SMN2 exon 7 inclusion is risdiplam.

Images